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 remain 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 employed the RGD-containing fibronectin fragment, RetroNectin (CH-296) and a variety of recombinant hematopoietic growth factors, such as stem cell factor (SCF), interleukin-6, megakaryocyte growth and differentiation factor (MGDF or thrombopoietin) and the human Flt-3 (fms-like tyrosine kinase) ligand in either serum containing or serum free media. Viral vectors evaluated include retroviral vectors, such as third generation chimeric human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors. Our efforts over the past year have resulted in publications evaluating the use of vectors in tracking lineage contributions of over time, immune recovery, and the identification of genetic factors important in efficient gene transduction of CD34+ cells. Efforts continue to be made to improve the level of gene marking, targeting gene expression to specific cell types, such as red blood cells, 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 adult mesenchymal stem/progenitor cells and induced pluripotential stem cells and evaluate their safety in this in vivo model system. Recent success in developing induced pluripotential stem cells (iPS cells) as well as mesenchymal stromal cells (MSC) have continued to be made over this past year. Attempts are also being made to improve methodology and the technology behind stem cell mobilization and collection, and myeloablation. Alternative approaches are being evaluated, for example, in both the instrumentation and methodology of leukapheresis procedures in small subjects and the use of chemotherapeutic agents such as busulfan, respectively. Despite continued improvements in methodology, questions remain. How can consistent high levels of expression be obtained using therapeutic genes? This is being evaluated by modifying globin expression in red blood cells both with intramural and extramural collaborative studies.. Can other stem cells either derived from bone marrow or other easily accessible tissues be targeted to assist in either the contribution or repair of other organs? This is being evaluated through the generation of iPS cells and MSC. How best to evaluate stem cells and their progeny therapeutically? Future studies are aimed to evaluate therapeutic vectors, improve hematopoietic stem cell recovery and transduction efficiency, further delineate the nature and clonality of populations contributing to the reconstitution using genetic tracking methodologies, and to isolate or induce and characterize primitive cell populations which may contribute to organogenesis or the repair of damaged tissues. In addition, the use of CrispR-Cas9 methodology is being explored which may allow correction or alteration of gene expression in hematopoietic CD34+ stem cells,more mature progenitors, or terminally differentiated cells such as granulocytes or lymphocytes. Collaborative studies both within the intramural and extramural programs have been initiated in order to determine the validity of the technology and its safety in the NHP model system.
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