RNA-based therapeutics
The Human Genome Project (HGP) has revealed a wealth of information about the human genome and has greatly enhanced its role in the development of biomedical research. Thanks to advances in next-generation sequencing technology, researchers have been able to reveal the role of a number of genetic factors in many diseases, such as cancer, rheumatoid arthritis, Parkinson’s disease and Alzheimer’s disease.
In addition, the results of many studies have revealed the important roles of coding and non-coding RNAs (ncRNAs), such as miRNAs, long ncRNAs (lncRNAs), circRNAs, and siRNAs. This lays the foundation for the development of possible treatments for various diseases by introducing nucleic acids into cells to permanently or transiently control gene expression. In addition, the development of various delivery systems has made it possible to transport RNA to the target site, solving the problems posed by the inherent instability of RNA. As a result, many RNA-based therapeutics involving siRNAs, ASOs, ribonuclease, mRNAs, aptamers, and CRISPR/Cas have been developed and are undergoing clinical trials for a variety of diseases. RNA-based drugs show excellent potential for applications.
| Drug | Molecule | Target | Disease | Reference |
| mRNA-1273 | mRNA | Spike (S) proten of SARS-COV-2 | COVID-19 | USFDA, (2021a) |
| BNT162b1 | mRNA | RBD of Spike glycoprotein | USFDA, (2021b) | |
| Patisiran | siRNA | Polyneuropathy | Hereditary transthyetin-mediated amyoidosis | Adams et al. (2018) |
| Givosiran | siRNA | ALAS1 | AHP | USFDA, (2019) |
| Pegaptanib | Aptamer | VEGF | AMD | Vinores, (2006) |
siRNA-based therapeutics
siRNA is produced through an endonuclease process by the ribonuclease Dicer, which is a RNase Family III endonucleases can produce 21-25 nucleotide double-stranded RNA. Once the siRNA is produced, Dicer will transfer it to the RNA-induced silencing complex (RISC), which contains Argonaute 2 that degrades the target mRNA molecule. Because of this ability of siRNAs, they are being used as a possible therapeutic approach.
siRNA therapies are being tested in a variety of cancer treatments. siG12D-LODER is a biodegradable polymer matrix containing siRNA against KRASG12D, which was shown in clinical studies (NCT01188785) to target tumors and inhibit tumor progression. A further study (NCT01676259) is currently underway to test the efficacy of siG12D-LODER in combination with chemotherapeutic agents, such as gemcitabine and nab paclitaxel, in treating patients with locally advanced pancreatic cancer.
Polo-like kinase (PLK) is involved in cell cycle regulation and cell proliferation, it is usually overexpressed in cancer cells and prevents cancer cell proliferation by inhibiting Plk. TKM-080301 is a lipid nanoparticle (LNP) formulation consisting of four lipids and a synthetic double-stranded siRNA targeting human PLK1 mRNA. Intravenous infusion of TKM-080301 showed good tolerability but only limited antitumor effects in patients with advanced hepatocellular carcinoma and no overall survival effect. However, TKM-080301 showed some tumor suppression in adrenocortical carcinoma (ACC).
Protein kinase N3 (PKN3) is a downstream effector of the phosphatidylinositol-3-kinase (PI3K) pathway, and blocking PKN3 inhibits tumor progression and lymph node metastasis.Atu027, a liposomal PKN3 siRNA formulation, has a favorable safety profile in patients with advanced solid tumors, with 41% of subjects in stable condition at the end of treatment. In subsequent studies, the combination of Atu027 and gemcitabine for the treatment of locally advanced or metastatic pancreatic cancer showed a favorable safety and tolerability profile.
| Name | Disease | Target | ClinicalTrials.gov Identifier | Recruitment Status | Phase | Reference |
| Inclisiran | Homozygous Famial | PCSK9 | NCT02597127 | Completed | Ⅱ | Ray etal. (2017) |
| Inclisiran | Hypercholesterole | PCSK9 | NCT03397121 | Completed | Ⅲ | Raal et al. (2020) |
| AGN211745 | CNV-AMD | VEGFR1 | NCT00363714 | Completed | Ⅰ&Ⅱ | Kaiser et al. (2010) |
| PF-04523655 | CNV-AMD | VEGFR1 | NCT00713518 | Completed | Ⅱ | Nguyen et al. (2012a) |
| PF-04523655 | Diabetic Macular Edema | RTP801 | NCT00701181 | Terminated | Ⅱ | Nguyen et al. (2012b) |
| QPI-1007 | Non-Arteritic Anterior Ischemic Optic Neuropathy | Caspase 2 | NCT01064505 | Completed | Ⅰ | Antoszyk et al. (2013) |
| siG12D LODER | Pancreatic Cancer | KRASG12D | NCT01188785 | Completed | Ⅰ | Golan et al. (2015) |
| siG12D LODER | NCT01676259 | Unknown | Ⅱ | Varghese et al. (2020) | ||
| TKM-080301 | Hepatocellular Carcinoma | PLK1 | NCT02191878 | Completed | Ⅶ | EI Dika et al. (2019) |
| TKM-080301 | Sold Cancer | NCT01262235 | Completed | Ⅶ | Demeure et al., (2016) | |
| Atu027 | Advanced Solid Tumors | Protein kinase N3 | NCT00938574 | Completed | Ⅰ | Schutheis et al. (2014) |
| Atu027 | Pancreatic Cancer | Protein kinase N3 | NCT01808638 | Completed | Ⅶ | Schultheis et al. (2016) |
| DCR-MYC | Solid Tumors, Multiple Myeloma, or Lymphoma | MYC | NCT02110563 | Terminated | Ⅰ | Tolcher et al. (2015) |
| CALAA-01 | Sold Tumor | RRM2 | NCT00689065 | Terminated | Ⅰ | Davis et al. (2010) |
| TD101 | Pachyonychia congenita | KRT6A | NCTOO7 16014 | Completed | Ⅰ | Leachman et al. (2010) |
| ARC-520 | Healthy | cocDNA-derived viral | NCT01872065 | Comploted | Ⅰ | Schluep et al. (2017) |
| ARC-520 | Chronic Hepatitis B | cocDNA-derived viral | NCT02452528 | Temminated | Ⅱ | Yuen et al. (2020) |
| QPI-1002 | Acute Renal Failure | p53 | NCT00554359 | Completed | Ⅰ | Demirjian et al. (2017) |
| QPI-1002 | Acute kidney injury | NCT00802347 | Completed | Ⅶ | Peddi et al. (2014) |
In addition to the field of cancer, siRNA applications in other disease areas are being actively developed. Inclisiran, a phosphorothioate, 2′-O-methylnucleotide, and 2′-fluoronucleotide-modified siRNA targeting PCSK9, subcutaneous injection has been shown to reduce PCSK9 levels and LDL cholesterol levels in patients at high cardiovascular risk. It is injected only once every six months and shows excellent patient compliance.On December 11, 2020, Inclisiran was approved for marketing in the European Union.
Many siRNA studies targeting choroidal neovascularization due to age-related macular degeneration (CNV-AMD), targeting vascular endothelial growth factor receptor-1 (VEGFR1) or RTP801, have shown promising results in various phases of clinical trials, such as AGN211745 and PF-04523655. In addition, QPI-1007, an siRNA that inhibits caspase 2 expression, showed good tolerability and improved vision in patients with optic nerve atrophy.
Furthermore, siRNA therapies, including autosomal recessive primary hyperoxaluria (PHs), hepatitis B virus (HBV) and in liver and idiopathic pulmonary fibrosis, have shown promising applications.
ASO-based therapeutics
Antisense oligonucleotide (ASO) are single-stranded RNA/DNA molecules of 18-30 base pairs designed to specifically inhibit mRNA function. They bind complementarily to specific mRNAs, blocking mRNA translation or degrading mRNAs via RNase H.
Many preclinical and clinical trials related to ASO in the treatment of ocular diseases have shown promising results. Leber congenital amaurosis (LCA) causes blindness or severe visual impairment in adolescents due to retinal dystrophy caused by intronic mutations in various genes including centrosomal protein 290 (CEP290). QR-110 is a single-stranded, thiophosphorylation, 2′O-methyl-modified splicing regulatory RNA oligonucleotide that is used to target CEP290. Clinical studies (NCT03140969 and NCT03913143) showed that intravitreal injection of QR-110 reconstituted CEP290 levels in fibroblasts from patients with LCA with good tolerability and safety.
Aptamer-based therapeutics
Aptamers are single-stranded nucleic acid (DNA or RNA) molecules that bind and inhibit proteins. The aptamer’s ability to form shapes provides high affinity and excellent specificity for the target, and because their mode of action is similar to that of antibodies, aptamers are also known as chemical antibodies. A variety of aptamers are now in clinical trials for various diseases such as macular degeneration, diabetic macular edema, and chronic inflammatory diseases.
| Name | Disease | Target | ClinicalTrials.gov ldentifier | Recruitment Status | Phase |
| Zimura | Macular Degeneration | C5 | NCTO2686658 | Completed | Ⅱ/Ⅲ |
| Pegcetacoplan | Geographic atrophy | C3 | NCT02503332 | Completed | Ⅱ |
| E10030 | Macular Degeneration | PDGF | NCT00569140 | Completed | Ⅰ |
| E10030 | Macular Degeneration | PDGF | NCTO1089517 | Completed | Ⅱ |
| ARC1779 | von Willebrand Disease | von Willebrand Factor | NCT00432770 | Completed | Ⅱ |
| NOX-H94 | Anemia of Chronic Disease | (WF) A1 | NCT01691040 | Completed | Ⅱ |
| NOX-E36 | Type 2 diabetes mellitus, Albuminuria | C-C motif-ligand 2 | NCT01547897 | Completed | Ⅱ |
EYE001, a polyethylene glycol aptamer of VEGF, has shown the ability to completely reduce VEGF-mediated vascular leakage and inhibit retinal neovascularization in an in vitro model, resulting in improved vision. Studies of EYE001 alone or in combination with photodynamic therapy in patients with CNV-AMD have shown significant visual stabilization or improvement.
In addition to VEGF, the complement pathway plays an important role in AMD. Clinical studies of Zimura, a polyethylene glycol single-stranded nucleic acid aptamer targeting complement factor C5 and pegcetacoplan targeting C3 and C3b, have demonstrated the ability of subcutaneous injections to reduce geographic atrophy (GA) secondary to AMD without adverse events.
In addition, ARC1779, an aptamer that targets the structural domain of vWF A1, showed good tolerability and the ability to inhibit vWF-dependent platelet function in patients with thrombotic thrombocytopenic purpura in a clinical trial (NCT00632242). Emapticap-pegol (NOX-E36), an aptamer that targets CCL2, has shown good tolerability and the ability to inhibit the CCL2/CCL2 receptor axis when administered subcutaneously in type 2 diabetic patients with proteinuria.
Ribonuclease-based therapeutics
Ribonuclease is the molecule that catalyzes RNA. It can hybridize with the target RNA and cause RNA degradation, thereby inhibiting the production of specific proteins. Importantly, ribonucleases can function in the absence of cellular proteins.
RPI.4610 (Angiozyme), a chemically stable anti-VEGFR-1 nuclease, has been shown to have a favorable safety profile, bioavailability, and tumor localization in combination with carboplatin and paclitaxel in patients with advanced solid tumors. However, it failed to demonstrate clinical efficacy in patients with metastatic breast cancer.
OZ1, a tat-vpr-specific anti-HIV ribonuclease delivered via autologous CD34+ cells, showed a significant increase in CD4+ lymphocytes in a clinical trial (NCT00074997), suggesting that cell-delivered gene transfer is equally reliable in maintaining the safety and activity of the ribonuclease.
Despite success in many clinical trials, some studies have reported a lack of efficacy and safety, such as Ad5CRT targeting human telomerase reverse transcriptase (hTERT)-encoding RNA in patients with gastrointestinal tract cancers, and vasomotor enzymes or chimeric ribozymes in patients with metastatic breast cancer.
Therefore, the ribonuclease still needs to be improved in terms of stability, in vivo activity, co-localization, delivery to specific cells, and sustained stable and long-term expression.
mRNA-based therapeutics
mRNA-based therapies represent a relatively novel and efficient class of drugs. Several recently published studies have highlighted the potential efficacy of mRNA vaccines in the treatment of different types of malignancies and infectious diseases where conventional vaccine strategies fail to elicit a protective immune response.
Infectious disease vaccines are currently the leading application of mRNA therapeutics. Moderma and Pfizer’s new crown vaccine has already demonstrated safety and efficacy in billions of people, showing strong promise for future applications, which will not be repeated here.
| Funding source | Name | Target | Vaccine type | Route of administration | Clinical trial phase | Clinical trial identifier |
| Moderna | mRNA-1647 | CMV | Nucleoside modified mRNA-LNP | Intramuscular | Phase II | NCT04232280, NCT03382405 |
| Moderna | mRNA-1443 | CMV | Nucleoside modified mRNA-LNP | Intramuscular | Phase I | NCT03382405 |
| Moderna | mRNA 1893 | Zika | Nucleoside- modified mRNA-LNP | Intramuscular | Phase I | NCT04064905 |
| Moderma | mRNA-1325 | Zika | Nucleoside modified mRNA _LNP | Intramuscular | Phase I | NCT03014089 |
| Moderna | mRNA-1653 | hMPV/PIV3 | Nucleoside modified mRNA-LNP | Intramuscular | Phase I | NCT04144348, NCT03392389 |
| Moderma | mRNA-1345 | RSV | Nucleoside- modified mRNA-LNP | Intramuscular | Phase I | NCT04528719 |
| Moderna, Merck | mRNA-1777 (V171) | RSV | Nucleoside modified mRNA-LNP | Intramuscular | Phase I | Unregistered |
| Moderna, Merck | mRNA-1172 (V172) | RSV | Nucleoside -modified mRNA LNP | Intramuscular | Phase I | Unregistered |
| Moderna | mRNA-1851 (VAL-339851) | Influenza A (H7N9) | Nucleoside modifed mRNA-LNP | Intramuscular | Phase I | NCT03345043 |
| Moderna | mRNA-1440 (VAL-506440) | Infuenza A(H10N8) | Nucleoside modified mRNA-LNP | Intramuscular | Phase I | NCT03076385 |
| Moderna | mRNA 1010 | Influenza A(H1N1, H3N2). influenza B (Yamagata lineage, Victoria lineage) | Unknown | Intramuscular | Phase V/II | NCT04956575 |
| Translate Bio, Sanofi | MRT5400 | Influenza A(H3N2) | Unknown | Intramuscular | Phase I | Unregistered |
| Translate Bio, Sanofi | MRT5401 | Infuenza A (H3N2) | Unknown | Intramuscular | Phase I | Unregistered |
| Moderna | mRNA- 1944 | Chikungunya | Nucleoside modified mRNA- _LNP | Intramuscular | Phase I | NCT03829384 |
| Moderna | mRNA-1388 (VAL-181388) | Chikungunya | Nucleoside modified mRNA-LNP | Intramuscular | Phase I | NCT03325075 |
| CureVac | CV7201 | Rabies | Unmodified mRNA complexed in RNActive | Intradermal, intramuscular | Phase I | NCT02241135 |
| CureVac | CV7202 | Rabies | Unmodified mRNA _LNP | Intramuscular | Phase I | NCT03713086 |
| GSK | GSK3903133A | Rabies | Self- amplifying mRNA in cationic nanoemulsion | Intramuscular | Phase I | NCT04062669 |
Currently, mRNA vaccines against tumors are available in 2 ways: i) using ex vivo loaded or electroporated DCs, and ii) by direct injection of mRNA with or without a carrier.
Several clinical trials of DC-based mRNA vaccines applied in cancer patients with mRNA encoding melanoma-associated antigens co-inoculated with TriMix led to impressive tumor regression in advanced melanoma. Currently mRNA vaccines are moving toward combination therapies, including chemotherapy, radiotherapy, and checkpoint inhibitors. Some of these studies have produced durable tumor growth inhibition. Although these DC-based approaches are effective, they are also limited by cumbersome procedures and high costs.
In contrast, direct injection of naked mRNA or complex mRNA is considered a fast and effective feasible method. For example, a phase I/II trial repeatedly applied an mRNA vaccine encoding six different TAAs (MUC1, CEA, Her2/neu, telomerase, survivin, MAGE-A1) intradermally in 30 patients with metastatic renal cell carcinoma. Long-term results after 10 years indicate that the mRNA vaccine is safe and effective. It delayed tumor growth and improved survival, which was closely linked to the detected immune response against TAAs.
In another study, 7 patients with locally advanced and 39 patients with metastatic NSCLC received 5 intradermal injections of CV9201, an active vaccine encoding 5 NSCLC antigens (NY-ESO-1, MAGE-C1/2, survivin, 5T4). 63% of patients produced an antigen-specific immune response against at least one antigen, and 60% showed an increase in activated IgD+CD38high B-cells. 31% of patients had stable disease (SD), and the other two-thirds had progressive disease. A similar study aimed at improving anti-tumor immunity tested the vaccination of patients with advanced NSCLC with CV9202 in combination with local radiotherapy.
CV9202 is an active RNA-based vaccine encoding six NSCLC TAAs (NY-ESO-1, MAGE-C1, MAGE-C2, 5T4, survivin, and MUC-1). Most patients had enhanced antigen-specific cellular and humoral immunity compared to baseline. One patient who received vaccine, radiation, and chemotherapy experienced partial remission (PR), and 46.2% achieved SD.
The mRNA-4157 vaccine is an mRNA vaccine developed by Moderna, Inc. in the U.S. It is an individualized tumor vaccine tailored to each tumor patient. mRNA-4157 cancer vaccines are currently able to accommodate up to 34 mRNA sequences encoding neoantigens. In the phase I clinical trial of mRNA-4157 in combination with Keytruda, 11 of 13 patients receiving monotherapy maintained stable disease. In the combination therapy group, 6 of 20 patients responded, including 1 complete and 5 partial remissions. Six patients had stable disease and eight deteriorated. All adverse events associated with mRNA-4157 were reversible and mild. No treatment-related grade ≥3 adverse events were observed, demonstrating the safety and tolerability of the vaccine. Several tumor mRNA vaccines are currently in the clinical recruitment phase.
CRISPR/Cas9-based therapeutics
The CRISPR- Cas9 system is a bacterial defense mechanism that uses guide RNA (gRNA) to mediate the introduction of site-specific breaks in target DNA by the DNA endonuclease Cas9. The CRISPR- Cas9 system is a bacterial defense mechanism that uses guide RNA (gRNA) to mediate the introduction of site-specific breaks in target DNA by the DNA endonuclease Cas9.
Many studies have shown that CRISPR/Cas9 can be used to treat genetic diseases such as cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), hemoglobinopathies as well as HIV and beta-thalassemia. In one study, CRISPR/Cas9 was able to increase the number of utrophin in myotubes and remove duplicated DMD exons 18-30, resulting in the production of full-length dystrophin in DMD.
In β-thalassemia, mutations in the human hemoglobin β (HBB) gene can be corrected by CRISPR/Cas9. CRISPR/Cas9 in patient-derived IPSCs efficiently corrected HBB mutations without affecting IPSC pluripotency and restored HBB expression upon differentiation into erythrocytes via piggyBac transposons. In addition, similar applications of CRISPR-Cas9 have been demonstrated in immune diseases such as AIDS.
While these studies provide a foundation for future CRISPR-Cas9 clinical trials, a number of challenges remain to be addressed before CRISPR/Cas9 can be further utilized in clinical trials and subsequent therapies. This includes the possible off-target effects of delivering gene editing tools to target cells. In addition, ethical issues and germline applications of CRISPR-Cas9 need to be considered before translating CRISPR-Cas9 into therapeutic applications.
Summary
In recent years, RNA-based therapies have emerged as potential intervention strategies for various diseases. In general, RNA therapy is divided into different categories based on its mode of action and the molecules used. Many RNA therapies have been developed for a variety of diseases and have yielded very promising results in many preclinical and clinical studies, with mRNA vaccines in particular becoming one of the hottest areas of research today. Currently, there are still many obstacles to RNA-based therapies, and many aspects, including delivery systems, still need to be further researched and developed. However, it is undeniable that the era of RNA-based therapies will eventually come in the next decade.
References:
1.RNA Therapeutics – Research and Clinical Advancements. Front Mol Biosci. 2021; 8: 710738.
2. mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nat RevDrugDiscov. 2021 Aug 25 : 1–22.
3. Clinical and immunological effects of mRNA vaccines inmalignant diseases. Mol Cancer. 2021 Mar 15;20(1):52.