Optimizing Modified mRNA In Vitro Synthesis Protocol for Heart Gene Therapy

Abstract

Synthetic modified RNA (modRNA) is a novel vector for gene transfer to the heart and other organs. modRNA can mediate strong, transient protein expression with minimal induction of the innate immune response and risk for genome integration. modRNA is already being used in several human clinical trials, and its use in basic and translational science is growing. Due to the complexity of preparing modRNA and the high cost of its reagents, there is a need for an improved, cost-efficient protocol to make modRNA. Here we show that changing the ratio between anti-reverse cap analog (ARCA) and N1-methyl-pseudouridine (N1mΨ), favoring ARCA over N1mΨ, significantly increases the yield per reaction, improves modRNA translation, and reduces its immunogenicity in vitro . This protocol will make modRNA preparation more accessible and financially affordable for basic and translational research.

Impact of mRNA chemistry and manufacturing process on innate immune activation

“…Treatment with ribonuclease III (RNase III), an RNase that recognizes and cleaves dsRNA of approximately 22 nucleotides in length, has also been shown to reduce immune-stimulatory response (23). It has also been observed that alteration of IVT components affects the purity of the mRNA, including dsRNA content, leading to greater or less innate immune activation (22,24).…”

Modified mRNA as a Therapeutic Tool for the Heart

“…1), particularly changing uridine with naturally occurring pseudouridine and 5-methyl-cytosine for cytosine [25], combined with advances in in vitro transcription technologies led to less recognition by TLRs and nucleases. Additionally, the stability and in vivo translation efficiency of modRNA have [26,27] and replacing 5′UTR with one from the fatty acid metabolism gene carboxylesterase 1D (Ces1d) [28].…”

“…They also optimized the nucleotide concentration to 31.6 mM per reaction, resulting in a 290% higher modRNA yield. This nucleotide optimization led to increased percentage of the capped modRNA, thus reducing its immunogenicity in both human cell lines and primary cardiac cells [27].…”

“…1 ), particularly changing uridine with naturally occurring pseudouridine and 5-methyl-cytosine for cytosine [ 25 ], combined with advances in in vitro transcription technologies led to less recognition by TLRs and nucleases. Additionally, the stability and in vivo translation efficiency of modRNA have been increased by capping the molecule with the 3′-O-Mem7G(5′)ppp(5′)G Anti Reverse Cap Analog (ARCA) at its 5′end [ 26 , 27 ] and replacing 5′UTR with one from the fatty acid metabolism gene carboxylesterase 1D (Ces1d) [ 28 ]. ModRNA has the capacity to target the gene of interest via multiple mechanisms including: (i) gain of function by overexpressing a target molecule [ 13 , 29 ], (ii) loss of function by either using dominant negative molecules or introducing miRNA [ 30 ], (iii) correcting gene deletions at the mRNA level [ 31 ], and (iv) inhibiting intracellular signaling pathways by introducing decoy receptors to dilute the ligands [ 30 ].…”

A Simplified Method to Produce Mrnas and Functional Proteins From Synthetic Double-Stranded DNA Templates

“…With advances in in vitro transcription (IVT) technology from either plasmid-based DNA templates or DNA PCR amplified from plasmids, as well as in delivery vehicles including polymeric-and lipid-based nanoparticles, interest has increased in the use of RNA molecules, including mRNA molecules as therapeutics for human disease [3][4][5][6][7][8]. Examples include loss-of-function mutations such as adenosine deaminase deficiency, ornithine transcarbamylase deficiency, phenylketonuria, hemophilia B and cystic fibrosis.…”