Retroviral Mediated Gene Transfer Into Primate Hematopoietic Cells

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

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 (NHP) are being undertaken to optimize gene transfer to NHP 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. Our efforts over the past year have resulted in several publications. We evaluated busulfan, a chemotherapeutic agent in combination with immuosuppressives to develop an alternative to total body irradiation for myeloablation. We also completed a study to monitor transplantation-related changes in fetal hemoglobin and F-cell percentages in NHP models to understand the relationship between both parameters for better prediction of possible clinical outcomes in sickle cell patients. We also evaluated the platform to assess vector genotoxicity and reported the first case of lentiviral insertion-induced genotoxicity, resulting in aberrant clonal hematopoiesis in the NHP model. These reports raised important issues for designing vectors for clinical gene therapy trials. We conducted a study developing an animal model for paroxysmal nocturnal hemoglobinuria (PNH) via CRISPR/Cas9 gene editing to study the disease mechanisms in a clinically relevant model. We continued to evaluate the use of vectors in tracking lineage contributions over time, in this case to demonstrate clonal expansion and compartmentalized maintenance of rhesus macaque NK cell subsets after transplantation. In this fiscal year, we have initiated studies involving antibody-conditioning methods as non-toxic transplant conditioning regimens to permit efficient engraftment of genetically-modified rhesus CD34+ cells. We also continued to evaluate 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 erythroid development. Efforts continue to be made to improve the level of gene marking, confine gene expression to specific cell types, such as red blood cells, evaluate immune reconstitution following transplant, the contribution of genetically marked cells to reconstitution post-transplantation. Studies are in progress aimed to improve CD34+ collection efficiency using a new apheresis machine; improve hematopoietic stem cell 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.