运用CRISPY系统治疗由于放射治疗引起的唾液腺功能损伤

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.

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。

本课题经过四年的研究从理论和细胞实验水平上验证了本课题的初步构想，即利用CRISPR系统利用病毒将AQP1重组进入细胞AAVS1位点，来达到既能够短期高效表达AQP1，又能够将其插入特定的基因组位点，从而达到长期表达的效果。同时我们的小鼠和小型化猪的实验结果都证明了其虽然能够在细胞水平达到我们的预期，但是在动物水平还有待提高重组效率。课题组还首次发现了Cas9单个蛋白在细胞内的表达，可以改变包括多个转录因子的多个蛋白的表达变化，相关结果为科研界利用CRISPR系统进行这些相关基因的调控研究，提供了很好的借鉴作用。同时我们的结果也证实通过CRISPR activation系统，加上DNM2融合蛋白，我们可以对AQP1启动子进行去甲基化，从而提高AQP1的表达量，进而提高唾液的分泌，这些结果，为今后增加唾液分泌的基因治疗提供了另外一条新的通路。因此，本课题的实施，培养了实验室人员，发表了文章，更为重要的是为今后进行进一步的研究提供了坚定的基础。