Self-amplifying RNA

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Self-amplifying RNA (saRNA), also termed self-replicating RNA (srRNA), is a type of mRNA molecule engineered to replicate itself within host cells, enhancing protein expression and boosting the immune response, making it a promising tool for vaccines and other therapeutic applications. As a "next-generation" mRNA, saRNA is designed to achieve greater protein expression with a reduced dose compared to conventional mRNA. [1] [2] [3] Unlike conventional mRNA, which has a short half-life and limited ability to express proteins for an extended time, saRNA can sustain protein expression for longer periods. saRNA are based on positive single stranded RNA viruses most commonly alphaviruses such as Venezuelan equine encephalitis virus.

Contents

Conventional messenger RNA (mRNA) vaccines only produce a finite amount of protein due to the short mRNA half-life. saRNA extends the kinetics of expression by a second ORF encoding the protein machinery necessary for its own replication. This self-replication dramatically increases both the amount of RNA and the time of expression. Consequently, the amount of protein produced from the initial dose is reduced as compared to conventional mRNA. [1] [2]

Structure and mechanism of action

The structure of saRNA includes two key components: [4]

Replicon region

Comparison of coding sequence structure of conventional mRNA and self-amplifying RNA (saRNA) Conventional mRNA and self-amplifying RNA (saRNA).png
Comparison of coding sequence structure of conventional mRNA and self-amplifying RNA (saRNA)

saRNA encode for the machinery to replicate and amplify the mRNA in its open reading frame (shown in orange), which is the viral RNA dependent RNA polymerase (RdRp). This is a single polypeptide of viral non-structural proteins that is processed into the four protein components of the RNA dependent RNA polymerase (nsp1, nsp2, nsp3 and nsp4).

Gene of interest

Mechanism of self-amplifying mRNA (saRNA) used for antigen production. The ORF encoding the antigen can also be substituted with a protein for use in protein replacement therapy Mechanism of self-amplifying mRNA (saRNA).png
Mechanism of self-amplifying mRNA (saRNA) used for antigen production. The ORF encoding the antigen can also be substituted with a protein for use in protein replacement therapy

This sequence encodes the protein of interest, used as an antigen in the case of vaccines or for protein replacement therapies. The gene of interest replaces the viral structural proteins. The RNA polymerase encoded by the non-structural proteins, transcribes the gene of interest from a specific promoter (the subgenomic promoter). This subgenomic mRNA encoding the gene of interest is produced at high levels and is capped by a protein component of the non-structural proteins.

Advantages

The self-replicating and amplifying nature of saRNA results in high levels of protein expression even at small doses, significantly enhancing the immune response. Additionally, saRNA vaccines can be manufactured more rapidly and at a lower cost compared to traditional vaccines. saRNA also offers stability by inducing a prolonged immune response, potentially providing longer-lasting protection. Furthermore, this versatile technology can be adapted for a wide range of applications, including infectious diseases, cancer immunotherapy, and genetic disorders.

Applications and research

The COVID-19 pandemic has accelerated research into RNA-based technologies, including saRNA. For instance, saRNA vaccines targeting SARS-CoV-2 have shown promising results in preclinical studies, indicating strong and durable immune responses with minimal adverse effects. [5] [6] [7] Recently an saRNA COVID booster vaccine developed by Arcturus (ARCT-154) has received full approval for use in adults by Japan's Ministry of Health, Labour and Welfare. [8]

saRNA is also being explored for gene therapy. Its ability to produce high levels of therapeutic proteins makes it a promising candidate for treating genetic disorders where protein replacement is needed. [9]

Challenges and future directions

While saRNA technology holds great promise, it also faces several challenges. Efficient and safe delivery of saRNA into target cells remains a critical hurdle, with lipid nanoparticles (LNPs) and other delivery systems currently being optimized to address this issue. Ensuring the long-term safety of saRNA is also important, and ongoing research is focused on minimizing potential side effects and immune reactions. Other delivery vehicles have been used in clinical trials to promote inflammation helpful for antibody production, such as the LION cationic nanocarrier formulation. [10] This has been used in the GEMCOVAC-19 vaccine with the saRNA being adsorbed on the surface of the LION nano-lipid emulsion and has received emergency licensure in India. [11]

A challenge with saRNAs as a therapeutic remains interferon production from the innate immune response. [12] It has been asserted that modified nucleosides are incompatible with the saRNA replication. [13] Nevertheless, to circumvent the induction of innate immune response, newer saRNA formats have been developed that incorporate modified nucleoside substitutions such as 5-methylcytosine, 5-methyluridine, N1-methylpseudouridine (the same nucleoside used in the Moderna and Pfizer/Biontech COVID mRNA vaccines) with varying degree of efficacy. [12] [14] [15] At low doses (10 ng/mouse), one study found use of the 5-methylcytosine nucleoside in synthesis having 5-fold higher protein expression than unmodified saRNA, which had in turn over 100x higher expression than N1-methylpseudouridine substituted saRNA. [12] Concomitantly, this study found that use of modified unmodified saRNA resulted in significant increases in the expression of IFNα and IFNβ after 6 h. [12] In contrast modified saRNA had reduced interferon expression. Specifically, modified saRNA with 5-methylcytosine and 5-hydroxymethylcytidine had reduced expression of IFNα1 8.5-fold and IFNβ1 3-fold respectively.

Related Research Articles

<span class="mw-page-title-main">RNA</span> Family of large biological molecules

Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself or by forming a template for the production of proteins. RNA and deoxyribonucleic acid (DNA) are nucleic acids. The nucleic acids constitute one of the four major macromolecules essential for all known forms of life. RNA is assembled as a chain of nucleotides. Cellular organisms use messenger RNA (mRNA) to convey genetic information that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.

<span class="mw-page-title-main">DNA vaccine</span> Vaccine containing DNA

A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.

Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, are an immortalised cell line derived from HEK cells isolated from a female fetus in the 1970s.

<span class="mw-page-title-main">Pseudouridine</span> Chemical compound

Pseudouridine is an isomer of the nucleoside uridine in which the uracil is attached via a carbon-carbon instead of a nitrogen-carbon glycosidic bond.

<span class="mw-page-title-main">Viral vector</span> Biotechnology to deliver genetic material into a cell

Viral vectors are modified viruses designed to deliver genetic material into cells. This process can be performed inside an organism or in cell culture. Viral vectors have widespread applications in basic research, agriculture, and medicine.

<i>Murine respirovirus</i> Sendai virus, virus of rodents

Murine respirovirus, formerly Sendai virus (SeV) and previously also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan (HVJ), is an enveloped, 150-200 nm–diameter, negative sense, single-stranded RNA virus of the family Paramyxoviridae. It typically infects rodents and it is not pathogenic for humans or domestic animals.

<span class="mw-page-title-main">Toll-like receptor 7</span> Protein found in humans

Toll-like receptor 7, also known as TLR7, is a protein that in humans is encoded by the TLR7 gene. Orthologs are found in mammals and birds. It is a member of the toll-like receptor (TLR) family and detects single stranded RNA.

<span class="mw-page-title-main">Virus</span> Infectious agent that replicates in cells

A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Viruses are found in almost every ecosystem on Earth and are the most numerous type of biological entity. Since Dmitri Ivanovsky's 1892 article describing a non-bacterial pathogen infecting tobacco plants and the discovery of the tobacco mosaic virus by Martinus Beijerinck in 1898, more than 11,000 of the millions of virus species have been described in detail. The study of viruses is known as virology, a subspeciality of microbiology.

Herpes simplex research includes all medical research that attempts to prevent, treat, or cure herpes, as well as fundamental research about the nature of herpes. Examples of particular herpes research include drug development, vaccines and genome editing. HSV-1 and HSV-2 are commonly thought of as oral and genital herpes respectively, but other members in the herpes family include chickenpox (varicella/zoster), cytomegalovirus, and Epstein-Barr virus. There are many more virus members that infect animals other than humans, some of which cause disease in companion animals or have economic impacts in the agriculture industry.

<span class="mw-page-title-main">Hepatitis C virus nonstructural protein 5B</span>

Nonstructural protein 5B (NS5B) is a viral protein found in the hepatitis C virus (HCV). It is an RNA-dependent RNA polymerase, having the key function of replicating HCV's viral RNA by using the viral positive RNA strand as a template to catalyze the polymerization of ribonucleoside triphosphates (rNTP) during RNA replication. Several crystal structures of NS5B polymerase in several crystalline forms have been determined based on the same consensus sequence BK. The structure can be represented by a right hand shape with fingers, palm, and thumb. The encircled active site, unique to NS5B, is contained within the palm structure of the protein. Recent studies on NS5B protein genotype 1b strain J4's (HC-J4) structure indicate a presence of an active site where possible control of nucleotide binding occurs and initiation of de-novo RNA synthesis. De-novo adds necessary primers for initiation of RNA replication.

Arcturus Therapeutics Holdings Inc. is an American RNA medicines biotechnology company focused on the discovery, development and commercialization of therapeutics for rare diseases and infectious diseases. Arcturus has developed proprietary lipid nanoparticle RNA therapeutics for nucleic acid medicines including small interfering RNA (siRNA), messenger RNA (mRNA), gene editing RNA, DNA, antisense oligonucleotides, and microRNA.

mRNA vaccine Type of vaccine

An mRNAvaccine is a type of vaccine that uses a copy of a molecule called messenger RNA (mRNA) to produce an immune response. The vaccine delivers molecules of antigen-encoding mRNA into cells, which use the designed mRNA as a blueprint to build foreign protein that would normally be produced by a pathogen or by a cancer cell. These protein molecules stimulate an adaptive immune response that teaches the body to identify and destroy the corresponding pathogen or cancer cells. The mRNA is delivered by a co-formulation of the RNA encapsulated in lipid nanoparticles that protect the RNA strands and help their absorption into the cells.

RNA therapeutics are a new class of medications based on ribonucleic acid (RNA). Research has been working on clinical use since the 1990s, with significant success in cancer therapy in the early 2010s. In 2020 and 2021, mRNA vaccines have been developed globally for use in combating the coronavirus disease. The Pfizer–BioNTech COVID-19 vaccine was the first mRNA vaccine approved by a medicines regulator, followed by the Moderna COVID-19 vaccine, and others.

A nucleoside-modified messenger RNA (modRNA) is a synthetic messenger RNA (mRNA) in which some nucleosides are replaced by other naturally modified nucleosides or by synthetic nucleoside analogues. modRNA is used to induce the production of a desired protein in certain cells. An important application is the development of mRNA vaccines, of which the first authorized were COVID-19 vaccines.

<span class="mw-page-title-main">CureVac COVID-19 vaccine</span> Vaccine candidate against COVID-19

The CureVac COVID-19 vaccine was a COVID-19 vaccine candidate developed by CureVac N.V. and the Coalition for Epidemic Preparedness Innovations (CEPI). The vaccine showed inadequate results in its Phase III trials with only 47% efficacy. In October 2021 CureVac abandoned further development and production plans for CVnCoV and refocused efforts on a cooperation with GlaxoSmithKline.

<span class="mw-page-title-main">ARCT-021</span> Vaccine candidate against COVID-19

ARCT-021, also known as LUNAR-COV19, is a COVID-19 vaccine candidate developed by Arcturus Therapeutics.

<span class="mw-page-title-main">Viral vector vaccine</span> Type of vaccine

A viral vector vaccine is a vaccine that uses a viral vector to deliver genetic material (DNA) that can be transcribed by the recipient's host cells as mRNA coding for a desired protein, or antigen, to elicit an immune response. As of April 2021, six viral vector vaccines, four COVID-19 vaccines and two Ebola vaccines, have been authorized for use in humans.

<span class="mw-page-title-main">N1-Methylpseudouridine</span> Chemical compound

N1-Methylpseudouridine is a natural archaeal tRNA component, and "hypermodified" pyrimidine nucleoside used in biochemistry and molecular biology for in vitro transcription and is found in the SARS-CoV-2 mRNA vaccines tozinameran (Pfizer–BioNTech) and elasomeran (Moderna).

<span class="mw-page-title-main">ARCT-154</span> Vaccine candidate against COVID-19

ARCT-154, also known as VBC-COV19-154 in Vietnam, is a COVID-19 vaccine candidate developed by Arcturus Therapeutics. For its development, Arcturus collaborated with Vinbiocare, a Vietnamese company, for support with clinical trials and manufacturing. The vaccine was authorised in Japan in November 2023.

References

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  5. ↑ Saraf A, Gurjar R, Kaviraj S, Kulkarni A, Kumar D, Kulkarni R, et al. (May 2024). "An Omicron-specific, self-amplifying mRNA booster vaccine for COVID-19: a phase 2/3 randomized trial". Nature Medicine. 30 (5): 1363–1372. doi:10.1038/s41591-024-02955-2. PMC   11108772 . PMID   38637636.
  6. ↑ Oda Y, Kumagai Y, Kanai M, Iwama Y, Okura I, Minamida T, et al. (April 2024). "Persistence of immune responses of a self-amplifying RNA COVID-19 vaccine (ARCT-154) versus BNT162b2". The Lancet. Infectious Diseases. 24 (4): 341–343. doi:10.1016/S1473-3099(24)00060-4. PMID   38310906.
  7. ↑ Oda Y, Kumagai Y, Kanai M, Iwama Y, Okura I, Minamida T, et al. (April 2024). "Immunogenicity and safety of a booster dose of a self-amplifying RNA COVID-19 vaccine (ARCT-154) versus BNT162b2 mRNA COVID-19 vaccine: a double-blind, multicentre, randomised, controlled, phase 3, non-inferiority trial". The Lancet. Infectious Diseases. 24 (4): 351–360. doi:10.1016/S1473-3099(23)00650-3. PMID   38141632.
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  9. ↑ Papukashvili D, Rcheulishvili N, Liu C, Ji Y, He Y, Wang PG (October 2022). "Self-Amplifying RNA Approach for Protein Replacement Therapy". International Journal of Molecular Sciences. 23 (21): 12884. doi: 10.3390/ijms232112884 . PMC   9655356 . PMID   36361673.
  10. ↑ Tregoning JS (2023-08-03). "LION: Taming RNA vaccine inflammation". Molecular Therapy. 31 (9): 2557. doi:10.1016/j.ymthe.2023.07.006. ISSN   1525-0016. PMC   10492017 . PMID   37541255.
  11. ↑ Saraf A, Gurjar R, Kaviraj S, Kulkarni A, Kumar D, Kulkarni R, et al. (2024-04-18). "An Omicron-specific, self-amplifying mRNA booster vaccine for COVID-19: a phase 2/3 randomized trial". Nature Medicine. 30 (5): 1363–1372. doi:10.1038/s41591-024-02955-2. ISSN   1546-170X. PMC   11108772 . PMID   38637636.
  12. 1 2 3 4 McGee JE, Kirsch JR, Kenney D, Cerbo F, Chavez EC, Shih TY, et al. (2024-07-08). "Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency". Nature Biotechnology: 1–7. doi:10.1038/s41587-024-02306-z. ISSN   1546-1696.
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  14. ↑ Azizi H, Renner TM, Agbayani G, Simard B, Dudani R, Harrison BA, et al. (2024-04-01). "Self-amplifying RNAs generated with the modified nucleotides 5-methylcytidine and 5-methyluridine mediate strong expression and immunogenicity in vivo". NAR Molecular Medicine. 1 (2). doi: 10.1093/narmme/ugae004 . ISSN   2976-856X.
  15. ↑ Miyazato P, Noguchi T, Ogawa F, Sugimoto T, Fauzyah Y, Sasaki R, et al. (2024-07-31). "1mΨ influences the performance of various positive-stranded RNA virus-based replicons". Scientific Reports. 14 (1): 17634. doi:10.1038/s41598-024-68617-y. ISSN   2045-2322. PMC   11292005 . PMID   39085360.
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