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We have been making good progress in our development of methods to generate pure, functional, mature human muscle tissue in mice. Our approach is based on our published method (Sakellariou et al., Skeletal Muscle 6:4 ), in which we start with immunodeficient NRG mice, X-irradiate their hind limbs, intoxicate the Tibialis anterior (TA) muscle to kill off the murine muscle tissue, and then inject the TA compartment with immortalized human myogenic precursor cells (hMPCs) from healthy donors or individuals with FSHD. Our best grafts to date, which we have generated by adding a regular regimen of electrical stimulation of the engrafted limbs to our protocol (see Sakellariou et al.) have ~1400 muscle fibers that are >98% of human origin. (A healthy mouse TA muscle has ~2500 muscle fibers). These muscles are innervated by murine motor neurons, are contractile, and are fully differentiated. Thus, we have so far been able to generate mice with mature, functional human muscles that are almost 60% the size of the murine muscle that they are replacing.
We are continuing to improve our methods in order to deal with one ongoing problem – variability – and a second problem that has arisen in the past 1.5 years – the lack of availability of the toxin that we have used to intoxicate the murine muscle before we inject the hMPCs. We are also adapting our methods to generate human muscle fibers that can be cultured for studies at the single fiber level in vitro. This should greatly facilitate cell biological and physiological studies of the changes that occur in FSHD. We have been working closely with Fulcrum Therapeutics (Cambridge MA) to address these challenges. Fulcrum has provided expertise and resources that we would otherwise have been hard pressed to access.
Deliverables and Progress in their Completion
Aim (i): Optimized methods for creating xenografts from isogenic control cells: 1 year (months 1-12).
These experiments are proceeding well and we anticipate significant progress by the end of year 1.
Aim (ii): Apply optimized methods to isogenic, D4Z4 contracted cells: 6 months (months 10-16)
We are ahead of schedule in beginning these studies.
Aim (iii): Assay biomarkers and contraction of FSHD and control grafts; initiate testing of therapeutic drug: 1 year (months 8-20 for controls; months 12-24 for FSHD)
We will be starting these studies with Fulcrum shortly and before the end of the funding period anticipate assessing compounds from Fulcrum for potential further development into FSHD patients.