title: Complexation of retrovirus with cationic and anionic polymers increases the efficiency of gene transfer.
abstract: Previously, we have demonstrated that chondroitin sulfate proteoglycans and glycosaminoglycans inhibit retrovirus transduction. While studying the mechanism of inhibition, we found that the combined addition of equal-weight concentrations (80 microg/ml) of Polybrene and chondroitin sulfate C to retrovirus stocks resulted in the formation of a high-molecular-weight retrovirus-polymer complex that could be pelleted by low-speed centrifugation. The pelleted complex contained more than 80% of the virus particles, but less than 0.3% of the proteins that were originally present in the virus stock. Surprisingly, the virus in the complex remained active and could be used to transduce cells. The titer of the pelleted virus, when resuspended in cell culture medium to the starting volume, was three-fold greater than the original virus stock. The selectivity (CFU/mg protein) of the process with respect to virus activity was more than 1000-fold. When the pelleted virus-polymer complex was resuspended in one-eighth of the original volume and used to transduce NIH 3T3 murine fibroblasts and primary human fibroblasts, gene transfer was increased 10- to 20-fold over the original unconcentrated retrovirus stock. The implications of our findings for the production, processing, and use of retrovirus stocks for human gene therapy protocols are discussed.
authors.affiliation: Georgia Tech/Emory Department of Biomedical Engineering, Atlanta, GA 30332-0535, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Le Doux
authors.forename: N
authors.initials: N
authors.lastname: Landazuri
authors.forename: M L
authors.initials: ML
authors.lastname: Yarmush
authors.forename: J R
authors.initials: JR
authors.lastname: Morgan
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000838:Anions / Q000378:metabolism; D016466:CHO Cells; D002412:Cations / Q000378:metabolism; D002498:Centrifugation; D011232:Chemical Precipitation; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D005347:Fibroblasts / Q000821:virology; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006583:Hexadimethrine Bromide / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009928:Organ Specificity; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: D000838:Anions; D002412:Cations; D010982:Platelet-Derived Growth Factor; D011108:Polymers; C075278:platelet-derived growth factor A; D006583:Hexadimethrine Bromide; D002809:Chondroitin Sulfates