mRNA Vaccines: Current Trends and Perspectives
The COVID-19 pandemic brought mRNA vaccines to the spotlight with the rapid release of highly efficacious (94-95%) vaccines by Pfizer/BioNTech and Moderna. Once the sequence of SARS-CoV-2 was published, Moderna had its first candidates available in only 28 days.1 Full phase 1-3 trials and release of millions of doses were completed in months – much shorter than the years of time typically required for other vaccines.2
Besides shorted development time and high efficacy (at least for COVID), there are other advantages for the use of mRNA for both prophylactic and therapeutic vaccines.3 One is the safety profile, which includes that the antigen is typically expressed for only a matter of days and can be modulated by the design of the mRNA. mRNA vaccines are also much more controllable than attenuated and vector vaccines. Unlike DNA-based approaches, mRNA vaccines do not require nuclear entry so there is less risk of genomic integration and mutagenesis. Lastly, mRNA vaccines offer the robust development of both cellular and antibody responses, which can be somewhat shifted between the two by the design of the mRNA, the choice of delivery method, or other approaches.
There are also advantages in the production method, as synthesis is based on well-established in vitro transcription processes in a cell-free system. The cell-free system helps reduce cost, timeline, and manufacturing footprint. One note, the pDNA used as a template for the mRNA vaccine does require a cell-based fermentation step, however this is not considered to be a highly costly or time consuming step. Moreover, mRNA presents flexibility as vaccines for variants or multivalent vaccines can typically be manufactured without a significant change of production process.
Nevertheless, there are some areas that need improvement.
Due to its negative charge, mRNA struggles to enter cells and it can be rapidly degraded by nucleases such as the enzyme RNase. This can be mitigated to a certain degree by methods such as lipid nanoparticles (LNP) encapsulation (which is used in the current mRNA based COVID vaccines), substitution of modified bases, design of the mRNA, or other approaches. Alternatively, physical methods, such as electroporation can be employed, and this approach has gained traction in ex-vivo administered therapeutic cancer vaccines but is not highly efficient.
Moreover, distribution of vaccine is an issue, as frozen storage is currently required. Alternative methods, such as lyophilization, are under study.4 Thus, mRNA therapeutics is an exciting emerging therapeutic modality, but there are areas that need improvement and maturation, such as manufacturing, administration and supply chain aspects.
From a manufacturing perspective there are a number of challenges. One of the main challenges in mRNA processing is the lack of dedicated equipment and consumables fit for the relatively small volumes and large size of the mRNA compared to traditional recombinant proteins. There is also room for improvement in technology development in many of the steps to improve scalability and process consistency.
Trends in RNA-based therapeutics
The potential of mRNA vaccines gained scientific attention in 1990 after the in vivo expression of a protein was observed after injection of naked mRNA into the skeletal muscle of a mouse.5 Since that time, the industry has seen rapid development and expansion. Today, more than 140 clinical trials have been launched that use mRNA to address various conditions such as infectious disease, cancer and a variety of other possible application areas.
Two forms of mRNA structure and currently being developed: conventional non-replicating mRNA and self-amplifying mRNA. Non-replicating mRNA vaccines have the conventional mRNA form and do not have replication capability built into the mRNA sequence. The sequence of the antigen is flanked by untranslated (UTR) regions, a 3’ poly (A) tail, and a 5’ cap. The cap, UTR, ORF, and tail can be customed designed to up or down regulate expression levels, or to modulate immune response.4 Modified nucleotides, such as pseudouridine and 5-methylcytidine can be used to lessen undesirable innate immune system responses and to increase translation efficiency.2,4 Thus, there are many aspects of the clinical response that can be modulated simply by the design of the mRNA.
Non-replicating mRNA vaccines are transient by nature and typically express antigen for hours or a few days (the cellular half-life of Pfizer and Modern vaccines is estimated to be 8-10 hours). For some applications this can be beneficial, however for others such as systemic protein therapies, extended expression of a protein would be beneficial.
Self-amplifying mRNA (saRNA) approaches are under development that enable the mRNA to replicate. This, in turn, can extend the expression window to weeks.4–6 Typically, saRNA is based on the addition of viral replicase genes, in cis or trans configuration, from alphavirus, flavivirus or picornavirus. These strategies can either increase expression level or lower mRNA dose requirements 10-100-fold. Self-replicating mRNA could potentially expand mRNA technology across many applications while lowering manufacturing demand. There are many areas of mRNA technology that are under development and optimization and mRNA design and optimization are important aspects of current efforts.
There are other RNA therapies outside of mRNA that are being developed or have been approved. Among these are antisense oligos, which modify gene expression; small interfering RNA (siRNA), which also modify gene expression via a different mechanism; aptamers, which can bind other ligands, including RNA; guide RNAs, used for CRISPR targeting; and other functional RNAs. Many of these RNA therapeutics share overlapping technology with mRNA vaccines. An example is the approved siRNA therapeutic Onpattro© that uses LNP technology.2,3 Thus, the entire RNA therapeutic space is advancing rapidly in addition to mRNA vaccines.
Different Types of mRNA-based Therapies
The COVID vaccines are prophylactic vaccines for infectious diseases There are numerous other prophylactic vaccines in development which include, influenza, Zika, dengue, rabies, Venezuelan equine encephalitis, in addition to bacterial infections such as staphylococcus and TB.4,6 Unique approaches include expression of a neutralizing monoclonal antibody for chikungunya virus.4
mRNA vaccines have also gained traction as a therapeutic approach for cancer. mRNA can be used to elicit immune responses to mutated oncogenes or regulatory cancer genes, such as p53, that are shared across many cancers in a therapeutic pan-cancer approach.
Other approaches for cancer include personalized therapy where vaccines are developed against a person’s individual cancer mutations. In this regard, a patient’s mutanome would be identified by next generation sequence and a handful of custom mRNA vaccines would be developed targeting the individual’s particular neoantigens.7
Therapeutic cancer vaccines are advancing quickly in development with over 70 completed clinical trials and additional ongoing results expected in the next 2-3 years.5 Many techniques are being evaluated including the direct stimulation of antigen presenting cells (APCs), via ex vivo electroporation of mRNA. Other approaches include direct intra-tumor injection, whole body approaches, and targeted organ approaches. Currently over 50% of clinical trials using mRNA focus on the treatment of melanomas, prostate, and brain cancer.5 Thus, there are numerous applications of mRNA vaccines which are in various stages of development from concept to clinical trials, although targeting specific organs, tissues and cells with LNP is still under research.
Scales of mRNA Production and Manufacturing Bottlenecks
Scales of mRNA needed for an application vary on the indication, the potency of the approach, market demand, and other factors. Customized, individualized applications may require the production of only milligram amounts of mRNA. Global needs may require much more mRNA production capacity. For example, the current Pfizer and Moderna COVID vaccines contain 30 and 100 micrograms of non-replicating mRNA respectively.1 In that case, a production campaign of a billion doses would require the manufacture of 30-100 kg of highly purified cGMP mRNA, preferably produced via production batches of at least several grams.
One of the most common bottlenecks in current manufacturing of mRNA is scaling. There is certainly a need for larger scale production technology now that COVID products are reaching scales of billions of doses. We see a large interest in Cytiva’s FlexFactoryTM and KUBioTM solutions where a full start to finish solution can be tailored and delivered to customers. These solutions have been developed and delivered for mAb applications as well as for plasmids and viral vectors already.” However, there is also needed improvement in smaller scale cGMP manufacturing as much of the current equipment is repurposed from the biotech industry and is designed for scales of manufacturing much larger than needed for mRNA. The industry could benefit from equipment specifically designed for mRNA cGMP manufacturing, including smaller scales.
The upstream manufacturing process of mRNA is rather mature. cGMP quality plasmid, polymerases, and enzymes needed for in vitro synthesis of mRNA are available but can be costly.5 Poly A tails can be created by inclusion in the template, or by use of enzyme.4 There are high efficiency, co-synthesis, capping options such as CleanCap© or alternatively capping can be performed by enzyme treatment with high efficiency as well.4 mRNA vaccines have the potential to be less costly than other vaccine approaches due to the cell free nature, but currently they are more expensive to produce. To improve the overall cost profile, costs for GMP reagents, capping reagents, for propriety LNP components, and other propriety components need to be reduced. The capacity constraints on the plasmids used as the starting template in the mRNA process is also a challenge which this application area shares with growing viral vector field. Also here companies are looking into ways to remove this bottleneck and new technologies around cell free processes for plasmid production could potentially improve this initial process step”
Downstream manufacturing, however, needs improvement. High purity of mRNA is required for efficient translation and to reduce undesirable immune responses.5 There are multiple impurities include enzymes, nucleotides, plasmid template, and aberrant RNA species and others which currently necessitate a multistep purification process.5 These multistep processes are varied and in a state of development. Techniques such as precipitation, affinity oligo dT, Ion Pair Chromatography (IPC) with or without cellulose, ion exchange, Tangential Flow Filtration (TFF), and others may be used.5 Thus, alternative purification ligands and refined purification approaches would benefit the industry greatly. Analytics could be improved as well.
While mRNA production is certainly amenable to standardization and platforming, as has occurred in the monoclonal therapeutics industry, much of the current production is performed in multiple steps using fit-for-purpose equipment. Other aspects such as single-use items and continuous processing strategies would benefit this emerging industry.
The speed and potential cost gains with the mRNA technology makes it an interesting technology for personalized medicine. Where vaccines are developed against a person’s individual cancer mutations. Many companies are working on integrated system mRNA processing solutions for this. Although many steps in the process are identical there are a number of added challenges due to scale and cost. But we are monitoring and contributing to development of this area which in a few years can have reached commercial stage.
Finally, there is a need for greater understanding of the science of the process. For example, LNPs are typically formed in rapid mixing process using microfluidic devices2 which is more of an art than an established method. Greater understanding of the influences of the LNP ingredients, and their effect on LNP stability, delivery, efficiency, immune response, and ultimately patient outcome would benefit the industry.2 The optimization of LNP and other delivery technology is critical attribute that can determine the ultimate success or failure of a therapeutic.
Encapsulation and Delivery Technologies
The use of nanostructures, such a LNPs, is common in mRNA therapeutic as these generally deliver higher efficiency than naked mRNA and allow for a broad variety of administration routes. A challenge with nanostructure technology is that it is complex by nature, and it involves many potential ingredients with many possible clinical outcomes. There is incomplete understanding in this regard. Nanostructure properties are critically important to clinical outcome and include: protection of nucleic acids, controlled release of RNA inside the cell, cell and tissue selectivity, translation efficiency, toxicity, and long term stability, and others.2
Nanostructure structure is sophisticated, and they may be composed from several components, such as common lipids, polymers (PEG, PEI, Poly-lysine, etc), proteins, cholesterol, or custom proprietary components such as ionizable lipids.3,4 Often conjugates are used such as PEG-lipid. Each of these can result a dramatic effect on its property. For example, polymer content can control particle size and affect efficiency and cell tropism. Structural lipids, such as cholesterol, can affect particle stability. Empty nanoparticles without payload can formed if not mixed correctly. Thus, nanostructure composition and formation are critical for desired clinical effect.2 Currently, LNPs are the landing non-viral delivery system for many systems, including gene therapy.2
There are other delivery methods under study and development. Exosomes are thought to use a receptor, and may offer more efficient uptake, greater specificity, and fewer side effects.8 This a promising early area of research. Other areas include conjugated RNA, such as GalNac-siRNA which has been shown to target liver hepatocytes.9 Likewise, GALA-peptide conjugated mRNA has been shown to improve uptake in APCs.10 There are other approaches under evaluation to increase target specificity or to improve cellular uptake.
Naked mRNA has been evaluated for cancer therapy by direct injection to the tumor or other approaches. Generally naked RNA is considered less efficient than other methods but has advantages in that it is easy to prepare since it only requires a buffer.4,11 In some application the intrinsic high immunogenicity of naked mRNA may provide benefit via boosted adjuvant activity.11
mRNA Industry Perspectives
With the arrival of the COVID-19 pandemic, mRNA for prophylactic vaccines took the public spotlight due to their urgent need. These vaccines demonstrated the promise of mRNA therapeutics through their quick developmental timeline and high efficacy.
While COVID vaccines are notable, the majority of mRNA vaccines to date have been focused on cancer therapy, and there have been dozens of complete or ongoing clinical trials to date. In the next 2-4 years, many of these trials should be completed. Many of these are personalized therapeutic cancer vaccines. Promising results in this area could further advance the mRNA industry.
Moreover, there are many therapeutic in early development, across diverse, areas that will have high impact if successful. Success in mRNA therapies could potentially displace less effective therapeutic approaches such as vaccines for influenza, TB, or other applications in the future.
mRNA therapy is a growing field that is experiencing rapid development and expansion. There are many applications in development that are too numerous to detail in this brief report. The technology offers great benefit and potentials for infectious diseases and personalized medicines due to its advantages in flexibility, cost and speed of development. There are of course still challenges to overcome to fully realize the potential of this technology including lack of experience and knowledge of scaling up mRNA processes, perceived regulatory uncertainties and targeted delivery technologies. The COVID-19 pandemic publicly demonstrated the promise of mRNA therapies, and the future looks bright for this emerging industry.
Learn more on mRNA processing
Henrik Ihre, Strategic Technology Partnerships Leader at Cytiva
Henrik Ihre has been the Director of Strategic Technologies since March 2020 with a specific knowledge and background in the Downstream purification of biopharmaceuticals since 20 years back in time.
Henrik joined legacy Amersham Pharmacia Biotech in August 2000, following its acquisition by GE Healthcare in 2004. His career has been devoted to the design and development of several purification products enabling the manufacturing of life changing drugs on the market. Henrik started as a Senior Scientist in the R&D chromatography resin department and later transferred into the role of Product Manager for several key products such as the Protein A portfolio. For more than ten year he headed and expanded the custom consumables organization offering customization within segments such as Chromatography resins, Ready To Process columns, Small scale pre-packed columns and Primer supports for oligo synthesis. During his time in the custom consumable’s organization more than 30 key products where brought to the market which now are a part of the Cytiva downstream offering. The year with the custom consumables organization gave valuable insight to the key elements of product design and development and also gave an excellent opportunity to develop a broad network within the biopharma customer community and better understand their needs.
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