title: Functional assessment of the engraftment potential of gammaretrovirus-modified CD34+ cells, using a short serum-free transduction protocol.
abstract: The successful transduction and engraftment of human mobilized peripheral blood (MBP) CD34(+) cells are determined to a large extent by the ex vivo cell-processing conditions. In preparation for upcoming clinical trials, we investigated essential culture parameters and devised a short and efficient gammaretroviral transduction protocol entailing minimal manipulation of MBP CD34(+) cells. The engraftment potential and in vivo transgene expression in the progeny of repopulating CD34(+) cells were measured to assess the functionality of CD34(+) cells transduced under these conditions. Using a competitive in vivo repopulation assay in nonobese diabetic/severe combined immunodeficient mice, we demonstrate equivalent engraftment of CD34(+) cells transduced under serum-free conditions as compared with CD34(+) cells cultured with serum. We also took advantage of this in vivo model to demonstrate that ex vivo manipulation of CD34(+) cells can be shortened to 60 hr, using 36 hr of prestimulation and two cycles of transduction 12 hr apart. These minimally manipulated CD34(+) cells engraft in a manner similar to cells transduced under longer protocols and the vector-encoded transgene is expressed at the same frequency in cells derived from repopulating CD34(+) cells in vivo. We have thus developed a short and efficient human MBP CD34(+) transduction protocol under serum-free conditions that is suitable and broadly applicable for phase I clinical trials.
authors.affiliation: Department of Medicine, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, 08903, USA.
authors.forename: Tulin
authors.initials: T
authors.lastname: Budak-Alpdogan
authors.forename: Mark
authors.initials: M
authors.lastname: Przybylowski
authors.forename: Mithat
authors.initials: M
authors.lastname: Gonen
authors.forename: Michel
authors.initials: M
authors.lastname: Sadelain
authors.forename: Joseph
authors.initials: J
authors.lastname: Bertino
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rivière
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D001773:Blood Cells / Q000737:chemistry / Q000637:transplantation*; D002417:Cattle; D018929:Cell Culture Techniques; D005260:Female; D017988:Gammaretrovirus / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D044967:Serum / Q000166:cytology; D019089:Stem Cell Factor / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D018952:Antigens, CD34; D019089:Stem Cell Factor; D013762:Tetrahydrofolate Dehydrogenase