Vector Identification and Gene Delivery Approach in Pigs

超出所提供的空间。本提案的目标是开发一种分子性肌力变性的新疗法，特别是病毒介导的钙调节蛋白SERCA 2a（肌浆网ca2¿atp酶）的心肌递送，以恢复心力衰竭的大型动物的功能并提高生存率，同时不恶化能量参数。充血性心力衰竭（CHF）是一个巨大的临床问题，需要有效的治疗方法。尽管传统的治疗方法取得了进展，包括药物治疗、心肌血运重建术、机械辅助装置和移植，但CHF仍然是世界范围内的主要死亡原因。因此，一种旨在降低CHF发病率和死亡率并改善数百万患者生活质量的新疗法尤其具有吸引力。心脏基因疗法在早期动物研究中显示出前景，并有助于治疗心力衰竭。细胞内钙处理缺陷被认为是人类和实验CHF的关键异常。在人类和动物模型中，衰竭心脏松弛期间肌浆网（SR）缺乏Ca2¿摄取与SR Ca2+ atp酶（SERCA2a）的表达和活性降低有关。我们在过去五年的初步工作表明，1)基因转移是将SERCA2a基因引入体外和体内肌细胞的有效手段，2)在啮齿动物CHF模型中，增加SERCA2a的表达可以恢复收缩性并使细胞内钙循环正常化。我们现在将实验从啮齿类动物扩展到猪模型。我们的目标是1)确定病毒载体在猪心脏分离的心肌细胞中的转导效率；2)测定AAV载体和El-E4缺失重组腺病毒在猪体内基因转移后的转导效率；3)测试人脑近端利钠（hBNP）启动子（相对于转录起始位点-408至+100）在压力过载和缺血诱导下的能力。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. The goal of this proposal is to develop a novel therapy for molecular inotropy, specifically, viral-mediated myocardial delivery of the calcium regulatory protein SERCA 2a (sarcoplasmic reticulum Ca 2¿ATPase) to large animals exhibiting heart failure in an attempt to restore function and improve survival without worsening energetic parameters. Congestive heart failure (CHF) represents an enormous clinical problem demanding effective therapeutic approaches. Despite advances in traditional approaches to its treatment, including pharmacologic management, myocardial revascularization, mechanical assist devices, and transplantation, CHF remains a leading cause of death worldwide. Therefore, a novel therapy aimed at decreasing the morbidity and mortality of CHF and improving the quality of life for millions of patients is particularly attractive. Cardiac gene therapy has shown promise in early animal studies and lends itself to the treatment of heart failure. A defect in intracellular calcium handling is known to be a key abnormality in both human and experimental CHF. Deficient Ca 2¿uptake by the sarcoplasmic reticulum (SR) during relaxation in failing hearts from humans and animal models has been associated with a decrease in the expression and activity of SR Ca2+ATPase (SERCA2a). Our preliminary work over the last five years have shown that 1) Gene transfer is an effective means of introducing the SERCA2a gene into myocytes in vitro and in vivo and 2) that increasing the expression of SERCA2a restores contractility and normalizes intracellular calcium cycling in a rodent model of CHF. We are now extending our experiments from rodents to porcine models. We aim to 1) determine the efficiency of transduction of viral vectors in cardiomyocytes isolated from pig hearts; 2) determine the efficiency of transduction of the AAV vectors and El-E4 deleted recombinant adenovirus following gene transfer in vivo in pigs; 3) test the proximal human brain natriuretic (hBNP) promoter (-408 to +100 relative to transcription start site) for the ability to be induced by pressure-overload versus ischemia. PERFORMANCE SITE ========================================Section End===========================================