Introduction to the History of in vitro transcription

Messenger RNA (mRNA) is the intermediate product necessary for the translation of protein-encoding DNA and the ribosomal production of proteins in the cytoplasm [1]. The mRNA is generated from DNA product using the in vitro transcription process. The product is purified and treated with phosphatase to remove 5'-triphosphates. After the additional purification and quality control of generated mRNA, the mRNA transfections can be performed. The currently accessible methods of in vitro transcription (IVT) have been built upon research dating back more than 50 years. The enormous cascade of experiments from then to now have supplied insight into how to best use in vitro and synthetic methods to produce mRNA. Using this knowledge has led to major breakthroughs and has shown potential in the treatment of various diseases.

1961–1995: Foundational discoveries

Jean Brachet, a Belgium biologist, first incorporated RNA in eukaryotic cells as an integral product of his studies in cell biology [2]. He showed that an enucleated cell, which holds most of the basophilic staining material (RNA), could continue to incorporate labeled amino acids for several days [2]. This was an important discovery regarding nucleic acids, as only DNA was accepted as seat of genetic information at this time after the Watson-Crick structure [2,3].

It was a few years after this that researchers began to describe the presence of unstable intermediate molecules that utilized information from DNA to the direct synthesis of proteins, which lead to the discovery of mRNA in 1961 by Sydney Brenner [4]. They initially hypothesized and later proved that ribosomal RNA did not contain the protein-encoding information, but rather were non-specialized structures that received information from the gene in the form of an unstable intermediate or “messenger”. They infected bacteria with a virulent bacteriophage and followed the distribution of new RNA and new protein with radioisotope labelling. They concluded that a transient RNA molecule must contain the transcript of the genetic code. They also described three distinct roles and species of RNA in bacteria. Bacterial mRNA appeared to be an unstable minor fraction of the total mRNA and ranged between 1 and 2 kb [4].

It became important to understand the relationship between nuclear mRNA and its involvement in the protein synthesis that occurs in the cytoplasm. In 1969, Lingrel and Lockard worked together and were successful in the first in vitro translation of mRNA [5]. In this study, they showed the first demonstration of protein synthesis in a mammalian cell-free system under the direction of mRNA, which was isolated from a different mammalian species. They isolated mouse 9S RNA from mouse reticulocytes and added it to the cell-free system prepared from rabbit reticulocytes. They found that the isolated 9S RNA could direct hemoglobin beta-chain production in the rabbit reticulocytes [5].

In the early 1970s, a 3’ unitary-sized polyA segment in the polyribosomal mRNA was discovered [6]. Researchers found that adding this unit of adenylic-acid residues to nuclear RNA by enzymes showed a strong suggestion for the processing of large nuclear molecules in the formation of mRNA [2]. A few years after the discovery of the polyA addition, the addition of 5’-mehtylated GpppXmP cap at the end 5’ end of mRNA was also discovered [6].

One of the first major developments in the field came from a method of synthesis for microgram quantities of eukaryotic mRNAs. Researchers utilized cDNAs that were cloned into SP6 vectors, which were then injected into the cytoplasm of frog oocytes [7]. They were able to use these synthetic mRNAs as part of IVT to show the production of copious quantities of mRNA and protein from any cDNA clone. They also confirmed the importance of a 5’ cap on the mRNA for translation while showing that 3’ flanking region could be removed without blocking protein synthesis [7]. This discovery led to more research into mRNA delivery and commercialization, such as that of SP6 and T7 RNA polymerases in 1986.

Even with the instability of RNA, early researchers still considered the possibility of using mRNA to develop therapies for various disease models. One of the first experiments to promote the idea came in 1989 after the development of a universally applicable in vitro transfection technique [8]. In this study, researchers used a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome. They found that transfecting with luciferase mRNA into NIH 3T3 mouse cells led to an increased linear response of luciferase activity and was reproducible in human, and rat cells. They also found that capped mRNAs with beta-globin untranslated sequences led to 1000x more luciferase protein that mRNA without these elements [8]. In the 1980's, Ambion was created, offering customers RNA-focused lab supplies for the first time which enabled researchers to use mRNA more readily. Ambion quickly became a leader in the development and supply of innovative RNA-based life science research and molecular diagnostic products and services.

In the 1990s, a group of researchers showed a unique application of mRNA delivery in vivo where they were able to temporarily correct diabetes insipidus in Brattleboro rats [9]. These rats are known to have a genetic mutation which makes them incapable of expressing and secreting vasopressin, which leads to diabetes insipidus. In this study, researchers were able to inject purified mRNAs from normal rats or synthetic copies of vasopressin mRNA into the hypothalamus of the Brattleboro rats. This injection led to selective uptake, transport, and downstream expression of vasopressin, thus temporarily reversing diabetes insipidus within hours [9].

The first mRNA therapeutics company was founded in 1997 after Eli Gilboa proposed taking immune cells from the blood and coaxing them to take up synthetic mRNA that encoded tumour proteins [10]. This California-based company, Merix Bioscience—which was later renamed to Argos Therapeutics and is now called CoImmune—inspired work across the globe. It also inspired the foundation of mRNA work with the creation of CureVac and, later in the early 2000’s, BioNTech. These companies continued the work of Gilboa by striving to administer mRNA directly into the body [11]. BioNTech later went on to partner with Pfizer to create one of the two COVID-19 vaccines in 2020.

With mRNA being utilized in various disease study-models, Conry et al. wanted to evaluate the use of mRNA transcripts in tumor vaccines [12]. This group of researchers constructed mRNA transcripts encoding the human carcinoembryonic antigen (CEA) which were capped and stabilized by human beta globin 5’ and 3’ untranslated region. They transfected CEA-encoding mRNA in vitro using liposome-mediated transfection and in vivo. Out of the seven mice injected, five of them showed anti-CEA antibodies 3 weeks post-tumor challenge compared to control mice with no antibody response. This was one of the first studies to incorporate mRNA as a therapeutic against cancer [12].

2005–2018: Corporate innovation

During the developing work with mRNA as a therapeutic, one massive hinderance was the immunogenicity of synthetic mRNA by the innate immune system [13]. In 2005, Karikó et al. found mRNA signals through toll-like receptors, but that the incorporation of modified nucleosides such as pseudo-uridine could lead to decreased immunogenicity and reduced cytokine expression. They found that when dendritic cells (DCs) were exposed to these modified RNAs, they expressed significantly less cytokines and had decreased activation of immune markers compared to unaltered RNA [13]. Understanding and using the effects of these modifications were important for better mRNA stability and delivery for in vitro transcription.

In the past two decades, companies like Ambion and Moderna became leaders in mRNA-based research. Ambion was foundational in supplying all-in-one kits for mRNA research. This was important as it enabled researchers to have all the components necessary to better study mRNA. However, in 2006, Ambion was acquired by Applied Biosciences, which later merged with Invitrogen to become Life Technologies. In 2008, the ETS product line which included MEGA, mMessage mMachine, and others became a part of the Thermo Fisher brand and started reaching wider scientific audiences to make synthetic mRNA research more accessible.

In 2014, this synthetic mRNA research continued and uncovered exact methods for allowing the clinical applicability of modified mRNA without worry about low stability and strong immunogenicity [2]. Avci-Adali et al demonstrated how to produce stabilized, modified mRNA for the induction of protein in cells, and go on to show that using this protocol can generate other desired mRNA as well. Since the in vitro synthesis of modified mRNA allows the transfection of cells with desired mRNAs to induce expression of target proteins, the researchers expressed the desired protein transiently under physiological conditions until the exogenously delivered mRNA was completely degraded. To keep the mRNA’s integrity, researchers determined that repeated freezing and thawing of the mRNA should be avoided and, thus, working aliquots can be prepared. Additionally, after PCR and IVT, they found that only a single specific band should be detected. Otherwise, the number of PCR cycles, the primer annealing temperature, and/or the amount of plasmid DNA should be optimized to obtain the specific DNA product for IVT. Finally, they found that the IVT time and the amount of DNA template for IVT can be optimized to obtain mRNA of a specific length in sufficient amounts [2].

On top of being a potential vaccine candidate, mRNA was influencing other fields like regenerative medicine [14]. Differentiated cells were reprogrammed into induced pluripotent stem cells (IPSCs) which can differentiate into other cell types in the body. In 2010, researchers transfected somatic cells with several transcription factors and converted them to have embryonic stem cell (ESC) states. To reprogram fibroblasts into IPSCs, they used RNA synthesized in vitro from cDNA of four ESC-specific transcription factors to show intracellular expression and localization of their respective proteins. This method resulted in consistent protein expression and the formation of IPSC (Induced pluripotent stem cells) colonies [14].

2019 and beyond: worldwide implications

Synthetic mRNA has crossed over into immunology in a larger way in recent years, building upon the mountains of research that’s come before now. Researchers began to use mRNA with a broad range of pharmaceutical applications, including different modalities of cancer immunotherapy. With the current rapid and large-scale manufacturing of mRNA, researchers like Beck et al. in 2019 aimed for not only-off-the-shelf cancer vaccines but also personalized neoantigen vaccination [15]. Since nucleoside modification and elimination of double-stranded RNA can reduce the immunomodulatory activity of mRNA and increase and prolong protein production, Beck et al hypothesized that mRNA could be harnessed for applications such as chimeric antigen receptor-modified adoptive T-cell based cancer therapeutics. Due to mRNA’s versatility, researchers are able to look beyond therapeutic cancer vaccination and towards CAR-T cell therapy, which in Beck’s case, successfully moved into clinical testing [15]. This leap from vaccine discoveries by Conry et al is a testament to the dedication of researchers as well as to the current state of research technology [12].

It was this same dedication and technology that allowed for the quick manufacturing of the COVID-19 vaccine. In late 2019, the first cases of COVID-19 began popping up across the globe. What was initially thought to be a small-scale issue soon grew into a global pandemic that required timely and effective therapeutics. Synthetic mRNA in liposomes became the focus as they can be seen as the most refined and safe options. The COVID-19 mRNA vaccine developed by BioNTech and Moderna—which was modeled after vaccines such as Mumps, Measles, and Rubella—became the third RNA therapeutic ever approved in December 2020 [16,17]. In the next few years, based on the development and success of SARS-CoV-2 vaccine, research will be amplified to use mRNA-based therapies for other disease models. As of March 10, 2022, more than 10.9 billion doses of COVID vaccines have been administered worldwide, with 4.45 billion people fully vaccinated [18].

A promise of growth

Thermo Fisher Scientific and Moderna have recently agreed to a 15-year production pact for further manufacturing of COVID vaccines and more. TFS has also proven to be a leader in supporting the development of plasmid DNA, protein subunits and mRNA-based research. Thermo Fisher Scientific offers products such as MEGAscript T7 ultra-high yield in vitro transcription kit, which modifies transcription reaction conditions to ensure high nucleotide concentration use. The best transcription reaction conditions and a patented, high yield technology leads to 10–50x the amount of RNA production compared to conventional transcription reactions. Thermo Fisher Scientific also allows users to change nucleotides and utilize their own caps. Thermo Fisher Scientific continues to support researchers to push the field of mRNA-based research, including in vitro transfection, and to supply tools for all the steps in the process.

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