Gene therapy for rare genetic disease Keratitis-Ichthyosis-Deafness Syndrome - Correction of Faults of the Disease-Associated Gene GJB2 by Prime Gene

角膜炎-鱼鳞病-耳聋（KID）综合征是一种罕见的常染色体显性遗传疾病，86%的病例由复发性突变引起：c。148 G>A，其编码蛋白质连接蛋白26。它是一种影响表皮、角膜上皮和内耳的外胚层疾病，导致角膜炎症、听力损失和鱼鳞病的临床症状。此外，由于鳞状细胞癌的发展，20-40岁的KID患者的预期寿命减少了20%。KID综合征目前无法治疗;因此，开发针对这种疾病的新疗法至关重要。通过引物编辑技术纠正GJB 2突变具有潜力。与替代基因编辑选项相比，例如具有相对较低编辑效率的核酸酶介导的同源定向修复和需要距离靶DNA序列约10个碱基对的前间隔区邻近基序（PAM）的碱基编辑技术，新的引物编辑技术具有的优点是，可以从PAM安装超过30个碱基对的点突变，在这个项目中，我将开发针对热突变c的主要基因编辑平台。KID综合征中的148 G>A。我将设计并克隆突变c的pegRNA。148 G>A插入载体pAAC-PE 2-N中。该载体与载体pAAVPE 2-C一起将通过AAV病毒系统递送到携带c.148G>A的患者细胞中。为了确定基因编辑和基因编辑脱靶的频率，将应用桑格测序、Edit-R分析和下一代测序。此外，连接蛋白26的表达和功能恢复将被进一步检查。最后，这种治疗策略将在魏立迪博士的小组新开发的KID疾病小鼠模型中进行测试。这将揭示基因治疗这种疾病的可行性。

Keratitis-Ichthyosis-Deafness (KID) syndrome is a rare autosomal dominant caused in 86% of cases by a recurrent mutation: c. 148G>A in the gene GJB2, which encodes the protein connexin 26. It is an ectodermal disorder that affects the epidermis, corneal epithelium, and inner ear, which lead to the clinical symptoms of corneal inflammation, hearing loss and ichthyosis. Furthermore, the life expectancy of KID patients aged between 20-40 is reduced by 20% due to the development of squamous cell carcinoma.KID syndrome is currently untreatable; therefore, it is vital to develop a new therapy for this disease.There is a potential in correcting mutations in GJB2 via primer edit technology. Compared to alternative gene editing options such as nuclease-mediated homology-directed repair which has relatively low editing efficiency and base-editing technologies which requires protospacer-adjacent motif (PAM) to be around 10 base pairs from the target DNA sequence, the novel prime editing technology has the advantage that can install point mutations over 30 base pairs from the PAM which offer a greater degree of flexibility when correcting, inserting, or deleting >20 nucleotides in a precise and targeted manner.In this project, I will develop the prime gene editing platform for the hot mutation c. 148G>A in KID syndrome. I will design and clone pegRNAs for the mutation c. 148G>A into the vector pAAC-PE2-N. This vector alongside the vector pAAVPE2-C will be delivered into patient cells harboring the c.148G>A by AAV viral system. To determine the frequencies of gene editing and off-target of the gene editing, Sanger sequencing, Edit-R analysis and next generation sequencing will be applied. In addition, the expression and functional recovery of connexin 26 will be further examined. Finally, this therapy strategy will be tested in a KID disease mouse model which was newly developed by Dr Wei Li-Di's group. This will reveal the feasibility of the gene therapy for this condition.