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

编码3-羟基酰基-Co-A脱水酶1(HACD1)的基因突变会导致人类、狗和小鼠的常染色体隐性遗传性先天性肌病。在狗身上，这种自然发生的疾病在拉布拉多犬中很普遍，在过去的15年里，人们选择性地培育了品系，以产生受影响的狗群，用于研究。HACD1缺陷的先天性肌病是最近在人类中被描述的，转基因小鼠也已经被开发出来--所有这些都有许多临床和病理特征。患者和受影响的动物表现出明显的虚弱、运动耐力差、肌肉质量减少和进食困难。目前还没有治疗方法，疾病机制也知之甚少。HACD1酶在发育和成熟的肌肉中特异表达，被认为在脂质生物合成中起重要作用。研究证明，正常肌肉功能所需的小管网状和线粒体膜系统在肌肉生长、发育、修复和维持方面存在缺陷。细胞模型缺乏成熟的有组织的膜系统，现有的狗和鼠动物模型从3Rs的角度来看尤其有缺点。我们之前的工作已经确定了斑马鱼HACD1的等价物，并证实HACD1在发育中的肌肉中的表达与哺乳动物相似，将HACD1突变引入胚胎斑马鱼，并证明它们表现出肌肉异常，复制了受影响的狗和人类的肌肉异常。我们正在利用CRISPR-Cas9基因组编辑技术开发和鉴定3种新的斑马鱼胚胎Hacd1缺乏症模型：一种是脆性模型(注射Cas9和结合在hacd1基因不同部分的3个导引的鸡尾酒)，另一种是携带遗传缺陷的两个突变系(即育种动物不受影响)，分别影响hacd1基因外显子1的起始密码子和hacd1基因外显子6的酶活性部位。这些突变的表型和对肌肉发育和功能的影响将在受精后5天的预保护胚胎中进行研究。此外，我们将评估肌肉发育过程中的脂肪酸水平以及Hacd1缺乏对脂类的影响，并演示该模型在包括HACD酶和脂肪替代在内的治疗试验中的应用。然后，将在曼彻斯特大学和利物浦大学的水族馆建立进一步工作所需的模型、经验和技术，并向该领域的其他研究人员提供。我们与巴黎的合作者计划对通过酵母研究确定的候选分子进行治疗性筛选，这将极大地影响对哺乳动物模型的需求。发展中的斑马鱼被提倡用于应用3R原理的研究应用，是一个成熟的实验系统，用于研究肌肉发育和疾病，包括其他一些先天性肌病。斑马鱼肌肉发育迅速，在母体外发育，易于注射进行遗传操作，易于成像。因此，hacd1突变斑马鱼将成为这项研究的主要和立即的成果，它将减少并部分取代哺乳动物HACD1缺乏症模型的使用，同时使我们能够回答不容易使用细胞和哺乳动物动物模型来探索的问题。这项工作旨在回答一个基本的生物学问题，并提供对脂肪在肌肉中的作用的洞察。这将提高对HACD1-CNM疾病机制的理解，这是未来开发可能最终造福于狗和人类的治疗策略的关键一步。

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 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.