Silencing HPV: the rise of RNA therapeutics in cervical cancer

Abstract

Despite the availability of preventative HPV vaccinations, cervical cancer remains a worldwide health concern. It is mostly caused by persistent infection with high-risk human papillomaviruses. Therapeutic techniques targeting the viral oncogenes E6 and E7, which are constitutively expressed in HPV-positive cervical cancers and inactivate the important tumor suppressors, p53 and Rb, offer intriguing molecular treatment options. RNA-based techniques, such as tiny interfering RNA, short hairpin RNA, antisense oligonucleotides, and mRNA-based vaccines for the selective silencing of E6/E7 genes, have emerged as leaders in targeted therapeutics. Preclinical studies have shown that RNA-mediated suppression of E6/E7 can restore p53 and Rb activity, causing apoptosis or senescence in cervical cancer cells and inhibiting tumor growth in animal models. Similarly, mRNA vaccination platforms encoding E6/E7 have been found to potently induce HPV T-cell responses and full tumor regression in animal models. RNA-based therapeutics in patients are now being evaluated in early-stage clinical studies, including novel mRNA vaccines for HPV-positive malignancies in combination with immunotherapies. While no RNA-based treatment for cervical cancer has yet achieved regulatory approval, this review summarizes the significant progress in this field that has been effective in therapies for cervical cancer using new strategies, such as advanced delivery systems, combinatorial treatments, and genome editing strategies.

Introduction

Cervical cancer is one of the most common malignancies in women worldwide, with over half a million new cases and hundreds of thousands of deaths annually [1–3]. Persistent infection with oncogenic human papillomavirus, especially types 16 and 18, is the established cause of the vast majority (~ 90%) of cervical cancers [1–4]. Prophylactic HPV vaccines, such as Gardasil and Cervarix, are highly effective in preventing new HPV infections and high-grade precancerous lesions, particularly when administered before exposure to the virus [5–7]. Cervical cancer continues to be widespread in areas with low vaccination and screening rates, as well as among individuals infected with HPV before vaccination [5]. Critically, current prophylactic vaccines prevent new infections but do not treat existing HPV infections or established diseases [8, 9]. Standard treatments, such as surgery, radiation, and cytotoxic chemotherapy, are effective but invasive and can result in substantial acute and late morbidity [10–12]. Despite advancements in immunotherapy, the prognosis for metastatic cervical cancer remains poor. Although PD-1 blockade enhances outcomes, many patients experience disease progression and limited survival [1, 12–14]. These realities underscore the need for strategies that target the root cause of HPV-driven malignancy: viral oncogenes that drive malignant transformation [15, 16]. HPV is a small double-stranded DNA virus [17]. In high-risk HPV types, oncogenesis is driven primarily by sustained expression of the early oncogenes E6 and E7, which cooperate to disable key tumor suppressor pathways and reprogram multiple host signaling networks that support proliferation, survival, and malignant progression [18]. E6 promotes ubiquitin-mediated degradation of p53, and E7 functionally inactivates the pRb pathway and other cell cycle regulators, permitting unchecked proliferation [10, 11, 19]. During carcinogenesis, viral DNA frequently integrates into the host genome, disrupting the viral E2 repressor and upregulating E6/E7 expression [10, 11, 20]. Continued E6/E7 expression is essential for both the initiation and maintenance of oncogenic addiction of the malignant phenotype in cervical epithelial cells, making E6/E7 a near-ideal, tumor-specific therapeutic target [10, 11, 20]. Molecular therapies targeting HPV oncoproteins aim to restore the normal tumor-suppressor functions of E6/E7 and abrogate or stimulate an immune response against HPV-expressing cells [21–25]. RNA-based approaches are particularly attractive because they can (І) suppress viral or host oncogenic pathways through small interfering RNA/short hairpin RNA or antisense oligonucleotides and (П) deliver immunostimulatory payloads, such as mRNA vaccines encoding HPV antigens, without permanently modifying the genome [26–31]. In contrast to small-molecule drugs or conventional chemotherapy, RNA therapeutics can be designed to specifically target viral mRNA transcripts or prompt the patient’s immune system to recognize HPV-derived peptides [32]. Early proof-of-concept studies over the past two decades using technologies such as antisense oligonucleotides and RNA interference showed that silencing E6/E7 can halt the growth of HPV-transformed cells in vitro and suppress tumor growth in vivo xenograft models [33–37]. Through this detailed discussion, we aim to illustrate how molecular therapy directed at HPV using RNA-based methods could become a paradigm shift in the treatment of cervical cancer, translating the specificity of molecular medicine into tangible clinical benefits.

Background on HPV and cervical cancer pathogenesis

HPV infection and oncogenesis

Human papillomaviruses are non-enveloped DNA viruses with a circular double-stranded genome of approximately 8 kb encoding early proteins E1, E2, E5, E6, and E7, including the E8^E2 fusion repressor and late capsid proteins L1 and L2 [38, 39]. Although the E4 ORF lies within the early region, the E1^E4 protein is predominantly expressed during the productive (late) phase and is widely used as a marker of productive HPV infection [40, 41]. Among the > 200 HPV genotypes identified, a subset of mucosal high-risk types, notably HPV16, 18, 31, 33, 45, and others, are classified as group 1 carcinogens because of their causative role in cervical and other anogenital cancers [42]. HPV16 alone is found in over half of cervical cancers globally, with HPV18 contributing to another 15% [43, 44]. High-risk HPV infection occurs in the basal layer of the cervical epithelium, typically through microabrasion. The virus initially maintains its genome as an episome, expressing early genes E1 and E2 to replicate viral DNA at a low copy number [45–49]. During multistep pathogenesis, the integration of HPV DNA into the host genome is a critical event in many cancers [50–52]. Viral integration frequently disrupts the E2 gene, a negative regulator of E6/E7 transcription, thereby unleashing the overexpression of E6/E7 in the affected cell clone [50–52]. As infected cells differentiate and migrate upwards into the epithelium, viral gene expression shifts to produce high levels of late proteins and infectious virions [42]. Invasive cervical cancer is typically preceded by a long phase of pre-invasive disease known as cervical intraepithelial neoplasia (CIN). CIN is classified into three categories: CIN1, CIN2, and CIN3, based on the severity of dysplasia, which reflects both the degree of disease severity and the proportion of abnormal cells present in the cervical epithelium [53]. CIN1, categorized as a low-grade squamous intraepithelial lesion (LSIL), involves approximately one-third of the basal epithelium and usually regresses to normal within two years after infection. In these cases, surgical treatment is not necessary, and Pap smear results often indicate LSIL. In contrast, CIN2 and CIN3 are classified as high-grade squamous intraepithelial lesions (HSIL) [54]. CIN2 represents moderate dysplasia affecting nearly two-thirds of the epithelial thickness, while CIN3 is characterized by severe dysplasia in which abnormal cells occupy the entire epithelial layer, although the basal membrane remains intact. This condition confers a significantly higher risk of progression to invasive cancer (Fig. 1) [55, 56]. Approximately 60% of CIN1 lesions regress to normal within one year; however, women with CIN2 and CIN3 require treatment due to their elevated risk of progression to invasive cancer, even though the average time for progression is typically several years [57]. In most cases, the immune system clears HPV infection within months [42]. In the cervix, HPV infection can follow a productive or transforming course, depending on the epithelial context. During productive infection, the viral genome is episomally maintained, differentiation-dependent late gene expression occurs, and virions are produced in the upper epithelium. In abortive transforming infection, the productive program is not completed, late gene expression and virion production are minimal, and E6/E7 remains persistently expressed, promoting progression to HSIL and, in some cases, causing invasive carcinoma [58]. Progression to high-grade lesions and invasive cancer is closely linked to the actions of HPV oncogenes E6 and E7 [3, 21, 59]. These oncoproteins are master drivers of cellular transformation [3, 21, 59]. E6 binds to E6-AP ubiquitin ligase, leading to the degradation of p53, which usually triggers cell cycle arrest or apoptosis in the presence of DNA damage [60]. Loss of p53 function permits the accumulation of genetic damage in host cells [61]. In parallel, E7 binds to the hypophosphorylated form of the retinoblastoma protein pRb and related pocket proteins, releasing E2F transcription factors, thereby improperly pushing the cell into the S phase [62]. E7 can also inactivate cyclin-dependent kinase inhibitors such as p21Cip1 and p27, further abrogating cell cycle checkpoints [63, 64]. Beyond cell cycle deregulation, HPV E6 and E7 also promote viral persistence and tumor immune evasion. They can attenuate innate antiviral signaling through interferon pathways, disrupt antigen processing and presentation including reduced MHC class I expression, and reshape cytokine or chemokine networks in the infected epithelium. These immunoregulatory effects provide an additional rationale for combining E6/E7-silencing strategies with therapeutic vaccination and immune checkpoint blockade [65–69]. The concerted effect of E6 and E7 is to induce genomic instability and continuous proliferation, which are key hallmarks of cancer. Notably, E6 and E7 oncoproteins can cause virtually all the classic hallmarks of cancer, including resistance to cell death, enabling replicative immortality through activation of telomerase, induction of angiogenesis, and activation of invasion and metastasis pathways [70–75]. This results in clonal expansion of cells with a growth advantage and accumulation of additional host genomic alterations over time [76–80]. By the time high-grade cervical intraepithelial neoplasia CIN3 or invasive carcinoma develops, E6 and E7 expression is uniformly retained and necessary for the survival of cancer cells. Experimental silencing of E6/E7 causes HPV-positive cancer cells to undergo growth arrest or apoptosis [76–80]. This oncogene addiction to viral gene function is unique to HPV-associated cancers [81].

Fig. 1.

Human papillomavirus (HPV) infection and progression to cervical cancer. This figure shows the uterine cervix and histologic change in the cervix from infection. Pre-Cancer and Cancer.Genital HPV infection is the most common sexually transmitted disease. And more than ~ 90% of new infections clear or become undetectable within 1 to 2 years (Step1). But a small percentage persists and progresses to pre-cancer over periods of months to years. (Step2). And progress to invasive cancer (Step3)

Current treatment landscape

Early-stage cervical cancer is often curable with radical surgery, hysterectomy, or chemoradiation, but these can have devastating effects on fertility and the quality of life [82, 83]. For persistent, recurrent, or metastatic cervical cancer, first-line therapy is now pembrolizumab plus platinum and taxane chemotherapy ± bevacizumab for PD-L1 positive disease, which improved median overall survival with chemotherapy or bevacizumab alone for 26.4 months and 16.8 months, respectively; in the pre-immunotherapy era, chemotherapy alone yielded about 13.3 months and improved to about 17 months with bevacizumab [84–86]. Immune checkpoint inhibitors, such as aspembrolizumab, have shown activity in a subset of PD-L1-positive cervical cancers, leading to regulatory approvals for advanced disease. Despite the efficacy of immune checkpoint blockades, many tumors exhibit primary or acquired resistance, highlighting the critical need for novel therapeutic strategies [87]. Moreover, none of the current therapies directly eliminates HPV infection or viral oncogenes. In contrast, molecular therapy targeting HPV could theoretically eradicate the root cause of the malignant behavior of cancer cells [87].

RNA-based therapeutic approaches

RNA-based therapies encompass a range of modalities that use nucleic acids or their analogs to modulate gene expression or produce therapeutic proteins [87]. In the context of HPV-positive cervical cancer, RNA therapies are primarily aimed at knocking down HPV oncogene expression or mounting an immune response against HPV-infected cells [26, 27, 88–90]. The major categories include (1) RNA interference techniques, such as siRNA and shRNA, to degrade HPV mRNAs; (2) antisense oligonucleotides to block the translation of HPV transcripts; and (3) mRNA-based vaccines that encode HPV antigens to stimulate anti-tumor immunity [91, 92]. Moderna’s mRNA-based therapeutic vaccine, mRNA4157/V940, is currently being tested for HPV-related cancers. This personalized vaccine is typically used in conjunction with the immunotherapy drug pembrolizumab, and early results indicate an effective immune response and a reduced risk of disease recurrence [93–100].

RNA interference: siRNA and shRNA

RNA interference (RNAi) is a natural cellular process that can be harnessed to silence specific genes using short double-stranded RNAs [101]. In therapy, the two main RNAi effectors are small interfering RNAs (siRNAs), typically 21–22 nucleotide duplexes, and short hairpin RNAs (shRNAs) expressed by vectors [102]. Both ultimately yield an antisense guide strand that is incorporated into the RNA-induced silencing complex (RISC) and directs it to complementary mRNA, leading to mRNA cleavage and degradation. For cervical cancer, the obvious RNAi targets are E6 and E7 mRNAs in high-risk HPV [102]. By designing siRNA sequences that match nucleotide stretches that are highly conserved across clinically important high-risk HPV lineages and ideally conserved across prevalent HPV16/18 variants, researchers can selectively knock down E6/E7 transcripts in HPV-positive cells to reactivate the p53 and Rb tumor suppressor pathways [78, 84]. Because naturally occurring HPV polymorphisms can introduce mismatches that reduce RNAi potency, candidate target sites should be selected using multisequence alignment of circulating variants and empirically screened for activity [101, 103, 104]. Preclinical studies have provided proof that RNAi against HPV oncogenes is effective. Transfection of HPV16 E6/E7 siRNAs or shRNAs into cancer cell lines, such as HeLa HPV18-positive or SiHa/CaSki HPV16-positive cells, leads to restoration of p53 and pRb levels, induction of p21Cip1, and consequent cell cycle arrest or apoptosis [105–107]. For example, silencing of E6/E7 causes the accumulation of p53 and hypophosphorylated Rb, triggering apoptotic cell death in vitro [91]. When the HPV16-E7 gene is silenced with shRNA, the expression of the E6 gene also decreases simultaneously [91]. Although E6 and E7 are transcribed from the same early promoter as a polycistronic pre-mRNA, the early transcript is alternatively spliced extensively. Full-length (unspliced) E6/E7 mRNA supports E6 translation, whereas spliced E6 transcripts are the major templates that enable efficient E7 production. Therefore, RNAi strategies aimed at suppressing both oncoproteins should be designed against RNA sequences shared by full-length and spliced E6 transcripts and/or against splice-junction-proximal regions, rather than assuming a single monocistronic mRNA [91–109]. A broad survey of siRNA designs targeting various regions of HPV16 E6 found that the most potent siRNAs could reduce E6 mRNA by ~ 80%, although the efficacy varied with the sequence and delivery method [110]. Importantly, numerous studies have demonstrated that RNAi-mediated E6/E7 knockdown in xenograft models of cervical cancer significantly slows down tumor growth [37, 90, 111]. In one of the earliest in vivo demonstrations, Yamato et al. achieved the suppression of established HPV16-positive tumors in mice using repeated E6/E7 siRNA injections, accompanied by the restoration of p53 and p21 expression in tumor cells [112]. A key advantage of RNAi therapy for HPV infections is its specificity. Because E6 and E7 are viral oncogenes, siRNAs can be designed with perfect complementarity to viral mRNA, minimizing off-target hits on human transcripts [113]. Furthermore, these viral genes are only expressed in infected or transformed cells, so normal tissues should be unaffected, in stark contrast to traditional chemotherapy, which reduces cell division [113]. However, achieving specific and efficient delivery of siRNAs to tumor sites has been challenging [113]. Deng et al. enhanced siRNA stability and delivery by employing 2’-O-methyl and 2’-fluoro modifications, phosphorothioate backbones, and nanoparticle carriers. The siRNA-loaded lipoplexes effectively silenced E6 in HPV16-positive CaSki cells, confirming that loss of E6 function can reactivate p53, thereby suppressing the aggressive behavior of cancer cells [113]. Another novel approach involves the use of cell-penetrating or tumor-homing peptides as carriers. Deng et al. 2023 engineered a transdermal peptide (PKU12) to facilitate delivery of siRNA through tissue layers: applying PKU12-siRNA complexes targeting oncoprotein HPV16 L1, E6, and E7 achieved significant gene knockdown in SiHa xenograft tumors and suppressed tumor growth in mice [114]. Peptide-based systems hold significant promise for topical treatment of cervical lesions [114]. For example, preclinical studies have shown that direct delivery of siRNA to cancerous tissue, with simultaneous inhibition of viral E6 and E7 proteins in conjunction with radiotherapy, generates stronger antitumor effects than either treatment alone [114]. These findings suggest that RNAi-based therapies, when combined with standard methods, can improve patient outcomes, which warrants further investigation in future clinical trials [114]. As HPV E6-mediated p53 loss confers resistance to DNA damage, E6 knockdown has been found to enhance the efficacy of chemotherapy and radiotherapy [27, 92, 115]. Shiri Aghbash et al. showed that E6 siRNA significantly increased the sensitivity of HPV16-positive cervical cancer cells to radiation, resulting in more pronounced growth inhibition than chemotherapy alone [27, 92, 115]. Similarly, Tan et al. illustrated that siRNA-mediated E6/E7 silencing increased the susceptibility of cervical carcinoma cells to cisplatin-induced apoptosis [36]. A variant of the RNAi approach uses shRNAs delivered by DNA or viral vectors, such as lentiviruses, to achieve long-term E6/E7 suppression. This method essentially programs cancer cells to produce their own siRNAs after the vector enters the cell. Although effective in laboratory experiments, vector-based shRNA therapy faces additional hurdles, including the potential for genomic integration and induction of antiviral immune responses against the vector [116, 117]. Thus, most current efforts focus on directly delivering synthetic siRNA oligonucleotides or using transient expression systems [116, 117]. In summary, RNA interference offers a precise method for targeting essential oncogenes of HPV. Extensive data has shown that silencing E6/E7 can reverse the cancerous phenotype at the cellular level [116, 117].

Antisense oligonucleotides (ASOs)

Antisense oligonucleotides are short, single-stranded DNA or RNA analogs, typically 15–25 nucleotides long, designed to complement a target mRNA [118, 119]. By binding to the target mRNA via Watson-Crick base pairing, ASO can interfere with gene expression in multiple ways [118, 119]. Some ASOs block translation by preventing ribosome binding or correct splicing, whereas others recruit RNase H, an enzyme that degrades RNA in RNA-DNA hybrids, thereby causing degradation of the target mRNA [120, 121]. For HPV therapy, antisense oligos complementary to E6 or E7 mRNA have been investigated to inhibit the production of these oncoproteins [120, 121]. Early antisense studies in the 1990s and the 2000s used unmodified phosphodiester or phosphorothioate DNA oligonucleotides that targeted HPV16 or HPV18 E6/E7 [122]. These results show that antisense molecules could reduce E6/E7 expression and restore some p53 functions in cervical cancer cell lines, resulting in inhibited growth and induction of apoptosis [123]. However, unmodified oligonucleotides are unstable, rapidly degraded by nucleases, and have limited cellular uptake [120, 124]. Chemical modifications have been introduced to create next-generation ASOs with greater stability and affinity, such as peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligomers (PMOs), and locked nucleic acids (LNAs), all of which resist nuclease digestion and form stable duplexes with RNA [125–130]. These modifications can also be combined with partial DNA content so that RNase H can still recognize the RNA/DNA hybrid and cleave the target [125–130]. Using these improved ASOs, researchers have achieved a more potent inhibition of HPV gene expression. For example, Bharti et al. reviewed numerous studies demonstrating that antisense oligos targeting various regions of E6/E7 or related transcripts such as E6-AP, the cellular partner of E6, could induce apoptosis in HPV-16-positive cells by upregulating the p53 and pRb pathways [123]. Some antisense sequences overlapped with the start codons or splice junctions of E6/E7, which proved effective in reducing the corresponding protein levels [131, 132]. Notably, in a three-dimensional cervical cancer tissue culture model, an ASO targeting HPV18 E6 led to the restoration of normal epithelial differentiation and a reduction in the neoplastic appearance of the tissue, indicating reversion of the cancerous phenotype [108, 133]. In vivo application of antisense therapy for HPV has also shown promise. In a study conducted by Gutiérrez et al., an antisense oligodeoxynucleotide (ODN) against HPV16 E7 was delivered intratumorally in mice with CaSki cell xenografts, resulting in suppressed tumor growth compared with controls [134]. Additionally, Reschner et al. introduced an innovative strategy of conjugating an HPV16-E6-targeting antisense oligonucleotide with a photoresponsive ruthenium complex [135]. Upon light activation, this design improved cellular uptake and enhanced E6 knockdown, resulting in significant growth inhibition in both monolayer and 3D cultures of SiHa cervical cancer cells [135]. This theranostic strategy could potentially allow for the spatial and temporal control of antisense activity [135]. Although no antisense drug has yet been approved for HPV, several ASO-based therapeutics have successfully reached the market, including mipomersen for hypercholesterolemia and multiple agents for genetic neurological disorders. These successes demonstrate the feasibility of developing ASOs as clinically applicable therapeutics [135]. In the context of HPV, an antisense-based strategy could potentially be administered topically for cervical intraepithelial neoplasia to achieve nonsurgical lesion clearance or systemically as an adjunctive treatment for cervical cancer [135]. One challenge is that HPV E6/E7 mRNAs are confined to the nucleus for part of their life cycle because of viral splicing and polycistronic transcripts, which may reduce ASO binding opportunities in the cytoplasm. Nevertheless, ongoing advances in ASO chemistry and delivery, such as ligand-conjugated designs that selectively target tumor cells, may help overcome these limitations [135].

Regulatory non-coding RNAs that modulate E6/E7 expression

In addition to engineered RNA therapeutics, endogenous non-coding RNAs, particularly microRNAs, are frequently dysregulated in HPV-associated cancers and can modulate E6/E7 expression directly or indirectly. For example, restoration of miR-375 expression suppresses HPV oncogene expression and inhibits telomerase activity and proliferation in HPV-positive cancer models [136]. More recently, HPV E7-mediated repression of miR-203 has been shown to promote proliferation through derepression of cellular targets, highlighting how HPV oncogenes actively rewire host miRNA networks during transformation [137]. These and other regulatory RNA mechanisms have been reviewed in detail by Obanya et al. [138].

mRNA-based therapeutic vaccines

Another powerful RNA-based strategy involves the use of mRNA as a vehicle for vaccination and immunotherapy against HPV-driven cancers [139, 140]. The basic principle is to deliver a synthetic messenger RNA encoding one or more tumor-associated antigens such that host cells transiently produce the antigen, which is then processed and presented to the immune system, particularly by activating cytotoxic CD8⁺ T cells [139, 140]. Consistent with this rationale, therapeutic HPV mRNA vaccines encoding E6/E7 have entered clinical testing, Draper et al. trials in CIN2/3- and HPV16-positive cancers, aiming to elicit robust antigen-specific T-cell responses against infected tumor cells [141]. Therapeutic vaccines for HPV-related diseases have been investigated for many years using various platforms, such as peptide vaccines, protein vaccines, DNA vaccines, and viral vector vaccines [142]. Until recently, mRNA vaccines had not been widely tested in this domain, partly because of concerns regarding mRNA stability and delivery [142]. The clinical success of mRNA-based COVID-19 vaccines in 2020–2021 demonstrated that mRNA-LNP (lipid nanoparticle) technology can effectively deliver mRNAs in vivo, leading to robust immune responses [143]. This success has revitalized the interest in mRNA vaccines for cancer, including cervical cancer [144, 145]. Modern mRNA vaccine formulations use chemically modified nucleosides, such as pseudouridine, to reduce innate immune sensing, maximize translation, and encapsulate mRNA in lipid nanoparticles to protect them from RNases and facilitate cellular uptake [144, 145]. The preclinical results of mRNA vaccines against HPV are highly encouraging for tumors [146]. A therapeutic study in mice with HPV-16 tumors using an mRNA-boosted vaccine resulted in a single-dose injection eliciting potent and activated T cells, eliminating mouse tumors [146]. Notably, these mRNA vaccines outperformed parallel DNA and protein vaccines in the same model, highlighting the potency of the mRNA platform [146]. The induced immunity is primarily mediated by CD8 + cytotoxic T lymphocytes recognizing the E7 antigen, which is crucial for killing HPV-expressing cells [146]. Another research team developed an mRNA-LNP vaccine targeting both HPV16 and HPV18 E6/E7 and reported that it elicited strong antigen-specific T cell responses in mice and rhesus macaques, demonstrating promising immunogenicity that could translate to humans [1]. In these animal studies, vaccinated subjects experienced a tumor re-challenge, indicating the formation of immune memory [1]. The mRNA vaccine approach for HPV-related cancers is advancing towards clinical evaluation. For instance, BioNTech’s BNT113 is a lipoplex-formulated nucleoside-modified mRNA vaccine that encodes the HPV16 E6 and E7 oncoproteins. It is currently being tested in a Phase I/II trial named HARE-40 in patients with HPV16-positive cancers, including cervical and/or oropharyngeal cancers [147]. Interim reports have shown that BNT113 is well tolerated and can generate CD4⁺ and CD8⁺ T cell responses against E6/E7 in patients. The trial is also exploring combination strategies; one arm combines BNT113 with an anti-CD40 agonist antibody to potentiate dendritic cell activation [148]. Another study by Merck, Co., and Moderna combined BNT113 with the PD-1 checkpoint inhibitor pembrolizumab in patients with advanced HPV-positive head and neck cancer. The rationale is that the vaccine will prime an HPV-specific T cell response, and the checkpoint inhibitor will prevent tumor-induced T cell suppression, thereby enhancing the overall efficacy [149]. Early clinical data from analogous peptide vaccine trials supports this approach. Another notable effort is Moderna’s mRNA-4157, also called V940, which is a personalized mRNA vaccine that can include patient-specific neoantigens and is being tested in HPV-positive head and neck cancer patients in combination with pembrolizumab [149]. mRNA vaccine technology allows the development of customized vaccines tailored to individual patients. For example, mRNA-4157 is a vaccine designed based on a patient’s specific HPV-16 tumor mutations, targeting E6/E7 oncoproteins [149]. mRNA vaccines offer some advantages over DNA or protein vaccines: they can drive both MHC class I and II presentations and activate CD8 and CD4 T cells, do not require nuclear entry, and can be rapidly updated or customized [149]. They also inherently activate innate immune mRNA vaccines and have certain benefits over DNA or protein vaccines. They can stimulate both MHC class I and II pathways, activating CD8 and CD4 T cells [149]. Unlike DNA vaccines, they do not need to enter the nucleus and can be modified or customized quickly. Additionally, they naturally activate innate immune sensors such as Toll-like receptors, which can boost the immune response. However, this activation must be carefully balanced because excessive stimulation can impair mRNA translation [149]. Cell-free production of mRNA vaccines allows for large-scale manufacturing, activates the immune system, and functions as an adjuvant [149] (Fig. 2).

Fig. 2.

RNA-based therapeutic approaches targeting HPV-associated carcinogenesis. (Left) High-risk HPV oncoproteins E6 and E7 promote degradation of the tumor suppressors p53 and retinoblastoma protein (pRb), respectively, resulting in impaired apoptosis, E2F release, and dysregulated cell cycle progression. (Right) RNA-based therapeutic strategies counteract these oncogenic effects through multiple mechanisms. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) induce RNA-induced silencing complex (RISC)-mediated degradation of E6/E7 transcripts, leading to restoration of p53 and pRb activity and induction of apoptosis or cell cycle arrest. Antisense oligonucleotides (ASOs) promote RNase H–dependent cleavage of target mRNA and inhibit translation. mRNA vaccines, delivered via lipid nanoparticles (LNPs), stimulate antigen expression and presentation via MHC class I and II pathways, activating CD8⁺ cytotoxic and CD4⁺ helper T-cell responses and promoting tumor cell elimination

Preclinical studies

Preclinical investigations of RNA-based therapies for cervical cancer, ranging from cell culture studies to animal models, have provided strong evidence to support the feasibility of targeting HPV with RNA molecules. The evidence from these studies underpins the rationale for clinical trials and sheds light on practical considerations, such as delivery efficacy and safety.

In vitro efficacy

HPV-positive cancer cells are highly sensitive to the suppression of E6/E7 genes. Yamato et al. The introduction of a specific siRNA against E6/E7 in the SiHa cell line resulted in cell growth arrest and apoptosis [112]. Restoration of p53 was observed in HPV-positive cells, confirming its on-target result is to choose reported antisense oligonucleotides; one study using an antisense morpholino against HPV16 E6 showed a sharp decline in cell proliferation and an increase in apoptotic markers in CaSki cells compared to scrambled controls. These experiments validated that E6/E7 is required for the survival of these cancer cells, a concept often described as oncogenic addiction [112]. Beyond measuring cell proliferation or death, researchers have examined the changes in cellular pathways after RNA therapy. For example, upon siRNA-mediated E6/E7 silencing, cervical cancer cells exhibit increased p21 expression owing to p53 activation and hypophosphorylated Rb, leading to the accumulation of cells in the G1 phase [150, 151]. Suppression of E6/E7 expression has been associated with the induction of senescence markers such as senescence-associated β-galactosidase, indicating that a subset of cells permanently withdraws from the cell cycle rather than undergoing apoptosis. This observation aligns with the concept that HPV oncogenes prolong cellular lifespan and their inhibition permits the reactivation of intrinsic aging programs [150, 151]. Targeting HPV can also affect the characteristics of cancer metastasis. In a study conducted by Serrano-Bello et al. using liposomal E6-siRNA, it was observed that treated cells not only exhibited reduced proliferation but also a decreased ability to migrate and invade, as assessed by wound healing and Matrigel invasion assays [113]. Specifically, E6 knockdown led to a ~ 96% reduction in migration and a ~ 98% reduction in the invasion of HeLa cells in vitro. This suggests that, beyond halting growth, RNA therapies might also reduce the aggressive behavior of cancer cells, possibly by reversing E6/E7’s effects on molecules such as cadherins, matrix metalloproteinases, or cytokine signaling. HPV oncoproteins are known to modulate several factors [113].

Animal model efficacy

Mouse models are crucial to demonstrate that RNA therapeutics can suppress tumor growth in vivo. Two main types of models were used [1]. Kang et al. used a xenograft model in which human cervical cancer cells HeLa, SiHa, and CaSki were implanted into immunodeficient mice [2]. Transgenic mouse models that express HPV oncogenes and spontaneously develop neoplasia, such as K14-HPV16 transgenic mice, develop premalignant lesions in the skin and cervix. In xenograft models, the local or systemic administration of anti-HPV siRNAs or ASOs has yielded tumor control. For example, one study used atelocollagen, a peptide-based carrier, to deliver HPV18 E6 siRNA to HeLa cell tumors in mice, which resulted in significant tumor growth suppression compared to controls, accompanied by increased p53 levels in the excised tissue. Another research group administered HPV16 E7 siRNA encapsulated in PEGylated nanoparticles, specifically targeting the transferrin receptor, resulting in approximately 50% suppression of SiHa xenograft tumor growth and significantly prolonged survival in treated mice compared to untreated controls. Immunocompetent mouse models are required to develop therapeutic vaccines. Researchers frequently employ tumor cell lines engineered to express HPV antigens, such as TC-1 cells, a murine cell line that expresses HPV16 E6/E7 and reliably forms tumors in syngeneic C57BL/6 mice. Qiu et al. reported that an mRNA-LNP vaccine targeting the HPV16 E7 oncoprotein elicited robust CD8 + T cell responses and controlled tumor growth in a murine model of HPV-positive oropharyngeal cancer. The vaccine not only slowed tumor progression but, in many cases, cleared the tumors, and the mice remained tumor-free upon re-challenge, indicating durable immune memory. Other research groups have demonstrated that the addition of immunostimulatory elements can significantly enhance vaccine efficacy. For instance, integrating self-amplifying replicon sequences or combining mRNA vaccination with adjuvant cytokines can substantially augment T-cell responses, surpassing the immunogenicity achieved by conventional mRNA vaccines alone. A particularly relevant preclinical study on cervical cancer demonstrated that therapeutic vaccination could eliminate precancerous lesions. In a rabbit model of oral papillomavirus, Trimble et al. showed that immunotherapy can cause regression of high-grade dysplastic lesions caused by the papillomavirus. In mice, a combination of an HPV16 E7 vaccine with a CpG adjuvant led to the clearance of established CIN-like lesions in the cervix. These findings suggest that RNA-based immunotherapy might be applied not only to treat cancer but also at earlier stages to eradicate lesions before they develop into invasive cancer [152–155]. Taken together, preclinical evidence paints a very optimistic picture: HPV oncogenes are weak, and RNA-based methods can exploit the weakness of disabled tumors in model systems. These consistent results across many laboratories have built momentum to test these approaches in humans.

Clinical translation and trial landscape

Clinically, HPV-directed immunotherapy has advanced rapidly through therapeutic vaccination strategies, frequently evaluated in combination with PD-1 blockade. For example, HPV therapeutic vaccination combined with pembrolizumab has been reported in HPV16/18-positive advanced cervical cancer in clinical testing, illustrating the translational potential of antigen-directed approaches even when the modality itself is not oligonucleotide knockdown therapy [156]. Importantly, mRNA therapeutic vaccines are now entering the early phase evaluation for HPV-associated cervical precancer, including a phase 1/2 study of an mRNA therapeutic vaccine, RG002, for HPV16/18-associated CIN2/3 (NCT06273553) [157]. In parallel, the clinical development of E6/E7-targeting mRNA immunotherapy is also being explored for HPV16-associated malignancies outside the cervix –for instance, BNT113 in HPV16 + head and neck cancer (NCT04534205), providing proof-of-platform maturity while underscoring that cervical-cancer–specific evidence may still be emerging [158]. In contrast, direct RNAi/ASO-mediated E6/E7 silencing in patients remains comparatively early, with the key translational hurdles being delivery to the tumor tissue, intracellular bioavailability, durability of knockdown, and safety. Therefore, the current state of play supports a balanced conclusion: the field shows a strong mechanistic rationale and promising preclinical efficacy, but substantial work remains to translate targeted E6/E7 suppression into broadly effective clinical therapy.

Therapeutic vaccine trials

Several clinical trials have assessed vaccines targeting HPV16/18 E6 and E7 in patients with cervical intraepithelial neoplasia CIN or cervical carcinoma. Earlier generation vaccines, such as peptide cocktails, such as ISA101 and HPV16-SLP, or DNA plasmid vaccines, such as VGX-3100 by Inovio and GX-188E by Genexine, have demonstrated that it is possible to generate HPV-specific immune responses and achieve clinical efficacy [152–155]. For instance, VGX-3100, as a DNA vaccine delivered by electroporation, showed in a Phase II trial that it could induce regression of CIN2/3 lesions in about 49% of patients vs. 30% in placebo, along with clearance of the HPV infection in many cases [152–155]. A new wave of trials has involved mRNA vaccines and other innovative RNA platforms. BioNTech BNT113 is currently under clinical development. The Phase I part of the HARE-40 trial NCT03418480 tested BNT113 as monotherapy in advanced HPV16-positive cancers, including cervical and anal cancers, as well as head and neck cancers [147]. Interim data presented in late 2023 showed that BNT113 induced measurable CD8⁺ T cell responses against E6/E7 in a significant fraction of patients, and there were indications of anti-tumor activity such as stable disease or minor tumor regression in some participants [149]. The safety profile was acceptable, with mostly grade 1–2 flu-like symptoms, typical of immune activation. Building on this, the ongoing Phase II Merck & Co./Moderna trial combines BNT113 with pembrolizumab for HPV16 + recurrent/metastatic head and neck squamous cell carcinoma. This trial will clarify whether the vaccine can improve outcomes compared with checkpoint inhibitors alone [147]. Similarly, Moderna’s personalized mRNA-4157 cancer vaccine, after showing positive results in melanoma, is currently being tested for HPV-associated cancers in combination with pembrolizumab. While personalized vaccines are not specific to HPV, they cover all neoantigens in a tumor. HPV peptides can be included in HPV + patients, and monoclonal antibodies, such as pembrolizumab, which is a monoclonal antibody, have been able to target inhibitory pathways of the immune system and enhance the activity of T cells against tumors [159–161] (Table 1).

Table 1.

Clinical trials currently evaluating therapeutic HPV vaccines

Vaccine	Target	Trial Phase	Clinical Symptoms	Reference
VGX-3100- DNA vaccine	HPV16/18 E6/E7	II	Regression of CIN2/3 ,~49% of patients, HPV clearance, mild AEs (grade 1–2)	[143–146]
ISA101 / HPV16-SLP- Peptide vaccine	HPV16 E6/E7	Pre- clinical	HPV-specific immune response induction, acceptable safety	[143–146]
GX-188E- DNA plasmid vaccine	HPV16-18 E6/E7	Pre- clinical	Immune response induction, acceptable safety	[143–146]
BNT113 (BioNTech)- mRNA vaccine	HPV16 E6/E7	II	CD8 + T cell responses, partial anti-tumor activity, flu-like symptoms grade 1–2	[137, 139]
mRNA-4157 -Moderna, personalized mRNA	Tumor neoantigens + HPV peptides	III	Immune and anti-tumor responses, acceptable safety	[115, 150]
BNT113 + Pembrolizumab-mRNA vaccine + immune checkpoint inhibitor	HPV16 E6/E7	II	Evaluating improved outcomes vs. ICI alone	[137]

Translational challenges and future directions

Finally, it has emphasized that the most clinically mature HPV-directed immunotherapy evidence currently comes from therapeutic vaccine approaches often combined with checkpoint blockade, while direct E6/E7 knockdown via RNAi/ASO remains largely preclinical. A realistic forward path likely involves (І) improved delivery technologies, including LNP and polymeric systems, and (П) combination regimens that exploit mechanistic complementarity for instance, knockdown to increase antigen presentation or apoptosis + PD-1 blockade [130].

Conclusion

RNA-based therapeutics offer a compelling route to target HPV-driven diseases by either directly suppressing E6/E7 expression or amplifying HPV-specific immunity. However, the gap between robust preclinical proof-of-concept and consistent clinical benefits remains largely defined by delivery, intracellular bioavailability, safety, and the need for rational combinations. The most clinically advanced evidence currently comes from antigen-directed immunotherapy strategies (including emerging mRNA vaccine programs), while direct E6/E7 knockdown approaches remain in translation. Continued progress will depend on improved delivery technologies, careful management of off-target and immunostimulatory effects, and well-designed trials with clinically meaningful end points.

Author contributions

SMKh: Literature search, design of the table, manuscript preparation; NE: Literature search, Manuscript preparation; PSA: Review and editing of the manuscript; ZZR: Figure design; HBB is the corresponding author of the manuscript, study conception, and contribution to the revision of the manuscript’s main text.   All the authors have read and approved the final manuscript.

Funding

This study did not receive any funding.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

None.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.