Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA

Abstract

mRNA based therapies hold great promise for the treatment of genetic diseases. However, this therapeutic approach suffers from multiple challenges including the short half-life of exogenously administered mRNA and subsequent protein production. Modulation of untranslated regions (UTR) represents one approach to enhance both mRNA stability and translation efficiency. The current studies describe and validate screening methods using a diverse set of 5'UTR and 3'UTR combinations for improved expression of the Arginase 1 (ARG1) protein, a potential therapeutic mRNA target. Data revealed a number of critical aspects which need to be considered when developing a screening approach for engineering mRNA improvements. First, plasmid-based screening methods do not correlate with protein expression driven by exogenously expressed mRNA. Second, improved ARG1 protein production was driven by increased translation and not improved mRNA stability. Finally, the 5' UTR appears to be the key driver in protein expression for exogenously delivered mRNA. From the testing of the combinatorial library, the 5'UTR for complement factor 3 (C3) and cytochrome p4502E1 (CYP2E1) showed the largest and most consistent increase in protein expression relative to a reference UTR. Collectively, these data provide important information for the development and optimization of therapeutic mRNAs.

Keywords: ARG1; UTR; mRNA; mRNA stabllity; mRNA translation.

Figure 1.

Arginase I protein expression 72hr following plasmid transfection in HeLa (Panel A) or HepG2 cells (Panel B). Columns represent the mean area under the curve as determined by capillary electrophoresis (± 1STDEV). * = p<0.05 vs reference UTR.

Figure 2.

Arginase I protein expression 72 hr following mRNA transfection in HepG2 cells. Columns represent the mean area under the curve as determined by capillary electrophoresis (± 1STDEV). * = p<0.05 vs reference UTR.

Figure 3.

Arginase I protein expression 24 hrs following mRNA or plasmid transfection in HepG2 cells. Columns represent the mean area under the curve as determined by capillary electrophoresis (± 1STDEV). * = p<0.05 vs reference UTR.

Figure 4.

Stability of ARG-1 constructs containing ASL or C3 UTRs. Cells were transfected (see Methods Section) with mRNA and incubated for 1 hr. mRNA was removed and cell incubated for indicated time points. mRNA half-life for Arg1 containing C3 (32 min) and ASL (40 min) UTRs was calculated using GraphPad prism. Lines represent Percent change from time 0 (after removal of mRNA from cells) ± 1STDEV.