Advancements in clinical RNA therapeutics: Present developments and prospective outlooks

Summary

RNA molecules have emerged as promising clinical therapeutics due to their ability to target “undruggable” proteins or molecules with high precision and minimal side effects. Nevertheless, the primary challenge in RNA therapeutics lies in rapid degradation and clearance from systemic circulation, the inability to traverse cell membranes, and the efficient intracellular delivery of bioactive RNA molecules. In this review, we explore the implications of RNAs in diseases and provide a chronological overview of the development of RNA therapeutics. Additionally, we summarize the technological advances in RNA-screening design, encompassing various RNA databases and design platforms. The paper then presents an update on FDA-approved RNA therapeutics and those currently undergoing clinical trials for various diseases, with a specific emphasis on RNA medicine and RNA vaccines.

Graphical abstract

In this review, we highlight the implications of RNAs in diseases, providing a detailed overview of the development of RNA therapeutics, including recent advancements in RNA-based vaccines and current RNA-based drugs in the clinic and in clinical trials.

Introduction

RNA plays a pivotal role in numerous cellular processes, including the translation of genetic information, regulation cellular activities, cellular differentiation, and more. While the RNAs within the coding genome are well understood, constituting less than 20% of total cellular RNAs, a substantial portion of RNAs within the non-coding genome remains enigmatic. Long non-coding RNAs (lncRNAs), for instance, exhibit distinct behaviors based on their sub-cellular localization.¹ Other types, such as 7SK RNA, contribute to transcription; small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) participate in RNA processing; while ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and microRNAs (miRNAs) are integral to the translation process. Additionally, there are various others, such as circular RNAs (circRNAs), RNase P RNAs, and those with less well-characterized functions or entirely unknown roles (e.g., vault RNAs, Y RNAs, Piwi-interacting RNAs; piRNAs, glycan RNAs).^2,3,4,5

To date, only approximately 15% of human proteins, out of around 20,000 human proteins, are deemed druggable.⁶ Data suggest that the Food and Drug Administration (FDA) has approved only about 700 small-molecule drugs targeting human proteins,⁷ underscoring the vast potential for the development of novel drugs independent of traditionally “druggable targets.” Since 2018, nearly 30% of FDA-approved drugs have been biologics,^8,9 with a subset based on nucleic acid, including antisense oligonucleotides (ASOs), aptamers, messenger RNAs (mRNAs), siRNAs, and miRNAs.^{10,11,12,13,14,15} This marks the emergence of a new era in RNA-based therapeutics. The consecutive FDA approvals of siRNA drugs, Patisiran and Givosiran, have translated the promise of siRNA into clinical reality.¹⁶ With numerous siRNA and mRNA-based therapeutics in the pipeline, the clinical translation of RNA therapeutics has transitioned from mere “hype” to a tangible “hope.”

In the first part of this review, we delve into the role of RNA in diseases and explore the technological advances in RNA therapeutic development. Subsequently, we will focus on RNA medicine and RNA vaccines, recognizing their immense potential in disease prevention and diagnosis.

Part 1: The role of RNA in diseases

Coding RNAs: Beyond the central dogma

Since the elucidation of DNA and the establishment of the central dogma, attention has been predominantly centered around three key RNA types crucial for protein synthesis: mRNAs, rRNAs, and tRNAs. mRNAs serve as blueprints for protein transcription, while rRNA forms the structural and enzymatic scaffold within ribosomes, facilitating the sequential matching of mRNAs that leads to protein synthesis. Simultaneously, tRNAs act as substrates, bringing complementary “3-amino acid words” that match specific mRNA sequences for incorporation into growing protein chains. However, advancements in molecular biology and imaging techniques have revealed that the translation process involves the orchestrated collaboration of numerous other RNAs, which chemically or structurally modify protein chains before they become fully functional. For instance, spliceosomal RNAs, in conjunction with their protein partners, splice out introns from pre-mRNA transcripts, subsequently aligning exons to construct distinct protein-coding genes. In the assembly and functioning of ribosomes, diverse protein enzymes collaborate with snoRNAs to guide modifying enzymes precisely to rRNAs. RNAse P, an enzyme present in all cells, plays a specific role in trimming the ends of tRNA precursors. Consequently, the incorporation of various ncRNA classes is evident in their involvement in gene regulation across multiple levels, directly influencing mRNA transcription, translation, production, and stability. Dysregulation of RNAs can be detrimental, disrupting normal cellular homeostasis and contributing to various human diseases, including autoimmune diseases, cancer, and other chronic conditions. The historical timeline of RNA discovery and RNA therapy are provided in Figure 1.

Figure 1.

A concise historical timeline outlining the discoveries in RNA biology and their subsequent contributions to RNA therapy development

ncRNAs: Emerging roles in major diseases

Numerous ncRNAs function by facilitating the recruitment of multi-subunit chromatin-modifying complexes to specific DNA regions, thereby exerting regulatory control over chromatin epigenetic marks, nucleosome positioning, histone modifications, or transcription.¹⁷ This control can either enhance or suppress gene expression. For instance, lncRNAs nuclear enriched abundant transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) are located in the same genomic region but have distinct regulatory roles. Another class of ncRNAs, miRNAs, interact with gene promoter regions. For example, miR-24-1 promotes RNA polymerase II activity by inducing the generation of enhancer RNAs (eRNAs) and binding to promoters. Similarly, hY1.RNA serves to suppress the proliferation of cells. Human YRNAs, which do not encode proteins, have emerged as potential cancer biomarkers, underscoring the diverse roles played by ncRNAs in governing cellular processes.¹⁸

Certain ncRNAs exert significant influence over essential cellular processes and disease progression by interacting with specific protein complexes that hold pivotal roles in gene expression regulation. Notably, spliceosomes, intricate molecular assemblies responsible for mRNA splicing, consist of snRNAs along with an array of associated proteins.¹⁹ Ribonucleoprotein (RNP) complexes further facilitate post-transcriptional modifications of precursor snRNA, tRNA, and rRNA. circRNA and lncRNAs also contribute to these regulatory processes by serving as recruiting platforms for various proteins.²⁰ For instance, circRNA-Foxo3, which exhibits increased expression during cancer cell apoptosis, holds potential as a therapeutic intervention for restraining tumor growth. Similarly, the involvement of lncRNA Xist in the female developmental framework is of significant importance due to its interaction with SAF-A and Lamin B receptor (LBR) proteins.²¹

The utilization of lncRNA from the opposite strand to repress specific target genes shows promise in addressing genetic abnormalities such as Spinocerebellar Ataxia Type 7 and Angelman syndrome. Additionally, interactions between miRNAs and mRNAs can lead to the silencing of mRNA expression. Elevated levels of certain miRNAs can function as oncogenes by effectively suppressing mRNA. In the context of osteosarcoma, the gene for cell adhesion molecule 1 (CADM1) gene serves as an miRNA sponge, thereby offering promising prospects for the development of novel therapeutic approaches.^22,23

Part 2: Technological advances in RNA screening development

The discovery of diverse RNA types has significantly expanded the landscape of RNA drug development.^24,25,26 However, the quest for drug development targeting RNA presents unique challenges, and advancements in this domain have been comparatively gradual when juxtaposed with protein targets.²⁷ Beyond the well-established distinctions in chemical and structural properties between protein and RNA ligands, the optimization of screening strategies for RNA is equally paramount. As depicted in Figure 2, this factor stands as a pivotal determinant for the success of RNA drug discovery.²⁸

Figure 2.

Schematic representation of the RNA screens development workflow

(1) Identification of RNAs of interest: Genetic methods, encompassing genome-wide association studies and knockout models, alongside cell-based strategies like immunoprecipitation (CLIP)-based methods and crosslinking, are employed to identify RNAs of interest.

(2) Characterization of RNA functions: RNA functions are elucidated through biochemical assays, such as electrophoretic mobility shift assays (EMSA).

(3) Design of RNA target construct: Insights derived from functional and structural analyses guide the development of the RNA target construct for the initial high-throughput screening (HTS).

(4) HTS: Entails affinity or mechanism-based screens to identify potential hits.

(5) Hit validation: Selected hits from HTS are validated using orthogonal secondary assays.

(6) Lead compound optimization: Hits undergo iterative modifications to yield a lead compound, optimizing for both efficacy and specificity.

Screening design: Optimal selection of RNA target

RNA-targeting screens are strategically designed to discover compounds with the ability to directly interact with RNA or function within ribonucleoprotein (RNP) complexes. The therapeutic potential of drugs exhibiting specificity for RNA is particularly noteworthy, especially in cases where the targeting of proteins proves challenging, such as in genetic disorders associated with abnormal transcripts or infectious diseases. However, direct RNA targeting may pose challenges when dealing with transcripts expressed at lower levels or lacking structured regions. The advent of proteomics advancements and techniques like crosslinking and immunoprecipitation (CLIP) has facilitated the discovery of RNPs, presenting them as alternative viable targets for modulating RNA functions and life cycles.²⁹ Traditionally perceived as challenging drug targets due to their often extensive, intrinsically disordered protein-RNA interfaces, RNPs are now becoming more accessible. This shift is attributed to drugs that employ allosteric or competitive inhibition of RNA-recognition motifs, as well as those targeting the degradation of RNPs.³⁰ Given that RNA-binding proteins often bind to multiple transcripts, Targeting RNP thus holds particular promise in the treatment of systemic diseases such as autoimmune disorders and cancers, where RNA-binding proteins play intricate roles across diverse transcripts.³¹

Screening implementation: Optimal selection of RNA-screening methods

RNA/RNP screens operate akin to protein screens, enabling the observation of compound impacts in both cellular or cell-free environments (mechanistic screens) and the assessment of direct binding to the molecular target (affinity screens). Notably, the refinement of compound libraries, validation assays, and screening platforms has been tailored to align with the biochemical and biophysical characteristics of RNAs (Figure 2). This optimization, coupled with the identification of new, druggable RNA/RNP targets, significantly influences the efficacy of the screening process.

Mechanistic screens: Capturing active compounds in RNA/RNP interactions

Mechanistic screens, which capture active compounds involved in RNA/RNP interactions, commonly employ cellular gene-reporter assays to study gene expression modulation, including splicing and translation machineries.³² These assays leverage the expression of chemiluminescent or fluorescent proteins, offering simplicity, speed, and precision. These attributes render gene-reporter assays invaluable for mechanistic screenings. However, while reporter screens are effective, they may detect compounds operating through unintended mechanisms. Therefore, it is critical to focus on disease-relevant systems that are well-characterized and amenable to mechanistic assays. One approach to mitigate off-target effects in mechanistic screens involves using purified factors, as illustrated in Figure 2. Nonetheless, this approach may pose challenges such as reduced in vivo reproducibility and an increased likelihood of false-positives.

For non-catalytic RNPs, immobilization on beads followed by assays like AlphaScreen,³³ scintillation proximity assay (SPA),³⁴ homogeneous time-resolved fluorescence (HTRF),³⁵ or technologies such as molecular beacons and catalytic enzyme-linked click chemistry assay (cat-ELCCA)^36,37 are more suitable. Alternatively, rather than immobilizing the RNA/RNP target, a method known as small-molecule microarrays (SMMs) can utilize beads to immobilize small compounds. SMMs offer the advantage of adaptability for use with either purified probes or cell lysates, requiring minimal materials, and can be seamlessly integrated into robotics infrastructure.³⁸

Exploration of low-affinity fragments in affinity screens: Broadening the chemical space of RNA binders

Affinity screens stand out as highly effective tools for measuring ligand binding, particularly in vitro (Figure 2). In contrast to mechanistic screens, they demonstrate superior efficiency in detecting target-ligand complexes, making them ideal for initial high-throughput screening or secondary validation. Recent advancements in sample injection and detection techniques³⁹ have markedly improved the precision and effectiveness of affinity screens, especially when coupled with mass spectrometry (MS). MS affinity screens come in two types: direct and indirect. Direct MS screens identify hit compounds, including those with low-affinity targets, providing insights into binding stoichiometry and affinities. This proves advantageous, particularly when targeting RNA/RNPs known to be druggable, such as ribosomal complexes, as it expands the chemical space of small-molecule interactors beyond known compounds. On the other hand, indirect MS screens separate target-bound compounds from non-bound ones before detection, typically through size-exclusion chromatography. These label-free screens, exemplified by the automated ligand identification system (ALIS), are suitable for detecting large transcripts that may be challenging to identify via MS. Additionally, detecting natural compounds, which are often utilized in RNA screens for their chemical diversity, can be challenging with MS. To address these challenges, fragment-based affinity screens serve as valuable complements to MS screens. These screens utilize simple molecules that may not exhibit strong binding affinity but can be optimized through fragment combinations. While fragment-based screens, particularly those using NMR (nuclear magnetic resonance), have a lower throughput rate, their ability to detect hits with millimolar dissociation constants (KD) and provide high-resolution visualization of binding is advantageous for hit optimization.^40,41 Notably, NMR-based fragment screens demonstrate remarkable efficacy against small (<30 kDa) and highly structured RNAs.

Computational screens: Identifying novel druggable RNA motifs

Despite notable progress in identifying compounds that target RNA/RNP through mechanistic or affinity screens, the advancement in RNA/RNP drug screening remains hindered primarily by the limited accessibility of high-resolution 3D structures for RNA/RNP. This limitation impedes the recognition of potential druggable RNA pockets. Furthermore, the intricate nature of RNA conformational dynamics presents a significant challenge, precluding the straightforward application of rational structure-based design in compound optimization.⁴² Within this framework, recent strides in RNA computational biology offer valuable insights for guiding the design of RNA-targeted drugs, particularly when integrated with experimental analyses.^43,44 Molecular dynamics simulations, when complemented by X-ray, NMR, or cryoelectron microscopy (cryo-EM), effectively capture the conformational flexibility of RNA, thereby augmenting the likelihood of discovering potential binding entities through virtual screening.⁴² In addition, in silico docking and molecular dynamics investigations play an essential role in optimizing and prioritizing hit compounds, predicting how compounds bind to RNA targets and elucidating their interaction mechanisms.

Part 3: RNA-based medicine

Starting with ASOs, the realm of RNA-based medicine has evolved to encompass RNAi-based therapies, mRNA replacement, ncRNA-based interventions, and RNA-based vaccines. At present, a diverse array of RNA therapeutics is advancing through preclinical and clinical development, underscoring the viability of harnessing RNA as a standalone medicinal entity. This section explores RNA-based medicines that have received approval from the FDA and provides insights into the current status of RNA therapeutics undergoing clinical trials. Additionally, we delve into a significant clinical development, anticipating the potential FDA approval of a CRISPR-based RNA medication by 2024.

Antisense oligonucleotides

In recent years, the exploration of ASO-based therapies has manifested in 100 phase I trials, with 25% progressing to phase II/III trials. These investigations have spanned the treatment landscape for both common and rare diseases, encompassing conditions like cancer and orphan genetic alterations.⁴⁵ Notably, the ASO drug fomivirsen achieved FDA approval for the treatment of cytomegalovirus retinitis (CMV) in individuals with AIDS.⁴⁶ Operating by complementing human CMV immediate-early mRNA with phosphorothioate (PS) ASOs, Fomivirsen obstructs viral protein synthesis, ultimately disrupting viral replication.⁴⁷ However, due to a decline in CMV prevalence with the advent of highly active antiretroviral therapy, Fomivirsen was discontinued from US and European markets in 2002. The second generation of ASO drugs, termed “Gapmers” or chimeric ASOs, includes mipomersen and inotersen. Mipomersen targets the degradation of apolipoprotein B (ApoB) mRNA, while Inotersen facilitates hepatic transthyretin (TTR) mRNA degradation through RNase H1-mediated means, thereby decreasing TTR protein synthesis and serum TTR levels.⁴⁸ Notably, nusinersen and eteplirsen are two splice-modulating ASOs that received approval for splicing defect treatment in 2016.⁴⁹ Nusinersen, a pioneering splicing-correcting ASO approved for treating spinal muscular atrophy, operates by promoting the inclusion of exon 7 and augmenting survival motor neuron (SMN) protein expression through intronic splicing inhibitor-binding at intron 7.⁵⁰ Duchenne muscular dystrophy (DMD), a debilitating global condition, is addressed by eteplirsen targeting a sequence in exon 51 to enhance splicing. This allows the spliceosome to read exon 52 in-frame by bypassing exon 51, resulting in the generation of semi-functional dystrophin proteins with shorter lengths.⁵¹ To address specific splicing defects in DMD, golodirsen,⁵² viltolarsen,⁵³ and casimersen⁵⁴ have been approved, as eteplirsen caters to only 13%–14% of DMD patients. These medications facilitate dystrophin protein expression by inducing the skipping of exon 45 or 53, thereby promoting the expression of dystrophin proteins.

Milasen represents a groundbreaking patient-customized ASO designed for the treatment of neuronal ceroid lipofuscinosis, a condition leading to neurodegeneration and eventual fatality. Importantly, this treatment has demonstrated acceptable tolerability with minimal side effects during therapeutic administration.⁵⁵ For instance, RPI.4610, also known as angiozyme, is a ribozyme engineered to impede angiogenesis by targeting the mRNA of the vascular endothelial growth factor receptor 1 (VEGFR1). Nonetheless, its clinical development has faced challenges due to suboptimal efficacy.⁵⁶ Another ribozyme, OZ1, has been designed to target specific sequences in the HIV genome, specifically the tat and vpr genes. The unique feature of OZ1 lies in its potential application in HIV therapy by targeting the virus at the genetic level. When administered to autologous CD34⁺ hematopoietic progenitor cells, these cells can differentiate into various blood cell types, including CD4⁺ lymphocytes, a primary target of HIV. The goal of OZ1 in this context is to disrupt HIV replication within the host’s immune cells, ultimately leading to an increase in the count of CD4⁺ lymphocytes, which are crucial for the immune system.⁵⁷ Despite positive outcomes in preliminary stages of a clinical trial, further investigation is imperative to elucidate critical aspects, including the stability of ribozymes, tissue delivery specificity, and sustained expression.⁵⁸

RNAi-based therapy

The emergence of Patisiran, among other GalNAc-conjugated siRNAs, signifies a significant leap forward in RNAi therapeutics. Presently, Lumasiran, Patisiran, and Givosiran, three siRNA-based pharmaceuticals, have received FDA approval. Concurrently, seven siRNA candidates—Teprasiran, Inclisiran, Nedosiran, Vutrisiran, Cosdosiran, Fitusiran, and Tivanisiranare—are undergoing phase III clinical trials. Patisiran, in particular, stands as a groundbreaking FDA-approved RNAi-based drug transforming the landscape for hTTR-associated polyneuropathy.⁵⁹ Targeting the 3′ UTR of the TTR gene, Patisiran suppresses the expression of all potential mRNAs carrying mutations at coding regions,⁵⁹ a mechanism akin to Inotersen.⁶⁰ The development of GalNAc delivery platforms by Alnylam has played a pivotal role in enhancing the effectiveness of siRNA-based pharmaceuticals in clinical applications. Approximately 30% of RNAi-based medications currently in clinical trials employ GalNAc-conjugated siRNAs. The initial exploration into GalNAc-siRNA drugs, as exemplified by Revusiran, exhibited heightened uptake by asialoglycoprotein receptors for hepatic delivery.⁶¹ However, its progress was halted due to unfavorable outcomes in the phase III clinical trial called ENDEAVOR (NCT02319005).⁶² Despite this setback, the commitment of Alnylam to the development of GalNAc-siRNA conjugates remain strong. Researchers strategically embed chemical modifications within the siRNA structure to enhance stability against nuclease activity.⁶³ Subsequent FDA approvals of Givosiran⁶⁴ and Lumasiran,⁶⁵ marking the second and third siRNA drugs in this category, serve as corroborative evidence affirming the viability and safety of GalNAc-conjugated, subcutaneously delivered siRNAs. These drugs not only demonstrate tolerability but also lead to a significant reduction in target mRNA levels, establishing a favorable hazard profile for therapeutic applications. Quark Pharmaceuticals, for example, has been at the forefront of therapeutic interventions for kidney injury (QPI-1002) and ocular conditions (QPI-1007). Presently, a notable paradigm shift is occurring within the pharmaceutical industry, with a swift redirection of focus toward RNAi-mediated drugs tailored for treating cancers. The advent of SiG12D LODER (Local Drug EluteR), characterized by a biodegradable polymeric matrix encapsulating KRASG12D siRNA (siG12D), represents a significant development for pancreatic ductal adenocarcinoma (NCT01188785) treatment.⁶⁶ Additionally, the development of TKM-08030, functioning as an inhibitor for Plk1, is tailored for treating hepatocellular carcinoma, while Atu027, targeting protein kinase N3, is designated for the treatment of solid tumors at advanced stages.⁶⁷

miRNAs and mimics

miRNAs can be regulated through the use of miRNA inhibitors, also known as anti-miRs, and miRNA mimics. These tools can either downregulate or upregulate miRNAs. Promising anti-miRs such as Miravirsen (SPC3649) and RG-101 have been developed to target miR-122, showing potential in treating infections caused by the hepatitis C virus.⁶⁸ In the realm of cancer therapeutics, MRX34 has emerged as an miR-34a mimic, representing a promising miRNA medication tailored for cancer targeting.⁶⁹ Despite their promise, none of these agents are currently in clinical use. A significant breakthrough has been achieved with the introduction of MTL-CEBPA, the first self-amplifying RNA (saRNA) to progress into clinical trial stages. This innovative therapeutic modality has the capacity to regulate hepatic and myeloid functions, influencing various cancer-related mechanisms via the upregulation of the CCAAT/enhancer-binding protein alpha (C/EBP-α) transcription factor.^70,71 The promising results from these trials have set the stage for exploring the synergy between MTL-CEBPA and an anti-PD-1 checkpoint inhibitor or radiofrequency ablation in the treatment of solid tumors, thereby propelling the initiation of a clinical trial to investigate the efficacy of this combinatorial approach.⁷²

Aptamers

Aptamer-based therapeutics employ two primary strategies. The first strategy involves utilizing antagonist aptamers to disrupt interactions between disease-linked targets, such as protein-protein or receptor-ligand interactions. Notably, this is the approach adopted by all current aptamers in clinical trials.⁷³ The second strategy involves aptamers with specificity based on cell type, employing them as vehicles to deliver other pharmacologic substances to specific tissues or cells.⁷⁴ A significant milestone in the field of aptamer drugs is Pegaptanib, also known as Macugen. It stands as the first aptamer drug to receive FDA approval, specifically designed to target VEGF in treating age-related macular degeneration.⁷⁵ The successful development of Pegaptanib has paved the way for numerous aptamers currently progressing through preclinical or clinical development phases. These aptamers show potential applications across a spectrum of diseases, including coagulation disorders, visual disorders, inflammatory conditions, and oncology.⁷³ This highlights the expanding scope of aptamer-based therapeutics in the medical landscape. For a comprehensive list of FDA-approved biologics (update until 2023), refer to Table S1.

CRISPR-Cas-based genome therapy

The application of CRISPR-associated protein (Cas) systems involves the utilization of an RNA-guided Cas nuclease along with a designed guide RNA (gRNA). The formation of the Cas-gRNA ribonucleoprotein complex occurs as the gRNA binds to Cas, recognizing a 20-nucleotide within the target and the protospacer-adjacent motif (PAM) element. Subsequently, the Cas nuclease cleaves either the dsDNA or an ssRNA at the specific site, enabling efficient genome editing.⁷⁶ These early successes have paved the way for innovative methods targeting and manipulating nucleic acids, including derivatives of Cas13 and Cas9 orthologs.⁷⁷ The Cas9 system can target both ssDNA and dsRNA. For example, the Streptococcus pyogenes Cas9 (RCas9) system necessitates a corresponding gRNA and a PAM-presenting oligonucleotide (PAMmer) that is complementary.⁷⁸ Other Cas9 orthologs, such as those from Staphylococcus aureus and Campylobacter jejuni, can cleave ssRNA in the absence of a PAM.⁷⁹ Conversely, RNA is the sole target of the Cas13-based systems. This is achieved by guiding Cas13 to specific RNA sequences using a CRISPR RNA (crRNA). In vitro experiments validated the ability of Cas13a, Cas13b, and Cas13d to interfere with and silence target RNA in mammalian cells. Among these, a subtype of Cas13d called CasRx (RfxCas13d) demonstrated the greatest efficiency in RNA knockdown in HEK293T cells.⁸⁰ To cleave RNA molecules that are both target and non-target, two conserved higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains may require a protospacer flanking sequence (PFS).⁸¹ Cas13d stands as a unique PFS-independent Cas variant. Meanwhile, a non-catalytic iteration of Cas13b, devoid of endonuclease activity, can elicit the A-to-I base switch when fused with the ADAR2 deaminase domain (ADAR2DD).⁸²

The first-ever gene-editing trial clinically conducted on humans involved the CRISPR-Cas system, focusing on the ex vivo application of Cas9 to knock out PD-1 expression in autologous T cells (NCT03399448).⁸³ In the realm of genetic diseases, the first-ever trial on humans for treating β-thalassemia with the CRISPR-Cas system (NCT03655678) was initiated. In the domain of retinal defects, the pioneering trial leveraging the gene-editing capability of CRISPR-Cas was conducted under the identifier EDIT-101 (NCT03872479).⁸⁴ These clinical trials employing CRISPR-Cas-based gene editing have laid crucial groundwork, serving as a cornerstone for subsequent genome-editing endeavors, including those utilizing zinc-finger nucleases, as reflected in the continuously evolving landscape of clinical research.⁸⁵ Current updates of CRISPR-Cas-based gene-editing medicine are summarized in Table S2.

Part 4: RNA-based vaccine

The use of traditional vaccine strategies, encompassing approaches such as utilizing inactivated or attenuated pathogens, and vaccines developed from pathogenic subunits, has proven highly effective in conferring long-lasting protection against various formidable diseases.⁸⁶ However, despite these achievements, significant challenges persist in the development of vaccines targeting certain infectious pathogens, particularly those adept at evading the adaptive immune responses.⁸⁷

The utilization of mRNA offers several advantages over traditional vaccine approaches and vaccines mediated by DNA, particularly in terms of safety. One key advantage of this platform is its non-infectious nature, eliminating the risk of insertional mutagenesis or infections as it does not integrate into the host genome. Additionally, the in vivo half-life of mRNA can be regulated through various modifications and delivery methods, providing greater control over its degradation through normal cellular processes.^88,89,90,91 Furthermore, the intrinsic immunogenicity of mRNA can be further reduced to enhance its safety profile. Moreover, the efficacy of mRNA vaccines is improved through a variety of modifications that enhance mRNA stability and translatability.^88,91,92 Efficient in vivo delivery of mRNA is facilitated by its formulation into carrier molecules, enabling rapid uptake and expression in the cytoplasm.^89,90 Being a minimal genetic vector, mRNA vaccines circumvent concerns related to anti-vector immunity, allowing for repeated administration. Last, production is a notable advantage of mRNA vaccines due to their potential for scalable manufacturing at a rapid rate and reduced cost, primarily due to the high outputs achievable through in vitro transcription reactions.

mRNAs and mRNA-based vaccine

The exploration of mRNA-encoded drugs traces its origins to the 1990s, marked by the demonstration of protein expression through the direct administration of in vitro transcribed (IVT) mRNA into the skeletal muscles of mice.⁹³ Subsequent to this discovery, extensive preclinical investigations into IVT mRNA laid the foundation for the clinical development of mRNA vaccines targeting both cancer and infectious diseases.⁹⁴ Mechanistically, mRNA vaccines are administered by introducing mRNA into the cytoplasm of host cells, typically antigen-presenting cells (APCs). Upon translation within these cells, targeted antigens are produced and presented on the surface of APCs by major histocompatibility complexes (MHCs). This process activates humoral immunity mediated by B cells and antibodies, as well as cellular immunity mediated by CD4⁺ T and CD8⁺ cytotoxic T cells.⁹⁵ Additionally, mRNA encoding immunostimulants, such as cytokines and chemokines, can facilitate the maturation and activation of APCs, thereby triggering a T cell-mediated response and enhancing the immune tumor microenvironment.⁹⁶ The application of mRNA-based cancer vaccines has gained prominence, with over 20 vaccines undergoing clinical trials to explore their potential in preventing solid tumors, including non-small cell lung cancer, colorectal carcinoma, and melanoma. These clinical trials often involve cytokine cocktails or the co-administration of mRNA cancer vaccines alongside checkpoint modulators (CTLA-4, TIM3, and PD-1) to enhance efficacy in their antitumor activities. The majority of cancer vaccines are therapeutic, aiming to stimulate cell-mediated responses, particularly from cytotoxic T lymphocytes (CTLs), which can eliminate or reduce tumor burden.⁹⁷ The concept of RNA cancer vaccines and their feasibility were first demonstrated over 2 decades ago in the initial proof-of-concept studies.^98,99 Since then, a wealth of preclinical and clinical research has substantiated the feasibility of mRNA vaccines as a promising avenue to combat various forms of cancer (please refer to Box 3 for technological advances in mRNA-lipid nanoparticle [LNP] vaccine development).

Dendritic-cell-based mRNA cancer vaccines

Dendritic cells (DCs) play a pivotal role in initiating antigen-specific immune responses, making them a rational choice for cancer immunotherapy. Pioneering work by Boczkowski and colleagues in 1996 demonstrated that electroporation of mRNA into DCs could elicit robust immune responses against tumor antigens.⁹⁹ In this seminal study, mRNA encoding ovalbumin (OVA) or RNA derived from tumors stimulated immune responses leading to reduced tumor growth in melanoma mouse models expressing OVA. The efficacy of DC cancer vaccines has been significantly augmented by incorporating mRNA-encoded adjuvants, acting as immune regulatory proteins. Numerous studies have shown that electroporating DCs with mRNA encoding co-stimulatory molecules, such as tumor necrosis factor receptor superfamily member 4 (TNFRSF4), 4-1BB ligand (4-1BBL), and CD83, enhances the immune stimulatory activity of DCs.^{65,100,101,102} Additionally, DC functions can be modulated by pro-inflammatory cytokines (i.e., interleukin [IL]-12) or trafficking-associated molecules encoded by mRNA.^103,104,105 Preclinical studies indicate that TriMix, a combination of mRNA-encoded adjuvants (constitutively active TLR4, CD40L, and CD40L), effectively enhances DC activation and phenotypical transformation of CD4⁺ T cell toward T helper 1 (TH1)-like cells.^{106,107,108,109} Administration of DCs loaded with mRNA encoding melanoma-associated antigens and TriMix adjuvant to patients with advanced melanoma achieved tumor regression in 27% of treated individuals, as reported in a study.¹¹⁰ Clinical trials have been conducted utilizing DC vaccines targeting various cancer types, including brain cancers, pancreatic cancer, acute myeloid leukemia, metastatic lung cancer, renal cell carcinoma, metastatic prostate cancer, melanoma, and others.^111,112 In one study, ipilimumab (a monoclonal antibody against CTL antigen 4, CTLA4) and DCs loaded with mRNA encoding melanoma-associated antigens, combined with TriMix, were administered to patients with advanced melanoma. This approach resulted in sustained tumor reduction in a subset of individuals with recurrent or refractory melanoma.¹¹³ Current clinical trials of mRNA-based vaccines are summarized in Table 1.

Table 1.

Clinical trials involving mRNA vaccines for cancer therapy

Vaccine type	Targets	Trial numbers (phase)
Non-loaded mRNA
Naked tumor-associated antigens (TAA) or neo-Ag mRNA	Melanoma	NCT01684241 (I) NCT02035956 (I)
RNActive TAA mRNA	Non-small-cell lung cancer	NCT00923312 (I/II) NCT01915524 (I)
BNT111	Stage 4 melanoma, unresectable melanoma	NCT04526899 (II)
Protamine-complexed TAA mRNA with GM-CSF protein	Melanoma	NCT00204607 (I/II)
RNActive∗ TAA mRNA	Prostate cancer	NCT00906243 (I/II)
Autologous tumor mRNA with GM-CSF protein	Melanoma	NCT00204516 (I/II)
DC-loaded mRNA vaccine
DC (electroporated [EP]) with TAA mRNA	Acute myeloid leukemia (AML)	NCT00834002 (I) NCT01686334 (II)
	AML, CML, multiple myeloma	NCT00965224 (II)
	Multiple solid tumors	NCT01291420 (I/II)
	Mesothelioma	NCT02649829 (I/II)
	Glioblastoma	NCT02649582 (I/II)
DC loaded with TAA mRNA	AML	NCT00510133 (II)
DC (pulsed) with human CMV pp65-LAMP mRNA	Glioblastoma	NCT03688178 (II)
DC, matured, loaded with TAA mRNA	Melanoma	NCT01216436 (I)
DC loaded with TAA mRNA	Glioblastoma	NCT02808364 (I/II) NCT02709616 (I/II)
	Brain metastases	NCT02808416 (I/II)
DC loaded with TAA mRNA	Breast cancer, melanoma	NCT00978913 (I)
	Prostate cancer	NCT01446731 (II)
DC, matured, loaded with TAA mRNA	Ovarian cancer	NCT01456065 (I)
DC loaded with TAA and CMV Ag mRNA	AML	NCT01734304 (I/II)
DC (Langerhans) EP with TAA mRNA	Melanoma	NCT01456104 (I)
	Multiple myeloma	NCT01995708 (I)
DC loaded with autologous tumor or TAA mRNA	Melanoma	NCT00961844 (I/II) NCT01278940 (I/II)
	Prostate cancer	NCT01197625 (I/II) NCT01278914 (I/II)
	Glioblastoma	NCT00846456 (I/II)
	Ovarian cancer	NCT01334047 (I/II)
DC EP with TAA mRNA	Colorectal cancer	NCT00228189 (I/II)
	Melanoma	NCT00929019 (I/II) NCT00243529 (I/II) NCT00940004 (I/II) NCT01530698 (I/II) NCT02285413 (II)
DC EP with TAA and TriMix mRNA	Melanoma	NCT01066390 (I) NCT01302496 (II) NCT01676779 (II)
DC loaded with TAA mRNA	AML, myelodysplastic syndromes	NCT03083054 (I/II)
DC (electroporated) with autologous tumor mRNA with or without CD40L mRNA	Renal cell carcinoma	NCT01482949 (II) NCT00678119 (II) NCT00272649 (I/II) NCT01582672 (III) NCT00087984 (I/II)
	Pancreatic cancer	NCT00664482 (NA)
	Prostate cancer	NCT02140138 (II) NCT00831467 (I/II) NCT01817738 (I/II)
DC loaded with CMV Ag mRNA	Glioblastoma, malignant glioma	NCT00626483 (I) NCT00639639 (I) NCT02529072 (I) NCT02366728 (II)
DC loaded with autologous tumor mRNA	Glioblastoma	NCT00890032 (I)
DC loaded with AML lysate and mRNA	AML	NCT00514189 (I)
DC, matured, loaded with autologous tumor RNA	Melanoma	NCT01983748 (III)
DC loaded with CMV Ag mRNA with GM-CSF protein	Glioblastoma, malignant glioma	NCT02465268 (II)
Autologous monocyte-derived DC-loaded WT1 mRNA	High grade Glioma	NCT04911621 (I/II)
Liposome-based mRNA vaccine
Liposome-complexed TAA mRNA (Lipo-MERIT)	Melanoma	NCT02410733 (I)
Liposome-formulated TAA and neo-Ag mRNA	Breast cancer	NCT02316457 (I)
Liposome-formulated W-ova1	Ovarian cancer	NCT04163094 (1)
Liposome-formulated (NCI)-4650	Melanoma, colon, gastrointestinal, genitourinary, Hepatocellular carcinoma, Squamous Cell, Head and neck cancer	NCT03480152 (I/II)
Liposome-formulated mRNA-4157	Melanoma	NCT03897881 (II)
RNA-lipid particle (RNA-LP)	Adult Glioblastoma	NCT05473140 (I)

Circular RNA-based vaccine

The in vitro synthesis of circRNA has witnessed notable advancements, paving the way for the development of next-generation RNA-based vaccines. In 2022, circRNAs were engineered to express pertinent antigens, eliciting adaptive immune responses and demonstrating therapeutic effects in various diseases. Notable instances include the encoding of chicken OVA for the treatment of melanoma malignancy¹¹⁴ and the encoding of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IL-12, and IL-15 to enhance anti-PD-1 antibody-mediated tumor suppression.¹¹⁵ It is noteworthy that, beyond their translation into proteins for disease treatment and prevention, artificial circRNAs can also exert an antitumor role through their non-coding functions. The details of the discovery of a circRNA vaccine are elucidated in Figure 3.¹¹⁶

Figure 3.

Development of a circRNA vaccine production pipeline and induction of immune response through disease-specific targeted antigen strategies

The circRNA vaccine production pipeline entails the design of encoding sequences for peptides/proteins, subsequently cloned into a plasmid DNA construct. Plasmid DNA is transcribed into a linear RNA precursor (pre-circRNA) using in vitro transcribed (IVT) technology. The pre-circRNA is then cyclized in vitro to form circRNA, followed by purification using high-performance liquid chromatography (HPLC). The purified circRNA is encapsulated in various vehicles (i.e., viral-like particles, liposomes). Prior to clinical trials, comprehensive biosafety and pharmacodynamic evaluations are imperative. The scale-up manufacturing of circRNA vaccine precedes clinical trials. Upon initiation of antigen-specific immune responses, intracellular processes of circRNA vaccines focus on immune initiation, antigen encoding, and endosome escape within antigen-presenting cells (APCs): (1) Endosomes are formed by circRNA vaccines-containing LNPs in the cytoplasm. (2) Subsequently, the circRNA vaccine is released from the endosomes. (3) The encoding sequences in circRNA then undergo translation, yielding proteins or peptides with antigenic properties. (4) Endogenous antigens undergo degradation by proteasomes, resulting in the formation of polypeptides subsequently presented by MHC I, (5) ultimately activating cytotoxic CD8⁺ T cells. Humoral immunity initiated by circRNA is as crucial, (6) due to the secretion of endogenous antigens eventually presented to helper T cells by MHC class II proteins. In turn, these helper T cells (CD4⁺ T cells) stimulate the production of neutralizing antibodies by B cells.

In contrast to conventional linear mRNA vaccines, circRNAs offer several advantages. Notably, circRNAs exhibit superior stability and storage characteristics when compared with mRNA vaccines, which are prone to degradation by RNases during delivery, transportation, and storage.¹¹⁷ While nucleotide modifications can enhance mRNA stability, they concurrently escalate costs and manufacturing complexities. Additionally, mRNA vaccines still necessitate a low-temperature cold chain for storage due to suboptimal thermostability.¹¹⁸ In contrast, unmodified circRNAs demonstrate high stability and resistance to RNase, allowing for storage at room temperature or under repeated freeze‒thaw conditions.^119,120,121 Unmodified circRNAs manifest fewer side effects compared with mRNA vaccines, which can induce cytotoxicity and side effects attributed to their high immunogenicity.^122,123 Even modified mRNA, designed to mitigate excessive immunogenicity, exhibits higher immunogenicity and cytotoxicity than unmodified circRNA.^124,125 The extended antigen-yielding capabilities of circRNAs contribute to prolonged antigen production and retention in APCs.^126,127 This prolonged antigen presentation enhances the triggering of adaptive immune responses, leading to increased neutralizing antibody production.^{114,119,128,129,130} This feature contributes to the sustained efficacy of circRNA-based vaccines. Another interesting fact about circRNA is that they are capable of encoding cryptic peptide that could serve as TSAs.¹³¹ However, it is important to note that while these circRNA-based therapeutic platforms show promise, they do not confer a sustained therapeutic effect and lack the prophylactic efficacy characteristic of vaccines. Consequently, the development and advancement of circRNA vaccines remain in the early stages of development.

Part 5: Challenges and advancements in RNA therapeutics

Challenges in precision RNA screening

Precision in RNA screening poses inherent challenges that are integral to the analytical validity of laboratory testing. The accuracy of a test is established through the comparison of a calculated or measured value against a reference value, commonly known as the “gold standard.” Reproducibility, a crucial aspect, mandates the test to consistently yield identical or comparable results upon repeated testing. Moreover, the test must demonstrate robust performance by withstanding minor, intentional alterations in pre-analytic or analytic factors associated with the testing process. These criteria collectively ensure the analytical soundness and reliability of laboratory tests.

The integration of RNA-seq into clinical laboratories encounters substantial hurdles in the realm of bioinformatics. Three predominant themes contributing to what can be termed as “analysis paralysis” in the development of bioinformatics-based solutions for RNA-seq are highlighted: (1) lack of consensus on best practices—the absence of consensus among governing bodies regarding reference standards and optimal methodologies for validating RNA-seq pipelines creates uncertainty and impedes progress; (2) abundance of software tools—the plethora of available software options, tools, or even combinations for RNA-seq analysis presents a challenge, whereby the sheer number of choices can be overwhelming and may contribute to difficulties in decision-making; and (3) complexity of pipelines—highly intricate pipelines, involving the concatenation of multiple independently developed, maintained, and licensed tools, contribute to the challenge, wherein coordinating these tools and ensuring compatibility can be a daunting task. Addressing these challenges is imperative for streamlining RNA-seq analysis and fostering its broader adoption in clinical laboratories.

Challenges in enhancing RNA stability

The effective delivery of RNAs is a vital aspect of engineering them for therapeutic applications. Various modifications have been developed to enhance cellular uptake, intracellular distribution, and stability. These modifications fall into two broad categories: formulation-based modifications and chemical modifications. Formulation-based modifications involve the utilization of delivery vehicles to encapsulate RNAs or adjusting their formulation to augment delivery efficiency. On the other hand, chemical modifications entail structural alterations to the molecular composition of RNAs.¹³² The goal of these modifications is to overcome barriers such as suboptimal cellular uptake, off-target effects, and enzymatic degradation, thereby making RNA-based therapies more effective and specific. This field is dynamic, with ongoing research aimed at optimizing delivery strategies for diverse therapeutic applications. However, the clinical application of siRNA molecules has faced limitations due to challenges associated with their immunogenicity, cellular uptake, and stability. To overcome these obstacles, chemical modifications have been introduced to enhance the properties of siRNA. These modifications can be broadly classified into two categories: phosphate backbone modifications and nucleotide modifications.¹³³ Collectively, these chemical modifications contribute to improved siRNA properties, addressing challenges related to stability, immunogenicity, and cellular uptake. The selection of specific modifications is contingent on the intended therapeutic outcome and the targeted application.

Advancements in RNA therapeutics: Targeting the RNA “life cycle”

The production of RNA binders presents a daunting challenge and may not consistently elicit the desired biological effects. Therefore, a novel approach involves targeting various stages of the RNA life cycle, encompassing biogenesis, maturation, localization, activity, or degradation, as proposed by Martin and his colleagues.²⁸ This strategy serves as a supplementary avenue to the direct development of RNA binders, offering the potential to influence cellular processes associated with “undruggable” RNAs or proteins that are challenging to target directly. The focus is directed toward mRNA molecules associated with such RNAs, providing a promising alternative for therapeutic intervention.

Modulation of RNA biogenesis

FDA-approved compounds have demonstrated notable efficacy in the precise targeting of spliceosomal components. Additionally, modulators of transcription that selectively bind RNA G-quadruplexes (G4) or RNA/DNA complexes, denoted as R-loops, have been successfully identified through the implementation of affinity, computational, and mechanistic-based screening approaches.^134,135,136 It is imperative to underscore that medications directed toward RNA/RNP interactions may exert a secondary influence on transcriptional processes. A case in point is the discernible impact on the localization and processing of HIV-1 RNA brought about by Rev-RRE inhibitors, consequently leading to concurrent transcriptional inhibition. Finally, the manipulation of transcriptional regulation can be accomplished through CRISPR-Cas9 RNP inhibition or potentiation.¹³⁷

Modulation of RNA maturation

The RNA epigenome has recently emerged as a potential target for therapeutic intervention, showcasing more than 170 chemical modifications associated with various diseases.^138,139 Current screening strategies focus on RNA-modifying enzymes, with the goal of inhibiting physiological RNA modifications. In addition, the use of ectopic ribonucleoproteins, exemplified by CRISPR-Cas13a, allows for the introduction of novel RNA modifications.^140,141 These approaches present promising avenues for the manipulation of the RNA epigenome and the formulation of efficacious therapeutic interventions.

Modulation of RNA localization and activity

The proper organization of RNA within nuclear bodies and its subsequent transport to the cytoplasm play crucial roles in upholding chromatin stability and ensuring efficient translation, respectively. Recent screening endeavors have strategically focused on machineries governing both nuclear export and nuclear retention. These efforts have led to the identification of novel compounds capable of influencing a range of functional RNA interactions, including viral RNPs, riboswitches, or internal ribosome entry site (IRES) motifs. Additionally, compounds that bind to the untranslated regions of coding transcripts may exert an impact on their folding, thereby inducing alterations in ribosome recognition and, ultimately, translation.¹⁴²

Modulation of RNA degradation

Precise regulation of transcript levels is achieved through the indispensable process of RNA degradation. In this context, screening efforts have revealed mimics of ribonuclease catalysis designed to drive effective RNA targeting and degradation. One such mimic, designed against RNA triplet repeats, is based on ribonuclease A (RNase A) and has demonstrated substantial efficacy in targeting and degrading target-specific mRNAs.¹⁴³ Additionally, compounds derived from Inforna, when coupled with synthetic RNase L mimics, have been shown to exhibit enhanced miRNA inhibition.^144,145 Furthermore, the use of bleomycin A5 glycopeptide, coupled with RNA inhibitors, holds promise for enhancing their efficacy via metal-based catalysis.^146,147 It is worth emphasizing that the mimics of RNase L selectively function within the cytoplasm, where RNase L is present, while conjugates involving bleomycin A5 operate actively both in the nucleus and the cytoplasm.¹⁴⁸ This distinction underscores the potential versatility and targeted action of these RNA degradation strategies.

Conclusion and future prospects

RNA molecules exhibit remarkable versatility, holding immense, largely unexplored potential for clinical applications as therapeutics and vaccines. The lower production cost and simplified manufacturing processes associated with RNA-based drugs facilitate rapid development compared with traditional protein or small molecule-based drugs. Nevertheless, the successful production of optimal RNA therapeutics necessitates the attainment of selective on-target specificity while mitigating undesirable off-target effect. Besides, it is imperative to prioritize early-stage design considerations to detect acute toxicity of RNA molecules, complementing assessments of immune tolerance, pharmacokinetics, and pharmacodynamics. A comprehensive approach, integrating multi-omics and interdisciplinary techniques such as single-cell sequencing and high-throughput screening, can refine RNA therapeutics to meet the unique requirements of each patient. This personalized therapeutic approach instils significant promise for the utilization of RNA medicines and vaccines in addressing various human diseases.