RNA-based Therapeutics and The Future of RNA Therapy

Dilan Ağırbaş^A., Ebru Kavaklı^E.

RNA therapy is a type of treatment that regulates biological pathways with RNA molecules to remedy a particular disease¹.  Manipulating gene expression and producing therapeutic proteins are fundamental functions of RNA therapies. Moreover, it can convert the therapeutic proteins appropriate for cancers, infectious diseases, immune system disorders, and pathologies with defined genetic targets². Compared to the drugs that can only target a limited number of proteins contributing to disease pathways, RNA-based therapeutics show promise in increasing the number of ‘druggable’ targets³. Inhibitors of mRNA translation (antisense), RNA interference (RNAi) agents, ribozymes, messenger RNA (mRNA), and aptamers are the main types of RNA therapeutics that will be clarified in this paper^4,5.

RNAi (Small Interfering RNA, MicroRNA)

The internal cellular process that utilizes double-stranded RNAs (dsRNAs), which are a crucial trigger in the body’s natural immune system against viral infections, to silence genes in the gene expression is called RNAi^6-8.

Small Interfering RNA (siRNA)

siRNAs that have 5′-phosphate/3′-hydroxyl ends and two 3′-overhang ribonucleotides on each duplex strand belong to the group of short dsRNAs^9,10. Based on the complementarity of the sequences, siRNAs bind to their target transcript and bring out an RNAi response¹¹. Dicer proteins associated with RNase III initiate the RNAi mechanism by cleaving a dsRNA into shorter duplexes¹². RNA-induced silencing complex (RISC), a multi-protein component complex, binds to the resulting siRNAs¹³. The siRNA is then cleaved into strands within the RISC complex, and the stable 5′ end strand amalgamates with the active RISC complex¹⁴. The RISC complex located on the target mRNA is positioned by antisense single-stranded siRNA. Next, mRNA cleavage occurs by the action of the catalytic RISC protein, a member of the argonaute family (Ago2) (Figure 1)¹⁵. siRNAs have been used to study the effect of different oncogenic long non-coding RNAs (lncRNAs) on loss of function. As a result of the studies, it was found that siRNAs have a silencing property against lncRNA HOX transcript antisense RNA (HOTAIR), whose role is to foster metastasis in cell lung cancer ¹⁶.

Figure 1. Mechanism of gene silencing via siRNA in eukaryotic cells with different pathways¹⁵. 1a) DICER cleaves dsRNA/shRNA into mature siRNA (Endogenous pathway). 1b) Synthetic siRNA enters via endosome, released into the cytoplasm (Exogenous pathway). 2) AGO protein separates strands. 3) The passenger strand is cleaved. 4) RISC complex selects guide strand for mRNA alignment. 5) mRNA is cleaved to silence the target gene.

miRNA

This RNA strain, which belongs to the class of non-coding RNA and is ~22-nt in length, controls gene expression and causes gene silencing by binding to target mRNAs after transcription^17,18. Primary miRNA (pri-miRNA) is formed from miRNA genes transcribed by the enzyme RNA polymerase II in the nucleus. Then, RNase III Drosha and microprocessor complex subunit DiGeorge syndrome critical region 8 (DGCR8) cleave the pri-miRNA, resulting in a precursor miRNA (pre-miRNA) containing 20–25 nucleotides^19,20. Subsequently, Exportin 5 delivers the pre-miRNA from the nucleus to the cytoplasm, and the Dicer enzyme converts the pre-miRNAs transported into the cytoplasm into an 18-25 nucleotide miRNA duplex^21,22. The miRNA duplex then joins the RISC to create the miRISC complex. In contrast to the processing of siRNA, where the RISC’s AGO2 leads to the cleavage of the siRNA’s passenger strand, the miRNA duplex opens, releasing and discarding the passenger strand. miRISC complex is directed to target mRNAs by mature single-stranded miRNAs²³. The miRNA connects to the target mRNAs, silencing the target gene with translational repression, degradation, and/or cleavage through partial complementary base pairing (Figure 2)²⁴.

In the process of the growth and spread of tumors, miRNAs either behave as tumor suppressors or oncogenes. The research found that controlling miRNA modifications via miRNA mimics can restore the gene regulatory network and alter the phenotypes in cancer cells²⁵. Numerous miRNA-targeted therapeutics are now in the clinical development stage. These include miR-122 antagonists that have entered phase II trials for treating hepatitis and a mimic of the tumor suppressor miRNA miR-34 that has reached phase I clinical trials²⁶.

Figure 2. Biogenesis and mechanism of action of microRNAs²⁴.

Antisense Oligonucleotides (ASOs)

ASOs are complementary nucleic acid fragments created to hybridize to a complementary endogenous pre-mRNA or mRNA through Watson-Crick base pairing²⁷. There are two discrete ways that ASOs function. The first involves RNA degradation, while the second entails RNA inhibition or modulation via steric hindrance^28-30.

In the RNA degradation, target RNA is bound by ASO, resulting in RNA/ASO duplexes. RNaseH1, an endonuclease that cleaves the phosphodiester bonds of RNA, is recruited by these duplexes. This process occurs for mRNA in the cytoplasm^31,32. 5′-3′ exoribonucleases (XRNs) and the exosome complex cleave the RNA fragments after RNaseH1 cleavage. Researchers discovered that ASO gene silencing was abolished by observing that the pre-RNA levels of the same gene increased with increased transcription as the target mRNA in the ribosomes was cleaved by the recruited RNase H1³³. In the RNA steric hindrance, RNA/ASO duplexes are rendered resistant to endonuclease cleavage by added sugar modifications to phosphorothioate-modified ASOs³⁴. Failure to assemble the initiation complex at the AUG start codon occurs because of the binding of such duplexes within the 5′ untranslated region of the mRNA, and mRNA translation is sterically stopped (Figure 3)³⁵. Furthermore, by averting 5′ capping or directing splice site selection, steric hindrance can control how pre-mRNA is processed in the nucleus. Steric inhibitory ASOs can also be used to increase the expression of therapeutically effective proteins³⁶. The interaction of the preinitiation complex with the primary open reading frame (ORF) is enhanced by ASOs that block upstream open reading frames in mRNA. In consequence, protein expression is increased³⁵.

Figure 3. Mechanism of action of ASOs³⁵. ASOs facilitate cleavage of pre/mRNAs via RNaseH1. Additionally, exon splicing, interaction of mRNA with ribosomal subunits (40S and 60S) and 5′ capping of the pre-RNA processes are blocked by ASOs.

Aptamers

Oligonucleotide sequences that behave similarly to monoclonal antibodies and are 25-80 bases in length are called Aptamers³⁷. Usually, they bind to specific protein targets by transforming into various 3D structures^38,39. A conceptually simple iterative selection procedure known as SELEX is used to isolate Aptamers from a pool of nucleic acids^40-42.

The US Food and Drug Administration (FDA) has approved only one RNA-based aptamer drug to date. This drug is pegaptanib, a 28-nucleotide construct with two polyethylene glycol (PEG) fragments attached to the end^43,44. To suppress cell proliferation, the main downstream effect of vascular endothelial growth factor (VEGF) signaling, pegaptanib binds to the 165 isoform of VEGF and blocks its interaction with the receptor protein (Figure 4)⁴⁵. In the context of this effect, pegaptanib was designed as a therapeutic agent to treat wet-type (neovascular) age-related macular degeneration (AMD), an illness that causes damage to the central retina^46,47. Pegaptanib is a good example of how RNA-based aptamers can be used as therapeutics, despite being infrequently used nowadays owing to competition from numerous antibody-based drugs with comparable efficacy⁴⁸.

Ribozymes

Catalytic RNA molecules that can reduce the expression of targeted genes by breaking down certain RNA sequences are called ribozymes⁴⁹. Although there are several classes of ribozymes, hammerhead ribozyme (HHRz) has received significant attention due to being one of the smallest ribozymes⁵⁰.

Figure 4. Interaction of Macugen (pegaptanib) with its target protein and its structural model⁴⁵. a) Based on the secondary structure determined by NMR, the pegaptanib structure model was presented. b) Interface of target protein VEGF165 and aptamer. Yellow parts were used to depict the VEGF165 surface and structure. The blue areas are residues that have undergone conformational change on pegaptanib. The VEGF165 residues that were involved in the complex’s interaction were highlighted in red and identified in black (R13, R14, K15, H16).

The catalytic core (22 nt-long) that controls the cleavage reaction and three hybridizing helices, two of which surround the catalytic core and utilize Watson-Crick base pairing to bind the target RNA in an antisense approach are the parts that make up the HHRz^51,52. Target RNA that contains NUH sequences, where N is any base and H is A, U, or C, is typically recognized by HHRz (Figure 5)⁵³. Target sites with the GUA, GUC, GUU, CUC, and UUC sequences are preferred the most. By immediate 3′ cleavage of the selected RNA into the NUH sequence, hammerhead ribozymes’ primary mechanism of action in repressing target gene expression is activated. This action results in two shorter types of RNA that lack stabilizing sequences, undergo rapid degradation, and cause a progressive decrease in protein synthesis^50,54. Compared to other antisense molecules in RNAi systems, a single ribozyme can recognize and bind more than one target mRNA^55,56. Ribozymes typically mediate multiple-turnover and sequence-specific reactions, and both features make these molecules appropriate candidates for therapeutic use⁵⁷. Targeting the 5′ URT of the hepatitis C virus (HCV) RNA genome, Heptazyme is one of the antiviral ribozymes that have made it to the clinical stage⁵⁸. Despite showing success in clinical trials, additional research is needed to enhance the stability, effectiveness, and safety of ribozymes⁵⁹.

Figure 5. The secondary structure of a hammerhead ribozyme model⁵³.

mRNA

It is a type of RNA obtained by copying from genomic DNA and whose function is to code proteins. The 5′ cap, 5′ untranslated region (UTR), coding region, 3′ UTR, and poly(A) tail are the components of mRNAs^60,61.

Direct injection of mRNA into mice resulted in successful transfection and the production of an immune response, allowing the expression of therapeutic proteins^61,62. This success led to the development of mRNA-based therapeutics. Infectious disease vaccines, genetic engineering, cancer immunotherapy, protein replacement therapy, and mRNA-based gene editing are the main application areas where mRNA is used today⁶³. mRNA-based treatments can be divided into two categories according to their purposes: enzyme replacement therapy and vaccine against infectious diseases or cancer antigens^60,64. Lipid nanoparticles (LNPs), which are lipid-based carrier systems, encapsulate mRNAs that will encode the target protein⁶⁵. The resulting LNP-mRNAs reach the cytosol via endocytosis. Based on the mRNAs, proteins are translated inside the ribosomes (Figure 6)⁶⁶. In vitro transcribed (IVT) mRNA protein replacement is functional in non-genetic disease conditions. IVT mRNA that encodes vascular endothelial growth factor-A (VEGF-A) could enhance blood flow, offering ischemic cardiovascular disease patients a regenerative treatment option⁶⁷. For cancer immunotherapy, mRNA-based dendritic cell (DC) vaccines are applied. These vaccines can direct cytotoxic T lymphocytes and natural killer cells into powerful anti-tumor weapons that can target tumor cells^68,69. In infectious diseases, IVT mRNA is delivered to the human body to translate antigenic proteins and stimulate the immune system against the pathogen^70,71.

Figure 6. Peptide epitopes are cleaved by proteasome antigenic proteins⁶⁶. To achieve cell-mediated immunity, CD8+ T cells are activated by loading these epitopes onto MHC I molecules. Secreted and endocytosed antigenic proteins are loaded onto MHC II located within endosomes. To help B cells produce antigen-specific antibodies for antibody-mediated immunity, MHC II-peptide complexes stimulate CD4+ T cells.

Several mRNA vaccine candidates for virus infections, including RSV (Respiratory Syncytial virus), influenza, Zika virus, and Cytomegalovirus (CMV), have entered clinical trials⁶⁶.

Future Prospects and Conclusion

The field of RNA therapeutics is rapidly expanding and is still in its early stages⁷². RNA drugs are currently approved for cardiovascular, metabolic, liver, infectious, neurological, neuromuscular, renal, and eye diseases, and therapeutic intervention has improved^73,74. The number of RNA medications in development and clinical trials is increasing quickly⁷⁵. As a result of clinical studies, it was discovered that ASOs demonstrate a high affinity toward targets based on one-dimensional sequence matches⁷⁶. Moreover, numerous ASO medications are currently being tested for genetic diseases and preventing complications from disease⁷⁷. The ASO drugs mipomersen, eteplirsen nusinersen, inotersen, golodirsen, viltolarsen, and casimersen, and the siRNA drugs patisiran, givosiran and

lumasiran are among the therapeutics that have been successfully marketed since 2013⁷⁸. Researchers expect that siRNA and mRNA will soon be utilized in clinical trials to treat respiratory diseases owing to recent successes with siRNA therapeutics and technological advancements in RNA modification⁷⁹. RNA therapeutics are poised to make an industry impact in a way diseases can be treated, thanks to approved treatment modalities in each of the major RNA therapeutic categories and significant research to improve their clinical use⁸⁰.

Drugs and treatment methods mentioned in the review article are based only on the information obtained from the articles. Please consult a specialist physician for diagnosis, treatment, and drug use.

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