RESUMEN
Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.
Asunto(s)
Sistemas CRISPR-Cas , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Edición Génica , Terapia Genética , Elastasa de Leucocito , Neutropenia , Regiones Promotoras Genéticas , Edición Génica/métodos , Humanos , Neutropenia/congénito , Neutropenia/terapia , Neutropenia/genética , Terapia Genética/métodos , Síndromes Congénitos de Insuficiencia de la Médula Ósea/terapia , Síndromes Congénitos de Insuficiencia de la Médula Ósea/genética , Elastasa de Leucocito/genética , Elastasa de Leucocito/metabolismo , Animales , Ratones , Neutrófilos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mutación , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Enfermedades Genéticas Ligadas al Cromosoma X/genéticaRESUMEN
Most studies on leukemia focus on leukemia cells as isolated objects without considering the bone marrow niche. Pal et al. have recreated the bone marrow niche using induced pluripotent stem cells (iPSCs), identifying CDH2 as a therapeutically druggable leukemia-promoting factor.1.