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

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    Authors
    Iyer, Sukanya
    Suresh, Sneha
    Guo, Dongsheng
    Daman, Katelyn
    Chen, Jennifer C. J.
    Zieger, Marina
    Luk, Kevin
    Roscoe, Benjamin P.
    Mueller, Christian
    King, Oliver D.
    Emerson, Charles P. Jr.
    Wolfe, Scot A.
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    UMass Chan Affiliations
    Department of Biochemistry and Molecular Pharmacology
    Li Weibo Institute for Rare Diseases Research
    Horae Gene Therapy Center
    Emerson Lab
    King Lab
    Wellstone Muscular Dystrophy Program
    Department of Neurology
    Department of Molecular, Cell and Cancer Biology
    Document Type
    Journal Article
    Publication Date
    2019-04-03
    Keywords
    UMCCTS funding
    Computational Biology
    Congenital, Hereditary, and Neonatal Diseases and Abnormalities
    Enzymes and Coenzymes
    Genetic Phenomena
    Genetics and Genomics
    Musculoskeletal Diseases
    Nervous System Diseases
    Therapeutics
    Translational Medical Research
    
    Metadata
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    Link to Full Text
    https://doi.org/10.1038/s41586-019-1076-8
    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.
    Source

    Nature. 2019 Apr;568(7753):561-565. doi: 10.1038/s41586-019-1076-8. Epub 2019 Apr 3. Link to article on publisher's site

    DOI
    10.1038/s41586-019-1076-8
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/50375
    PubMed ID
    30944467
    Related Resources

    Link to Article in PubMed

    ae974a485f413a2113503eed53cd6c53
    10.1038/s41586-019-1076-8
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    Wellstone Center for FSHD Publications
    UMass Center for Clinical and Translational Science Supported Publications

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