Targeted gene delivery and activation by ultrasound

描述(申请人提供)：基因治疗的成功关键依赖于新技术的开发，这些新技术可以用于产生部位特异性基因传递，而不会引起全身毒性和免疫原性反应，以及调节体内转基因表达的能力。尽管过去进行了广泛的努力，但基因治疗中的这些主要障碍仍然存在。这项提议旨在开发新的超声系统，通过提供新的技术来克服这些障碍：1)将基因定向输送到体内器官；2)体内转基因表达的时空调节。在初步研究中，我们已经证明了利用体外培养的HeLa细胞和碎石器冲击波(LSW)介导的猪肾脏部位特异性基因转移在体外进行基因转移的可行性。我们还表明，在热休克蛋白(HSP)70B启动子的控制下，超声波可以作为一种简单方便的物理手段来激活转基因表达。该提案中概述的工作将是对这些初步研究的重大扩展，并将包括一项多学科的全面调查，将创新的工程方法与细胞和分子生物学的现代技术以及动物实验相结合。我们的具体目标是：1.开发新型的超声系统，用于靶向基因的传递和激活。2.新型超声系统在水和组织模体中产生的声场和相关空化气泡动力学的物理特征。3.体外超声基因传递条件的优化。4.猪肾超声靶向基因传递；5.超声调控的骨骼肌基因传递和激活--慢性肾衰竭大鼠促红细胞生成素(EPO)基因治疗的新途径。由于超声波可以深入组织并聚焦于体内特定的器官，因此在体内超声靶向基因传递和激活的前景非常诱人。这种用于基因传递和激活的通用物理方法可能对包括癌症、心血管疾病和各种肾脏疾病(多囊肾、肾癌、急性肾小球肾炎和慢性间质性疾病)在内的广泛疾病的分子治疗具有重要价值。

DESCRIPTION (provided by applicant): The success of gene therapy relies critically on the development of new technologies that can be used to produce site-specific gene delivery without causing systemic toxicity and immunogenic response, as well as the ability to regulate transgene expression in vivo. Despite extensive efforts in the past, these primary obstacles in gene therapy still exist. This proposal aims to develop novel ultrasound systems that have the potential to overcome these obstacles by providing new techniques for 1) targeted gene delivery to internal organs and 2) spatial and temporal regulation of transgene expression in vivo. In preliminary studies, we have already demonstrated the feasibility of ultrasound-mediated gene transfer in vitro using cultured HeLa cells and lithotripter shock wave (LSW)-mediated site-specific gene delivery in vivo in porcine kidney. We have also shown that ultrasound can be used as a simple and convenient physical means to activate transgene expression under the control of heat shock protein (hsp) 70B promoter. The work outlined in this proposal will be a significant expansion of these preliminary studies and will encompass a multidisciplinary and comprehensive investigation combining innovative engineering approaches with modem techniques in cell and molecular biology, and animal experimentations. Our specific aims are: 1. Development of novel ultrasound systems for targeted gene delivery and activation. 2. Physical characterization of the acoustic fields and associated cavitation bubble dynamics produced by the novel ultrasound systems both in water and in tissue phantoms. 3. Optimization of ultrasound-mediated gene delivery in vitro. 4. Ultrasound-targeted gene delivery in porcine kidney, and 5. Ultrasound-regulated gene delivery and activation in skeletal muscle -a novel approach for erythropoietin (Epo) gene therapy in a rat model with chronic renal failure. Because ultrasound can penetrate deep into tissue and be focused on specific organs of the body, the prospect for ultrasound-targeted gene delivery and activation in vivo is very appealing. Such a versatile physical method for gene delivery and activation could be of great value for molecular therapies of a wide range of diseases, including cancer, cardiovascular disease, and various renal disorders (polycystic kidney, renal cancer, acute glomerulonephritis, and chronic interstitial disease).