Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity.

Satoshi Uchida,Kazunori Kataoka,Keiji Itaka

doi:10.3390/pharmaceutics7030137

Abstract

Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes.

Highlights

Therapeutic delivery of messenger RNA is a new method of providing therapeutic proteins and peptides for cells [1,2]
With ψU100% resulted in Gaussia luciferase (GLuc) expression comparable to unmodified messenger RNA (mRNA), but it induced a much stronger inflammatory response. In accordance with these results, cell viability was positively correlated with luciferase expression efficiency, some formulations, especially mC25% sU25%, exhibited only a small increase in luciferase expression despite remarkable increase in cell viability (Figure S2). These results indicate that the reduced inflammatory responses, as well as the increase in cell viability, are not always correlated with an increase in protein expression efficiency, motivating us to analyze the effect of mRNA modification using other cell types
Translational activity tended to decrease after mRNA modification compared to that of unmodified mRNA, the extent of the decrease was considerably different depending on the formulation (Figure 6)

Summary

Introduction

Therapeutic delivery of messenger RNA (mRNA) is a new method of providing therapeutic proteins and peptides for cells [1,2]. MRNA delivery is a safe option for introducing programmable nucleases, such as zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR/Cas, for genome editing [5,6,7]. In addition to these examples of in vitro delivery, we and other groups have shown the feasibility of in vivo mRNA delivery for treating disease in animal models, such as surfactant B deficiency, cancer, myocardial infarction, and sensory nerve dysfunction [8,9,10,11]. Toll-like receptor 3, 7, 8, and retinoic acid-inducible gene I (RIG-I), resulting in a strong inflammatory response To date, this has limited the application of mRNA mainly to vaccine therapy [14,15,16]

Results

Discussion

Conclusion