The beginning: Discovery of mRNA and its translation in the lab
It started in 1961, when Brenner and colleagues described the presence of an unstable intermediate molecule that copies the information encoded by the DNA and directs the synthesis of proteins: RNA. The group around Brenner worked with virus-infected cells and analysed the gene expression (Brenner et al., 1961).
In 1963, Issac et al. demonstrated that viral nucleic acids induce the production of interferon in infected cells (Isaacs et al., 1963).
In 1969, mRNA was translated in the lab for the first time. Lockard and Lingrel, who worked together at the University of Cincinnati, Ohio, provided the first evidence of in-vitro translation of mRNA. They used a mammalian (rabbit) cell-free system to demonstrate the translation of an mRNA transcript from another mammalian species (Lockard and Lingrel, 1969).
Moreover, in 1984, experimental work showed that any desired cDNA can be utilised for the synthesis of functional mRNAs and, consequently, the synthesis of proteins (Krieg and Melton, 1984). In light of this work, SP6 RNA polymerase was eventually commercialised. These pioneer experiments lead to an unstoppable series of practical work concerning mRNA delivery and commercialisation.
In 1978, liposomes were utilised for the delivery of mRNA to eukaryotic cells (Dimitriadis, 1978). By the end of the following decade, a cationic liposome mRNA delivery system, DOTMA, was described and commercialised (Malone et al., 1989).
The rise: mRNA as a therapeutic agent
The foundation of the concept of mRNA as a therapeutic agent was laid by Wolff J. and colleagues in 1990. The team injected naked RNA into mice muscles to provide proof of principle for direct gene transfer in vivo (Wolff et al., 1990).
In 1992, a team of scientists working at Scripps Research Institute used mRNA to transiently reverse diabetes insipidus in Brattleboro rats that do not produce the hormone vasopressin (Jirikowski et al., 1992).
Even though the concept of mRNA vaccines sounds relatively advanced, it dates back to 1995, when Robert and his team designed the first mRNA vaccine that encoded cancer antigens (Conry et al., 1995).
All this work in mRNA therapeutics laid the cornerstone of the first mRNA company ever founded: Merix Bioscience (1997). In 2004, the company changed its name to Argos Therapeutics as a sign of its evolution.
The climax: Challenges vs solutions
The idea of using mRNA as a therapeutic agent sounds straightforward, but it is not. Two major challenges had to be overcome: 1) Successful delivery of mRNA, and 2) Prevention of an immune response against the mRNA molecule.
Isolation of RNA is difficult as ever present RNase easily degrades RNA. In order to prevent this degradation, different measures to inhibit RNase function have to be taken when RNA is isolated from cells/tissue.
When mRNA is injected into the body it triggers an immune response. As a result, the injected mRNA is degraded and the synthesis of the desired proteins is shut down.
For these reasons, little funding was granted to studies focusing on mRNA based therapeutics. Even Nobel laureate Philip A. Sharp found it to be a concept with little practicality for the above mentioned reasons. Katalin Kariko, a researcher at the University of Pennsylvania, struggled to get funds for her research on mRNA application. “I thought of going somewhere else, or doing something else,” Kariko said. “I also thought maybe I’m not good enough, not smart enough. I tried to imagine: Everything is here, and I just have to do better experiments.” Even after all the setbacks, she continued her search for that one better experiment (Leah Asmelash and AJ Willingham, 2020).
In 2015, Katalin Kariko and her colleague Drew Weissmann found the solution to prevent activation of the immune response against the injected mRNA. It has been found that mRNA activates the toll-like receptors (TLR) on immune cells. Karikó and Weissman modified the RNA by inserting a naturally occurring modified nucleoside: pseudouridine. This modified RNA transcript does not activate the TLR-mediated immune response and even provides a higher translational capacity (Karikó et al., 2005). A milestone for mRNA based therapeutics was reached. Even Sharp, the former critic, eventually said about the work done at Moderna: “They’ve totally convinced me it is possible to do”.
Applications of mRNA technology
During the following years, various pre-clinical and clinical trials on mRNA-based vaccines against infectious diseases, hypersensitivities and cancer were completed (Sahin et al., 2014; Weissman, 2015).
In 2009, researchers conducted the first-ever trial on cancer immunotherapy using mRNA-based vaccines in human subjects with metastatic melanoma. The results of the trial showed an increase in the number of vaccine-directed T cells against melanoma (Weide et al., 2009).
In 2020, the FDA approved the first mRNA-based vaccines against an infectious disease SARS-CoV-2. This was only made possible by decades of research on mRNA-based therapeutics.
mRNA has not only been a subject of interest for vaccine development, it has also influenced other fields including stem cell science and protein replacement therapies. Induced pluripotent stem cells (IPSCs) are a highly interesting addition to the tool-box of regenerative medicine, as IPSCs can differentiate into every other cell type in the body. The Japanese scientist Shinya Yamanaka transfected somatic cells by introducing several transcription factors and converted them to an embryonic stem cell state. That’s the dream of regenerative medicine. However, this process has the inherited danger of DNA integration into random sites of the genome and thereby potentially leading to adverse mutations, and unpredictable results. To counteract this issue, Yakubov et al. reprogrammed fibroblasts (a kind of skin cells) into IPSCs using mRNA transfection. This method completely avoids DNA integration and could be further developed to replace already available methods to generate IPSCs (Yakubov et al., 2010).
Protein replacement therapies
Fundamentally, mRNA therapeutics can be considered as a transient form of gene therapy that bypasses the complications of “conventional” gene therapy where DNA is inserted in the genome, including insertional mutagenesis and toxicity associated with viral vectors. Currently, researchers are working on introducing mRNA-based protein replacement therapies for treating myocardial infarction (heart attack) (Weissman, 2015).
Bringing about post-transcriptional changes with RNA interference
Another class of naturally occurring RNA called small interfering RNA (siRNA) or micro RNA (miRNA) was discovered in 1998 (MK and Kostas, 1998). They are part of the post-transcriptional machinery in the cell. In principal, siRNA and miRNA target specific mRNAs to form double stranded RNA molecules that are quickly degraded (gene silencing). In this way, gene expression in a cell can be controlled at a post-transcriptional level. This approach is used to develop treatments for HIV, cancer and melanoma. Moreover, in 2018, FDA approved mRNA-based therapeutics against hereditary ATTR amyloidosis (DeWeerdt, 2019).
What does RNA hold for us in the future?
The fascinating dream of RNA therapeutics has transitioned to practical reality. In the future, RNA holds the potential to disrupt the field of biopharmaceuticals, vaccine development, cell reprogramming, RNA interference and much more. Stay tuned to find out where RNA is steering in the future in our upcoming article.
By Tamseel Fatima and Dr Andreas Ebertz
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