IN-VITRO T-LYMPHOPOIESIS - A MODEL SYSTEM FOR STEM-CELL GENE-THERAPY FOR AIDS

Stable introduction of therapeutic genes into hematopoietic stem cells has the potential to reconstitute immunity in individuals with HIV in fection. However, many important questions regarding the safety and ef ficacy of this approach remain unanswered and may be addressed in a no n-human primate model. To facilitate evaluation of expression of forei gn genes in T cells derived from transduced hematopoietic progenitor c ells, we have established a culture system that supports the different iation of rhesus macaque and human CD34(+) bone marrow derived cells i nto mature T cells. Thymic stromal monolayers were prepared from the a dherent cell fraction of collagenase digested fetal or neonatal thymus . After 10-14 days, purified rhesus CD34(+) bone marrow-derived cells cultured on thymic stromal monolayers yielded CD3(+)CD4(+)CD8(+), CD3( +)CD4(+)CD8(-), and CD3(+)CD4(-)CD8(+) cells. Following stimulation wi th mitogens, these T cells derived from CD34(+) cells could be expande d over 1,000-fold and maintained in culture for up to 20 weeks. We nex t evaluated the ability of rhesus CD34(+) cells transduced with a retr oviral vector containing the marker gene neo to undergo in vitro T cel l differentiation. CD34(+) cells transduced in the presence of bone ma rrow stroma and then cultured on rhesus thymic stroma resulted in T ce lls containing the retroviral marker gene. These studies should facili tate both in vitro and in vivo studies of hematopoietic stem cell ther apeutic strategies for AIDS.