Engineering of a stable retroviral gene delivery vector by directed evolution

The lack of safe and effective delivery vectors continues to be a critical limitation facing human gene therapy. Viruses offer excellent efficiency but can be difficult and expensive to produce and purify. For example, the production and efficiency of murine leukemia virus (MLV) are limited by its inherent instability; the half-life of infectivity is 5-8 hours at 37 °C. In order to generate a stable MLV, we randomly mutated the virus genome and selected for infectivity after prolonged incubation at 37 °C. After seven rounds of incubation and infection, we isolated a pool of MLV variants with double the half-life of wild-type MLV. Remarkably, a single mutation in the viral protease (PR), G119E, was responsible for the enhanced stability. Saturation mutagenesis at residue 119 revealed variants with half-lives of ∼24 hours at 37 °C. Double mutants combining the changes at position 119 of the PR and substitutions in the PR substrate-binding pocket exhibited half-lives of up to ∼40 hours. MLV variants provided two- to fourfold higher viral titers and exhibited increased stability with various wild-type envelope proteins. The improved stability of the variant MLVs will provide more facile virus production and increased transduction efficiency.