Efficient in vivo marking of primary CD4+ T lymphocytes in nonhuman primates using a gibbon ape leukemia virus-derived retroviral vector.
High efficiency retroviral-mediated gene transfer to rhesus CD4+ peripheral blood lymphocytes (PBL) was accomplished using an optimized transduction protocol using a gibbon ape leukemia virus (GaLV) envelope-containing packaging cell line PG13. Engineered CD4+ PBL were administered to three nonmyeloablated animals in three or four separate infusions over 9 months. Polymerase chain reaction (PCR) demonstrated in vivo reconstitution of the genetically engineered CD4+ PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene marking indicates that up to 30% of endogenous circulating CD4+ cells can be genetically engineered. The high levels of marked lymphocytes persist for the first 3 weeks following reinfusion then decline to < or = 0.1% over the next 21 weeks. Lymph node (LN) biopsies were performed to determine if the engineered CD4+ lymphocytes could traffic to lymphoid tissues. Marked lymphocytes were detected in LN biopsies 100 days following reinfusion of the transduced cells. Expression of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that were activated by culturing in the presence of recombinant interleukin-2 (rlL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expanded CD4+ lymphocytes. No antibody response was detected to the neomycin-resistance (Neo(R)) transgene, the murine retroviral group-specific antigen (gag), or GaLV envelope (env) proteins.
Bunnell
B A
BA
Clinical Gene Therapy Branch, National Center for Human Genome Research, Bethesda, MD 20850, USA.
Metzger
M
M
Byrne
E
E
Morgan
R A
RA
Donahue
R E
RE
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000637:transplantation; D002465:Cell Movement / Q000235:genetics / Q000276:immunology; D002470:Cell Survival / Q000235:genetics / Q000276:immunology; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000276:immunology*; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics* / Q000276:immunology; D008198:Lymph Nodes / Q000276:immunology; D008253:Macaca mulatta
D000914:Antibodies, Viral