A systems approach for upregulating cardiac (fetal) α-actin as a therapeutic strategy for treating skeletal muscle actin diseases

Globally, this thesis research explores upregulation of cardiac alpha-actin (encoded byACTC1) as a therapeutic approach for treating skeletal muscle myopathies caused by mutations of ACTA1, the gene encoding skeletal alpha-actin. Since these diseases are often very severe and currently are untreatable, the findings presented in this body of work have relevant implications both for basic science and for future translational applications.

The primary motivation for this thesis research stems from previous studies that support the notion that ACTC1, a gene predominantly expressed in the heart, may be used as a ‘replacement’ (upregulation) therapy to ‘fix’ (make less severe) skeletal muscle deficiencies in humans that result from ACTA1 mutations. With that focus, I investigated the factors regulating cardiac actin expression in skeletal muscle (and other non-heart tissues), which until now has been poorly understood.

Overall, this body of work involves a multi-pronged approach for investigating potential therapeutic targets and exploring strategies for treating ACTA1-related diseases; specifically by:
(1) the selection of highly diverse animal models that generate much more robust and generalizable results with better translational utility;
(2) the use of sophisticated computational workflows that exploit the best of "exploratory " genomic and genetic techniques; along with
(3) the use of new tools in the field to explored RNA-guided activation by a CRISPR-Cas9-based transcription factor.

Based on these approaches I was able to obtain relevant original data, including identification of a single region (locus) in the mouse genome that is critically involved in controlling Actc1 expression, also known as an expression quantitative trait locus (eQTL).An in-depth survey of Actc1 expression across multiple tissues, strains and ages revealed developmental differences in the control of cardiac actin that mapped to the same eQTL in the gene promoter. Furthermore, proof of principle work showed that the cardiac actin promoter can be activated in human cells using the CRISPR/dCas9 platform, validating this technique as a rational therapeutic approach to pursue for treatments of patients withACTA1 myopathies.

Although the crux of the thesis relates to regulation of cardiac actin (a known modifier of skeletal muscle actin diseases), in Chapter 5 a similar approach was taken to help identify novel modifier genes (different from Actc1). One putative modifier locus was identified, suggesting other genes might exist that are able reduce the severity of the lethal myopathic phenotype associated with skeletal muscle actin deficiency.

Overall, my thesis research harnessed cutting edge resources that are currently being developed at UWA by Prof Grant Morahan and colleagues. For example, the Collaborative Cross (a mouse genetics resource of international significance) along with customized bioinformatic tools were extended and improved as the result of this thesis. One key idea that I tried to impress throughout the writing of this thesis relates to the generalizability of these approaches for treating other diseases. Indeed, the ability to selectively upregulate and modulate the expression of the paralogs of mutated genes is a promising therapeutic approach that can be applied outside the context of skeletal muscle disease.