运用CRISPY系统治疗由于放射治疗引起的唾液腺功能损伤

项目名称： 运用CRISPY系统治疗由于放射治疗引起的唾液腺功能损伤

项目编号： No.81472854

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 王志民

作者单位： 郑州大学

项目金额： 75万元

中文摘要： 每年全球有超过50万的头颈癌新患者。多数患者的放射性疗导致半数以上的患者产生不可逆的腮腺损伤，造成唾液分泌显著减少等症状。本研究利用最新的CRISPY系统，首次运用腺病毒、腺相关病毒。将人水通道渗透蛋白在唾液腺细胞中表达并将其基因重组至细胞基因组，提高细胞膜的渗透性，增加唾液分泌。本研究的前期工作完成了腺病毒及腺相关病毒的构建，验证了腺病毒表达CRISPY系统对细胞基因组进行剪切，建立了小鼠的唾液腺表达模型和小型猪的疾病模型。本研究将在前期工作的基础上，利用两种病毒载体实现在细胞和小鼠体内瞬时和长期表达渗透蛋白；尝试用该双病毒系统治疗唾液腺损伤的小型猪。本研究将会为唾液腺的功能治疗提供新的途径，提高头颈癌患者的生活质量。

中文关键词： 头颈部肿瘤；唾液腺损伤；CRISPY；病毒载体；功能拯救

英文摘要： Each year, more than half million patients develop head and neck cancers worldwide. Typical and efficient treatment for these patients includes irradiation (IR). After IR, about half of all surviving patients experience irreversible damage to salivary glands in the IR field, thus dramatically reduces saliva secretion. As a result, these patients suffer considerable morbidity, including dyaphagia, oral infection, mucositis and frank discomfort (xerostomia), with a marked decline in quality of life. So far, the only available treatment for these xerostomia is minimally effective wetting agents. This absence of conventional therapy prompted our efforts to use gene therapy to increase fluid secretion. In the field of gene therapy, genome modification has laid the basement of functional studies in modern biolody. Scientists have been perusing effective method to manipulate genetic materials in different organisms. Recent work on the regularly interspaced, short palindromic repeats (CRISPR) system have shown its potential function as a new effective tool to direct genome DNA cleavage in culture cells. In this study, we will apply this new technology to deliver human aquaporin 1 (AQP1) gene in salivary gland to restore salivary flow in patients with radiation treatment (RT) damaged salivary gland, and attempt to induce temperary expression of AQP1 and cure the xerostomia by cleavage specific genome DNA and insert the target cDNA of AQP1 in salivary gland cells. In our previous work, we have developed adenovirus (Adv) CRISPY delivery system, also we developed an adeno-associate virus (AAV) system which contains both AQP1 expression cassette and CRISPY homologous recombinate template. Our preliminary data have shown that by using Adv deliver system, we can achieve specific genome editing at two different sites, and the cleavage efficiency is higher than that of sole plasmids induction. Mouse model was established to test target gene expression in salivary gland using Adv and AAV system.In addition, the xerostomia mini pig model was established to validate the corrective treatment for RT-induced salivary hypofunction. In this study, we will insert AQP1 cDNA in salivary gland cell genome using the two virus system, in the mean time, transit expression AQP1. We will try to cure the salivary hypofunction using this system in the well established min pig model. If the mini pig results are positive, it will not only show the potential using of CRISPY technology to cure radiation caused salivary gland damage but also place upon proof-of-principle demonstration of CRISPY technology in curing genetic disorder and virus arise diseases.

英文关键词： head and neck cancer;hypofunction of salivary gland;CRISPY;virus vector;function rescue