Advances in RNA-based cancer therapeutics: pre-clinical and clinical implications

Abstract

Cancer therapy has been revolutionised by the emergence of RNA-based therapeutics, providing several strategies and mechanisms to regulate gene expression via messenger RNA (mRNA), small interfering RNA (siRNA), microRNAs (miRNA), antisense oligonucleotides (ASOs), and RNA aptamers. The present review highlights the recent advances in the preclinical development and clinical applications of RNA-based therapeutics, focusing on the delivery strategies, biological targets, and pharmacological optimisation, together with key clinical data. mRNA therapeutics, especially those adapted from vaccine platforms are being developed for the cancer immunotherapy and protein replacement, while siRNAs and ASOs enable highly specific gene silencing and splice correction. miRNA therapies show potential for diverse oncogenic pathway control, despite ongoing challenges in the delivery and specificity. RNA aptamers are obtaining attention as tumor-targeting agents in the drug delivery systems. Progress in lipid nanoparticles, chemical modifications, and tissue-specific delivery has improved the stability and efficacy of these agents. Early-phase clinical trials report encouraging outcomes in both solid tumours and haematologic malignancies, particularly in overcoming resistance and modulating the tumor microenvironment (TME). Although challenges remain in scalability, immune activation, and deep-tumour penetration, RNA-based strategies are advancing towards integration into clinical oncology. Continued refinement of delivery technologies and targeted trial designs will be critical for translating these therapies into effective, personalized cancer treatments.

Graphical Abstract

Highlights

• RNA-based therapies allow for precise intervention at the genetic and molecular levels of cancer. RNA-based therapies enable targeted intervention at the genetic and molecular levels of cancer.

• Distinct RNA modalities including mRNA, siRNA, miRNA, ASOs, and aptamers offer provide complementary mechanisms for tumor modulation.

• Advances in delivery technologies, particularly lipid nanoparticles (LNPs), have significantly improved RNA stability, targeting, and intracellular uptake.

• Clinical trials report encouraging promising efficacy and tolerability stability of RNA therapeutics in both solid tumours and haematologic malignancies.

• Novel approaches such as self-amplifying RNA (saRNA) and synthetic lethality are emerging as precision strategies to address tumour heterogeneity and drug resistance.

Questions

• How do different types of RNA therapeutics function in cancer treatment?

• What are the major challenges in delivering the delivery of RNA molecules effectively to tumor sites?

• How do chemical modifications improve the performance of RNA-based drugs?

• What clinical evidence supports the use of RNA therapeutics in oncology?

• In what ways can RNA therapies be integrated into personalized cancer care strategies?

Introduction

Cancer is considered a hereditary disorder, and tumour progression is enhanced by clonal selection and evolutionary changes within neoplastic cell populations. This is a kind of concept that is corroborated by the sequential mutation analyses in the human colorectal cancer and other cancer varieties. Studies show that somatic mutations accumulate with age in both human and murine cells and tissues, mainly because of lifelong exposure to endogenous and exogenous DNA-damaging agents, as well as cumulative replication errors including defects in proofreading and mismatch repair [1]. Tumor heterogeneity denotes the coexistence of distinct subpopulations within a single tumour, its metastases, or among histologically identical tumours. These subpopulations demonstrate differences in the genetic composition and phenotypic characteristics, leading to the diverse biological behaviors. The progress in deep sequencing technology has progressively uncovered the pervasive nature of both intra-tumor and inter-tumor heterogeneity. Although some aspects of intra-tumour heterogeneity are captured in the routine pathological assessments, they have yet to be incorporated into therapeutic decision-making [2]. Regarding the significant tumor heterogeneity and poor efficacy of conventional therapeutics, more attention should be directed towards the new therapeutics affecting specific aspects of cancer progression.

For over two centuries, the specific environmental exposures and Lifestyle factors have been associated with the increased cancer risk. Despite this long-standing awareness, a mechanistic understanding of carcinogenesis only emerged in the latter half of the 20th century. Nowdays, the prevention is widely regarded as a practical and effective means of reducing cancer incidence. Yet cancer remains a major global health and economic burden. The World Health Organization (WHO) projects 12 million cancer-related deaths worldwide by 2030 And estimates that 30–40% of these fatalities are avoidable. In 2004 alone, cancer caused 7.4 million deaths 13% of all global mortality of which An estimated 2.2–2.9 million were potentially preventable [3]. In according to the advances in the diagnosis and treatment of cancer, this disease is still incurable and therefore, novel methods such as RNA therapeutics should be developed.

After the elucidation of DNA’s double Helix in 1953, it was focused on deciphering the genetic code and comprehending the mechanisms by which genes control the protein synthesis, culminating in the identification of mRNA, tRNA, and rRNA as essential constituents in this process. Crick’s central dogma, introduced in 1958, delineates the flow of genetic information from DNA to RNA to protein and remains a foundational principle. Early research confirmed the roles of rRNA, tRNA, and mRNA and defined the transcriptional control of gene expression. Initially, RNA was viewed as a passive intermediary or structural component, while proteins were considered the primary functional molecules, including those responsible for regulation. This perspective produced the transcription-factor model of gene regulation, positing that protein-based mechanisms could account for human complexity. Emerging evidence, including data from genome-wide association studies, reveals that several regulatory elements lie outside of protein-coding regions, challenging traditional views and highlighting the broader regulatory role of noncoding parts of the genome [4]. RNA molecules are highly diverse and perform multiple essential cellular functions. They are thought to have been instrumental in the origin of life, as their ability to store genetic information, carry out enzymatic functions, and form complex structures reflects the core features of living organisms. In eukaryotes, RNA is synthesized by three distinct RNA polymerases: RNA polymerase I, II, and III. Each RNA polymerase is responsible for producing specific types of RNA: RNA polymerase I primarily generates ribosomal RNA (rRNA), RNA polymerase II mainly produces mRNA, and RNA polymerase III synthesizes transfer RNA (tRNA), 5 S rRNA, and various other small RNAs. After transcription, newly formed RNA molecules typically undergo processing steps that influence their function, stability, and localization within the cell. Additionally, RNA transcript levels are tightly regulated, both at the synthesis stage and through carefully controlled degradation mechanisms [5]. The therapeutic use of RNA is a relatively recent development, gaining momentum over the past decade as insights into RNA structure and function have expanded. RNA molecules can adopt intricate three-dimensional shapes, enabling them to catalyze biochemical reactions or bind selectively to other RNA, DNA, or proteins. These unique properties have inspired efforts to harness RNA for disease treatment, leading to the development of RNA-based therapies currently in clinical trials for conditions such as cancer and infectious diseases. Therapeutic RNAs are broadly categorized into four types: gene inhibitors, gene modifiers, protein inhibitors, and immunostimulatory agents. Antisense RNAs, first discovered in prokaryotes, work by binding to specific mRNAs, thereby blocking their translation or promoting their degradation. Early studies in bacterial and eukaryotic models showed promise but also revealed the need for high doses of antisense RNA to achieve significant effects. This challenge spurred efforts to enhance RNA expression and engineer catalytic RNAs, such as trans-cleaving ribozymes. Hammerhead and hairpin ribozymes have shown consistent ability to cleave target RNAs and are currently being explored for their therapeutic potential [6].

This review comprehensively examines the recent advances in RNA-based therapeutics, emphasizing their preclinical and clinical implications for cancer treatment. By exploring the molecular basis and functional versatility of mRNA, siRNA, miRNA, ASOs, and RNA aptamers, it elucidates how these strategies are engineered to overcome challenges in delivery, stability, specificity, and immunogenicity. This review highlights both the biological basis and underlying mechanisms making RNA molecules as promising therapeutic agents but also critically examines their translational journey from laboratory research to clinical application, as demonstrated by preclinical models and early-phase clinical trials. It shows how RNA-based strategies are tailored to addres key challenges in the cancer treatment including tumour heterogeneity, drug resistance, and immune evasion and presents a framework for advancing and integrating RNA technologies into precision oncology and personalized medicine. Different kinds of RNA-based therapeutics.

mRNA therapies

Basic information

The mRNA therapeutics represent a promising and versatile approach in the treatment of a wide range of diseases by the delivery of mRNA sequences instructing cells to produce therapeutic proteins directly within the body. Unlike traditional protein-based drugs, mRNA therapeutics provide the potential for the rapid development, customization, and scalable manufacturing. However, their effectiveness relies on addressing several critical challenges, including achieving efficient and targeted delivery to the specific tissues or organs, maintaining adequate and long-lasting protein production, and controlling immune responses that may arise from repeated dosing, particularly in the context of chronic treatments. Advances in the delivery systems, such as lipid-based nanoparticles (LNPs), chemical modifications of mRNA, and tissue-targeting strategies, are actively being developed to unlock the full therapeutic potential of this technology across diverse indications for diseases Such as cancer, metabolic or inflammatory disorders, and genetic diseases. Advancing mRNA therapies faces greater challenges than developing mRNA vaccines, as treatments May require up to 1,000-fold more protein to achieve therapeutic efficacy, in contrast to vaccinations that depend on the immune system’s amplification. The effective mRNA treatments should accurately target specific cells, tissues, or organs, rendering transport efficiency, tissue bioavailability, half-life in bloodstream, and LNP performance significant limiting factors, particularly beyond the liver, which is more readily targeted. Moreover, chronic disorders frequently require several doses; nevertheless, despite enhanced mRNA alterations and complicated LNPs, recurrent administration may stimulate innate immune responses, thereby reducing protein production. Current research and developing technologies explore to tackle these difficulties [7].

The mRNA has the potential to revolutionize areas Such as immunization, protein replacement therapy, and the Management of Hereditary diseases. Since the early preclinical investigations in the 1990s, significant progress has been made in advancing mRNA treatments toward clinical use, mainly due to improvements in mRNA design, synthesis, and delivery mechanisms. In case of therapeutic application, a number of mRNA parameters such as stability, Translatability and immunogenicity should be considered. Multiple factors affect the efficiency of mRNA translation And its stability within the organism. The 5 And 3′untranslated regions (UTRs) are crucial because they interact with RNA-binding proteins and miRNAs, thereby significantly affecting Translation efficiency. Furthermore, codon optimization, which involves replacing rare codons in the coding sequence with more prevalent, synonymous codons, can significantly enhance protein production Levels. Augmenting the 5′ mRNA cap can enhance translation by reducing RNA decapping and increasing the molecule’s resistance to the enzymatic degradation. Chemical modifications to RNA bases can affect the molecule’s ability to stimulate the immune system. Adjusting this immune response can be beneficial depending on the therapeutic goal, for example, in vaccine development, a moderate level of immune activation may enhance effectiveness [8].

mRNA Functions as a temporary conduit for the genetic instructions in protein synthesis, rendering it a viable alternative to DNA or recombinant proteins for therapeutic applications. The preliminary research in the 1990s demonstrated that injected mRNA could elicit measurable protein production and confer therapeutic benefits in animal models. Synthetic mRNA is typically produced through in vitro transcription (IVT); however, this process can generate contaminants such as aberrant transcripts and double-stranded RNA (dsRNA), which may trigger immune responses and reduce the efficiency of protein translation. Although the incorporation of modified nucleosides, such as pseudouridine, into mRNA can diminish immunostimulation and enhance protein production, these alterations are often inadequate on their own. Rigorous purification techniques, especially high-performance liquid chromatography (HPLC), are essential for eliminating impurities and improving mRNA efficacy. The recent studies indicate that integrating HPLC purification with refined sequence design can occasionally obviate the need for nucleotide changes, resulting in highly effective and less immunogenic mRNA. Both modified and unmodified pure mRNAs have demonstrated substantial therapeutic value, including applications including passive vaccination via the administration of antibody-encoding mRNA [9]. Figure 1 illustrates the key scientific advances in the field of mRNA therapeutics and the mechanisms for producing mRNA.

Fig. 1.

A The advancement of mRNA-based therapeutics has progressed through distinct phases. From 1961 to 1990, foundational discoveries laid the groundwork, including the identification of mRNA and early delivery techniques Such as Liposomes. Between 1990 And 2019, the focus shifted to experimental applications in vaccines, immunotherapy, and gene editing, highlighted by key milestones such as the first clinical trials using mRNA-transfected dendritic cells (DCs) and improvements in LNP delivery systems. The COVID-19 pandemic (2019–present) marked a pivotal moment, bringing global recognition to mRNA technology with the rapid rollout of vaccines such as mRNA-1273 and BNT162b. This evolution highlights the platform’s versatility, rapid development capabilities, and broad therapeutic potential in treating infectious diseases, cancer, and genetic conditions. Inpisrted and redrawn from Ref [10]. B Conventional mRNA constructs consist of a 5’ cap, 5’ UTR, gene of interest (GOI), 3’ UTR, and a poly(A) tail. Upon reaching the cytoplasm, this mRNA is directly translated into the target protein. In contrast, saRNA includes additional viral-derived elements, such as non-structural proteins (nsP1–4) and a subgenomic promoter (SGP). After cellular entry, saRNA first produces nsP1–4, which assemble into a replication complex that synthesizes complementary negative-sense RNA. This complex subsequently transitions into a replicase that generates multiple saRNA copies and subgenomic RNAs. These subgenomic RNAs are then efficiently translated, enabling sustained and amplified protein production compared to conventional mRNA. Inpisrted and redrawn from Ref [11]. (Created with Biorender.com)

mRNA therapeutics in cancer

The recent advances in mRNA-based immunotherapy have demonstrated promising potential in oncology, particularly in leveraging DC vaccines and neoantigen-targeted approaches to elicit robust antitumor immune responses. These strategies highlight the ability of mRNA to encode tumor-associated antigens (CEA, KRAS mutations) or pro-apoptotic proteins (apoptin), enabling precise immune activation or direct tumor cell killing. Below, the key preclinical and clinical findings are summarized that highlight the feasibility, safety, and efficacy of these platforms, highlighting their Translational relevance in pancreatic cancer and other malignancies. Autologous DCs loaded with CEA mRNA were effectively produced from three pancreatic cancer patients who had received neoadjuvant chemoradiotherapy and Surgical resection, followed by monthly intradermal immunizations over six months. This process resulted in 1.9–7.1 × 10⁸ DCs, with purity Levels between 20% And 85%, fresh viability ranging from 85 to 94%, And post-thaw viability between 74% And 78%. The vaccines were well tolerated, with no acute toxicities or adverse effects directly associated with the therapy, except for anticipated injection site reactions noted in all individuals. Subtle yet discernible delayed-type hypersensitivity reactions were observed at the injection sites, with histological examination indicating perivascular infiltrates of CD⁴ and CD⁸ T-cells, supporting the activation of DCs. All three patients remained alive without signs of disease recurrence over follow-up periods of three to four years’ post-surgery, which is favorable compared to previous survival rates for similar patient groups. Hence, DC-based immunotherapy in the postoperative setting was both viable and safe, presenting a promising approach that may enhance long-term outcomes, especially regarding minimal residual disease after intensive conventional treatments [12].

Combining a KRAS G12V mRNA vaccine with pembrolizumab resulted in the partial tumor reduction in two end-stage cancer patients carrying KRAS G12V mutations and the HLA-A11:01 allele. The treatment was associated with mild to moderate side effects, Such as fever And injection site discomfort. According to RECIST 1.1 criteria, tumor shrinkage was observed after three treatment cycles in both a patient with pancreatic head cancer and another with non-small-cell lung cancer. Post-vaccination, HLA-A11:01-restricted KRAS G12V-specific CD⁸⁺ T cells were identified, demonstrating polyfunctional cytotoxic activity via the production of IFN-γ and TNF-α. TCR sequencing demonstrated the proliferation of specific T-cell clones that recognized KRAS G12V, and functional studies validated their specificity. The results highlight the efficacy of off-the-shelf, single-neoantigen mRNA vaccines in delivering clinical benefits to the late-stage cancer patients, especially when aligned with high-frequency HLA subtypes such as HLA-A*11:01, indicating a viable approach for expanding immune treatment options [13].

Nonenzymatic 3′-end extension of mRNA using imidazole-activated natural and modified guanosine derivatives has been shown to enhance Translation efficiency, mRNA stability, and apoptosis in cancer cells. Among these, the Sugar-modified 2′O-Me-GMP-2-amino-IM was particularly effective. It led to significantly higher expression of GFP, luciferase, and apoptin compared to control mRNAs. Sanger sequencing confirmed Successful 4G extensions at the mRNA’s 3′ end. Time-course Analyses showed that these modified mRNAs had a longer half-life And higher resistance to exonuclease degradation. In cancer cell viability assays, apoptin mRNA treated with 2′O-Me-GMP-2-amino-IM showed the strongest apoptotic effect, with the lowest IC₅₀ value (1.5 µg). This was accompanied by significant downregulation of EZH2, a key epigenetic regulator involved in suppressing oncogenes. These results suggest that nonenzymatically modified apoptin mRNA could be a promising therapeutic tool. It provides enhanced protein expression, increased mRNA stability, and targeted modulation of cancer-related epigenetic mechanisms [14]. Therefore, these studies illustrate the versatility of mRNA-based strategies in cancer immunotherapy, from DC vaccines eliciting durable T-cell responses to neoantigen-specific vaccines synergizing with checkpoint inhibitors. The observed clinical benefits, including prolonged Survival in Pancreatic cancer and tumor regression in KRAS-mutated cases, highlight the potential of mRNA platforms to address unmet needs in oncology. Furthermore, advancements in mRNA modification, Such as 3′-end extensions, provide additional tools to enhance therapeutic efficacy by optimizing stability and translational capacity. Future research should focus on larger cohorts to validate these findings, explore combination therapies, and refine mRNA design to broaden applicability across diverse cancer types and mutational profiles.

Emerging mRNA-based cancer therapies and vaccines continue to show strong promise. These strategies can modulate immune responses, boost tumor-specific cytotoxicity, and help overcome the immunosuppressive TME. Approaches such as intratumoral mRNA delivery, adoptive T-cell therapies, and DC vaccines are being refined to improve both safety and effectiveness. Progress is being made in preclinical and clinical trials. Research on mRNA vaccine responses in immunocompromised individuals, including cancer patients, reveals key challenges and areas for improvement. In one study, intratumoral injection of survivin mRNA combined with the STAT3 inhibitor stattic significantly improved antitumor effects in mice with colon cancer. This combination triggered strong CD⁸⁺ T cell responses, increased apoptosis, and reduced tumor growth. Tumor volume and weight dropped notably, and survival improved compared to monotherapies. The treatment also lowered levels of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the TME. At the same time, it raised levels of IFN-γ, TNF-α, and IL-12, while decreasing IL-6, IL-10, VEGF, and CCL2. Mechanistic studies showed that stattic suppressed RANKL-induced MDSCs by blocking STAT3 and NF-κB signaling. Immunohistochemistry revealed the lowest Ki67 index and the highest number of apoptotic cells in the combination group. These findings point to a strong immune-activating and tumor-suppressing effect [15]. Elevated expression of GABAA receptor alpha3 (GABRA3), typically confined to the adult brain, was observed to correlate with diminished breast cancer survival, as it activated the AKT pathway to enhance cell migration, invasion, and metastasis. Conversely, the knockdown of GABRA3 significantly reduced these metastatic characteristics without affecting cellular proliferation. An A-to-I RNA-edited form of Gabra3, found only in non-invasive breast cancer cells, was shown to reduce metastasis by lowering Gabra3 surface levels and suppressing AKT signaling. In mouse models, unedited Gabra3 promoted lung metastases, while the edited variant significantly limited spread. Flow cytometry showed that unedited Gabra3 increased cancer stem cell (CD44^high/CD24^low) populations, whereas the edited form did not, suggesting a dominant-negative effect. These results suggest that targeting Gabra3 and enhancing its RNA editing could help prevent breast cancer metastasis [16].

The mRNA electroporation effectively reprogrammed CD⁴⁺ and CD⁸⁺ T cells to transiently express chimeric immunoreceptors targeting ErbB2 And CEA, with expression exceeding 80% of cells within 4–24 h And complete deletion by day 9. Upon encountering antigens, these T cells generated proinflammatory cytokines (IFN-γ, IL-2, TNF-α) and demonstrated selective, dose-dependent lysis of ErbB2 + and CEA + tumor cells, as validated by blocking tests using soluble antigens. Compared to retrovirally transduced T cells, RNA-electroporated T cells showed comparable antigen-specific cytotoxicity after two days, though approximately twice as many were needed to achieve the same level of lysis. The transient nature of RNA-based receptor expression was highlighted as a safety advantage, acting as a built-in “off switch” to reduce the risk of autoimmune reactions. These findings suggest that RNA electroporation provides a rapid, efficient, and safer approach for generating tumor-specific T cells for adoptive immunotherapy [17].

Vaccination using DCs Transfected with autologous tumor mRNA proved feasible And safe in 22 patients with advanced melanoma, with no Major side effects. Vaccine-specific T-cell responses were observed in about 50% of patients. Both intradermal and intranodal injections triggered immune responses, though intranodal delivery offered no clear advantage. Enhanced responses, such as T-cell proliferation, IFN-γ ELISPOT, and delayed-type hypersensitivity reactions, were highlighted in certain patients, with prolonged Survival exceeding 20 months found in many immune responders relative to non-responders. Cytokine tests revealed the release of IFN-γ, TNF-α, IL-5, IL-13, and chemokines such as MCP-1 and MIP-1β, indicating a combination of Th1- and Th2-type immune responses. Although there is evidence of immune activity, objective tumor regressions were limited, highlighting the necessity for further optimization in future studies [18].

Patients with hematologic cancers showed significantly weaker antibody and T cell responses to mRNA COVID-19 vaccines compared to those with solid tumors. Seropositivity rates were 66% vs. 93%, respectively, 36 days after the second dose. By three months, antibody Levels declined sharply, with about 10% of previously seropositive patients, mainly those with blood cancers, becoming seronegative. Most seronegative patients also lacked CD⁴⁺/CD⁸⁺ T cell responses. Seronegativity was linked to factors such as steroid use, anti-CD20 or BTK inhibitor therapy, chemotherapy, and diagnoses such as chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma. These results highlight that while patients with solid tumors mount strong immune responses, those with hematologic malignancies exhibit weaker and shorter-lived immunity, underscoring the need for improved vaccination strategies in this high-risk group [19].

Antibody responses to SARS-CoV-2 following two doses of mRNA COVID-19 vaccines were found to be insufficient in patients with hematologic Malignancies, as 46% of the 67 individuals studied had no detectable Antibodies, classifying them as vaccine non-responders. Particularly poor responses were observed in patients with B-cell CLL, where only 23% developed Antibodies, even though nearly 70% were not undergoing active cancer treatment. Non-responsiveness was associated with age, while no significant differences were observed between responders and non-responders in terms of sex, IgG levels, vaccine type, or treatment status. Quantitative research revealed that non-responders with CLL had significantly lower extinction coefficient ratios compared to non-CLL patients, indicating an almost complete absence of antibody response. These results highlight the heightened vulnerability of individuals with hematologic malignancies to COVID-19 despite vaccination and reinforce the importance of continued non-pharmaceutical precautions, routine post-vaccination antibody monitoring, and exploration of alternative preventive strategies such as booster shots or monoclonal antibody prophylaxis significantly [20].

Preclinical studies using in vitro-transcribed mRNA chimeric antigen receptor T cells (IVT mRNA CAR T) have shown effective, targeted cytotoxicity against various hematologic cancers, including CLL, acute lymphoblastic leukemia, and acute myeloid leukemia (AML), as well as solid tumors including mesothelioma, ovarian cancer, breast cancer, neuroblastoma, glioblastoma (GBM), and melanoma. These studies demonstrated significant tumor reduction, cytokine release, and improved survival in both cell culture and animal models. Clinical trials have further shown that IVT mRNA CAR T cells can induce temporary antitumor responses in both hematologic malignancies and solid tumors such as mesothelioma, pancreatic adenocarcinoma, and metastatic breast cancer. These responses were characterized by partial tumor regression, low incidence of severe toxicity, and reduced on-target off-tumor effects, primarily due to the short-lived nature of mRNA expression. Key limitations included short-lived T cell persistence, poor tumor infiltration, and manufacturing challenges. Future efforts aim to optimize antigen targeting, increase mRNA stability, enhance homing to the TME, and expand clinical trials to assess effectiveness across different cancer types [21].

A synthetic lethal interaction was discovered between the PELO–HBS1L ribosomal rescue complex and the SKI mRNA quality control complex in multiple cancer types. This interaction suggests that disruption of the SKI complex, due to factors such as FOCAD (9p21.3) deletion or microsatellite instability-high (MSI-H) status, creates a dependency on PELO and HBS1L for tumor cell Survival. Knocking down PELO significantly reduced viability in cancer cell Lines with 9p21.3 deletions or SKI complex Mutations, Leading to a 70–74% decrease in tumor growth in xenograft models. The combined loss of PELO and SKI components impaired cell cycle progression, particularly during the S-phase, activated unfolded protein response pathways via IRE1α, and ultimately caused late-stage apoptosis. Functional rescue experiments revealed that only full restoration of the SKI complex, not partial reconstitution of its core subunits, was sufficient to relieve dependence on PELO. These findings highlight PELO and HBS1L as promising therapeutic targets and present a strategy to exploit synthetic lethality in SKI-deficient cancers across diverse genetic backgrounds [22].

Vaccination using autologous DCs transfected with allogeneic prostate cancer (PCa) mRNA was found to be safe And well-tolerated in a cohort of 19 patients with androgen-resistant PCa, with no serious toxicities and only mild local reactions observed. More than half of the patients developed specific T-cell immune responses, as confirmed through ELISPOT, proliferation assays, and T-cell cloning, involving both CD⁴⁺ and CD⁸⁺ populations. Thirteen patients experienced a reduction in prostate-specific antigen (PSA) Levels, And disease stabilization was observed in 11, showing a strong correlation between immune activation and early clinical benefit (P = 0.002, r = 0.68). Booster doses significantly enhanced both immune and biochemical responses, indicating that prolonged or repeated vaccination may be vital for the optimal therapeutic impact. These results suggest that mRNA-loaded DC vaccines represent a promising treatment strategy for PCa by inducing broad, tumor-specific immune responses [23]. A detailed description of using mRNA therapeutics in cancer has been summarized in Table 1. (Figure 2).

Table 1.

Applications of mRNA therapeutics in cancer treatment

Study Focus	Cancer Type/Disease	Therapeutic Approach	Sample Size	Immune Response	Clinical Outcomes	Key Findings	Limitations/Next Steps	Refs
Two-component mRNA vaccines inducing balanced immune responses and combination therapies	PCa, NSCLC, preclinical mouse models	Self-adjuvating two-component mRNA vaccine with free and protamine-complexed mRNA	Preclinical (mice), Phase I/IIa (patients)	Induced antigen-specific CD4+ and CD8+ T cells, B cell responses, cytokines, memory T cells, NK cell activation	Strong anti-tumor effect in mice, complete protection against antigen-positive tumors, delay in tumor growth, synergy with chemo and anti-CTLA-4	mRNA vaccines induce rapid, boostable, balanced innate and adaptive immune responses, can combine synergistically with chemo (docetaxel, cisplatin) and anti-CTLA-4	Need optimization of vaccination schedule, exploration of combination protocols in clinical trials, and understanding NK cell-mediated effects in humans	[24]
Development of STING-activating polymers (PD18 LNPs) to enhance lymphatic delivery and immune activation of mRNA vaccines	Melanoma (OVA-B16F10 mouse model)	STING-activating PD18 polymer in LNPs carrying mRNA vaccine (OVA-mRNA)	Preclinical (mice, cell lines)	Strong CD8+ T cell, effector memory T cell, DC activation, increased cytokines (IFN-β, TNF-α, IL-6), STING pathway activation	Significant tumor growth inhibition, 42% Survival at day 50, complete tumor-free survival in some mice, protection on tumor rechallenge, minimal toxicity	Incorporation of PD18 polymer improved mRNA delivery, endosomal escape, and STING-dependent immune activation, outperforming conventional LNP formulations	Need to test clinically relevant tumor antigens, optimize polymer structures and ratios, and advance toward clinical translation	[25]
Vaccination with autologous DCs transfected with GBM stem cell (GSC) mRNA to target CSCs	GBM (brain cancer)	Autologous DC vaccine loaded with amplified mRNA from patient-derived GSCs	7 patients (compared to 10 matched historical controls)	Specific lymphocyte proliferation responses to GSC lysates, hTERT, and survivin peptides; positive delayed-type hypersensitivity in some	Median progression-free Survival 694 days (vs. 236 in controls), median overall survival (OS) 759 days (vs. 585 in controls), no serious autoimmune events	First clinical demonstration that GSC-targeted immunotherapy is feasible, safe, and potentially improves progression-free survival; personalized vaccines possible using GMP-grown autologous spheres	Limited by small sample size, technical challenges of sphere culture, need for larger phase II trials; further testing needed for clinical validation and optimization	[26]
Systemic vaccination with MART1 mRNA histidylated lipopolyplexes to prevent melanoma progression and metastasis	Melanoma (B16F10 mouse model)	Intravenous injection of MART1 mRNA formulated in histidylated lipopolyplexes (PEG-HpK + HDHE: chol liposomes)	Preclinical (mice, multiple groups)	Induced CD4+ and CD8+ T cell responses, IFN-γ secretion, strong cytotoxic T lymphocyte activity, specific anti-B16 immune response	Significant inhibition of tumor growth, ~ 75% reduction in lung metastases, dose-dependent effect (optimal at 12.5 µg), synergistic effect when targeting both MHC I and II	Demonstrated that optimized mRNA formulation and delivery enhanced immune responses, combining MHC class I and II targeting increased tumor protection	Needs further exploration of delivery routes, translation to human models, testing in more complex or clinical settings, evaluation of long-term immunity and safety	[27]
Impact of timing between systemic cancer treatment and mRNA vaccination on immune responses	Broad: hematologic malignancies, solid tumors	SARS-CoV-2 mRNA vaccine (BNT162b2, BioNTech/Pfizer)	237 oncology patients + 21 healthy controls	IgG spike antibody titers, CD4+ and CD8+ T cell IFN-γ responses, stratified by last treatment (chemo, ICI, TKIs, anti-CD20, cortisone, antibody therapy)	Strongest responses in checkpoint inhibitor patients; poor responses if mRNA vaccine given < 6–12 months after chemo, cortisone, or antibody therapies	Timing of mRNA vaccination is critical: ≥6–12 months post-treatment improves immune responses; B cell depletion strongly impairs humoral responses, though CD8+ T cells remain active	Larger cohort validation needed; recommend waiting intervals for mRNA cancer vaccine trials; explore passive immunization strategies for B cell-depleted patients	[28]
Phase I/IIa trial of self-adjuvanted mRNA vaccine CV9103 targeting multiple PCa antigens	Castration-resistant PCa (CRPC)	Intradermal injection of RNActive® mRNA vaccine encoding PSA, PSCA, PSMA, STEAP1	44 patients (12 phase I, 32 phase IIa)	Cellular responses in 76% (CD4, CD8 T cells), Multi-antigen responses in 58% of responders; humoral responses to PSA in subset	Median overall Survival: 29.3 months (all), 31.4 months (metastatic subgroup); one patient showed > 80% PSA reduction	CV9103 was safe, well-tolerated, and highly immunogenic; multi-antigen targeting reduced immune escape risk; promising survival correlation with immune responders	Larger controlled phase IIb trial initiated (CV9104); need to assess clinical efficacy, biomarker correlation, and combination strategies with immune modulators	[29]
In situ engineering of mRNA-CAR T cells using spleen-targeted LNPs to eliminate melanoma	Melanoma (B16F10 mouse model, pulmonary metastasis model)	Ionizable LNPs (CD3-LNPs) delivering CAR and IL-7 mRNA to generate CAR T And 7CAR T cells in vivo, combined with PD-1 blockade	Preclinical (mice, in vitro & in vivo)	Strong CD8+ T cell activation, IL-7–enhanced T cell proliferation, cytokine secretion (IFN-γ, TNF-α, IL-2), improved tumor infiltration, reduced exhaustion (PD-1)	Significant tumor regression, prolonged survival, reduced pulmonary metastases, no severe cytokine release syndrome (CRS), no acute toxicity	Demonstrated safe, scalable, and effective in vivo CAR T cell generation targeting TRP1 in melanoma; IL-7 co-expression and PD-1 blockade synergistically enhanced efficacy	Needs further testing in larger animal models and human settings; exploration of B cell roles; long-term immunity and biosafety assessments	[30]
Evaluation of CpG-B class oligodeoxynucleotide (CpG2018B) combined with neoantigen mRNA vaccine for antitumor effects	Melanoma (B16-F10 mouse model)	Intratumoral injection of CpG2018B and LNP-encapsulated mRNA neoantigen vaccine	Preclinical (mice, in vitro/ex vivo)	Strong CD4+ and CD8+ T cell activation, IFN-γ, TNF-α, IL-6 secretion, TLR9 pathway activation, B cell stimulation, tumor infiltration	Significant tumor suppression, nearly complete tumor regression with combination therapy, no severe toxicity, stable body weights	CpG2018B promoted innate and adaptive immunity via TLR9; combined with an mRNA vaccine, it enhanced immune infiltration, turned cold tumors hot, and achieved robust tumor control	Requires further mechanistic exploration, long-term safety studies, larger animal models, and translation to human clinical settings	[31]
Combining ImmunER (mRNA adjuvant) with Tetra (multi-TAA mRNA vaccine) to enhance antitumor immunity	PCa (RM-1-PSMA mouse model), plus B16-OVA and TC-1 models	mRNA LNPs encoding TAAs (PSMA, PAP, PSCA, TGM4) + immune-enhancers (4-1BBL, OX40L, CCR7)	Preclinical (mice, in vitro, in vivo)	Increased DC maturation, migration, JAK-STAT1 activation, IL-12, TNF-α, IFN-γ secretion, CD8+ T cell proliferation and memory, stronger cytotoxicity	Significant tumor growth inhibition, prolonged survival, complete remissions in some models, enhanced immune memory, minimal toxicity	Demonstrated that combining ImmunER and Tetra boosts antigen-specific T cell responses, cross-presentation, and durable tumor control; effective across multiple tumor models	Needs further testing in larger animal models, human trials, exploration of optimal dosing and combination strategies for translation to clinical settings	[32]
Safety and immunogenicity of a third (booster) dose of mRNA-1273 COVID-19 vaccine	Solid tumors and hematologic malignancies (diverse cancers)	The third 100 µg dose of the mRNA-1273 vaccine was administered ~ 7–9 months after the primary series	284 cancer patients (130 in detailed subcohort)	Increased SARS-CoV-2 IgG seropositivity from 81.7–94.4%; 19-fold rise in GMTs overall; weaker responses in lymphoid cancers, CD19 CAR-T, anti-CD20-treated patients	Mild, Transient adverse events in 70.4%; very few severe events (< 2%); strong seroconversion (69.2%) in previously negative patients	The booster dose was well tolerated, significantly improved humoral immunity, especially in previously weak responders; highlights the need for tailored boosting in immunocompromised patients	Larger follow-up needed for durability of response, cellular immunity assessment, and optimized booster timing for specific cancer subgroups	[33]
Development of lantern-shaped flexible RNA origami for Smad4 mRNA delivery to suppress tumor growth	Colorectal cancer (SW480, SW620, HT29 mouse models)	Lantern-shaped RNA origami using two RGD-modified circular RNA (circRNA) staples to deliver Smad4 mRNA	Preclinical (in vitro, subcutaneous, and orthotopic mouse models)	High intracellular uptake, strong Smad4 protein translation, downregulation of oncogenes (MYC, VEGFC, CXCL5), reduced cell proliferation, migration, and invasion	Significant tumor growth suppression in vitro and in vivo, reduced clonogenicity and metastasis, low toxicity, enhanced tumor targeting, and lysosomal escape	Demonstrated that flexible RNA origami balances nanolization and translation efficiency, enabling safe, targeted delivery of therapeutic mRNA for solid tumor suppression	Further optimization of stability, exploration of diverse mRNA cargos, development of computer-aided and AI-guided origami designs, translation to clinical studies	[34]
mRNA-LNP vaccination to boost CD8+ T cell responses against HPV-positive oropharyngeal cancer	HPV-positive oropharyngeal squamous cell carcinoma (OPSCC)	Intravenous HPV16 E7 mRNA-LNP vaccine, combined with LAG3/CTLA4 immune checkpoint blockade	Preclinical (C57BL/6J mice, tumor-bearing models, in vitro, scRNA-seq, TCR-seq)	Strong systemic and HPV-specific CD8+ T cell activation; IFN-stimulated, proliferative, effector, and exhausted T cell trajectories identified; robust TCR clonal expansion	Significant tumor suppression, enhanced antitumor activity when combined with checkpoint inhibitors, minimal systemic toxicity, stable body weights, and no organ damage	Demonstrated that combining mRNA-LNP vaccine with LAG3/CTLA4 blockade synergistically boosts effector CD8+ T cells, overcomes exhaustion, and promotes tumor regression	Requires translation to human trials, long-term memory assessment, exploration of optimal vaccine–checkpoint inhibitor combinations for clinical application	[35]
Development of enveloped self-amplifying mRNA CARG-2020 targeting IL-12, IL-17, and PD-L1 pathways to prevent tumor recurrence	Colorectal cancer (MC38), hepatocellular carcinoma (BNL-T3), liver cancer, melanoma (B16-F10), ovarian cancer (models)	Intratumoral delivery of CARG-2020 virus-like vesicle carrying IL-12, IL-17RA antagonist, and PD-L1 shRNA	Preclinical (mice, multiple tumor models)	Strong Th1 activation, increased CD8+ and CD4+ IFN-γ + T cells, reduced Tregs, reduced exhaustion markers, systemic immune activation, long-term memory	Complete eradication of tumors, prevention of recurrence, protection upon tumor rechallenge, abscopal effects on distal tumors, superior survival compared to PD-1 blockade	Combining IL-12, IL-17 inhibition, and PD-L1 knockdown in one delivery system synergistically improves antitumor response, overcomes immune suppression, and prevents relapse	Needs further investigation of tumor-infiltrating cell roles, biomarker identification, and protein/cytokine dissemination safety before human translation	[36]
Phase I/IIa trial of CV9201 mRNA-based cancer immunotherapy in advanced NSCLC	Stage IIIB/IV non-small cell lung cancer (NSCLC)	Intradermal administration of RNActive® mRNA vaccine CV9201 encoding five tumor-associated antigens	46 patients (9 phase I, 37 phase IIa)	Antigen-specific immune responses detected in 63% of evaluable patients (T cells, antibodies); increases in activated IgD + CD38hi B cells; modest and variable T cell responses	Median progression-free survival (PFS) 5.0 months; median OS 10.8 months; 1-, 2-, 3-year OS rates 44.4%, 26.7%, 20.7%; stable disease in 31%	CV9201 was well-tolerated, induced immune responses against multiple NSCLC antigens, but showed limited objective tumor responses, supporting further development and combination strategies	Needs improvement in immunogenicity, possible use of LNP formulations or needle-free injection; future studies combining with checkpoint inhibitors underway (CV9202)	[37]
Development of piperazine-derived ionizable LNPs for enhanced mRNA delivery and cancer immunotherapy	Colorectal cancer (CT26 mouse model), breast cancer (4T1), cervical cancer (HeLa, in vitro)	Intratumoral delivery of IL-12 mRNA loaded in L10-LNP or L7-LNP formulations	Preclinical (mice, in vitro, multiple cell lines)	High CD8+ T cell activation, elevated IFN-γ production, enhanced intracellular uptake, and endosomal escape; improved mRNA expression in tumors	Significant tumor growth inhibition (up to 98.6% with L10-LNP), tumor regression in some mice, minimal toxicity, stable body weight, no major organ damage	Piperazine-based LNPs (especially L10-LNP) showed superior mRNA delivery, transfection, and antitumor efficacy compared to MC3-LNP; a promising candidate for clinical mRNA immunotherapy	Requires further pharmacokinetics, long-term safety studies, larger animal testing, and translational work toward clinical application	[38]

Limitations: Despite their promise, mRNA therapeutics in cancer therapy face several significant limitations. A major hurdle is the efficient and targeted delivery of mRNA to tumor cells, as mRNA is inherently unstable and can be rapidly degraded by nucleases in the bloodstream. LNPs, the most common delivery vehicles, often accumulate in the liver and may elicit immune responses, limiting their use in targeting solid tumors. Additionally, the TME can be immunosuppressive and hostile to mRNA-based interventions, reducing therapeutic efficacy. There is also the challenge of ensuring sustained and controlled protein expression, as mRNA-induced expression is transient. Finally, variability in patient response, potential off-target effects, and manufacturing scalability further complicate clinical translation and widespread adoption.

Fig. 2.

This figure presents the diverse strategies employed in mRNA-based cancer therapy, emphasizing key mechanisms and clinical outcomes. DC vaccines encoding tumor-associated antigens such as CEA and KRAS G12V stimulate strong T-cell responses, contributing to tumor regression and extended Survival. mRNA modifications, Such as 3′-end extensions, improve transcript stability and enhance the expression of pro-apoptotic proteins like apoptin for selective tumor cell killing. Combination therapies, for example survivin mRNA paired with STAT3 inhibitors, help reshape the immunosuppressive TME. Adoptive T-cell therapies using mRNA-electroporated chimeric antigen receptors (CARs) deliver transient but powerful antitumor activity. However, limited vaccine efficacy in hematologic malignancies highlights the need for tailored strategies in immunocompromised patients. Synthetic lethality approaches, such as targeting PELO-HBS1L in SKI-deficient tumors, and promising clinical outcomes in PCa, highlight the adaptability of mRNA platforms in oncology. Despite these advances, challenges remain, Such as the short-lived nature of CAR T-cell effects, inconsistent immune responses in blood cancers, and the suppressive TME. Additional obstacles include complex Manufacturing processes, selecting optimal antigens, and developing personalized neoantigen vaccines. Future directions should prioritize enhancing mRNA stability through advanced 3′ modifications, boosting TME infiltration via checkpoint inhibitors or cytokines, and expanding clinical trials to validate efficacy across cancer types. Strategies such as booster dosing, synthetic lethality targeting, and customized approaches for patients with impaired immunity may further optimize the potential of this evolving therapeutic platform. (Created with biorender.com)

Current challenges and future novelties

Despite the transformative potential of mRNA-based therapies in oncology, several critical challenges remain. First, the delivery of mRNA to the target tissues and cells continues to be a significant challenge. Although LNPs offer efficient delivery to the liver, they are far less effective at delivering mRNA to other organs, particularly solid tumors. TMEs are often hypoxic, immunosuppressive, and physically dense, which further impairs the bioavailability and penetration of mRNA constructs. Moreover, mRNA therapies demand significantly higher levels of protein expression compared to vaccines, which further intensifies delivery challenges. In chronic conditions such as cancer, repeated dosing is often required but is limited by the activation of the innate immune system, which can diminish therapeutic effectiveness with each administration, even when using chemically modified mRNA and advanced delivery platforms. Immunogenicity, often triggered by double-stranded RNA contaminants or incomplete purification, adds another layer of complexity. This is particularly problematic in immunocompromised patients, such as those with hematologic cancers, where responses to mRNA-based therapies and vaccines are often significantly reduced. To overcome these barriers, future research must prioritize the advancement of delivery technologies and the enhancement of mRNA construct stability at the molecular level. saRNA platforms are gaining traction for their ability to produce high protein levels with smaller doses, potentially addressing challenges related to mRNA quantity and immune activation. Their safety and effectiveness have already been demonstrated in the development of COVID-19 vaccines. Unlike conventional non-replicating mRNA (nrmRNA), saRNA’s self-replicating nature enables more efficient protein production and significant dose reduction, sparking growing interest in improving saRNA expression. In an effort, in vitro adaptive passaging of saRNA under exogenous interferon pressure has revealed numerous mutations in nonstructural proteins. Two stable mutations, Q48P and I113F, in the NSP3 macrodomain were found to impair mono-ADP ribose hydrolysis. These changes reduced saRNA replication but increased protein expression compared to wild-type saRNA (wt saRNA). Transcriptome analysis showed reduced activation of dsRNA sensors, leading to a weaker innate immune response. The modified saRNA also caused less translational inhibition and cell death, improving protein expression both in vitro and in vivo. Overall, these findings highlight the potential of genetic modifications to reduce saRNA-induced innate immune responses and cytotoxicity, offering a promising strategy to boost translational efficiency in saRNA-based applications [39].

Incorporating advanced purification methods Such as HPLC, And engineering highly stable 3′-end modifications, including sugar-modified guanosine analogs, can improve half-life and protein expression while reducing immunostimulation. Combinatorial strategies using mRNA vaccines alongside immune checkpoint inhibitors, STAT3 blockers, or agents that remodel the TME have shown superior efficacy in the preclinical models, such as increased apoptosis, reduction of regulatory T cells, and enhancement of CD⁸⁺ T-cell activity. These approaches hold the potential to increase therapeutic depth and broaden applicability to resistant and poorly immunogenic tumors. Moreover, identifying shared or personalized neoantigens and tailoring vaccines based on patients’ HLA types and mutational profiles, such as the KRAS G12V in HLA-A*11:01 individuals, represents a key step toward personalized and precise immunotherapy. To fully harness the potential of mRNA in cancer therapy, several innovative directions should be explored in future studies. First, developing non-viral, tissue-targeted mRNA delivery vehicles that can bypass the liver and effectively home to specific tumors or immune cells (such as DCs or tumor-infiltrating lymphocytes) is crucial. Nanoparticles responsive to tumor-specific stimuli (pH, enzymes) or conjugated with tumor-homing ligands may enhance targeting accuracy. Second, synthetic lethality approaches, such as targeting PELO-HBS1L dependencies in SKI-deficient tumors, provide a precision oncology pathway where mRNA-based systems can be employed to selectively induce tumor cell death. Third, using mRNA platforms in adoptive T-cell therapies, through transient CAR expression via electroporation, provides a safer and more flexible approach for treating solid tumors. This strategy could be further improved by co-delivering cytokine mRNA to locally boost immune responses. Additionally, RNA editing therapies that harness natural A-to-I modifications hold potential for regulating oncogenic pathways, such as Gabra3 in metastatic breast cancer. Finally, longitudinal studies in immunocompromised populations should evaluate immune reconstitution timelines post-vaccination and explore mRNA-based adjuvants or co-delivered immune boosters to optimize vaccine efficacy. These avenues promise to expand the reach, safety, and effectiveness of mRNA therapeutics in cancer care.

Table 3.

The RNA modifications in cancer

RNA Modification Type	Key Molecular Targets	Cancer Type	Main Findings	Clinical Impact	Associated Outcomes	Refs
Adenosine-to-inosine editing	miR-376a*, RAP2A, AMFR	GBM	Editing of miR-376a* was significantly reduced in GBMs, leading to unedited miR-376a* accumulation which promotes invasion by repressing RAP2A and failing to suppress AMFR.	Editing frequency inversely correlated with tumor invasiveness and could serve as a biomarker.	Increased invasiveness, tumor spread, and shorter survival in animal models and patient data.	[179]
Adenosine-to-inosine editing	miR-455-5p, ADAR1, CPEB1	Melanoma	Reduced ADAR1 expression driven by CREB leads to hypo-editing of miR-455-5p, promoting melanoma growth and metastasis via repression of tumor suppressor CPEB1 by unedited miR-455-5p.	Restoration of ADAR1 or edited miR-455-5p suppresses tumor growth and metastasis, potential therapeutic strategy.	Increased tumor volume, lung and lymph node metastasis with unedited miR-455-5p; inhibition observed with edited form.	[180]
Adenosine-to-inosine editing	AZIN1, ADAR1	Gastric Cancer	AZIN1 RNA editing and ADAR1 expression were significantly elevated in tumor tissues and positively correlated; hyper-editing of AZIN1 was associated with advanced tumor stage, lymph node/distant metastasis, and higher TNM stage.	AZIN1 hyper-editing emerged as an independent prognostic factor for overall and DFS; it may guide treatment decisions.	Poor OS and DFS, higher risk of lymph node metastasis (HR for DFS = 4.55; P = 0.0001).	[181]
Adenosine-to-inosine editing	ADAR1, AZIN1, FLNB	Esophageal Squamous Cell Carcinoma (ESCC)	ADAR1 was significantly overexpressed due to gene amplification; it induced hyperediting of AZIN1 and FLNB, promoting tumor growth, invasion, and migration. Edited AZIN1 conferred a gain-of-function phenotype.	ADAR1 overexpression was associated with poor prognosis and was an independent predictor of reduced overall survival.	Enhanced tumorigenicity, increased editing in 69.57% of cases, and shorter survival in patients with high ADAR1 (P = 0.017).	[182]
Adenosine-to-inosine editing	ADAR1, DHFR, miR-25-3p, miR-125a-3p	Breast Cancer	ADAR1-mediated A-to-I editing in the 3′-UTR of DHFR disrupted miRNA binding, upregulated DHFR expression, enhanced mRNA stability, and promoted cell proliferation and methotrexate resistance.	Elevated ADAR1 and editing levels in tumor tissues, a potential target for increasing chemotherapy sensitivity.	Increased proliferation and drug resistance; ADAR1 knockdown reduced cell viability and sensitized cells to methotrexate.	[183]
Adenosine-to-inosine editing	ADAR3, GRIA2, ADAR2	GBM	ADAR3 was shown to inhibit A-to-I editing at the Q/R site of GRIA2 by competitively binding its pre-mRNA, displacing the editing enzyme ADAR2; ADAR3 was significantly overexpressed in tumor tissues compared to adjacent brain.	Elevated ADAR3 correlated with reduced GRIA2 editing and may contribute to increased calcium-permeable AMPA receptors, potentially enhancing tumor invasion.	Reduced GRIA2 editing (73–97%) was observed in all tumors vs. 100% in adjacent tissue; overexpression of ADAR3 correlated with reduced editing and may be a target for therapeutic modulation.	[184]
Adenosine-to-inosine editing	ADAR2, CDC14B, Skp2, p21, p27	GBM	ADAR2 editing was found to inhibit GBM growth by increasing editing and expression of CDC14B, which leads to Skp2 degradation and subsequent stabilization of p21 and p27, causing G1 arrest.	ADAR2 was significantly downregulated in GBMs, and its restoration reduced tumor growth and extended survival in vivo; ADAR2 acts as a tumor suppressor.	Decreased tumor proliferation (~ 40%), reduced Skp2, increased p21/p27, extended survival in mice injected with ADAR2-overexpressing cells.	[185]
tRNA Demethylation	ALKBH3, 1-meA, 3-meC, N6-meA	Pancreatic Cancer	ALKBH3 was shown to demethylate 1-meA, 3-meC, and N6-meA in tRNA, enhancing translation efficiency; knockdown led to RNA methylation accumulation and reduced nascent protein synthesis.	ALKBH3 overexpression supports tumor growth via increased protein synthesis, a potential target for therapy.	Decreased protein synthesis and cell proliferation upon ALKBH3 knockdown in PANC-1 cells.	[186]
m6A Demethylation	ALKBH5, FOXM1, FOXM1-AS	GBM	ALKBH5 was highly expressed in GBM stem-like cells (GSCs) and maintained tumorigenicity by demethylating FOXM1 nascent transcripts; the antisense lncRNA FOXM1-AS facilitated this regulation.	High ALKBH5 expression predicted poor prognosis; ALKBH5 depletion suppressed tumor growth, GSC self-renewal, and expression of FOXM1.	Impaired proliferation, self-renewal, and tumorigenesis upon ALKBH5 or FOXM1-AS knockdown; FOXM1 overexpression rescued these effects.	[187]
m6A Methylation	METTL3, FXR1, TET1	ESCC	METTL3-mediated m6A RNA methylation was shown to guide DNA demethylation via recruitment of TET1 by m6A reader FXR1, leading to altered chromatin accessibility and transcriptional reprogramming.	High expression of METTL3, FXR1, and TET1 correlated with poor prognosis; their expression modulates oncogene and tumor suppressor activity through epigenetic reprogramming.	Enhanced tumor growth altered chromatin states, dysregulation of oncogenes (WNT7B) and tumor suppressors (FAT4), and poorer patient survival.	[188]
m6A Methylation	METTL3, CD40, CD80, Tirap	DC–related Immunity/Cancer Immunotherapy	METTL3-catalyzed m6A modification enhanced translation of immune transcripts (CD40, CD80, Tirap), promoting DC maturation, NF-κB signaling, and T cell activation in a catalytic activity-dependent manner.	METTL3 deficiency impaired DC maturation and function, suggesting its potential as a therapeutic target or adjuvant for cancer immunotherapy.	Impaired co-stimulatory molecule expression and cytokine secretion, decreased T cell activation, reduced NF-κB signaling, and defective DC-based immune responses.	[189]
m6A Methylation	METTL3, METTL14, YTHDC1, KDM3B	-	m6A methylation was shown to co-transcriptionally recruit the histone demethylase KDM3B via YTHDC1 to remove H3K9me2, promoting gene expression through chromatin remodeling.	Demonstrated a direct mechanistic link between m6A RNA modification and histone demethylation, suggesting a new layer of epigenetic control.	Altered m6A dynamics affected chromatin states and transcription, potential implications for developmental diseases and cancer.	[190]
m6A Methylation	METTL3, SPRED2, YTHDF1, CCL22	Melanoma (B16 model), Lung Cancer (LLC model)	Myeloid-specific deletion of METTL3 in macrophages promoted tumor growth, metastasis, and immunosuppression by impairing SPRED2 translation via YTHDF1, activating NF-κB/STAT3, and enhancing M1/M2-like TAM and Treg infiltration.	Loss of METTL3 diminished efficacy of anti–PD–1 therapy and created an immunosuppressive TME, suggesting METTL3 as a therapeutic target to improve immunotherapy.	Increased tumor burden and lung metastasis, reduced survival, elevated cytokine signaling, and resistance to PD-1 checkpoint blockade.	[191]
m6A Methylation	METTL3, METTL14, WTAP, YTHDF2, FTO	-	A novel method, GLORI, enables absolute quantification of single-base m6A methylation across the transcriptome; shows dynamic m6A regulation under stress, enrichment of clustered m6A in regulatory regions, and inverse correlation with translation efficiency.	Establishes GLORI as a high-resolution, quantitative tool for studying m6A-mediated regulation in development, stress, and potentially cancer.	Dynamic and clustered m6A associated with reduced mRNA stability and translation under normal conditions, but enhanced translation under stress.	[192]
m6A Demethylation	FTO, PD-1 (PDCD1), CXCR4, SOX10, YTHDF2	Melanoma	FTO was shown to promote melanoma tumorigenesis and resistance to anti–PD–1 immunotherapy by demethylating m6A in mRNAs of tumor-promoting genes (PDCD1, CXCR4, SOX10), reducing their degradation via YTHDF2.	FTO overexpression correlates with aggressive melanoma; its knockdown sensitizes tumors to anti–PD–1 treatment; combining FTO inhibition with checkpoint blockade may enhance immunotherapy efficacy.	Enhanced tumor growth, metastasis, immune evasion, and resistance to immunotherapy; reversed by FTO knockdown.	[193]
m6A Methylation	METTL3, METTL14, FTO, ADAM19	GBM	m6A RNA methylation was shown to regulate GSC self-renewal and tumorigenesis; knockdown of METTL3 or METTL14 increased GSC growth and tumor formation, while overexpression or FTO inhibition suppressed tumor progression.	m6A machinery components are potential therapeutic targets; FTO inhibitor MA2 suppressed tumorigenesis and extended survival in GSC-xenograft mice.	Enhanced tumor growth and shortened survival upon METTL3/METTL14 knockdown; MA2 treatment reversed effects and improved lifespan.	[194]
m6A Methylation	METTL14, DGCR8, pri-miR126	HCC	METTL14 was shown to suppress HCC metastasis by promoting m6A-dependent processing of pri-miR126 via interaction with DGCR8; reduced METTL14 leads to decreased miR126, enhancing tumor invasion and metastasis.	METTL14 downregulation was significantly correlated with poor prognosis, increased metastasis, and lower recurrence-free and overall survival; METTL14 is a potential prognostic biomarker.	Increased intrahepatic and lung metastasis in METTL14-silenced models; reduced tumor progression with METTL14 overexpression; correlation between METTL14 and miR126 expression in patient tissues.	[195]
m6A Demethylation	FTO, MZF1	Lung cancer	FTO was found to be overexpressed in LUSC and acted as an oncogene by demethylating MZF1 mRNA, enhancing its stability, and promoting malignant behaviors such as proliferation, invasion, and reduced apoptosis.	High FTO expression predicted poor prognosis and contributed to aberrant m6A levels; targeting FTO could represent a therapeutic strategy.	Increased proliferation, invasion, and tumor progression; reduced m6A levels correlated with elevated MZF1 and aggressive tumor phenotype.	[196]
m6A Methylation	METTL3, AXL	Ovarian Carcinoma	METTL3 was upregulated in ovarian carcinoma and promoted proliferation, invasion, EMT, and tumor formation by enhancing AXL translation independent of catalytic activity.	High METTL3 expression correlated with advanced tumor stage, nodal/metastatic spread, and poor overall survival; METTL3 identified as an independent prognostic factor.	Enhanced cell growth, invasion, EMT marker shift, increased tumor burden in vivo; reduced survival in patients with high METTL3 expression.	[197]
m6A Methylation	METTL3, EGFR, TAZ	LUAD	METTL3 promotes translation of oncogenes like EGFR and TAZ in a manner independent of its catalytic activity or m6A reader proteins by recruiting eIF3 to the translation initiation complex.	METTL3 is overexpressed in LUAD; its depletion suppresses cancer cell growth, survival, and invasion; it presents a therapeutic target.	Reduced proliferation, increased apoptosis, and decreased invasion in METTL3 knockdown models; enhanced tumorigenesis with overexpression.	[198]
m6A Methylation	METTL3, HBXIP, let-7 g	Breast Cancer	METTL3 was upregulated by the oncoprotein HBXIP via suppression of the tumor suppressor miRNA let-7 g; in turn, METTL3 promoted HBXIP expression via m6A modification, forming a positive feedback loop that enhanced cancer cell proliferation.	METTL3 and HBXIP were significantly elevated in breast cancer tissues; their expression correlated with malignancy; targeting this feedback loop offers a potential therapeutic avenue.	Increased m6A levels, elevated METTL3 and HBXIP expression, accelerated cell proliferation, and reduced apoptosis in breast cancer cells.	[199]
m6A Methylation	METTL3, SOCS2, YTHDF2	HCC	METTL3 was significantly upregulated in HCC and promoted tumor growth and metastasis by inducing m6A-dependent degradation of SOCS2 mRNA via YTHDF2.	METTL3 overexpression correlated with poor prognosis; targeting METTL3 suppressed proliferation and metastasis in vitro and in vivo.	Increased tumor size, lung metastasis, reduced SOCS2 expression, shorter OS, and DFS in patients with high METTL3.	[200]
m6A Methylation	WTAP, ETS1, HuR, p21/p27	HCC	WTAP was significantly upregulated in HCC and promoted tumor proliferation, invasion, and progression by suppressing ETS1 expression via m6A methylation and HuR-mediated destabilization; ETS1 regulated p21/p27-mediated cell cycle control.	WTAP expression served as an independent predictor of poor prognosis; its inhibition suppressed tumor growth and reversed malignant phenotypes via ETS1-p21/p27 axis.	Increased tumor growth, recurrence, and lower survival in high WTAP/low ETS1 patients; G2/M arrest upon WTAP knockdown; restoration of ETS1 reversed oncogenic effects.	[201]
m6A Methylation	WTAP, mTOR pathway, CD11b/CD14	AML	WTAP was upregulated in ~ 32% of AML patients and promoted leukemic proliferation, inhibited differentiation, and contributed to chemoresistance; WTAP knockdown reduced tumor growth, promoted differentiation, and suppressed mTOR signaling.	WTAP was identified as an oncogenic factor and novel Hsp90 client; its degradation by ganetespib suggests potential therapeutic targeting in AML.	Reduced tumor volume in vivo, increased apoptosis with etoposide, reversal of differentiation block, mTOR signaling inhibition, and enhanced ubiquitination with proteasome inhibition.	[202]
m6A Methylation	METTL14, MYB, MYC, SPI1	AML	METTL14 was highly expressed in AML and inhibited differentiation while promoting proliferation and self-renewal of leukemia stem cells by stabilizing MYB and MYC transcripts via m6A modification; its expression was negatively regulated by SPI1.	Targeting METTL14 reversed myeloid differentiation block and suppressed AML progression, identified as a potential therapeutic target.	Enhanced leukemogenesis, increased MYB/MYC levels, impaired differentiation; METTL14 knockdown prolonged survival and reduced leukemia stem cell frequency.	[203]

Limitations: RNA modifications in cancer, such as N6-methyladenosine (m6A), pseudouridine, And 5-methylcytosine (m5C), present complex limitations due to their context-dependent effects and lack of mechanistic clarity. For example, m6A modifications can both promote and suppress tumorigenesis depending on the cancer type and cellular context, making therapeutic targeting risky and unpredictable. Moreover, the enzymes that add (writers), remove (erasers), or recognize (readers) these modifications often have pleiotropic roles, so modulating their activity can lead to unintended off-target effects across various cellular pathways. Technically, detecting and quantifying RNA modifications with high resolution remains challenging due to the lack of robust, single-base resolution tools, hindering biomarker development and precision targeting. Additionally, the dynamic and reversible nature of RNA modifications complicates efforts to achieve sustained therapeutic effects in cancer treatment

SiRNA

Basics and principles

Following the identification of RNA interference (RNAi) in mammalian cells, there has been much excitement regarding the use of this process for the development of novel disease therapies. RNAi is a natural biological mechanism repressing gene expression post-transcription, triggered by dsRNA molecules, including endogenous miRNAs or synthetic short interfering RNAs (siRNAs). This pathway’s activation enables siRNAs to specifically and effectively suppress the expression of almost any gene, even those previously deemed ‘undruggable’. RNAi possesses significant therapeutic potential, and siRNA-based pharmaceuticals are currently being formulated to target several ailments, including viral infections, genetic disorders, and Malignancies. Significant effort, research, and financial resources have been allocated to the advancement of siRNA therapeutics for clinical use. To date, at Least 22 RNAi-based therapeutics have advanced to clinical trials, with several other candidates undergoing preliminary development phases [40]. siRNA primarily functions within the cytoplasm. The enzyme Dicer, an endoribonuclease, cleaves both endogenous and exogenous dsRNA or short hairpin RNA (shRNA) into small siRNA fragments. These siRNA fragments are then incorporated into an inactive RISC, comprising elements such as Dicer, Argonaute, and the TAR RNA-binding protein (TRBP). Upon entering RISC, the sense strand of the siRNA is eliminated, facilitating the Maturation of the complex into its active state. siRNA typically has a structure of around 21–23 nucleotides in Length, characterized by two-nucleotide overhangs at the 3’ terminus And a phosphorylated 5’ terminus. The silencing mechanism commences when the antisense strand of siRNA aligns properly with its target mRNA. Argonaute, an essential component of RISC, subsequently cleaves the mRNA into fragments, thereby inhibiting the translation of the gene into a functional protein [41].

RNAi has three primary forms: siRNA, shRNA, and miRNA, all of which utilize the RISC complex for mRNA degradation and post-transcriptional gene silencing, albeit with variations in the effectiveness and specificity. miRNAs are produced by RNA polymerase II and then processed into mature forms, which bind imperfectly to target mRNAs, enabling extensive regulatory effects, including translational suppression without mRNA destruction. Conversely, siRNA associates with perfect complementarity, resulting in the precise mRNA cleavage. shRNA, encoded on expression vectors and transcribed by RNA polymerase III, is processed into siRNA-like molecules. Despite these distinctions, both siRNA and shRNA can achieve efficient gene knockdown in vivo, though shRNA is often more potent on a molar basis [42]. Figure 3illustrates the mechanism of action of siRNA.

Fig. 3.

A From Discovery to Regulatory Approval: The Timeline of Small Interfering RNA (siRNA). GalNAc, N-acetylgalactosamine; RISC, RNA-induced silencing complex; RNAi, RNA interference; US FDA, US Food and Drug Administration. Reprinted with permission from Wiley [43]. B The mechanism of action of siRNA in the suppression of gene expression (Created with Biorender.com)

SiRNA therapeutics in cancer

Substantial decreases in EphA2 protein levels and tumor cell proliferation were observed after the restoration of miR-520d-3p, accompanied by pronounced reductions in cell motility, invasion, and intraperitoneal metastatic load in both in vitro and in vivo models. The concurrent application of miR-520d-3p and EphA2-targeting siRNA resulted in the synergistic anti-tumor effects, characterized by a higher reduction in tumor weight, angiogenesis, and proliferation, as well as a significant increase in apoptosis relative to monotherapies. Moreover, miR-520d-3p was identified as a target of EphB2, with elevated expression associated with a positive prognosis, highlighting its dual therapeutic and prognostic significance [44]. Significant decreases in colony formation and invasive potential were observed after Wnt2B silencing using siRNA in A2780 and C13K ovarian cancer cells, alongside increased sensitivity to paclitaxel and cisplatin, respectively. Downregulation of Wnt2B during chemotherapy led to a significant increase in apoptosis and a decrease in cell viability, accompanied by significant suppression of the caspase-9/BCL2/BCL-xL and EMT/p-AKT. Inhibiting Wnt2B was also shown to reduce metastatic potential and drug resistance, highlighting its critical role in promoting ovarian cancer progression and chemoresistance [45].

Substantial tumor suppression was achieved both in vitro and in vivo using the photoresponsive antibody–siRNA combination (PARC), wherein photoirradiation facilitated the controlled release of siRNA, leading to a reduction in intracellular PD-L1 mRNA And an 86% decrease in protein levels. Enhanced immune cell infiltration and activation were observed, Marked by a 2.6-fold increase in CD⁴⁵⁺ lymphocytes, a 3.6-fold increase in CD³⁺ T cells, and elevated levels of IFN-γ and TNFα. These immune responses Led to An approximate 62% reduction in tumor volume following light irradiation, with no notable toxicity or changes in body weight, confirming both strong therapeutic efficacy and favorable biosafety [46].

Ligand–siRNA conjugates targeting CD47, PD-L1, And EGFR achieved high cellular binding, uptake, And effective gene silencing, with conjugation efficiencies reaching up to 90%. These conjugates significantly reduced mRNA and protein expression across multiple cancer cell lines. CD47 targeting enhanced macrophage-mediated phagocytosis, while PD-L1 and EGFR conjugates led to sustained receptor downregulation and reduced cell migration, respectively, without causing toxicity even at higher doses. These bioconjugates outperformed treatments using either ligand or siRNA alone, highlighting the therapeutic potential of this dual-targeting approach in cancer treatment [47].

Following siRNA-mediated knockdown of RhoA and RhoC, a significant downregulation of both mRNA and protein levels was observed in SGC7901 cells. This suppression significantly inhibited cell proliferation and invasion, as evidenced by reduced viability, decreased invasion counts, and an increased apoptosis rate. Tumor growth was remarkably suppressed in BALB/C nude Mice treated with combined RhoA And RhoC siRNA, showing a 51.7% reduction in tumor volume And a 56.8% decrease in tumor weight. These effects were mediated through the PI3K/Akt pathway, demonstrating the therapeutic potential of targeting RhoA and RhoC to inhibit gastric cancer progression [48].

Substantial downregulation of EGFR expression was achieved in EphA2-positive ovarian cancer cells using siRNA-loaded, peptide-functionalized nanogels. This intervention led to a pronounced enhancement in sensitivity to docetaxel, with cytotoxicity increasing approximately eightfold compared to untreated controls. Targeted specificity was confirmed, as EphA2-negative cells showed no significant changes in EGFR expression or chemosensitivity. The nanogel-based delivery system achieved high siRNA encapsulation efficiency (~ 93%) and sustained retention, enabling prolonged gene silencing. It also exhibited minimal nonspecific uptake and low toxicity, highlighting its potential to improve chemotherapy outcomes through targeted gene delivery [49].

Following siRNA transfection in MGC-803 gastric cancer cells, a significant downregulation of survivin mRNA and protein expression was observed. This resulted in a substantial increase in apoptosis and a pronounced accumulation of cells in the G2/M phase, along with reduced G0/G1 phase ratios. Cell proliferation was signficantly inhibited, with growth diminished to approximately 30%. In vivo experiments revealed that tumor sizes in nude mice inoculated with transfected cells were substantially smaller than those in control groups, indicating that survivin silencing effectively curtailed tumor formation and growth [50]. Substantial growth suppression in A549 cancer cells was achieved through siRNA-mediated knockdown of RRM1, RRM2, and PLK1, with efficacy varying based on siRNA sequence, target location, and chemical design. Stealth™ siRNAs have consistently demonstrated enhanced inhibitory effects compared to regular siRNAs and Dicer substrates, delivering greater growth inhibition even at low nanomolar or picomolar doses. The transformation of active conventional siRNAs into Stealth™ duplexes often increased efficacy, But blunt 19-bp siRNAs, regardless of sense strand inactivation, exhibited sequence-dependent effects. These findings highlight the importance of concurrently optimizing siRNA length, chemical modifications, and structural characteristics to enhance silencing efficacy and growth suppression [51].

Folate receptor-targeted combinatorial siRNA polyplexes (TCPs) achieved effective gene silencing and tumor-specific cytotoxicity. These polyplexes formed spherical, uniform nanoparticles with strong siRNA binding, ideal size (~ 104 nm), and reduced Surface charge. Receptor-mediated endocytosis enabled efficient cellular uptake, resulting in 75–94% luciferase silencing in vitro, while transfection with antitumoral EG5 siRNA Led to significant cell death in folate receptor-positive cancer cells. In vivo, intravenous administration of TCPs produced a 46% decrease in EG5 mRNA levels within tumors, demonstrated prolonged systemic circulation, selective tumor accumulation, and no significant changes in body weight or liver and kidney function markers, confirming the safety and effectiveness of this tumor-targeted siRNA delivery approach [52]. Figure 4 illustrates some of the mechanisms employed by siRNA-based therapeutics in cancer suppression.

Fig. 4.

Mechanisms of siRNA-Based Therapeutics in Cancer Therapy. The downregulation of TPX2, EphA2, Wnt2B, RhoA/RhoC, survivin, and RRM1/RRM2/PLK1 by siRNA can effectively suppress tumor cell proliferation. Additionally, siRNA-induced gene silencing can cause cell cycle arrest in the S phase and induce a G2/M phase shift. Increased apoptosis can also be achieved through siRNA. The downregulation of Wnt2B and RhoA/RhoC by siRNA can inhibit the EMT/PI3K/AKT axis, thereby disrupting tumor metastasis. Combining miR-520d-3p with EphA2 siRNA can exert a synergistic effect in suppressing tumor invasion. Moreover, the use of EGFR and Wnt2B siRNA can enhance sensitivity to chemotherapy. Application of PARC, as photoactivated siRNA promotes PD-L1 downregulation to increase T cell infiltration in cancer therapy. (Created with biorender.com)

Current challenges and future novelties

The siRNA-induced immune stimulation remains a concern, especially for the systemic delivery. The activation of Toll-like receptors and subsequent inflammatory cascades can compromise therapeutic efficacy and patient safety. Achieving targeted delivery without nonspecific uptake, especially in non-hepatic tissues, remains a key challenge. Additionally, siRNA therapeutics must overcome cellular barriers, such as endosomal entrapment following internalization. Emerging light-activated or enzyme-responsive carriers show promise for the controlled endosomal escape [53, 54], which can be further leveraged for cancer treatment through the delivery of siRNA. The future development of siRNA therapeutics will significantly depend on the continued innovation of delivery technologies and molecular engineering strategies. Combining biomaterials science with molecular biology holds promise for developing next-generation delivery systems that are more targeted, responsive, and efficient. Smart carriers, triggered by stimuli Such as Light, pH, or enzymes, enable controlled release And site-specific activity, enhancing therapeutic effectiveness while reducing systemic toxicity. Moreover, the optimization of siRNA chemical structures through modifications Such as 2’-O-methylation, phosphorothioate linkages, and locked nucleic acids (LNAs) can significantly improve their stability, reduce immunogenicity, and fine-tune their silencing potency.

The clinical translation of siRNA therapeutics will also benefit significantly from personalized medicine approaches that tailor siRNA sequences to the specific genetic profiles of a patient’s tumor, potentially enhancing therapeutic responses. To further amplify the impact of siRNA-based cancer therapies, future research should explore innovative strategies that integrate siRNA with immunotherapy, gene editing, or cell-based delivery systems. Using engineered immune cells, such as macrophages or T cells, as delivery vehicles for tumor-homing siRNAs could significantly improve target specificity. Combining siRNA with CRISPR-Cas gene editing tools may enable both transient silencing and permanent gene knockouts, yielding synergistic anti-tumor effects. Additionally, the development of multiplexed siRNA cocktails designed to simultaneously target multiple oncogenic pathways or resistance mechanisms could help overcome redundancy in tumor signaling networks. Real-time tracking of siRNA biodistribution and therapeutic response using molecular imaging techniques could offer critical insights into efficacy and pharmacodynamics. Lastly, expanding research into siRNA delivery across the blood–brain barrier and exploring applications in rare or treatment-resistant cancers would significantly broaden the therapeutic landscape and address major unmet clinical needs.

MiRNA modulators

Basics and principles

MicroRNAs (miRNAs) are approximately 22-nucleotide non-coding RNAs that modulate gene expression post-transcriptionally by facilitating mRNA cleavage or inhibiting translation. Initially overlooked due to technical constraints, their significance became apparent with the identification of lin-4 and let-7, which regulate developmental timing in Caenorhabditis elegans by binding imperfectly to the 3′ UTRs of target mRNAs. Subsequent research, including findings in Arabidopsis, indicated that miRNAs originated early in eukaryotic evolution. Since then, numerous miRNAs have been identified in animals, plants, and viruses, with their regulatory roles linked to key biological processes such as developmental timing, cell differentiation, proliferation, apoptosis, cancer progression, and host-pathogen interactions [55]. The synthesis of miRNAs involves multiple cleavage stages occurring in both the nucleus and the cytoplasm. The primary miRNA transcript (pri-miRNA) is cleaved in the nucleus by the Microprocessor complex, which comprises the catalytic proteins Drosha and DGCR8. The stem-loop structure of the pri-miRNA is well-suited for cleavage via interactions between Drosha and the basal UG motif, while the DGCR8 dimer aligns with the apical UGU motif to ensure precise processing. This cleavage produces precursor miRNA (pre-miRNA), which is then transported to the cytoplasm by exportin-5. Once in the cytoplasm, the enzyme Dicer cleaves the pre-miRNA, yielding a double-stranded mature miRNA. One strand, referred to as the guide strand, interacts with the Argonaute protein to form the miRNA-induced silencing complex (miRISC), while the complementary strand, termed the passenger strand or miRNA*, is degraded and destroyed [56].

miRNAs are vital for posttranscriptional regulation, targeting multiple genes often within the same signaling pathway. Their targets include not only mRNAs but also long noncoding RNAs (lncRNAs), pseudogenes, and circular RNAs (circRNAs), through mechanisms such as competing endogenous RNA (ceRNA) interactions. By modulating critical biological processes such as growth, tissue differentiation, proliferation, embryonic development, and apoptosis, miRNAs profoundly influence the progression of various diseases, including aging, cardiovascular disorders, and cancer. Moreover, miRNAs can be encapsulated within exosomes or microvesicles and released into physiological fluids, facilitating long-range intercellular communication. Circulating miRNAs are increasingly recognized as potential biomarkers for cancer diagnosis and prognosis, as well as for other diseases [57].

MiRNAs in cancers

miRNAs have emerged as highly tissue-specific biomarkers capable of accurately determining the tissue origin of cancers, including those of unknown primary origin. Through a decision-tree classification algorithm utilizing the expression profiles of 48 MiRNAs assessed in over 400 tumor samples, this method achieved approximately 90% classification accuracy, with over 85% sensitivity on blinded metastatic samples. This performance surpassed previous mRNA-based classifiers and was maintained across independent validation platforms. Thus, miRNA profiles can function as robust and precise diagnostic tools for identifying tumor origin without reliance on numerous gene markers or complex multifeatured models [58]. A bead-based flow cytometric profiling technique was developed to systematically examine the expression of 217 MiRNAs across 334 human tissue and cancer samples. The analysis revealed that MiRNA expression patterns reflect tumor Lineage And differentiation status, with a general trend of downregulation in tumors compared to normal tissues. Notably, MiRNA profiles were able to accurately classify poorly differentiated tumors where mRNA-based methods fell short. Hierarchical clustering grouped cancers according to their tissue of origin, while cross-species classifiers achieved 100% accuracy in distinguishing normal from tumor samples. Remarkably, only a small subset of miRNAs was required for reliable cancer classification, highlighting their unexpected diagnostic value and linking global miRNA expression to cellular differentiation states [59]. miRNAs miR-373 and miR-520c have been found to enhance tumor cell migration, invasion, and metastasis both in vitro and in vivo by directly inhibiting CD44, a cell surface receptor recognized as a metastasis suppressor. The enforced expression of these miRNAs in non-metastatic breast cancer cells resulted in migratory and invasive characteristics, whereas the inhibition of endogenous miR-373 significantly diminished migration in aggressive cancer cell lines. Furthermore, elevated levels of miR-373 were noted in metastatic breast cancer samples, inversely correlating with CD44 expression, thereby highlighting the role of these miRNAs in facilitating metastatic progression [60].

Serum concentrations of miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a, and miR-429 were measured in patients with epithelial ovarian cancer (EOC) and healthy women. The results indicated that miR-7 and miR-429 were significantly upregulated, while miR-25 and miR-93 were downregulated in EOC. A panel comprising four miRNAs (miR-7, miR-25, miR-93, miR-429) demonstrated 93% sensitivity And 92% specificity in differentiating patients from controls. Elevated serum levels of miR-429 were associated with increased CA125, advanced FIGO stages, and diminished overall survival, serving as an independent prognostic marker. miR-429 inhibited, while miR-7 promoted, the migration and invasion of ovarian cancer cells without influencing proliferation or apoptosis, indicating the diagnostic and therapeutic potential of these circulating miRNAs [61]. The discovery of circulating miRNAs as biomarkers not only enables early and precise diagnosis but also establishes a basis for treatment development by clarifying their functional roles in tumor growth. The fact that miR-429 inhibits while miR-7 enhances the migration and invasion of ovarian cancer cells indicates that these miRNAs may be directly targeted to alter tumor aggressiveness. This combination of diagnostic and functional knowledge facilitates the advancement of miRNA-based therapies, such as miRNA mimics or inhibitors, to limit metastasis or improve therapy response. Consequently, the integration of biomarker identification with mechanistic understanding closes the gap toward miRNA-directed therapy approaches in EOC. A novel delivery platform for antimiR-155 was developed by conjugating peptide nucleic acid antimiRs to a pH (low) insertion peptide (pHLIP). This approach facilitates targeted delivery to the acidic TME while evading systemic clearance. In lymphoma models, this method effectively silenced miR-155, significantly reduced spleen size, decreased liver metastases by twelvefold, preserved normal splenic architecture, and enhanced motor function in mice, all without inducing systemic toxicity or impairing liver and kidney function. Gene expression analyses indicated upregulation of cancer and cell adhesion pathways following miR-155 withdrawal, highlighting the therapeutic potential of pHLIP-conjugated antimiRs in targeting oncomiR-addicted cancers [62].

A unique delivery platform involves the use of pHLIP (pH [low] insertion peptide) conjugates for the targeted administration of antimiRs, including antimiR-155. This technique leverages the acidic TME to specifically deliver therapeutic drugs while avoiding systemic clearance. Significantly, pHLIP-conjugated peptide nucleic acid antimiRs demonstrated potent silencing of miR-155 in lymphoma models, leading to the substantial therapeutic outcomes, including significant reductions in spleen size and liver metastases, preservation of splenic architecture, and enhanced motor function, all without causing systemic toxicity or compromising liver and kidney function. These findings highlight the clinical potential of pHLIP-based delivery systems and highlight their unique advantages over traditional methods, positioning them at the forefront of next-generation RNA therapy platforms alongside other innovative systems like exosome-mimetic vesicles.

miR-221/222 facilitates the EMT in basal-like breast cancer by directly targeting and downregulating ADIPOR1, a receptor predominantly expressed in the less aggressive luminal subtypes. The absence of ADIPOR1 activates the NF-κB/IL6/STAT3 signaling pathway, promoting EMT and invasion. Conversely, overexpression of ADIPOR1 mitigates these effects, indicating its potential as a therapeutic target [63]. Basal-like breast cancer, characterized by its more aggressive clinical behavior compared to the luminal subtype, exhibits elevated levels of the miRNAs including miR-221 and miR-222. These miRNAs promote EMT by downregulating epithelial genes and upregulating mesenchymal markers, thereby enhancing cell migration and invasion. They are transcriptionally induced by the basal-specific transcription factor FOSL1 and are located downstream of the RAS pathway, as their expression diminishes with EGFR or MEK inhibition. miR-221/222 targets the 3′-UTR of TRPS1, a transcriptional regulator of the GATA family that typically suppresses EMT by inhibiting ZEB2. By inhibiting TRPS1 and thereby elevating ZEB2 levels, miR-221/222 reduce E-cadherin expression and promote EMT, contributing to the aggressive phenotype of basal-like breast tumors [64, 65]. Beyond breast cancer, the role of this miRNA has also been evaluated in colorectal cancer. miR-221* and miR-224 are downregulated in metastatic colorectal cancer, with their diminished levels associated with advanced tumor stage, lymph node metastasis, and reduced patient survival. These miRNAs inhibit metastasis by targeting MBD2, which typically represses the metastasis suppressor maspin. Reestablishing miR-221*/224 expression diminishes tumor growth and metastasis, indicating promise for therapeutic and biomarker advancement [66]. Additionally, there is a notable association between the expression of miR-221 and the progression of liver cancer. miR-221 is significantly elevated during the transition from normal liver tissue to hepatocellular carcinoma (HCC) and facilitates tumor proliferation by targeting tumor suppressors such as p27 and DDIT4, the latter serving as a regulator of the mTOR pathway. Inhibition of miR-221 using antagomiRs diminishes tumor cell growth, underscoring its potential as a therapeutic target in hepatic carcinoma [67].

It has been revealed that miRNAs play essential roles in tumor suppression, metastasis, and angiogenesis across several malignancies. miR-34a, miR-34b, and miR-34c have been identified as direct targets of p53, which induce cell cycle arrest and tumor suppression by downregulating genes enhancing proliferation [68]. Concurrently, miR-335 and miR-126 have demonstrated their roles as metastasis suppressors in breast cancer [69]. Specifically, miR-335 suppresses SOX4 and tenascin C, while miR-126 reduces proliferation, both of which are strongly associated with enhanced metastasis-free survival. Furthermore, miR-10b, which is generated by Twist, has been identified as a promoter of metastasis [70]. It accomplishes this by inhibiting HOXD10 and stimulating RHOC. Research using animal models has shown that inhibiting miR-10b with antagomirs can significantly reduce lung metastases while leaving primary tumor development unaffected, underscoring its therapeutic promise. A regulatory network consisting of miR-192, EGR1, and HOXB9 has been demonstrated to regulate the angiogenic switch in ovarian and renal malignancies. The administration of miR-192 via nanoliposomes has shown a significant capacity to block angiogenesis and tumor proliferation, surpassing the efficacy of anti-VEGF antibodies. These findings establish correlations among p53-regulated miRNA networks (miR-34 family), metastasis-inhibiting miRNAs (miR-335, miR-126), pro-metastatic miRNAs (miR-10b), and angiogenesis-inhibitory miRNAs (miR-192). These distinct miRNA pathways collectively regulate essential processes in tumor suppression, metastatic progression, and treatment response. Hence, these discoveries provide a complex, interconnected framework for prospective miRNA-based cancer therapies.

A comprehensive understanding has been achieved regarding the roles of miRNAs in cancer therapy resistance, metastasis, angiogenesis, and oncogenesis, facilitated by diverse molecular processes. For instance, miR-205 has been identified as a radiosensitizer that targets ZEB1 and Ubc13, thereby enhancing the effectiveness of radiation in breast cancer by impeding DNA damage repair [71]. Simultaneously, miR-126 has demonstrated the ability to block metastasis by obstructing endothelial recruitment, angiogenesis, and colonization through the coordinated targeting of IGFBP2, PITPNC1, and MERTK, thus highlighting non-cell-autonomous pathways [72]. Cetuximab resistance in colorectal cancer is mediated by the lncRNA MIR100HG and its associated miRNAs, miR-100 and miR-125b [73]. These miRNAs inhibit negative regulators of Wnt/β-catenin signaling, thereby enhancing Wnt activity and rendering cancers resistant to EGFR-targeted treatment. The miR-17–92 cluster has been identified as a potential human oncogene that synergizes with c-Myc to accelerate B-cell lymphoma progression and inhibit apoptosis, demonstrating that miRNA polycistrons can act as oncogenic drivers [74]. These studies have integrated radiosensitization, metastatic suppression, treatment resistance, and oncogenic acceleration into a cohesive paradigm. Within this framework, miRNAs and their host loci, whether functioning independently or interdependently, have been identified as pivotal elements influencing therapy response, metastatic efficacy, and tumor progression across various cancer models. This indicates that targeting these miRNA-driven regulatory networks may effectively address resistance and enhance cancer therapy outcomes. Figure 5 illustrates the functions of these miRNAs in cancer therapy.

Fig. 5.

The Essential Role of miRNAs in Cancer Therapy. miRNAs are central to cancer biology, acting as both oncogenes and tumor suppressors by regulating crucial processes such as metastasis, angiogenesis, and EMT. Pro-metastatic miRNAs, such as miR-373 and miR-10b, promote tumor invasion by downregulating targets such as CD44 and HOXD10, respectively. In contrast, tumor-suppressive miRNAs like miR-34a and the miR-200 family inhibit EMT and reduce cancer stemness by targeting CD44 and ZEB1. Circulating miRNAs, including miR-7 and miR-429, serve as highly specific and sensitive biomarkers, aiding in accurate tumor classification and prognosis. Furthermore, exosomal miRNAs such as miR-1247-3p and miR-25-3p influence the TME by activating fibroblasts and enhancing vascular permeability, highlighting their key role in cancer progression and the establishment of metastatic niches. (Created with biorender.com)

Current challenges and future perspectives

Despite growing insight into miRNA biology and its impact on cancer progression, several obstacles hinder the translation of miRNA-based discoveries into clinical practice. The pleiotropic and context-dependent nature of miRNA function is a major barrier: a single miRNA can regulate multiple transcripts, and its effects vary across tissues and pathologies, complicating outcome prediction and the development of universal therapies. Tumour heterogeneity and dynamic changes in miRNA expression during disease evolution or treatment further undermine their utility as stable biomarkers or therapeutic targets. Future research should integrate miRNA profiles with additional molecular markers to refine cancer classification, predict therapeutic response, and guide personalised treatment. Advanced computational modelling and artificial intelligence can help decode miRNA-regulatory networks by analysing interactions with coding and non-coding RNAs, transcription factors, and epigenetic modifiers. Multi-dimensional diagnostic platforms that combine circulating miRNAs, exosomal cargo, and gene-expression data could enhance early detection and real-time monitoring of disease progression and treatment efficacy. Identifying miRNAs that drive treatment resistance such as those influencing DNA-repair or drug-metabolism pathways may enable combination regimens in which miRNA modulators are administered alongside chemotherapy, radiotherapy, or immunotherapy. Such strategies could reverse resistance and restore sensitivity, particularly in tumours prone to recurrence or poor prognosis. Additional avenues include exploring non-canonical miRNA biogenesis pathways in rare or treatment-refractory cancers, and elucidating miRNA-mediated communication within the TME via exosomes and interactions with stromal, immune, and endothelial cells. Investigating synergistic networks such as the co-regulation of epithelial–mesenchymal transition and angiogenesis by the miR-200 family, miR-126, and miR-192 may uncover new therapeutic targets. CRISPR-based editing or epigenetic modulation could precisely alter miRNA loci in vivo, allowing rigorous functional testing in pre-clinical models. Future clinical trials must stratify patients according to miRNA-expression signatures, assess miRNA therapeutics in combination with standard-of-care treatments, and monitor long-term safety. Such efforts could elevate miRNAs from molecular novelties to central components of personalised oncology. Importantly, the dual role of miRNAs as oncogenes or tumour suppressors necessitates delivery systems that minimise off-target gene silencing and ensure therapeutic precision.

Antisense oligonucleotides (ASOs)

Basics and principles

ASOs, initially established as a therapeutic strategy by Stephenson And Zamecnik in 1978, are synthetic entities capable of modulating the expression of nearly any RNA target, providing a versatile and promising therapeutic option. Advancements such as next-generation sequencing and automated manufacturing have made ASOs relatively cost-effective to produce, with decades of research contributing to improved pharmacological efficacy and safety. While ASOs are primarily used to treat neurological and metabolic disorders, their role in cancer therapy is expanding, especially following the FDA approval of imetelstat for specific myelodysplastic syndromes. However, broader application in oncology still faces key challenges, including target selection, overcoming biological barriers, ensuring adequate tumor biodistribution, preventing enzymatic degradation, and achieving efficient cellular uptake [75]. Recent advancements in ASO-based treatments have garnered considerable interest in pharmacological research for several disorders. As of now, the U.S. Food and Drug Administration (FDA) has sanctioned four ASO therapeutics: Vitravene, indicated for cytomegalovirus (CMV) retinitis in immunocompromised patients; Kynamro, for the treatment of homozygous familial hypercholesterolemia; Exondys51, designed for Duchenne muscular dystrophy (DMD); and Spinraza, intended for spinal muscular atrophy (SMA). These successful clinical translations demonstrate the extensive potential of ASOs, which function by causing specific mRNA degradation, exon skipping, or exon retention at either the mature or pre-mRNA level to address diverse disorders [76].

ASOs can modulate gene expression via several ways. One key mechanism of action for ASOs involves RNase H-mediated cleavage, where ASOs bind to complementary mRNA sequences to form DNA-mRNA duplexes that recruit RNase H, leading to selective mRNA degradation and reduced gene expression, particularly when using oligodeoxyribonucleotides. Alternatively, ASOs can act as steric blockers, physically preventing proteins from interacting with critical mRNA regions Such as the 5′ cap, ribosome binding site, or Translation initiation site. ASOs can also bind to pre-mRNA to modulate splicing, enabling exon skipping, exon inclusion, or intron retention. These splicing modifications can Shift the reading frame, allowing production of partially functional proteins, as demonstrated in treatments for DMD. FDA-approved drugs Like Exondys 51 And Vyondys 53 use this strategy to skip mutated exons and restore protein function. Additionally, ASOs can target exonic or intronic silencers to recover exons lost due to disease-associated exon skipping [77].

ASOs in cancer

HER-2 ASOs (AS HER-2 ODNs) was utilized alongside chemotherapeutic drugs to treat breast cancer cells, indicating that HER-2 overexpression was unnecessary for chemosensitization. Multiple breast cancer cell lines, exhibiting both elevated and diminished HER-2 expression, were subjected to AS HER-2 ODNs aimed at the HER-2 mRNA start codon, leading to substantial decreases in HER-2 protein levels. Notable improvements in drug sensitivity were observed when combined with agents like doxorubicin, taxol, and vinblastine, with the degree of fold sensitization varying among different cell lines. The combination of AS HER-2 ODNs and taxol synergistically increased apoptosis, as shown by flow cytometry and protein studies of apoptosis markers. These data highlight that successful therapy is not confined to tumors with heightened HER-2 levels, hence expanding the potential use of AS HER-2 ODN-based treatments to a broader spectrum of breast malignancies [78]. An ASO aimed at cIAP-1 was produced to diminish this inhibitor of apoptosis protein in PCa cells, especially in the PC3 and DU145 cell lines. Using a liposomal transfection method, efficient uptake of ASOs was achieved, leading to a marked reduction in cIAP-1 expression, as confirmed by Western blot analysis, without affecting the levels of cIAP-2 or XIAP. Although spontaneous apoptosis increased in both tested cell lines, overall cell viability remained relatively unchanged. Notably, treatment with paclitaxel or caffeic acid phenethyl ester did not enhance apoptosis. However, AO pre-treatment sensitized PC3 cells to apoptosis triggered by Fas antibody and TNFα. These findings suggest that cIAP-1 downregulation can overcome resistance to receptor-mediated apoptotic pathways, though it does not influence resistance to mitochondrial-mediated apoptosis, highlighting a promising strategy to sensitize hormone-resistant prostate cancer cells to specific apoptotic signals [79].

Substantial sensitization of head and neck cancer cells to chemotherapeutic drugs was accomplished with HER-2-targeted ASOs. ASO therapy alone was shown to suppress HER-2 protein expression and improve the efficacy of medicines Like taxol, doxorubicin, cisplatin, vinblastine, And 5-fluorouracil, irrespective of intrinsic HER-2 levels. Cell lines with decreased HER-2 expression showed notable improvements in drug sensitivity, with the degree of enhancement varying depending on the drug and cell type. When ASOs were combined with chemotherapy, apoptosis was significantly induced, as indicated by increased poly(ADP-ribose) polymerase (PARP) cleavage and reduced Bcl-2 levels, suggesting a synergistic apoptotic effect. Importantly, HER-2 overexpression was not required for effective sensitization, highlighting the therapeutic potential of ASO-based combination therapies in breast cancer. This approach may enable lower chemotherapy doses, potentially reducing associated toxicities while maintaining efficacy [80].

Nusinersen (Spinraza) is the inaugural FDA-approved therapy for SMA, a hereditary condition resulting from Mutations on chromosome 5q that create a lack in survival motor neuron (SMN) protein. Nusinersen, as a modified ASO, has shown effectiveness in enhancing motor function across different forms of SMA, according to evidence from open-label and randomized controlled studies. It is often well tolerated, with prevalent side effects comprising respiratory infections, headaches, back discomfort, constipation, and post-lumbar puncture syndrome. Although phase III study outcomes endorse its therapeutic efficacy, obstacles persist regarding its intrathecal delivery and elevated expense. Nusinersen is a safe and effective treatment for SMA patients of all ages [81]. The success of nusinersen in treating SMA highlights the growing clinical potential of ASOs as targeted genetic therapies. By modulating RNA splicing to restore functional protein production, nusinersen exemplifies how ASOs can precisely correct pathogenic gene expression. This same principle is increasingly being applied in oncology, where ASOs can silence oncogenes, inhibit miRNAs like miR-155, or alter splicing in cancer-driving transcripts. The therapeutic promise of ASOs in cancer is further enhanced by advances in delivery platforms such as pHLIP conjugates and exosome-mimetic vesicles, offering tumor-specific targeting and minimizing off-target effects. Thus, lessons learned from nusinersen’s development and clinical use pave the way for ASO-based cancer therapies.

An ASO targeting Livin was developed and applied to Human bladder cancer 5637 cells, resulting in significant downregulation of Livin mRNA and protein levels, as confirmed by RT-PCR, Western blotting, and immunofluorescence. Cell growth was significantly inhibited in a dose-dependent manner, with the highest inhibition rate Surpassing 90%. Apoptosis was notably increased, as demonstrated by flow cytometry and ultrastructural changes observed via electron microscopy, along with elevated caspase-3 activity, indicating that apoptosis was mediated through the caspase-3 pathway. In vivo, Livin ASO-treated xenografts in nude mice showed reduced tumor volume and weight, increased apoptotic activity as revealed by TUNEL staining, and higher caspase-3 expression. These results confirm the anticancer potential of Livin ASO, suggesting it effectively suppresses bladder cancer growth by promoting caspase-3–dependent apoptosis and may offer a promising strategy for gene therapy in bladder cancer [82]. Substantial anti-tumor effects were attained with the use of ASOs to facilitate the insertion of the TRA2β poison exon (TRA2β-PE), resulting in reduced TRA2β protein levels and enhanced production of TRA2β–PE–containing lncRNAs that sequester nuclear proteins. Significant transcriptome changes were noted, affecting RNA processing, mTOR, and p53 signaling pathways, resulting in reduced cancer cell viability in Many tumor types, including breast, glioma, And prostate Malignancies. Enhanced apoptosis And decreased proliferation were seen in 2D cultures, 3D organoids, and in vivo xenograft models, with negligible effects noted upon direct TRA2β knockdown by siRNAs, suggesting that both protein depletion and lncRNA function play a role in the therapeutic impact. These findings highlight TRA2β-PE–targeting ASOs as a viable approach to inhibit oncogenic splicing factors and influence cancer cell viability [83].

A Generation-2.5 ASO, ISIS581088, was engineered to target the androgen receptor (AR) in PCa, demonstrating a substantial reduction in AR mRNA and protein levels in both castration-naive and CRPC models. Systemic administration of the ASO effectively suppressed both full-length AR and its splice variants, leading to reduced tumor growth, decreased cell proliferation, and increased apoptosis. These antitumor effects were comparable to those achieved with surgical castration and exceeded the efficacy of second-generation antiandrogens such as enzalutamide and abiraterone. When combined with the pan-AKT inhibitor AZD5363, the treatment significantly enhanced antitumor activity, further promoting apoptosis and improving survival in advanced CRPC mouse models. These findings suggest that co-targeting the AR and PI3K/AKT pathways may overcome resistance mechanisms and offer a promising therapeutic strategy for advanced, PTEN-deficient prostate cancer [84].

An ASO modified with LNA, ISTH0047, was designed to specifically decrease TGF-β2 mRNA and protein, while sparing TGF-β1 and TGF-β3, demonstrating notable anticancer efficacy in preclinical animals. Through gymnotic delivery in vitro and systemic dosing in vivo, ISTH0047 significantly reduced lung metastasis in syngeneic mice models of renal cell carcinoma (RCC) and breast cancer (4T1), without affecting initial tumor development. In a human lung cancer xenograft model (CRL5807), characterized by predominant TGF-β2, ISTH0047 downregulated TGF-β2 and reduced the formation of lung lesions mostly through stromal mechanisms. The treatment altered tumor-associated macrophages (TAMs) by enhancing CD86 expression, a tumor-suppressive factor, underscoring the significance of the microenvironment in facilitating the antitumor response. The findings indicate that targeting stromal TGF-β2 with ISTH0047 is a viable therapeutic approach to inhibit lung metastasis by altering the TMEand immune landscape [85]. ASOs targeting the lncRNA AC104041.1 were created and shown a substantial reduction in tumor development and metastasis in head and neck squamous cell carcinoma (HNSCC). Increased expression of AC104041.1 was recognized as an oncogenic driver associated with reduced patient survival, functioning as a ceRNA by sequestering miR-6817-3p, which in turn stabilizes Wnt2B expression and activates the Wnt/β-catenin signaling pathway. Silencing of AC104041.1 decreased tumor proliferation, migration, and β-catenin nuclear translocation in both in vitro and in vivo models. The combination of AC104041.1-specific LNA-ASOs with salinomycin, a Wnt pathway inhibitor, enhanced anticancer effects by further diminishing cell survival, migration, and β-catenin activity. The data indicate that targeting AC104041.1 with ASOs, particularly in conjunction with salinomycin, presents a viable therapeutic approach for the treatment of HNSCC [86]. Figure 6 highlights the function of ASOs in cancer therapy.

Fig. 6.

A The therapeutic application of ASOs in cancer therapy. B ASOs can inhibit the synthesis of protein products by obstructing the addition of the 5′ cap or the poly-A tail, or by inducing alternative splicing of newly transcribed mRNA. ASOs can impede translation by obstructing ribosome binding to mRNA or by enlisting RNase, leading to RNA breakdown [87]

Current challenges and future novelties

Despite the remarkable therapeutic promise of ASOs in cancer therapy, several formidable challenges continue to hinder their widespread clinical adoption. ASOs are susceptible to rapid degradation by nucleases in the bloodstream and often suffer from poor cellular uptake, which reduces their bioavailability and therapeutic efficacy. Even when ASOs reach their intended intracellular targets, tumor heterogeneity, including variable gene expression and resistance mechanisms, can significantly limit their effectiveness. Specificity remains another major concern, as off-target effects and unintended interactions with non-target transcripts can lead to toxicity or reduced precision. ASO technology is poised to play an increasingly central role in precision medicine, particularly within personalized oncology. The widespread use of next-generation sequencing allows for precise identification of patient-specific mutations, splice variants, and non-coding RNAs, enabling the design of customized ASOs tailored to individual tumor profiles. Moreover, combining ASOs with existing therapies, including chemotherapy, immunotherapy, or kinase inhibitors, offers the potential for synergistic effects, enhanced efficacy, and reduced resistance, while also allowing for lower drug doses and minimized side effects. For instance, dual targeting of the AR and the PI3K/AKT pathway in prostate cancer or HER-2 in combination with taxol in breast cancer has demonstrated improved tumor suppression and increased apoptosis. ASOs that modulate the TME or influence immune signaling, such as those targeting TGF-β2, also hold great promise for reshaping cancer treatment paradigms. Additionally, ASOs’ ability to alter splicing events or inhibit oncogenic lncRNAs opens new avenues for Transcriptome-level reprogramming, particularly in resistant or previously untreatable cancers. Future development of ASO therapies can be accelerated by integrating artificial intelligence and machine learning to predict optimal ASO sequences with high specificity and minimal off-target effects, thereby streamlining the design process. Research should also focus on discovering novel RNA biomarkers, including tissue-specific lncRNAs and circular RNAs, which can serve as unique, cancer-selective targets. The development of smart ASO systems that respond to tumor-specific conditions, Such as hypoxia, acidic pH, or distinct enzymatic environments, could Further improve selectivity And therapeutic precision. Advances in preclinical modeling, including the use of organoid cultures, 3D-bioprinted tumors, and humanized mouse models, will enhance the physiological relevance of testing platforms and support more reliable translation into clinical settings. Unlocking the full therapeutic potential of ASOs in cancer will require a multidisciplinary, integrated approach that combines innovations in genomics, delivery technologies, immunotherapy, and oncology.

RNA aptamers

Basics and principles

Aptamers are single-stranded, non-coding oligonucleotides, either DNA or RNA, generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. Like antibodies, they exhibit great affinity and selectivity for certain targets, positioning them as a primary focus in disease-targeted treatment. Their non-immunogenic characteristics, along with exceptional specificity, stability, and ease of chemical modification, have led to their designation as “chemical antibodies.” Aptamers are economically viable to manufacture and can be directly conjugated to therapeutic agents or affixed to nanocarriers, therefore mitigating systemic toxicity and improving targeted medication delivery. These advantages make aptamers highly promising candidates for future therapeutic applications [88]. The production and separation of aptamers predominantly depend on the SELEX, an in vitro method that facilitates the selection of high-affinity, high-specificity aptamers from extensive Libraries of random oligonucleotide sequences. Classical SELEX entails repetitive cycles of binding, separation, And amplification, utilizing RNA or DNA Libraries often including 10¹⁴ sequences, to enrich aptamers specific to a target molecule, followed by cloning and sequencing to select optimal candidates. Numerous SELEX modifications have developed over time to enhance efficiency, minimize selection rounds, and customize aptamers for specific applications or intricate targets. CE-SELEX utilizes capillary electrophoresis for fast separation, negative SELEX removes nonspecific binders, and Cell-SELEX uses live cells to identify aptamers that target cell surface markers, facilitating the finding of cancer biomarkers. Furthermore, in vivo SELEX administers libraries directly into animal models to isolate aptamers capable of permeating tissues or traversing biological barriers such as the blood-brain barrier. Modified SELEX techniques, including mirror-image SELEX, photo-SELEX, toggle-SELEX, and microfluidic SELEX, optimize the selection process, augment aptamer stability, or improve functional diversity. Some approaches involve immobilizing targets on matrices and incorporating counter-selection steps to reduce nonspecific binding, while others, like CE-SELEX, eliminate the amplification step and significantly shorten the selection process, though they face limitations such as smaller library sizes. The effectiveness of SELEX depends on optimizing selection stringency, maintaining structural diversity, and applying appropriate quality control and sequencing methods, making it a powerful and adaptable platform for developing aptamers in research, diagnostics, and therapeutic applications [89].

RNA aptamers are naturally prone to degradation and instability; however, their durability can be significantly enhanced through chemical base modifications. Without these modifications, RNA aptamers degrade quickly and lose their ability to recognize or bind target molecules. One of the key reasons for introducing such modifications is to protect the single-stranded regions, particularly hairpin loops crucial for target interaction, from degradation by RNase A enzymes. These modifications help preserve the structural elements necessary for binding, thereby maintaining the aptamer’s affinity. Additionally, chemically modified aptamers show greatly improved resistance to RNase-mediated degradation, remaining stable for more than two hours in human blood. Beyond protecting against enzymatic cleavage, these alterations also improve the aptamer’s thermodynamic stability, helping it retain its functional conformation and enhancing its binding performance. During IVT, modified bases are commonly introduced by substituting native pyrimidines with analogs such as fluoropyrimidines (F-Py), O-methyl (OMe) groups, or amino groups at the 2′ position of deoxyribose. These modifications mimic RNA base properties while improving overall stability [90].

RNA aptamers in cancer therapy

RNA aptamers are short, single-stranded RNA molecules that fold into unique three-dimensional shapes, allowing them to bind with high specificity and affinity to various targets, including proteins, small molecules, and even entire cells. In cancer treatment, RNA aptamers are used for targeted drug delivery, tumor imaging, and inhibition of cancer-associated proteins. By binding to tumor-specific markers or receptors, they can interfere with key signaling pathways that drive cancer progression and metastasis, or act as carriers for chemotherapeutic drugs, minimizing off-target toxicity and improving treatment outcomes. They also enhance diagnostic sensitivity in cancer detection and can be combined with nanoparticles or other therapeutic platforms to enable multifunctional treatment strategies. Their high target specificity, low immunogenicity, and straightforward synthesis make them strong candidates for precision oncology applications. A notable example is the development of CG3, a prostate-specific RNA aptamer engineered to recognize the folded structure of PCA3 long non-coding RNA (lncRNA), which is overexpressed in over 95% of PCa tissues. Using genome SELEX, six high-affinity aptamers were identified, with CG3 showing the strongest binding to PCA3’s structured RNA. CG3 was incorporated into an apta-qPCR assay that amplified PCA3 Transcripts with sensitivities 97 times higher in blood And 7 times higher in tissue compared to conventional qPCR. Histopathological analysis of biotin-labeled CG3 in 161 tissue microarray samples revealed mild nuclear and cytoplasmic staining in PCa cells, strong nuclear staining in benign prostatic hyperplasia (BPH), and no staining in stromal or non-PCa tissues, confirming its specificity. Moreover, CG3 detected multiple transcript variants of PCA3, suggesting it can recognize post-transcriptional modifications. These results highlight CG3’s diagnostic potential in prostate cancer and suggest future applications in targeted therapy and drug development [91].

An RNA aptamer, Apt-1, was developed to specifically target the extracellular sheddase domain of the metalloproteinase Adam8, which plays a critical role in maintaining the myofibroblast-like cancer-associated fibroblast (myCAF) phenotype and cancer stemness in breast (MDA-MB-231) and liver (HepG2) cancer models. Apt-1 exhibited high binding affinity (Kd ~ 29.7 nM), a long in vitro and in vivo half-life (~ 278 min), and selectively blocked Adam8 metalloproteinase activity without affecting related enzymes like Adam10 or Adam17. In vitro, Apt-1 disrupted the maintenance (but not initiation) of the myCAF phenotype and reduced cancer stemness markers (Sox2, Oct4, Nanog), leading to a significant decrease in tumorsphere formation. In vivo, intravenous administration of Apt-1-26nt in NOD-SCID mouse xenograft models led to stabilization or reduction of tumor growth, blocked metastasis to the liver and lungs, and significantly lowered the number of myCAFs and cancer cells within tumors. These findings reveal that extracellular Adam8 activity is essential for sustaining tumor-promoting interactions between cancer cells and the TME, and that Apt-1 offers a promising therapeutic approach to inhibit cancer growth and metastasis by targeting this newly characterized pathway [92]. Modified DNA aptamers, GLB-G25 and GLB-A04, were created by transforming an RNA aptamer that targets phospho-AXL (p-AXL) to enhance stability, bioavailability, And Antitumor efficacy. The aptamers had modifications of 2′-fluoro pyrimidine (2′-F Py), dithiophosphate, and polyethylene glycol (PEG), which significantly increased their resistance to nuclease degradation, decreased renal clearance, and enhanced pharmacokinetic characteristics. In ovarian cancer cell lines, GLB-G25 and GLB-A04 exhibited robust affinity for the AXL receptor, effectively downregulating p-AXL, diminishing cell invasion and migration, and decreasing levels of phosphorylated focal adhesion kinase (p-FAK). In vivo studies showed that PEGylated versions of these aptamers, administered intravenously, significantly reduced tumor Weight, by as Much as 91%, and lowered metastasis rates in orthotopic ovarian cancer mouse models. These effects were observed both when the aptamers were used alone and in combination with paclitaxel, all without causing toxicity. Biodistribution analyses confirmed strong tumor-specific accumulation, and mathematical modeling further supported the synergistic inhibition of tumor growth, particularly when combined with chemotherapy. These findings indicate that chemically modified DNA aptamers can surpass previous RNA aptamers, presenting a viable approach for targeted cancer therapy and potential for further clinical use [93].

The recurrence of GBM following radiation therapy is primarily influenced by vasculogenesis, as irradiation suppresses local angiogenesis. This study evaluated NOX-A12, a high-affinity inhibitor of SDF-1 that obstructs both CXCR4 and CXCR7, in GBM models, building on prior research indicating that inhibiting the CXCR4-SDF-1 connection postpones tumor recurrence. In U251 human GBM-bearing mice and ENU-induced brain tumor-bearing rats, the combination of NOX-A12 with post-irradiation therapy significantly improved tumor control and survival, with effectiveness contingent upon dosage and treatment duration. MRI results further validated a decrease in tumor size that surpasses normal temozolomide therapy. These results corroborate the clinical assessment of NOX-A12 in patients with newly diagnosed GBM [94].

NOX-A12 is an inhibitor of CXCL12 that impedes chemotactic signaling by obstructing receptor interaction and gradient formation. The OPERA study (NCT03168139), a Phase 1/2 clinical trial, assessed the pharmacodynamics, safety, and efficacy of NOX-A12 both as a monotherapy and in conjunction with pembrolizumab in patients with metastatic microsatellite-stable (MSS) colorectal (CRC) and pancreatic (PaC) cancers, where PD-1 inhibitors have demonstrated restricted efficacy. Patients were administered 300 mg of NOX-A12 biweekly for two weeks prior to initiating combination treatment with 200 mg pembrolizumab in 21-day cycles. All Subjects had extensive pretreatment, with the Majority exhibiting worsening illness as their optimal response to previous treatments. Initial biopsies indicated a Limited T cell infiltration at the tumor Margin, averaging 327 cells/mm², significantly below the 600 cells/mm² threshold linked to positive results. NOX-A12 monotherapy elicited Th1 cytokines (IFNγ, IL-2, IL-16) in around 50% of the patients. No objective responses were noted; nevertheless, 25% of patients attained stable illness, And 35% experienced an extended duration on this medication relative to prior regimens. The median progression-free Survival was 1.87 months, with OS rates of 42% at 6 months And 19% at 12 months. The safety profile of the combination treatment corresponded with the established effects of pembrolizumab and the associated illnesses. The results suggest that NOX-A12, particularly in conjunction with pembrolizumab, may enhance immune responses and achieve disease stability in a specific group of extensively pretreated MSS CRC and PaC patients [95].

A phase II single-arm study delivered the DNA aptamer AS1411, which targets nucleolin, to 35 patients with metastatic clear-cell RCC who had previously failed at Least one tyrosine kinase inhibitor. Patients were administered 40 mg/kg/day with continuous intravenous infusion on days 1 to 4 of a 28-day cycle, for two cycles. The overall response rate was minimal, with just one patient (2.9%) achieving a substantial and sustained tumor decrease (84% per RECIST 1.0) and staying progression-free two years after therapy. Whole exome sequencing of this patient’s tumor identified missense mutations in the mTOR and FGFR2 genes, which may be significant due to nucleolin’s function in augmenting mTOR pathway activity through AKT1 mRNA translation. No other objective replies were noted. Adverse events associated with AS1411 occurred in 34% of patients, all classified as mild or severe. The data indicate that AS1411 has modest effectiveness in unselected metastatic RCC patients; nonetheless, its promise is highlightd by rare, significant responses and low toxicity, especially in biomarker-selected cohorts [96].

A 2′-fluoropyrimidine-modified RNA aptamer, P12FR2, was engineered to selectively bind pancreatic adenocarcinoma up-regulated factor (PAUF), a secreted protein that is significantly overexpressed in pancreatic cancer and is recognized for its role in tumor proliferation and metastasis. P12FR2 was chosen using SELEX for its strong affinity (Kd ~ 77 nM) and specificity towards human PAUF. P12FR2 significantly prevented PAUF-induced migration of PANC-1 pancreatic cancer cells in vitro, as demonstrated by wound-healing experiments, whereas control aptamers had no impact. In vivo, intraperitoneal administration of P12FR2 in nude mice bearing CFPAC-1 Pancreatic cancer xenografts Led to approximately a 60% decrease in tumor development over 20 days, as assessed by bioluminescence imaging, without inducing weight loss or toxicity. These findings highlight P12FR2’s potential as a therapeutic drug aimed at PAUF to impede pancreatic cancer progression, presenting a promising strategy for the development of innovative anti-cancer therapeutics [97].

A chemically produced RNA aptamer, syn-RA16, along with its shortened form S3, was evaluated for its capacity to target and inhibit NSCLC NCI-H460 cells both in vitro and in vivo. Syn-RA16 had a high binding affinity (Kd ~ 24.75 nM), akin to the previously documented RA16 aptamer, and displayed dose-dependent suppression of cell viability, with IC50 values around 118.4 nM. Internalization experiments validated receptor-mediated endocytosis, resulting in cytoplasmic accumulation, while in vivo imaging demonstrated significant tumor targeting and retention, with aptamer Levels in tumors being 50 to 1000 times greater than in other tissues. A shortened segment, S3, exhibited partial binding (Kd ~ 63.2 nM) And reduced cell growth by around 39% at 150 nM, but with less efficacy than full-length RA16. These results endorse syn-RA16 as a viable therapeutic or diagnostic agent for NSCLC, emphasizing its chemical stability, tumor selectivity, and potential for large-scale production and clinical use [98]. An RNA aptamer, A9g, was engineered to limit the enzymatic activity of prostate-specific membrane antigen (PSMA), demonstrating significant suppression of PCa metastasis in preclinical models. A9g preferentially bonded to PSMA in vitro, significantly inhibiting cell migration and invasion without impacting proliferation, and substantially reduced PSMA enzymatic activity in PCa cells. Systemic injection of A9g in a mouse model of metastatic PCa significantly decreased the incidence And dissemination of bone metastases, with just 2 of 18 treated Animals exhibiting metastases, in contrast to 80–90% in control groups. Pharmacokinetic and biodistribution investigations utilizing near-infrared–labeled A9g demonstrated preferential retention in PSMA + tumors for a duration of up to 72 h, devoid of nonspecific accumulation. Safety evaluations indicated no detrimental impacts on murine behavior, organ integrity, hematological parameters, or immunological activation, while human peripheral blood mononuclear cells (PBMCs) showed no cytotoxicity or immune stimulation upon exposure to A9g. The findings highlight A9g as a secure, efficacious, and tumor-targeted RNA-based smart medication, presenting a promising therapeutic approach for the management of metastatic CRPC (mCRPC), with potential for future integration into combination therapies or tailored treatment regimens [99].

Current challenges and future novelties

Despite the significant advancements in the field of RNA aptamers, several challenges remain that impede their full clinical integration. A principal concern is the inherent instability of RNA aptamers in biological environments due to rapid degradation by nucleases Such as RNase A. Although chemical modifications Like 2′-fluoropyrimidine substitution or O-methylation have been employed to enhance stability, these adaptations may compromise the aptamer’s natural binding affinity or introduce synthesis complexities. Another bottleneck is the delivery of RNA aptamers in vivo, where issues such as rapid renal clearance, limited tissue penetration, and off-target interactions reduce therapeutic efficiency. The SELEX process, although highly effective, can be time-consuming and labor-intensive, with success heavily reliant on optimal stringency and sequence diversity maintenance throughout selection rounds. Furthermore, while aptamers can be engineered to exhibit high specificity, the potential for immunogenicity or unintended interactions with non-target biomolecules remains a concern, particularly in the context of complex TMEs. The development of RNA aptamers in cancer therapy holds vast potential, particularly when integrated with nanotechnology, targeted delivery platforms, or immunotherapy. Future work will possibly focus on refining chemical modifications to achieve a balance between enhanced stability and preserved binding functionality. Additionally, advancements in high-throughput SELEX variants, such as microfluidic and in vivo SELEX, promise to accelerate the discovery of functionally robust aptamers for intricate targets like tumor-associated RNAs or proteins. In cancer diagnostics, RNA aptamers can be further incorporated into biosensor platforms and imaging systems for early detection, especially given their success in recognizing cancer-specific lncRNAs, such as PCA3 in prostate cancer. The ability to engineer aptamers with multi-domain structures could open the door to multifunctional therapeutics agents that not only bind to cancer markers but also deliver cytotoxic agents or modulate immune responses in real time. The combination of aptamers with CRISPR-based systems or RNAi technologies could also enhance gene regulation precision in tumor cells. To expand the translational potential of RNA aptamers, future studies could focus on several novel strategies. One key direction involves the design of “smart” aptamer-drug conjugates that release payloads only upon binding to their specific targets, reducing systemic toxicity and improving treatment precision. Incorporating aptamers into exosome-based delivery systems or LNPs could improve cellular uptake and protect them from degradation. Another promising avenue is the development of bispecific or multivalent aptamers capable of targeting multiple tumor antigens simultaneously, thereby overcoming tumor heterogeneity and resistance mechanisms. Researchers might also explore aptamer scaffolds that mimic antibody functions while remaining non-immunogenic, enabling repeated dosing without immune activation. Finally, integrating artificial intelligence and machine learning into the aptamer discovery process could drastically improve sequence prediction and structural modeling, expediting the development of clinically viable candidates. Overall, the synergy of biochemical innovation, computational tools, and delivery technology heralds a transformative era for RNA aptamer research in oncology and beyond. In addition to serving as delivery ligands, RNA aptamers also function as direct antagonists by binding with high specificity and affinity to cancer-associated targets, thereby inhibiting their biological activity. This dual functionality is exemplified by aptamers such as Apt-1, which directly inhibits Adam8 metalloproteinase activity, and A9g, which suppresses PSMA enzymatic function and metastasis in prostate cancer models. Such aptamers disrupt tumor-promoting pathways, block protein interactions, or interfere with receptor signaling without the need for a therapeutic payload, highlighting their intrinsic therapeutic potential beyond targeted delivery applications.

RNA aptamers exert their anticancer effects by directly targeting and modulating key molecular pathways involved in tumor progression, metastasis, and therapy resistance. For example, Apt-1 binds to the extracellular domain of the metalloproteinase Adam8, inhibiting its activity and consequently disrupting the maintenance of the myofibroblast-like cancer-associated fibroblast (myCAF) phenotype and reducing expression of stemness markers such as Sox2, Oct4, and Nanog, which are critical for tumor renewal and invasiveness. GLB-G25 and GLB-A04, modified DNA aptamers derived from an RNA aptamer, effectively downregulate phospho-AXL, thereby inhibiting the AXL/FAK signaling axis that drives cancer cell migration and invasion in ovarian cancer. Similarly, the A9g RNA aptamer targets PSMA, suppressing its enzymatic function and significantly reducing cell migration and bone metastasis in prostate cancer models. NOX-A12 targets the CXCL12 chemokine and blocks its interaction with CXCR4 and CXCR7 receptors, thereby disrupting vasculogenesis and immune evasion pathways, particularly in GBM and MSS colorectal and pancreatic cancers. These examples illustrate how RNA aptamers function by interfering with specific molecular circuits that underlie cancer development and therapeutic resistance, highlighting their potential as precise modulators of oncogenic signaling networks (Fig. 7).

Fig. 7.

A visual roadmap summarizing emerging strategies to enhance RNA aptamer applications in oncology. Future efforts focus on improving stability, enabling smart delivery systems, developing multiplexed aptamer cocktails, integrating AI for faster discovery, and combining aptamers with cutting-edge therapeutic platforms. (Created with Biorender.com)

CRISPR/Cas9-guided gene editing

Basics and principles

The CRISPR–Cas9 system, initially a bacterial defensive mechanism against phages and plasmids, has been adapted as a potent RNA-guided DNA editing instrument with extensive applications in genome editing, transcriptional control, epigenetic modification, and genome imaging. Utilizing a short guide RNA, researchers may accurately modify nearly any genomic sequence to investigate gene activities, rectify pathogenic mutations, and modulate oncogenes or tumor suppressor genes via nuclease-deficient Cas9 fusion proteins. Moreover, CRISPR enables the concurrent targeting of numerous genes, significantly enhancing our comprehension of intricate disease mechanisms such as cancer. Genome-wide CRISPR screens using sgRNA libraries enable the discovery of key drug targets and genes linked to various diseases, making this technology a powerful tool for advancing treatments and potential cures for genetic disorders. These include cancer, neurodegenerative diseases, sickle cell anemia, cystic fibrosis, DMD, viral infections, immune disorders, and cardiovascular conditions [100]. The CRISPR–Cas system, initially recognized as an adaptive immunological mechanism in bacteria and archaea, has been refined into RNA-guided endonucleases (RGENs) for accurate genome editing purposes. In a conventional type II CRISPR system, the CRISPR RNA (crRNA) associates with a trans-activating crRNA (tracrRNA) to create a duplex, commonly substituted in practice by a single guide RNA (sgRNA), which directs the Streptococcus pyogenes Cas9 (SpyCas9) enzyme to specific DNA sequences, leading to targeted DNA strand cleavage. Unlike zinc finger nucleases or transcription activator-like effector nucleases, CRISPR–Cas9 does not require the creation of custom protein pairs for each target. Instead, it relies on simple RNA-DNA base-pairing rules to recognize specific sequences. This streamlined design has dramatically transformed genome engineering across a wide range of cell types and species [101]. The CRISPR/Cas9 system operates by directing the Cas9 nuclease to specific DNA sequences via complementary base-pairing with an attached single-guide RNA (sgRNA), necessitating An adjacent 3′ PAM sequence (NGG or NAG) for optimal binding and cleavage. A double-stranded DNA break can be repaired using either non-homologous end joining (NHEJ) or homology-directed repair (HDR). The sgRNA comprises a 20-nucleotide segment that specifies the target sequence and a tracrRNA-derived handle for Cas9 attachment, supplemented with scaffold modifications for improved efficacy. Additionally, engineered SpCas9 variants, such as the nuclease-deficient dCas9 fused to different effector domains, enable diverse applications. These include transcriptional activation (with VP64), transcriptional repression (with KRAB), epigenetic modification (using enzymes like DNMT3A), and precise base editing (with enzymes such as AID) [102].

CRISPR/Cas9 in cancer therapy

A humanized mouse model of HCC was established by transplanting human liver parenchymal, non-parenchymal, and hematopoietic cells into immunodeficient Fah^−/−; Rag2^−/−; Il2rgc^−/− (FRG) mice, facilitating the investigation of tumor-promoting gene-environment interactions pertinent to metabolic liver diseases. CRISPR-Cas9 technology was employed to create a deletion of ARID1A and a constitutively active knock-in of CTNNB1 in human hepatic progenitors before transplantation. Following the induction of elevated human cell chimerism, an alcohol Western diet (AWD) was implemented for six months, resulting in a Marked increase in Liver tumor prevalence: 25% in CTNNB1 Mutant Mice, 50% in ARID1A Mutant Mice, And 70% in mice harboring both Mutations, with approximately 50% of tumors histologically verified as HCC. This model offers a comprehensive framework for assessing the impacts of personalized therapies aimed at specific cancer-driver mutations within clinically pertinent environmental contexts, addressing the constraints of interspecies variability and facilitating accurate gene-environment interaction analyses for pharmacological evaluation and mechanistic understanding of HCC progression [103]. CRISPR-Cas9 knockdown screening revealed KIAA1429 as a critical gene in Ewing sarcoma (ES), a notably aggressive pediatric bone and soft tissue malignancy. Functional genomic and transcriptome investigations revealed that the genetic reduction of KIAA1429 significantly reduced embryonic stem cell proliferation, viability, and colony formation, and triggered apoptosis both in vitro and in subcutaneous xenograft mouse models. Transcriptome analysis indicated that KIAA1429 promotes ES carcinogenesis by modulating cancer-related pathways, including cell cycle regulation, ribosomal activity, and inflammatory responses. An interaction was observed in which STAT3, a crucial oncogenic transcription factor, was diminished following KIAA1429 knockdown; notably, the reduction of STAT3 also decreased KIAA1429 levels, indicating a positive feedback loop. Additionally, NKX2-2, a recognized embryonic stem cell transcription factor, was demonstrated to transcriptionally activate KIAA1429 and other m6A RNA methylation writers, hence amplifying oncogenic signaling. These findings highlight KIAA1429 as a critical epigenetic regulator in ES and position it as a promising therapeutic target. They also suggest potential strategies for targeting the KIAA1429-STAT3 axis to interfere with the malignant signaling network driving ES progression (Fig. 8) [104].

Fig. 8.

A prospective NKX2-2–mediated m6A regulation network implicating KIAA1429 in ES: A Venn diagram indicates nine transcripts in ES that are both highly expressed and positively linked with KIAA1429 expression. The nine ES-specific transcripts are prioritized according on their expression fold change. Analysis of DepMap cancer cell line data indicates a unique transcriptional profile for NKX2-2 in embryonic stem cells. Gene track data from publically accessible ES ChIP-seq datasets reveal pronounced NKX2-2 binding peaks in the promoter regions of KIAA1429 (top panel) and METTL3 (bottom panel), indicated by promoter-associated histone modifications H3K27ac and H3K4me3. (E) RNA-seq analysis following NKX2-2 knockdown showed a decrease in numerous m6A methylation-related genes. A correlation matrix demonstrates co-expression patterns among KIAA1429, METTL3, WTAP, and YTHDF3 across many ES tumor datasets. A diagram illustrating the KIAA1429-centered m6A regulation pathway and its possible influence on essential phenotypic characteristics in embryonic stem cells. *Abbreviations: ChIP – chromatin immunoprecipitation; ChIP-seq – chromatin immunoprecipitation sequencing; ES; FC – fold change. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001. Reprinted with permission from Springer BMC Nature [104]

An effective and scalable system was created to produce isogenic cancer models by integrating AAV-intron-trap vectors, CRISPR/Cas9 genome editing, And inducible Cre-recombinase. This approach achieved over 90% efficiency in introducing the oncogenic K700E mutation into the SF3B1 gene, a common splicing factor mutation in cancers, by leveraging the homologous recombination (HDR) capability of AAV alongside CRISPR-induced double-strand breaks (DSBs) at the target site. The combined use of AAV and CRISPR enabled precise integration of the mutation on a single allele, as confirmed by PCR and Sanger sequencing, whereas each method alone resulted in lower editing efficiency and failed to introduce the exact point mutation. To mitigate toxicity from mutant SF3B1 expression, an inducible Cre-recombinase system was employed, allowing edited cells to expand before activating mutant allele expression. Functional validation confirmed the expression of the mutant protein and its characteristic splicing defects. The system was also successfully adapted to other cell types and mutations, including U2AF1 S34F and FLAG-tagged SF3B1 variants. Overall, this platform provides a powerful and flexible tool for generating precise, single-allele cancer models, enabling detailed studies of harmful mutations and complex molecular pathways in clonal diseases, with potential applications in biochemical research, therapeutic testing, and functional genomics [105]. The CRISPR/Cas9-mediated deletion of CD73 in pancreatic cancer cells greatly inhibited cell proliferation, reduced colony formation, and arrested the cell cycle in the G1 phase without triggering apoptosis. Elevated CD73 expression, validated in both human (PANC1) and murine (TB32047) pancreatic cancer cell lines, correlated with diminished overall and disease-free lifespan. Post-CD73 deletion, less cell movement was seen by wound healing experiments, accompanied by lower phosphorylation of ERK and STAT3, and elevated E-cadherin expression, indicating the inhibition of critical oncogenic pathways and the amplification of tumor-suppressor signaling. A CRISPR/Cas9-based kinase library screen discovered possible regulators of CD73 expression, including Pbk, Fastk, Cdk19, Adck5, Trim28, and Pfkp, which were enriched in CD73-high cells and exhibited differential expression in pancreatic cancers. The findings highlight the critical role of CD73 in promoting pancreatic cancer cell growth and migration, suggesting that its targeted inhibition, alongside modulation of upstream regulatory genes, offers a promising therapeutic strategy for treating pancreatic cancer [106].

RNA and DNA aptamers were engineered to target critical oncogenic pathways, cancer biomarkers, and molecular processes, exhibiting considerable therapeutic and diagnostic promise. An RNA aptamer targeting NF-κB in NSCLC has proven effective in reducing doxorubicin resistance, inhibiting tumor growth, and inducing apoptosis both in vitro and in vivo [107]. Additionally, a one-pot CRISPR/Cas12a cis-cleavage fluorescent RNA aptamer assay has been developed for the ultrasensitive, label-free detection of miRNA-21 in breast cancer, offering rapid and highly specific diagnostics [108]. A glutathione-binding RNA aptamer, selected through SELEX, triggered ROS-mediated apoptosis in MCF-7 breast cancer cells by depleting intracellular glutathione and activating caspase-3, providing a novel strategy for inducing cell death [109]. In colon cancer, an RNA aptamer targeting β-catenin was used to suppress the transcription of oncogenic targets such as cyclin D1 and c-myc, interfere with β-catenin-driven alternative splicing, induce cell cycle arrest, and reduce tumorigenicity [110]. These aptamer-based systems showcase advanced capabilities in modulating transcription factors, splicing regulators, oxidative stress responses, and miRNA biomarkers, demonstrating their versatility as therapeutic agents, chemosensitizers, and diagnostic tools across various cancer types.

CRISPR/Cas9 screening methodologies constitute an effective way for elucidating in vivo cancer dependencies. Hematopoietic malignancies are genetically intricate disorders marked by the progressive accumulation of somatic mutations that promote clonal diversity. As the illness advances, supplementary cooperative mutations may facilitate further transformation. A pooled in vivo gene-editing screen aimed at epigenetic regulators in primary murine hematopoietic stem and progenitor cells (HSPCs) was utilized to find previously unknown genes implicated in leukemia development. A myeloid leukemia model was created in mice by functionally disrupting both Tet2 and Tet3 in HSPCs, subsequently followed by transplantation. Subsequent pooled CRISPR/Cas9 gene editing of epigenetic factors identified Pbrm1/Baf180, a component of the polybromo BRG1/BRM-associated factor (PBAF) SWI/SNF chromatin-remodeling complex, as a negative regulator of illness development. The absence of Pbrm1 expedited leukemogenesis, leading to a significantly reduced illness latency. Leukemia cells lacking Pbrm1 had decreased immunogenicity, impaired interferon signaling, and reduced expression of major histocompatibility complex class II (MHC II) molecules. The role of PBRM1 in regulating components of the interferon pathway was assessed for its relevance to human leukemia. PBRM1 was identified as binding to the promoters of many genes within this pathway, including IRF1, a crucial regulator of MHC II expression [111].

Chromosomal translocations, especially those involving the MLL/KMT2A gene, are significant contributors to childhood leukemias. To evaluate their genuine leukemogenic potential, researchers engineered a lentiviral CRISPR-Cas9 system that can induce the t(11;19)/MLL-ENL translocation in human CD³⁴⁺ HSPCs. This translocation provided HSPCs with a temporary growth advantage in vitro, facilitating long-term engraftment and triggering monocytic leukemia-like illness in vivo. Notably, subsequent transplants resulted in acute lymphoblastic leukemia in certain instances, indicating that environmental variables affect both disease phenotype and transformation. The research emphasizes the significance of microenvironmental signals in the progression of juvenile leukemia and prospective therapeutic approaches [112].

Adult T-cell leukemia/lymphoma (ATLL) is a highly aggressive T-cell neoplasm with a restricted response to existing treatments. After investigating environmental factors affecting juvenile leukemia, genome-wide CRISPR-Cas9 screening in ATLL models revealed numerous essential genes necessary for ATLL cell proliferation and survival, including CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB. CDK6 has emerged as a significant therapeutic target. Palbociclib-mediated inhibition of CDK6 resulted in cell cycle arrest and death in ATLL models with wild-type TP53. Models with genetically inactivated TP53 exhibited resistance attributed to compensatory CDK2 activity, a constraint that might be mitigated by APR-246, a small chemical that reactivates mutant TP53. The CRISPR-Cas9 screen demonstrated a significant reliance of ATLL cells on mTORC1 signaling. The combination of palbociclib and mTORC1 inhibitors resulted in synergistic cytotoxicity independent of TP53 status. These findings endorse CDK6 as a feasible therapeutic target and indicate that simultaneous inhibition of CDK6 and mTORC1 is a promising method for addressing this challenging malignancy [113].

CRISPR/Cas9-based technologies have enabled the identification of novel therapeutic vulnerabilities and mechanistic insights across various hematologic and solid pediatric Malignancies. In CLL, biallelic loss of the ATM gene in cases with 11q deletion (del(11q)) is associated with poor prognosis, and CRISPR-generated isogenic models revealed a therapeutic vulnerability to combined BCR and PARP inhibition, with synergistic effects confirmed ex vivo in primary patient samples, suggesting a promising strategy for high-risk CLL [114]. Further CRISPR screening in MYCN-amplified neuroblastoma uncovered a dependency on polycomb repressive complex 2 (PRC2) components, particularly EZH2, which is transcriptionally activated by MYCN and suppresses neuronal differentiation [115]. Pharmacological inhibition of EZH2 suppressed tumor growth, and synergy with histone deacetylase inhibitors highlighted its therapeutic relevance in high-risk neuroblastoma. Additionally, in pediatric rhabdomyosarcoma (RMS), a CRISPR-based screen identified HDAC3 as a key repressor of myogenic differentiation through its interaction with nuclear receptor corepressors (NCORs) and inhibition of MYOD1 function [116]. Targeting HDAC3 induced terminal differentiation, albeit with a transient upregulation of growth-promoting genes, indicating a cancer-specific adaptation. These findings highlight the utility of CRISPR/Cas9 in uncovering cancer dependencies and advancing targeted and differentiation-based therapies across pediatric cancers.

CRISPR/Cas9 technology and small-molecule screening continue to reveal critical insights and therapeutic targets across various cancer and disease models. In the human rhabdomyosarcoma TE671 cell line, typically used in muscle biology studies, muscle differentiation was successfully induced using the MAPK inhibitor U0126, resulting in myotube formation and upregulation of differentiation markers such as dysferlin and myogenin, while pax7 was downregulated. CRISPR-mediated knockout of the DYSF gene (Dysf-KO TE671) eliminated dysferlin expression, yet myogenin induction remained possible with U0126 treatment, supporting the use of this model for studying dysferlin-deficient muscle disorders and for drug screening [117]. In pediatric rhabdoid tumors (RTs), which often lack targetable mutations, a combined high-throughput drug screen and genome-scale CRISPR-Cas9 knockout study identified receptor tyrosine kinases (RTKs) and the downstream signaling protein SHP2 (encoded by PTPN11) as essential for tumor survival despite the absence of genetic alterations, highlighting non-mutational dependencies [118]. Similarly, in AML, An optimized genome-wide CRISPR screen revealed 492 AML-specific essential genes, including both known and novel therapeutic targets. Among these, KAT2A emerged as a promising candidate, with its inhibition inducing differentiation and apoptosis in AML cells while sparing normal hematopoietic stem-progenitor cells [119]. These findings highlight the power of CRISPR-based functional genomics in uncovering disease-specific vulnerabilities and shaping the future of targeted therapy for cancers and muscle-related diseases.

Current challenges and future perspectives

Despite the transformative potential of CRISPR/Cas9 technology in cancer therapy, several challenges must be overcome for widespread clinical implementation. The potential for off-target effects remains a pressing concern, as unintended genome edits could lead to mutagenesis or oncogenesis. Although engineered high-fidelity Cas9 variants and base editors have reduced off-target activity, achieving complete specificity remains elusive. Another significant barrier is tumor heterogeneity, which complicates the identification and targeting of universal driver mutations across patient populations. Moreover, the immune response against Cas9 proteins derived from bacterial sources could limit repeated therapeutic applications, highlighting the need for immune-evasive strategies or development of orthologous Cas9 systems with reduced immunogenicity. Looking forward, the future of CRISPR/Cas9 in oncology lies in the refinement of multiplexed and context-aware gene editing strategies. Synthetic biology tools such as CRISPR-based logic gates show substantial promise in increasing tumor specificity, as evidenced by dual-promoter systems that activate therapeutic genes only in specific cancer cell contexts, such as bladder cancer. Expanding this concept to include Boolean logic circuits using more tumor-specific promoters could enable even greater precision in targeting malignant cells while sparing normal tissue. Furthermore, the integration of CRISPR technologies with multi-omics platforms (transcriptomics, proteomics, and epigenomics) could allow for the real-time monitoring of cellular states and the tailoring of gene editing interventions accordingly. Another vital area for future exploration is temporal control over gene editing activity, which could be achieved using inducible Cas9 systems responsive to small molecules or external stimuli (light or ultrasound), thereby adding a layer of control to enhance safety and efficacy. In terms of novel study directions, future research could explore the use of humanized organoid or tissue-engineered cancer models combined with CRISPR to more accurately mimic human TME s and evaluate therapeutic outcomes. Another innovative approach would involve coupling CRISPR systems with RNA-targeting capabilities, such as Cas13, to transiently modulate oncogenic RNA transcripts without altering the DNA, providing a reversible and less permanent therapeutic option. Additionally, the role of epigenetic modifiers, such as KIAA1429 and other RNA methylation regulators, opens new avenues for investigating non-mutational contributors to oncogenesis. Targeting these epigenetic regulators with CRISPR interference (CRISPRi) or activation (CRISPRa) could allow precise modulation of gene expression without DNA cleavage. Finally, expanding the scope of CRISPR-based screens in patient-derived xenografts (PDX) or 3D bioprinted tumor constructs could enhance the discovery of novel drug targets and resistance mechanisms, ultimately leading to more personalized and adaptive cancer therapies. (Table 2).

Table 2.

The application of RNA therapeutics in cancer

Study Focus	Cancer Type/Disease	Therapeutic Approach	Sample Size	Immune Response	Clinical Outcomes	Key Findings	Limitations/Next Steps	Refs
Silencing FGFR3 via siRNA to reduce invasion and migration in LUAD cells	NSCLC, LUAD (A549 cell line)	siRNA targeting FGFR3 (three siRNAs: siRNA-855, siRNA-1447, siRNA-2076) transfected into A549 cells	Preclinical (in vitro cell culture, Transwell assays)	Downregulation of FGFR3 and MMP9 expression; upregulation of E-cadherin; reduced EMT markers	Significant reduction in cell invasion (~ 62–64% inhibition), reduced MMP9, increased E-cadherin levels, no change in negative controls, suggesting specific FGFR3 silencing effects	Demonstrated that FGFR3 silencing via siRNA inhibits A549 lung cancer cell invasion by modulating EMT and ECM degradation pathways, identifying FGFR3 as a therapeutic target	Requires further in vivo validation, mechanistic studies on downstream signaling, exploration of delivery methods, and safety assessment before clinical translation	[120]
Inhibition of gastric cancer-associated angiogenesis by CIAPIN1 siRNA	Gastric cancer (SGC7901 cell line, nude mice)	Stable transfection of CIAPIN1 siRNA into gastric cancer cells (SGC7901) to reduce angiogenesis and tumor growth	Preclinical (in vitro: HUVEC assays; in vivo: nude mice tumor models)	Reduced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs); arrested HUVECs in G1 phase	Significant in vivo reduction in tumor volume, vascularization, and microvascular density; suppressed tumor angiogenesis in mice	Demonstrated that CIAPIN1 silencing inhibits gastric cancer angiogenesis and tumorigenicity by downregulating endothelial activity, identifying CIAPIN1 as a therapeutic target	Needs further exploration in larger animal models, mechanistic studies on downstream pathways, and investigation into delivery systems for clinical application	[121]
Knockdown of KRT17 by siRNA to investigate its antitumoral effects	Gastric adenocarcinoma (AGS, NCI-N87 cell lines)	RNAi (siRNA) targeting KRT17 to reduce proliferation, migration, and invasion	Preclinical (in vitro cell lines; in vivo xenograft mouse models)	Reduced AKT/mTOR phosphorylation, increased AMPKα1/CREB activation, cell cycle arrest (increased G0/G1, reduced S phase), decreased CD44 and VEGFA expression	~ 40–70% reduced proliferation and Migration; 69–84% reduction in tumor weight in xenograft models; maintained KRT17 knockdown in tumors over 9 weeks	Demonstrated that KRT17 promotes gastric cancer cell proliferation, migration, and tumor growth, suggesting its value as a therapeutic target and biomarker	Requires further clinical validation, mechanistic studies on signaling pathways, and exploration of KRT17 detection in patient serum as a noninvasive biomarker	[122]
Downregulation of UHRF1 by shRNA lentivirus to inhibit breast cancer cell growth	Breast cancer (MDA-MB-231, MCF-7 cell lines)	shRNA lentiviral system targeting UHRF1 to suppress gene expression and proliferation	Preclinical (in vitro cell lines)	~ 80–90% knockdown of UHRF1 mRNA and protein; reduced colony formation (> 50%), reduced cell proliferation (MTT, BrdU assays)	Significant inhibition of breast cancer cell proliferation and colony formation; no change in adriamycin sensitivity in apoptosis assays	Demonstrated that UHRF1 plays a critical role in breast cancer proliferation and colony formation; RNAi-mediated silencing shows potential as a therapeutic approach	Requires investigation of molecular mechanisms, in vivo validation, assessment of effects on drug resistance, and potential combination strategies	[123]
Isoform-specific RNAi targeting PKM2 to induce apoptosis and tumor regression	Multiple cancers (colon, liver, ovarian; cell lines and xenograft models)	siRNA (siPKM2) specifically silencing pyruvate kinase M2 (PKM2) isoform, delivered via LNPs (lipidoids)	Preclinical (in vitro cell lines, in vivo SCID mice xenografts)	Strong apoptosis induction (Caspase 3/7), robust PKM2 knockdown (> 95%), reduced lactate production, minimal effect on normal fibroblasts and endothelial cells	Significant tumor regression (~ 75–85% reduction), some tumors undetectable post-treatment, no major toxicity, specific to cancer cell metabolism	Demonstrated that siRNA can selectively target cancer-specific isoforms like PKM2, triggering metabolic disruption and apoptosis; broad applicability across tumor types	Needs investigation of long-term effects, combinatorial treatments, delivery optimization, and progression toward clinical testing	[124]
Systematic investigation of siRNA-mediated knockdown of oncogenes to inhibit tumor growth	Multiple cancers (HeLa, LUAD, hepatoma, melanoma, ovarian carcinoma cells)	Chemically synthesized and vector-driven siRNAs targeting oncogenes (bcl-2, cdk-2, mdm-2, H-ras, pkc-alpha, vegf)	Preclinical (in vitro cell lines, transfection assays, combinatorial siRNA treatments)	Efficient knockdown of target mRNAs and proteins, dose-dependent effects, increased p53 levels via mdm2 knockdown, additive inhibitory effects with combinatorial siRNAs	Significant inhibition of proliferation (up to ~ 80% reduction), additive effects with multi-target siRNA combinations, cell-type-specific responses, no effect with control siRNAs	Demonstrated that targeting multiple oncogenes simultaneously via siRNA effectively suppresses tumor cell proliferation, offering a promising gene-silencing therapeutic approach	Requires in vivo animal model validation, optimization of delivery systems, investigation of long-term and systemic effects, and exploration of combinatorial therapeutic strategies	[125]
Enhancing pancreatic cancer chemosensitivity to gemcitabine via survivin knockdown	Pancreatic cancer (Panc-1, BxPC3 cell lines)	siRNA plasmid expression vector targeting survivin (psiRNA-survivin)	Preclinical (in vitro Panc-1, BxPC3; apoptosis, cell cycle, cytotoxicity assays)	Significant reduction in survivin mRNA (~ 68%) and protein (~ 76%), G0/G1 cell cycle arrest, increased spontaneous apoptosis, enhanced gemcitabine-induced apoptosis	Reduced cell proliferation (~ 55–58% inhibition), enhanced chemosensitivity (apoptosis increased from ~ 26–31% to ~ 43–48% post-gemcitabine), higher cytotoxicity by MTT assay	Demonstrated that survivin silencing effectively sensitizes pancreatic cancer cells to gemcitabine, suggesting RNAi as a potential chemosensitization strategy	Needs in vivo validation, delivery optimization, investigation of off-target effects, and evaluation of combination strategies for translation to clinical settings	[126]
Evaluation of atelocollagen-mediated in vivo siRNA delivery targeting Bcl-xL and Mcl-1	Ovarian cancer (SKOV3 xenograft models)	Intraperitoneal (i.p.) or intravenous (i.v.) delivery of siRNA complexed with atelocollagen targeting Bcl-xL, Mcl-1, or luciferase	Preclinical (nude mice, s.c. and peritoneal tumor models)	Significant luciferase signal reduction (70%) in s.c. tumors after i.v. siRNA delivery; no detectable Bcl-xL or Mcl-1 silencing; no apoptosis induction or target downregulation	Transient luciferase signal suppression; no therapeutic effect with Bcl-xL or Mcl-1 siRNAs; no significant tumor regression or growth inhibition	Demonstrated that atelocollagen enables transient siRNA delivery to s.c. tumors but not to peritoneal carcinomatosis; Bcl-xL and Mcl-1 siRNAs failed to achieve target knockdown in vivo	Requires alternative delivery systems, enhanced siRNA formulations, and optimized dosing strategies to achieve durable gene silencing and therapeutic effects in ovarian cancer models	[127]
Silencing VEGFR-2 via siRNA to inhibit LPA-induced invasion in ovarian cancer	EOC, SKOV3, DOV13 cell lines)	siRNA targeting VEGFR-2 to suppress VEGF–VEGFR signaling and reduce invasion and metastasis	Preclinical (in vitro, tissue qPCR array, invasion assays)	Reduced VEGFR-2 mRNA and protein expression by ~ 70–80%; decreased LPA-induced expression of VEGF121, VEGF165, and MMP activity; suppressed cell invasion	Significant reduction in LPA-stimulated invasion in both SKOV3 and DOV13 cells; ~90–95% reduction in invasion after VEGFR-2 knockdown; correlation of VEGFR-2 with tumor grade in tissues	Demonstrated that VEGFR-2 is a key mediator of LPA-induced ovarian cancer invasion; silencing VEGFR-2 reduces metastasis potential, suggesting therapeutic potential	Requires further in vivo validation, exploration of delivery mechanisms, assessment of long-term effects, and evaluation in patient-derived models for clinical translation	[128]
Combining paclitaxel with siRNA targeting HPV16 oncogenes (E6/E7) to enhance cytotoxicity	Cervical carcinoma (CC) a (HPV16-positive; SiHa, CaSki cell lines, xenograft mouse models)	Intronic and exonic siRNAs targeting HPV16 E6/E7 combined with systemic paclitaxel treatment	Preclinical (in vitro CaSki, SiHa; in vivo SiHa xenograft mice, n = 4–5/group)	~ 80% reduction in full-length E6 transcripts, ~ 60% reduction in total E6/E7 transcripts; increased p53 and hypophosphorylated pRb; restored apoptosis and cell cycle arrest	Significant tumor volume reduction, synergistic cytotoxicity with paclitaxel and intronic siRNA, enhanced survival vs. controls, no major toxicity	Demonstrated that targeting E6 alone (restoring p53) provides greater synergy with paclitaxel than combined E6/E7 targeting; supports E6-focused RNAi as a chemosensitizer	Requires further optimization of siRNA delivery systems, investigation of systemic administration, long-term efficacy studies, and translation to clinical trials	[129]
Inhibition of HPV16E7 expression via siRNA to suppress cervical cancer cell proliferation	Cervical cancer (HPV16-positive CaSki cell line)	Chemically synthesized siRNA targeting HPV16E7, delivered via liposomal transfection	Preclinical (in vitro cell line study)	Significant reduction in HPV16E7 mRNA (~ 66%) and protein levels (~ 39%); increased G0–G1 phase arrest; reduced S phase cell population after 24–72 h	Marked inhibition of CaSki cell proliferation, induction of cell cycle arrest, no effect observed in nonsense siRNA control groups	Demonstrated that HPV16E7-specific siRNA effectively suppresses cervical cancer cell growth and represents a promising genetic therapy target	Requires in vivo validation, investigation of long-term effects, optimization of delivery systems, and evaluation in combination with other therapeutic approaches	[130]
siRNA-mediated downregulation of TC21 to sensitize esophageal cancer cells to cisplatin	Esophageal squamous cell carcinoma (ESCC; TE13 cell line)	siRNA targeting TC21 combined with cisplatin chemotherapy	Preclinical (in vitro TE13 cells)	~ 80% reduction in TC21 mRNA and ~ 95% protein knockdown; decreased PI3K/Akt/NF-κB/cyclin D1 signaling; increased pPTEN; minor effect on Raf pathway	TC21 knockdown alone induced ~ 14% cell death; cisplatin alone ~ 18% cell death; combined treatment led to ~ 38% cell death; significant S-phase reduction, enhanced sensitivity	Demonstrated that TC21 knockdown sensitizes ESCC cells to cisplatin by suppressing survival pathways; highlights TC21 as a predictive marker for chemosensitivity	Requires in vivo validation, exploration of delivery methods, identification of additional downstream effectors, and clinical translation for patient stratification	[131]
siRNA-mediated knockdown of Keap1 to activate Nrf2-regulated cytoprotective genes	Chemoprevention model (human HaCaT keratinocytes, in vitro)	Duplex siRNA targeting human Keap1 mRNA to relieve Nrf2 inhibition and induce antioxidant response	Preclinical (in vitro cell culture)	~ 70% reduction of Keap1 mRNA, marked increase in Nrf2 protein, ~ 2.3-fold rise in ARE-driven reporter activity, 5–14-fold increase in detoxifying gene expression, 1.75-fold rise in intracellular glutathione	-	Provided proof of concept that Keap1 siRNA selectively upregulates Nrf2-mediated antioxidant defense genes, offering a potential non-toxic chemopreventive approach	Requires in vivo validation, exploration of delivery systems, long-term safety and efficacy assessments, and investigation of off-target effects in complex systems [132]	[132]
Use of Bcl-xL ASOs to induce apoptosis and enhance chemosensitivity	Pancreatic cancer (Panc-1, MIA-PaCa-2, Capan-1, ASPC-1, T3M4 cell lines)	Antisense phosphorothioate oligonucleotides targeting Bcl-xL, combined with gemcitabine chemotherapy	Preclinical (in vitro pancreatic cancer cell lines)	Significant downregulation of Bcl-xL mRNA and protein; increased apoptosis (confirmed by DAPI staining); dose- and time-dependent effects observed; enhanced gemcitabine sensitivity	Marked reduction in cell viability across all tested lines; maximal effects when combined with gemcitabine; induced apoptotic morphological changes	Demonstrated that targeting Bcl-xL effectively induces apoptosis and enhances chemotherapy response, suggesting potential for antisense-based therapeutic strategies in pancreatic cancer	Needs in vivo validation, exploration of delivery optimization, long-term efficacy and safety assessment, and progression toward clinical testing	[133]
Direct targeting of FOXP3 in Tregs with AZD8701 ASO to relieve immunosuppression	Various cancers (preclinical models, including ovarian, colon, breast, and syngeneic murine models)	Systemic administration of constrained ethyl (cEt)-modified ASOs targeting FOXP3 (AZD8701)	Preclinical (human PBMCs, humanized mice, multiple mouse tumor models)	> 70% FOXP3 knockdown in Tregs; reduced immunosuppressive markers (CTLA4, ICOS, GITR); enhanced CD8+ T cell infiltration; increased effector/memory T cell populations	Significant tumor growth inhibition; 25–50% complete responses (CR) in some models; additive effects when combined with PD-1/PD-L1 blockade; minimal autoimmune toxicity observed	Demonstrated that FOXP3 ASOs can reprogram Tregs to a less suppressive state, boost antitumor immunity, and enhance immune checkpoint therapy without depleting Tregs	Requires further clinical validation (currently in Phase 1a/b, NCT04504669), detailed investigation of long-term effects, Treg subset-specific responses, and biomarker identification	[134]
Development and clinical evaluation of AZD9150, a next-generation ASO targeting STAT3	Lymphoma (including DLBCL, Hodgkin’s), NSCLC, various solid tumors	Systemic administration of cEt-modified ASO AZD9150 targeting STAT3	Preclinical (multiple cancer models, xenografts) and phase I clinical trial (25 patients)	> 70% STAT3 knockdown, selective target inhibition, enhanced CD8+ T cell infiltration, reduced tumor-associated survival signals (MCL-1, c-MYC, VEGF)	Preclinical: significant tumor inhibition; Clinical: 44% stable disease or partial responses (PRs), 2/6 DLBCL patients with durable PR, activity at doses as low as 2–3 mg/kg	Demonstrated that AZD9150 selectively inhibits STAT3, reduces tumor growth in models, and shows early clinical activity in refractory lymphomas and NSCLC	Needs phase II/III trials, investigation of patient selection biomarkers, long-term safety, optimal combination strategies, and detailed stromal vs. tumor cell contributions	[135]
Enhanced potency of GalNAc-conjugated ASOs) in HCC models	HCC human cell lines, DEN-induced mouse models, patient-derived circulating tumor cells)	Systemic administration of GalNAc-conjugated ASOs targeting MyD88, SR-B1, YAP1, or MALAT1	Preclinical (in vitro HCC cell lines, in vivo mouse models, ex vivo human CTC spheroids)	Up to 10–20-fold increased ASO activity in ASGR-positive HCC cells, improved mRNA knockdown (MyD88, SR-B1, YAP1), increased apoptosis markers, enhanced ASO accumulation in tumors	Significant tumor Burden reduction, 63% of animals tumor-free after GalNAc-MyD88 ASO, superior efficacy vs. parental ASO, minimal hepatotoxicity or systemic toxicity	Demonstrated that GalNAc-conjugated ASOs improve hepatocyte-specific delivery even in partially ASGR-reduced HCC, providing a potent strategy for targeting hard-to-drug pathways like MyD88	Needs patient selection strategies based on ASGR functionality, further clinical translation, determination of ASGR lower threshold, and long-term efficacy and safety studies	[136]
Downregulation of SRSF3 by ASOs to sensitize cancer cells to paclitaxel (PTX)	Oral squamous cell carcinoma (CAL 27, SCC-9) and breast cancer (MCF-7)	ASO SR-3 targeting SRSF3, combined with PTX chemotherapy	Preclinical (in vitro cell lines, tissue samples, TCGA data analysis)	Significant reduction in SRSF3 mRNA And protein, altered alternative splicing of exon 4, enhanced apoptosis, suppressed proliferation, restored PTX sensitivity	Marked increase in cancer cell sensitivity to PTX, higher apoptosis rates with combined ASO + PTX, potential prognostic value Linked to exon 4 splicing ratios	Demonstrated that targeting SRSF3 improves PTX efficacy by modulating splicing and sensitizing resistant cancer cells; Links between exon 4 splicing and survival identified	Requires in vivo validation, exploration of clinical translation, optimization of ASO delivery, and investigation of long-term treatment effects	[137]
Customized design of ASOs targeting EGFR driver mutations for NSCLC	NSCLC with EGFR L858R, T790M mutations	Patient-specific ASOs delivered via red blood cell-derived extracellular vesicles (RBCEVs), including nanobody surface modification for targeting	Preclinical (cell lines, xenograft Mice, PDX models, 3 patient tumor samples)	Selective knockdown of EGFR mutants (L858R, T790M) with minimal effect on wild-type EGFR; reduced AKT, ERK1/2 signaling; enhanced apoptosis, reduced tumor cell proliferation	Marked tumor growth inhibition in xenografts; effective suppression of TKI-resistant patient-derived tumors; superior efficacy compared to first-, second, and third-generation TKIs	Demonstrated a precision medicine platform using mutation-specific ASOs and EV delivery, achieving potent, selective inhibition of drug-resistant EGFR mutations in NSCLC	Needs clinical translation, large-scale validation, assessment of long-term safety, optimized delivery strategies, and exploration of combination therapies	[138]
Delivery of ASOs for splice correction of AR pre-mRNA using cell-penetrating peptides (CPPs)	CRPC 22Rv1, DuCaP, VCaP cell lines	Amphipathic CPP PepFect 14 (PF14)-mediated delivery of splice-correcting AONs targeting AR-V7 generation	Preclinical (in vitro cell lines, confocal microscopy, flow cytometry, RT-qPCR, western blot)	~ 37–59% reduction in AR-V7 mRNA, confirmed protein-level downregulation; reduced androgen-independent proliferation in DuCaP, VCaP cells; uniform uptake across cell populations	Significant inhibition of androgen-independent proliferation in DuCaP, VCaP; lower But significant reduction in 22Rv1; less efficient than lipid-based delivery (X-tremeGENE 9)	Demonstrated that PF14 enables uniform, effective AON delivery for AR-V7 splice correction; showed potential to complement AON technology for PCa targeted therapy	Needs further in vivo validation, optimization of CPP formulations, exploration of targeting ligands (PSMA), and development for clinical translation	[139]
Investigating compensatory gene expression following BCL2 ASO treatment in PCa	PCa (LNCaP cell line)	Monospecific and bispecific ASOs targeting BCL2 (and EGFR) delivered in lipofectin nanoparticles	Preclinical (in vitro; gene expression analysis, RT-PCR, gel electrophoresis)	Significant upregulation of non-targeted genes, notably KI-67 (~ 350–430% increase), cyclin D1 (33–73% increase), AR, AKT1, IL6, STAT3, PD1/PDL1; suppressed caspase-3 expression	-	Demonstrated that BCL2-targeted ASOs induce broad compensatory responses enhancing proliferation and survival pathways; identified KI-67 as a promising co-target	Requires in vivo validation, testing of dual or multispecific oligonucleotides, exploration of delivery improvements, and assessment of long-term therapeutic efficacy and resistance mechanisms	[140]
Development of cathepsin B (CB)-activatable cyclic ASOs (cASOs) for cell-specific gene knockdown	PCa (PC-3 cells), inflammation-related cancer (LTBR target)	CB-sensitive cASOs targeting TCTP or LTBR, activated in CB-rich environments, compared with linear ASOs and non-CB-sensitive controls	Preclinical (PC-3, HUVEC cell lines; xenograft mouse models)	> 70% knockdown of TCTP and LTBR in CB-rich PC-3 cells; minimal effect in CB-deficient HUVECs; reduced nonspecific immune markers (IL-6, IFN-β) compared to linear ASOs	Significant tumor volume and weight reduction in PC-3 xenograft mice; ~71% TCTP protein downregulation; comparable efficacy to linear ASOs but with enhanced specificity	Demonstrated that cASOs allow enzyme-triggered, cell-specific gene silencing with reduced off-target effects and immunostimulation, offering a promising antisense prodrug platform	Needs scalable synthesis strategies, improved delivery systems, long-term in vivo safety evaluation, and translation to systemic application for clinical use	[141]
Targeting GLI2 with ASOs to inhibit the Sonic Hedgehog pathway in bladder cancer	Bladder cancer (UM-UC-3, 253 J-BV cell lines; orthotopic mouse models)	Intravesical administration of GLI2-targeted ASOs (Ionis 183652) vs. scrambled controls	Preclinical (in vitro cell lines; in vivo orthotopic bladder cancer mouse models, n = 18 mice)	Significant GLI2 mRNA and protein knockdown; enhanced apoptosis (cleaved PARP, TUNEL staining); reduced proliferation (Ki67) in tumors; variable SHH pathway responsiveness across cell lines	Marked tumor growth inhibition in GLI2 ASO-treated mice (no net growth over 40 days vs. ~589-fold increase in controls); significantly reduced growth rates (p = 0.018)	Demonstrated that direct inhibition of GLI2 bypasses upstream pathway variability, effectively reducing tumor growth even in SHH inhibitor-resistant cells; highlights bladder as a uniquely accessible site for local ASO delivery	Requires larger-scale in vivo testing, assessment of potential off-target effects, exploration of combinatorial strategies, and eventual clinical translation for intravesical therapy	[142]
ASOs targeting miRNA-21 to inhibit the proliferation and migration of colon cancer cells	Colon carcinoma (HCT116, SW620 cell lines)	Transfection with p-miR-21-ASO vector encoding ASOs against miR-21	Preclinical (in vitro colon cancer cell lines)	Significant reduction of miR-21 levels; increased PTEN expression; decreased phospho-AKT and phospho-ERK1/2; reduced VEGF expression	Reduced proliferation, colony formation, invasion, and migration in vitro; impaired metastatic potential; specific downregulation of oncogenic pathways	Demonstrated that miR-21 ASO effectively suppresses proliferation and migration via PTEN upregulation and downstream AKT/ERK pathway modulation, offering a potential therapeutic approach	Needs in vivo validation, exploration of delivery strategies, assessment of other miR-21 targets (like PDCD4), and evaluation in combination with other therapies	[143]
Development of dual-modified ASOs (AmNA-ASOs) targeting PCDHA11 in gastric cancer	Gastric cancer, also tested in pancreatic, colon, breast, lung, esophageal cancers	AmNA-modified ASOs targeting PCDHA11, delivered intraperitoneally and systemically	Preclinical (in vitro cell lines; mouse models of peritoneal metastasis, systemic metastasis, subcutaneous tumors; Pcdha11-deficient mice)	Significant knockdown of PCDHA11 mRNA and protein, suppression of AKT/mTOR, Wnt/β-catenin, JAK/STAT signaling; increased apoptosis; reduced stemness (ALDH, spheroid formation); impaired adhesion to mesothelial cells	Strong inhibition of tumor proliferation, invasion, migration, peritoneal and systemic metastasis; reduced subcutaneous tumor growth; reversible, dose-dependent liver toxicity; no gross abnormalities in knockout mice	Demonstrated that PCDHA11 is an oncogenic driver in gastric and other solid tumors; AmNA-ASOs provide a promising targeted therapy with potential as a biomarker-guided treatment approach	Requires further systemic delivery studies, combination therapy trials, clinical translation, optimization of dosing to minimize liver toxicity, and companion diagnostic development	[144]
Targeting TRIM34 to enhance ferroptosis sensitivity and improve immunotherapy efficacy	HCC	CRISPR/Cas9 screening; TRIM34 knockdown to disrupt TRIM34/UPF1/GPX4 axis; anti-PD-1 immunotherapy combination	Preclinical (HCC cell lines, xenograft models, patient samples; n = 90 paired tissues, multiple mouse groups)	Increased lipid peroxidation, iron accumulation, reactive oxygen species (ROS), and mitochondrial damage upon TRIM34 knockdown; boosted CD8+ T cell infiltration and activity	Significant tumor growth inhibition, reduced metastasis, prolonged survival in mice; enhanced anti-PD-1 efficacy when combined with TRIM34 knockdown	Identified TRIM34 as a key suppressor of ferroptosis by promoting UPF1 degradation and stabilizing GPX4 mRNA; demonstrated TRIM34 targeting as a strategy to sensitize HCC to immunotherapy	Needs further validation in clinical settings, exploration of delivery methods for TRIM34 inhibition, long-term safety assessment, and combination strategy optimization	[145]
Identification of KRAS-induced COX2 as a driver of immunotherapy resistance	KRAS-mutant LUAD	CRISPR–Cas9 screening; pharmacologic inhibition of COX2/PGE2 axis; combination with KRASG12C inhibitors and anti-PD1 therapy	Preclinical (in vitro KRAS-mutant lines, orthotopic mouse models, human patient cohorts)	KRAS-induced COX2 upregulation suppresses antitumor immunity; COX2 knockout/inhibition increases CD8+ T-cell and NK cell infiltration, shifts macrophage polarization, and boosts effector function	COX2 deletion or inhibition (celecoxib, EP2–EP4 antagonist) sensitizes tumors to anti-PD1; combination delays tumor relapse after KRASG12C inhibition, improves survival; high COX-IS predicts poor response in patients	Demonstrated that KRAS drives immune evasion via COX2/PGE2, contributing to immunotherapy and KRAS inhibitor resistance; COX2 inhibition reverses immunosuppression and improves outcomes	Requires clinical trials of COX2/PGE2 inhibitors combined with KRAS-targeted or immunotherapy agents; investigation of toxicity, long-term safety, and patient stratification using COX-IS signatures	[146]
Computational correction of copy-number effects to improve specificity in CRISPR-Cas9 essentiality screens	Pan-cancer (342 cancer cell Lines from 27 lineages)	Genome-scale CRISPR-Cas9 loss-of-function screens corrected using CERES, a computational model estimating gene dependency independent of copy-number artifacts	Preclinical (342 cell Lines; additional independent datasets with 33 And 14 cell lines reanalyzed)	-	Improved identification of true essential genes, reduced false positives, preserved known cancer-specific dependencies (KRAS, TRPS1, GRHL2) even in amplified regions	Demonstrated that CERES correction significantly improves the accuracy of CRISPR-Cas9 screens, enabling better mapping of cancer dependencies across genomic contexts	Requires broader application across more datasets, integration with clinical tumor samples, further refinement of guide activity prediction, and combination with chemical screening data	[147]
Scaffold-mediated CRISPR-Cas9 delivery targeting IL1RAP to eliminate leukemia stem cells (LSCs)	AML	Bioreducible lipidoid nanoparticle (LNP)-encapsulated Cas9/sgRNA RNP loaded on mesenchymal stem cell membrane–coated nanofibril scaffolds (MSCM-NF)	Preclinical (THP-1 leukemia cells, mouse xenotransplantation model, in vitro and in vivo studies)	Efficient IL1RAP gene knockout (~ 53% editing efficiency), reduced IL1RAP expression, enhanced apoptosis, decreased colony formation, increased local retention of Cas9 RNP	Significant reduction in leukemic burden in NSG mice, impaired colony-forming ability of LSCs, sustained local delivery, and improved editing efficiency compared to free LNPs	Demonstrated that local scaffold-mediated delivery of CRISPR-Cas9 efficiently targets AML LSCs, overcomes delivery challenges, and reduces leukemic progression	Requires further optimization of scaffold degradation rate, scaling up for in vivo use, clinical translation, and exploration of combination strategies with systemic therapies	[148]
CRISPR–Cas9–mediated selective targeting of mutant KRAS to inhibit tumor growth	KRAS-mutant cancers (colorectal, lung, pancreatic; various cell lines)	Lentiviral and AAV delivery of Cas9 and sgRNAs targeting specific KRAS mutations (G12V, G12D, G13D)	Preclinical (multiple KRAS-mutant and wild-type cancer cell lines, mouse xenograft models)	Selective disruption of mutant KRAS alleles; minimal effect on wild-type KRAS; significant reduction in KRAS signaling pathways; increased apoptosis in mutant cells	Strong inhibition of colony formation (up to ~ 94%), reduced soft agar growth, 7–10-fold reduction in tumor weights in vivo, no impact on wild-type KRAS tumors	Demonstrated proof-of-concept that CRISPR-Cas9 can selectively target and disrupt mutant KRAS, controlling tumor proliferation both in vitro and in vivo	Requires improved delivery efficiency, assessment of potential off-target effects or resistance, long-term safety evaluation, and exploration of combination with other therapies	[149]
CRISPR/Cas9 targeting of GPRC6A to suppress PCa progression	PCa (PC-3, DU145, LNCap, 22Rv1 cell lines; xenograft mouse models)	CRISPR/Cas9-mediated deletion of GPRC6A in PC-3 cells; in vitro and in vivo testing with osteocalcin stimulation	Preclinical (in vitro assays; xenograft nude mice, n = 5 per group)	Significant reduction of ERK, AKT, mTOR signaling after GPRC6A knockout; decreased PCNA, RUNX2, c-Fos, MMP9, VEGF, BMP3, PSA expression; attenuated proliferation, migration	Reduced tumor growth and weight in xenografts; osteocalcin-driven tumor progression blocked by GPRC6A knockout; impaired androgen biosynthesis-related gene expression	Demonstrated that GPRC6A promotes PCa progression via ligand-dependent activation (osteocalcin, testosterone); validated GPRC6A as a potential therapeutic target	Needs clinical translation, further exploration of GPRC6A antagonists, investigation into racial disparities (polymorphisms), and evaluation in AR-independent resistance models	[150]
Cancer-specific CRISPR-Cas9 targeting of InDel mutations (CINDELA approach) to induce selective cancer cell death	Multiple cancers (osteosarcoma, colon, GBM, lung cancer; PDXs)	CRISPR-Cas9 system delivering multiple sgRNAs targeting cancer-specific InDels to induce high-load DNA DSBs	Preclinical (in vitro U2OS, HCT116, patient-derived GBM; in vivo xenografts and PDX models in mice)	Selective induction of γ-H2AX, TUNEL-positive apoptosis, > 50 cancer-specific DSBs, no effect on normal cells; enhanced by combining with DNA repair inhibitors	Significant inhibition of tumor growth in xenografts and PDXs; delayed tumor progression; minimal off-target toxicity in normal cells or tissues	Demonstrated that leveraging cancer-specific InDels for targeted CRISPR-Cas9 DSB induction (CINDELA) achieves potent, selective killing of cancer cells, offering a novel precision therapeutic platform	Needs development of high-fidelity Cas9 systems, delivery optimization for clinical translation, investigation of resistance mechanisms, and combination therapy strategies	[151]
In vivo CRISPR/Cas9 multiplex-mutagenesis for high-throughput functional genomics	HCC, intrahepatic cholangiocarcinoma (ICC) in mice	Hydrodynamic tail vein injection (HTVI) of multiplexed CRISPR/Cas9 vectors targeting 10–18 genes in mouse livers	Preclinical (mouse models, 41 tumors analyzed, multi-region sampling, cell lines, patient-derived data comparison)	High-efficiency biallelic mutations (~ 79%), positive selection for functional mutations (Pten, Arid1a), increased clonal diversity, minimal off-target effects; reshaped hepatobiliary tumorigenesis landscape	Robust induction of HCC and ICC in mice; tumor growth driven by chromatin modifier and PI3K pathway alterations; metastasis tracking and subclonal heterogeneity captured; validated in Kras-mutant and CCl4-damaged livers	Demonstrated feasibility of large-scale, in vivo CRISPR/Cas9 multiplexing for cancer gene discovery, revealing key functional drivers and chromosomal alterations; identified chromatin modifiers as central players	Requires delivery optimization, comprehensive off-target profiling, exploration of long-term effects, and clinical translation potential for functional cancer genomics and precision targeting	[152]
Investigating the role of CD133 in melanoma invasion, metastasis, and therapeutic resistance	Melanoma (BAK-P, BAK-R, POT, SK-Mel2 cell lines; xenograft and zebrafish models)	CRISPR-Cas9 and siRNA knockdown of CD133; doxycycline-inducible CD133 overexpression; transwell and zebrafish assays	Preclinical (in vitro cell lines, xenografts, zebrafish metastasis models)	CD133 overexpression increased MMP2/MMP9 expression, stem cell markers, EMT traits, chemoresistance; CD133 knockdown reduced invasion, metastasis, MMP levels	CD133 knockdown significantly reduced invasion and metastasis in vitro and in vivo; CD133 overexpression promoted invasion; CD133 linked to poor survival and recurrence	Demonstrated that CD133 plays an essential role in melanoma invasion and metastasis via MMP upregulation, establishing CD133 as a potential therapeutic target	Requires further mechanistic exploration of CD133-MMP pathways, in vivo validation in additional models, and development of CD133-targeted therapeutic strategies	[153]
Efficient CRISPR-Cas9/HDR-based somatic precision gene editing for in vivo cancer modeling	Breast cancer (mouse mammary gland models), tested with Kras and Pik3ca mutations	AAV9 vector–delivered CRISPR-Cas9 system introducing HDR-mediated precise mutations (KrasG12D, Pik3caH1047R) in somatic mammary cells	Preclinical (normal and MMTV-Wnt1 precancerous mice; xenograft models; comparative lentivirus controls)	High editing efficiency (~ 50% target mutation rate), minimal off-target effects, preserved native gene regulation, robust tumor induction, enhanced apoptosis and oncogenic signaling	Fast and consistent tumor induction (as short as 9–28 days for Kras editing; ~3.9 months for Pik3ca), histologically distinct tumors compared to lentivirus models, efficient modeling of tumor evolution	Demonstrated that precise in vivo CRISPR-HDR editing yields high-fidelity mouse tumor models, better mimicking natural tumor evolution and signaling compared to viral oncogene overexpression	Requires extension to other tissues/organs, optimization of delivery methods, exploration of multi-gene editing, and long-term safety assessment for translational applications	[154]
CRISPR-Cas9 targeting of somatic mutation-derived protospacer adjacent motifs (PAMs) in cancer cells	Pancreatic cancer (with comparison to lung, esophageal cancers)	Whole genome sequencing (WGS)-guided design of sgRNAs targeting novel PAMs generated by somatic mutations; multiplexed CRISPR-Cas9 killing strategy	Preclinical (3 patient-derived Pancreatic cancer Lines, 591 ICGC tumors analyzed, in vitro co-culture, mouse-human models)	Selective induction of DNA DSBs in tumor cells; minimal off-target effects; increased γH2A.X foci in target cells; enhanced apoptosis; no damage to normal cells or irrelevant cancer lines	Up to 99% selective killing in pancreatic cancer cell lines; >83–88% reduction of target cells over 14–21 days; hundreds to thousands of targetable PAMs identified per tumor; validation across multiple tumor types	Demonstrated a novel precision genome-editing approach leveraging somatic PAMs for highly selective cancer cell killing; expanded CRISPR target repertoire beyond coding mutations	Requires optimization of in vivo delivery systems (nanoparticles), assessment of safety and immunogenicity in animal models, clinical translation strategies, and scalability for patient-specific applications	[155]
Systematic assessment of functional relevance of gene fusions in cancer cell fitness	41 cancer types (1011 human cancer cell lines, including solid and hematologic tumors)	Integration of RNA-seq, pharmacological drug screening (> 350 anti-cancer compounds), and whole-genome CRISPR-Cas9 loss-of-fitness screens	Preclinical (1011 cell Lines; CRISPR in 371 Lines; drug sensitivity in 982 lines)	-	Most gene fusions were non-functional for cell fitness; however, actionable fusions (RAF1, BRD4-NUTM1, ROS1, YAP1-MAML2) were identified, revealing new therapeutic targets across cancers	Provided a functional map distinguishing driver vs. passenger fusions, uncovering rare but targetable fusions with potential for clinical repurposing of existing drugs, including in histologies where not previously recognized	Requires in vivo validation of therapeutic targets, exploration of tissue-specific effects, evaluation of subclonal fusions, and development of targeted clinical trials or basket trials for identified fusion-positive tumors	[156]
CRISPR-Cas9 screening to identify synthetic lethality (SL) between LIG1 and PARP in PCa	CRPC, extended to lung, breast, colorectal cancers	CRISPR-Cas9 knockout screens targeting 356 DNA repair genes + PARPi (olaparib, talazoparib); pharmacologic co-inhibition using LIG1 inhibitors (L82-G17) + PARPi	Preclinical (PCa cell lines, xenografts; other tumor models; TCGA and SU2C-PCF datasets)	Induced replication stress, DSBs, γH2AX accumulation, apoptosis in LIG1-deficient cells treated with PARPi; conserved across tumor types; no major immune focus reported	Strong reduction in tumor cell viability in vitro; significant tumor growth inhibition in vivo; effective combination therapy even in BRCA2-deficient cells; selective killing of LIG1-deficient tumors	Identified LIG1 as a novel biomarker predicting PARPi sensitivity; validated SL interaction with PARP; proposed combined pharmacologic inhibition as a new therapeutic strategy	Needs development of potent in vivo LIG1 inhibitors, clinical translation for patient stratification, expanded testing across cancers, assessment of long-term safety and resistance mechanisms	[157]
Inhibition of HSF1 DNA binding using a potent RNA aptamer (iaRNAHSF1) to induce cancer cell apoptosis	Cervical cancer (HeLa), transformed kidney (293T), non-transformed lung fibroblasts (IMR-90), breast cancer, GBM, neuroblastoma	Synthetic gene-delivered dimeric RNA aptamer (iaRNAHSF1) blocking HSF1 DNA binding; blasticidin selection; functional validation	Preclinical (in vitro HeLa, IMR-90, 293 T, MCF7, U87, MDA-MB-231, SK-N-BE(2)-M17 cell lines; soft agar assays)	Significant inhibition of HSF1 and HSF2 promoter occupancy; reduction in Hsp70, Hsp90, Grp78, calnexin, TGM2 levels; reduced MAPK signaling (EGFR, Erk1/2); apoptosis induction (~ 7–9-fold increase)	Abolished colony formation in soft agar; ~63% apoptosis in HeLa, ~ 7-fold apoptosis Increases in 293 T; phenotypes rescued by HSF1 or Hsp90 overexpression; minimal effect on IMR-90 normal cells	Demonstrated that RNA aptamer-mediated HSF1 inhibition induces selective apoptosis and impairs transformation, validating HSF1 as a therapeutic target in cancer	Requires optimization for in vivo delivery, exploration of aptamer modifications for stability, broader cancer applicability testing, and preclinical safety/efficacy assessment	[158]
Screening and characterization of a novel RNA aptamer targeting human prostatic acid phosphatase (PAP)	PCa (PC-3, LNCaP cell lines; comparison with non-cancer IMR-90 cells)	SELEX-derived 2’-fluoropyrimidine (FY)-modified RNA aptamer (6 N) with PAP binding specificity; minimization and structure-function studies	Preclinical (in vitro cell binding assays, fluorescence imaging, EMSA)	Specific binding to PAP-expressing PCa cells; no binding to PAP-negative control cells; localization on cell membranes; not internalized	Identified high-affinity binding (Kd ~ 118 nM); confirmed aptamer specificity; minimal size (~ 50 nt) with two essential hairpin structures for binding; potential theranostic applications	Developed a stable, specific RNA aptamer platform for targeting PAP, opening opportunities for conjugation with anti-cancer drugs or imaging agents for PCa diagnosis and therapy	Needs in vivo validation, assessment of binding to native glycosylated PAP forms, further functional testing (antagonistic/agonistic roles), and development of therapeutic or diagnostic conjugates	[159]
Development of RNA aptamer (PDR3) targeting PDGFRα to inhibit GBM via STAT3 and p53 pathways	GBM	SELEX-derived RNA aptamer PDR3 targeting PDGFRα; also tested as PDR3-siSTAT3 chimera for targeted siRNA delivery	Preclinical (U251-MG GBM cells; in vitro assays; confocal, qPCR, western blot, apoptosis arrays, DNA methylation analysis)	Significant downregulation of STAT3, upregulation of JMJD3 and p53, increased p53 phosphorylation, activation of death receptors (TRAIL-R1/R2, FADD, Fas), induction of apoptosis	Dose-dependent reduction in GBM cell viability, suppression of proliferation, nuclear translocation, and DNA hypomethylation at tumor-related gene promoters (CCND2)	Demonstrated that PDGFRα-targeting aptamer induces extrinsic apoptosis, regulates epigenetic states, and enhances STAT3 silencing when combined with siRNA; promising targeted therapeutic strategy for GBM	Needs in vivo efficacy testing, optimization of delivery and stability, further exploration of nuclear mechanisms, and evaluation of innate immune activation effects	[160]
Development of an RNA aptamer (YJ-1) targeting the metastasis-inducing domain of CEA to inhibit liver metastasis	Colon cancer (CEA-positive lines; LS174T, LoVo, CAPAN-1; in vivo mouse liver metastasis model)	SELEX-derived RNA aptamer YJ-1 targeting the PELPK sequence of CEA; PEGylated for in vivo use; tested in vitro and in vivo	Preclinical (in vitro cell lines; n = 7 mice per group in liver metastasis model)	Specific disruption of CEA–heterogeneous nuclear ribonucleoprotein M4 and CEA–DR5 interactions; induced apoptosis (anoikis); inhibited homotypic aggregation, migration, and invasion	91% reduction in hepatic metastasis tumor volume in mice; significant inhibition of cancer cell aggregation, migration, and invasion; minimal hepatotoxicity	Demonstrated that the YJ-1 aptamer selectively blocks CEA-mediated metastasis processes, induces anoikis, and significantly reduces liver metastasis in vivo, offering diagnostic and therapeutic potential	Requires further pharmacokinetic optimization, in vivo imaging validation, clinical translation, and assessment of long-term safety and efficacy in diverse cancer models	[161]
Selection and characterization of RNA aptamers targeting ErbB2 for imaging ErbB2-overexpressing breast cancer cells	Breast cancer (ErbB2-positive and negative lines; MDA-MB-453, T47D, KPL-4, MCF-7, A431)	SELEX-derived 2′-fluorine-modified RNA aptamers (mini-aptamer SE15-8) binding extracellular ErbB2 domain	Preclinical (in vitro binding assays, surface plasmon resonance, immunocytochemistry)	High-affinity binding (Kd ~ 3.49 nM) to extracellular ErbB2; specific recognition of ErbB2-positive breast cancer cells; no cross-reactivity with EGFR-positive or ErbB2-negative lines	Demonstrated specific staining of ErbB2-positive cells, strong membrane-bound signals, and potential utility as an imaging agent for ErbB2-expressing tumors	Identified and optimized a small, stable RNA aptamer platform for ErbB2-specific targeting, opening potential for non-antibody-based imaging and therapeutic applications	Requires in vivo imaging validation, assessment of pharmacokinetics and biodistribution, exploration of therapeutic conjugates, and preclinical safety evaluation	[162]
RNA aptamer (OPN-R3) targeting osteopontin (OPN) to inhibit breast cancer growth and metastasis	Breast cancer (MDA-MB231 human breast cancer cells)	SELEX-derived RNA aptamer (OPN-R3) binding OPN, blocking interactions with CD44 and αvβ3 integrin receptors; modified aptamer used in vivo	Preclinical (in vitro adhesion, migration, invasion assays; in vivo xenograft mouse models)	Significant downregulation of PI3K, JNK1/2, Src, Akt signaling; reduced MMP2 and uPA levels; blocked OPN binding on cell surface; enhanced apoptosis	60% reduction in adhesion, 50% reduction in Migration, 65% reduction in invasion in vitro; 4–12-fold decrease in tumor bioluminescence; 18–20-fold reduction in tumor volume; >99% reduction in lung metastases in mice	Demonstrated that OPN-R3 effectively inhibits OPN-mediated signaling, invasion, and metastasis, offering a novel RNA aptamer–based therapeutic strategy against breast cancer	Requires optimization of delivery and stability for clinical use, long-term safety evaluation, and exploration of combinatorial treatments with existing therapies	[163]
Development of RNA aptamers targeting PPAR-δ to inhibit tumor-promoting gene transcription	Colon cancer (HCT116, SW480, DLD-1 cell lines)	Nuclear-localized RNA aptamers (intramers) against PPAR-δ, expressed via U6 promoter, inhibiting VEGF-A and COX-2 expression	Preclinical (in vitro transcriptional assays, luciferase reporters, colony formation assays)	Significant reduction in VEGF-A and COX-2 mRNA levels; suppression of PPRE-driven luciferase activity; inhibition of β-catenin-mediated transcription when combined with β-catenin aptamer	Marked reduction in colony formation (tumorigenic potential) in HCT116 cells; 20% suppression of cell proliferation; potent modulation of PPAR-δ and β-catenin targets	Demonstrated that PPAR-δ RNA aptamers effectively inhibit transcription of tumor-promoting genes, reduce colon cancer cell tumorigenicity, and offer a platform for combined signaling inhibition	Needs in vivo validation, exploration of aptamer delivery and stability, mechanistic dissection of PPAR-δ inhibition, and development toward therapeutic applications	[164]
Development of a nuclease-resistant RNA aptamer (CL4) targeting EGFR to induce selective apoptosis	Non-small-cell lung cancer (NSCLC), glioma, breast cancer, EGFR-positive lines (A549, Calu1, A431)	29-fluoro-pyrimidine RNA aptamer CL4 targeting EGFR; in vitro and in vivo xenograft models; combined with cetuximab	Preclinical (in vitro cell viability, apoptosis assays; mouse xenograft model; A549, Calu1, A431, H460, MCF7 cell lines)	Inhibition of EGFR phosphorylation and downstream signaling (STAT3, ERK1/2); activation of caspase-3, caspase-8, PARP cleavage; apoptosis induction; no effect in EGFR-negative cells	~ 60% reduction in cell viability; ~57% tumor growth inhibition in A549 xenografts over 16 days; synergy with cetuximab; no added benefit when combined with gefitinib	Demonstrated that CL4 selectively targets and inhibits EGFR homodimers and heterodimers, blocks survival signaling, and induces apoptosis even in EGFR–inhibitor–resistant cells	Requires pharmacokinetic optimization, epitope mapping, long-term in vivo safety evaluation, and exploration of clinical translation and aptamer–antibody combinations	[165]
Investigation of miR-128-3p in driving chemoresistance and metastasis via Wnt/β-catenin and TGF-β pathways	NSCLC, A549, Calu-3, H520, LL/2-luc-M38 cell lines; patient samples)	Overexpression or antagonism of miR-128-3p; use of miRNA sponge, antagomir, and pathway inhibitors; in vitro and in vivo xenograft/metastasis models	Preclinical (in vitro assays; xenograft, spontaneous, experimental metastasis models; cohorts of 153 And 234 NSCLC patients for expression analysis)	Induction of EMT, CSC traits, anti-apoptotic signaling, drug transporter expression (ABCG2, CTR2), Wnt and TGF-β pathway overactivation	High miR-128-3p levels correlated with poor survival (median OS 19 vs. 68 months), lower chemotherapy response (PFS 2.7 vs. 5.7 months); inhibition of miR-128-3p reduced tumor growth and metastasis, resensitized cells to chemotherapy	Identified miR-128-3p as a driver of chemoresistance-associated metastasis in NSCLC by co-activating Wnt/β-catenin and TGF-β pathways; antagonism reversed malignancy and drug resistance	Requires further investigation into clinical targeting strategies, long-term efficacy of antagomir delivery, potential combination therapies, and patient stratification for miR-128-3p targeting	[166]
Investigation of miR-126 and miR-126* roles in suppressing breast cancer metastasis by modulating the TME	Breast cancer (4T1 murine mammary tumors; human breast cancer patient samples)	Ectopic expression or inhibition of pri-miR-126/miR-126*; RNA sponges; luciferase assays; tail-vein injections; syngeneic mouse models	Preclinical (4T1 cells in BALB/c Mice; 240 human patient samples; in vitro migration, invasion, qPCR, immunostaining)	Suppressed recruitment of mesenchymal stem cells (MSCs) and inflammatory monocytes via downregulation of Sdf-1α and Ccl2; reduced stromal support; no major impact on endothelial cell recruitment	Significant reduction in lung metastases (~ 60–70% decrease); correlation between low miR-126/126* expression and poor metastasis-free survival in patients; Sdf-1α rescue experiments restored metastasis	Identified miR-126/126* as key suppressors of stromal cell recruitment and metastatic spread by targeting Sdf-1α/Ccl2 axis; showed epigenetic silencing of miR-126 via Egfl7 promoter methylation in patient tumors	Requires development of therapeutic delivery strategies for miR-126 restoration, exploration of combinatorial treatments, and clinical trials to validate stromal-targeted metastasis prevention	[167]

Limitations: RNA-based therapeutics in cancer treatment encounter several specific challenges. Although RNA aptamers offer high target specificity, they are prone to rapid degradation by nucleases and exhibit limited in vivo stability, restricting their systemic use unless chemically modified. ASOs may cause off-target effects due to partial sequence complementarity and can elicit immune responses, particularly through activation of toll-like receptors. siRNAs must be efficiently delivered to the cytoplasm, yet they are often sequestered in endosomes or degraded before reaching their targets, with unintended gene silencing from off-target interactions remaining a significant issue. miRNAs, because of their inherent role in regulating numerous genes, present a risk of widespread and unpredictable changes in gene expression when altered, making it difficult to achieve therapeutic specificity without unintended side effects

Innovations in the stability of RNA therapeutics

Enhancing the stability of RNA therapies in oncological treatment is a fundamental problem that directly influences their clinical efficacy. RNA molecules, including siRNAs, mRNAs, and miRNAs, exhibit intrinsic instability owing to their vulnerability to destruction by pervasive ribonucleases (RNases) present in biological fluids and cellular environments. Chemical changes to the RNA backbone, Sugar, or bases have been extensively utilized to improve stability. Incorporating 2’-O-methyl, 2’-fluoro, or LNA alterations can enhance nuclease resistance while maintaining the molecule’s capacity to bind its target. Furthermore, phosphorothioate linkages, where sulfur atoms replace non-bridging oxygen in the phosphate backbone, significantly improve the stability and bioavailability of RNA molecules. These chemical modifications are often tailored for RNA-based therapies to enhance stability, preserve biological function, and minimize immunogenicity, which is crucial for avoiding unwanted immune responses in cancer patients. In addition to chemical alterations, delivery mechanisms are crucial for safeguarding RNA therapies and enhancing their stability in vivo. LNPs, polymeric nanoparticles, and exosome-based carriers have become significant methods for encapsulating RNA molecules, protecting them from enzymatic destruction, and enabling targeted delivery to tumor cells. LNPs have transformed RNA treatment by offering a stable, biocompatible delivery system that can be tailored for tumor-specific absorption by surface changes, including the attachment of ligands or antibodies that target cancer-specific markers. Polymeric systems, such as polyethyleneimine (PEI)-based nanoparticles, can augment cellular absorption via endocytosis while offering a safe encasement for the RNA. Moreover, hybrid systems that integrate lipids and polymers or utilize natural carriers such as exosomes provide novel methods to enhance stability, extend circulation duration, and guarantee the intact delivery of RNA cargo to targeted cancer cells. Alongside advancements in chemistry and delivery mechanisms, enhancing the intracellular stability and release of RNA therapies is crucial for successful cancer treatment. Upon entering the target cell, RNA molecules encounter other obstacles, such as trapping in endosomes and destruction within the lysosomal compartment. Researchers are creating intelligent delivery vehicles equipped with endosomal escape mechanisms, utilizing pH-sensitive lipids or polymers that destabilize endosomal membranes in acidic environments, therefore releasing the RNA payload into the cytoplasm to facilitate its therapeutic action. Furthermore, the integration of RNA therapeutics with small-molecule agents or gene-editing technologies (such as CRISPR-Cas systems) might augment the functional stability of RNA-based therapies, resulting in synergistic anticancer effects. An integrated approach that incorporates chemical modifications, advanced delivery systems, and controlled intracellular release is essential to overcome the inherent instability of RNA therapeutics, paving the way for more effective and long-lasting cancer treatments.

Although recent advances, particularly the use of LNPs and targeted conjugates like GalNAc, have improved the delivery of RNA therapies, significant challenges remain in the design, production, and application of nanoparticle-based delivery systems. A major hurdle lies in the material design of these nanoparticles, requiring precise engineering to achieve an optimal balance between stability, biocompatibility, and delivery efficiency. Key physical characteristics such as size, surface charge, lipid composition, hydrophobicity, and shape critically influence their biodistribution, cellular uptake, endosomal escape, and clearance from the body. Ionizable lipids can improve endosomal escape, but they also carry the risk of increased toxicity or immune activation. Additionally, an overly positive surface charge can lead to rapid clearance from the body or unintended off-target effects. While the enhanced permeability and retention (EPR) effect is commonly leveraged for tumor targeting, its variability in human tumors, unlike in consistent preclinical mouse models, poses a challenge for reliable drug delivery to cancerous tissues.

One of the major hurdles lies in the large-scale manufacturing and reproducibility of nanoparticle formulations. Producing nanoparticles for clinical or commercial use requires precise control over formulation parameters, such as lipid composition, encapsulation efficiency, particle uniformity, and sterility, all while meeting strict Good Manufacturing Practice (GMP) standards. Even minor inconsistencies during production can cause batch-to-batch variation, potentially affecting therapeutic efficacy, safety, and regulatory approval. Additionally, ensuring long-term stability and shelf-life is critical; nanoparticles must remain stable during extended storage and under various environmental conditions without aggregating or losing their payload. Lyophilization and reconstitution are sometimes employed to improve stability, but these techniques introduce further material design and stability challenges. Scaling up production demands cost-effective and scalable processes that utilize pharmaceutical-grade equipment and materials, which can present both technical and economic difficulties. Beyond manufacturing concerns, biological and translational challenges further complicate the implementation of nanoparticle-based RNA therapies. Immune recognition, complement activation, and the risk of cytokine storms, especially with repeated or systemic dosing, remain significant concerns. Achieving precise targeting of diseased tissues while sparing healthy ones requires the development of advanced ligand-receptor targeting systems or “smart” nanoparticles that respond to local microenvironmental cues such as pH or enzymatic activity. Moreover, the emergence of personalized medicine adds an additional layer of complexity, as delivery systems may need to be tailored to individual tumor profiles or genetic backgrounds. Altogether, these challenges highlight that while significant progress has been made in RNA chemistry and nanoparticle delivery, realizing the full clinical potential of RNA therapeutics depends on coordinated advances in materials science, scalable and consistent manufacturing, and a deeper understanding of biological systems to develop safe, effective, and reproducible delivery platforms.

RNA modifications in cancer

To improve the therapeutic efficacy of RNA molecules, it is essential to alter them to inhibit nuclease destruction and quick renal clearance, while also enhancing target binding and minimizing immune recognition. A diverse array of biological and chemical RNA modifications, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), pseudouridine, 5-hydroxymethylcytosine (hm5C), and N1-methyladenosine (m1A), are pivotal in modulating mRNA stability. The interaction of writer proteins (including METTL3, METTL14, WTAP, KIAA1429, NSUN2, and PUSs), RNA-binding proteins (RBPs) such as IGF2BPs, YTH domain-containing proteins, and YBX1, alongside eraser proteins like ALKBH5, shapes the dynamic modification landscape and ultimately affects RNA destiny and Functionality. Chemical modifications at the 2′ position of the ribose Sugar specifically 2′-O-methyl (2′-O-me), 2′-fluoro (2′-F), 2′-O-methoxyethyl (2′-MOE), And 2′-O-guanidinopropyl (2′-O-GP) have been demonstrated to augment oligonucleotide thermal stability, enhance serum resistance, and maintain or improve gene-silencing efficacy, as evidenced in siRNA studies, including those involving hepatitis B virus models. CircRNAs, frequently utilized in gene therapy because of their intrinsic stability, may elicit innate immune responses unless suitably modified; specifically, the addition of m6A tags might enable circRNAs to elude immune recognition by designating them as “self” RNA. These advancements highlight the intricate yet vital function of accurate RNA alterations in enhancing the stability, effectiveness, and immunological compatibility of RNA-based therapies [168]. RNA modifications are essential in regulating gene expression and RNA metabolism. m6A is one of the most extensively studied RNA modifications. It is a reversible alteration found in mRNAs and various other RNA types, added by the METTL3/METTL14 complex, removed by FTO and ALKBH5, and primarily recognized by YTH family proteins N1-methyladenosine (m1A), concentrated at start codons, is modulated by TRMT10 and TRM6-TRM61, and reversed by ALKBH1/3. m5C is present in diverse RNAs, installed by NSUN/DNMT2, reversed by TET/ALKBH1, and identified by ALYREF and YBX1. N7-methylguanosine (m7G), predominantly located at the mRNA 5’ cap and inside other RNAs, is synthesized by METTL1-WDR4 and RNMT-RAM, however no particular demethylases or binding proteins have been identified. Pseudouridine, the predominant RNA alteration, arises from the isomerization of uridine through enzymes such as DKC1, and now lacks identified erasers or readers. Finally, adenosine-to-inosine (A-to-I) editing, an irreversible change facilitated by ADAR enzymes, mostly affects Alu-derived dsRNAs, influencing RNA stability and functionality [169].

Recent advancements in RNA sequencing and mass spectrometry have shown the critical importance of RNA alterations in cancer, redirecting attention from prevalent RNAs such as tRNAs and rRNAs to less common species such as mRNAs and miRNAs. Modifications such as m6A, m5C, pseudouridine, and adenosine-to-inosine (A-to-I) editing have become essential regulators of RNA metabolism, including stability, splicing, translation, and localization. These modifications affect gene expression and may function as oncogenes or tumor suppressors, contingent upon the cellular environment. For instance, m6A promotes the translation of oncogenes in AML, but its absence facilitates carcinogenesis in endometrial cancer; m5C stabilizes oncogenic mRNAs in bladder cancer, while its reduction exacerbates GBM. RNA alterations at key tRNA locations (34 And 37) are associated with translational dysregulation in cancer. The intricate interaction among RNA alterations, modifying enzymes, and cellular environments highlights their multifaceted functions in tumorigenesis, positioning them as prospective targets for cancer diagnostics and therapies [170]. RNA alterations have been increasingly associated with cancer development owing to their functions in RNA metabolism and gene regulation. Facilitated by technical advancements, these chemical modifications such as m6A, m5C, and pseudouridine have been detected in both prevalent RNAs like rRNA and tRNA, as well as in less abundant mRNAs and miRNAs. m6A has been demonstrated to affect mRNA splicing, export, translation, and degradation, with its dysregulation either facilitating or inhibiting cancer based on the environment. Likewise, m5C has been identified as a stabilizing agent for carcinogenic transcripts, and its absence may lead to a failure in tumor suppression. Ψ improves RNA stability and functionality, and its urinary excretion is a possible diagnostic indicator. The alteration or dysregulation of RNA at pivotal sites, particularly in tRNAs, has been linked to impaired protein translation and cancer advancement. Epitranscriptomic alterations are facilitated by certain writer, reader, and eraser proteins, and their dysregulated expression or function has been associated with many human malignancies [171]. RNA modifications, including m6A, m5C, and pseudouridine, are increasingly acknowledged as vital regulators of gene expression and cancer biology. These modifications are added, deleted, and interpreted by designated writer, eraser, and reader proteins, affecting RNA metabolism, including splicing, translation, stability, and localization. Altered expression or activity of these RNA-modifying proteins has been noted in several malignancies. For example, m6A has been linked to either tumor promotion or suppression based on the cell type, with METTL3 and METTL14 frequently upregulated to augment oncogene translation, whereas demethylases such as FTO and ALKBH5 have been associated with maintaining cancer cell proliferation and resistance to therapy. Likewise, m5C changes introduced by NSUN and DNMT2 family members are associated with increased mRNA stability and oncogene expression, whereas their absence may result in translational alterations that promote stress responses and tumor advancement. Pseudouridine modifications, mostly mediated by pseudouridine synthases, are present in mRNAs and non-coding RNAs, enhancing RNA stability and translational efficiency, with changes in their deposition linked to worse outcomes in malignancies like GBM. Dysregulated RNA modification landscapes are linked to several cancer hallmarks, prompting efforts to target these pathways for therapeutic intervention [172].

The H/ACA small nucleolar RNA SNORA24 is considered to play a crucial role in tumor Suppression by directing two pseudouridine modifications on 18 S rRNA. Its expression is significantly diminished in human HCC tumors relative to adjacent non-tumor tissue, and lower levels of SNORA24 are associated with reduced patient survival. In murine models, the deletion of SNORA24 was found to bypass oncogene-induced senescence and promote the development of fatty liver cancer resembling human steatohepatitic HCC. Ribosomes lacking SNORA24-guided modifications exhibited altered aminoacyl-tRNA selection dynamics, leading to increased rates of translational miscoding (~ 10–20%) and stop codon readthrough (~ 15%), without affecting overall protein production. Additionally, these modified ribosomes showed greater resistance to the Translation inhibitor Anisomycin And demonstrated approximately 50% improved aminoacyl-tRNA selection efficiency for tRNAPhe [173].

In urothelial carcinoma of the bladder (UCB), a widespread increase in mRNA m5C methylation was observed, with 4,126 hypermethylated m5C sites across 2,041 mRNAs in tumor tissues compared to normal controls. This elevated methylation was particularly enriched in key oncogenic pathways, including JAK–STAT, PI3K–AKT, VEGF, and EMT. Among the hypermethylated genes, HDGF showed a strong correlation with elevated mRNA expression, and increased expression of HDGF, YBX1, and NSUN2 was linked to reduced disease-free survival (DFS). Silencing either NSUN2 or YBX1 resulted in a marked decrease in tumor growth, invasion, and metastasis in both in vitro and in vivo models, with these effects being reversible by the overexpression of the corresponding wild-type proteins, but not their mutants. Furthermore, HDGF was identified as a direct m5C-modified target, with its mRNA stability and expression dependent on the methyltransferase activity of NSUN2 and the binding capability of YBX1. This reinforces the role of the m5C-dependent oncogenic pathway in UCB [174].

ADAR1-mediated adenosine-to-inosine RNA editing targets the 3′ UTRs of the antiapoptotic genes XIAP and MDM2, leading to reduced protein translation without affecting mRNA stability or miRNA binding. This editing predominantly occurs within inverted Alu elements, where ADAR1 binding competes with the RNA export factor STAU1, resulting in nuclear retention and diminished cytoplasmic translation of the edited transcripts. Loss of ADAR1 increases XIAP and MDM2 protein levels, enhances ribosomal association of their mRNAs, and reduces apoptosis in response to staurosporine, as evidenced by decreased PARP cleavage and Annexin V/PI staining. Conversely, ADAR1 overexpression promotes apoptosis in U87 and HepG2 cells, highlighting its role in modulating cell death through post-transcriptional regulation of antiapoptotic genes [175]. In LUAD, A-to-I editing levels of specific miRNAs were significantly altered. Notably, miR-99a-5p, miR-379-5p, and miR-497-5p showed a loss of editing in 100%, 80%, And 87% of matched tumor samples, respectively, while miR-200b-3p exhibited increased editing in 46% of cases. The most pronounced difference in editing between tumor and normal tissues was observed for miR-99a-5p. In An independent cohort of 50 resected lung cancer cases, reduced editing of miR-99a-5p was detected in 38% of patients and was associated with poorer OS (P = 0.047, HR = 3.25) and a trend toward shorter recurrence-free survival. Tumor samples also showed decreased expression of ADAR2, and editing levels were positively correlated with ADAR2 expression (rs = 0.424, P < 0.001), supporting a mechanistic link between reduced ADAR2 levels and impaired miRNA editing [176].

RNA modifications have been progressively associated with cancer via changes in mRNA stability, splicing, translation, and localization, influenced by the dysregulation of “writer,” “eraser,” and “reader” enzymes. The m6A alteration, catalyzed by METTL3-METTL14 and eliminated by FTO or ALKBH5, has been demonstrated to either facilitate or inhibit tumorigenesis, contingent upon the cancer type and specific transcripts involved. Pseudouridine, predominantly catalyzed by dyskerin and PUS enzymes, is linked to tumor growth through the enhancement of RNA stability and translation. The m5C alteration, facilitated by NSUN and DNMT2 family members and identified by proteins like YBX1, has been associated with the stability of carcinogenic RNAs. Likewise, aberrant m1A methylation modulated by TRMT6/61A and ALKBH3 has facilitated cell proliferation and metastasis in several malignancies. The m7G cap, facilitated by METTL1-WDR4 and RNMT-RAM complexes, has impacted mRNA processing and translation, hence leading to cancer. Additional changes, such as ac4C and A-to-I editing, have been linked to cancer through their impact on transcript stability and coding capacity. These changes regulate the expression of critical oncogenes or tumor suppressors across many tumor types, rendering them significant therapeutic targets [177]. RNA alterations are becoming acknowledged as vital regulators inside the TME, affecting every phase of the cancer-immunity cycle. Modifications including m6A, m5C, m1A, m7G, pseudouridine, and A-to-I editing have been demonstrated to influence RNA stability, translation, antigen presentation, immune cell trafficking, and immune checkpoint expression. The alterations have been facilitated by certain enzymes such as METTL3, ALKBH5, FTO, and PUS7, with their dysregulation associated with immune evasion, tumor advancement, and resistance to treatment. For example, m6A methylation has been shown to modify chemokine production and antigen presentation, affecting T cell infiltration and cytotoxicity, whereas demethylases such as ALKBH5 and FTO have regulated PD-L1 expression and immune suppression. TAMs and fibroblasts have been modulated by RNA alterations to either enhance or suppress T cell activity. Exosomal RNAs with modifications and microbiome-induced epitranscriptomic alterations have been associated with the reconfiguration of the TME. The therapeutic promise has been proven by inhibitors and mimetics that target RNA-modifying enzymes, underscoring RNA alterations as both biomarkers and prospective targets for the enhancement of immunotherapy (Fig. 9) [178]. Table 3 demonstrates the RNA modifications in cancer.

Fig. 9.

RNA changes are essential in modulating every phase of the cancer-immunity cycle, which includes the fundamental processes necessary for T cells to accurately identify and eradicate cancer cells. These alterations affect several cell types and molecular mechanisms inside the TME. The commencement of an immune response is initiated by APCs, including DCs, which process antigens and display co-stimulatory chemicals. The m6A methylation of mRNA, facilitated by METTL3, augments the production of the co-stimulatory molecule CD80, hence enhancing antigen presentation and T cell priming. B Upon activation, CTLs traverse the circulation and lymphatic system, guided by chemokines. RNA changes can modulate the expression of these chemokines. In tumor cells, METTL3 and METTL14 inhibit the transcription of CXCL9 and CXCL10 via m6A alterations, facilitating immunological exclusion in colorectal cancer. Furthermore, the enzyme PUS7, which facilitates pseudouridylation a common RNA alteration in GBM has been demonstrated to reduce CXCL10 levels. The infiltration of T cells into tumor tissue is influenced by the extracellular matrix, especially via collagen buildup. CAFs synthesize collagens such as COL10A1. In lung squamous cell carcinoma (LUSC), METTL3-mediated m6A methylation enhances the stability of COL10A1 mRNA, hence increasing its production and secretion by CAFs. Furthermore, VEGFA released by CAFs stimulates angiogenesis and can enhance METTL3 expression in NSCLC cells. CAFs produce extracellular vesicles that contain PIATs, which depend on m5C methylation to augment YBX1 protein binding, hence promoting neural remodeling in the TME. As cancer cells perish, they emit tumor antigens, including neoantigens, into the adjacent microenvironment. Tumors characterized by reduced m6A and m1A scores typically have an elevated neoantigen burden. Modifications including GPX4 m6A (facilitated by RBM15B and IGFBP2) and m5C (catalyzed by NSUN5) are associated with the activation of the STING pathway, which contributes to anti-tumor immunity. E Effective antigen presentation through MHC I molecules is required for T lymphocytes to detect tumor cells. In glioma stem cells, reduced expression of METTL3 and YTHDF2 diminishes m6A levels, which is associated with elevated MHC I expression. Concurrently, the immunosuppressive lncRNA LINC00624 stabilizes ADAR1, an enzyme responsible for adenosine-to-inosine RNA editing. This modification hinders MHC I antigen presentation and diminishes CD⁸⁺ T cell infiltration. In ICC, the m6A demethylase ALKBH5 diminishes m6A modifications on PD-L1 mRNA, enhancing its stability and facilitating immune evasion. In AML, the inhibition of the demethylase FTO reduces the production of immunological checkpoint proteins such as PD-L1, hence increasing the tumor’s vulnerability to T cell-mediated eradication. Reprinted with permission from Springer BMC Nature [178]

Despite significant advances in the understanding of RNA modifications in cancer, several key challenges persist that hinder the full therapeutic potential of epitranscriptomic interventions. First, the functional redundancy and context-dependent behavior of many RNA-modifying enzymes such as METTL3, ALKBH5, NSUN2, and ADAR1 pose difficulties in interpreting their roles across various cancer types. For instance, m6A may promote oncogenesis in AML while acting as a tumor suppressor in endometrial cancer, complicating the development of universal therapeutic strategies. Additionally, the dynamic and reversible nature of most RNA modifications adds another layer of complexity in pinpointing causality and stability in tumorigenic processes. Another challenge lies in the lack of high-resolution, transcriptome-wide mapping tools that can accurately and consistently identify and quantify specific RNA modifications at single-nucleotide resolution. Current sequencing methods and mass spectrometry technologies, while promising, still face limitations in sensitivity, specificity, and scalability, especially when applied to rare transcripts or tumor subpopulations. Future research is poised to benefit from advancements in multi-omics integration, artificial intelligence, and single-cell analysis to decode the temporal and spatial distribution of RNA modifications in cancer. There is a pressing need to delineate the interplay between different types of RNA modifications such as m6A, m5C, Ψ, and A-to-I editing and how their crosstalk influences gene expression and protein translation in the TME. The development of specific inhibitors, mimetics, or CRISPR-based editing systems targeting writer, reader, or eraser proteins offers a potential avenue for precision medicine. These tools could allow researchers to manipulate RNA modifications in a targeted, reversible, and tissue-specific manner, enabling the validation of epitranscriptomic pathways as viable therapeutic targets. Given the crucial role of RNA modifications in immune regulation, incorporating them into immuno-oncology approaches, such as checkpoint inhibition, adoptive cell therapy, and cancer vaccines, holds great promise for enhancing treatment precision and effectiveness. To advance the field further, future research should prioritize the development of highly sensitive, quantitative, and real-time imaging tools to track RNA modifications in live cells. A particularly promising direction involves the use of nanopore-based sequencing technologies, paired with machine learning algorithms, to detect RNA modifications without requiring chemical derivatization. Additionally, engineered circRNAs with customized m6A modification patterns could be explored to evade immune surveillance while preserving therapeutic efficacy, especially in gene therapy and vaccine delivery. Further investigation is also needed into the impact of microbiome-driven epitranscriptomic alterations and their potential effects on cancer progression and immune modulation. Finally, personalized epitranscriptomic profiling in clinical cohorts could uncover patient-specific RNA modification patterns, paving the way for predictive biomarkers and tailored therapeutic interventions. As the field progresses, a comprehensive catalogue of cancer-specific RNA modifications, their regulatory proteins, and downstream effects will be critical for the rational design of next-generation cancer therapies.

Innovations in RNA delivery

In the recent years, there has been focus towards the development of nanoparticles for the specific delivery of cargo. In addition to the efficacy, the delivery systems should be safe and biocompatible. The development of such nanoparticles is dependent on understanding the biological landscape of tumors. Targeting can occur via passive mechanisms, exploiting the enhanced permeability and retention (EPR) effect, or active mechanisms using ligands such as folic acid (FA) to selectively bind to the specific cancer cell receptors such as folate receptor α (FRα). However, there are still a number of challenges, especially in case of reaching and targeting metastatic cells in the different normal and healthy tissues such as liver. To overcome these barriers, RNA nanotechnology has been introduced, specifically the stable three-way junction (3WJ) motif derived from bacteriophage phi29 pRNA to develop versatile and multifunctional nanocarriers. These nanoparticles can be precisely engineered to deliver therapeutic agents, target ligands, and provide imaging molecules while they remain structurally stable, non-toxic, and resistant to enzymatic degradation, making a promising strategy for the targeted cancer therapy [204]. In order to further elaborate on delivery mechanism, specific kinds of nanoparticles have been developed [205, 206]. Notably, the liposomes are among the most common and efficient carriers in the field of cancer therapy. The liposomes have been beneficial in the different sectors including lymph node immune microenvironment remodelling [207], accelerating cancer immunotherapy [208–210], drug and gene delivery in response to X-ray radiation [211], increase in the anti-cancer activity of chemotherapeutics and reducing their adverse impacts [212], among others. However, a promising application of liposomes can be followed in the delivery of RNAs to facilitate the treatment of cancer. In this regard, a nanoscale liposomal delivery system, modified with a PSMA-specific RNA aptamer, was developed to facilitate targeted doxorubicin delivery to PCa cells, therefore improving treatment effectiveness and reducing off-target damage. Aptamer-conjugated liposomes (aptamosomes), roughly 97.6 nm in diameter with a zeta potential of − 10.5 mV, demonstrated improved stability and specific affinity for PSMA-positive LNCaP cells, resulting in higher cellular uptake and cytotoxicity compared to the standard Liposomes. In vitro, the cell viability in LNCaP cells treated with aptamosomal doxorubicin decreased to 50.4%, in contrast to 104.5% with non-targeted liposomes, with no increased toxicity demonstrated in PSMA-negative PC3 cells. In vivo fluorescence imaging revealed that aptamosomes provided significant tumor formation for up to 24 h post-injection, while non-targeted liposomes Mainly localized in the Liver And kidneys. The tumor volume in treated Animals decreased to 331 mm³ with aptamosomes, Much Less than 725 mm³ with Liposomal Dox And 1531 mm³ with free Dox, demonstrating the enhanced transport efficiency and anticancer efficacy of the aptamosomal approach [213].

The integration of biology and engineering can further improve the potential in cancer therapy. Xkr8, a phospholipid scramblase that facilitates phosphatidylserine (PS) exposure on apoptotic cells, has been shown to be upregulated in case of exposure to chemotherapeutic agents such as FuOXP, doxorubicin, and paclitaxel, both in vitro and in vivo. This upregulation contributes to the immunosuppressive effects via enhanced PS-mediated macrophage polarization and immune evasion. Therefore, Xkr8 demonstrates abnormal levels in cancer. To address this issue, an innovative nanoparticle delivery system PMBOP-CP NPs co-loaded with FuOXP and siXkr8 was developed to provide tumor-specific delivery through CD44-mediated targeting, utilizing chondroitin sulfate (CS) and PEG-CS coating to improve tumor accumulation while minimizing hepatic uptake. These nanoparticles demonstrated good tumor targeting, extended circulation duration, and efficient transport of both the drug and siRNA to the tumor tissues, while protecting siRNA from degradation. The silencing of Xkr8 via siRNA inhibited chemotherapy-induced phosphatidylserine exposure and diminished the release of immunosuppressive extracellular vesicles, thereby averting the polarization of macrophages towards the M2 phenotype and providing a more pro-inflammatory, anti-tumor immune microenvironment, characterized by increased CD⁴⁵⁺ immune cell infiltration, enhanced IFN-γ + and granzyme B + CD⁸⁺ T cells, and decreased Treg presence. In vivo, the co-delivery of FuOXP and siXkr8 nanoparticles resulted in significantly enhanced tumor suppression and survival in colorectal and pancreatic tumor models, with further therapeutic improvement observed when combined with anti-PD-1 checkpoint inhibition. The observed effects were achieved without considerable systemic toxicity or the induction of inflammatory cytokines, suggesting that local Xkr8 knockdown enhances tumor sensitivity to chemotherapy while mitigating drug-induced immunosuppression, thereby presenting a promising approach for improving the efficacy of anticancer treatments via immune modulation [214]. Although this study provides a promising strategy in cancer therapy, some suggestions can be considered for the future studies. The study focused on colorectal and pancreatic tumors, and it is suggested that other kinds of solid tumors are also tested. Moreover, the main focus was on macrophages, while the impact on other kinds of cells such as natural killer cells and DCs should be evaluated.

The present understanding highlights the efficacy of delivery systems in improving efficacy of RNA therapeutics. One of the strength points of the studies is the focus on the application of the different kinds of nanostructures. In this case, the micelles have been suggested as promising candidates. Notably, the thermo-sensitive micelles have been demonstrated to combine chemotherapy and immunotherapy in synergistic tumor suppression [215]. One of the factors in the induction of anti-cancer immunity is related to the release of an adenylate cyclase (AC) inhibitor from polymeric micelles to suppress melanoma [216]. In addition, micelles are promising for the delivery of genes in cancer therapy and they can be light-responsive [217]. Ultralow-CMC micelles with zwitterionic and lipid domains provide enhanced stability and bioavailability, facilitating efficient tumor elimination in vivo relative to traditional micellar medication formulations [218]. A therapeutic micellar nanocomplex formed by self-assembly of EGCG derivatives and the anticancer protein Herceptin enhances anticancer efficacy, tumor targeting, and circulation time while reducing the need for inert carriers [219]. Tranilast-loaded micelles improve the reprogramming of cCAFs and enhance the efficiency of nano-immunotherapy at much reduced dosages, facilitating cures and immunological memory in resistant breast cancer models, while tumor stiffness reduction via SWE acts as a predictive biomarker [220]. Therefore, application of micelles for the delivery of RNA therapeutics can further improve the treatment of cancer. A multifunctional micelle-like nanoparticle (MNP) platform was developed by hydrophobizing different ribonucleic acids with DOTAP and DLin-MC3-DMA lipids, Subsequently assembling with mPEG-b-PLGA polymer to produce stable nanoparticles around 100 nm in size, exhibiting near-neutral charge And enhanced serum stability. These MNPs facilitated effective RNA encapsulation, cellular internalization, And fast endosomal release, resulting in over 82% CD47 and significant PD-L1 gene silencing in melanoma cells, hence promoting phagocytosis by macrophages and cytotoxicity from CD^8⁺ T cells. In vivo, MNP treatment led to a substantial decrease in tumor volume, with the combined siRNA therapy (CD47/PD-L1) Yielding the most pronounced tumor Suppression And immune activation, as demonstrated by a 2.1-fold increase in Mature DCs, a 3.3-fold enhancement in macrophage-mediated phagocytosis, and significantly elevated CD^8⁺ T cell responses. Moreover, the method demonstrated compatibility with mRNA and plasmid DNA, resulting in effective antigen presentation and protective immunity in a B16-OVA melanoma model, while ensuring superior biocompatibility and systemic safety [221].

Efficient mRNA delivery remains a major challenge in advancing cancer immunotherapy, primarily due to the toxicity and instability associated with conventional mRNA treatments such as IL-12. Although these therapies show considerable promise, their systemic administration has often led to serious adverse effects. Intratumoral injection and LNPs have enhanced targeted delivery and decreased systemic toxicity. Innovations such as self-replicating mRNA prolong expression length yet encounter challenges related to the stability and immunogenicity. CircRNA presents a viable alternative owing to its superior stability, extended half-life, and reduced immunogenicity, all without requiring chemical changes. The efficacy of RNA therapeutics is significantly contingent upon the design of LNP delivery methods, especially the ionizable lipid component, which influences encapsulation efficiency, cellular uptake, and endosomal escape. Factors particular to tumors, such as hypoxia and tissue heterogeneity, demand customized LNP designs to enhance RNA transport and treatment efficacy across various cancer types [222]. Since the RNA therapeutics can significantly improve cancer immunotherapy [223–225], development of delivery systems can open a new gate in this regard. A study elaborates towards the development of of iDR-NCs intertwining DNA-RNA nanocomplexes as effective nanoadjuvants for individualized tumor immunotherapy. iDR-NCs were manufactured by a four-step procedure incorporating rolling circle replication (RCR) and transcription (RCT) to produce tandem CpG and Stat3 shRNA sequences, which self-assembled into micrometer-scale DNA-RNA microfibers (MFs). The MFs were further reduced to nanoscale iDR-NCs utilizing a biocompatible PEGylated cationic polypeptide (PPT-g-(PEG)6), therefore enhancing intracellular transport and reducing cytotoxicity. The resultant iDR-NCs, analyzed using SEM, DLS, and further methodologies, exhibited significant biostability and effective absorption by DCs and macrophages, predominantly localizing inside endolysosomes. Functional experiments demonstrated that iDR-NCs enabled the co-delivery of CpG and shRNA, facilitating TLR9 activation and Stat3 silencing, which collectively augmented DC activation, increased CD80 expression, and stimulated the release of pro-inflammatory cytokines (IL-6, IL-12p40, TNFα). The co-loading of iDR-NCs with the model antigen (CSIINFEKL) or neoantigen (Adpgk) through hydrophobic interactions facilitated strong and prolonged antigen presentation in antigen-presenting cells (APCs). Subcutaneously administered iDR-NC/Adpgk nanovaccines demonstrated effective targeting of lymph nodes (LNs), high co-delivery efficiency to LN-resident APCs, and robust immunostimulatory effects, including upregulation of CD80 and significant induction of Adpgk-specific CD⁸⁺ T cells exhibiting elevated PD-1 expression and a central memory phenotype. In a syngeneic MC38 tumor model, iDR-NC/Adpgk nanovaccines significantly decreased tumor burden and metabolic activity in the lungs, indicating systemic therapeutic effectiveness and sustained, neoantigen-specific immunological memory. The findings indicate that iDR-NCs offer a reliable, biocompatible platform for the combinatorial delivery of genes and antigens, hence improving the effectiveness of cancer nanovaccines (Fig. 10) [226].

Fig. 10.

Schematics of iDR-NC/neoantigen Nanovaccines for Improved Tumor Immunotherapy. a In a unified reaction system, rolling circle replication (RCR) and rolling circle transcription (RCT) were executed concurrently, yielding concatenated CpG motifs and Stat3 shRNA strands. These nucleic acid products self-assembled into hybrid DNA-RNA microfibers (MFs) exhibiting an interwoven topology. b The resultant MFs were condensed with PPT-g-PEG, yielding iDR-NCs. The nanocomplexes were further loaded with tumor-specific neoantigens via hydrophobic interactions between the peptide antigens and the hydrophobic domains of PPT. c When administered to immunocompetent mice, the iDR-NC/neoantigen complexes specifically targeted antigen-presenting cells (APCs) within the draining lymph nodes. This elicited robust and prolonged neoantigen-specific T cell responses, resulting in efficient tumor growth suppression. Reprinted with permission from Springer Nature [226]

During cancer immunotherapy, DCs have been always under attention. DCs are a heterogeneous and essential category of antigen-presenting cells that modulate both innate and adaptive immune responses, serving pivotal functions in cancer immunology and immunotherapy. Their capacity to stimulate cytotoxic T cells renders them significant candidates for augmenting anti-tumor responses, particularly via techniques such as DC-based vaccinations or the regulation of endogenous DCs. Within DC subsets, stimulatory DCs (SDCs) in the TME are notably significant, with their prevalence associated with FLT3LG generated by NK cells. These NK–SDC interactions augment T cell responses and are associated with favorable results in checkpoint immunotherapy, including anti-PD-1 treatment. Furthermore, DCs are increasingly acknowledged as pivotal components in the PD-1/PD-L1 pathway, with their PD-L1 expression significantly affecting T cell suppression and tumor advancement, surpassing the effect of macrophages. Progress in single-cell research is revealing the intricacies of DC functional states, highlighting their dual capacity to either promote or impede anti-tumor immunity. Consequently, leveraging and optimizing DC activities is a promising yet underexploited approach for cancer immunotherapy [227–231]. A multifunctional lipid-based nanoparticle system (R8/GALA-MEND_SUV) was engineered to ex vivo deliver siRNA to DCs, therefore augmenting cancer vaccine effectiveness through enhanced gene silencing, endosomal escape, And cytoplasmic release. The nanoparticles, measuring around 131.5 nm with a zeta potential of + 42.6 mV and a low polydispersity index (PDI = 0.17), were designed using a fusogenic peptide (GALA) and an improved lipid formulation (DOPE/PA) to enhance membrane fusion and intracellular delivery. In contrast to conventional hydration-based methods, the MEND_SUV demonstrated over 90% siRNA dissociation efficiency And around 70% endosomal escape, resulting in over 70% gene knockdown at one-tenth the dosage. Suppression of SOCS1 in bone marrow-derived DCs led to a reduction in SOCS1 mRNA expression to 21.5% of control levels, which resulted in enhanced phosphorylation of STAT1 and increased production of TNF-α and IL-6. Mice vaccinated with SOCS1-silenced, antigen-pulsed DCs demonstrated significantly improved tumor suppression in vivo, validating the efficacy of this carrier in augmenting DC-based immunotherapy [232].

Although the specific targets of RNA-loaded nanoparticles and their efficacy in cancer therapy have been highlighted, it would be beneficial to also focus on the development of safe carriers.A biocompatible and biodegradable nanoparticle system utilizing PEG-PLA and a cationic lipid (BHEM-Chol) was developed to deliver CDK1-targeted siRNA (siCDK1) for the treatment of triple-negative breast cancer (TNBC) through a synthetic Lethality approach. The nanoparticles measured An average size of 146.4 nm, exhibiting a siRNA encapsulation effectiveness of 90.9%. They displayed RNase protection, effective intracellular transport, and CDK1 knockdown in c–Myc–overexpressing TNBC cells (SUM149, BT549), but not in normal mammary epithelial cells (MCF-10 A). In TNBC cells, the administration of siCDK1 resulted in decreased cell viability (49.2% in SUM149, 56.8% in BT549 at 200 nM), G2/M cell cycle arrest, and apoptosis (~ 32.2% in SUM149), while sparing normal cells. Systemic treatment in mice significantly inhibited tumor development in SUM149 and BT549 xenografts without eliciting immunological activation or systemic toxicity, while augmenting siRNA tumor accumulation via the improved permeability and retention effect [233]. A research offers an extensive optimization and assessment of RNA-triple-helix nanoconjugates, which are created through the self-assembly of three modified RNA oligonucleotides miR-205 sense, antisense, and antagomiR-221 combined with PAMAM G5 dendrimers to produce nanoscale particles (~ 50–60 nm) that subsequently aggregate into microscale structures (~ 3–4 μm) and are incorporated into dextran-aldehyde hydrogels for macroscopic tumor-adherent scaffolds. The triplex structure is validated using quenching experiments, gel electrophoresis, and Tm measurement (Tm = 74.5 °C), demonstrating remarkable stability under physiological stresses (temperature, urea, pH, serum). In contrast to naked PAMAM dendrimers that utilize caveolae-mediated endocytosis, dendrimer–triplex nanoparticles are ingested by cancer cells by macropinocytosis, due to their modified size and charge (− 19.6 mV zeta potential). In vitro investigations in MDA-MB-231 TNBC cells demonstrate nearly 100% cellular uptake of triplex nanoconjugates, significantly surpassing RNA double helices, and effectively downregulating oncogenic miR-221 while upregulating tumor-suppressive miR-205, thereby inhibiting cell proliferation, migration, and colony formation. In vivo, RNA-triple-helix hydrogel scaffolds implanted adjacent to TNBC xenografts in murine models facilitate localized, Sustained release of functional MiRs without off-target accumulation or toxicity, resulting in approximately 90% tumor reduction, diminished Ki67 expression, reduced vascularization, and significantly enhanced survival relative to controls and conventional chemotherapeutics (DOX, PTX, Avastin). Gene expression analysis verifies the miR-205-induced downregulation of LAMC1 and E2F1, the overexpression of p53, and the inhibition of miR-221, resulting in the restoration of E-cadherin and a reduction in Snail1/Slug, all of which correlate with diminished tumor growth and metastasis [234].

Exosomes, tiny vesicles secreted by cells, are integral to cancer advancement, detection, and treatment. Exosomes produced from tumors can stimulate axonogenesis by facilitating neurite outgrowth through molecules such as EphrinB1, hence augmenting tumor innervation and metastasis. Diagnostic methodologies employing artificial intelligence and Raman spectroscopy of plasma exosomes provide precise early-stage cancer identification and tissue origin categorization. Cancer-derived exosomes may be designed to selectively transport palladium catalysts to tumor cells, facilitating bioorthogonal catalysis for targeted drug activation, therefore underscoring its promise in precision oncology. More information on exosomes in cancer can be found in these studies [235–237]. An exosome-based nanoparticle approach targeting folate was developed to provide effective cytosolic siRNA delivery, circumventing endosomal trapping And consequently improving gene silencing And therapeutic effectiveness. The nanoparticles, around 130 nm in diameter with an 80% siRNA loading efficiency, employed exosomes adorned with folate-conjugated RNA nanoparticles to selectively target folate receptor-positive cancer cells. Confocal imaging And 3D reconstruction demonstrated that siRNA conveyed by folate-displaying exosomes was uniformly distributed within the cytoplasm, whereas folate-siRNA alone was confined to endosomes and lysosomes (colocalization coefficient M = 0.86 for endosomes versus negligible overlap for exosome-loaded siRNA). In vitro, Survivin gene expression was reduced by 60% with the application of FA/exosome/siRNA, whereas folate-siRNA alone, without exosomes, exhibited minimal silence. In a colorectal cancer xenograft model, repeated intravenous administrations of FA/exosome/siRNA (0.5 mg/kg every two days) significantly diminished tumor volume and weight relative to control groups (p < 0.0001), substantiating improved in vivo delivery and therapeutic efficacy via folate-mediated membrane fusion rather than endocytic trafficking [238].

Exosomal miRNAs are essential in cancer biology and provide potential as biomarkers for cancer detection. Nonetheless, their limited prevalence in exosomes poses a considerable obstacle for straightforward and efficient identification. A liposome-mediated membrane fusion technique (MFS) was utilized to transport CRISPR/Cas13a into exosomes, termed MFS-CRISPR, allowing the direct detection of exosomal miRNAs in plasma. The MFS-CRISPR technology, when utilized for the detection of exosomal miR-21, exhibited a linear detection range of four orders of magnitude (10⁴–10⁸ particles/mL) and could identify miR-21 concentrations as low as 1.2 × 10³ particles/mL. The liposome-mediated fusion technique concentrated fluorescent signals within merged vesicles, enabling the examination of exosome heterogeneity. The study of clinical samples with the MFS-CRISPR test demonstrated a notable disparity in miR-21 expression between breast cancer patients and healthy subjects. This approach has significant potential for clinical applications in cancer detection and therapy monitoring due to its high sensitivity and operational simplicity [239]. The inherent tendency of neural stem cells (NSCs) to migrate towards hypoxic tumor areas presents a viable strategy for targeted medication administration. In this context, NSCs have demonstrated the ability to effectively deliver ASOs intended to reduce the oncogenic and immunosuppressive STAT3 protein into the glioma microenvironment. CpG-STAT3ASO conjugates were used to promote spontaneous absorption via scavenger receptor-mediated endocytosis. Following internalization and release from endosomes, CpG-STAT3ASO was shown to colocalize with CD⁶³⁺ vesicles and subsequently with CD⁶³⁺CD⁸¹⁺ exosomes. During a three-day interval, NSCs released exosomes comprising up to 80% of the CpG-STAT3ASO payload. The CpG-STAT3ASO-loaded exosomes demonstrated significantly enhanced immunostimulatory effects on human DCs and murine macrophages relative to native NSC-derived exosomes, stimulating NF-κB signaling and facilitating interleukin-12 (IL-12) production. In an orthotopic GL261 tumor model, NSC-mediated Transport significantly improved the transfer of oligonucleotides from distant injection sites into the glioma Milieu compared to unmodified oligonucleotides. This tailored delivery method also enhanced the activation of glioma-associated Microglia. Moreover, peritumoral delivery of 5× 10⁵ NSCs loaded with CpG-STAT3ASO significantly inhibited the development of subcutaneous tumors in mice, surpassing the anticancer effectiveness of equal dosages of free oligonucleotides [240].

Exosomes have emerged as intriguing natural delivery vehicles in cancer therapy owing to their low immunogenicity, superior biocompatibility, and inherent tumor-targeting ability. Diverse engineering methodologies have been devised to improve delivery efficiency and targeted precision. One method entailed the embellishment of exosomes derived from HepG2 cells with cell-penetrating peptides (CPPs), which enhanced cellular uptake and enabled the incorporation of ASOs, leading to effective intracellular delivery and the downregulation of the antiapoptotic protein Bcl-2 in tumor cells [241]. A further technique employed T7 peptide-modified exosomes (T7-exo) to target the transferrin receptor, which is overexpressed in GBM cells, for the systemic delivery of antisense miRNA oligonucleotides aimed at miR-21 (AMO-21) [242]. The T7-exo particles exhibited greater targeting of the brain and GBM, resulting in efficient inhibition of miR-21, increased expression of tumor suppressors PDCD4 and PTEN, and substantial decrease in tumor growth in vivo. Furthermore, exosome–dendrimer hybrid nanoparticles amalgamate the advantages of natural exosomes with synthetic poly(amidoamine) (PAMAM) dendrimers, establishing a multifunctional platform for gene delivery that enhances cellular uptake and diminishes cytotoxicity relative to free dendrimers, while significantly improving siRNA delivery and the downregulation of the immune checkpoint PD-L1 [243]. Exosomes obtained from patient primary cells were utilized in a separate application to deliver siRNAs aimed at CCDC80, a protein associated with liver metastases and chemoresistance in colorectal cancer. Inhibition of CCDC80 enhanced the sensitivity of OXA-resistant colorectal cancer cells to chemotherapy across many models, including patient-derived organoids and xenografts, hence enhancing survival in tumor-bearing mice [244]. These results collectively highlight the adaptability and therapeutic promise of modified exosomes and hybrid nanoplatforms in surmounting delivery obstacles, improving nucleic acid-based treatments, and providing individualized treatment approaches for diverse malignancies.

Small RNA-based treatments, including as siRNAs, miRNAs, and artificial circular RNAs (acircRNAs), have considerable potential in cancer treatment; nevertheless, their clinical efficacy is frequently constrained by inadequate delivery efficiency and specificity. Exosomes, owing to their natural origin, little immunogenicity, and superior biocompatibility, have emerged as extremely efficient delivery vehicles capable of traversing cellular barriers. Numerous creative ways have been devised to use and manipulate exosomes for improved RNA delivery. Protocols have been developed to produce specific exosomes using ligand-peptide fusion proteins, allowing effective siRNA administration both in vitro and in vivo, including to the brain [245]. Cancer-derived exosomes have demonstrated a natural affinity for malignancies and may efficiently transport CRISPR/Cas9 plasmids to ovarian cancer, augmenting apoptosis and chemosensitivity through the modification of the PARP-1 gene [246]. Likewise, EGFR-targeted exosomes altered with the GE11 peptide effectively transported tumor-suppressive let-7a miRNA to breast cancer cells in vivo [247]. The innovative exosome-mediated delivery of acircRNAs that emulate the CRISPR system has exhibited enhanced gene silencing and anti-tumor efficacy in bladder cancer cells relative to CRISPR-dCas9-KRAB, by precisely targeting the β-catenin and NF-κB pathways [248]. A light-inducible exosome system has been created for the effective and controlled loading of long RNAs, such as miR-21 sponges, into exosomes, facilitating targeted transport to leukemia cells and causing death by decreasing miR-21 activity [249]. The improvements in exosome engineering and RNA loading techniques highlight the significant therapeutic promise of exosome-based RNA delivery systems for individualized and targeted cancer therapies. Table 4 discusses the delivery of RNA therapeutics by nanoparticles in cancer therapy.

Table 4.

The delivery of RNA molecules/modification in cancer therapy

Carrier Type	Targeting Strategy	Cargo Type	Size (nm)	Delivery Mechanism	Gene Silencing Efficiency	In Vivo Efficacy	Refs
CCLA-based liposome	Passive (non-targeted)	siRNA (c-raf), DNA	110–120	Endocytosis & endosomal release	Up to 62% cytotoxicity in vitro (PC-3); ~54–34% in others	73% tumor growth suppression in SCID mice (MDA-MB-231)	[250]
PMBOP-CP micelle-based NP	CD44-mediated (CS/PEG-CS coated)	siRNA (siXkr8) + FuOXP	119 (coated)	Endocytosis + endosomal escape	~ 75.8% knockdown (luciferase); significant Xkr8 mRNA silencing	Significant tumor growth suppression; enhanced CD8⁺ T cell activity; reduced Tregs in colon and pancreatic cancer models	[214]
DNA cross-linked polymeric nanoframework (DPNF)	Phenylboronic acid (targets sialic acid on cancer cells)	siRNA	~ 266 (DPNF-20); increased to ~ 326 after loading	Endocytosis, ATP-triggered cytoplasmic release, lysosomal escape	PLK1 mRNA reduced by 44.36%; protein by ~ 80% (at 300 nM siRNA); ~63% protein knockdown at 200 nM	Tumor Weight reduced by 90.47%; high tumor accumulation; minimal off-target distribution or toxicity	[251]
Gold nanocluster (GNC)	Passive (EPR effect)	siRNA (NGF)	~ 70 (complex); 2.6 (core)	Endocytosis, lysosomal escape	75% NGF mRNA knockdown; 69% in tumor tissue	Tumor volume reduced by up to 52% (subcutaneous), 85.7% neurite reduction; effective in PDX, orthotopic, and subcutaneous models with minimal toxicity	[252]
HEK293-derived exosome (IL-12-Exo)	Passive inhalation + tumor tropism	IL-12 mRNA	151.0 ± 5.7	Inhalation, endocytosis, and cytoplasmic translation	Robust IL-12 and IFN-γ expression in lungs, superior to liposomes; strong immune activation	Tumor growth suppression, survival extended from ~ 21 to ~ 50 days, immune memory formed, minimal systemic toxicity	[253]
Magnetic nanoparticle (MN-NIRF)	Passive (EPR effect + MPAP peptide for membrane translocation)	siRNA (GFP, survivin)	~ 30–50 (core); ~70 (with surface mods)	Endocytosis and cytoplasmic release via MPAP	85–97% mRNA knockdown (GFP, survivin); ~80% protein reduction	Tumor signal reduction by MRI and NIRF; survivin silencing led to apoptosis, necrosis, and significant tumor inhibition with no systemic toxicity	[254]
7C1 (polymeric NP from low-MW PEI and lipids)	Passive (endothelial tropism via formulation)	siRNA	35–60	Caveolae- and clathrin-mediated endocytosis	> 90% silencing of ICAM-2 mRNA in Lung; 60–80% knockdown of five genes with multi-siRNA dose	Suppressed tumor growth by 40–70%, reduced Lung metastases by 52–63%, induced emphysema-like phenotype via VEGFR2 knockdown, well tolerated at high doses	[255]
iDR-NC (DNA-RNA nanocapsule)	Passive (LN targeting via size, SC injection)	CpG DNA, Stat3 shRNA, peptide neoantigen	~ 252	Endocytosis, acid-triggered endosomal escape	Stat3 mRNA reduced to ~ 50%; p-STAT3 and immunosuppressive signaling downregulated	Induced 8× more neoantigen-specific CD8⁺ T cells vs. CpG alone; suppressed metastatic tumor growth; strong immune memory	[226]
RNA 4WJ-X nanostructure	EGFR aptamer-mediated targeting	Paclitaxel (chemotherapy)	~ 9.1	Endocytosis, aptamer-directed targeting, ester cleavage	-	Significant tumor growth inhibition; 45.1% apoptosis in vitro; no organ toxicity; increased survival and reduced cytokine response	[256]
RNA-LPX (lipoplex)	Passive targeting via charge tuning (no ligand)	mRNA (tumor antigens: OVA, gp70, TRP-1)	~ 200–320	Macropinocytosis by DCs and pDCs	High antigen expression; >30–60% of CD8⁺ T cells specific to antigen	Complete rejection of lung and subcutaneous tumors in multiple models; memory T cell formation; IFNα-mediated immunity	[257]
RNAtr NPs (self-assembled RNA/DNA hybrids)	Folate-receptor-mediated (FA–DNA)	siRNA (RFP)	~ 190.1 ± 37.2	Endocytosis and Dicer-mediated cytoplasmic processing	RFP mRNA reduced to 25%; protein reduced to ~ 55% in vitro	Tumor-specific accumulation; RFP expression reduced by 55–80%; no INF-α or TNF-α induction; low toxicity	[258]
RNAi-microsponge (RCT-derived)	Passive (PEI condensation for cellular uptake)	siRNA	~ 2,000 (uncondensed), ~ 200 (condensed with PEI)	Dicer-mediated siRNA generation post-endocytosis	Luciferase expression reduced to 42.4% at 980 fM siRNA-equivalent dose	Significant Luciferase knockdown in vivo with 2.1 fmol RNAi-MS/PEI; 1,000× lower dose vs. conventional carriers	[259]
Extracellular vesicle (EV) decorated with RNA-3WJ	RNA aptamers: PSMA (prostate), EGFR (breast), folate (colon)	siRNA	~ 96–120	Endocytosis, EV membrane fusion, aptamer targeting	Survivin mRNA reduced by ~ 62.3%; protein down by ~ 63%; 71% cell viability	Complete tumor suppression in prostate model; significant tumor reduction in breast and colorectal models; no toxicity	[260]
LNP (Synthetic RNA virus)	Passive tumor tropism via vRNA tropism	vRNA (SVV, CVA21)	~ 85	LNP-mediated cytoplasmic release, in situ viral replication	-	Complete tumor regression in SCLC and melanoma; efficacy sustained despite neutralizing antibodies; increased CD8⁺ T cells, prolonged survival	[261]
RNA-triple-helix hydrogel scaffold	Local tumor adhesion (via dextran aldehyde)	miR-205 mimic + miR-221 antagomiR	~ 56.6 (NP); ~3.4 μm (aggregates)	Macropinocytosis, endosomal escape, sustained local release	miR-221 downregulated, miR-205 upregulated; 95% reduction in cell survival; complete migration block	Nearly 90% tumor shrinkage in TNBC mouse model; increased p53, reduced Ki67 and VEGF; no off-target organ accumulation	[234]
Red blood cell extracellular vesicles (RBCEVs)	Passive (heparan sulfate-mediated uptake)	ASOs, Cas9 mRNA, gRNA	~ 140	Endocytosis, electroporation-based RNA loading	80–95% knockdown of miR-125b; ~98% knockdown via Cas9-gRNA system	Suppressed tumor growth in breast cancer and leukemia models; no toxicity; widespread organ uptake; effective CRISPR editing	[262]
PLGA polymer nanoparticle	Passive topical delivery to mucosal tissue	siRNA (EGFP, MAPK1)	< 200	Endocytosis, sustained release via pH-sensitive degradation	~ 50–60% EGFP silencing in vaginal, cervical, and uterine tissues; >14 days sustained effect	Significant gene silencing throughout the reproductive tract; deeper tissue penetration (up to 120 μm); minimal inflammation or toxicity compared to lipoplexes	[263]
DNA tetrahedron ONP (self-assembled)	Folate ligand (3 per particle optimal)	siRNA	~ 28.6	Endocytosis, spatially controlled ligand orientation	~ 60% silencing of luciferase in tumors; >60% GFP knockdown in KB cells	Significant luciferase knockdown via IV and IT injection; tumor accumulation confirmed; no immune response observed	[264]

Limitations: Nanoparticles for RNA delivery in cancer therapy face significant limitations related to stability, targeting specificity, and safety. Systemically administered nanoparticles often undergo rapid clearance by the mononuclear phagocyte system or are sequestered in non-target tissues like the liver and spleen, reducing therapeutic efficacy. Their ability to penetrate solid tumors is hindered by the dense extracellular matrix and abnormal vasculature, leading to poor intratumoral distribution. Additionally, many nanoparticles require surface modifications (PEGylation or targeting ligands) to improve circulation and specificity, which can complicate manufacturing and lead to immune reactions such as the accelerated blood clearance (ABC) phenomenon. Furthermore, endosomal entrapment after cellular uptake remains a major barrier, as inefficient endosomal escape prevents RNA molecules from reaching the cytoplasm. Lastly, potential toxicity from the nanoparticle components themselves, especially cationic lipids or polymers, can trigger inflammation or cytotoxicity, limiting their clinical translation

The advancement of nucleic acid ligands for immune activation of the TME via the stimulation of pattern recognition receptors (PRRs) has garnered considerable attention, especially for addressing immunologically “cold” cancers. A significant difficulty in this domain has been attaining tumor-specific delivery of these ligands to reduce systemic toxicity. Contemporary methods frequently depend on direct intra-tumoral injection of RNAs or other immune-stimulatory agents, a technique that is inadequate for addressing metastatic illness. Recent investigations assessed the efficacy of a lupus-derived, cell-penetrating antibody for in vivo nucleic acid delivery to Malignancies. The Antibody, a modified variation of 3E10-D31N known as GMAB, creates non-covalent interactions with RNA and facilitates highly selective tumor delivery by intravenous injection. The technique targets the nucleoside transporter ENT2, which is overexpressed in neoplastic cells. Imaging experiments with labeled RNAs verified tumor-specific delivery and functional RNA production, with little dispersion to healthy regions. Subsequent inquiry concentrated on employing the antibody to Transport RIG-I agonists to neoplasms. The RIG-I agonist 3p-hpRNA demonstrated single-agent anti-tumor efficacy in GMAB/RNA complexes across many types, including a B16 melanoma model and an orthotopic pancreatic cancer model (KPC). Quantitative RT-PCR research revealed a 1000-fold greater absorption of 3p-hpRNA in KPC tumor cells relative to CD⁴⁵⁺ immune cells inside the TME. Furthermore, GMAB/RNA complexes exhibited synergistic effects with anti-PD-1 treatment in breast (EMT6) and colon (MC38) cancer mice. Considering ENT2 expression in the blood-brain barrier, GMAB/RNA complexes demonstrated effectiveness in an orthotopic medulloblastoma model, inhibiting both primary tumor proliferation and spinal metastases. These findings confirm GMAB as a multifaceted platform that can localize to orthotopic and flank cancers, traverse the blood-brain barrier, and systemically distribute RNA-based therapeutics, such as immunogenic RNAs, mRNAs, and siRNAs, with excellent tumor selectivity [265].

The creation of secure and efficient nanoprobes for precise imaging and targeted treatment of in-situ stomach cancer continues to pose a considerable difficulty. A multifunctional nanoprobe, consisting of HER2 monoclonal antibody-conjugated, RNase A-associated CdTe quantum dot clusters (HER2-RQDs), was developed, and its cytotoxic effects were evaluated. Subcutaneous gastric cancer models in nude mice and in-situ gastric cancer models in SCID mice were developed and treated with HER2-RQDs by intravenous injection to assess biodistribution and therapeutic effectiveness in vivo. The HER2-RQDs nanoprobes exhibited specific cytotoxicity towards MGC803 gastric cancer cells, facilitated targeted imaging of Subcutaneous tumors within 3 h post-injection, And localized to in-situ gastric tumors within 6 h. These nanoprobes significantly suppressed tumor proliferation and extended survival in animal models with stomach cancer. The therapeutic effect was ascribed to RNase A produced from the nanoprobes, which destroyed cytoplasmic functional RNAs, inhibited protein synthesis, and triggered death. The high-performance HER2-RQDs nanoprobes exhibit significant potential for future applications in targeted imaging and selective treatment of in-situ gastric cancer [266].

The targeted administration of chemotherapeutics can enhance drug concentration in tumors while minimizing systemic exposure. Incorporating antibodies into a micellar framework provides a method for targeted delivery of anticancer drugs, resulting in “immunomicelles” that augment intratumoral drug concentration and improve cytotoxicity against cancer cells. This method involves mixed dendrimer micelles (MDM) composed of PEG2k-DOPE-conjugated generation 4 polyamidoamine dendrimer (G4-PAMAM-PEG2k-DOPE) and PEG5k-DOPE, which were co-loaded with doxorubicin and siMDR-1. The formulation was then altered using monoclonal Antibody 2C5, which exhibits nucleosome-restricted specificity and identifies cancer cells through cell-surface-bound nucleosomes. Micelles containing 2C5 antibodies demonstrated significantly enhanced cellular association and tumor cell cytotoxicity in both monolayer and spheroid models, along with better anticancer activity in vivo compared to non-targeted counterparts [267].

In the recent years, RNA-based cancer immunotherapy has made significant advances, particularly with the advancement of nanoparticle-mediated delivery systems. Despite notable progress, several biological and technical barriers persist that limit the efficacy and clinical translation of RNA therapeutics. One of the foremost challenges is navigating the complex and heterogeneous TME, which poses physical barriers such as abnormal vasculature, dense extracellular matrix, and high interstitial pressure that hinder nanoparticle penetration and distribution. Although there are studies highlighting the application of nanoparticles for the delivery of RNA therapeutics and improving anti-cancer immunity, those studies rarely addressed the presence of specific physical barriers (above mentioned). Moreover, some of the tumors have a dense TME such as pancreatic cancer and a comparison with other cancers in terms of RNA delivery and cancer immunotherapy should be performed. For other types of tumors such as brain cancers, there is blood-brain barrier (BBB) that further challenges therapy and therefore, more focus should be on the delivery systems for crossing over BBB and how such delivery systems can improve efficacy of RNA therapeutics. Additionally, the immunosuppressive milieu, characterized by Tregs, TAMs, and high levels of ROS can compromise the function of RNA cargoes. Notably, studies have also rarely evaluated the efficacy of RNA-loaded delivery systems in affecting TAMs and CAFs in cancer therapy that can be focus of future experiment. Moreover, the off-target accumulation of nanoparticles in healthy organs such as the liver and spleen, driven by the mononuclear phagocyte system (MPS), reduces therapeutic concentration at tumor sites and raises toxicity concerns. A major intracellular barrier is endosomal entrapment, where the internalized RNA payloads fail to escape into the cytosol and are instead degraded. Furthermore, the innate immune system can recognize and respond to foreign RNA sequences through toll-like receptors (TLRs) and cytosolic sensors such as RIG-I and MDA5, leading to unintended immunostimulation or reduced RNA stability. The development of targeted, biocompatible, and scalable nanocarriers that can effectively encapsulate and protect RNA, navigate the TME, achieve organ- and cell-specific delivery, and ensure cytosolic release remains a pivotal challenge in the field.

Several innovations in delivery system composition and design offer promising solutions to these limitations. One area of active exploration is the development of multi-stimuli-responsive nanoparticles that respond to pH, redox conditions, enzymes, and hypoxia to release RNA selectively within tumors. Although stimuli-responsive nanoparticles have been significantly applied in cancer therapy [268–272], more focus should be on the delivery of RNA therapeutics. Advanced polymeric systems such as disulfide-bridged poly(disulfide amide) (PDSA) and matrix metalloproteinase (MMP)-cleavable hydrogels exemplify these intelligent release mechanisms. Another frontier is the use of RNA nanostructures such as RNA origami and three-way junction (3WJ) motifs that self-assemble into modular, programmable scaffolds capable of displaying targeting ligands, immune adjuvants, and therapeutic RNA molecules with nanometer precision. Synthetic exosome-mimetic systems and fusogenic LNPs, engineered with surface ligands such as aptamers or tumor-homing peptides, enable direct cytosolic RNA delivery by bypassing the endosomal route. Organelle-targeted RNA delivery is emerging as a strategy to enhance therapeutic effects, for example, directing RNA payloads to mitochondria using triphenylphosphonium modifications to trigger apoptosis via BCL2 silencing. Additionally, integrating multiple RNA types within a single delivery system such as combining mRNA encoding immune-stimulating cytokines with siRNA targeting immune checkpoints like PD-L1 could synergistically activate antitumor immunity. To enable rapid, personalized immunotherapy, click-chemistry strategies for post-synthetic conjugation of patient-specific neoantigens to RNA scaffolds are under development. Meanwhile, coating nanoparticles with zwitterionic polymers or employing PEGylated hybrid designs can improve systemic circulation and reduce immunogenicity. These advances, coupled with improved manufacturing methods and real-time tumor profiling via single-cell and spatial transcriptomics, could revolutionize RNA delivery systems and firmly establish them as a cornerstone in precision cancer immunotherapy.

Combination of RNA therapy with chemotherapy/radiotherapy

Chemotherapy

Focusing on anti-apoptotic systems in cancer cells has become a potential approach to surmount treatment resistance and improve the effectiveness of chemotherapy and radiation. The overexpression of proteins including Bcl-2, Bcl-xL, and survivin facilitates chemoresistance in several malignancies, encompassing acute leukemia and solid tumors. Clinical and experimental investigations have demonstrated that ASOs and siRNAs targeting these anti-apoptotic genes might enhance the susceptibility of tumor cells to treatment drugs. A phase I study with the Bcl-2 antisense G3139 (Genasense) in conjunction with FLAG treatment for relapsed or refractory acute leukemia shown safety, target downregulation, and promising response rates, with Bcl-2 mRNA decreased in 75% of assessable patients [273]. Subsequent investigations verified that the inhibition of Bcl-2, Bcl-xL, or survivin amplifies apoptosis via both intrinsic and extrinsic pathways and increases the cytotoxicity of chemotherapeutic agents and death ligands such as TRAIL, with Bcl-2/Bcl-xL inhibition demonstrating more pronounced effects than survivin silencing [274]. Innovative delivery techniques, including PEGylated liposomes, have been created to enhance the intracellular transport of ASOs and pharmaceuticals, facilitating effective cytoplasmic and nuclear delivery, surmounting multidrug resistance, and significantly augmenting the antitumor efficacy of doxorubicin in ovarian cancer models [275]. Aptamer-siRNA conjugates targeting HER2 + breast cancer cells demonstrate preferential internalization and gene silencing of Bcl-2, thereby restoring chemosensitivity to cisplatin [276]. These findings collectively highlight the increasing potential of antisense and siRNA-based therapeutics, particularly when combined with tailored delivery methods, to enhance treatment responses in apoptosis-resistant malignancies.

Cisplatin and analogous DNA-damaging chemotherapies frequently encounter constraints owing to tumor resistance, mostly attributed to error-prone translesion DNA synthesis mediated by proteins such as REV1 and REV3L. Targeting these proteins with siRNAs has demonstrated promise to surmount resistance and improve therapeutic success. A nanoparticle approach was created to concurrently deliver a cisplatin prodrug and siRNAs aimed at REV1 and REV3L directly to tumor cells. This method accomplished prolonged gene silence and significantly enhanced tumor suppression in a prostate cancer murine model relative to cisplatin alone, illustrating a viable option to augment chemotherapy through the integration of siRNA-mediated gene targeting [277].

Recent advancements in cancer research have shown the possibility of integrating gene-targeted strategies with traditional chemotherapy to surmount drug resistance and improve therapeutic effectiveness across many malignancies. In hepatic cancer, multifunctional folate-targeted nanoparticles co-delivering BCL-2 siRNA and DOX accomplished targeted gene silencing, triggered apoptosis, and improved the therapeutic efficacy of DOX via synergistic RNAi and chemotherapy [278]. In chemo-resistant SCLC, CRISPR/Cas9 screening revealed CDC7 as a critical vulnerability, where its suppression with XL413 rendered resistant cells more susceptible to cisplatin and etoposide by enhancing DNA damage and death, while leaving chemo-sensitive cells unaffected [279]. In HCC, genome-wide CRISPR screening identified phosphoseryl-tRNA kinase (PSTK) as an inhibitor of chemotherapy-induced ferroptosis. Targeting PSTK impaired glutathione metabolism, increased ferroptosis, and rendered HCC cells more susceptible to Sorafenib, with punicalin recognized as a possible PSTK inhibitor [280]. A kinome-wide CRISPR knockout screen in malignant pleural mesothelioma (MPM) revealed WEE1 as a G2–M checkpoint kinase, whose suppression mitigated chemotherapy resistance by averting cell-cycle arrest, facilitating DNA damage buildup, and augmenting cisplatin/pemetrexed-induced death [281]. Collectively, these findings highlight the efficacy of CRISPR-based functional genomics in elucidating critical resistance mechanisms and corroborating synergistic therapeutic targets, thereby endorsing the incorporation of gene silencing, kinase inhibition, and ferroptosis induction into innovative combinatorial cancer treatment strategies.

The CRISPR-associated Cas9 (CRISPR/Cas9) system has emerged as a potent gene-editing instrument with significant potential in cancer treatment. Nonetheless, constraints such as the biological hazards linked to viral vectors and the instability of exogenous plasmid DNA in systemic circulation impede its practical use. A stimuli-responsive chitosan-based nanocomplex functionalized with lactobionic acid was created to co-deliver sgVEGFR2/Cas9 plasmid and the chemotherapeutic drug paclitaxel (PTX) for the treatment of HCC. This nanoplatform attained a genome editing effectiveness of 38.6% in vitro in HepG2 cells And 33.4% in vivo in tumor tissues. It also significantly reduced VEGFR2 protein expression by more than 60% in HepG2 cells And prevented tumor growth by 70% in animals with HCC. in vivo investigations revealed targeted tumor accumulation and superior biosafety of the nanocomplex. The co-delivered gene and medication combination stimulated anti-tumorigenic pathways by diminishing the production of pro-inflammatory cytokines IL-6 and IL-8, together with the angiogenesis-related protein NF-κB p65. The data indicate that paclitaxel may augment the therapeutic efficacy of gene therapy aimed at VEGFR2 in HCC. This gene-drug co-delivery technology provides an efficient and secure method for synergistic CRISPR/Cas9-mediated gene therapy and chemotherapy (Fig. 11) [282]. In addition, CRISPR/Cas9 system can be utilized to provide the synergistic impact in cancer chemotherapy, suggesting that PARP1 downregulation can increase the response of breast tumor cells to chemotherapy [283].

Fig. 11.

An in vitro assessment of the therapeutic effectiveness of CLPV nanoparticles (NPs) against HCC was performed. A schematic diagram depicts the therapy plan of CLPV nanoparticles for HCC. T7 endonuclease I (T7E1) tests were conducted to evaluate VEGFR2 gene editing in HepG2 and HeLa cells subjected to CLPV nanoparticles and VC liposome-mediated transfection. Western blot analysis was employed to assess VEGFR2 protein expression in HepG2 cells after treatment with liposome-transfected and nanoparticle formulations (CLP and CLPV NPs). The cytotoxic effects of various nanoparticle groups were evaluated using a CCK-8 test and compared to free paclitaxel (PTX) on SMMC-7721 and HepG2 cell lines. Data are expressed as mean ± standard deviation (SD, N = 6), with statistical significance indicated (* P < 0.05, ** P < 0.01). Quantitative RT-PCR and Western blot analyses were employed to assess IL-6 and IL-8 mRNA expression and NF-κB p65 protein levels, respectively. Data are presented as mean ± SD (N = 6), with * P < 0.05 denoting statistical significance. Western blot analysis was performed to assess the expression of NF-κB p65 and Ser276-phosphorylated proteins in HepG2 cells following treatment with PTX and NP formulations. Reprinted with permission from Elsevier [282]

AS ODNs have arisen as a viable approach to circumvent chemoresistance by specifically targeting anti-apoptotic or oncogenic genes implicated in tumor viability. Survivin, an apoptosis inhibitor does not present in normal tissues but abundant in Malignancies, was significantly downregulated by a 20-mer antisense oligodeoxynucleotide (4003) in A549 LuAD cells, resulting in a 70% reduction in mRNA and an increase in etoposide-induced apoptosis, without impacting normal leukocytes [284]. In NSCLC H460 cells, the targeting of XIAP using G4 AS ODN decreased mRNA and protein Levels by more than 50%, triggered substantial apoptosis, and enhanced cellular sensitivity to several chemotherapeutics, such as doxorubicin and vinorelbine [285]. In vivo, G4 AS ODN inhibited tumor proliferation and increased the efficacy of chemotherapy in xenograft models. In malignant pleural mesothelioma (MPM), BCL-XL-targeted antisense oligodeoxynucleotides (AS ODNs) significantly enhanced the apoptotic response to cisplatin, with isobologram analysis validating synergistic effects [286]. Research on breast cancer focusing on HER-2 mRNA using AS HER-2 ODNs revealed enhanced sensitivity to many chemotherapeutics, independent of HER-2 expression levels, with combination therapy exhibiting synergistic apoptosis, especially in conjunction with taxol [78]. These data together endorse the efficacy of antisense-based medicines to enhance conventional chemotherapy across several cancer types by silencing genes that promote survival, even in tumors with modest target expression.

The function of stroma-targeting treatments in altering tumor-induced immune suppression is predominantly under-researched. STNM01, an RNA oligonucleotide engineered to inhibit carbohydrate Sulfotransferase 15 (CHST15), a critical enzyme in proteoglycan synthesis and extracellular matrix remodeling, was assessed in a phase I/IIa open-label, dose-escalation trial involving patients with unresectable PDAC who were resistant to gemcitabine plus nab-paclitaxel. STNM01 was delivered using endoscopic ultrasound-guided locoregional injection biweekly, in conjunction with oral S-1 chemotherapy. Doses varied from 250 to 10,000 nM, with patients undergoing a maximum of three treatment cycles. The main outcome was the occurrence of dose-limiting toxicity (DLT), whilst secondary endpoints were OS, tumor response, immunohistopathological alterations in the TME, And safety. Among the 22 recruited patients, no dose-limiting toxicities were seen, And the medication was well tolerated. The median OS was 7.8 months, accompanied by a disease control rate of 77.3%. One patient demonstrated complete resolution of lesions in the pancreas and lymph nodes associated with the tumor. Baseline results indicated that elevated CHST15 expression was associated with reduced infiltration of CD³⁺ and CD⁸⁺ T cells. After administration of STNM01 and S-1, a notable decrease in CHST15 expression was recorded, accompanied by heightened infiltration of CD³⁺ and CD⁸⁺ T cells, especially during the initial treatment cycle. A significant elevation in CD³⁺ T cells correlated with extended Survival. Eight grade 3 adverse events were recorded in total. The locoregional administration of STNM01 with S-1 shown safety and potential effectiveness by altering the TME to bolster T cell-mediated immune responses and improved clinical outcomes in advanced PDAC [287].

Chemotherapy-induced tumor RNA nanoparticles (C-RNA-NPs), created by condensing RNA derived from chemotherapy-treated cancer cells with protamine, efficiently enhance DC maturation and provoke robust antitumor immune responses. C-RNA-NPs, when used in conjunction with immune checkpoint blockade treatment, augment T cell infiltration, elevate the CD⁸⁺/Treg ratio, and boost the synergistic effectiveness of chemoimmunotherapy [288]. Furthermore, chemotherapy-induced RNA disruption in tumors, measured by the RNA Disruption Assay (RDA), shown a substantial correlation with both pathologic CR (pCR) and long-term DFS in breast cancer patients. RDA more precisely identified chemotherapy non-responders compared to clinical evaluation and indicated that elevated RNA disruption levels forecasted enhanced DFS, even in patients who did not attain pathological CR, underscoring its potential as a better biomarker for directing chemotherapy response [289]. Based on these findings, self-assembled apoptin mRNA nanospheres (mRNA-NSs) were synthesized using rolling circle transcription, resulting in uniform nanostructures that facilitate efficient apoptin expression and substantial DOX loading. The mRNA-NSs@DOX exhibited significant synergistic gene-chemotherapy effects by inducing apoptosis in breast cancer cells and augmenting tumor necrosis in vivo, presenting a viable approach for integrated mRNA-based cancer treatment [290]. Building upon this therapeutic strategy, maintenance therapy with an autologous RNA-loaded DC vaccine that expresses leukemia-associated antigens (FDC101) was provided to AML patients in their first complete remission (CR1) who were not suitable for allogeneic stem cell Transplantation. This method was well tolerated And resulted in a 75% five-year OS rate, indicating a viable low-toxicity option for maintaining remission and enhancing long-term outcomes in elderly or transplant-ineligible AML patients [291].

Radiotherapy

Human papillomavirus (HPV) infection is a recognized etiological factor for several cancer types, including vaginal, anal, and head and neck malignancies. The viral oncoproteins E6 and E7 are principal facilitators of carcinogenesis, rendering them compelling candidates for therapeutic vaccination. A new RNA lipoplex (RNA-LPX)-based HPV16 vaccination targeting E7, termed E7 RNA-LPX, has recently demonstrated the ability to induce regression of HPV16-positive tumors in mice and to elicit enduring protective T cell memory. A vaccination targeting HPV16 E6/E7 RNA-LPX is presently undergoing evaluation in phase I and II clinical trials for several HPV-related malignancies. Nonetheless, a considerable unmet demand persists for efficacious treatments for individuals with radiosensitive HPV16-positive malignancies. The therapeutic potential of combining the E7 RNA-LPX vaccination with conventional local radiotherapy (LRT) was assessed. The combination had a synergistic impact in HPV16-positive murine tumor models, with therapeutic results exceeding those of each therapy individually. Mechanistic investigations demonstrated that the E7 RNA-LPX vaccination elicited substantial infiltration of E7-specific CD⁸⁺ T cells into tumors, successfully transforming immunologically “cold” tumors into “hot” ones. Simultaneously, LRT mostly induced lethal effects by diminishing tumor volume and hypoxia, hence increasing the vulnerability of tumor cells to antigen-specific T cell-mediated elimination. The apparent synergy between the two treatments depended on the overall radiation dosage delivered, rather than on dose-fractionation methods [292].

The integration of RNA-based treatments with radiation is a potential approach to augment anticancer effectiveness by addressing both tumor-intrinsic pathways and immune activation. RNAi directed at hypoxia-inducible factor-1α (HIF-1α) has demonstrated the ability to enhance tumor cell sensitivity to hypoxia-induced apoptosis and diminish tumor proliferation, particularly when combined with ionizing radiation. This indicates that inhibiting essential survival pathways using RNA-based methods can amplify the harmful effects of radiation. In microsatellite stable (MSS) colorectal tumors, which generally exhibit resistance to immune checkpoint inhibitors, chemoradiation treatment (CRT) that includes oxaliplatin (OX) has been shown to promote RNA editing through the upregulation of the RNA editing enzyme ADAR1. This process produces new neoantigens, enhancing tumor immunogenicity and perhaps augmenting the efficacy of immunotherapy. These findings highlight the possibility of integrating RNA therapies, such as siRNAs or RNA editing modulation, with radiation to augment tumor cell apoptosis, change the TME, and improve immune recognition [293, 294].

The amalgamation of CRISPR-mediated gene editing with radiation constitutes a viable therapeutic approach to surmount tumor radioresistance and augment treatment effectiveness across diverse cancer types. In GBM, genome-wide CRISPR loss-of-function screening has revealed glutathione synthetase (GSS) as a crucial regulator of radiation resistance via inhibiting ferroptosis [295]. Targeting GSS impedes glutathione production, inactivates GPX4, and facilitates iron buildup, hence augmenting radiotherapy-induced ferroptotic cell death. A unique extracellular vesicle system, modified with Angiopep-2 and TAT peptides, has been created to overcome delivery hurdles of CRISPR treatments, facilitating effective Cas9-sgRNA distribution across the BBB and attaining high GSS editing efficiency with low off-target consequences. Likewise, CRISPR activation screening in GBM has identified CARHSP1, a cold-shock domain protein, as an additional driver of radioresistance, with its knockdown enhancing tumor sensitivity to radiation through the inhibition of TNF-α–mediated inflammatory signalling [296]. In laryngeal cancer, the deletion of HIF-1α and Glut-1 by gene editing has been demonstrated to improve radiosensitivity by downregulating the PI3K/Akt/mTOR pathway, therefore alleviating hypoxia-induced resistance and metabolic adaptation [297]. Furthermore, novel RNA-based approaches, including siRNA delivery via metabolizable nanocluster platforms (BSC_gal), have been utilized to target immunosuppressive agents such as galectin-1, thereby enhancing the efficacy of stereotactic ablative radiotherapy (SABR) to augment both local and systemic anti-tumor immune responses [298]. These findings highlight the potential of CRISPR and RNAi technologies, in conjunction with radiation, to address critical resistance mechanisms, alter TME s, and provide a tailored and more successful cancer treatment strategy.

A study introduces the creation And validation of a new boron-containing nanoparticle platform, 10B/siPD-L1, aimed at improving boron neutron capture treatment (BNCT) via accurate tumor targeting And immune regulation. The nanoparticles were synthesized using the self-assembly of a 10B-enriched triblock copolymer containing PD-L1 siRNA, subsequently stabilized by disulfide crosslinking. These particles exhibited remarkable serum stability, siRNA protection, biocompatibility, and reactivity to tumor-specific intracellular conditions (elevated GSH and ATP), facilitating controlled disintegration and selective payload release within tumor cells. The cRGD ligand enhanced tumor-specific uptake by targeting integrin αvβ3, whereas intracellular glycoconjugate binding increased 10B retention. In comparison to traditional treatments, these nanoparticles demonstrated significantly enhanced tumor formation and tumor-to-normal tissue (T/N) ratios, facilitating accurate BNCT-induced DNA damage while minimizing off-target effects. In vitro and in vivo investigations shown that PD-L1 siRNA not only suppressed radiation-induced PD-L1 overexpression but also disrupted DNA repair mechanisms by downregulating BRCA1, RAD50, and MRE11, thereby exacerbating DNA damage and death. This dual-therapy strategy facilitated ICD, DC maturation, and significant CD⁸⁺ T cell infiltration, altering the tumor immunological Microenvironment by diminishing Treg populations and augmenting Lethal gene expression. In 4T1 tumor-bearing and metastatic murine models, the integration of BNCT and PD-L1 silencing resulted in significant tumor growth inhibition, pronounced abscopal effects, reduction of lung metastasis, and prolonged life, significantly Surpassing the efficacy of either treatment independently. These findings highlight the therapeutic promise of 10B/siPD-L1 nanoparticles as a novel BNCT agent with synergistic immunotherapeutic effects, facilitating clinical translation and wider applicability in the treatment of solid malignancies (Fig. 12) [299].

Fig. 12.

Design And characterization of 10B/siPD-L1 nanoparticles. a A diagram illustrating the production of 10B/siPD-L1 nanoparticles and their application in the integrated approach of BNCT and immunotherapy. b ICP-MS spectra of the cRGD-PEG-PME-PBOB copolymer used for ascertaining the boron isotope composition. c Acid-base titration profile of the cRGD-PEG-PME-PBOB copolymer to evaluate its pKa. d Gel electrophoresis findings demonstrating siRNA loading efficiency at different Mass ratios of 10B polymer to siRNA. e Assessments of particle size, zeta potential, and transmission electron microscopy (TEM) Pictures of 10B/siPD-L1 nanoparticles before to and after to disulfide crosslinking. Reprinted with permission from Wiley [299]

Apurinic/apyrimidinic endonuclease 1 (APE1), a bifunctional enzyme engaged in DNA repair and redox control, has been associated with the development of tumor radioresistance. A chimeric adenoviral vector, Ad5/F35, expressing human APE1 siRNA (Ad5/F35-APE1 siRNA), was developed to investigate the potential of targeting APE1 to augment radiosensitivity. This vector exhibited enhanced infectivity in LOVO colon cancer cells relative to the conventional Ad5 vector. In LOVO cells, APE1 exhibited elevated expression, and its downstream target, nuclear factor κB (NF-κB), a recognized role in radioresistance, was persistently active. Treatment with Ad5/F35-APE1 siRNA resulted in a dose-dependent decrease in APE1 protein levels and AP endonuclease activity in vitro. This inhibition significantly enhanced the susceptibility of LOVO cells to irradiation in clonogenic survival experiments, correlated with higher apoptotic levels. Irradiation stimulated APE1 expression and augmented NF-κB DNA-binding activity in a dose-dependent manner, both of which were significantly inhibited by Ad5/F35-APE1 siRNA. In a subcutaneous colon cancer xenograft model utilizing nude mice, intratumoral administration of Ad5/F35-APE1 siRNA significantly decreased APE1 expression and enhanced the tumor growth-inhibitory effects of irradiation. The findings indicate that APE1 is crucial in facilitating radioresistance, and its targeted restriction may enhance the effectiveness of radiotherapy [300].

Recent studies have shown the crucial roles of circRNAs, RNA alterations, and immunological regulation in facilitating resistance to radiation in diverse malignancies and in improving treatment outcomes by targeted therapies. In nasopharyngeal cancer (NPC), the increased circRNA circADARB1 inhibits ferroptosis and imparts radioresistance by stabilizing critical antioxidant proteins such as SLC7A11 and GPX4 through HSP90B1, hence reducing radiotherapy-induced lipid peroxidation [301]. A biomimetic nanomaterial co-delivering siRNA targeting circADARB1 and iron ions significantly mitigated this resistance, enhancing the sensitivity of NPC cells to radiation by facilitating ferroptosis. In LUAD, circNEIL3 was recognized as a suppressor of pyroptosis during irradiation by functioning as a miR-1184 sponge, therefore releasing PIF1 and activating the AIM2 inflammasome pathway; inhibiting circNEIL3 improved the efficiency of radiotherapy in vivo [302]. In CC, the m6A-modified circRNF13 is associated with radioresistance, where METTL3/YTHDF2-mediated degradation of circRNF13 results in the instability of CXCL1 and enhanced radiosensitivity upon knockdown [303]. Furthermore, in NSCLC, an immunosuppressive TME, influenced by M2 TAMs and STAT6 signaling, was identified as a limiting factor for the efficacy of radiation and immunotherapy [304]. The integration of STAT6-targeting ASOs with hypofractionated radiation significantly decreased tumor burden and metastasis, improved T cell and macrophage activity, and stimulated a pro-inflammatory immune response. These findings collectively highlight the therapeutic potential of targeting non-coding RNAs, RNA alterations, and immunosuppressive pathways to mitigate radioresistance and enhance cancer therapy outcomes.

The overexpression of O6-methylguanine DNA methyltransferase (MGMT) significantly contributes to resistance to chemoradiation treatment (CRT) in brain malignancies. Prior research indicated that non-ablative radiation could improve the administration of anti-MGMT morpholino oligonucleotides (AMONs), successfully reducing MGMT levels in subcutaneous tumor xenografts. This research examined the capacity of this technique to enhance CRT effectiveness in rat brain tumor xenograft models. The impact of radiation on targeted delivery was evaluated using fluorescently tagged oligonucleotides (f-ON). Confocal microscopy examination in T98G glioma cells demonstrated f-ON localization within clathrin-coated vesicles, endosomes, and lysosomes. Fluorescent signals were detected in the irradiated brains of Long Evans rats, but they were absent in non-irradiated controls, indicating improved delivery following radiation exposure. Cranial radiation (2 Gy), succeeded by intravenous delivery of AMONs (10.5 mg/kg), Led to a 50% decrease in MGMT expression in both orthotopic cerebellar D283 medulloblastoma and intracerebral H460 NSCLC xenograft mice. Administration of AMONs in conjunction with CRT (2 Gy radiation And 20 mg/kg oral temozolomide for four days) resulted in a substantial decrease of tumor sizes in the medulloblastoma model (p = 0.012), with a comparable reduction trend noted in the NSCLC brain metastasis model [305].

Hypoxia-induced radioresistance poses a considerable obstacle to the therapeutic efficacy of radiation. Carbonic anhydrase IX (CA IX), a cell-surface enzyme sensitive to hypoxia that regulates the acidic microenvironment of solid tumors, has become a prospective therapeutic target. A dual exogenous/endogenous inhibitory method for CA IX was devised using core/satellite-structured nanohybrids made of metal-organic framework (UiO-66) and gold nanoparticles (Au NPs) to improve radiation effectiveness in TNBC under hypoxic settings. In this approach, gold nanoparticles (Au NPs) were synthesized in situ on the UiO-66 matrix, which disintegrated in tumor cells due to elevated phosphate levels, releasing p-phthalic acid (PTA), a structural constituent of UiO-66, to externally limit carbonic anhydrase IX (CA IX) activity. The Au nanoparticles served both as radiosensitizers and as carriers for CA IX ASOs, which were incorporated through stable Au–S bonds to facilitate gene silencing of CA IX. This combined inhibition, via pharmacologic inhibition by PTA and genetic knockdown by ASO effectively alleviated hypoxia-induced radioresistance. The method also complemented Au NP-mediated radiosensitization, leading to improved therapeutic results both in vitro and in vivo, highlighting the potential of this strategy to address hypoxia-related treatment resistance in solid tumors (Fig. 13) [306].

Fig. 13.

Schematic diagram illustrating the synthesis pathway for the multifunctional UAAP nanoparticles and the suggested mechanism for dual inhibition of CA IX and Au-mediated improved irradiation. Reprinted with permission from Elsevier [306]

Innovative techniques that integrate radiation (RT) with molecular-targeted therapies are shown encouraging outcomes in improving treatment effectiveness for diverse cancer types. In GBM, the CXCL12-neutralizing aptamer NOX-A12, administered in conjunction with radiotherapy in the GLORIA phase I/II trial, exhibited a favorable safety profile devoid of dose-limiting toxicities and revealed radiographic responses in most patients, encompassing partial remissions and diminished tumor perfusion, suggesting potential efficacy. Increased CXCL12 expression was associated with extended progression-free survival, warranting future exploration of CXCL12-targeted radiosensitization. Concurrent initiatives have utilized siRNAs to inhibit critical DNA damage response proteins, ATM, ATR, and DNA-PKcs, achieving substantial target suppression (~ 90%) and increased radiosensitivity surpassing conventional chemical inhibitors, along with enhanced chemosensitivity to agents such as methyl methanesulfonate. Moreover, the suppression of telomerase using siRNA directed at human telomerase reverse transcriptase (hTERT) in cervical cancer cells decreased proliferation, triggered replicative senescence, and heightened susceptibility to DNA-damaging treatments, including ionizing radiation and chemotherapeutic agents. These findings collectively highlight the increasing potential of integrating radiotherapy with aptamer- or siRNA-based strategies to surmount therapeutic resistance, enhance tumor sensitivity to radiation and chemotherapy, and improve clinical outcomes across many cancer models [307–309].

Advanced NSCLC continues to pose a significant therapeutic challenge, with Less than 20% of patients surviving beyond two years, despite progress in targeted therapy for select patient subgroups. Cancer vaccines have emerged as a viable approach to elicit enduring immune responses by stimulating the patient’s immune system. CV9202 is a self-adjuvanting mRNA vaccine aimed against six tumor-associated antigens commonly expressed in NSCLC: NY-ESO-1, MAGEC1, MAGEC2, 5T4, survivin, and MUC1. A clinical trial has been established to assess the safety, tolerability, and immunogenic potential of CV9202 in conjunction with targeted irradiation to augment immune activation. The trial will recruit stage IV NSCLC patients who have attained either stable disease or a response after first-line chemotherapy or EGFR tyrosine kinase inhibitor treatment. Participants representing three histological and molecular subtypes of NSCLC, squamous and non-squamous, with or without EGFR mutations, will receive two initial doses of CV9202, followed by localized radiotherapy (5 Gy/day for four days), and will continue vaccine administration until disease progression occurs. The main outcome measure is the occurrence of Grade > 3 treatment-related adverse events. Secondary analyses will investigate antigen-specific immune responses to both CV9202-encoded antigens and unrelated tumor antigens (indicative of antigen dissemination), in conjunction with routine clinical effectiveness assessments. This study seeks to assess the feasibility of integrating RNActive mRNA vaccination with radiation as an innovative treatment strategy for advanced NSCLC [310].

RNA therapy in cold and hot TME

Transforming “cold” cancers into “hot” tumors is essential for enhancing the effectiveness of anticancer immunotherapy. A study introduces an innovative cancer treatment approach employing an MMP-2-responsive, peptide-based micelleplex (P^A7R@siPD-L1) that combines PDT, ICB, and vascular normalization to address solid tumors and enhance antitumor immunity. The chimeric peptide P^A7R, consisting of PEG-R9K(Ce6)-LLGPLGVRG-A7R, was synthesized and self-assembled with lecithin and siPD-L1 to create stable, homogenous micelleplexes, which were characterized using TEM, DLS, and zeta potential analysis. These micelleplexes exhibited MMP-2-responsive disintegration, pH-sensitive morphological alterations, and effective singlet oxygen formation, therefore improving Ce6 accessibility and ROS production during laser irradiation. P^A7R@siPD-L1 demonstrated enhanced cellular uptake, siRNA protection, And phototoxicity in 4T1 tumor cells, while also displaying antiangiogenic properties through the A7R section, particularly following MMP-2 cleavage. The medication successfully inhibited PD-L1, triggered ICD, facilitated DC maturation, and increased the infiltration of CD⁴⁺ and CD⁸⁺ T cells, while diminishing regulatory T cells and M2 macrophages. In vivo investigations validated biosafety, tumor accumulation, prolonged circulation duration, and substantial suppression of both primary and abscopal tumor development in mouse breast cancer models, with no damage to healthy tissues. P^A7R@siPD-L1 reduced lung metastases and restored tumor vasculature, mitigating hypoxia and restructuring the TME to promote immune activation. The increased secretion of immune-stimulatory cytokines (IFN-γ, TNF-α, IL-2) and the reduced levels of immunosuppressive IL-10 further confirmed the vigorous immunological response (Fig. 14) [311].

Fig. 14.

Depiction of MMP-2-responsive, peptide-assembled micelleplexes for improved photoimmunotherapy. Reprinted with permission from Elsevier [311]

Chemoimmunotherapy holds considerable potential for eliciting antitumor immune responses; yet, surmounting the immunosuppressive TME and T-cell depletion is a substantial challenge in cancer therapy. An efficacious immune-modulatory strategy necessitates simultaneous modulation of T-cell infiltration and fatigue. A novel carrier-free nanoparticle system has been developed to co-deliver a chemotherapeutic agent (DOX), a cytolytic peptide (melittin, MPI), and siRNA targeting TOX (siTOX), a crucial regulator of T-cell exhaustion, utilizing a fluorinated prodrug strategy. This multifunctional design allows DOX and MPI to collaboratively induce significant ICD, serving as a stimulatory “offense” signal to augment CD⁸⁺ T-cell infiltration, while the siTOX component offers a complementary “defense” signal by downregulating TOX expression, thus mitigating T-cell exhaustion. The coordinated function of these components provoked a robust antitumor immune response in liver cancer and metastatic models, illustrating the system’s capacity as an effective delivery platform for converting immunologically “cold” tumors into “hot” ones [312].

Inducing robust immune responses against tumors continues to pose a considerable difficulty in cancer immunotherapy, which generally advantages only a few group of patients with “hot” tumors marked by pre-existing effector immune cell infiltration. A nanoparticle-based strategy has been devised to transform immunologically “cold” cancers into “hot” tumors by augmenting the recruitment and activation of T and NK cells, while inhibiting tumor-derived TGF-β1 release. This method entails the co-encapsulation of LTX-315, a pioneering oncolytic cationic peptide, and TGF-β1-targeting siRNA within a polymer-lipid hybrid nanoparticle formed from PLGA, DSPE-mPEG, and DSPE-PEG coupled with a cRGD peptide (designated as LTX/siR-NPs). In vitro investigations revealed that LTX/siR-NPs significantly suppressed TGF-β1 expression, enhanced type I interferon secretion, and induced ICD, as shown by increased levels of damage-associated molecular patterns. The nanoparticle formulation in vivo safeguarded LTX-315 against serum breakdown and enhanced tumor formation. This led to significant inhibition of TGF-β1 production and the alteration of the TME into an immunological-active condition, characterized by enhanced infiltration of antitumor effector immune cells. The combination of NKG2A immune checkpoint suppression with LTX/siR-NPs significantly increased the presence of CD⁸⁺NKG2D + and NK1.1 + NKG2D + cells in tumor tissues, resulting in substantial tumor growth reduction and extended life in the treated animals [313].

A research presents a cryo-immunochemotherapeutic approach, designated ICIE, that integrates cryosurgery with cold-responsive nanoparticles (CRNPs) co-loaded with the chemotherapeutic agent irinotecan (CPT) and PD-L1-targeting siRNA (siR) to induce robust and lasting antitumor immunity. The CRNPs are constructed from a polymer with a low critical solution temperature (~ − 4 °C), allowing them to disintegrate and release their therapeutic agents precisely under the cold conditions produced during cryosurgery. The technology effectively administers CPT and siR into tumor cells, facilitates endo/lysosomal escape, and induces ICD by cold-triggered release, characterized by elevated production of DAMPs (HMGB1, CRT, HSP-70, HSP-90). This results in DC maturation and efficient CD⁸⁺ T cell activation, culminating in strong cytotoxic T lymphocyte (CTL) responses against tumor cells. In murine models of breast cancer, ICIE significantly transformed the TME from immunologically “cold” to “hot” by diminishing immunosuppressive cells (Tregs, M2-TAMs, MDSCs), augmenting the infiltration of CTLs, and elevating the CD⁸⁺/Treg ratio. The medication successfully impeded both primary and metastatic tumor proliferation, curtailed lung metastasis, and extended longevity, all without any systemic toxicity. Moreover, ICIE elicited robust short-term and long-term memory immune responses, characterized by increased numbers of effector and central memory T cells, underscoring its potential for enduring protection against tumor recurrence and metastasis. ICIE offers a robust, synergistic platform that combines cryosurgery, controlled drug and gene delivery, and immune regulation to address the shortcomings of traditional cancer treatments, presenting a viable avenue for the treatment of aggressive and metastatic malignancies (Fig. 15) [314].

Fig. 15.

ICIE combines cryosurgery with the systemic administration of cold-responsive nanoparticles (CRNPs) co-encapsulated with irinotecan (camptothecin or CPT) and PD-L1-targeting siRNA (siR) to restructure the TME. This method promotes ICD and inhibits PD-L1 expression in tumor cells, successfully converting an immunosuppressive (“cold”) TME into an immuno-active (“hot”) one. Consequently, CD⁸⁺ T lymphocytes are stimulated to facilitate both local and systemic tumor eradication. The CPT&siR CRNPs are produced utilizing a double-emulsion method, which includes CPT, siR, poly(D, L-lactide-co-glycolide) (PLGA), poly(N-isopropylacrylamide-co-butyl acrylate) (pNIPAAm-BA), chitosan-modified Pluronic F-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). Reprinted with permission from Springer Nature [314]

Elevated levels of TGFβ and Cox2 in the TME impede T-cell infiltration, obstructing immune cells from reaching tumor antigens and thereby diminishing the efficacy of immune checkpoint treatments. A polypeptide-based nanoparticle was created for intravenous administration of two siRNAs aimed at TGFβ and Cox2 to Surmount this obstacle. In a syngeneic orthotopic animal model of HCC, systemic treatment of this nanoparticle effectively delivered siRNAs to Hepatic cells And significantly inhibited tumor development. At a dosage of 2 mg/kg given biweekly, the nanoparticle demonstrated significant monotherapy activity, reducing tumor burden to undetectable Levels after just five administrations. Reducing the siRNA dosage to 1 mg/kg BIW resulted in enhanced tumor suppression when administered alongside PD-L1 monoclonal antibodies. Following only three treatments, remaining tumors exhibited significant reduction in size, and histological examination demonstrated enhanced infiltration of CD⁴⁺ and CD⁸⁺ T cells, a response not observed in mice administered with control (non-silencing) siRNA. The findings indicate that the simultaneous silencing of TGFβ and Cox2 by polypeptide nanoparticles functions as a successful monotherapy for HCC and improves the effectiveness of immune checkpoint inhibition by converting immune-excluded (cool) tumors into T-cell inflamed (hot) tumors [315].

Increasing data suggests that physical exercise significantly inhibits the onset and advancement of several malignancies, partly through the action of exercise-responsive miRNAs, which are essential in facilitating these antitumor effects. This study examined the expression patterns and prognostic relevance of exercise-responsive miRNAs in breast cancer (BRCA) and broadened the analysis to include other cancer types. The tumor-suppressive effect of miR-29a-3p was thoroughly assessed using several independent public datasets, proprietary patient cohorts, in vitro tests using macrophages, fibroblasts, and tumor cells, and in vivo models. Research indicated that miR-29a-3p, a significant exercise-responsive miRNA, was downregulated in tumor tissues and associated with worse prognosis in BRCA. Functionally, miR-29a-3p targeted many elements of the TME, such as macrophages, fibroblasts, and cancer cells, to inhibit the production of B7 Homolog 3 (B7-H3). Single-cell RNA sequencing (scRNA-seq) and time-of-flight cytometry (CyTOF) analysis demonstrated that miR-29a-3p efficiently disrupted immune-resistant and immunologically “cold” tumors, promoting the change to an immuno-active or “hot” TME. Liposomal nanoparticles encapsulating miR-29a-3p (lipo@miR-29a-3p) were engineered for translational use, exhibiting significant anticancer efficacy and excellent biocompatibility in a mouse model [316].

Treatment options for advanced HCC are constrained, with immunotherapy becoming a significant element in multidisciplinary strategies for holistic care. A bio-responsive delivery system, Au@miR-183i, was created to selectively target liver cancer stem cells (LCSCs) by the administration of a miR-183 inhibitor. This approach was integrated with αPD-L1 immune checkpoint treatment to augment therapeutic effectiveness. The multifunctional Au@miR-183i nanocomplexes (NCs) are engineered to self-assemble in the TME, disrupting redox homeostasis by depleting NADPH and H₂O₂, resulting in increased ROS levels, modulation of the LCSC niche, and diminished cancer cell stemness. These NCs facilitate the precise delivery of miR-183i to LCSCs, triggering ICD, fostering DC maturation, augmenting CD⁸⁺ T cell infiltration, and converting immunologically “cold” tumors into “hot” tumors. In vivo investigations corroborated the in vitro results, demonstrating that Au@miR-183i NCs effectively targeted tumors, induced robust ICD, and significantly enhanced αPD-L1 immunotherapy, finally eliciting a vigorous systemic anticancer immune response in mice models with HCC [317].

Advanced colorectal cancer is characterized by a highly immunosuppressive TME and often has limited response to immunotherapy. Inducing cancer ICD has become a potential approach to address this constraint. Research has identified the lncRNA VPS9D1-AS1 as primarily prevalent in consensus molecular subtype (CMS)−2 colorectal cancer, with its overexpression associated with enhanced susceptibility to immune checkpoint blockade (ICB). A LNP-based delivery system containing ASOs was designed to therapeutically target VPS9D1-AS1. This nanodrug efficiently suppresses VPS9D1-AS1 in tumor cells and PDX models, leading to significant reduction in tumor growth and spread. Suppression of VPS9D1-AS1 mechanistically increases MLKL-mediated ICD, facilitating tumor antigen exposure and augmenting immune recognition. Furthermore, the inhibition of VPS9D1-AS1 reduces HLA-G expression, hence enhancing the susceptibility of CRC cells to immunotherapy. The LNP-ASO formulation alters the TME by enhancing the invasion of CD⁸⁺ T cells and DCs, and promotes better interaction between tumor cells and DCs via the AXL/GAS6 signaling pathway. In vivo investigations shown that co-targeting PD-1 and VPS9D1-AS1 significantly enhances immune checkpoint blockade effectiveness, underscoring the promise of VPS9D1-AS1-targeting nanotherapies as a prospective therapeutic strategy for advanced colorectal cancer therapy [318].

A research sought to evaluate the in vivo Anticancer effectiveness of Cantide, both as a standalone treatment And in conjunction with 5-fluorouracil, utilizing an orthotopic human HCC mice model. Neoplasms were induced in the liver and then treated with intravenous infusion of Cantide at different dosages. Tumor growth was assessed using caliper measures, and plasma alpha-fetoprotein (AFP) levels were evaluated by radioimmunoassay. Histological investigation was conducted utilizing hematoxylin-eosin (H&E) staining, alongside Western blotting to assess human telomerase reverse transcriptase (hTERT) protein quantities. The results indicated that Cantide significantly suppressed tumor development in a dose-dependent fashion, with higher dosages (75 And 50 mg·kg⁻¹·d⁻¹) Yielding Superior tumor suppression relative to saline controls. Furthermore, Cantide therapy decreased the recurrence of liver tumors and the incidence of Lung metastasis. The combination of Cantide And 5-fluorouracil significantly decreased tumor size more than either medication used independently. Both monotherapy and combination treatment resulted in a dose-dependent reduction in plasma AFP levels. These data demonstrate that Cantide is a powerful ASO with significant anticancer efficacy and considerable promise for clinical advancement as a treatment agent for HCC [319].

Clinical applications and trials

Individuals with cancer are at an elevated risk of adverse outcomes from SARS-CoV-2 infection. Although COVID-19 immunization is advised for this demographic, there is little data regarding the immune response and safety of these vaccinations in patients with solid tumors receiving systemic cancer treatments. The Vaccination Against COVID in Cancer (VOICE) experiment aims to assess the impact of immunotherapy, chemotherapy, and chemoimmunotherapy on the immunogenicity and safety of the mRNA-1273 (Moderna Biotech, Madrid, Spain) COVID-19 vaccine in this at-risk group. The VOICE trial is a prospective, multicenter, investigator-initiated non-inferiority research to assess the immunogenicity, safety, and immunological responses to two doses of the mRNA-1273 (Moderna) COVID-19 vaccine in individuals with and without cancer. Participants are categorized into four groups: healthy persons (cohort A), and patients with solid tumors undergoing immunotherapy (cohort B), chemotherapy (cohort C), or chemoimmunotherapy (cohort D). Eligible individuals Must be 18 Years of age or older, possess a Life expectancy beyond 12 months, and have no history of SARS-CoV-2 infection. They get two intramuscular injections of the vaccine, each containing 100 µg, administered 28 days apart. The main endpoint, seroconversion, characterized by a SARS-CoV-2-binding Antibody concentration exceeding 10 BAU/mL on day 28 post-second vaccination, is assessed centrally via a fluorescent bead-based technique. The study satisfies its non-inferiority criteria: seroconversion is observed in 100% of cohort A, 99% of cohort B, 97% of cohort C, And 100% of cohort D. Post-hoc Analysis establishes a threshold of 300 BAU/mL to differentiate between Sufficient And inadequate responders, with 99% of healthy persons Surpassing this Level, in contrast to 93% in the immunotherapy group, 84% in the chemotherapy group, And 89% in the chemoimmunotherapy group. Spike-specific T-cell responses, assessed using IFNγ ELISpot, are seen in 53–69% of chosen individuals, with reduced frequency among poor responders and missing in a minority of chemotherapy patients. Neutralizing antibody titers, evaluated using PRNT, exhibit a high correlation with binding antibody levels, reinforcing the Sufficiency threshold of 300 BAU/mL based on a protective titer of 40. Exploratory biomarker studies reveal associations between immunological response and lymphocyte/neutrophil numbers, treatment scheduling in relation to vaccination, and the utilization of immunosuppressive medications. The pharmacodynamic (pD) studies are carried out by comprehensive examinations of antibody and T-cell responses. Proposed exploratory objectives including functional and phenotypic characterization of SARS-CoV-2-specific immune cells, cytokine synthesis, cellular proliferation, and mucosal antibody concentrations. Safety data indicate that systemic side effects, including weariness, fever, and headache, are more prevalent with the second dosage, whereas local responses are minor, with discomfort at the injection site being the most common. Severe adverse effects are few, with a limited number probably associated with immunization (fever, diarrhea, febrile neutropenia, Stevens-Johnson syndrome). Immune-related side effects Manifest in around 4% of patients undergoing immunotherapy or chemoimmunotherapy, predominantly minor, but one instance of deadly pneumonitis has been documented. The experiment indicates that the mRNA-1273 vaccination elicits robust humoral and cellular immune responses in most cancer patients, endorsing its administration even with ongoing systemic therapy [320].

Research examines the immunogenicity and safety of the initial dose of the BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine in 16 adult patients with chronic phase chronic myeloid leukaemia (CML) undergoing treatment with tyrosine kinase inhibitors (TKIs) at Guy’s and St Thomas’ NHS Foundation Trust. Participants are administered a 30 µg dosage of BNT162b2 And undergo peripheral blood collection before to And about three weeks following immunization for immunological examination. The vaccination is often well tolerated, with localized inflammation occurring in 56.3% of patients And flu-like symptoms in 23.5%, although no SARS-CoV-2 infections were recorded throughout the research period. Serological testing using ELISA indicates that 87.5% (14/16) have measurable anti-Spike IgG responses, with two non-responders, one having a history of immune suppression and the other on ponatinib with inadequate disease management. Neutralising antibodies are identified in all post-vaccination samples, with a median ID50 of 445.5, with responses varying across low, medium, and high titres, showing no significant variations across TKI types. One patient who received a second vaccination dose has a significant rise in IgG EC50 And neutralizing titers. T-cell responses, evaluated through intracellular cytokine staining, indicate that 93.3% (14/15) of assessable patients exhibit SARS-CoV-2-specific T-cell responses following the initial dose, comprising CD⁴⁺ responses in 80% and CD⁸⁺ responses in 60%, with 80% demonstrating polyfunctional responses involving TNF-α, IFN-γ, and IL-2. Patients administered nilotinib have significantly elevated CD⁴⁺ T-cell TNF-α expression and dual TNF-α/IFN-γ responses in comparison to those receiving other TKIs. The findings indicate that a single dosage of BNT162b2 generates a robust humoral and cellular immune response in most of CML patients, and nilotinib may correlate with improved T-cell functioning [321].

A multicenter, open-label, exploratory phase Ib study assesses the safety, immunogenicity, and initial efficacy of the mRNA-based cancer immunotherapy BI1361849 (CV9202) in conjunction with local radiotherapy for patients with stage IV non-small-cell lung cancer (NSCLC) who exhibit stable disease or PR following first-line chemotherapy or EGFR-TKI therapy. Conducted at 13 locations in Germany, Austria, And Switzerland, 26 patients are categorized into three strata based on histology and treatment history. Patients have intradermal BI1361849 vaccines following a priming and Maintenance regimen, in conjunction with 20 Gy of fractionated radiotherapy; some also receive pemetrexed or ongoing EGFR-TKI as maintenance therapy. Safety outcomes indicate that BI1361849 and/or radiation-associated grade ≥ 3 adverse events Manifest in 15.4% of patients, which is below the established 30% safety threshold, with no significant immune-related or fatal treatment-emergent adverse events recorded. Immunomonitoring in 25 assessable patients indicates that 84% demonstrate at Least a two-fold enhancement in immune responses to one or more of the six vaccine-encoded tumor Antigens, including 40% with Functional T-cell responses And 80% with elevated antigen-specific antibody levels. CD⁴⁺ T-cell responses are more pronounced, with 52% of patients exhibiting responses to several Antigens. Significantly, 50% exhibit indications of Humoral immunological widening against non-vaccine tumor-associated Antigens, implying a possible Antigen cascade impact. Efficacy results indicate that 46.2% of patients attain stable illness, while one patient exhibits a PR. In six individuals, non-irradiated Lesion shrinking over 15% is seen, suggesting possible systemic effects. The median progression-free survival (PFS) is 2.87 months, whereas the median OS is 13.95 months. The data indicate that the combination of BI1361849 with localized irradiation is safe, immunologically effective, and demonstrates first evidence of disease control and enhanced immune response in advanced NSCLC [322].

A prospective, multicenter, open-label phase I/IIa trial assesses the safety, immunogenicity, and efficacy of CV9201, an mRNA-based cancer vaccine targeting five tumor-associated antigens, in patients with advanced NSCLC who have attained at Least stable disease following first-line treatment. During the dosage-escalation phase, nine patients were administered doses of 400, 800, or 1600 µg mRNA without encountering dose-limiting toxicities, Hence designating 1600 µg as the recommended phase II dose (RP2D). Thirty-seven individuals received treatment at the RP2D during phase IIa. In summary, CV9201 is generally well tolerated, with no significant side effects or autoimmune disorders connected to the medication documented. Most of adverse effects are Mild to severe, encompassing injection site reactions, Lethargy, And flu-like symptoms. Immunologically, 63% of assessable patients reveal at Least one Antigen-specific immune response, with 27% displaying T-cell responses using ICS or ELISPOT And 47% presenting Humoral IgM or IgG responses. T-cell responses, predominantly mono-functional, are often low And variable between ICS And ELISPOT assays. Significantly, 60% of patients have a ≥ 2-fold elevation in activated IgD + CD38 B cells, signifying B cell activation. Despite constrained T-cell responses, lymphocyte proliferation in one patient with negative ex vivo testing indicates the existence of antigen-specific cells, implying Limits in assay sensitivity. Nine of the 29 evaluable patients exhibit disease stability, with a median progression-free survival (PFS) of 5.0 months And a median OS of 10.8 months in the phase IIa group. The one-, two-, And three-year OS rates are 44.4%, 26.7%, And 20.7%, respectively. The findings indicate that CV9201 is safe and immunologically effective, provoking humoral and restricted cellular responses in a portion of NSCLC patients, and may provide minor therapeutic advantage [37].

A phase I clinical trial assessed the safety and immunogenicity of a tailored mRNA vaccine (mRNA-4650) that encodes specific neoantigens, mutations in driver genes, and HLA-I–predicted epitopes in four extensively pretreated patients with metastatic gastrointestinal Malignancies. Patients were administered four to seven intramuscular vaccines at dosages of either 0.13 mg or 0.39 mg, with no serious side effects observed, indicating excellent tolerability. No clinical responses were noted; however, neoantigen-specific CD^4⁺ and CD^8⁺ T cell responses were identified in three out of four patients, mostly following in vitro stimulation (IVS) of PBMCs. T cell responses were predominantly CD^4⁺ rather than CD^8⁺, despite the HLA-I-targeted design, indicating a preferred activation of CD^4⁺ T cells, potentially attributable to mRNA vaccine expression levels and antigen presentation dynamics. Neoantigens, which were defined and functionally verified using tumor-infiltrating lymphocytes (TILs), did not provoke enhanced responses following vaccination, and preexisting TIL-derived reactivities exhibited no augmentation, maybe attributable to terminal T cell differentiation or immune fatigue. TCR sequencing validated the proliferation of neoantigen-specific T cell clones post-vaccination in certain individuals; however, no intratumoral investigation was conducted. One patient (4303) exhibited no discernible reaction, potentially due to an absence of immunogenic epitopes. The personalized vaccination technique proved technically viable and elicited unique T cell responses; nevertheless, the lack of therapeutic benefit and the complexities of production highlight limits in scalability. Future techniques may prioritize expedited, economical antigen identification and combinatorial approaches with checkpoint inhibitors or adoptive T cell treatments to augment therapeutic efficacy [323].

PDAC is a fatal malignancy characterized by restricted therapeutic alternatives, elevated recurrence rates, and dismal long-term survival, necessitating the development of innovative treatments. Despite PDACs often exhibiting limited reactivity to immune checkpoint inhibitors attributed to a putative low neoantigen load, current studies indicate they possess a greater number of immunogenic neoantigens than previously recognized. A phase I clinical trial evaluated a personalized mRNA vaccine, autogene cevumeran, in conjunction with the PD-L1 inhibitor atezolizumab and chemotherapy (mFOLFIRINOX) in patients with resected PDAC, based on observations that long-term PDAC Survivors have neoantigen-specific T cell responses. Among the 34 recruited patients, 19 were administered atezolizumab And 16 got the vaccination, with no significant adverse effects reported. 50% of the vaccinated patients exhibited strong de novo neoantigen-specific T cell responses, validated by IFNγ ELISpot tests and the innovative CloneTrack technique. The responses comprised polyfunctional, cytotoxic CD^8⁺ T cells that exhibited long-term persistence and clonal re-expansion following stimulation. Vaccine responders exhibited significantly prolonged recurrence-free survival (RFS), without any confounding clinical or immunological disparities as compared to non-responders. Analysis indicated that vaccination responders had a greater number of clonal tumors and superior neoantigens, implying that the quality of neoantigens and tumor clonality affect immunogenicity. Notably, in one patient, vaccine-expanded T cells invaded and eradicated a suspicious liver lesion harboring tumor-specific mutations, suggesting the vaccine’s capacity to remove micrometastatic illness. The findings indicate that personalized mRNA vaccines are safe, viable, and able to elicit long-lasting, functional neoantigen-specific T cell responses that might enhance outcomes in PDAC, hence endorsing the continued advancement of this strategy alongside immune modulation [324].

A prolonged follow-up of a phase I trial examines the enduring clinical And immunological impacts of autogene cevumeran, a customized uridine-based mRNA vaccine encoding up to 20 neoantigens, administered alongside anti-PD-L1 (atezolizumab) And mFOLFIRINOX chemotherapy in patients with resected PDAC. At a median follow-up of 3.2 years, vaccine-induced neoantigen-specific CD^8⁺ T cell responses are Substantially associated with extended RFS, with responders exhibiting a median RFS that has not been achieved, compared to 13.4 months in non-responders. Employing CloneTrack, researchers identify 79 CD^8⁺ T cell clones generated de novo by the vaccine, predominantly undetected in blood or tissue prior to vaccination, many of which have long-term persistence, projected lifespans extending to several years (up to 100 years), and polyfunctionality. A solitary vaccine boost significantly improves clone lifetime, with these enduring clones achieving a stable effector and tissue-resident memory-like (TRM-like) state devoid of depletion, Maintaining vigorous cytokine production And cytotoxic activity for up to 3.6 years following immunization. CloneTrack clones demonstrate phenotypic and functional stability over time and possess great avidity for their corresponding neoepitopes. In two instances of recurrence, diminished or postponed T cell responses and abbreviated clone lifetime correlate with disease relapse, and vaccine-induced T cells are observed infiltrating recurrent tumors. Clonal development of recurring cancers demonstrates immune editing through the elimination of immunogenic neoantigen-bearing clones, indicating vaccine-induced immunological pressure. Although there is little evidence of neoantigen dissemination, the vaccine stimulates naïve T cells to recognize immunogenic neoantigens instead of amplifying existing responses, highlighting its ability to surmount immunological tolerance in a malignancy with minimal mutational load, such as PDAC. This study demonstrates the viability of generating long-lived, functional neoantigen-specific CD^8⁺ T cells by an mRNA vaccination in an adjuvant context, underscoring the promise of autogene cevumeran in postponing PDAC recurrence and providing a framework for forthcoming neoantigen-targeted immunotherapies [325].

A research assesses the longevity of immunity conferred by COVID-19 vaccines and the effectiveness of booster doses in cancer patients. After standard vaccination, the Majority of patients with solid tumors exhibited strong Anti-spike IgG responses, while around 20% of patients with hematologic malignancies, especially those who underwent anti-CD20 therapy, CAR T cell therapy, or stem cell Transplantation, demonstrated diminished seroconversion. Antibody titers diminished considerably after a period of 4 to 6 months, while the Majority of patients continued to test seropositive. In a booster vaccination cohort, 56% of originally seronegative patients achieved seroconversion, with T cell responses seen in several individuals who remained seronegative, indicating that supplementary vaccine doses can restore or augment immunity. Seroconversion rates were decreased in patients with B cell malignancies, those who recently had anti-CD20 treatment, or those receiving Bruton Tyrosine Kinase inhibitor therapy. The results emphasize the significance of booster immunizations in cancer patients, especially those with hematologic malignancies, while highlighting the necessity for continuous measures, including passive immunization, for patients who do not achieve a serological response [326].

Researchers assessed vaccine safety And immunogenicity, particularly seroconversion And spike IgG Antibody titers, in a trial including 233 cancer patients who completed an FDA-authorized COVID-19 immunization regimen. In the immunogenicity investigation of 200 individuals, the overall seroconversion rate was high at 94%, but considerably lower in patients with hematologic malignancies (85%) compared to those with solid tumors (98%). Antibody titers were maximal in individuals administered the mRNA-1273 vaccination, followed by BNT162b2, with the Ad26.COV2.S vaccine exhibiting the lowest titers. Patients with solid tumors exhibited significantly elevated titers compared to those with hematologic malignancies, a disparity that persisted after controlling for the duration after immunization. Immunosuppressive treatments, such as anti-CD20 antibodies, stem cell transplantation, and CAR-T cell therapy, were significantly correlated with diminished seroconversion rates and lowered antibody titers. Conversely, medicines such as hormone therapy and immune checkpoint inhibitors exhibited elevated seroconversion rates. No substantial correlations were seen between seroconversion and age, ethnicity, steroid use, or the timing of cancer treatment in relation to immunization. Patients with a history of COVID-19 infection demonstrated significantly elevated antibody titers following vaccination, underscoring the advantages of hybrid immunity. The research confirmed the general safety of vaccinations, with most side effects being moderate. These findings emphasize the effectiveness of COVID-19 vaccinations in cancer patients, while underscoring the necessity for customized immunization regimens for immunocompromised populations, particularly individuals with hematologic malignancies and those undergoing treatments that compromise B-cell function [327].

Checkpoint inhibitors constitute a conventional adjuvant therapy for individuals with resected stage IIB–IV melanoma; Yet recurrence is prevalent. This phase 2b open-label, randomized study assessed the efficacy of mRNA-4157 (V940), a personalized mRNA-based neoantigen vaccine, in conjunction with pembrolizumab, compared to pembrolizumab monotherapy in patients with fully resected high-risk cutaneous melanoma (stage IIIB–IV). A total of 157 patients from the USA And Australia were randomly allocated in a 2:1 ratio to receive either mRNA-4157 in conjunction with pembrolizumab (n = 107) or pembrolizumab alone (n = 50). mRNA-4157 was administered intramuscularly (up to 9 doses) and pembrolizumab intravenously (up to 18 doses) at 3-week intervals. At a median follow-up of 23–24 months, the combination treatment cohort exhibited prolonged RFS (HR 0.561, 95% CI 0.309–1.017; p = 0.053) and reduced rates of recurrence or mortality (22% vs. 40%). The 18-month RFS rate was significantly elevated with the combination treatment (79%) in contrast to monotherapy (62%). The safety profile was acceptable, with most adverse events classified as mild to moderate (grade 1–2); grade ≥ 3 treatment-related adverse events were observed in 25% of the combination group compared to 18% in the monotherapy group, And no grade 4–5 occurrences were associated with mRNA-4157. Immune-mediated adverse events manifested at an equivalent prevalence in both cohorts (36%). The incorporation of mRNA-4157 with pembrolizumab exhibited significant enhancement in recurrence-free survival, underscoring the promise of tailored mRNA-based neoantigen vaccines as efficacious adjuvant treatment for high-risk melanoma [328].

mRNA-4157 is a tailored, mRNA-based neoantigen treatment intended to enhance a patient’s antitumor T-cell responses by focusing on specific tumor mutations. In the KEYNOTE-942 phase 2 study, patients with fully resected high-risk stage IIIB–IV cutaneous melanoma were randomized in a 2:1 ratio to receive either mRNA-4157 (up to 9 intramuscular doses) in conjunction with pembrolizumab (up to 18 intravenous cycles) or pembrolizumab monotherapy. The preliminary research with a median follow-up of 23 months indicated a substantial enhancement in RFS and distant metastasis-free survival (DMFS) in the combination group. This revised research, with a median follow-up of around 35 months, corroborated And expanded upon these findings. The combined treatment decreased the risk of recurrence or mortality by 49% (HR 0.510; p = 0.019), Yielding a 2.5-year RFS rate of 74.8% versus 55.6% for pembrolizumab monotherapy. DMFS shown significant enhancement (HR 0.384; p = 0.0154), while OS demonstrated a positive trend, with 2.5-year rates of 96.0% for the combined treatment compared to 90.2% for monotherapy. The RFS advantage of the combination was uniform across biomarker-defined subgroups, encompassing both TMB-high and TMB-non-high, PD-L1-positive and negative, as well as ctDNA-negative individuals. Heterozygosity at HLA class I loci was correlated with enhanced RFS in the pembrolizumab-only group, but not in the combination group, indicating that the inclusion of mRNA-4157 may alleviate genetic constraints on immunological response. No novel safety issues arose, and there was no increase in immune-related adverse events associated with the combination. This prolonged follow-up substantiates the sustained therapeutic advantage and acceptable safety profile of mRNA-4157 in conjunction with pembrolizumab in surgically excised high-risk melanoma, reinforcing its potential to enhance the efficacy of adjuvant immunotherapy [329].

In the phase 2 KEYNOTE-942 trial (NCT03897881), the combination of mRNA-4157, a personalized mRNA-based neoantigen vaccine, with pembrolizumab significantly enhanced RFS and DMFS relative to pembrolizumab monotherapy in patients with resected high-risk stage IIIB–IV melanoma. This investigation examined the significance of minimum residual disease (MRD), assessed using circulating tumor DNA (ctDNA), in forecasting treatment outcomes and investigated the impact of BRAF mutant status. ctDNA levels were longitudinally evaluated during and after therapy using the RaDaR^® test. Of the 157 study patients, 142 were assessable for ctDNA dynamics, comprising 100 who received combination treatment And 42 who received pembrolizumab monotherapy. Patients were designated as molecular responders (MRs) if ctDNA levels were undetectable throughout therapy or exhibited fluctuations before resolution, and as molecular non-responders (MNRs) if ctDNA remained detectable or deteriorated. Recurrence was observed in 57% of MRs And 94% of MNRs, with MNRs exhibiting a higher propensity for distant metastases, whilst MRs encountered a greater incidence of local recurrences. This tendency was seen in a more significant distinction in DMFS compared to RFS across the groups. Analysis based on BRAF mutation status revealed a more pronounced therapeutic advantage of mRNA-4157 combined with pembrolizumab in BRAF-mutant patients (HR 0.332) relative to wild-type patients (HR 0.808). However, in ctDNA-negative patients, treatment efficacy appeared more uniform across both BRAF wild-type and mutant subgroups. These findings emphasize the prognostic significance of ctDNA dynamics in assessing treatment response and recurrence risk, and they highlight the potential of ctDNA to inform future treatment options in resected high-risk melanoma [330].

mRNA-4157 (V940) is a personalized neoantigen treatment intended to target as Many as 34 patient-specific tumor neoantigens, with the objective of eliciting T-cell responses and augmenting anticancer efficacy. Mechanistic insights into the immunogenicity of mRNA-4157 were assessed through T-cell response characterization in the first-in-human, phase 1 KEYNOTE-603 study (NCT03313778), which included patients with resected NSCLC (Part A: 1 mg mRNA-4157, n = 4) or resected cutaneous melanoma (Part D: 1 mg mRNA-4157 in conjunction with 200 mg pembrolizumab, n = 12). The research evaluated safety, tolerability, and immunogenicity. All participants had at Least one treatment-emergent adverse event; However, no grade 4 or 5 occurrences or dose-limiting toxicities were documented. mRNA-4157 monotherapy reliably elicited de novo and augmented preexisting T-cell responses to the targeted neoantigens. The medication, in conjunction with pembrolizumab, resulted in prolonged neoantigen-specific T-cell responses and enhanced proliferation of cytotoxic CD⁸ and CD⁴ T cells [331].

mRNA-4157, a lipid-encapsulated personalized vaccine that encodes various neoantigens identified through a proprietary algorithm, was assessed as adjuvant monotherapy in patients with resected solid tumors (including melanoma, bladder carcinoma, HPV-negative HNSCC, NSCLC, SCLC, MSI-High, or TMB-High cancers) and in conjunction with pembrolizumab in patients with advanced or metastatic disease. Patients had a maximum of nine cycles of mRNA-4157 (0.04–1 mg, administered intramuscularly) at three-week intervals. In the combination arm, pembrolizumab (200 mg) was delivered for two cycles prior to mRNA-4157 and may be maintained Subsequently. A total of 33 individuals received treatment: 13 with monotherapy And 20 with combination therapy. No dose-limiting effects were detected. The treatment-related adverse events were predominantly mild and transitory, with no significant adverse events or grade ≥ 3 occurrences associated with the medication. Of the patients undergoing adjuvant monotherapy (3 with melanoma, 8 with NSCLC, And 2 with MSI-High), 12 were disease-free at a median follow-up of 8 months. Within the combination cohort (1 TMB-High, 4 bladder, 2 HNSCC, 1 melanoma, 7 NSCLC, 2 SCLC, 3 MSI-High), 12 individuals had previously had progression on checkpoint inhibitors. Among the 16 patients that underwent restaging, there was 1 full response (noted during pembrolizumab treatment prior to vaccination), 2 partial responses, 5 instances of stable illness maintained for a Minimum of five combo cycles, 5 cases of progressive disease, 2 instances of indeterminate/unconfirmed progressive disease, And 1 non-evaluable patient. Neoantigen-specific T cell responses were identified in peripheral blood mononuclear cells by IFN-γ ELISpot assay. mRNA-4157 demonstrated safety And tolerability at all administered dosages. Clinical responses observed with pembrolizumab, including the activation of neoantigen-specific T cells, warrant additional investigation in phase 2 studies [332].

MTL-CEBPA, the inaugural saRNA to undergo clinical trials, targets the transcription factor C/EBPα, a crucial regulator of myeloid cell development. There was a positive safety profile for MTL-CEBPA delivered as a monotherapy at a dosage of 130 mg/m² QWx3 every 28 days in patients with HCC. After the cessation of MTL-CEBPA, three of five patients subsequently administered sorafenib outside the trial sustained a complete radiological response (CR) for a duration of 7 to 18 months, with two patients demonstrating complete clearance of lung metastases for more than one Year. This study presents updated Outcomes from phase 1 patients administered sorafenib outside of the trial, together with supplementary combination cohorts. MTL-CEBPA (130 mg/m² QWx3 or BIW) and sorafenib (400 mg bi-daily) were delivered through both combination and sequential regimens in cohorts that were either naïve to tyrosine kinase inhibitors (TKIs) or resistant. Liver biopsies obtained during therapy were examined for variations in M2 macrophages (CD163), while peripheral blood samples were evaluated using flow cytometry for changes in myeloid cell populations. A total of 26 patients (23 Male, 3 female; median age 65.5 Years, range 44–83; ECOG PS 0/1: 18/8) had therapy, 12 got concurrent MTL-CEBPA And sorafenib, whereas 14 were administered medication sequentially. The frequently noted treatment-related adverse effects (AEs), including all grades And grade 3, included face flushing (4/0), raised AST (3/1), elevated ALT (2/1), tiredness (5/0), increased ALP (2/0), anemia (2/2), diarrhea (3/0), rash (2/0), and anorexia (1/0). One TKI-naïve patient in the combination group Sustained a Full response for 7 months, But two patients in the sequential cohort had stable disease after 3 And 4 months, respectively. Immunohistochemistry (IHC) in the patient exhibiting a Full response demonstrated a 95% drop in M2 macrophages, alongside substantial declines in immature CD10- neutrophils (−85.7%; p = 0.0078), PMN-MDSCs (−49.3%; p = 0.00145), and M-MDSCs (−18.4%; p = 0.0072). All individuals demonstrating a clinical response had underlying HBV or HCV infections. The findings indicate that MTL-CEBPA, an innovative saRNA that modulates myeloid cell populations, may substantially improve anti-tumor responses in virally associated HCC [333].

Challenges and limitations

The specificity of RNA therapies is crucial, as their effectiveness depends on strong on-target activity with minimal off-target or unintended on-target effects; however, several challenges persist despite notable advancements. Chemical modifications, including second-generation and third generation LNA designs, have enhanced potency and specificity; however, siRNAs may still downregulate off-target genes via partial complementarity, and improper RISC loading can result in passenger strand activity. This necessitates strategies such as passenger strand modifications or Dicer-substrate siRNA designs to improve guide-strand precision. Protein interactions also lead to off-target effects, shown by phosphorothioate-modified ASOs such as oblimersen, which unintentionally associate with mitochondrial proteins and induce death in non-target cells. Moreover, systemically delivered RNA therapeutics may concentrate in non-tumor cells, such as leukocytes, possibly modifying immunological or neurological functioning, as seen in trials with MRX34 or AEG35156. Dosing complicates specificity, as excessive siRNA or shRNA can saturate the RNAi machinery, disrupt endogenous miRNA regulation, and result in extensive gene expression alterations. Additionally, the intricate combinatorial nature of miRNA-mRNA interactions renders the concentration of mimics or inhibitors a crucial yet poorly comprehended factor affecting their “targetome.” Consequently, meticulously refining chemical design, delivery mechanisms, and dosage protocols while examining the intricate impacts of miRNA or siRNA concentrations on gene networks will be crucial for surmounting specificity challenges and completely harnessing the therapeutic potential of RNA-based cancer therapies [334]. A primary constraint of RNA treatments is the significant delivery obstacles presented by biological barriers that have developed over billions of years to safeguard cells from exogenous RNA molecules. Although tiny, neutral molecules may permeate lipid bilayers, RNA therapeutics, such as siRNAs, ASOs, and CRISPR components, are substantial, charged macromolecules that cannot traverse these membranes independently and are instead internalized by endocytosis, subsequently being sequestered within endosomes. The issue is exacerbated by evolutionary defences such as RNases, toll-like receptors, and the rapid elimination by the kidneys and liver, which further diminish bioavailability. Notwithstanding progress in stabilizing RNA molecules and augmenting their on-target efficacy, the primary problem is still in developing safe and highly effective strategies for endosomal escape to deliver RNA therapies into the cytoplasm for functional action. Current methodologies, like LNPs and GalNAc conjugates, demonstrate efficacy primarily in hepatocytes, leaving the distribution to extra-hepatic tissues and systemic applications unaddressed. The necessity for non-toxic, highly effective endosomal escape agents, enhanced targeting systems, and methods to prolong RNA therapeutic circulation in the bloodstream is essential for surmounting these biological obstacles and achieving the complete clinical potential of RNA-based therapies [335].

The quantitative assessment of RNAi effector molecules, including siRNA and shRNA, is crucial for the progression of RNAi-based treatments, as it evaluates delivery efficiency and pharmacokinetics, hence serving as a vital element in both preclinical and clinical research. Among various bioanalytical techniques Such as quantitative RT-PCR, hybridization assays, HPLC, And LC-MS stem-loop quantitative RT-PCR has become the favored method due to its Superior sensitivity, specificity, extensive dynamic range, And capacity to detect small RNAs as short as 19 bases, rendering it optimal for evaluating the biodistribution and kinetics of RNAi therapeutics. In a study utilizing LNPs for systemic delivery, chemically stabilized siRNA was identified in the Liver, spleen, And kidney within 0.5 h, exhibiting decreasing Levels over 24 h, with negligible presence in other organs, whereas synthetic miR-124 Mimics administered through neutral lipid emulsion demonstrated significant accumulation in the Lung. Moreover, comprehending the molecular mechanism of RNAi-mediated gene silencing is essential, And the 5′ RACE-PCR test has emerged as a standard method for identifying siRNA-induced cleavage products across many systems, albeit it does not facilitate the quantification of cleavage levels. RNA sequencing is poised to be a valuable future technique, enabling concurrent investigation of transcript identification and abundance, hence advancing the comprehension and optimization of RNAi therapies [336].

Preliminary first-in-human studies of systemically given siRNA-based anticancer medicines have shown that these treatments may be safely provided to people, representing a significant milestone in RNAi-based medication development. These preliminary studies have yielded significant data and established a foundation for enhanced formulations and trial designs, especially by connecting human and animal data to better prediction models. siRNA molecules successfully activate the RNAi pathway at both RISC and Dicer; nevertheless, heterogeneity in Dicer expression across cancer types may affect clinical outcomes. In addition to siRNAs, therapeutic RNAs, including miRNAs and perhaps CRISPR-based RNAs, are also undergoing clinical trials. Nanoparticle-based delivery methods are pivotal to siRNA treatment, with an increasing comprehension of their biodistribution and clearance in humans; uniform and biodegradable formulations provide potential safety and effectiveness benefits, although introducing regulatory complexities. Tumor vasculature and nanoparticle trafficking provide significant problems, as delivery to and within tumors differs significantly across animal models and people. The uneven distribution of nanoparticles in tumors indicates that siRNA therapy could depend on bystander effects, potentially facilitated by exosome-mediated intercellular communication. Future nanoparticles are anticipated to be multifunctional, transporting numerous siRNAs and targeting agents to improve effectiveness. Significantly, preliminary clinical studies lacked patient genetic pre-screening, so constraining the evaluation of pharmacodynamics and effectiveness; further trials ought to stratify patients based on tumor genetics to enhance the assessment of siRNA effects. Target selection is essential; siRNAs should effectively silence genes that induce cancer cell apoptosis, such as EGFR and NRAS, with the latter being especially promising in NRAS-mutant melanoma, where therapy options are limited. Despite current delivery problems, these preliminary data affirm the potential of RNAi treatments and establish a basis for more focused and effective clinical research [337].

Conclusion and perspectives

The advancement of RNA therapeutics, encompassing ASOs and siRNAs, has significant potential as a novel category of cancer therapies, enabling the targeting of untreatable cancer drivers. Innovations such as next-generation ASO chemistries (cEt-containing ASOs) have significantly enhanced efficacy, particularly in difficult-to-access tissues, and provide safety benefits by eliminating the necessity for nanoparticle delivery. Nonetheless, significant hurdles persist, including inadequate absorption, distribution, metabolism, and excretion (ADME) characteristics, restricted tumor growth, and obstacles to intracellular delivery. Advancements in the targeted delivery systems akin to the efficacy of GalNAc conjugation for liver-targeted therapies may revolutionize cancer treatments. Ongoing innovation in RNA therapies is anticipated to be pivotal in personalized cancer treatment, utilizing patient-specific genetic profiles to inform the selection of customized RNA medications that address resistance mechanisms and enhance clinical results [338]. Following more than twenty years of advancement, RNA therapeutics has materialized as a clinical reality, with enhancements in the design and chemistry of siRNAs, ASOs, and mRNAs attaining adequate stability, immune evasion, and therapeutic efficacy, while delivery technologies have progressed through effective, biocompatible materials and high-throughput screening. Notwithstanding several setbacks, such as the cessation of Alnylam’s siRNA–GalNAc conjugate and CureVac’s mRNA vaccination studies, the field persists in its advancement, evidenced by FDA approval of many ASOs and a plethora of RNA-based therapeutics with improved modifications advancing through late-stage trials. The emergence of CRISPR–Cas genome editing has accelerated advancements in RNA delivery, with firms such as CRISPR Therapeutics, Editas Medicine, and Intellia Therapeutics creating sophisticated preclinical programs aimed at the liver, lung, and hematopoietic systems through ex vivo and in vivo methodologies, including AAVs, LNPs, and RNPs. Nonetheless, safety and delivery continue to pose significant problems, particularly for RNA–protein and CRISPR-based treatments and will influence the trajectory of forthcoming clinical trials. The sector is seeing swift development, propelled by the potential of RNA therapeutics for personalized medicine, immunotherapy, and the treatment of hereditary, viral, and chronic ailments, guaranteeing its ongoing advancement in the future [339]. RNAi has developed into a viable therapeutic approach for addressing numerous pathological disorders, with significant progress achieved in gene targeting and delivery technologies. Improvements have significantly increased the efficiency of gene silencing and the efficacy of delivery strategies throughout time. Notwithstanding these advancements, several challenges continue to impede the effective implementation of RNAi therapeutics in clinical settings. Safety concerns, especially about the activation of the innate immune system by RNAi drugs and their nanocarriers, have resulted in the cessation of specific clinical trials. To achieve efficient and precise gene silencing with reduced toxicity in vivo, it is crucial to create appropriately engineered delivery systems. Future research endeavours will likely focus on addressing these obstacles, particularly through the utilization of tissue-specific ligands that enhance stability and binding affinity to optimize targeting. Current insights into RNAi processes and delivery developments suggest that these innovations will usher in a new age of molecular therapeutics, providing safer, more efficient, and tailored therapy alternatives for patients [340].

RNA therapeutics have progressed swiftly due to chemical modifications that improve RNA stability, safeguard against enzymatic degradation, and facilitate more efficient delivery systems such as GalNAc conjugates. Additionally, the advent of bioengineered RNA agents provides even more resilient and biologically authentic therapeutic alternatives. Notwithstanding advancements, obstacles persist, especially concerning the possible detrimental impacts of chemical alterations on RNA activity, the necessity to target inaccessible tissues such as leukocytes, and surmounting the significant impediment of endosomal escape. Although liver- and kidney-targeted therapies have been predominant, future objectives should emphasize extrahepatic delivery utilizing sophisticated carriers such as LNPs or dendrimers modified with targeting moieties like peptides or antibodies, which can enhance specificity, internalization, and therapeutic efficacy. These tactics are particularly crucial for illnesses like as cancer, autoimmune disorders, or chronic leukocyte-related ailments, where accurate targeting may diminish costs and enhance therapeutic efficacy, guaranteeing that the RNA revolution transitions from ambition to broad practical implementation [341]. RNAi is regarded as a promising therapeutic strategy because of its ability to specifically silence genes linked with diseases; yet its practical use has been obstructed by many physiological and technical challenges. The effective distribution of naked siRNA is hindered by its instability in circulation, immunological detection, inadequate tissue penetration, and intracellular destruction. Nanoparticle-based carriers have been utilized to mitigate these challenges; however, they may elicit immunological responses or toxicity contingent upon their composition and surface charge. Off-target effects, RISC oversaturation, and heterogeneous RNAi processing across many tissues exacerbate treatment complexities. Moreover, resistance mechanisms, erratic pharmacokinetics, and difficulties in monitoring administration and response have hindered reliable treatment results. Notwithstanding these constraints, progress in nanoparticle design, targeting methodologies, and computer modeling indicates that several challenges may be surmounted via ongoing innovation [342].

A modular, non-cationic nanoparticle approach was developed to provide effective and targeted siRNA administration, while reducing toxicity and addressing challenges such as low tumor accumulation, serum breakdown, and inadequate endosomal escape. The nanoparticle, composed of an octameric ribonucleoprotein scaffold and endosomolytic peptides, was functionalized with targeting ligands and siRNAs, yielding a slightly negative charge that improved tumor penetration and biocompatibility. Gene silencing was accomplished in PCa models, with notable tumor growth inhibition occurring solely when all functional elements namely the targeting ligand, disulfide linker, and peptide were included. High specificity, little immune response, and prolonged tumor retention were evidenced; nonetheless, issues concerning scalability, off-target effects, and manufacturing complexity persist and require resolution [343].

RNAi-based therapies hold significant promise for treating cancer, particularly due to their potential to target disease at the molecular level. However, a major hurdle in cancer treatment remains the effective targeting of metastatic cells that have migrated from the primary tumor site. These metastatic cells often develop resistance to conventional chemotherapy, leading to disease relapse. It is still uncertain whether siRNAs can be successfully delivered to these dispersed cancer cells or whether viable molecular targets can be identified to eliminate them. Beyond cancer, neurodegenerative disorders like Huntington’s disease and ALS are also compelling candidates for RNAi treatment, given the current lack of effective therapies. In these cases, the central challenge lies in delivering RNAi agents specifically to affected neurons. To date, siRNA carriers have not demonstrated the ability to cross the blood–brain barrier, which limits their therapeutic reach. While experimental studies in animals have employed direct brain injections to bypass this barrier, this invasive technique is unlikely to be well-received for use in humans, especially considering the potential need for repeated administrations. However, the use of viral vectors to deliver shRNAs could offer a more sustainable solution by reducing the need for frequent dosing [344].

Although RNA therapy has been significantly beneficial in the treatment of diseases And both pre-clinical And clinical studies have provided promising results, it should be noted that some of them have faced also challenges And failure. An example is the case of Revusiran. On October 4, 2016, Alnylam Pharmaceuticals terminated revusiran, its RNAi therapeutic for Transthyretin amyloidosis with cardiomyopathy, following 19 patient fatalities in a phase 3 trial,, 17 in the treatment cohort compared to 2 in the placebo group,, resulting in a $3.6 billion decline in market capitalization and investor doubt regarding the RNAi platform. The precise explanation is uncertain; however, possible contributing variables including baseline patient variations, medication toxicity, either off-target or on-target, and difficulties connected to delivery. Although revusiran necessitated elevated dosages, Alnylam’s novel, more efficacious ESC-GalNAc conjugates may mitigate exposure-associated hazards. Nonetheless, recent toxicity seen with another ESC agent warrants caution. Analysts perceive the occurrence as a maturation point for RNAi rather than a fundamental failure, highlighting the necessity for indication-specific optimization in RNAi medication development [345]. Although this was an example of non-cancerous disease, it can provide insights that such failures can happen in the treatment of cancer using RNA therapeutics. The failure of RNA therapy for cancer treatment in patients can stem from a complex interplay of biological, delivery-related, and tumor-specific factors that undermine its effectiveness despite promising preclinical results. One of the primary challenges is the instability of RNA molecules in the bloodstream, as they are rapidly degraded by nucleases unless chemically modified or protected by specialized delivery systems like LNPs, which themselves may cause immune reactions or fail to penetrate solid tumors efficiently. Moreover, the TME often poses a physical and immunological barrier, hindering the uptake and activity of RNA-based therapeutics within the malignant cells. Heterogeneity among tumor cells, both genetically and epigenetically, can lead to differential responses, with some cells evading therapy through alternative signaling pathways or intrinsic resistance mechanisms. Additionally, RNA therapies typically rely on the presence of specific molecular targets or mutations, and their absence or alteration due to tumor evolution can render the treatment ineffective. Immune-related adverse events, off-target effects, and patient-specific differences in metabolism or immune status may further complicate outcomes, leading to suboptimal responses or treatment discontinuation. Altogether, while RNA therapy holds great potential, these multifaceted obstacles contribute to its clinical failure in certain cancer patients, necessitating improved targeting strategies, personalized approaches, and combination therapies to enhance efficacy and overcome resistance.

The utilization of RNA nanotechnology in medicine encounters several significant hurdles that must be resolved to achieve its complete therapeutic potential. A significant challenge is the precise prediction and assurance of the proper global folding of RNA constructs, which is crucial for preserving the structural integrity and functioning of RNA nanoparticles. Although tools such as Mfold, Sfold, RNA Designer, and NUPACK offer significant support, their emphasis on two-dimensional structures constrains forecast precision (~ 70%), especially in the context of intricate tertiary or quaternary structures, non-canonical base pairing, and intermolecular interactions. This highlights the necessity for more advanced software capable of simulating RNA folding in three-dimensional and even four-dimensional space. Another problem is improving the in vivo stability of RNA nanoparticles, not just increasing it, since excessively stable RNAs may have poor pharmacokinetics or inefficient therapeutic windows due to quick elimination or inadequate absorption. This necessitates meticulous regulation of the integration of chemically altered nucleotides to optimize RNA stability and functionality in specific tissues. Moreover, effective endosomal escape continues to be a limitation in ligand-mediated delivery methods for RNA therapies. Multivalent RNA nanoparticles can be co-formulated with acid-responsive linkers (hydrazones, acetals), protonatable groups (imidazoles, sulfonamides), or cell-penetrating peptides that utilize the proton sponge effect to deliver cargo into the cytoplasm. Ultimately, large-scale RNA manufacturing remains impeded by poor yields and elevated costs, although progress in bacterial fermentation systems employing tRNA scaffolds. Additional enhancements are required in the design of expression vectors and host strains to optimize RNA yield, facilitate modified nucleotide absorption, and inhibit nucleolytic degradation. Advancements in computational design, chemical stability, intracellular trafficking, and biomanufacturing will be crucial for converting RNA nanotechnology into effective therapeutic medicines [346].

The implementation of RNAi as a viable in vivo cancer treatment encounters several biological and technical obstacles, especially concerning transport, effectiveness, selectivity, immune activation, off-target effects, and resistance. siRNAs, regardless of delivery method, direct administration, plasmid introduction, or viral vector utilization, experience brief half-lives, variable cellular absorption, swift extracellular breakdown, and dilution in rapidly proliferating cells, hence constraining their prolonged gene silencing efficacy. Chemical modifications and encapsulation techniques, including LNPs, antibody conjugates, fusogenic peptides, and viral vectors, have enhanced RNAi stability and delivery specificity, however sometimes entail compromises in immunogenicity or toxicity. Viral vectors can facilitate prolonged expression but may exhibit insufficient cell-type selectivity and provide safety concerns. Notwithstanding progress in promoter designs and delivery mechanisms, sustained expression and selective absorption continue to pose challenges. Moreover, elevated levels of siRNA may elicit nonspecific immunological responses via interferon signaling or toll-like receptor activation, hence increasing apprehensions regarding inflammation and possible autoimmune repercussions. Off-target gene silencing remains a concern, since siRNAs with partial complementarity might inadvertently downregulate non-target mRNAs, while endogenous siRNAs processed by Dicer and RISC may exhibit less susceptibility to these inaccuracies. Computational techniques now facilitate the creation of more precise sequences; yet, resistance persists as a challenge, stemming from mutations in target locations, inaccessible RNA sections, or the targeting of long-lived proteins. Moreover, mutations in RNAi machinery, such as RISC, might hinder treatment efficacy. To surmount these obstacles, forthcoming techniques must include enhanced vector systems, stringent sequence selection, multi-target methodologies, and optimized formulations to improve delivery, reduce unintended consequences, and tackle the intricate and adaptable characteristics of cancer cells [347].

Notwithstanding significant progress in RNA therapies, their clinical efficacy is constrained by enduring obstacles, chiefly with delivery inefficiency, off-target effects, restricted tumor penetration, and hybridization-associated toxicities. Unprotected RNA molecules, including ASOs and miRNAs, often aggregate in clearance organs such as the liver and kidneys, resulting in diminished bioavailability and inadequate distribution. The diverse TME exacerbates the challenges of medication distribution and absorption. To resolve these challenges, several techniques have been developed, including chemical changes and nonviral carriers such as cationic peptides, lipids, polymers, antibodies, and nanoparticles, designed to enhance stability and cellular penetration. Nonetheless, these alterations may occasionally diminish target affinity and elicit unwanted immunological or coagulation reactions. Hybridization-dependent toxicity and off-target effects, exemplified by unintentional gene silencing observed in medications such as LY2275796, present considerable problems. Furthermore, the intricacy of cancer, characterized by repetitive and diverse genetic alterations, often makes single-target therapy inadequate, resulting in often transient responses. This highlights the justification for integrating RNA-based therapeutics with other modalities like as chemotherapy or radiation to improve effectiveness and mitigate resistance. Despite the presence of various RNA therapeutics in clinical trials and the promising results of some combinations, a substantial therapeutic advancement in cancer therapy utilizing antisense techniques remains anticipated. Nevertheless, continuous innovation and combinatorial strategies provide measured optimism for the future [348]. RNA therapeutics, despite its potential for accuracy and flexibility, encounter considerable obstacles concerning manufacturing scalability, regulatory problems, and cost-effectiveness. Enhancing RNA production to satisfy clinical and commercial requirements necessitates specialized facilities, rigorous quality control, and cold-chain logistics, particularly for LNP-encapsulated mRNA formulations, which are prone to degradation. These requirements augment manufacturing complexity and constrain the capacity for swift supply expansion. In the regulatory domain, RNA-based treatments, especially novel modalities such as saRNA or circular RNA, frequently lack well-defined routes, necessitating that developers traverse shifting standards and provide comprehensive safety and effectiveness data. This engenders ambiguity and may extend development schedules. Cost is a key obstacle: although the fundamental synthesis of RNA may be more economical than protein biologics, the requirement for sophisticated delivery methods, high-purity reagents, and stringent testing considerably increases the total expenditure. The characteristics collectively provide significant challenges to the extensive acceptance and commercialization of RNA therapeutics, necessitating synchronized innovation in technology, policy, and economics to realize their complete potential.

In addition to their therapeutic potential, RNA-based treatments offer notable advantages in terms of scalability, adaptability, and cost-effectiveness, which are especially relevant for low- and middle-income countries (LMICs). Unlike traditional biologics or small-molecule drugs that often require complex manufacturing and distribution infrastructures, RNA therapeutics can be synthesized rapidly and tailored to specific targets with relatively low production costs. This plasticity makes them attractive candidates for addressing the growing cancer burden in LMICs, where access to advanced therapies is often limited by economic and logistical constraints. As such, the development of RNA therapeutics holds promise not only for innovation in oncology but also for promoting greater global health equity.

The integration of artificial intelligence technologies, such as AI-driven target discovery and in vivo CRISPR screening, is poised to significantly accelerate and refine the development of RNA-based cancer therapeutics. AI-driven target discovery allows for the high-throughput identification of novel oncogenic drivers, noncoding RNA elements, and patient-specific mutational signatures by leveraging multi-omics data, thereby enabling the rational design of RNA therapeutics, including siRNAs, miRNAs, and mRNA vaccines, with enhanced specificity and therapeutic relevance. Simultaneously, in vivo CRISPR screens are increasingly being used to functionally validate these AI-predicted targets within physiologically relevant TMEs, including immune-competent models, offering insights into essential genes, resistance mechanisms, and synthetic lethality interactions. For instance, RNA-based therapeutics targeting the PELO–HBS1L axis in SKI-deficient tumors exemplify how CRISPR screens can uncover vulnerabilities that are exploitable with precision RNA modalities. These technologies also support the optimization of delivery strategies by identifying genes that regulate nanoparticle uptake or endosomal escape. Moreover, AI can streamline the customization of personalized mRNA cancer vaccines by matching tumor neoantigens to a patient’s HLA profile, as seen in KRAS G12V-targeted therapies. The convergence of AI and functional genomics is therefore transforming the landscape of RNA-based oncology, not only by accelerating discovery pipelines but also by enabling the real-time adaptation of therapeutic strategies based on tumor evolution and immune dynamics, ultimately fostering a new era of precision, efficacy, and scalability in cancer treatment.

RNA therapeutics for cancer therapy, while promising and innovative, are associated with several potential toxicities that may arise in patients due to the nature of the molecules, delivery systems, and their interactions with host biology. One major concern is off-target effects, where siRNAs, miRNAs, or mRNA-based therapies may unintentionally silence or alter the expression of non-target genes, leading to unintended cellular dysfunction. Immunogenicity is another significant issue, as synthetic RNA molecules and their delivery vehicles (LNPs or viral vectors) can activate innate immune responses through pattern recognition receptors such as Toll-like receptors (TLRs), RIG-I-like receptors, and others, causing cytokine release, inflammation, or systemic immune activation that could exacerbate disease or lead to autoimmune reactions. Additionally, some RNA molecules may induce hepatotoxicity or renal toxicity due to accumulation in or clearance through the liver and kidneys, respectively, especially with repeated dosing. The use of chemical modifications to increase RNA stability and reduce immunogenicity may themselves introduce new safety concerns, such as altering biodistribution or causing unanticipated toxicities. Furthermore, delivery systems like cationic lipids or polymers may damage cellular membranes, disrupt endosomal compartments, or exhibit intrinsic cytotoxicity. Altogether, while RNA-based cancer therapies hold great therapeutic potential, their clinical application requires careful assessment and mitigation of toxicity risks to ensure patient safety and treatment efficacy.

CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) are the most prevalent and possibly severe adverse effects linked to CAR T-cell treatment. CRS arises from the excessive activation of immune effector cells and a cytokine storm triggered by CAR T-cell-induced tumor pyroptosis, which stimulates macrophages and initiates a cascade of proinflammatory cytokine release (particularly IL-6, IL-1, IFN-γ, GM-CSF), endothelial activation, and systemic inflammation. ICANS, closely associated with CRS, results from the rupture of the BBB caused by cytokine-mediated endothelial activation, which leads to immune cell infiltration, stress in astrocytes and pericytes, excitotoxicity, and neuroinflammation. Management methods encompass an improved CAR structure (low-affinity scFv, 4-1BB costimulatory domains), dosage adjustment (split dosing), and the administration of anticytokine drugs such as tocilizumab and corticosteroids. Tocilizumab is beneficial for CRS but may exacerbate immune effector cell-associated neurotoxicity syndrome (ICANS) due to inadequate central nervous system penetration, whereas corticosteroids are favored for ICANS despite apprehensions over their potential to diminish CAR T-cell efficiency. Reversible and irreversible CAR “off-switches” and CAR engineering, such as GM-CSF knockdown, are being investigated to reduce toxicities while maintaining therapeutic advantages [349]. Therefore, if the RNA therapeutics want to be used for the regulation of immune system and affecting CAR-T cells, the toxicity aspect should be considered.

mRNA/LNP-based vaccines have shown effective in avoiding infectious illnesses and are under investigation for cancer therapy; nevertheless, their restricted capacity to elicit enduring immunological memory, particularly CD^8⁺ T cell memory, poses a significant obstacle. Recent research has explored the integration of microbiome-derived metabolites, such as short-chain fatty acids like butyrate, with lipid nanoparticles (mmi-LNPs or mbm-LNPs) to increase this. The modified LNPs significantly raised the production of stem cell-like, central, and effector memory T cells, as well as augmented antigen-specific CD^8⁺ T cell and B cell responses in both infectious and tumor models. Furthermore, longitudinal studies involving CLL and FL patients who received the BNT162b2 COVID-19 vaccine demonstrated that, despite undergoing immunosuppressive therapy, vaccination influenced both immune and microbiome profiles, resulting in the proliferation of memory T and NK cells and alterations in gut microbiota composition. These findings highlight the capacity of microbiome metabolite-enriched LNPs to enhance mRNA vaccination effectiveness by improving adaptive immunity and immunological memory [350–352]. RNA therapies provide potential opportunities for microbiome manipulation in cancer treatment by targeting microbial gene expression or host-microbiota interactions to alter the TME and improve immune responses. siRNAs or mRNAs administered using LNPs can be engineered to inhibit microbial virulence factors, regulate microbial metabolite synthesis, or promote advantageous microbial populations that bolster anti-tumor immunity. For instance, mRNA can facilitate the expression of enzymes or signaling molecules that enhance the synthesis of short-chain fatty acids such as butyrate, which is recognized for its role in supporting T cell memory and immunological control. Furthermore, RNA-based methodologies can modulate host genes associated with immune recognition of microbiota or barrier function, thereby affecting systemic immunity. The integration of RNA therapeutics with microbiome-targeted techniques has the potential to enhance cancer treatment results by concurrently altering the microbiota and improving the effectiveness of immunotherapies.

Authors’ contributions

Yubo Yan, Conceptualization, Writing-original draft; Shuang Liu, Conceptualization, Writing-original draft; Jie Wen, Conceptualization, Writing-original draft; Yunlong He, Conceptualization, Writing-original draft; Chenyang Duan, Conceptualization, Writing-original draft; Noushin Nabavi, Writing-review editing; Milad Ashrafizadeh, Writing-review editing; Gautam Sethi, Conceptualization, Writing-review editing; Lubin Liu, Conceptualization, Writing-review editing; Rong Ma, Conceptualization, Writing-review editing.

Funding

None.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.