Autologous hematopoietic stem cell (HSC) gene therapy is one of the most promising strategies for the treatment of genetic diseases affecting the blood and immune system. Currently, the only curative treatment for patients with diseases like hemoglobinopathies or immunodeficiencies is an allogeneic HSC transplant. While outcomes with an HLA-matched sibling donor are very encouraging, most patients will not have an HLA-matched family donor. HSC transplants from unrelated donors or partially matched donors can be associated with life- threatening side effects especially from graft-vs-host disease (GVHD) and related infectious complications. To avoid these complications, efforts have been made to use the patients own (autologous) HSCs and correct them with either lentivirus-mediated gene therapy or gene editing. A critical problem and bottleneck, however, has been the inability to identify and purify a ?true? HSC population and therefore optimize conditions for such cells that allow for rapid multi-lineage engraftment. A reliable strategy to determine the quality and quantity of such a HSC population would be a major advance for high-throughput screening of ex vivo gene editing and expansion conditions. Using our nonhuman primate (NHP) stem cell transplantation model, we have recently identified an exclusive HSC-enriched population capable of multi-lineage short-term and long-term engraftment that is evolutionary conserved between human and NHPs. This HSC-enriched population accounts for ~3-5% of the entire CD34+ cell population, reducing the number of cells for gene therapy approaches by ~25-fold. Most importantly, flow-cytometric quantification of this HSC-enriched phenotype allowed us to reliably predict engraftment success as well as the onset of neutrophil and platelet recovery after HSC transplantation regardless of the source, gene modification, or ex vivo expansion. Availability of this novel HSC-enriched phenotype in combination with the ability to easily enrich for this subpopulation will help overcome current limitations of autologous HSC gene therapy. We hypothesize that gene editing of this HSC-enriched population will improve targeting, enhance gene editing efficiency, and increase in vivo persistence of gene-modified and corrected blood and immune cells. Furthermore, we hypothesize that this phenotype will serve as a reliable read-out to develop ex vivo culture conditions for the expansion of gene-modified HSCs promoting improved engraftment, which in turn should enable us to reduce the intensity of currently used conditioning regimens for HSC gene therapy. Evolutionary conservation of this HSC-enriched phenotype between human and NHP cells will allow us to rapidly translate these findings to clinical HSC gene therapy studies.
We propose to develop strategies for more efficient, less toxic and more predictable gene therapy treatments for genetic diseases in blood-forming stem cells. This approach is not limited to a single disease but aims to overcome current limitations of blood stem cell gene therapy including costs, safety and overall outcome.