The TTN gene encodes the largest protein in nature, titin, a critical scaffolding component that provides elasticity and stability to skeletal and cardiac muscle. Pathogenic TTN variants cause a range of skeletal muscle disorders and cardiomyopathies collectively termed ‘titinopathies’, many of which result in significant disability and can be life-limiting. Currently, no effective titinopathy treatments exist. Over 130 pathogenic TTN variants have been reported in the literature to date, restricting the feasibility of developing mutation-corrective approaches. However, comprehensive analysis of these variants suggests that decreased titin expression contributes to the pathology in titinopathy patients.
Thus, the goal of this study was to investigate the use of CRISPR activation (CRISPRa), a technique employed to activate and increase gene expression in a targeted manner, as means of boosting titin expression and thereby improving titinopathy clinical outcomes. To achieve this, we designed guide RNAs mapping to the TTN promoter in CRISPRa vectors encoding deactivated Cas9 flanked by VP64, a strong transcriptional activator. We have identified two guide RNAs that synergistically induce potent upregulation of TTN in the HEK293 cell line, immortalised human myoblasts, and primary human myoblasts. Following further validation of this upregulation strategy in vitro, we will progress to in vivo disease models.
The in vivo efficacy of CRISPRa has previously been demonstrated in a mouse model of congenital muscular dystrophy type 1A, in which dystrophic symptoms were reversed upon upregulation of a compensatory disease-modifying gene. If CRISPRa-mediated restoration of titin levels ameliorates symptoms in our disease models, this will establish the groundwork for the development of a mutation-agnostic, much-needed therapeutic strategy for patients with titinopathies.