RESUMEN
ß-Thalassemia pathology is due not only to loss of ß-globin (HBB), but also to erythrotoxic accumulation and aggregation of the ß-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in ß-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize ß-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing ß-thalassemia.
Asunto(s)
Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Hemoglobinas/metabolismo , Globinas alfa/genética , Globinas beta/genética , Talasemia beta/genética , Talasemia beta/terapia , Anemia de Células Falciformes/patología , Animales , Antígenos CD34/metabolismo , Dependovirus/genética , Eritrocitos/metabolismo , Edición Génica , Genes Reporteros , Sitios Genéticos , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Regiones Promotoras Genéticas/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.