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.
Rationale Duchenne muscular dystrophy (DMD) 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 DMD 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 DMD 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 (AAV). 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/gRNA constructs into an AAV 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 to untreated controls. Furthermore, our targeted deep sequencing results confirmed that our AAV-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.
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 and 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. Micro-CT analyses demonstrated kyn-induced bone loss. Levels of serum markers of osteoclastic activity (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 co-factor 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.
Objectives Nutrition plays a key role in the maintenance of muscle and bone mass, and dietary protein deficiency has in particular been associated with catabolism of both muscle and bone tissue. One mechanism thought to link protein deficiency with loss of muscle mass is deficiency in specific amino acids that play a role in muscle metabolism. We tested the hypothesis that the essential amino acid tryptophan, and its metabolite kynurenine, might directly impact muscle metabolism in the setting of protein deficiency. Methods Adult mice (12 mo) were fed a normal diet (18% protein), as well as diets with low protein (8%) supplemented with increasing concentrations (50, 100, and 200 uM) of kynurenine (Kyn; or with tryptophan (Trp; 1.5 mM). Myoprogenitor cells were also treated with Trp and Kyn in vitro to determine their effects on cell proliferation and expression of myogenic differentiation markers. Results Results indicate that all mice on the low protein diets weighed less than the group fed normal protein (18%). Lean mass measured by DXA was lowest in mice on the high kynurenine diet, whereas percent lean mass was highest in mice receiving tryptophan supplementation and percent body fat was lowest in mice receiving tryptophan. ELISA assays showed significant increases in skeletal muscle IGF-1, leptin, and the myostatin antagonist follistatin with tryptophan supplementation. mRNA microarray and gene pathway analysis performed on muscle samples demonstrate that mTor/eif4/p70s6k pathway molecules are significantly up-regulated in muscles from mice on Kyn and Trp supplementation. In vitro, neither amino acid affected proliferation of myoprogenitors, but tryptophan increased the expression of the myogenic markers MyoD, myogenin, and myosin heavy chain. Conclusion Together, these findings suggest that dietary amino acids can directly impact molecular signaling in skeletal muscle, further indicating that dietary manipulation with specific amino acids could potentially attenuate muscle loss with dietary protein deficiency.
Age-induced bone loss is associated with greater bone resorption and decreased bone formation resulting in osteoporosis and osteoporosis-related fractures. The etiology of this age-induced bone loss is not clear but has been associated with increased generation of reactive oxygen species (ROS) from leaky mitochondria. ROS are known to oxidize/damage the surrounding proteins/amino acids/enzymes and thus impair their normal function. Among the amino acids, the aromatic amino acids are particularly prone to modification by oxidation. Since impaired osteoblastic differentiation from bone marrow mesenchymal stem cells (BMMSCs) plays a role in age-related bone loss, we wished to examine whether oxidized amino acids (in particular the aromatic amino acids) modulated BMMSC function. Using mouse BMMSCs, we examined the effects of the oxidized amino acids di-tyrosine and kynurenine on proliferation, differentiation and Mitogen-Activated Protein Kinase (MAPK) pathway. Our data demonstrate that amino acid oxides (in particular kynurenine) inhibited BMMSC proliferation, alkaline phosphatase expression and activity and the expression of osteogenic markers (Osteocalcin and Runx2). Taken together, our data are consistent with a potential pathogenic role for oxidized amino acids in age-induced bone loss.
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