Genome-wide CRISPR screen to identify pro-myogenic factors for cell therapy of DMD

Project Abstract Myos Incis developing a cell therapy treatment for patients with Duchenne muscular dystrophyDMDbased on HLA matched donor cells that are pretreated with small molecule cocktails ex vivo to modify genes that will enhance engraftment and fusion and enable muscle regenerationMyos is collaborating with DrLouis KunkelBoston Children s Hospitalto address the issues that led to low efficacy in previous clinical trialsGiven the focus on DMDthis project is highly relevant to the mission of the NIH and the NIAMSThe long term goal is to develop efficient muscle cell therapy for all DMD patients of all agesThe objective of this Phase I STTR project is to use genome wide screens to identify key factors that will promote myogenic fusion and cell extravasationwhich are required for effective muscle cell engraftmentThe Aims areAimIdentify genes whose perturbation results in altered myogenic fusionThe project will utilize genome wide screening libraries to generate gene edited myogenic cell populations with either single gene knock outs or single gene over expressionGene edited cells will be switched to media that promotes myogenic fusion into multinucleated myotubesNormal fused cells will be separated from abnormal un fusedmononuclearcells by filtration and analyzed by sequencing to identify CRISPR gene modifications that distinguish fused vs un fused cellsMilestoneThe milestone is that the project will generate a validated list ofgenes pathways involved in cell fusionThese findings will be used in a Phase II STTR to test efficacy in preclinical mice modelsAimIdentify genes whose perturbation results in increased efficiency of cellular extravasation and homing to musclesUsing a human myogenic cell line expressing the molecular marker green fluorescence proteinGFPthe project will separately transduce genome wide libraries to induce gene knock out or overexpressionGene edited cells will be intravenously injected into DBAJ mdx micea well characterized model of human DMDMuscles will be harvested and digested for cell culture expansion and selection of human cells that have successfully extravasated from the circulatory systemTheseengraftablecells will be expanded in culture and sequenced to identify their gene modificationsMilestoneThe project will generate a validated list ofgenes pathways involved in increased cell extravasation and muscle homingThe findings will be used in a Phase II STTR to test efficacy in mice modelsThis is a fundamentally novel approach to identify genes that promote muscle recolonization and growth to treat DMD regardless of disease mutationSuccessful completion of this project will lead to a future Phase II STTR application to conduct preclinical animal testing of DMD cell therapy in preparation for clinical trials Project Narrative This projectif successfulwill be the first step in developing a therapeutic that benefits the thousands of boys and young men suffering from Duchenne muscular dystrophyDMDa rare and fatal disease that is characterized by progressive muscle wastingThe critical research completed under the Phase I STTR will enable Myos to begin to develop a cell therapy treatment that would allow patients suffering from DMD to not only regain muscle butmore importantlyhave a significantly longer life expectancy than current projectionsThis therapeutic would have a significant return in that it would allow this group of patients to lead productive lives while significantly reducing the annual cost to care for the disease