Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Autor: Iyer S; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA., Suresh S; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA., Guo D; Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.; Wellstone Muscular Dystrophy Program, University of Massachusetts Medical School, Worcester, MA, USA., Daman K; Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.; Wellstone Muscular Dystrophy Program, University of Massachusetts Medical School, Worcester, MA, USA., Chen JCJ; Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.; Wellstone Muscular Dystrophy Program, University of Massachusetts Medical School, Worcester, MA, USA.; Office of the Vice-Principal (Research), Queen's University, Kingston, Ontario, Canada., Liu P; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA., Zieger M; Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA., Luk K; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA., Roscoe BP; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.; COGEN Therapeutics, Cambridge, MA, USA., Mueller C; Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA., King OD; Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.; Wellstone Muscular Dystrophy Program, University of Massachusetts Medical School, Worcester, MA, USA., Emerson CP Jr; Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA. charles.emersonjr@umassmed.edu.; Wellstone Muscular Dystrophy Program, University of Massachusetts Medical School, Worcester, MA, USA. charles.emersonjr@umassmed.edu.; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA. charles.emersonjr@umassmed.edu., Wolfe SA; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.
Jazyk: angličtina
Zdroj: Nature [Nature] 2019 Apr; Vol. 568 (7753), pp. 561-565. Date of Electronic Publication: 2019 Apr 03.
DOI: 10.1038/s41586-019-1076-8
Abstrakt: Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G) 1 and Hermansky-Pudlak syndrome type 1 (HPS1) 2 . Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.
Databáze: MEDLINE
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