Design, Off-Target Toxicity, and Intracellular Processing of Nucleotide-Modified Messenger RNAs for Human Therapies

Abstract

RNA therapy is a modality, analogous to gene therapy, in which in vitro-transcribed mRNA (IVT mRNA) is introduced to cells for therapeutic applications. IVT mRNA is transfected into target cells, translated, and the fully-synthesised encoded protein exerts a therapeutic effect via specific mechanisms. IVT mRNAs have been applied to treatment of inherited protein deficiencies, tissue reprogramming, cancer immunotherapy, genome editing, and vaccination against infectious diseases. The use of mRNA instead of DNA-based vectors eliminates the risk of insertional mutagenesis and has demonstrably high protein expression in vivo. However, using mRNAs for therapy has several problems. Cellular delivery of IVT mRNA activates the innate immune system via pattern recognition receptors leading to phosphorylation of eIF2α, global inhibition of translation, and upregulation of pro-inflammatory cytokines. These issues have been partly addressed by the use of nucleotide modifications in IVT mRNA, which increases the expression of the encoded protein and reduces innate immune activation compared to unmodified IVT mRNA. Despite this progress, there remain several potential issues with the use of modified nucleotides in mRNA therapy which may contribute to off-target and long-term toxicity. These include immunogenicity of IVT mRNA and off-target effects; the mutagenic potential of modified nucleotides that are introduced to cells by degradation of modified IVT mRNA; and the effect of modified nucleotides on mRNA translation and co-translational protein folding. Furthermore, while mRNA-based therapies are highly promising therapeutic agents, mode of delivery remains problematic – systemic delivery, in particular. Techniques for targeted mRNA delivery and increased transgene expression are lacking and vital for efficacious mRNA therapies. This study investigated the off-target toxicity of three commonly used IVT mRNA modified nucleosides –5-methoxyuridine, 5-methylcytidine, and 1-methylpseudouridine – in cultured human cells. 5-methoxyuridine exhibited cytotoxicity in A549 cells and HeLa cells at concentrations greater than 100 μM, while 1-methylpseudouridine was nontoxic at all tested concentrations (up to 1000 μM). 5-methylcytidine was more cytotoxic than other nucleosides (100 μM