- Recent Advances, Including TriLink’s CleanCap®, Have Streamlined mRNA Production
- There Are at Least Ten Efforts to Develop COVID-19 mRNA Vaccines
The current COVID-19 crisis has led to intense interest and unprecedented global hyperactivity aimed at combating this highly contagious virus. Like with other viral diseases, ongoing medical efforts against COVID-19 encompass either treatment or prevention, i.e. drugs or vaccines. Either of these two basic strategies can involve traditional nucleic acid-based approaches, such as nucleic acid analogs as antiviral drugs or DNA-encoded vaccines. Regarding DNA-based vaccines, there is a spectrum of strategies spanning live-attenuated viral vectors to synthetic plasmids, as reviewed elsewhere.
DNA plasmid vaccines have, however, not been as successful as initially anticipated two decades ago. There are currently no FDA-approved DNA vaccines for use in humans, and only a few have been approved by the USDA for veterinary use, for reasons reviewed in depth elsewhere. Consequently, much attention has been directed to mRNA vaccines as a possibly more successful and speedier route to protection. The speed factor in the development of mRNA vaccine candidates accounts for the spotlight in media coverage, given that “speed matters” in blunting the current global pandemic and future outbreaks of viral diseases. This blog will briefly introduce mRNA vaccines, summarize recent advances in mRNA vaccine technology—featuring TriLink’s CleanCap®—and comment on the mRNA vaccines being investigated for COVID-19.
Introduction to mRNA Vaccines
The simplified process diagram shown below depicts basic elements of the general strategy for using mRNA as a vaccine. A protein antigen (e.g. COVID-19 spike protein) is encoded in DNA that is then used to synthesize the corresponding mRNA by in vitro transcription (IVT). Unprotected (“naked”) mRNA, or mRNA that protected from degradation by nucleases, is administered to a person for delivery of the mRNA to the immunologically relevant, target cell-type (antigen presenting cells, e.g. dendritic cells). Therein, cytosolic mRNA is translated into the protein antigen, which ultimately triggers a complex protective immune response (e.g. against COVID-19), as reviewed in 2020 by Saylor et al.
Simplified process diagram for an mRNA vaccine. Drawn by Jerry Zon
3D cut-away illustration of a liposome.
In vivo demonstration of the feasibility of an mRNA vaccine was first reported in 1993 by Martinon et al., who induced anti‐influenza cytotoxic T lymphocytes (CTL) by immunizing mice with liposomes containing mRNA encoding the influenza virus nucleoprotein. In this case, the liposomes were comprised of a mixture of cholesterol, phosphatidylcholine, and phosphatidylserine.
Since 2004, the early history of mRNA vaccine investigations has been expertly chronicled by Pascolo. Hurdles stemming from mRNA’s instability, inefficient in vivo delivery, and stimulation of excessive inflammatory responses were key issues that had to be addressed. Although producing mRNA by IVT is a fairly straightforward process, making high-quality “therapeutic grade” efficiently translatable mRNA that does not induce serious inflammation has been a major limitation in the field, until recently. By the early 2010s, this problem was largely resolved by a number of critical innovations, including the incorporation of modified nucleosides (particularly modified uridine), optimization of coding sequences, and stringent purification of IVT mRNA by high performance liquid chromatography (HPLC) to remove double-stranded RNA (dsRNA) contaminants responsible for innate immune activation. All of these techniques served to dampen the innate sensing of synthetic mRNA, thus reducing toxicity and improving its translation, as expertly reviewed elsewhere.
The final impediment to the viability of mRNA therapeutics has been inefficient cytoplasmic delivery, according to a 2020 review by experts Pardi , Hogan and Weissman (hereafter referred to as Pardi et al.). They note that several approaches such as ex vivo-loaded dendritic cells (DCs), intranodal delivery of mRNA, and mechanical methods (e.g. ‘gene gun’, shown here, and electroporation) were developed to deliver naked mRNA for vaccination. However, according to Pardi et al., these approaches are either complicated and expensive (ex vivo loading of DCs) or difficult to use in humans (intranodal delivery, electroporation). Thus, “the ideal way to deliver mRNA would be with a material that protects it from degradation and facilitates efficient cellular uptake after simple injection”. Remarkable progress in this delivery method is among the recent advances in mRNA vaccine technology discussed in the next section.
Schematic for gene gun delivery of DNA into a plant protoplast and picture of the handheld gene gun. Taken from wikipedia.org and free to use.
Recent Advances in mRNA Vaccine Technology
According to Pardi et al., the most important innovations in mRNA vaccine technology in recent years have been in the areas of: (1) engineering of mRNA sequences, (2) development of methods that enable simple, rapid, and large-scale cGMP production of mRNA; and (3) development of highly efficient and safe mRNA vaccine delivery materials. The following sections cover each of these three areas.
Engineering of mRNA Sequences: As depicted here, the template for transcription of mRNA consists of five structural elements: (i) the optimized cap structure, (ii) the optimized 5′ untranslated region (UTR), (iii) the codon-optimized coding sequence, (iv) the optimized 3′ UTR, and (v) the polyA tail. These structural elements are discussed on the TriLink webpage titled Anatomy of an mRNA.
Schematic representation of optimized mRNA. Taken from Van Van Hoecke and Roose J. Transl. Med. (2019) 17:54. © The Authors 2019 (Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium).
The TriLink Education webpage features a number of posters on translation-optimization strategies, as well as a poster titled Strategies to Minimize Innate Immune Stimulation to Maximize Messenger RNA Bioavailability. For maximal expression in cells or target organs, transfected mRNAs must avoid detection by pattern recognition receptors (PRRs). PRRs sense pathogenic non-self RNAs, including improperly capped RNAs and double stranded RNA. Their activation leads to cytokine production, translational arrest, and cell toxicity or death.
As mentioned above, the vast majority of effective mRNA vaccines use some kind of a delivery material, but Pardi et al. note two recent publications (Beissert et al. and Blakney et al.) that report a new vaccine format, which shows high potency in the absence of a delivery material. This method utilizes the co-delivery of an mRNA encoding an alphavirus RNA-dependent RNA polymerase, plus a second mRNA encoding the antigen of interest and the alphavirus genomic features required for its replication in the cytoplasm. This so-called ‘transreplicon’ or ‘splitzicon’ system builds on the dose-sparing properties of self-amplifying mRNA (depicted here), as a very low dose (50 ng) was effective in inducing protective immune responses in mice, even when delivered as unformulated mRNA.
Scheme showing a self-amplifying RNA derived from an alphavirus, in which structural genes have been replaced by the gene of interest, patterned after Rodríguez Gascón et al., and drawn by Jerry Zon.
Pardi et al. say that “[t>
hese findings are particularly attractive, as the utilization of low doses decreases the cost of the vaccine production. The absence of a delivery material further decreases the cost, simplifies manufacturing, and raises the possibility of vaccine lyophilization and storage at ambient temperature”.
Schematic of 2017 vs. 2019 methods for mRNA vaccine preparation technology, patterned after Pardi et al., and drawn by Jerry Zon.
Optimization of mRNA Production: The development of methods that enable rapid, simple, large-scale, and inexpensive production of high-quality mRNA is a critical requirement for the future implementation of mRNA vaccines. The evolution of oligonucleotide drug production has evolved incrementally, and similar progress can be anticipated in the development of mRNA technologies, which has already been noted by Pardi et al. in their comparison of 2017 vs. 2019 methods for mRNA vaccine preparation technology, shown here. They observe that most current mRNA production protocols entail separate enzymatic reactions for DNA template preparation, IVT, and 5’ capping, with nucleic acid precipitation after each step to remove other reaction components. This iterative process involves sample loss at each step and increases the time and cost of production. Additionally, some current mRNA purification methods—notably, HPLC purification—are not easily scalable, further bottlenecking mass production.
While current approaches work well on a small scale, they are suboptimal for large-scale manufacturing. Ideally, the final mRNA product would be prepared in a ‘one-pot’ reaction with a highly scalable purification method. Two recently described innovations have made progress towards this end. The first advance, introduced by TriLink, involves a co-transcriptional capping strategy called CleanCap®, which adds a natural 5’ Cap1 structure (Figure 1) to a specific transcription start sequence during IVT, as reported by Vaidyanathan et al. and further discussed on TriLink’s website. According to Pardi et al., “this development is significant, because previous protocols used either enzymatic capping—adding additional reaction components and purification steps to manufacturing—or co-transcriptional capping with Cap0, resulting innate immune activation when the IVT mRNA preparation contains dsRNA contaminant”. This utility is echoed by the fact that the Zone found 61 publications indexed to CleanCap® in Google Scholar.
Figure 1. Eukaryotic Cap Structures and Cap Analogs (A) Eukaryotic cap structure. Presence of 2’-O-methyl groups at R1 and R2 determine if a cap structure is Cap 0, Cap 1, and Cap 2 as indicated. (B) Structure of anti-reverse cap analog used in standard co-transcriptional capping. (C) Structure of CleanCap AG Cap1 Trimer. (D) Proposed mechanism of CleanCap co-transcriptional initiation, in which the AmG dimer portion of CleanCap docks onto the +1 and +2 template nucleotides. Initiation occurs when CleanCap couples to an NTP occupying the +3 position. Taken from Vaidyanathan et al. Molecular Therapy: Nucleic Acids Vol. 12 September 2018 © 2018 The Authors. This is an open access article under the CC BY license and free to use.
The second methodological innovation is useful at both laboratory and industrial scales, and has provided an attractive alternative to HPLC purification. Baiersdorfer et al. described a simple method for mRNA purification via adsorption of double-stranded RNA contaminants to cellulose, a cheap and abundant polysaccharide, depicted here.
The authors demonstrated that this highly scalable and inexpensive method works just as well as HPLC to remove dsRNA contaminants from IVT mRNA samples. Pardi et al. conclude that “[t>
aken together, research in the past couple of years has greatly facilitated the large-scale manufacture of therapeutic mRNA, and it is likely that additional research in this vein will further improve the simplicity and cost-efficiency of mRNA production”.
Materials for Efficient In Vivo mRNA Delivery: As discussed above, the vast majority of mRNA vaccines are designed to be injected with a carrier molecule that protects mRNA from rapid degradation and delivers it to the cytoplasm without significant toxicity. Pardi et al. state that “no efficient broadly applicable in vivo mRNA delivery materials were available until very recently; thus, we believe that the most significant recent progress in the mRNA vaccine field has occurred in this area and that it has played a critical role in advancing mRNA vaccines through several landmark efficacy studies published within the last three years”. Briefly, these notable studies are grouped as follows.
(1.) Polymers—Haabeth et al. and McKinlay et al. have recently developed novel lipid-containing polymers called charge-altering releasable transporters (CARTs; see Zone blog) that have efficiently targeted T cells, which are important in immune response. Thus, say Pardi et al., “CARTs are very attractive delivery materials with great potential in the areas of mRNA vaccines…”. Also cited is a publication by Chahal et al., who achieved immunization with branched polyamine-based polymers called dendrimers (show here) formulated with a lipid-anchored polyethylene glycol (PEG) and an antigen-encoding, self-amplifying mRNA.
Depiction of an exemplary dendrimer. Taken from commons.wikimedia.org and free to use.
(2.) Peptides—Cell-penetrating peptides (CPPs) are now widely used for mRNA vaccines, and there has been recent progress in the field. Udhayakumar et al. developed CPPs containing amphipathic Arg-Ala-Leu-Ala motifs to condense mRNA into particles. These particles were able to disrupt and penetrate membranes, and demonstrated potent cytolytic T cell responses after mice were immunized with CPP-complexed mRNA.
(3.) Lipid Nanoparticles—According to Pardi et al., ionizable lipid-containing nanoparticles (LNPs), initially developed for siRNA delivery, are the most widely used in vivo mRNA delivery materials to date. After a proof-of-concept for the in vivo translation of mRNA-LNPs was demonstrated in 2015, multiple vaccine studies have used LNPs with unmodified or nucleoside-modified mRNA that induced durable, protective immune responses against multiple infectious pathogens, often after a single dose (see Pardi et al. for 13 citations). Several clinical trials using mRNA-LNPs are underway, and some published data from two Phase 1 influenza virus vaccine trials are available (Bahl et al. and Feldman et al.).
While additional examples using mRNA-LNP delivery can be found in Pardi et al., the Zone notes an April 2020 report by Cheng et al. that describes a new strategy termed selective organ targeting (SORT), wherein multiple classes of LNPs are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT LNPs were designed for selective delivery to immunologically relevant T cells or other therapeutically relevant cell types.
COVID-19 Vaccines in Development
For an April 2020 review of the strategies being pursued for the development of COVID-19 vaccines, readers can consult an Open Access perspective by Amanat & Krammer published in the journal Immunity, which provides technical details, as well as a status report. Given the unprecedented number and speed of reports appearing in this area, the Zone searched for an online source of reliable information that is also comprehensive and updated frequently. We found the COVID-19 Vaccine Tracker, which is updated weekly and can be accessed at this link. This informational tool is provided by the Regulatory Affairs Professionals Society (RAPS), the largest global organization of and for those involved with the regulation of healthcare and related products.
The tracker covers all types of candidate vaccines (e.g. live-attenuated virus, engineered adenovirus DNA vectors, mRNA, etc.). It provides designation (clinical Phase, pre-clinical or research), company/organization(s) involved, type of vaccine candidate, and miscellaneous details/status for each. As of April 13, 2020, perusal of the tracker—and other sources—by the Zone provided the following candidate mRNA vaccines.
Phase 1 Vaccine Candidate
Vaccine candidate: mRNA-1273
Misc. Details/Status: mRNA-1273 is a vaccine candidate developed using prior studies of related coronaviruses, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). This is an ongoing open-label, dose-ranging clinical trial of 45 healthy participants between 18-55 years old.
Vaccine candidate: BNT162
Misc. Details/Status: Pfizer and BioNtech have announced an agreement to collaborate on developing an mRNA-based COVID-19 vaccine originally developed by BioNTech. Clinical testing is expected to begin in April 2020.
Pre-Clinical Vaccine Candidates
Vaccine candidate: No name announced
Misc. Details/Status: CureVac announced the development of an mRNA-based COVID-19 vaccine “within a few months,” according to a press release. The company plans to start clinical trials in the summer and has identified two study centers.
Company: Imperial College London
Vaccine candidate: Self-amplifying RNA vaccine
Misc. Details/Status: Imperial College London researchers are developing a self-amplifying RNA vaccine for COVID-19. They developed a vaccine candidate within 14 days of receiving the sequence from China. Animal testing is underway, and the researchers aim to begin clinical trials in the summer of 2020.
Vaccine candidate: mRNA vaccine candidate
Misc. Details/Status: Russian biotech company BIOCAD announced it is developing an mRNA vaccine candidate for COVID-19. BIOCAD said it expects to begin animal testing in April 2020.
Vaccine candidate: mRNA vaccine candidate
Vaccine candidate: LUNAR-COV19
Misc. Details/Status: A vaccine candidate will be developed using Arcturus’ self-replicating RNA and nanoparticle non-viral delivery system. Arcturus announced that, under the guidance of the Singapore Health Sciences Authority, a trial is to begin this summer and will enroll up to 76 healthy adult volunteers, including elderly individuals, with follow-up over several months to evaluate extent and duration of immune response.
Organizations: An eTheRNA-led consortium
Vaccine candidate: mRNA vaccine candidate
Misc. Details/Status: On March 24, 2020, eTheRNA Immunotherapies NV (Niel, Belgium) announced that a consortium has been formed with North American and European partners to develop a novel mRNA vaccine against COVID-19. Preclinical development has started.
Organizations: Duke Human Vaccine Institute
Vaccine candidate: mRNA vaccine candidate
Misc. Details/Status: Prof. Greg Sempowski at DHVI states that “we’re able to use in-house resources to rapidly go from bench to bedside”, and researchers are pursuing the use of mRNA-encoded antibodies against the virus.
Vaccine candidate: mRNA vaccine candidate
Misc. Details/Status: Two methods are being evaluated to develop an mRNA-based vaccine: using mRNA to express the receptor-binding domain of the spike protein of COVID-19 to induce neutralizing-antibodies, and developing mRNAs that can instruct the host to produce virus-like particles. No details on further development or testing are available at this time.
Just as this blog’s title highlights speed, its concluding comments will too. In a March 11, 2020, news article in Chemistry World (published by the Royal Society of Chemistry) titled RNA Vaccines Are Coronavirus Frontrunners, Anthony King stated the following:
“On the vaccine front, RNA vaccines are leading the way because they’re particularly suited to speedy development. And while no RNA vaccine has ever been licensed, the threat of a pandemic is a great incentive to accelerate their progress.”
The number of mRNA vaccine candidates for COVID-19 supports a speedier route to an effective vaccine, in order to prevent further spread of this pandemic. The Zone agrees completely with King’s additional comment. Multiple companies and organizations are all pursing the same goal, a protective mRNA vaccine against COVID-19:
“It is a race, but a race against the virus rather than each other.”
As usual, your comments are welcomed.