Research Project
9679984
Project Abstract Myos Inc. is developing a cell therapy treatment for patients with Duchenne muscular dystrophy (DMD) based 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 regeneration. Myos is collaborating with Dr. Louis Kunkel (Boston Children?s Hospital) to address the issues that led to low efficacy in previous clinical trials. Given the focus on DMD, this project is highly relevant to the mission of the NIH and the NIAMS. The long-term goal is to develop efficient muscle cell therapy for all DMD patients of all ages. The objective of this Phase I STTR project is to use genome-wide screens to identify key factors that will promote myogenic fusion and cell extravasation, which are required for effective muscle cell engraftment. The Aims are: Aim 1: Identify genes whose perturbation results in altered myogenic fusion. The project will utilize genome-wide screening libraries to generate gene-edited myogenic cell populations with either single gene knock-outs or single-gene over-expression. Gene-edited cells will be switched to media that promotes myogenic fusion into multinucleated myotubes. Normal fused cells will be separated from abnormal un-fused (mononuclear) cells by filtration and analyzed by sequencing to identify CRISPR gene modifications that distinguish fused vs un-fused cells. Milestone: The milestone is that the project will generate a validated list of 20 genes/pathways involved in cell fusion. These findings will be used in a Phase II STTR to test efficacy in preclinical mice models. Aim 2: Identify genes whose perturbation results in increased efficiency of cellular extravasation and homing to muscles. Using a human myogenic cell line expressing the molecular marker green fluorescence protein (GFP), the project will separately transduce genome-wide libraries to induce gene knock-out or over- expression. Gene-edited cells will be intravenously injected into DBA/2J-mdx mice, a well characterized model of human DMD. Muscles will be harvested and digested for cell culture expansion and selection of human cells that have successfully extravasated from the circulatory system. These ?engraftable? cells will be expanded in culture and sequenced to identify their gene modifications. Milestone: The project will generate a validated list of 20 genes/pathways involved in increased cell extravasation and muscle homing. The findings will be used in a Phase II STTR to test efficacy in mice models. This is a fundamentally novel approach to identify genes that promote muscle recolonization and growth to treat DMD regardless of disease mutation. Successful 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.
