mRNA is a type of nucleic acid molecule that plays an essential role in the natural processing of genetic information. Using modern tools of molecular biology and genetic engineering, it is now possible to design synthetic mRNA molecules capable of executing an astonishing array of therapeutic functions. Advances in nanotechnology are starting to enable safe and effective delivery of therapeutic mRNA for the treatment of acquired and inherited conditions and for prevention of infectious disease.
In the field of infectious disease, a prophylactic vaccine is a product that prevents disease by inducing an immune response against an infectious agent or pathogen. Prophylactic vaccines have played a critical role in the public health response to diseases such as smallpox, polio, measles, and many others – most recently COVID-19. The coordinated response by government, academic, and commercial research teams to the COVID-19 epidemic raised public awareness of the potential for mRNA vaccines to play a significant role in public health and resulted in the first two FDA approvals for mRNA products. These results helped to reinvigorate ongoing efforts to develop prophylactic mRNA vaccines for a broad variety of infectious diseases. In the coming years, mRNA vaccines will play an important and complementary role alongside other approaches, such as those involving recombinant proteins, live attenuated viruses, and engineered viral vectors in protecting public health.
For pathogens that cause cancer, such as human papilloma virus (HPV), a vaccine against that infectious agent can also contribute to protection against cancer. HPV prophylactic vaccination programs have provided significant public health benefits by reducing the occurrence of cervical cancer.
The molecular target of an induced immune response is called an antigen. Many investigators are interested in developing prophylactic vaccines that may prime an immune response directly against a cancer antigen rather than an infectious agent; however, development of such prophylactic cancer vaccines is still in early stages.
Building on the science of prophylactic vaccines, much recent effort has focused on therapeutic vaccines. A therapeutic vaccine is intended to treat an existing disease by inducing an immune response against a disease-associated antigen. Some of this effort involves infectious disease, but most of the research is focused on treatment of cancer.
A defining feature of both prophylactic and therapeutic vaccines is the targeted induction of an immune response against one or more antigens associated with the disease. Most therapeutic drug products are not vaccines. However, modern gene transfer techniques provide shared platforms that are being used to develop (i) prophylactic vaccines, (ii) therapeutic vaccines and (iii) non-vaccine therapies across a broad range of therapeutic areas.
Gene transfer research constitutes a rapidly growing sector of drug/biologic product development and encompasses the closely related fields of cell and gene therapy (CGT), gene editing, molecular medicine, genetic medicines, and advanced therapy medicinal products.
In every nucleated cell, genetic information is stored as DNA in chromosomes in the nucleus, where that information is transcribed into RNA, which then exits the nucleus and passes into the cytoplasm. In the cytoplasm, information from this RNA is translated into proteins, which act as essential structural and enzymatic components of cells. RNA performing this function of carrying the message encoding instructions for the protein sequence from the nucleus to the cytoplasm is called messenger RNA (mRNA). Molecular interventions that add or alter genetic information in DNA or mRNA have the potential to affect almost any biological process controlling human health and disease. The key challenges are making the correct genetic changes and targeting them to the correct cells.
As part of the natural process of gene expression, individual mRNA molecules are generally short-lived in the cytoplasm. The transient nature of mRNA is an essential aspect of dynamic gene regulation. For some applications utilizing engineered mRNA, highly transient expression is desirable. For other applications, investigators have developed modifications to the basic mRNA design that result in mRNA persistence and prolonged translational activity. Some such modifications are based on naturally occurring sequence variations that signal the cell to protect and preserve the mRNA molecule. Other approaches use chemical modifications of the building blocks of mRNA not found in nature to protect the mRNA product from rapid degradation. Discovery, testing, and implementation of these modifications to basic mRNA chemistry have been critical steps in enhancing the efficacy of mRNA products destined for commercialization and clinical use.
One way to deliver new genetic information to a cell is by adding genetically modified DNA. For most systems, the engineered DNA must cross both the outer plasma membrane of the cell and the nuclear membrane to reach the nucleus, where the genetic information can be expressed. Often the engineered DNA is delivered by means of a viral vector—a biological construct derived from a wildtype virus that has been extensively modified to carry recombinant genetic material and not cause disease. Viral vectors are the key components in successful products including both prophylactic vaccines and gene therapies for cancer and other diseases. Many different families of wild viruses serve as the basis for engineered viral vectors in clinical and preclinical development. Various FDA-approved products utilize viral vectors based on adenoviruses, adeno-associated viruses (AAV), and herpes simplex viruses. Compared to most small molecules and many biologic products, viral vector products involve complex and resource-intensive efforts for design, testing, manufacturing, and quality control.
Rather than directing DNA to the nucleus, another way to deliver genetic information to a cell is to introduce synthetic mRNA into the cytoplasm. In contrast to most DNA-based approaches, genetic information encoded in synthetic mRNA can be expressed immediately upon entering the cell and does not need to cross the nuclear membrane to enter the nucleus, be transcribed, and then exit the nucleus to reach the cytoplasm as a translatable message. Thus, functional gene delivery by synthetic mRNA is simpler than that required for DNA in significant ways. Rather than viral vectors, many approaches to mRNA delivery utilize nanoparticles. The simplest nanoparticles for gene transfer use lipid or lipidoid compounds to form self-assembling nanoscale spheres around mRNA cargo. These particles serve the dual function of protecting mRNA from destructive enzymes in the environment and allowing the mRNA to cross the plasma membrane and enter the cell.
The DNA in our chromosomes is chemically very stable, with information stored in two complementary strands assembled as a double-helix (double-stranded DNA). mRNA is a single stranded molecule, inherently less stable and suitable for transient functions in the cell. Partly as a mechanism for protection against dangerous viruses, our bodies produce an excess of RNA-degrading enzymes, called RNAses. For these reasons, in the past, mRNA molecules were often regarded as too unstable to be very useful for drug development. Recent experience has shown that RNA prepared under strict RNAse-free conditions and packaged appropriately, as with lipid nanoparticles, is quite stable and very much suitable for use as a drug substance in many applications.
Non-biologic drugs such as small molecules can often be manufactured via an easily routinized process that reliably yields a product with predictable CMC (Chemistry, Manufacturing, and Control) output. In contrast, biological products in the CGT category often pose severe challenges for design, testing, and CMC, due to the complexity of products comprised of nucleic acids, proteins, lipid membranes, and in some cases living cells. With synthetic mRNA, manufacturing considerations are largely independent of the sequence of the genetic code, meaning that an established manufacturing process for one mRNA product can often be implemented broadly in many different applications. In this way, the production of mRNA drug products is more like non-biologic chemical drugs than most CGT products. Using advanced sequencing and bioinformatics techniques, it is possible to acquire genetic sequences from biological samples and generate new mRNA products within a very short timeframe – within days of sample acquisition for preclinical research. This allows mRNA products to be incorporated into rapid-response plans for public health emergencies for example. This flexibility also makes mRNA approaches suitable for personalized therapeutic cancer vaccines.
As discussed above, DNA-based gene transfer approaches usually rely on a viral vector for gene delivery. One of the most popular platforms uses AAVs as vectors for DNA transfer. An important issue with these vectors is that for any natural AAV serotype, a significant percent of the population will have circulating antibodies that neutralize the respective AAV vector. Seronegative individuals usually seroconvert after a single dose of AAV gene therapy, and thereby become ineligible for future treatment with the same AAV serotype. As a result, most AAV products are designed to be one-and-done treatments, intended to provide lifelong therapeutic effects without the possibility of re-dosing. The one-time nature of AAV dosing poses significant challenges for dose-finding studies, as well as for long-term follow up to assess safety and efficacy. This treatment approach is also a challenge for commercialization strategies given that payment models in many therapeutic areas are built around the assumption that the same drug will be administered in multiple doses over time.
In contrast to AAVs, mRNA lipid nanoparticles currently in use do not induce an immune response to the particle that interferes with redosing. Furthermore, as explained above, after administration, synthetic mRNA products are usually short-lived in the cell and result in transient protein expression. In combination, these factors make it easier to optimize dose-finding approaches with mRNA compared to AAVs. The ability to apply repeated doses allows for serial boosting in vaccination regimens. As a therapeutic approach for chronic disease, mRNA treatment plans would potentially allow continuous administration of the therapeutic in multiple doses for as long as clinically necessary; they would also allow for rapid cessation of treatment in case of unwanted side effects and adverse events. It is likely that commercialization plans for mRNA-based therapies would also be more compatible with standard reimbursement plans for the cost of drugs. While AAVs and other vector-based approaches will certainly be the basis for many valuable new therapies for the indefinite future, mRNA presents an opportunity to develop complementary modalities in many therapeutic areas.
One area where mRNA therapeutics show particular promise is the development of personalized vaccines. An essential challenge in cancer immunology comes from the fact that cancer cells originate from the patient’s own tissue. This means that most potential antigens found in tumor cells are shared with normal healthy tissue and cannot be the basis of a useful immune response. Over the course of oncogenesis, tumors usually undergo extensive mutations in tumor cell chromosomal DNA. Many of these changes encode proteins that contain altered, tumor-specific antigens, also known as neo antigens. A key feature of neoantigens is that they are sufficiently distinct from antigens in normal tissue to serve as targets for an immune response. For certain cancer types, immune responses directed against neoantigens have been shown to provide significant clinical benefits in preclinical and clinical testing. For most tumor types, the majority of neoantigens detected from a biopsy are “private” and specific to the respective individual patient.
As noted above, new mRNA products can be rapidly designed and manufactured from any genetic sequence. This makes mRNA a promising platform for personalized cancer vaccines. To create a personalized vaccine, investigators sequence genetic content of a tumor biopsy and use bioinformatic methods to identify potential neoantigen targets – representing protein sequences present in the tumor but not in healthy tissue. mRNA encoding those neoantigen targets can then be rapidly synthesized and administered to the originating patient in an immunogenic formulation to induce a protective immune response against the tumor.
As with any new treatment approach, development of neoantigen vaccines involves challenges and potential pitfalls. If the identified neoantigens are too similar to native host proteins, then the immune system may fail to see the neoantigens as “non-self,” and thus may fail to mount an effective response. Alternatively, if the neoantigens are too similar to native host proteins, an aggressive immune response could be primed against both the tumor antigen and normal healthy tissue, resulting in potentially serious autoimmune disease. Another potential issue is that neoantigens are often the result of extreme genetic instability of tumors. Therefore, as effective immune responses start to suppress the tumor, the tumor may rapidly evolve to cease expression of the most effective neoantigen targets. This process is called antigenic escape. One approach to minimize antigenic escape is to vaccinate with many neoepitopes at once. At the time of this writing, news reports indicate very promising results in a Phase 2b clinical trial of a personalized mRNA vaccine in combination with an anti-PD-1 immunotherapy, in participants with Stage III/IV melanoma.
To date, the majority of clinical trials of mRNA products have been for prophylactic vaccines for infectious disease. Most of the remaining trials are for the development of therapeutic cancer vaccines. Nevertheless, there are many preclinical and clinical efforts to apply mRNA in the area of nonvaccine therapeutics. One promising approach is a gene replacement therapy for inherited rare disease.
There are more than 7,000 rare diseases recognized, with a large percentage being congenital conditions affecting individuals that received two defective versions of the same gene, one from each parent (i.e., through autosomal recessive inheritance). In theory, each of these conditions should be treatable via molecular gene replacement therapy. Gene replacement refers to a variety of therapeutic approaches whereby the missing function of the defective gene is restored by delivering a corrected gene to some key tissue in the body. To date, most gene replacement therapy approaches have utilized AAV, an approach with great potential and significant drawbacks as described above. Some of those drawbacks may be avoided with mRNA-based gene replacement, and ongoing efforts in this area include clinical trials in cystic fibrosis and a variety of inborn errors of metabolism.
Aside from gene replacement, mRNA can potentially be utilized for the delivery of a next generation of products developed with synthetic biology. Synthetic biology uses systematic modular engineering approaches to create novel biomolecules, some of which perform new functions distinct from any natural molecules. Some products of synthetic biology may enable white blood cells to recognize and attack tumor cells. Some are designed to detect and correct hormonal imbalances in vivo in real time. For clinical purposes, the underlying genetic constructs may be delivered by a variety of gene transfer techniques, including mRNA. Thus, we can expect mRNA to be an important part of the drug development landscape in the coming decades.