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Nature. 2019 Apr 3. doi: 10.1038/s41586-019-1076-8. [Epub ahead of print]

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

Iyer S1, Suresh S1, Guo D2,3, Daman K2,3, Chen JCJ2,3,4, Liu P1, Zieger M5,6, Luk K1, Roscoe BP1,7, Mueller C5,6, King OD2,3, Emerson CP Jr8,9,10, Wolfe SA11,12,13.

Author information

1
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.
2
Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.
3
Wellstone Muscular Dystrophy Program, University of Massachusetts Medical School, Worcester, MA, USA.
4
Office of the Vice-Principal (Research), Queen's University, Kingston, Ontario, Canada.
5
Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.
6
Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA.
7
COGEN Therapeutics, Cambridge, MA, USA.
8
Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA. charles.emersonjr@umassmed.edu.
9
Wellstone Muscular Dystrophy Program, University of Massachusetts Medical School, Worcester, MA, USA. charles.emersonjr@umassmed.edu.
10
Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA. charles.emersonjr@umassmed.edu.
11
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.
12
Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.
13
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.

Abstract

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.

PMID:
30944467
DOI:
10.1038/s41586-019-1076-8

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