ABSTRACT
Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.
Subject(s)
Dystrophin/genetics , Frameshift Mutation , Gene Editing/methods , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , RNA, Guide, Kinetoplastida/genetics , Animals , Disease Models, Animal , Exons , Female , Gene Expression Regulation , Genetic Therapy , Genome , Heart Failure/genetics , Heart Failure/therapy , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Male , Mass Spectrometry , Muscle, Skeletal/metabolism , Muscles/metabolism , Myoblasts/metabolism , Myocytes, Cardiac/metabolism , Proteome , SwineABSTRACT
Prediabetes is a condition affecting 35% of US adults and about 50% of US adults age 65+. Foods rich in polyphenols, including flavanols and other flavonoids, have been studied for their putative beneficial effects on many different health conditions including type 2 diabetes mellitus and prediabetes. Studies have shown that some flavanols increase glucagon-like peptide 1 (GLP-1) secretion. GLP-1 is a feeding hormone that increases insulin secretion after carbohydrate consumption, and increased GLP-1 secretion may be responsible for some of the beneficial effects on glycemic control after flavanol consumption. The present study explored the effects of grape powder consumption on metrics of glycemic health in normoglycemic and prediabetic C57BL/6J mice; additionally, the mechanism of action of grape powder polyphenols was investigated. Grape powder significantly reduced (p < 0.01) blood glucose levels following oral glucose gavage after GLP-1 receptor antagonism by exendin-3 (9-39) compared to sugar-matched control, indicating that it was able to attenuate the hyperglycemic effects of GLP-1 receptor antagonism. Grape powder was employed in acute (1.6 g grape powder per kg bodyweight) and long-term high fat diet (grape powder incorporated into treatment diets at 5% w/w) feeding studies in normoglycemic and prediabetic (diet-induced obesity) mice; grape powder did not impove glycemic control in these studies versus sugar-matched control. The mechanisms by which grape powder ameliorates the deleterious effects of GLP-1 receptor antagonism warrant further study.
Subject(s)
Glucagon-Like Peptide-1 Receptor/metabolism , Peptides/pharmacology , Phytotherapy , Plant Preparations/pharmacology , Polyphenols/pharmacology , Vitis/chemistry , Animals , Blood Glucose/metabolism , Diet, High-Fat , Disease Models, Animal , Flavonoids/pharmacology , Glucagon-Like Peptide-1 Receptor/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Powders , Prediabetic State/drug therapyABSTRACT
The hydrolysis of whole casein and isolated casein components were investigated with the purpose of obtaining information concerning the kinetic and specifty of aspartic proteases in rennin, pepsin and 4 microbial rennet substitutes. The velocity of hydrolysis decreased rapidly within the first hour. However, the hydrolysis was not completed after 2 days. A mathematical description of the slope of hydrolysis is possible by use of exponential equations. More than 40 peptides were detected by capillary electrophoresis or PAGE. The characterization of the C- and N-terminal amino acids of peptides shows that the hydrolysis of any peptide bond depends mainly on the structure of the C-terminal side chains of the amino acids. The detection of the basic amino acids lysin and arginin in the C-terminal position of peptides is a new result, furthering the knowledge about the specificity of aspartic proteases. Differences in the reaction velocity or in the extent of hydrolysis are one of the possible explanations for the described differences in the rennet curd yield. It was concluded that the rennet enzymes are active also in the later phases of cheese ripening and are able to support the action of cheese ripening flora.
