Two point mutations increase targeted transduction and stabilize vector association of a modified retroviral envelope protein

The current strategy of targeting retroviral vector transduction by inserti ng a peptide ligand into the envelope protein has met with several obstacle s. These modified proteins redirected vector binding to a new cognate recep tor on a specific cell type but gave little or no gene transfer because the y did not fuse the vector and target cell membranes. They dissociated readi ly from vectors and often required coassembly of wild-type envelope protein . Here we report a novel strategy to overcome the fusion and stability defe cts of modified retroviral envelope proteins. We inserted a prototypic liga nd, the receptor binding domain of amphotropic murine leukemia virus, into an ecotropic murine leukemia virus envelope protein mutant containing gluta mine 227-to-arginine plus aspartate 243-to-tyrosine substitutions. This mod ified protein increased transduction redirected to human cells expressing t he amphotropic receptor to a level within 10-fold that of wild-type amphotr opic virus, an increase of as great as 2000-fold over transduction by modif ied protein lacking the mutations. In addition to suppressing the fusion de fect, these mutations unexpectedly stabilized the association of the modifi ed protein with vector particles. Insertion of clinically relevant ligands into this envelope mutant should improve the efficiency and reliability of retroviral transduction of specific cell types for gene therapy application s.