Adeno-associated virus for IGF-I cartilage gene therapy

描述(申请人提供)：腺相关病毒是一组新兴的病毒载体之一，可能为治疗关节疾病的治疗产品的病毒输送提供新的热情。腺病毒和逆转录病毒虽然在转导效率或转基因整合方面具有优势，但都有生产、安全或基因持久性的缺点，限制了它们在软骨修复和关节炎治疗中的应用。这项建议将允许候选人在AAV载体构建中使用当代技术来生成AAV-IGF-I载体，测试该复合材料对生长因子基因编码序列的有效整合和延长表达，并最终证明其对软骨细胞和滑膜细胞代谢和增殖的刺激作用。研究人员将使用当代的三质粒方法进行繁殖，并将使用实时聚合酶链式反应和微阵列技术检查目标细胞中的基因持久性。效应细胞的反应将通过放射免疫分析来测量，蛋白质的上调将通过染料结合和免疫细胞化学技术来评估。发起人在病媒开发方面拥有丰富的经验，他的实验室和同事都是AAV研究和生产的领导者。候选人将参与载体的生产，将自己的分子生物学背景整合到转基因构建中，并在这12个月的学习中开发可靠的载体克隆和生产熟练程度。

DESCRIPTION (provided by applicant): Adeno-associated virus is one of an emerging group of viral vectors that may provide renewed enthusiasm for viral delivery of therapeutic products to treat joint disease. Adenovirus and retrovirus, while providing advantages in either transduction efficiency or transgene integration, both have either production, safety, or gene persistence draw-backs that limit their application in cartilage repair and arthritis treatment. This proposal will allow the candidate to use contemporary techniques in AAV vector construction to generate an AAV-IGF-I vector, to test the composite for effective integration and prolonged expression of the growth factor gene coding sequence, and finally to document the stimulatory effects it has on chondrocyte and synoviocyte metabolism and proliferation. The investigators will use contemporary triple-plasmid methods for propagation, and the examination of gene persistence in target cells will use real-time PCR and microarray techniques. Effector cell responses will be gauged by radioimmunological assay, and the protein up-regulation will be assessed by dye-binding and immunocytochemical techniques. The sponsor has extensive experience in vector development, and his laboratory and colleagues are leaders in AAV research and production. The candidate will participate in the production of vectors, integrate his own molecular biology background into transgene construction, and develop reliable vector cloning and production proficiency during this 12-month study.