PILOT STUDY--GENE THERAPY OF CYSTIC FIBROSIS PANCREATIC DISEASE

这一试点项目提案中所包含的研究旨在 积累对开发动物模型有用的数据，以及， 最终，通过调查，为未来的临床试验提供临床前数据 优化胰腺基因转移的策略。胰腺- 定向基因转移是一种潜在的有用的治疗策略。 许多疾病，包括囊性纤维化、糖尿病、癌症和 异基因和异种移植的免疫调节。二 将使用的方法是：使用腺病毒进行体内基因转移，以及在 利用逆转录病毒进行活体基因转移。会注意保险箱的 将外来遗传物质送入胰腺，作为严重急性 胰腺炎是任何胰腺操作的主要问题。这个 拟议的研究将生理学研究与进展相结合 细胞和分子生物学，这一方法将产生显著的 基因治疗在胰腺疾病中应用的新见解 囊性纤维化的表现以及其他人类疾病。这个 有待检验的假设包括：1)腺病毒可成功用于 体内将外源遗传物质导入大鼠胰腺细胞。 1b)体内基因转移可以安全地进行，而一个主要的并发症， 胰腺炎，可以通过药物预防在治疗前减轻 病毒输注。逆转录病毒可以用来靶向特定的细胞 体内基因转移模型中胰腺的种群；2) 多肽生长因子可用于增加胰腺细胞 扩散并通过这样做增加逆转录病毒的比率 转导。将用来检验上述内容的具体目标 假设是：1)优化用腺病毒进行体内基因转移 含有细菌β-半乳糖苷酶的基因。1b)用来治疗老鼠 多种药理药物(如奥曲肽、长效 生长抑素或蛋白水解酶抑制剂的类似物)或饮食 预防或减轻体内基因中显著胰腺炎的治疗方案 2a)选择性地分离胰腺细胞群体 (胰岛、小叶、腺泡细胞)，并用逆转录病毒转导它们 在体外含有细菌β-半乳糖苷酶基因。；2b)至 将胰岛、小叶和腺泡暴露于转化生长因子α、胰岛素和 高血糖素，评估增殖反应和基因转移率。 各种细胞和分子生物学技术将被用于 这些实验包括Southern印迹分析、氚胸苷 放射自显影、组织化学染色、细胞和组织培养 病毒传播。为提高效率，将需要多个内核 这些实验的性能，包括向量核心、 形态核心和动物模型核心。

The studies contained in this pilot project proposal are designed to accumulate data useful in the development of animal models, and, ultimately, pre-clinical data for future clinical trials by investigating strategies for optimizing gene transfer into the pancreas. Pancreas- directed gene transfer is a potentially useful strategy for the treatment of a number of diseases, including cystic fibrosis, diabetes, cancer, and immunologic modulation of allogeneic and xenogeneic transplants. Two approaches will be used: in vivo gene transfer using adenoviruses, and in vivo gene transfer using retroviruses. Attention will be given to the safe delivery of foreign genetic material into the pancreas, as severe acute pancreatitis is a major concern with any manipulation of the pancreas. The proposed research integrates physiological studies with advances in cellular and molecular biology, an approach that will yield significant new insights for the application of genetic therapy to pancreatic manifestations of cystic fibrosis as well as other human diseases. The hypotheses to be tested include: 1a) Adenoviruses be successfully used to transfer foreign genetic material into cells of the rat pancreas in vivo. 1b) In vivo gene transfer can be done safely, and a major complication, pancreatitis, can be attenuated by pharmacologic prophylaxis prior to virus infusion. 2a) Retroviruses can be used to target specific cell populations of the pancreas in models of in vivo gene transfer; 2b) Peptide growth factors can be used to increase pancreatic cell proliferation and in doing so increase the rate of retroviral transduction. The specific aims which will be used to test the above hypotheses are: 1a) To optimize in vivo gene transfer with adenoviruses containing genes for bacterial beta-galactosidase. 1b) To treat rats with a variety of pharmacologic agents (such as octreotide, a long-acting analog of somatostatin or trasylol, a protease inhibitor) or dietary regimens to prevent or attenuate significant pancreatitis in in vivo gene transfer; 2a) To selectively isolate populations of pancreatic cells (islets, lobules, acinar cells) and transduce them with retroviruses containing the gene for bacterial beta-galactosidase in vitro. ; 2b) To expose pancreatic islets, lobules and acini to TGFalpha, insulin, and glucagon, assessing the proliferative response and rate of gene transfer. A variety of cell and molecular biological techniques will be used in these experiments, including Southern blot analysis, tritiated thymidine autoradiography, histochemical staining, cell and tissue culture, and virus propagation. A number of cores will be necessary for the efficient performance of these experiments, including the Vector Core, the Morphology Core, and the Animal Models Core.