For successful gene transfer to primitive hematopoietic cells several requirements need to be achieved. These include identification of the desired target cell population, identification of the appropriate vector to be used, and achieving desired levels of gene expression. To date, successful gene transfer in human subjects remains problematic. To address these problems as well as important safety issues, studies in non-human primates (NHPs) are being undertaken to optimize gene transfer to nonhuman primate hematopoietic cells prior to human clinical studies. Vectors that have been evaluated include self-inactivating (SIN) retroviral vectors, and lentiviral vectors constructed to optimally transduce rhesus CD34+ cells. These vectors have been constructed to express reporter genes, such as the enhanced green fluorescent protein (EGFP), or therapeutic genes, such as hemoglobin. Transduction conditions continue to be evaluated and optimized. The use of feeder layers, such as the human umbilical vein endothelial cells, are being evaluated. In addition new drugs for improving mobilization of hematopoietic stem cells are being considered. Our efforts over the past year have resulted in publications which evaluate the use of vectors in tracking lineage contributions over time, and characterize bone marrow as a hematopoietic stem cell source for gene therapy in sickle cell disease. Evaluation of immune recovery through T-cell immunosuppression utilizing immunotherapies directed specifically against CD3 expressing lymphocytes continues. We have also initiated studies involving vectors which express hemoglobin specifically in erythroid progenitors targeting hemoglobinopathies, as well as studies utilizing electroporation and CrispR/Cas9 to target genes potentially involved in leukemogenesis, and to develop an animal model for in paroxysmal nocturnal hemoglobinuria and the knockout of CD33, a transmembrane receptor expressed on myeloidlineage. Efforts continue to be made to improve the level of gene marking, targeting gene expression to specific cell types, such as red blood cells and granulocytes, evaluate immune reconstitution following transplant and the contribution of genetically marked cells to the recovery, and to derive stem cells from other tissues besides BM and cytokine mobilized PB, such as induced pluripotential stem cells, and evaluate their safety in this in vivo model system. Recent success in developing induced pluripotential stem cells (iPS cells) have continued to be made over this past year in rhesus macaques and imaging these cells through molecular imaging using the sodium iodide symporter gene. Attempts are also being made to develop an alternative to total body irradiation for myeloablation through the use of chemotherapeutic agents such as busulfan and immuosuppressives such as abatacept and sirolimus. Questions remain concerning how consistent high levels of expression might be obtained using therapeutic genes. This is being evaluated by modifying globin expression in red blood cells both with intramural and extramural collaborative studies, with a particular interest in targeting BCL11A using the CRISPR-Cas gene knockout system. Future studies are aimed to evaluate therapeutic vectors; improve hematopoietic stem cell mobilization, recovery, and transduction efficiency; further delineate the nature and clonality of populations contributing to reconstitution using genetic tracking methodologies; and to isolate or induce primitive cell populations which may contribute to organogenesis or the repair of damaged tissues. Collaborative studies both within the intramural and extramural programs continue to be initiated in order to determine the validity of the technology and its safety and efficacy in the non-human primate model system.
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