Nuclease mediated gene targeting of the sickle cell disease mutation utilizing non-viral delivery strategies
Cottle, Renee Nicole
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Sickle cell disease (SCD) is an autosomal recessive disorder caused by aberrant hemoglobin structure and is associated with global mortality and health disparities. Allogeneic hematopoietic stem cell transplantation is the only curative therapy, but is not available for the vast majority of patients. Gene therapy involving the insertion and forced expression of normal or anti-sickling β-globin (HBB) variants have been proposed, however, safety concerns due to non-targeted gene insertion impedes its clinical application. A novel curative therapeutic approach involves utilizing the newly developed precision gene-editing tools, including TALENs and CRISPR/Cas9 systems, to stimulate targeted correction of the disease-causing mutation in hematopoietic stem and progenitor cells (HSPC) isolated from the patient. Subsequent engraftment of gene corrected HSPCs will replace sickled cells with healthy red blood cells, thus curing SCD. One major hurdle for advancing a gene-editing based treatment for SCD is delivering nucleases along with donor template into HSPCs. This dissertation explores the use of different delivery strategies, including nucleofection and microinjection, and determines the efficiency and off-target effects of nuclease-based gene modification by completing the following aims: (1) developing and characterizing a glass needle-mediated microinjection method for the direct delivery of molecules into human hematopoietic cells, (2) demonstrating proof-of principle for microinjecting gene-editing tools for targeting the SCD mutation within hematopoietic cells, and (3) optimizing nucleofection of gene-editing reagents into human HSPCs. This work systematically evaluates microinjection and nucleofection as delivery methods for gene correction in human hematopoietic cells and provides insight into the clinical applicability of gene-editing tools for treating sickle cell disease.