INTRACELLULAR IMMUNIZATION OF RHESUS CD34(-CELLS AND MACROPHAGES FROMSIMIAN IMMUNODEFICIENCY VIRUS-INFECTION() HEMATOPOIETIC PROGENITOR CELLS WITH A HAIRPIN RIBOZYME PROTECTS T)

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified he matopoietic progenitor cells. Using a novel thymic stromal culture tec hnique, we evaluated the ability of a hairpin ribozyme specific for si mian immunodeficiency virus (SIV) and human immunodeficiency virus typ e 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34(+) progenitor cells. Retroviral transduction of rhesu s macaque CD34(+) progenitor cells with a retroviral vector (p9456t) e ncoding the SIV-specific ribozyme and the selectable marker neomycin p hosphotransferase in the presence of bone marrow stroma and in the, ab sence of exogenous cytokines resulted in efficient transduction of bot h colony-forming units and long-term culture-initiating cells. with tr ansduction efficiencies ranging between 21% and 56%. After transductio n, CD34(+) cells were cultured on rhesus thymic stromal culture (to su pport in vitro differentiation of T cells) or in the presence of cytok ines (to support differentiation of macrophage-like cells). After expa nsion end selection with the neomycin analog G418. cells derived from transduced progenitor cells were challenged with SIV. CD4(+) T cells d erived from CD34(+) hematopoietic cells transduced with the ribozyme v ector p9456t were highly resistant to challenge with SIV, exhibiting u p to a 500-fold decrease in SIV replication, even after high multiplic ities of infection. Macrophages derived from CD34(+) cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of S IV replication. These results show that a hairpin ribozyme introduced into CD34(+) hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and suppor t the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hemat opoietic lineages with the SIV-specific ribozyme should permit analysi s of stem cell gene therapy for AIDS in the SIV/macaque model. (C) 199 7 by The American Society of Hematology.