Using functional readouts from engineering models of innervated skeletal muscle to assess the efficacy of CRISPR-based c9orf72 ALS gene therapies

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PROJECT SUMMARY/ABSTRACT Gene therapies employing CRISPR-Cas9-mediated genetic editing techniques have the potential to cure a wide range of inheritable disorders, including amyotrophic lateral sclerosis (ALS). However, identifying edits capable of neutralizing disease-causing mutations is a pressing issue. A critical bottleneck in the translation of novel gene therapies to clinical trials is a lack of human models capable of producing functional metrics that correlate with patient outcomes and provide predictive data with which to guide subsequent in vivo experiments. For ALS and other neuromuscular disorders, the complexity associated with generating mature and functionally competent neuromuscular junctions (NMJs) in culture with sufficient throughput for screening purposes is a major hindrance to this effort. The development of a multiplexed platform capable of promoting NMJ development across a parallel array of engineered muscle tissues will have a substantial positive impact on advanced therapy development, drug efficacy/toxicity screening, and mechanistic studies of neuronal and NMJ pathophysiology in ALS. Building on the PI’s work as a KL2 scholar, this project seeks to combine optogenetic motor neurons derived from ALS patient induced pluripotent stem cells (iPSCs) with a magnet-based sensing platform for non-invasively detecting engineered muscle contractions to establish a system for real-time, continuous assessment of NMJ functional decline in ALS (Aim 1). Tests with cholinergic synaptic agonists and antagonists, in terms of their ability to alter synaptic communication between cultured muscle and neurons, will be used to demonstrate the suitability of this model for assaying changes in NMJ function in vitro. Once optimized, the described system will be used to investigate multiple gene editing strategies for restoring function in C9orf72-mutant ALS; the most common inheritable form of the disorder (Aim 2). ALS patient iPSC-derived motor neurons subjected to either bi-allelic repeat excision or allele-specific C9orf72 gene inactivation will be compared for their ability to maintain NMJ function over time in co-culture with engineered muscle tissues. The non-invasive nature of our magnetic sensing system enables continuous assessment of muscle performance in response to optogenetically-controlled neuronal activation, thereby enabling longitudinal study of therapeutic efficacy and parallel assessment of multiple tissues subjected to different treatment regimens. Results from these experiments will provide a framework for further preclinical validation of novel therapies targeting peripheral neuropathic diseases as well as data to aid in the selection of which gene editing technique has the best chance of success in C9orf72 ALS patients. Validation of the technologies outlined in this proposal will represent the culmination of work started by the PI as part of the KL2 program and will form the core of the PI’s independent research going forward. Results collected from this R03 funded program will provide valuable preliminary data for the continued development of the PI’s research program and will be central to the success of subsequent federal grant applications.