During the year 2022, more than 12,000 articles related to mRNA vaccines or mRNA therapeutics will have been added to the NIH PubMed database. This substantial volume of literature has provided many exciting topics to feature in this year’s Zone in with Zon blog posts. For our final post of 2022, we’re reflecting on the year to highlight major advancements in several broad areas of research covered in the blog:
- Using modified nucleosides for mRNA
- Production of mRNA
- Delivery of mRNA
- mRNA vaccines
- mRNA therapeutics
1. Modified Nucleosides
As highlighted in the first blog post of this year, pioneering findings by Katalin Karikó and Drew Weissman on the advantages of nucleoside modifications in mRNA for vaccines ushered in an exciting new era of vaccinology (Karikó et al., 2008). We discussed the benefits of 5-methoxy uridine (5-moU) and N1-methylpseudourine (m1Ψ) to replace uridine during the in vitro synthesis of mRNA. In conjunction with TriLink’s CleanCap® reagents, these modifications greatly improve expression and reduce host immunogenicity to mRNA. Importantly, this blog noted that CleanCap® and m1Ψ are incorporated into the world’s leading COVID-19 vaccine program! By September 2022, this vaccine had been used in more than 3 billion doses with approval in more than 140 countries.
Six months later, our June blog discussed nucleoside modifications in relation to COVID-19. The Korean pharmaceutical company EyeGene developed its vaccine against COVID-19 using 5-moU mRNA. As an EyeGene partner, TriLink supplied its proprietary CleanCap® mRNA capping technology and the modified nucleoside, uridine triphosphate, for vaccine production. This collaboration with EyeGene represented the first clinical-stage program to use GMP-grade modified uridine triphosphate, with an intended use for further processing.
Both nucleoside-modified vaccines against SARS-CoV-2 take advantage of TriLink’s high-purity, modified nucleoside triphosphates (NTPs) to optimize and enhance in vitro transcribed (IVT) mRNA performance and expression. From research use to the first-ever GMP-grade offering of nucleoside-modified NTPs, TriLink provides a portfolio of off-the-shelf, chemically synthesized NTPs that are ready to scale to production.
In June, we highlighted the importance of developing lyophilized mRNA vaccines to improve development and distribution. Lyophilization can increase the stability of mRNA to prevent degradation and obviate the problematic need for storing and shipping mRNA vaccines at ultra-low temperatures. Check out this blog post for further discussion on the various approaches and ongoing clinical trials using lyophilized mRNA vaccines against COVID-19.
Typically, commercial vaccines are manufactured, stored, and shipped from centralized facilities with large-scale production and purification according to GMP guidelines. However, as our August blog observed, this traditional paradigm is now being supplemented with decentralized “on-demand” IVT mRNA manufacturing to better cope with regional endemic infections involving lower numbers of doses or genetic variants. This paradigm shift was exemplified by the newly developed and implemented mobile modular BioNTainer®. These shipping container-sized units will become a hub for decentralized, end-to-end manufacturing of IVT mRNA vaccines for people residing around the world.
Backing up to streamline upstream manufacturing for the industry, in September 2022, TriLink announced the expansion of our CleanCap® reagent and modified nucleoside triphosphate (NTP) portfolios to include GMP grade, allowing clients to seamlessly scale-up mRNA drug substance manufacturing from research through clinical trials.
Many mRNA vaccines and mRNA therapeutics use lipid nanoparticles (LNPs) for protective encapsulation and delivery. These LNPs are comprised of mixtures of lipid components, the chemical details of which and precise ratios thereof are important variables. A recent review (Godbout et al., 2022) discusses the most effective compositions and proportions of lipids for targeting specific organs. Despite the success of synthetic LNP-mediated delivery of mRNA, several reports of LNP-associated adverse effects have led to an interest in alternative delivery vehicles.
One such example was discussed in the July blog—exosomes. Herein, exosomal lipid-bilayer particles are harvested from the cultures of producer cells and then loaded with IVT mRNA. Promising results were summarized from two pre-clinical studies: a mouse model to treat familial hypercholesterolemia by protein replacement and a mouse model for vaccination against COVID-19. Recently, an optimized exosome production strategy for enhanced yield without sacrificing cargo loading efficiency was published (Zhang et al., 2022).
Along the delivery challenge road, “needle phobia” continues to be a significant problem, with estimates that up to 20% of the world’s population have this fear at the level of a phobia. But, virtually all mRNA vaccines and therapeutics use needle injections to date. However, as highlighted in our May blog, studies of three jab-free routes of mRNA administration have begun: transdermal, oral, and intranasal. Since then, a high-pressure transdermal administration has entered clinical trials, and intranasal delivery of an mRNA COVID-19 vaccine was protective in a hamster model (Vanover et al., 2022).
COVID-19 Boosters. Since our January blog discussed a new era in mRNA vaccinology stemming from COVID-19 clinical trials, there has been significant progress in developing boosters. Currently, the FDA has authorized bivalent formulations of both the Moderna and the Pfizer BioNTech COVID-19 vaccines for use as a single booster dose at least 2 months after completing primary or booster vaccination.
As detailed elsewhere, the Pfizer BioNTech bivalent booster includes mRNA encoding the spike proteins of the wild-type SARS-CoV-2 and the Omicron BA.4/BA.5 subvariants. Because BA.4 and BA.5 contain identical spike protein amino acid sequences, both can be targeted with a single mRNA strand. Apart from adding the mRNA sequence of the BA.4/BA.5 spike protein, all other vaccine components remain unchanged.
Seasonal Flu. Human influenza A and B viruses cause seasonal epidemics of disease almost every winter. Only influenza A viruses are known to cause flu pandemics (i.e., global epidemics of flu disease) when a new and different influenza A virus subtype emerges that can both infect and spread efficiently among people. In past years, flu vaccines have only been approximately 40% to 60%, emphasizing the need for improved modalities.
Recently, McMahon et al., 2022 addressed some of the known conventional flu vaccine shortcomings. Using IVT mRNAs prepared with TriLink’s m1Ψ-5’-triphosphate and CleanCap® reagents, the group developed a quadrivalent influenza A vaccine for testing in mice. This vaccine provided greater protection than that of the four monovalent formulations. Including multiple conserved antigens considerably broadened the breadth and level of protection against a panel of heterologous influenza virus strains.
HIV/AIDS. Combating HIV/AIDS with mRNA vaccines and CRISPR gene editing was featured in the April blog. The vaccine approaches we discussed used mRNA encoding the HIV trimeric envelope (env) spike glycoprotein, including administration of an LNP-formulated IVT env mRNA trimer derived from m1Ψ-5’-triphosphate purchased from TriLink. Finding an effective prophylactic vaccine remains a central component of a multi-pronged strategy to end the HIV and AIDS pandemic. Explore the promising progress toward anti-HIV mRNA vaccines for prevention in this post.
Introduction of chimeric antigen receptor (CAR) T-cell modifications initially employed genetic transformation methods with plasmid or viral vectors. However, this process is now more safely achievable via the transient expression of IVT mRNA. Recent advances in cancer immunotherapy using the novel mRNA-based approach were discussed in our September blog, where we covered advances in CAR T-cell immunotherapy against select cancer types.
One such publication highlighted the use of TriLink-manufactured TALEN mRNA to edit allogeneic anti-CD70 CAR T cells ex vivo, preventing graft-versus-host disease in recipient cancer patients. Importantly, the resultant TALEN mRNA-edited T cells were produced at a large enough scale to enable a phase 1 clinical trial. The study, sponsored by Allogene Therapeutics, evaluates safety in adult patients with advanced or metastatic renal cell carcinoma. Notably, one of this year’s most downloaded (>106,000) Science articles in this field reported in vivo formation of CAR T cells using IVT mRNA that incorporated TriLink’s CleanCap® and m1Ψ reagents (Rurik et al., 2022).
Protein replacement therapy enabled by the delivery of IVT mRNA is a safer plasmid alternative over viral vector forms for treating rare monogenic diseases caused by the absence of a functioning wild-type protein. Awareness of Rare Disease Day on February 28 was highlighted in a special blog in February. Moreover, the September blog featured promising results for IVT mRNA treatment of hereditary tyrosinemia type 1 (HT1), a rare genetic metabolic disorder. The study used an established mouse model of HT1 caused by the loss of functional fumarylacetoacetate hydrolase (FAH). Repeated administration of LNP-formulated CleanCap® FAH mRNA prevented HT1 in a manner superior to the current standard of care.
Looking Ahead to 2023
The spectacular advances in vaccinology enabled by use of IVT mRNA will undoubtedly continue during the coming year. A forward-looking perspective from the National Cancer Institute adds that the success of the mRNA COVID-19 vaccines will help accelerate clinical research on mRNA vaccines to treat cancer. The mRNA-enabled cell and gene therapy approaches mentioned above are an indication of promising approaches to combating cancer and rare diseases.
TriLink reagents used for vaccines, therapeutics, and gene editing to be highlighted in 2023 will continue to exemplify TriLink’s leading the way in mRNA collectively!
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