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

罕见遗传病角膜炎-鱼鳞病-耳聋综合征的基因治疗 - Prime Gene 纠正疾病相关基因 GJB2 的缺陷

• 批准号: 2577719
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2577719
• 关键词: Gene; therapy; rare; genetic; disease

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.

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病，86%的病例是由编码连接蛋白26的基因GJB2中的一个经常性突变：C.148G和GT；A引起的。它是一种外胚层疾病，影响表皮、角膜上皮和内耳，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岁的儿童患者的预期寿命减少了20%。KID综合征目前无法治疗；因此，开发一种新的治疗方法至关重要。通过引物编辑技术纠正GJB2的突变是有潜力的。与其他基因编辑方法相比，例如编辑效率相对较低的核酸酶介导的同源定向修复和碱基编辑技术要求目标DNA序列中的Protspacer-相邻基序(PAM)约为10个碱基对，新的Prib编辑技术的优势是可以从PAM安装30多个碱基对的点突变，这为精确和有针对性地纠正、插入或删除&gt；20个核苷酸提供了更大的灵活性。在这个项目中，我将为儿童综合征中的热门突变c.148g&gt；A开发一个基础基因编辑平台。我将设计并克隆突变c.148G和gt；A的pegRNAs到载体PAAC-PE2-N中。该载体将与载体pAAVPE2-C一起通过AAV病毒系统进入携带C.148G和GT；A的患者细胞。为了确定基因编辑的频率和基因编辑的非靶标，将应用桑格测序、编辑-R分析和下一代测序。此外，还将进一步研究缝隙连接蛋白26的表达和功能恢复。最后，这种治疗策略将在魏丽-迪博士的团队新开发的儿童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。