Abstract
Genome editing with the CRISPR/Cas9 technology has emerged recently as a potential strategy for therapy in genetic diseases. For dominant mutations linked to gain-of-function effects, allele-specific correction may be the most suitable approach. In this study, we tested allele-specific inactivation or correction of a heterozygous mutation in the Dynamin 2 (DNM2) gene that causes the autosomal dominant form of centronuclear myopathies (CNMs), a rare muscle disorder belonging to the large group of congenital myopathies. Truncated single-guide RNAs targeting specifically the mutated allele were tested on cells derived from a mouse model and patients. The mutated allele was successfully targeted in patient fibroblasts and Dnm2R465W/+ mouse myoblasts, and clones were obtained with precise genome correction or inactivation. Dnm2R465W/+ myoblasts showed an alteration in transferrin uptake and autophagy. Specific inactivation or correction of the mutated allele rescued these phenotypes. These findings illustrate the potential of CRISPR/Cas9 to target and correct in an allele-specific manner heterozygous point mutations leading to a gain-of-function effect, and to rescue autosomal dominant CNM-related phenotypes. This strategy may be suitable for a large number of diseases caused by germline or somatic mutations resulting in a gain-of-function mechanism.
Highlights
Autosomal dominant diseases represent a challenge for development of effective therapies
Primary myoblasts were isolated from lower limb muscles of postnatal day 5 (P5) WT and Dnm2R465W/+ mice and transduced with a lentivirus expressing cyclindependent kinase 4 (CDK4)
In this study, we investigated the pathomechanisms of Dynamin 2 (DNM2)-related centronuclear myopathies (CNMs) in muscle cells from a mouse model and patients and identified an aberrant increase in endocytosis, supporting a gain-of-function effect of DNM2 mutations
Summary
Autosomal dominant diseases represent a challenge for development of effective therapies. Whereas haploinsufficiency may be treated by conventional gene replacement, diseases linked to gain-of-function or toxic effect in essential proteins require the targeting of the mutated allele. The mutated allele can be either inactivated, leading to haploinsufficiency, or corrected to revert to a wild-type (WT) genotype. In the case of disease-implicated genes essential for cellular functions, allelespecific correction would be preferred to avoid deleterious effects due to an overall decrease of the protein expression. We tested the potential of genome editing to rescue the phenotypes of a dominant disease through allele-specific inactivation or correction. Allele-specific gene inactivation can be achieved through gene silencing with shRNA1 or antisense oligonucleotides, leading to hap-
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