EFFICIENT IN-VIVO MARKING OF PRIMARY CD4(-LYMPHOCYTES IN NONHUMAN-PRIMATES USING A GIBBON APE LEUKEMIA VIRUS-DERIVED RETROVIRAL VECTOR() T)

High efficiency retroviral-mediated gene transfer to rhesus CD4(+) per ipheral blood lymphocytes (PBL) was accomplished using an optimized tr ansduction protocol using a gibbon ape leukemia virus (GaLV) envelope- containing packaging cell line PG13. Engineered CD4(+) PBL were admini stered to three nonmyeloablated animals in three or four separate infu sions over 9 months, Polymerase chain reaction (PCR) demonstrated in v ivo reconstitution of the genetically engineered CD4(+) PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene m arking 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 les s than or equal to 0.1% over the next 21 weeks. Lymph node (LN) biopsi es were performed to determine if the engineered CD4(+) lymphocytes co uld traffic to lymphoid tissues. Marked lymphocytes were detected in L N biopsies 100 days following reinfusion of the transduced cells, Expr ession of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that wer e activated by culturing in the presence of recombinant interleukin-2 (rlL-2). A humoral immune response to fetal bovine serum (FBS) was det ected in all animals following the second administration of the cultur e expanded CD4(+) lymphocytes. No antibody response was detected to th e neomycin-resistance (Neo(R)) transgene, the murine retroviral group- specific antigen (gag), or GaLV envelope (env) proteins. (C) 1997 by T he American Society of Hematology.