Gene Transfer into Nonhuman Primate CD34+CD11b–Bone Marrow Progenitor Cells Capable of Repopulating Lymphoid and Myeloid Lineages

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Abstract
We investigated whether rhesus monkey CD34+CD11b– hematopoietic stem cells can be transduced with recombinant retroviruses carrying the human adenosine deaminase (hADA) gene by co-cultivation with a virus-producing cell line. Following autologous transplantation, polymerase chain reaction (PCR) analysis on peripheral blood mononuclear cells and granulocytes showed that the hADA-retrovirus was present in approximately 0.1% of the cells for at least 400 days post transplantation in 2 monkeys. Bone marrow that was harvested 16 months after transplantation carried ADA-overexpressing myeloid progenitor cells capable of in vitro colony formation. In addition, hADA activity could be demonstrated in T lymphocytes that were harvested 9 months post transplantation. Thus, in vitro transduction of CD34+CD11b– cells led to long-term repopulation of the hematopoietic system with transduced cells of lymphoid and myeloid lineages expressing the hADA gene. To investigate whether infusion of virus-producing cells into a rhesus monkey undergoing autologous bone marow transplantation could lead to in vivo transfer of the recombinant retrovirus, 1 monkey was infused with CD34+CD11b– bone marrow cells (BMC) and a large quantity of virus-producing cells. Few provirus-carrying cells could temporarily be detected in this animal. This shows that in vivo gene transfer into a regenerating hemopoietic system can occur, albeit at very low efficiency. Retrovirus-mediated gene transfer into pluripotent hematopoietic stem cells may provide an efficacious therapy for many diseases of the hematopoietic system. Previously, we have transduced long-term repopulating hematopoietic stem cells in autologous rhesus monkey bone marrow grafts with recombinant retroviruses carrying the human adenosine deaminase (ADA) gene. Here we use the same procedure to modify genetically rhesus monkey bone marrow grafts that were first enriched for hematopoietic stem cells by means of immunomagnetic bead selection. Following transplantation, long-term multilineage genetic modification of the hematopoietic system was obtained. Moreover, functional expression of the human ADA gene was demonstrated in mature cells from lymphoid and myeloid lineages. This provided more convincing evidence for the pluripotency of the transduced stem cells and for the ability of the recombinant retrovirus to direct sustained expression of the ADA gene following maturation of transduced stem cells into multiple hematopoietic lineages.

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