CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia.
Mol Ther
; 28(12): 2621-2634, 2020 12 02.
Article
em En
| MEDLINE
| ID: mdl-32822592
ABSTRACT
Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANEL172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, â¼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Terapia Genética
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Transplante de Células-Tronco Hematopoéticas
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Elastase de Leucócito
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Sistemas CRISPR-Cas
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Síndrome Congênita de Insuficiência da Medula Óssea
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Mutação
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Neutropenia
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article