Chang Li, Aphrodite Georgakopoulou, Kiriaki Paschoudi, Anna K Anderson, Lishan Huang, Sucheol Gil, Maria Giannaki, Efthymia Vlachaki, Gregory A Newby, David R Liu, Evangelia Yannaki, Hans-Peter Kiem, André Lieber
{"title":"通过体内碱基编辑将 G-Makassar 变体引入造血干细胞,治疗小鼠镰状细胞病。","authors":"Chang Li, Aphrodite Georgakopoulou, Kiriaki Paschoudi, Anna K Anderson, Lishan Huang, Sucheol Gil, Maria Giannaki, Efthymia Vlachaki, Gregory A Newby, David R Liu, Evangelia Yannaki, Hans-Peter Kiem, André Lieber","doi":"10.1016/j.ymthe.2024.10.018","DOIUrl":null,"url":null,"abstract":"<p><p>Precise repair of the pathogenic mutation in hematopoietic stem cells (HSCs) represents an ideal cure for patients with sickle cell disease (SCD). Here, we demonstrate correction of the SCD phenotype by converting the sickle mutation codon (GTG) into a benign G-Makassar variant (GCG) using in vivo base editing in HSCs. We show successful production of helper-dependent adenoviral vectors expressing an all-in-one base editor mapping to the sickle mutation site. In HSC-enriched cells from SCD patients, transduction with the base editing vector in vitro resulted in 35% GTG > GCG conversion and phenotypic improvements in the derived red blood cells. After ex vivo transduction of HSCs from an SCD mouse model and subsequent transplantation, we achieved an average of 88% editing at the target site in transplanted mice. Importantly, in vivo HSC base editing followed by selection generated 24.5% Makassar variant in long-term repopulating HSCs of SCD mice. The treated animals demonstrated correction of disease hallmarks without any noticeable side effects. Off-target analyses at top-scored genomic sites revealed no off-target editing. This in vivo approach requires a single non-integrating vector, only intravenous/subcutaneous injections, and minimal in vivo selection. This technically simple approach holds potential for scalable applications in resource-limiting regions where SCD is prevalent.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"4353-4371"},"PeriodicalIF":12.1000,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11638829/pdf/","citationCount":"0","resultStr":"{\"title\":\"Introducing a hemoglobin G-Makassar variant in HSCs by in vivo base editing treats sickle cell disease in mice.\",\"authors\":\"Chang Li, Aphrodite Georgakopoulou, Kiriaki Paschoudi, Anna K Anderson, Lishan Huang, Sucheol Gil, Maria Giannaki, Efthymia Vlachaki, Gregory A Newby, David R Liu, Evangelia Yannaki, Hans-Peter Kiem, André Lieber\",\"doi\":\"10.1016/j.ymthe.2024.10.018\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Precise repair of the pathogenic mutation in hematopoietic stem cells (HSCs) represents an ideal cure for patients with sickle cell disease (SCD). Here, we demonstrate correction of the SCD phenotype by converting the sickle mutation codon (GTG) into a benign G-Makassar variant (GCG) using in vivo base editing in HSCs. We show successful production of helper-dependent adenoviral vectors expressing an all-in-one base editor mapping to the sickle mutation site. In HSC-enriched cells from SCD patients, transduction with the base editing vector in vitro resulted in 35% GTG > GCG conversion and phenotypic improvements in the derived red blood cells. After ex vivo transduction of HSCs from an SCD mouse model and subsequent transplantation, we achieved an average of 88% editing at the target site in transplanted mice. Importantly, in vivo HSC base editing followed by selection generated 24.5% Makassar variant in long-term repopulating HSCs of SCD mice. The treated animals demonstrated correction of disease hallmarks without any noticeable side effects. Off-target analyses at top-scored genomic sites revealed no off-target editing. This in vivo approach requires a single non-integrating vector, only intravenous/subcutaneous injections, and minimal in vivo selection. 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Introducing a hemoglobin G-Makassar variant in HSCs by in vivo base editing treats sickle cell disease in mice.
Precise repair of the pathogenic mutation in hematopoietic stem cells (HSCs) represents an ideal cure for patients with sickle cell disease (SCD). Here, we demonstrate correction of the SCD phenotype by converting the sickle mutation codon (GTG) into a benign G-Makassar variant (GCG) using in vivo base editing in HSCs. We show successful production of helper-dependent adenoviral vectors expressing an all-in-one base editor mapping to the sickle mutation site. In HSC-enriched cells from SCD patients, transduction with the base editing vector in vitro resulted in 35% GTG > GCG conversion and phenotypic improvements in the derived red blood cells. After ex vivo transduction of HSCs from an SCD mouse model and subsequent transplantation, we achieved an average of 88% editing at the target site in transplanted mice. Importantly, in vivo HSC base editing followed by selection generated 24.5% Makassar variant in long-term repopulating HSCs of SCD mice. The treated animals demonstrated correction of disease hallmarks without any noticeable side effects. Off-target analyses at top-scored genomic sites revealed no off-target editing. This in vivo approach requires a single non-integrating vector, only intravenous/subcutaneous injections, and minimal in vivo selection. This technically simple approach holds potential for scalable applications in resource-limiting regions where SCD is prevalent.
期刊介绍:
Molecular Therapy is the leading journal for research in gene transfer, vector development, stem cell manipulation, and therapeutic interventions. It covers a broad spectrum of topics including genetic and acquired disease correction, vaccine development, pre-clinical validation, safety/efficacy studies, and clinical trials. With a focus on advancing genetics, medicine, and biotechnology, Molecular Therapy publishes peer-reviewed research, reviews, and commentaries to showcase the latest advancements in the field. With an impressive impact factor of 12.4 in 2022, it continues to attract top-tier contributions.