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 less than or equal to 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. (C) 1997 by The American Society of Hematology.