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.

Keywords: gene therapy; in vitro mRNA synthesis; modified mRNA.

Figure 1

Effect of Nucleotide Composition in IVT Reaction on modRNA Integrity (A–F) modRNA integrity was evaluated using a bioanalyzer (Tap Station 2200) of compositions 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), and 6 (F).

Figure 2

Effect of Nucleotide Composition in IVT Reaction on Gene Expression in Human Cell Lines and Rat Cardiac Cells (A) GFP expression in rat cardiac cells 18 h post transfection with or without nGFP modRNA generated with the ARCA 5 protocol and ARCA 10 protocol. (B) Representative image of Luc activity level in neonatal rat cardiomyocytes 24 h post transfection with or without Luc modRNA generated using the ARCA 5 protocol and ARCA 10 protocol. (C and D) Quantification of GFP expression in neonatal rat cardiac cells (C) or cardiomyocytes (D) 18 h post transfection with nGFP modRNA generated using the ARCA 5 protocol and ARCA 10 protocol. (E–H) Quantification of Luc activity level in HUVECs (E), rat neonatal cardiomyocytes (F), HeLa (G), or Hek293 (H) cells 18 h post transfection with or without Luc modRNA generated using the ARCA 5 protocol and ARCA 10 protocol. One-way ANOVA, Tukey’s multiple comparison test; ***p < 0.001, **p < 0.01, *p < 0.05. n = 5 (A and B); n = 4 (C–F).

Figure 3

Immunogenicity of modRNA Compared with Unmodified mRNA (A) Luc mRNA or modRNA transfected into neonatal rat cardiomyocytes 8 h after transfection cells were collected. qPCR was performed to evaluate the gene expression of innate immune response markers. (B–D) Expression of innate immune response markers: RIG1 (B), IFNα (C), or IFNβ (D) 8 h post transfecting unmodified mRNA, Luc modRNA (ARCA 5 protocol), or Luc modRNA (ARCA 10 protocol). One-way ANOVA, Tukey’s multiple comparison test; ****p < 0.0001. n = 5.