Gene Therapy Targeting Peripheral Blood CD34+Hematopoietic Stem Cells of HIV-infected Individuals

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Abstract
Gene therapy is a promising treatment modality for acquired immunodeficiency syndrome (AIDS). Autologous transplantation with genetically altered pluripotent hematopoietic stem cells encoding anti-human immunodeficiency virus (HIV) genes could in theory completely and permanently reconstitute all blood lineages and immune functions with cells resistant to HIV. Recent studies showed that CD34+ stem cell can be mobilized in HIV-infected individuals after granulocyte colony-stimulating factor (G-CSF) administration without major side effects or increase of viral load. In this study, peripheral blood CD34+ cells of five HIV-infected individuals were mobilized with G-CSF and after leukapheresis and enrichment, subjected to retroviral transduction with genes encoding anti-HIV ribozyme-decoy fusion molecules. These cells were tested for the ability to give rise to progeny cells, for retroviral transduction efficiency, and for expression of the transgene. CD34+-derived macrophage-like cells were also challenged with HIV. Results showed that CD34+ cells from HIV-infected individuals gave rise to similar numbers of progeny colonies as cells from healthy donors. The transduction efficiency of these cells varied from 68.8 to 100% as assessed by DNA polymerase chain reaction (PCR) of the transgene in individual colonies. CD34+-derived macrophages expressed anti-HIV genes and displayed a substantial and sustained inhibition of HIV replication as compared to untransduced cells. Furthermore, we showed that after thawing, cryopreserved CD34+ cells from these individuals have survival, proliferation, and transduction parameters comparable to fresh cells. Thus, CD34+ cells from HIV-infected patients can be stored for further genetic manipulations with improved vectors or anti-HIV genes as they become available. Five HIV-infected individuals were treated with granulocyte colony-stimulating factor (G-CSF) to mobilize CD34+ cells to the periphery. The CD34+ cells were harvested by leukapharesis and transduced with a retroviral vector expressing anti-HIV ribozyme-decoy fusion molecules. The transduced cells from infected or uninfected donors gave rise to similar numbers of progeny colonies. The transduction efficiency of these cells varied from 68.8 to 100% as assessed by DNA polymerase chain reaction (PCR) of the transgene in individual colonies. Cryopreserved CD34+ cells from these individuals have survival, proliferation, and transduction parameters comparable to fresh cells. CD34+ derived macrophage progeny cells expressed the ribozyme-decoy molecules and displayed a substantial and sustained inhibition of HIV replication as compared to untransduced cells. These results suggest that gene targeting of progenitor cells may be a viable therapeutic regimen for HIV-infected patients.