Variant-agnostic modulation of gene expression to identify novel treatments for genetic neuromuscular disorders

Abstract

Genetic neuromuscular disorders (NMDs) that predominantly affect muscle tissue are individually rare but cumulatively are responsible for significant clinical and economic burdens on patients and healthcare systems. Few effective, targeted therapies exist outside of the Duchenne muscular dystrophy (DMD) landscape, highlighting an urgent need for further research into therapeutic options to improve patient outcomes. The clustered regularly interspaced short palindromic repeats (CRISPR) revolution presents new avenues to address this challenge. However, many genetic NMDs are associated with multiple pathogenic variants across various genes, limiting the feasibility of harnessing conventional CRISPR-based gene editing as individualised strategies would be required. Consequently, we sought to evaluate the therapeutic applicability of variant-agnostic CRISPR-based methods. Numerous NMDs are caused or exacerbated by dysregulation of gene and protein expression. Studies have demonstrated the efficacy of modulating the levels of affected or compensatory proteins through variant-agnostic CRISPR techniques in ameliorating symptoms in disease models. Thus, in this thesis, we investigated the suitability of CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), techniques to increase or repress gene expression respectively, as means of altering levels of a suite of previously untested NMD-associated genes. For each gene, we designed five guide RNAs mapping to the promoter, cloned them individually in CRISPRa/i vectors, and evaluated the transcriptional response to each construct via quantitative PCR (qPCR). Among the five genes tested in this pilot approach, we identified that TTN (titin), implicated in titinopathies, was responsive to CRISPRa, producing robust upregulation at the mRNA level in the HEK293 cell line, immortalised human myoblasts, and primary human myoblasts. In an attempt to assess this response at the protein level, we engineered a myoblast cell line that co-expressed EGFP with titin and demonstrated that CRISPRa-mediated TTN upregulation corresponded with a modest increase in EGFP fluorescence. Subsequently, we developed two titinopathy patient variant cell models in which we validated the efficacy of TTN CRISPRa at the mRNA level, with our data suggesting that CRISPRa for TTN may also be effective in patients’ cells. Following further validation of this strategy in vitro, we aim in the future to progress to in vivo disease models. If CRISPRa-mediated upregulation of titin levels ameliorates symptoms in these in vivo disease models, this could establish the groundwork for the development of a variant-independent, much-needed therapeutic strategy for patients with titinopathies.