Efficient in vivo marking of primary CD4⁺ T lymphocytes in nonhuman primates using a gibbon ape leukemia virus-derived retrovital 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 than decline 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 retrovital 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 (rIL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expended 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.