In Vivo Applications of mRNA Technology

Messenger RNA (mRNA) has emerged as one of the newest and most effective therapeutic modalities to prevent and treat various diseases by using the information in genes to create a blueprint for making proteins. For mRNA to function successfully in vivo, it requires a safe, effective, and stable delivery system that can protect nucleic acid from degradation and can allow cellular uptake and the release of mRNA. There are multiple types of applications for in vivo therapeutics, but the main modes for mRNA vaccines in vivo are induced pluripotent stem cells (iPSCs) and chimeric antigen receptor (CAR) therapies. Lipid nanoparticles (LNPs) have become the most successful component for delivery of mRNA [1]. Currently, LNP-mRNA vaccines are in clinical use against coronavirus disease 2019 (COVID-19), which marks a milestone for mRNA therapeutics [1]. With the help of LNPs, mRNA in vivo therapy can be used for a wide variety of therapeutic applications, such as cell and gene therapy, genome editing, etc. [2].

As previously mentioned, the two main application methods are CAR therapies and iPSCs. CAR-T cell therapy is an adoptive immunotherapy where T lymphocytes are engineered with synthetic receptors to recognize and eliminate specific cancer cells that are independent of major histocompatibility complex molecules [3]. In this methodology, the encapsulated mRNA molecules are taken up by T cells and function as templates to produce targeting receptors which are then programmed to attack. The CAR-T cell has been used predominantly in the treatment of hematological malignancies, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and multiple myeloma. Solid tumors including melanoma, breast cancer and sarcoma offer great promise in CAR-T cell research and development [3].

Pluripotent stem cell (PSC) technologies have opened possibilities for generation of patient-specific cells and tissues for the study of diseases and therapeutic applications. Typically, PSC differentiation protocols employ morphogens and growth factors to induce differentiation [4]. Recent advances in identification of key transcriptional regulators involved in cell fate commitment make it possible to directly convert somatic cells and PSCs to desired cell lineage simply by forced expression of transcription factors. Viral gene delivery systems for differentiation/reprogramming are associated with genome integration and reactivation of the viral proteins, making it unsafe for therapeutic purposes. In recent years, advances in mRNA synthesis have improved their use in differentiation/reprogramming [4].

In 2006, Yamanaka et al. first discovered and generated induced pluripotent stem cells (iPSCs) from mouse fibroblasts transduced with four transcription factors such as Oct3/4, Sox2, c-Myc, and Klf4 (OSKM), under ES cell culture conditions [5]. iPSCs are derived from a variety of somatic cell types using reprogramming, are more readily obtainable for stable research, and, due to their origin, have no ethical concerns around how they are harvested. iPSCs from either diseased or healthy cells, such as human ESCs or diseased organoids, can be used for the in vitro screening of drug candidates. Rowe and Daley used neural, gastrointestinal, and liver organoids derived from human PSCs to establish clear organoid-level readout, which allowed for the diseased organoids to stand out and thus be used in drug screenings and validation studies [6]. There are four types of reprogramming methods that can be used with iPSCs, including virus-based integrative reprogramming, RNA-based non-integrative reprogramming, plasmid-based non-integrative reprogramming, and mRNA-based non-integrative reprogramming [5]. RNA-based non-integrative reprogramming differs from mRNA-based non-integrative reprogramming in that the RNA-based reprogramming uses the Sendai virus to overexpress pluripotent transgenes and initiate iPSC generation, and the mRNA-based reprogramming uses mRNA-carrying pluripotent genes for iPSC generation. mRNA reprogramming technology is the most unambiguously footprint free and genomic integration free for iPSC generation. In recent years, researchers have gradually developed modified mRNA transcripts, which enhance their stability, reduce immunogenicity, and improve their delivery. In 2021, Wang focused on mRNA-based reprogramming, finding that modRNA can directly drive specific cell fate and cell reprogramming from various somatic cells [5]. Moreover, a modRNA cocktail can be designed to simultaneously induce multiple different proteins in somatic cells [7].

Since iPSCs hold enormous promise for various applications in regenerative medicine including disease modeling, drug discovery, and cell replacement therapy, they are also an option to consider when it comes to treating midbrain dopaminergic (mDA) neurons for individuals with diseases such as Parkinson’s. Xue et al. established the first mRNA-driven strategy for efficient iPSC differentiation to mDA neurons. They used mRNAs coding Atoh1 and Ngn2 with defined phosphosite modifications to the proteins to reach higher and more stable protein expression and found that these mRNAs induced more efficient neuron conversion, as compared to mRNAs coding wild type proteins [8]. Modifications in this study included blocking the phosphorylation of these serine sites, which has been shown to stabilize transcription factor (TF) proteins and/or enhance their transcriptional activity. They found that mRNA-induced mDA (miDA) neurons recapitulate key biochemical and electrophysiological features of primary mDA neurons and can provide high-content neuron cultures for drug discovery. Lastly, they provided a methodology to help facilitate the development of mRNA-driven differentiation strategies for generating iPSC-derived progenies widely applicable to disease modeling and cell replacement therapy [8].

LNPs have been found to be appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA using therapeutic proteins [9]. Since mRNA is highly unstable under physiological conditions, unprotected mRNA delivered by itself is unsuitable for broad therapeutic applications and was therefore ignored by the pharmaceutical industry for a long time. It was the development of RNA interference and its tremendous therapeutic potential that triggered intense efforts toward stabilization of RNA in vivo. Several strategies have been developed for RNA delivery, including RNA-conjugates, modified RNA, viral vectors and microparticles and nanoparticles [10]. Viral vectors were the obvious choice for delivery because viruses have naturally evolved to become highly efficient at nucleic-acid delivery. However, there are limitations with these vectors including immunogenicity, carcinogenesis, broad tropism, packaging capacity and production difficulties.

Nonviral vectors exhibit significantly reduced transfection efficiency but tend to have lower immunogenicity than viruses. Varying popular types of nonviral vectors include lipids, polymers, peptides, and inorganic nanoparticles. Currently, lipid nanoparticles are among the most frequently used vectors for in vivo RNA delivery. They can be synthesized with relative ease in a scalable manner, protect the mRNA against degradation, facilitate endosomal escape, can be targeted to the desired cell type by surface decoration with ligands, and as needed, can be co-delivered with adjuvants [10]. Also, in addition to functioning as a delivery component, lipids can have therapeutic effects synergistic with mRNA-encoded proteins [11]. LNPs are composed of helper lipids which come in many shapes—for example, cylindrical-shaped lipid phosphatidylcholine can provide greater bilayer stability, which is important for in vivo application of LNPs [12]. In a 2016 study from Pardi et al., researchers found that intramuscular and intradermal delivery of mRNA-LNPs resulted in the longest duration of translation [9].

Thermo Fisher Scientific is a stand-out leader in mRNA innovation. Among their many products, the MEGAscript T7 Transcription Kit has been used in many studies centering around the in vivo applications of mRNA. This ultra-high yield IVT kit optimizes transcription reaction conditions so that exceedingly high nucleotide concentrations can be effectively used. The MEGAscript T7 Kit is intended for the synthesis of RNA for a variety of uses, including in vivo and in vitro translation, antisense/microinjection studies, and isolation of RNA binding proteins.

Thermo Fisher Scientific can be considered a “one-stop shop” for transfection experiment supplies, with tools for all six steps in the process including target gene design and plasmid production, plasmid purification and linearization, mRNA synthesis, mRNA purification, mRNA analytics, and formulation, fill, and finish.

1. Cross R. Without these lipid shells, there would be no mRNA vaccines for COVID-19. 2021. Chemical and Engineering News, 8(99).
2. Melamed JR, Hajj KA, Chaudhary N, Strelkova D, Arral ML, Pardi N, Alameh MG, Miller JB, Farbiak L, Siegwart DJ, Weissman D, Whitehead KA. Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery. J Control Release 341, p. 206–214 (2022). doi: 10.1016/j.jconrel.2021.11.022.
3. Zhao Z, Chen Y, Francisco NM, Zhang Y, Wu M. The application of CAR-T cell therapy in hematological malignancies: advantages and challenges. 2018. Acta Pharmaceutica Sinica B, 8(4), p. 539–551. doi: 10.1016/j.apsb.2018.03.001.
4. Suknuntha K, Tao L, Brok-Volchanskaya V, D/Souza SS, Kumar A, Slukvin I. Optimization of synthetic mRNA for highly efficient translation and its application in the generation of endothelial and hematopoietic cells from human and primate pluripotent stem cells. 2018. Stem Cell Rev., 14(4), p. 525–534. doi: 10.1007/s12015-018-9805-1.
5. Wang AYL. Application of Modified mRNA in Somatic Reprogramming to Pluripotency and Directed Conversion of Cell Fate. 2021. International Journal of Molecular Sciences, 22(5), 8148. doi: 10.3390/ijms22158148.
6. Rowe RG, Daley GQ. Induced pluripotent stem cells in disease modeling and drug discovery. 2019. Nature Reviews Genetics, 20, p. 377–388. doi: 10.1038/s41576-019-0100-z.
7. Yanamaka S, Takahashi K. Introduction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. 2006. Cell, 126(4), p. 663–676. doi: 10.1016/j.cell.2006.07.024.
8. Xue Y, Zhan X, Sun S, Karuppagounder SS, Xia S, Dawson VL, Dawson TM, Laterra J, Zhang J, Ying M. Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. 2019. Stem Cells Translational Medicine, 8(2), p. 112-123. doi: 10.1002/sctm.18-0036.
9. Pardi N, Tuyishime S, Muramatsu H, Kariko K, Mui BL, Tam YK, Madden TD, Hope MJ, Weissman D. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. 2015. J Control Release 217, p. 345–351. doi: 10.1016/j.jconrel.2015.08.007.
10. Reichmuth AM, Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles. 2016. Therapeutic Delivery, 7(5), p. 319-334. doi: 10.4155/tde-2016-0006.
11. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. 2021. Nature Reviews Materials, 6, p. 1078-1094. doi: 10.1038/s41578-021-00358-0.
12. Cheng X, Lee RJ. The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery. 2016. Advanced Drug Delivery Reviews, 99 Part A, p. 129-137. doi: 10.1016/j.addr.2016.01.022.