Targeting Usher syndrome with CRISPR mediated base editing

Usher综合征是一种隐性遗传的遗传性聋盲综合征。有不同类型的Usher综合征表现出不同的症状严重程度：II型是最常见的，患者在耳聋发作后表现出进行性视网膜变性。Usher综合征通常是由USH2A基因点突变引起的，这种突变导致一种名为usherin的蛋白质的缺陷版本。人们认为，迎春素蛋白参与了光感受器的长期维持，光感受器参与了视网膜中的光检测。这种DPhil的目的是使用一种称为碱基编辑的技术来纠正USH2A基因的个别点突变。引导RNA将碱基编辑装置引导到与点突变相邻的DNA的特定片段，以便酶可以将突变的核苷酸改变为其原始形式，从而恢复视觉敏感度。碱基编辑系统将通过视网膜下注射，通过病毒载体传递给患者。这种给药方法已经在涉及其他遗传性视网膜疾病的人类临床试验中取得了成功。在设计好碱基编辑系统后，我将首先在体外测试其有效性，然后在哈威尔研究所创建的亚瑟综合征小鼠模型上进行测试。然后可以进行实验，以调查是否发生了任何生理救援。重要的是优化基本编辑系统，以减少任何不必要的副作用，并最大限度地提高其效率。DPhil所涉及的工作有可能对亚瑟综合征患者的生活产生重大影响。此外，如果研究成功，它将为未来旨在逆转视网膜内外有害基因突变的碱基编辑实验提供良好的基础。

Usher syndrome is an hereditary deaf-blindness syndrome inherited in a recessive manner. There are different types of Usher syndrome displaying a range in the severity of symptoms: Type II is the most common and patients demonstrate progressive retinal degeneration occurring after the onset of deafness. Usher syndrome is commonly caused by point mutations in the USH2A gene that result in a defective version of a protein called usherin. It is thought that the usherin protein is involved in long term maintenance of photoreceptors, the cells involved in light detection in the retina.The aim of this DPhil is to correct individual point mutations in the USH2A gene using a technique called base editing. A guide RNA directs the base editing apparatus to a specific section of DNA adjacent to the point mutation so that an enzyme can change the mutated nucleotide to its original form, thus restoring visual acuity. The base editing system will be delivered to patients inside a viral vector via an injection underneath the retina. This method of delivery has already had success in human clinical trials involving other genetic retinal diseases. After the base editing system has been designed, I will begin by testing its efficacy in vitro and will then progress to testing it on a mouse model of Usher syndrome created by the Harwell Institute. Experiments can then be performed to investigate whether any physiological rescue has occurred. It will be important to optimise the base editing system to reduce any unwanted side effects and to maximise its efficiency. The work involved in this DPhil has the potential to make a significant impact on the lives of patients with Usher syndrome. Additionally, if the study is successful it will provide an excellent basis for future base editing experiments directed at reversing harmful genetic mutations both inside and outside the retina.