Abstract
CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.
Highlights
Patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy
Having confirmed that we could perform precise gene targeting in functional mouse HSCs, we evaluated the functional consequences of HbS gene correction in the Townes-sickle cell disease (SCD) model[12]
We found that 9.5 Gy radiation was required to clear endogenous erythropoiesis within 4 weeks based on using the amount of sickle hemoglobin present in the peripheral blood (PB) as a surrogate for niche clearance with a dose-response of engraftment proportional to the amount of irradiation
Summary
Patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. We use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. 4 Division of Stem Cell Therapy, Distinguished Professor Unit, The Institute of Medical Science, The. even with the identification of immunologically well-matched donors (which most patients do not have), HSCT recipients can still suffer from graft-vs-host disease and/or the consequences of long-term immunosuppression. The advent of CRISPR/Cas[9] genome editing has opened the potential for efficient correction of disease-causing genetic mutations in patientderived HSCs, including the A-to-T base mutation within codon of the HBB gene in SCD patients that is responsible for the mutant HbS allele and the resulting glutamic acid to valine (E6V)
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