Genetic engineering strategy for generating a stable dsRNA virus vector using a virus-like codon-modified transgene

Deletion or truncation of transgenes encoded in RNA virus vectors frequently occur. Genetic instability of transgenes is the primary obstacle to establishing RNA virus vectors as tractable gene transduction tools. To improve transgene stability, we designed codon-modified genes that imitated codon usage bias in the genomes of rotavirus (RV) and mammalian orthoreovirus (MRV), both of which have segmented double-stranded (ds) RNA genomes. Synthesized, codon-modified NanoLuc, ZsGreen, AsRed, and Akaluc genes, which had RV genome-like and/or MRV genome-like codon usage biases, were used as transgenes in RV and MRV vectors. Here, we show that the genetic stabilities of these codon-modified genes in RV and MRV vectors were dramatically increased relative to their respective parental genes. Because genetic stabilities were improved independent of transgenes and insertion sites within the viral genome, virus-like codon modification could be essential for constructing dsRNA virus vectors and overcome the primary obstacle of transgene genetic instability. IMPORTANCE The stabilities of transgenes in RNA virus vectors differ between the genes of interest, but the molecular mechanisms determining genetic stability remain unknown. This study demonstrated that the stability of a transgene was affected by the nucleotide composition, and altering the codon usage of transgenes to resemble that of the viral genome significantly increased transgene stability in double-stranded RNA virus vectors. The virus-like codon modification strategy enabled generation of stable rotavirus and mammalian orthoreovirus vectors, which could be developed as machinery for gene delivery to the intestines and/or respiratory organs. This technology has further potential to be expanded to other RNA viruses.