title: Enhanced transduction of antigen-stimulated T lymphocytes with recombinant retroviruses concentrated by centrifugal filtration.
abstract: Retroviral gene transduction of antigen-specific T cells and reintroduction of the gene-modified T cells into animals or human subjects is attractive for experimental disease-modeling applications and gene therapy approaches for autoimmune or allergic diseases. However, retrovirus titers are often a limiting factor for the efficient gene transfer of mature T cells, which have proven to be relatively refractory to gene transduction. Retrovirus-containing supernatants with titers sufficient for effective transduction of immortalized T cell lines may fail to transduce peripheral T cells. The use of high-titer retroviruses pseudotyped with vesicular stomatitis virus G protein and concentrated by ultracentrifugation is limited by the loss of specific tropism, lower lymphocyte transduction efficiency on infectious particle basis and pseudotransduction. Herein, we present a simple method to concentrate retroviruses by centrifugal filtration at low g force. We compared the ability of unconcentrated and concentrated retroviruses to transduce immortalized fibroblasts as well as primary rat splenocytes activated with antigen and we evaluated transduction efficiency and mean fluorescence intensity of transgene expression in transduced cells. Our data demonstrate that, with this technique, retrovirus titers were increased nearly 10-fold without significant loss of infectious particles. Compared to unconcentrated retroviral preparations, the concentrated retrovirus supernatants more effectively transduced antigen-stimulated, primary rat T cells. This simple method of concentrating retroviruses may be exploited to generate gene-modified T cells for gene therapy applications in animal models of human autoimmune or allergic disease and may also be applicable for T lymphocyte-based gene therapy approaches in humans.
authors.affiliation: Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain, Montreal, Quebec, Canada H2X 2P2.
authors.forename: Margaret W
authors.initials: MW
authors.lastname: Kinyanjui
authors.forename: David
authors.initials: D
authors.lastname: Ramos-Barbón
authors.forename: Annie
authors.initials: A
authors.lastname: Villeneuve
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Fixman
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002460:Cell Line; D002498:Centrifugation / Q000379:methods*; D005374:Filtration / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D051381:Rats; D012190:Retroviridae / Q000502:physiology*; D013601:T-Lymphocytes; D014161:Transduction, Genetic; D014162:Transfection; D019065:Virus Assembly