Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD) The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2) After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal The treated dogs also showed improved muscle histology These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD

https://science.sciencemag.org/content/sci/362/6410/86.full.pdf

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis. Dystrophin-deficient mice are used to test corrective strategies for Duchenne muscular dystrophy, but evaluation of dystrophin expression requires collection of tissue samples from specific muscles and time points. Here, the authors generate mice in which dystrophin expression is coupled to luciferase, and show that bioluminescence allows non-invasive monitoring of dystrophin expression following genome editing.

/pdf/in-vivo-non-invasive-monitoring-of-dystrophin-correction-in-1aliozxolf.pdf

In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse.

Emerging evidence suggests that epigenetics regulates telomere dynamics in adults. However, the relationship between these pathways in children and youth remains unknown. Thus, we examined this association in 542 healthy adolescents aged 14 to 18 years old (44.8% African Americans; 55.2% females). Global DNA methylation level (%5-mC) was quantified using ELISA method. Leukocyte telomere length (LTL) was defined as relative telomere to single copy gene (T/S) ratio. Multiple linear regression models, adjusted for age, gender, ethnicity, Tanner stage, BMI, PA, and batch effect, revealed that %5 mC was associated with LTL (adjusted β = 0.17, p < 0.01). %5 mC accounted for 5.0% of the variation for LTL. A significant gender interaction was identified (p < 0.01). There was an association between %5 mC and LTL in females (all ps < 0.01), but not in males. Further sensitivity analyses by race revealed similar associations in African Americans and whites (all ps < 0.03). The present study, for the first time, shows that lower levels of global DNA methylation are associated with shorter telomere lengths in youth, which may decrease genome stability and augment the susceptibility to diseases. Longitudinal studies are warranted to establish the effects of global DNA methylation on LTL maintenance over time.

/pdf/associations-between-global-dna-methylation-and-telomere-x1y9lf12ca.pdf

Associations between Global DNA Methylation and Telomere Length in Healthy Adolescents

Age-related changes in methylation are involved in the occurrence and development of tumors, autoimmune disease, and nervous system disorders, including Alzheimer's disease (AD), in elderly individuals; hence, modulation of these methylation changes may be an effective strategy to delay the progression of AD pathology. In this study, the AD model rats were used to screen the main active extracts from the mushroom, Ganoderma lucidum, for anti-aging properties, and their effects on DNA methylation were evaluated. The results of evaluation of rats treated with 100 mg/kg/day of D-galactose to induce accelerated aging showed that alcohol extracts of G. lucidum contained the main active anti-aging extract. The effects on DNA methylation of these G. lucidum extracts were then evaluated using SAMP8 and APP/PS1 AD model mice by whole genome bisulfite sequencing, and some methylation regulators including Histone H3, DNMT3A, and DNMT3B in brain tissues were up-regulated after treatment with alcohol extracts from G. lucidum. Molecular docking analysis was carried out to screen for molecules regulated by specific components, including ganoderic acid Mk, ganoderic acid C6, and lucidone A, which may be active ingredients of G. lucidum, including the methylation regulators of Histone H3, MYT, DNMT3A, and DNMT3B. Auxiliary tests also demonstrated that G. lucidum alcohol extracts could improve learning and memory function, ameliorate neuronal apoptosis and brain atrophy, and down-regulate the expression of the AD intracellular marker, Aβ1-42. We concluded that alcohol extracts from G. lucidum, including ganoderic acid and lucidone A, are the main extracts involved in delaying AD progression.

/pdf/alcohol-extracts-from-ganoderma-lucidum-delay-the-progress-8p0hmioyq7.pdf

Alcohol Extracts From Ganoderma lucidum Delay the Progress of Alzheimer's Disease by Regulating DNA Methylation in Rodents.

ObjectiveTo describe the deletion patterns and distribution characteristics of the dystrophin gene in a Chinese population of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).MethodsPatients with DMD/BMD were recruited. Deletions in 19 exons of the dystrophin gene were evaluated using accurate multiplex polymerase chain reaction (PCR).ResultMultiplex PCR identified deletions in 238/401 (59.4%) patients with DMD/BMD. Of these, 196 (82.4%) were in the distal hotspot, 32 (13.4%) were in the proximal hotspot, five (2.1%) were in both regions and five (2.1%) were in neither hotspot. Deletions were classified into 54 patterns. Exon 49 was the most frequently deleted. The reading frame rule was upheld for 91.9% of cases.ConclusionAccurate multiplex PCR for 19 exons is an effective diagnostic tool.

https://journals.sagepub.com/doi/pdf/10.1177/0300060515613223

Distribution of dystrophin gene deletions in a Chinese population

Background: DNA hypomethylation of long interspersed nuclear elements-1 (LINEs-1) occurs during carcinogenesis, whereas information addressing LINE-1 methylation in Wilms tumor (WT) is limited. The main purpose of our study was to quantify LINE-1 methylation levels and evaluate their relationship with relative telomere length (TL) in WT. Methods: We investigated LINE-1 methylation and relative TL using bisulfite-polymerase chain reaction (PCR) pyrosequencing and quantitative PCR, respectively, in 20 WT tissues, 10 normal kidney tissues and a WT cell line. Significant changes were analyzed by t -tests. Results: LINE-1 methylation levels were significantly lower ( P P r = 0.671, P = 0.001). LINE-1 Methylation levels were significantly associated with global DNA methylation ( r = 0.332, P Conclusion: These results suggest that LINE-1 hypomethylation is common and may be linked to telomere shortening in WT.

Zhuo Liu

Papers

Distribution of dystrophin gene deletions in a Chinese population

Association between Long Interspersed Nuclear Element-1 Methylation and Relative Telomere Length in Wilms Tumor.

Hypermethylation of AKT2 gene is associated with neural-tube defects in fetus