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Introduction: α-thalassemia major (ATM) is an autosomal recessive disorder where all four copies of the α-globin gene are deleted. A traditionally lethal disease due to severe fetal hypoxia, this condition is now survivable due to the advent of in utero blood transfusions. However, patients still require life-long transfusions postnatally. While allogeneic hematopoietic stem cell transplant (HSCT) can provide a definitive cure, it is limited by the paucity of suitable donors, has a high morbidity and mortality due the prerequisite myeloablative regimens, and carries the risk of immune rejection and graft-versus-host disease. Here, we describe a CRISPR/AAV-mediated genome editing strategy to restore a full-length copy of the α-globin gene at the β-globin locus in ATM patient-derived HSCs to restore normal hemoglobin production to a patient's red blood cells (RBCs).

Methods: We identified a Cas9 gRNA that efficiently …