Upgrading the Code - RNA Beyond Conventional mRNA
Written by Jim Grady and Peter Bak, PhD
With Contributions by Mavra Nasir, PhD
Introduction
The field of RNA biology has seen significant clinical development activity and technological advancement in recent years. Coding RNAs, in particular, act as messengers on behalf of DNA to induce expression of the more than 20,000 proteins that provide most of the structure and function in human cell biology. These linear, non-self-replicating nucleic acid sequences, referred to as messenger RNA (mRNA), are the foundation for the first-generation of RNA-based medicines, deployed to express a therapeutic protein, be it an antigen in the context of vaccines, a cytokine for cancer immunotherapy, or the replacement of a defective, damaged, or missing endogenous copy of a critical gene. The most well-known examples of these approaches are the FDA-approved mRNA COVID-19 vaccines. Though first-generation or “conventional” mRNA molecules are indeed versatile tools for the development of new medicines, they come with certain limitations related to pharmacokinetics, delivery, unintended immunogenicity, manufacturing scale-up, and temperature sensitivity. Thus, many organizations are now aiming to overcome these limitations through the development of the next generation of coding RNA technologies, notably self-amplifying RNA (saRNA) and circular RNA (circRNA).
Overview: RNA as Medicines
Well before the approval of COVID-19 vaccines, the biopharmaceutical field understood the potential to harness mRNA as a therapeutic modality, given the molecules:
Leverage human cells as precise in vivo production “factories” of proteins, which can act as vaccine antigens or therapeutic proteins with high target specificity
Offer the potential to develop personalized medicines via the numerous possible permutations and varying lengths of coding RNA sequences
Require less time and expenditure to manufacture vs. traditional biologics, which typically require ex vivo cell-based expression systems or other complex, time-consuming, or more costly manufacturing processes (e.g., mAbs, ex vivo CAR-T therapies, AAV gene therapies)
Can be deployed across a diverse array of therapeutic areas, including infectious disease, oncology, rare genetic disease, metabolic disease, autoimmunity, and others
Nevertheless, limitations exist to conventional mRNA, which has made its advancement through clinical trials a challenging process. These limitations include:
Non-durable in vivo protein expression, limiting duration of therapeutic or prophylactic effect
Instability and shorter-than-desired shelf-life, complicating supply chain and inventory logistics
Challenges with large-scale production and relative expense of capping materials, which account for 20%-40% of raw material spend [1]
Off-target or otherwise unintended immunogenicity due to:
High dosing made necessary by low relative protein expression
Cationic LNP delivery molecules
mRNA sequences having suboptimal nucleotide design
Manufacturing impurities
Challenges with cell-specific delivery and avoiding sequestration in the liver
Temperature sensitivity requiring ultra-cold freezers between -90°C and -15°C, limiting geographic reach in low-income countries
Injectable routes of administration requiring clinicians to administer
To overcome the limitations of conventional mRNA, researchers and organizations are developing next-generation coding RNA technologies with the following aims: [2] [3] [4] [5]
Increase in vivo durability, whether through half-life improvements or self-amplification
Increase relative expression of protein to enable prolonged therapeutic or prophylactic effect and lower dosing to reduce off-target or unintended levels of immunogenicity
Avoid need for frequent re-dosing
Decrease need for capping materials and related manufacturing processes via RNA circularization
Increase shelf-life and max viable temperature for storage
Improved delivery methods in terms of cell-targeting, extra-hepatic distribution, and more convenient routes of administration (e.g., potential oral delivery with a safe, gram-negative bacterial vector)
Beyond Conventional: saRNA and circRNA
saRNA: Containing sequences derived from self-replicating, positive-strand RNA viruses, saRNA has emerged as a promising new technology in recent years. These virus-derived sequences produce enzymes termed replicases, which catalyze the replication (or amplification) of the sequence within the saRNA that encodes for the protein of interest, resulting in many additional transcripts. As a result, saRNA can produce greater absolute and relative levels of protein and for longer periods of time than conventional mRNA (“relative” defined as mass of target protein expressed per mass of RNA dosed). This distinguishing trait suggests saRNA may, in particular, enable the development of vaccines with greater immuno-protection and durability. It also suggests lower and less frequent dosing may be realized with saRNA over conventional mRNA, which may translate to improved patient tolerability without forfeiture of efficacy.
circRNA: Novel coding circRNA is formed by splicing the 5’ and 3’ ends of linear mRNA strands, which prevents cytosolic enzymes from accessing these otherwise exposed sites and rapidly degrading the molecule. As a result, these circular nucleotides with significantly longer half-life may enable prolonged therapeutic effect and/or lowering of requisite doses. Beyond prolonged expression of the desired protein, the ability to dose less frequently may be similarly realized with circRNA and saRNA. Beyond improvements to half-life, circularization of mRNA removes the need for costly raw materials and processes ordinarily required for the capping of conventional mRNA, making circRNA a more attractive modality from a cost of goods perspective. circRNA may also extend the shelf-life of coding RNA products, another current limitation of conventional mRNA.
Coding RNA Development Landscape
As of late August 2024, biotechnology companies were collectively developing more than 230 coding RNA programs, predominantly for infectious disease (ID) and oncology indications (Figure 1). Within the overall coding RNA landscape, 51% of programs are for infectious diseases (ID), 24% are for oncology indications, 7% are for metabolic diseases, and the remainder are for various other therapeutic areas (< 5% each) or program indications have yet to be disclosed (ND). Coronavirus vaccines account for 32% of all ID programs, though it is likely that only a minority are still being prioritized for development from an internal company perspective. Beyond Moderna’s and BioNTech’s approved COVID-19 vaccines, the lone launched product is Moderna’s vaccine for respiratory syncytial virus (RSV). These and other companies are also developing coding RNA-based ID vaccines for influenza virus, herpes zoster virus (shingles), human immunodeficiency virus (HIV), monkeypox virus, herpes simplex virus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV), among more than 15 other viruses. Cancer vaccines account for more than 50% of all oncology programs, while most of the remaining oncology programs encode cytokines, targeted therapeutic proteins, or in vivo CAR-T therapies. Non-small cell lung cancer, melanoma, and human papilloma virus-positive tumors are more popular indications for oncology programs, with multiple companies developing coding RNA therapies in each of those indications. Other indications such as leukemias and lymphomas, colorectal cancer, breast cancer and HER2+ tumors, Epstein-Barr virus-positive tumors, pancreatic cancer, prostate cancer, glioblastoma, and several other tumor-types are also indications for which development is active.
FIGURE 1
Coding RNA competitive landscape as of August 2024
“Other” includes respiratory, dermatology, hematology, musculoskeletal, immunology/autoimmune, genitourinary, neurology, ocular
ID = infectious disease, ND = not disclosed
Sources: Back Bay analysis, Biomedtracker, company websites
Within the circRNA and saRNA pipeline, there are a total of more than 35 disclosed programs, albeit at an overall earlier stage of clinical maturity (Figure 2). Though ID and oncology still lead the way in terms of most frequented therapeutic areas, they do not claim as large of a share as they do in the overall landscape, as the potential for longer gene transgene expression and lower immunogenicity are of critical importance for chronic diseases.
FIGURE 2
saRNA and circRNA pipeline as of August 2024
ID = infectious disease, ND = not disclosed
Sources: Back Bay analysis, Biomedtracker, company websites
Within Europe, Japan, and US markets, the furthest advanced saRNA programs are Arcturus’ and CSL Behring’s phase 3 vaccine for COVID-19 (approved in Japan) and for influenza. The phase 2 oncology program in Figure 2 is Strand Therapeutics’ saRNA-encoded IL-12 to treat melanoma, triple-negative breast cancer, and other solid tumors (technically, a combined phase 1/2 trial). The phase 1 Other ID saRNA programs are from Immorna, Arcturus Therapeutics, and Replicate Bioscience and are for shingles, influenza, and rabies, respectively.
Investment Trends
Given the commercial success of first-generation, conventional mRNA vaccines during the pandemic, venture capital has placed a number of bets on emerging RNA technologies. Indeed, multiple companies with circRNA technologies have each raised more than $300M in venture capital since 2019 (Table 1).
TABLE 1
circRNA landscape and selection of saRNA companies (financings source: Pitchbook)
Source: Pitchbook
A number of these companies anticipate high profile data readouts in the coming years, which will set the stage for further capital deployment in these technologies, or a retrenchment in venture capitalists’ perspectives on the field:
Strand Therapeutics phase 1/2 open label trial of saRNA-encoded IL-12 for melanoma, triple-negative breast cancer, and other solid tumors dosed its first patient in May 2024 and has a primary completion date of May 2027 (NCT06249048). The company also has an saRNA in vivo CAR-T program in preclinical stages of development, but with anticipated timeline for an IND submission undisclosed.
Venture-backed Orbital Therapeutics has several RNA-based technologies in development, including circRNA delivered using novel LNPs or virus-like particles, with plans for autoimmune disease and oncology programs, next-generation vaccines, and other targeted protein therapeutics, though specific indications have yet to be disclosed.
Orna aims to develop its circRNA platform and novel LNPs, which target extra-hepatic cells in a passive manner rather than a ligand-based active manner, for B-cell-driven autoimmune disease and B-cell cancers. In addition, they are pursuing sickle cell disease, beta thalassemia, and undisclosed infectious diseases. Orna’s ID program(s) are in partnership with Merck.
Sail Biomedicines formed as a result of the merger of Flagship Pioneering’s Laronde and Senda Biosciences. Sail’s platform combines programmable circRNA technology, generative AI, and proprietary nanoparticles to develop personalized medicines in indications yet to be announced.
Arcturus Therapeutics’ and partner CSL Behring’s saRNA vaccine (Kostaive) for COVID-19 is now approved in Japan, with launch planned for 4Q 2024. A Marketing Authorization Application has been submitted to the European Medicines Agency and is currently under review, with a decision anticipated prior to YE 2024. Despite the competitive nature of the field, analysts remain bullish on the prospects with estimated peak sales in excess of $500M by 2035 if it remains a standalone COVID vaccine and in excess of $2B by 2035 if it is converted to a dual COVID-influenza vaccine.
In May 2024, Replicate Bioscience announced phase 1 proof-of-concept data for its saRNA rabies vaccine, which met the WHO-established surrogate endpoint for protection at doses significantly lower than any other reported RNA-based vaccines. The company has focused its clinical programs on ID, including rabies and Epstein-Barr virus, with additional programs in high value applications such as breast cancer, lung cancer, and autoimmunity.
HDT Bio made headlines in 2022 when its saRNA COVID-19 vaccine (Gemcovac) was approved in India and became the first saRNA therapy to gain marketing approval. Gemcovac is now in phase 1 development in the US. In addition to enabling lower doses vs. conventional mRNA, HDT Bio’s approach allows saRNA therapies to be stored at refrigerator temperatures rather than the sub-zero temperatures required for the mRNA therapeutics approved in the US. Such improvement to temperature sensitivity was a major differentiating factor for the Indian market.
Summary
A total of more than 230 conventional mRNA and next-generation coding RNA technologies continue to advance toward marketing approval. Self-amplifying and circular RNAs have demonstrated valuable proofs-of-concept at the clinical and preclinical stages of development, respectively, indicating a strong likelihood of their ability to prolong protein expression across an array of therapeutic or prophylactic use-cases. How this will translate to clinical differentiation remains to be seen.
Beyond the key benefits directly brought by saRNA and circRNA, other limitations of conventional mRNA remain in terms of maximizing the potential utility of these technologies for patients across the globe. Challenges related to delivery, other causes of unintended immunogenicity, manufacturing, temperature sensitivity, and routes of administration are areas marked for improvement. To that end, novel polymer nanoparticle (PNP) delivery technologies are in development, which offer high biocompatibility and can be modified with cell-targeting ligands and may one day replace LNPs as the preferred method for coding RNA delivery. Improved selection and design of nucleotides to avoid unintended immunogenicity is a consistent and ongoing focus of the field. This becomes even more pertinent if coding RNA products are to become the widely deployed, personalized forms of medicine that many in the field have envisioned. Though coding RNAs are already manufactured using cell-free methods and are thus cheaper to manufacture than many other biological therapeutics, continuous improvements to manufacturing processes or decreases in raw materials expense make the modality more valuable. Whether through cheaper co-transcriptional capping methods or by circularization, which avoids the need for capping, research is ongoing to improve manufacturing efficiency and decrease the cost of coding RNA production (of key importance in a pandemic use-case). Last, temperature sensitivity and injectable route of administration (RoA) can be an onerous logistical challenge, particularly in low- and middle-income countries. Whether through pH-dependent nanoparticle technologies or microbial vectors that are less temperature sensitive and generally-recognized-as-safe (GRAS) for human use, the field would benefit from coding RNA therapeutics that can be delivered via oral RoA rather than injection.
FOOTER
[1] Strand Therapeutics, Orna Therapeutics, Orbital Therapeutics, Replicate Bioscience, Arcturus Therapeutics corporate materials
[2] Zhou, Jiang, et al., Viruses, 2023 Aug 18;15(8):1760. doi: 10.3390/v15081760
[3] Niu, Wu, Lian, Signal Transduct Target Ther. 2023 Sep 11;8(1):341. doi: 10.1038/s41392-023-01561-x
[4] Papukashvili, Rcheulishvili, et al., Int J Mol Sci, 2022 Oct 25;23(21):12884. doi: 10.3390/ijms232112884
[5] Liu, Zhang et al., J Control Release. 2022 Jun 2;348:84–94. doi: 10.1016/j.jconrel.2022.05.043
BACK BAY TEAM MEMBER
Jim Grady, Consultant
Jim Grady is a consultant at Back Bay Life Science Advisors. He has considerable experience working in immuno-oncology and across various therapeutic areas within the rare diseases space. During his career, Mr. Grady has supported a variety of projects that span business development and licensing, commercial strategy, search and evaluation, primary and secondary market research, startup operations, and financial analysis.
Before joining Back Bay, Mr. Grady worked in commercial and business development roles for Rallybio, a publicly traded rare disease-focused biotech, as a consultant for Boulder Biotech Launch Specialists, and as co-founder of IngenoVax, an immuno-oncology startup that had developed and patented a fully-synthetic cancer vaccine platform. Prior to his pivot to the biotech industry, Mr. Grady began his corporate career by working for several years as a Credit Analyst at CoBank, a large corporate lender.
Mr. Grady holds a BS in Chemical & Biological Engineering and a BS in Business Administration with an emphasis in Finance, both from University of Colorado Boulder. He also holds an MBA with emphasis in Corporate Finance & Consulting from University of Notre Dame.