Therapeutic Genome Editing On Human DM1 Induced Pluripotent Stem Cells And Establishment Of Outcome Measure For Function Evaluation Of Skeletal Muscle Precursor Cells For DM1

Myotonic Dystrophy 1(DM1)is the most common adult form of muscular dystrophy caused by expanded CTG nucleotide repeats in 3 prime untranslated region(3’-UTR).It is a fatal disease and no treatment is presently available.Patient Specific iPS cells carry the full set of genetic information and have the potential to multi-differentiation,could be used for exploring disease pathogenesis,drugs screening also provide disease model and theoretical basis for transplant therapy research.Gene editing techniques could efficiently correct the mutations of i PS cells provides a new strategy for the treatment of genetic diseases.As the rapid development of induced pluripotent stem cells(iPS)technology and therapeutic genome editing make patient-specific cell transplantation has broad prospects for development,and provides a new direction for DM1 treatment.We hypothesized that genome editing can correct the mutation in patient-specific DM1 iPS cells,which can circumvent the ethical and immune rejection issues for autologous cell transplantation.However,an unmet need is to find outcome measures in vitro to demonstrate the therapeutic effect before moving these therapies to in vivo clinical trials.The first part of the current study is to develop a strategy to completely eliminate expanded CUG mutant transcripts by precise incorporation of Poly A signalsupstream of DMPK CTG repeats.This will be accomplished by homologous recombination facilitated by double-strand bread induced by a pair of site-specific TALENs.We will examine whether the genome editing will affect the pluripotency of human DM1 iPS cells by in vitro linear differentiation and in vivo teratoma formation.We will further examine whether this approach can reverse DM1 phenotype in linear-differentiated neural stem cells,cardiomyocytes and teratoma tissues.The second part of the current study is to establish an easy and sensitive outcome measures to evaluate in vitro skeletal muscle regeneration.We will examine and compare the in vitro myotube formation in myoblasts isolated form normal and DM1 subjects to identify outcome measures.The outcome measure will be used in evaluating the function of skeletal muscle precursor cells derived from DM1 and genome-corrected DM1 iPS cells,which will pave the way for future autologous skeletal muscle precursor cell transplantation therapy.Objectives:1.To establish genome-corrected DM1 iPS cell lines.To accomplish this,we will incorporate PolyA signals upstream of CTG repeat to abolish mutant mRNA.We will examine the pluripotency and phenotype reversal in the genome-corrected iPS cell lines.2.To establish outcome measures to evaluate skeletal muscle regeneration.To accomplish this,we will study the whole process of myotube formation in vitro in normal and DM1 myoblasts in order to identify new pathologic features that are easy and sensitive to follow for monitoring therapeutic effects.Methods:1.Construction of donor containing homologous arms and polyA signals;designing and selection of site-specific TALENs to facilitate the homologousrecombination through double strand breaks;transfection of human DM1 iPS cells and identify positive clones that have PolyA signals inserted into designed TALEN cutting site.2.Teratoma formation assay to prove pluripotency of genome-corrected DM1 iPS clones.H&E and immunohistochemical staining to confirm the expression of three germ layer cell markers.RNA FISH to evaluate the loss of pathogenic intranuclear foci.3.In vitro differentiation of normal,parental and genome-corrected DM1 iPS cells into neural stem cells、 neurons、astrocytes and Cardiomyocytes to exam the phenotype reversal of RNA foci formation and alternative splicing.4.Generation of myoblast cells from muscle biopsy tissue in a normal and two DM1 subjects with CTG expansion.Studying the whole process of myotube formation,myoblast proliferation,early myotube formation and late myotube formation.5.RNA FISH and immunofluorescence staining to examine the morphology of myotube formation,nuclear aggregation and myofibril degeneration.6.Co-culture of DM1 myoblasts and normal myoblasts at differential ratios to validate nuclear aggregation index and myofibril degeneration as outcome measures for monitoring of therapeutic effects.7.All the data were acquired in there independent experiment.One-way ANOVA was used to compare the difference among group.P<0.05 was used as significant difference Results:1.Successful incorporation of PolyA signals upstream of CTG repeats of DMPK gene,which led to elimination of mutant intranuclear RNA foci.2.Genome-corrected DM1 iPS cells maintain pluripotency by teratoma assay.The loss of intranuclear RNA foci was also confirmed in teratoma tissues.3.Demonstrated the loss of pathogenic intranuclear RNA foci and reversal of alternative splicing in neural stem cells、neurons、astrocyte and cardiomyocytes derived from genome-corrected DM1 iPS cells.4.Successful establishment of normal and DM1 myoblasts from muscle biopsy tissues.5.Identification of nuclear aggregation during early stage of myotube formation in DM1.6.Identification of myofibril degeneration during late stage of myotube formation in DM1.7.Validation of nuclear aggregation index and myofibril degeneration as outcomemeasures as demonstrated by amelioration of nuclear aggregation and myofibril degeneration with introducing of normal myoblasts in the co-culture system.Conclusion:1.Genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny.These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.2.Nuclear aggregation and myofibril degeneration during myotube formation can be used to evaluate the function of skeletal muscle precursor cells.These outcome measures can be used in monitoring the therapeutic effects of genome editing and small molecules in vitro before moving them into clinical trials.