Introduction to mRNA research and oncology

Messenger RNA (mRNA), specifically synthetic mRNA, has emerged as a powerful tool for the transfer of genetic information, and it is being explored for a variety of therapeutic applications [1]. In recent years, scientists have explored the vast therapeutic potential of mRNA for many “undruggable” diseases. mRNA therapeutics offer some key advantages over traditional drugs or proteins. Primarily, it is easier to manufacture mRNA transcripts as opposed to folded and post-translationally modified proteins. Compared to DNA, mRNA is biologically active in both dividing and non-dividing cells. It does not need to enter the nucleus to be active and poses no risk of genome integration. Technological advances have made it possible to create engineered mRNA that mimics natural mRNA through in-vitro transcription (IVT). This can be used as a medical tool to target “undruggable” diseases such as certain cancers and muscular dystrophies caused by genetic defects or mutations [2].

mRNA vaccines are an ideal development platform. Compared to other therapeutic types, it is quicker and more efficient to design and screen multiple antigens. Producing mRNA therapeutics is easier, cheaper, and safer than other therapeutics. For example, a single facility can be used to produce multiple mRNA therapeutics because all materials are the same - the only difference is the sequence [3]. For the other therapeutic types, separate facilities are required to make different therapeutics. mRNA-based approach is being applied to multiple therapeutic modalities including cell therapy, gene editing, gene therapy, and protein replacement [1,2].

mRNA therapeutics and cancer therapy

Cell therapy

Synthetic in vitro-transcribed (IVT) mRNA can be engineered to mimic naturally occurring mRNA and thus be useful in cancer immunotherapy. There has been difficulty in the past with generating small molecules to enhance the activity of enzymes, but with mRNA, this issue disappears. Since mRNA encoding the wild-type enzyme is easily generated, mRNA can replace deficient enzymes when delivered to the appropriate cell type. Compared to DNA delivered gene vectors, RNA is biologically active in both dividing and non-dividing cells and does not need to enter the cell nucleus to generate its therapeutic effect. Furthermore, with standard mRNA, there is no risk of altering the host genome [2].

In cell therapy, mRNA is transfected into the cells ex vivo to therapeutically enhance cell survival, proliferation, and/or function. The success with this modality has given way for stem cell generation and enhancement, and with specific research from Chanda et al, for cardiovascular regeneration [2]. Another key approach is to transfect patient-derived T cells with mRNAs encoding chimeric antigen receptors (CAR), enabling T cells to directly identify specific antigens on tumors. CAR-T cell therapy is an important immunotherapy to treat certain types of leukemia and lymphoma. For example, this is an FDA approved therapy for relapsed or refractory B-cell acute lymphoblastic leukemia patients [4]. Patients with mantle cell lymphoma and follicular lymphoma have shown significant improvement with this therapy [5].

This progress in cancer research is based on the efficacy of COVID-19 vaccines, which are specialized to train the immune system to target and kill cancerous cells. Scientists at Pfizer-BioNTech and Moderna have been working on mRNA-based cancer treatments for over a decade and used that experience to create COVID vaccines. Although some trials for cancers such as pancreatic cancer, colorectal cancer, and melanoma have not yet been proven to improve the conditions of people with various cancers or enhance the body’s immune response to tumors, research is still ongoing.

Gene therapy

Gene therapy involves transfer of genetic material via a vector or a carrier for gene uptake into specific cells. DNA-based gene therapies were more prevalent in the past, but they have taken a back seat due to severe side-effects such as targeting of the wrong cells, infections caused by the virus, and possible tumor growth. mRNA-based gene therapies, however, are gaining more attention as mRNA is short lived, less stable and does not induce permanent genetic changes. mRNA treatments have been shown to help patients with glioblastoma for a host of reasons: mRNA can be used as a biomarker and target for cancer therapy; the potential of mRNA to evoke effective anti-tumor immunity; and the ease of regulating the mRNA of many proteins which would then regulate the expression of tumor-related proteins [6]. mRNA makes it possible to deliver all epitopes of entire antigens in one step together and makes manipulation as well as purification simple tasks. mRNA encoding versatile antigens combined with delivery to dendritic cells (DCs) has been found to be a strong and promising approach to induce immune response in cancer patients. For example, 1–10% of DCs are transfected by means of electroporation, cationic polymers, or cationic lipids. However, transfection efficiencies by electroporation of mRNA have been previously shown to reach up to 95% transfected cells. Since mRNA does not have to be transported into the nucleus, mRNA transfer for gene therapy is much more effective [7].

Challenges and potential solutions

Though mRNA therapies are promising and have great potential in oncology, they face challenges too. For one, mRNA is rapidly degraded by extracellular RNase reducing its half-life and hindering efficacy [8]. Bioavailability of mRNA is also limited due to lack of passive diffusion across plasma membrane, caused by high molecular weight and electrostatic repulsion between opposite charges of proteoglycan coated cell membrane and mRNA molecules [5].

Molecular stabilization techniques using engineering of sequence and/or structure are instrumental in protecting the mRNA molecule from degradation and increase protein expression levels [9]. Adding synthetic cap analogue at the 5’ end of mRNA serves multiple functions: It protects the mRNA degradation from exonucleases, plays a vital role during translation as eukaryotic initiation factor (eIF) binds to this cap, and it also prevents innate immune sensors from recognizing the mRNA [5]. Another approach is to modify or augment 3’ and 5’ end untranslated regions (UTRs) of mRNA. Incorporating α globin 3’ end UTRs makes mRNA more stable, whereas β globin at both 5’ and 3’ end UTR leads to increased translational efficiency [1]. Poly(A) tail has been shown to decrease exonuclease-based degradation of mRNA and enhance translation efficiency [10].

Role of Thermo Fisher Scientific

Dedicated to the goal of cancer free society, Thermo Fisher Scientific is at the forefront of immunotherapy development. They offer a variety of cutting-edge solutions for cell therapy and gene therapy whether it is single use consumable such as cell factory systems, fluid transfer assemblies) or bioprocessing equipment. They also offer in vitro transcription kits such as MEGAscript T7 Transcription Kit and mMessage mMachine ULTRA as well as mRNA clean-up products such as MEGAclear Transcription Clean-Up Kit.

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