Production and concentration of pseudotyped HIV-1-based gene transfer vectors

Strategies to generate highly concentrated HIV-1 vector pseudotypes involvi ng different envelope (Env) proteins including the vesicular stomatitis vir us (VSV) G glycoprotein, the Moloney murine leukemia virus (MLV) 4070A amph otropic Env and the rabies G glycoprotein were established. Virus stocks we re prepared by transient transfection using standard cell culture media or serum-free media. Such stocks were concentrated 50- to 300-fold by ultracen trifugation or by ultrafiltration using Centricon Plus-80 units yielding ti ters of up to 16 transducing units per milliliter. There was no loss in tit er with any of the pseudotypes tested. Thus, like lentiviral vectors pseudo typed with VSV-G, HIV-1-based vectors pseudotyped with the MLV 4070A amphot ropic Env and the rabies G glycoprotein resist inactivation during concentr ation. This opens up the possibility to generate highly concentrated HIV-1 vector stocks carrying alternative Env proteins on a large scale.