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

HACD1 缺陷的胚胎斑马鱼模型可替代哺乳动物进行先天性肌病和脂质组学研究

• 批准号: 2749044
• 金额: --
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2749044
• 关键词: Embryonic; zebrafish; models; HACD1; deficiency

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.

编码3-羟酰基-Co-A脂肪酶1（HACD 1）的基因突变可导致人类、犬和小鼠的常染色体隐性遗传性先天性肌病。在狗中，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，人们选择性地繁殖了这些品系，以产生受影响的狗的群体用于研究。最近在人类中描述了HACD 1缺陷型先天性肌病，并且还开发了转基因小鼠品系-所有这些都具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，运动耐量差，肌肉质量减少和进食困难。没有治疗方法，疾病机制知之甚少。HACD 1酶在发育和成熟的肌肉中特异性表达，并且被认为在脂质生物合成中很重要。研究已经记录了正常肌肉功能所需的肌肉生长、发育以及小管网状和线粒体膜系统的修复和维护方面的缺陷。细胞模型缺乏成熟的有组织的膜系统和现有的动物模型在狗和小鼠有缺点，至少从3Rs的角度来看，我们以前的工作已经确定了斑马鱼相当于HACD 1和证实HACD 1的表达在发育中的肌肉是类似的哺乳动物，引入HACD 1突变到胚胎斑马鱼，并证明他们显示肌肉异常复制那些在受影响的狗和人类。我们正在使用CRISPR-Cas9基因组编辑开发和表征胚胎斑马鱼中Hacd 1缺陷的3种新模型：轮廓分明的模特儿（注射Cas9和3种结合hacd 1基因不同部分的向导的混合物）以及两种携带遗传缺陷的突变株系。（即繁殖动物不受影响），分别影响hacd 1基因外显子1和外显子6的酶活性位点的起始密码子。然后将在受精后5天的预保护胚胎中研究突变对肌肉发育和功能的表型和影响。此外，我们还将评估肌肉发育过程中的脂肪酸水平以及Hacd 1缺乏对脂质组的影响，并展示该模型在包括HACD酶和脂质替代在内的治疗试验中的应用。模型、经验和进一步工作所需的技术将在曼彻斯特大学和利物浦大学的水族馆中建立，并提供给该领域的其他研究人员。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原则的研究应用，并且是用于研究肌肉发育和疾病（包括一些其他先天性肌病）的完善的实验系统。斑马鱼经历快速的肌肉发育，在母体外发育，易于注射进行遗传操作，易于成像。它们作为一个封闭的系统存在，直到5dpf开始进食时-因此它们不受外部因素的影响，例如细胞等培养基的差异或哺乳动物通过胎盘的母体输送营养。hacd 1突变斑马鱼因此将是一个主要的，直接的，本研究的结果将减少并部分取代哺乳动物模型的使用，这是一个很好的解决方法，同时允许我们回答使用细胞和哺乳动物模型无法轻易探索的问题。这项工作旨在回答一个基本的生物学问题，并深入了解脂质在肌肉中的作用。这将提高对HACD 1-CNM疾病机制的理解，这是未来开发治疗策略的关键一步，最终可能使狗和人类受益。