Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. Based on their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N, and T368A mutations enhanced serum resistance, and additionally K66T, T368A, and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N + T368A or K66T + S162T + T230N + T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy.