Poster Presentation 44th Lorne Genome Conference 2023

Investigation of Novel Gene Therapies for ACTA1-Related Nemaline Myopathy (#201)

Christina M Vo 1 2 , Gavin V Monahan 1 2 , Joshua S Clayton 1 2 , David Mowat 3 , Robert Smith 1 2 , Carolin Scriba 1 2 4 , Kristen J Nowak 2 5 6 , Nigel G Laing 1 2 , Gina Ravenscroft 1 2 , Rhonda L Taylor 1 2
  1. Harry Perkins Institute of Medical Research- UWA, Nedlands, WA, Australia
  2. Centre for Medical Research, University of Western Australia, QEII Medical Centre, Nedlands, WA, Australia
  3. Department of Medical Genetics, Sydney Children's Hospital, Sydney, NSW, Australia
  4. Neurogenetics Laboratory, Department of DIagnostic Genomics, PathWest, PP Block, QEII Medical Centre, Nedlands, WA, Australia
  5. Department of Pediatrics, University of Melbourne, Melbourne, VIC, Australia
  6. Office of Population Health Genomics, Public and Aboriginal Health Division, Western Australian Department of Health, East Perth, WA, Australia

The skeletal muscle α-actin gene (ACTA1) encodes a critical protein required for muscle contraction. Pathogenic variants in ACTA1 are associated with several congenital myopathies including nemaline myopathy (NEM). Heterozygous ACTA1 variants typically act via a dominant negative ‘poison protein’ mechanism, where the mutant protein interferes with the normal function of the wildtype protein. ACTA1 myopathies are usually severe, often leading to neonatal death. Milder forms of the disease have also been identified, with cases linked to reduced abundance of the toxic protein, including via low-level ACTA1 mosaicism.  

Here, we describe a mother presenting with mild NEM, and her stillborn child with severe NEM. Whole exome sequencing identified the same heterozygous ACTA1 missense variant (c.115C>G, p.Arg39Gly), however, the mother was found to be somatic mosaic with the variant present in only 6% of reads. The striking difference in disease severity between the mother and child exemplifies the link between muscle function and poison protein dosage in ACTA1 disease. To this end, we have developed a genetic treatment aimed at reducing poison protein abundance.  

Our therapeutic approach involves allele-specific CRISPR-Cas9 editing to remove a toxic ACTA1 allele, leaving a single functional ACTA1 allele. The treatment was tested on two dominant ACTA1 myopathy patient-derived induced pluripotent stem cell (iPSCs) lines. Edited lines with varying levels of mutant ACTA1 deletion (0-100%) were differentiated into skeletal muscle. Using allele-selective ddPCR assays, we showed that there is a strong concordance between editing efficiency and mutant ACTA1 transcript levels. Interestingly, following editing, we saw upregulation of the wildtype ACTA1 transcript suggesting concomitant upregulation of the wildtype allele in response to mutant allele deletion.  

This study highlights the link between ACTA1 mutant protein levels and disease severity and shows potential feasibility of an allele-selective deletion approach for ACTA1 myopathies with further applications in a multitude of genetic diseases.