An equimolar ratio of gag-pol mRNA and vector genomic RNA in producer cells is optimal for efficient genome packaging during lentiviral vector production.

Abstract

Abstract Lentiviral vectors are showing success in the clinic but producing enough vector to meet the growing demand is a major challenge. Furthermore, next generation gene therapy vectors encode multiple genes resulting in larger genome sizes which is reported to reduce titres. A packaging limit has not been defined. The aim of this work was to assess the impact of genome size on the production of lentiviral vectors with an emphasis on producer cell mRNA levels, packaging efficiency and infectivity measures. Consistent with work by others, vector titres reduced as genome size increased. While genomic infectivity accounted for much of this effect, genome sizes exceeding that of clinical HIV-1 isolates result in low titres due to a combination of both low genomic infectivity and decreased packaging efficiency. Manipulating the relative level of genomic RNA to gag-pol mRNA in the producer cells revealed a direct relationship between producer cell mRNA levels and packaging efficiency yet could not rescue packaging of oversized genomes implying a de facto packaging defect. However, independent of genome size, an equimolar ratio between wild type gag-pol mRNA and vector genomic RNA in producer cells was optimal for titre.