A Preliminary Study On The Differentiation Of Myoblasts Induced From H1cells And The Deletion Of Exon 50-51 Of The DMD Gene

BackgroundDuchenne muscular dystrophy(DMD)is the most common lethal X-linked recessive genetic disease in male children.The main cause of the disease is DMD gene mutation.There is no effective treatment for DMD in clinical practice,mainly through glucocorticoids therapy just to delay the progression of disease.At present,the treatment research on DMD has focused on gene therapy.In the presence of exon51,by knocking out or skipping the exon 51,13%of DMD patients can be corrected as BMD to delay the progression of disease.The iPSC derived from DMD patients is a cell model for DMD disease research and can evaluate the therapeutic effect.Genetic modification of iPSCs derived from patients with monogenic genetic diseases such as DMD by CRISPR/Cas9 can obtain the unmodified and modified diseased cells with the same genetic background from the same source.However,it is difficult to obtain the iPSC of fetal origin derivedfrom DMD suitable for Exon51 exon deletion.Therefore,it is a reasonable solution to genetically transform the myoblast cells induced by normal human H1 cells intomutant cell models of exon 51 knockout of DMD gene by CRISPR/Cas9 gene editing technology.In this study,the myoblast cell line derived from H1 cells of exon 50 deletion of DMD gene was established,exon 51 of DMD gene was knocked out by CRISPR/Cas9 technology and exon skipping reading after knocking was detected to determine the effectiveness of gene editing therapy at the level of human cell.Objectives1.To establish a directed differentiation system of H1 cells to myoblast cells.2.To construct a CRISPR/Cas9-mediated DMD gene exon50 and exon50-51deficient H1 cell-derived myoblast cell line.MethodsThe H1 cells were finally induced into myoblast cells by the STEMdiff~TMM Mesenchymal Progenitor Kit and the method of transfecting the MyoD expression vector through the MSC-like cell stage.The exon 50 of the DMD gene of myoblast cells induced by H1 cells was knocked out by using CRISPR/Cas9 technology,and a myoblast cell strain in which the stably inherited DMD gene exon50 was deleted was obtained.The exon 50-51 of the DMD gene was knocked out in the same manner to obtain a stable genetic model of knockdown and repair of the DMD gene.Results1.A directed differentiation system of H1 cells to myoblast cells was successfully established..2.The myoblast cell models with exon 50 and exon 50-51 deletion of the DMD gene were constructed.ConclusionIn this study,the existing H1 cells were used to successfully establish a stem cell-derived myoblast cell differentiation platform,and the CRISPR/Cas9 gene editing technology was used to successfully delete exon50 and exon50-51 of myoblast cell DMD gene,and obtained a stable cell model with DMD gene knockout and repairment,which contributed to the identification and safety evaluation of CRISPR/Cas9-mediated gene editing..