GENE TRANSFER TO CONNECTIVE TISSUES--RABBIT KNEE MODEL

基因转移至结缔组织--兔膝模型

• 批准号: 6239053
• 负责人: CHRISTOPHER Howard EVANS
• 金额: $ 12.71万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-15 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6239053
• 关键词: Herpesviridae; adeno associated virus group; biological models; cytokine receptors; fibroblasts; gene expression; gene therapy; genetic markers; inflammation; inhibitor /antagonist; interleukin 1; joint disorder; knee; laboratory rabbit; liposomes; model design /development; synovial fluid; tissue /cell culture; transfection /expression vector

Chronic inflammation and tissue breakdown are the major pathophysiological lesions in many connective tissue diseases. Treatment of these conditions remains unsatisfactory, partly because existing drugs are intrinsically inadequate and partly because these drugs cannot be specifically targeted to sites of disease. Furthermore, present therapeutic strategies fail to address the chronicity of these diseases. We propose to use gene therapy as a radical new approach to treating such conditions. In this approach, a gene, or gene(s) coding for protein(s) with anti-inflammatory or anti-erosive properties are stably introduced into resident, mesenchymal, connective tissue cells at the disease site. This procedure permits selective, local synthesis of the therapeutic agent(s) just at sites of disease activity. In this way, systemic ef- fects are minimized. Furthermore, stable incorporation and expression of the transduced genes ensures prolonged synthesis of their products, as is necessary for the treatment of chronic diseases. These ideas will be tested experimentally in a step-wise fashion with the rabbit knee as a model system. In short-term experiments, we have successfully transplanted lac Z+, neo+ synovial fibroblasts into the knee joint of recipient rabbits, where they survive for at least two weeks, and possibly as long as three months. During the present proposal, we will further refine this ex vivo procedure, attempting to achieve per- manent gene transfer to the synovium for the lifetime of the animal. As an alternative, in vivo approach, the possibility of direct, in situ transfection of synovial cells will be evaluated with emphasis on adenoassociated virus, herpes virus and liposomes as vectors. Having thus established a system of gene delivery to synovial fibroblasts, this delivery method will be used to antagonize the actions of interleukin-1 (IL-1), a cytokine which produces inflammation and tissue destruction. To do this, a gene coding for an IL-1 receptor antagonist (IRAP) will be transduced into the synovial cells lining the knee joint. The in situ production of IRAP will be confirmed, steps taken to maximize its in vivo expression. The ability of endogenously synthesized IRAP to block the actions of intraarticularly administered IL-1 will be tested. Finally, the therapeutic effect of transducing IRAP genes into synovium will be tested in an experimental model of articular inflammation and tissue destruction.

慢性炎症和组织破坏是主要原因 许多结缔组织疾病的病理生理病变。 治疗 其中一些情况仍然不能令人满意，部分原因是现有药物 本质上是不够的，部分原因是这些药物不能 专门针对疾病部位。 此外，目前 治疗策略无法解决这些疾病的慢性问题。 我们建议使用基因疗法作为治疗此类疾病的全新方法 状况。 在这种方法中，一个或多个编码蛋白质的基因 具有抗炎或抗侵蚀特性的稳定引入 进入疾病部位的常驻细胞、间充质细胞、结缔组织细胞。 该程序允许选择性、局部合成治疗药物 药剂仅位于疾病活动部位。 这样，系统性的效果 影响最小化。 此外，稳定掺入和表达 转导的基因确保了其产品的长时间合成， 治疗慢性疾病所必需的。 这些想法将以逐步的方式进行实验测试 兔膝作为模型系统。 在短期实验中，我们有 成功将lac Z+、neo+滑膜成纤维细胞移植到膝关节 受体兔子的关节，它们可以存活至少两周， 并且可能长达三个月。 在本提案中，我们 将进一步完善这一离体程序，试图实现 在动物的一生中，基因持续转移到滑膜。 作为 另一种体内方法，直接原位的可能性 评估滑膜细胞转染的重点是 腺相关病毒、疱疹病毒和脂质体作为载体。 由此建立了将基因递送至滑膜的系统 成纤维细胞，这种递送方法将用于拮抗作用 白细胞介素-1 (IL-1)，一种产生炎症和炎症的细胞因子 组织破坏。 为此，需要编码 IL-1 受体的基因 拮抗剂（IRAP）将被转导至滑膜细胞内 膝关节。 IRAP的原位生产将得到确认，步骤 采取最大化其体内表达。 内生能力 合成的 IRAP 可阻断关节内注射的作用 将测试 IL-1。 最后，转导IRAP的治疗效果 基因进入滑膜将在关节实验模型中进行测试 炎症和组织破坏。