1. CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.
- Author
-
Nasri M, Ritter MU, Mir P, Dannenmann B, Kaufmann MM, Arreba-Tutusaus P, Xu Y, Borbaran-Bravo N, Klimiankou M, Lengerke C, Zeidler C, Cathomen T, Welte K, and Skokowa J
- Subjects
- Humans, Animals, Mice, Neutrophils metabolism, Hematopoietic Stem Cells metabolism, Mutation, Disease Models, Animal, Granulocyte Colony-Stimulating Factor genetics, Genetic Diseases, X-Linked therapy, Genetic Diseases, X-Linked genetics, Gene Editing methods, Neutropenia congenital, Neutropenia therapy, Neutropenia genetics, Genetic Therapy methods, Congenital Bone Marrow Failure Syndromes therapy, Congenital Bone Marrow Failure Syndromes genetics, CRISPR-Cas Systems, Promoter Regions, Genetic, Leukocyte Elastase genetics, Leukocyte Elastase metabolism
- Abstract
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., Competing Interests: Declaration of interests M.N., P.M., and J.S. have filed a patent on the invention described in this study. T.C. is an advisor to Cimeio Therapeutics and Excision BioTherapeutics and holds a patent on CAST-Seq., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
- View/download PDF