Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD.

: Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. μCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research

Rationale:: Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr ^+/− mice after a single systemic delivery of recombinant adeno-associated virus. Objective:: To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. Methods and Results:: Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat–associated 9 from Staphylococcus aureus ) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr ^+/− neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. Conclusions:: This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal β-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and β-catenin. A pharmacological activator of the WNT/β-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and β-catenin, and evidence for targeted activation of the WNT/β-catenin pathway as a potential treatment for this disease.