Embryonic zebrafish models of HACD1-deficiency to replace mammals in congenital myopathy and lipidomic research

Mutations in the gene encoding 3-hydroxyacyl-Co-A dehydratase 1 (HACD1) cause autosomal recessive congenital myopathies in humans, dogs and mice. In dogs, the naturally-occurring condition is widespread in Labradors and lines have been selectively bred over the last 15 years to produce colonies of affected dogs for research. HACD1-deficient congenital myopathies are more recently described in humans and lines of transgenic mice have also been developed - all share many clinical and pathological features. Patients and affected animals display marked weakness, poor exercise tolerance, reduced muscle mass and difficulties eating. There is no treatment and the disease mechanisms are poorly understood. The HACD1 enzyme is specifically expressed in developing and mature muscles and is thought to be important in lipid biosynthesis. Research has documented defects in muscle growth, development and repair and maintenance of tubuloreticular and mitochondrial membrane systems required for normal muscle function. Cellular models lack mature organised membrane systems and existing animal models in dogs and mice have disadvantages not least from a 3Rs perspective.

Our previous work, including from our current NC3Rs funding (training fellowship for Rhiannon Morgan) has identified the zebrafish equivalent of HACD1 and confirmed hacd1 expression in developing muscle is similar to that in mammals, introduced hacd1 mutations into embryonic zebrafish and demonstrated that they display muscle abnormalities that replicate those seen in affected dogs and humans. We are in the process of developing and characterising 3 novel models of Hacd1-deficiency in embryonic zebrafish using CRISPR-Cas9 genome editing: a crispant model (injected with Cas9 and a cocktail of 3 guides that bind in separate parts of the hacd1 gene) and also two mutant lines carrying genetic defects (i.e. breeding animals are not affected) affecting the start codon in exon 1 and in the enzyme active site in exon 6 of the hacd1 gene respectively. The phenotype and impact of the mutations on muscle development and function will then be studied in preprotected embryos up to 5 days post fertilisation. In addition, we will evaluate fatty acid levels during muscle development and the impact of Hacd1 deficiency on the lipidome and demonstrate the use of the model in therapeutic trials including HACD enzyme and lipid replacement.

The models, the experience and techniques required for further work will then be established in the aquaria at the Universities of Manchester and Liverpool and made available to other researchers in the field. We have plans with our collaborators in Paris for therapeutic screens of candidate molecules identified through yeast studies, which will dramatically affect the requirement for mammalian models. The developing zebrafish is advocated for research applications where 3R principles are applied and is a well-established experimental system for the study of muscle development and diseases, including some other congenital myopathies. Zebrafish undergo rapid muscle development, develop outside the mother, are simple to inject for genetic manipulation and easy to image. They exist as a closed system until 5dpf when feeding starts - they are therefore unaffected by external factors such as differences in culture media like cells or maternal delivery of nutrients via the placenta as in mammals.

The hacd1-mutant zebrafish will hence be a major, and immediate, output of this study that will reduce and partially replace use of mammalian models of HACD1-deficiency whilst allowing us to answer questions that cannot easily be explored using cellular and mammalian animal models. This work aims to answer a fundamental biological question and provide insight into the roles of lipids in muscle. This will improve understanding of the disease mechanisms in HACD1-CNM, a critical step for future development of treatment strategies that may ultimately benefit both dogs and humans

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PhD Studentship

Status

Pending start

Institution

University of Liverpool

Grant reference number

NC/W001993/1

Award date

Oct 2022 - Sep 2026

Grant amount

£120,000