title: In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors.
abstract: We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated beta-galactosidase gene (beta-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.
authors.affiliation: Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Hanazono
authors.forename: K E
authors.initials: KE
authors.lastname: Brown
authors.forename: A
authors.initials: A
authors.lastname: Handa
authors.forename: M E
authors.initials: ME
authors.lastname: Metzger
authors.forename: D
authors.initials: D
authors.lastname: Heim
authors.forename: G J
authors.initials: GJ
authors.lastname: Kurtzman
authors.forename: R E
authors.initials: RE
authors.lastname: Donahue
authors.forename: C E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D017930:Genes, Reporter; D005822:Genetic Vectors; D017710:Lymphocyte Transfusion; D008253:Macaca mulatta; D009355:Neomycin; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001616:beta-Galactosidase; D009355:Neomycin