493. Concentration and Purification of Lentivirus Pseudotypes Bearing Alternative Envelope Glycoproteins

Abstract

Current problems that impact the use of lentiviral (LV) vectors for applications in large animals include low titers, poor purity and difficulties related to their large-scale production. A number of useful protocols are available to concentrate and purify LV vector pseudotypes bearing the traditional vesicular stomatitis virus G (VSV-G) glycoprotein. These protocols include centrifugation-based approaches, ultrafiltration, anion exchange chromatography, and size exclusion chromatography. There is an increasing demand for alternative (i.e. non-VSV-G-based) LV pseudotypes and thus there are needs for improved concentration/purification protocols for such pseudotypes. We have investigated scaleable protocols for the capture and purification of alternative LV pseudotypes based on anion exchange membrane chromatography. In one approach, HIV-1-based LV vectors pseudotyped with a lyssavirus (rabies-related) glycoprotein were generated by transient transfection. In another approach, amphotropic LV pseudotypes were generated using the STAR-A amphotropic packaging cell line. Virus-containing cell culture supernatants were subsequently captured onto chromatography membranes bearing functional quarternary (Q) ammonium groups and eluted with high salt and then desalted and further concentrated by ultrafiltration. Using this procedure, up to 87 % of the virus applied (up to 1.8 |[times]| 108 transducing units) could be recovered from a single anion exchange membrane unit with a membrane bed volume of 0.18 ml. Our results indicate that these purification procedures are useful for concentrating LV vectors bearing alternative envelope glycoproteins with a minimal loss of infectivity. We expect these approaches to facilitate the preparation of lentiviral vectors for large-scale preclinical studies. They may ultimately be applicable to generate clinical-grade LV vectors.