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Efficient in vivo marking of primary CD4+ T lymphocytes in nonhuman
primates using a gibbon ape leukemia virus-derived retroviral vector
BA Bunnell, M Metzger, E Byrne, RA Morgan and RE Donahue
Clinical Gene Therapy Branch, National Center for Human Genome Research,
Bethesda, MD 20850, USA.
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.
Volume 89,
Issue 6,
pp. 1987-1995,
03/15/1997
Copyright © 1997 by The American Society of Hematology
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