title: Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer.
abstract: Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency. Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates. We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity. Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles. We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells. We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus. We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells.
authors.affiliation: University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Yamada
authors.forename: Douglas M
authors.initials: DM
authors.lastname: McCarty
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Madden
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Walsh
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000302:isolation & purification*; D016086:Lentivirus / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure; D051379:Mice; D014161:Transduction, Genetic / Q000379:methods*