Precise therapeutic gene correction by a simple nuclease-induced double-stranded break
Department of Molecular, Cell and Cancer Biology; Department of Neurology; Wellstone Muscular Dystrophy Program; King Lab; Emerson Lab; Horae Gene Therapy Center; Li Weibo Institute for Rare Diseases Research; Department of Biochemistry and Molecular Pharmacology
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
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.
DOI of Published Version
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
Iyer, Sukanya; Suresh, Sneha; Guo, Dongsheng; Daman, Katelyn; Chen, Jennifer C. J.; Pengpeng Liu; Zieger, Marina; Luk, Kevin; Roscoe, Benjamin P.; Mueller, Christian; King, Oliver D.; Emerson, Charles P. Jr.; and Wolfe, Scot A., "Precise therapeutic gene correction by a simple nuclease-induced double-stranded break" (2019). UMass Center for Clinical and Translational Science Supported Publications. 202.