Vector Identification and Gene Delivery Approach in Pigs

猪的载体鉴定和基因传递方法

• 批准号: 7002035
• 负责人: Judith K Gwathmey
• 金额: $ 10.25万
• 依托单位: GWATHMEY, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-10 至 2005-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7002035
• 关键词: adeno associated virus group; bioenergetics; biotechnology; calcium flux; calcium transporting ATPase; cardiac myocytes; disease /disorder model; gene delivery system; gene expression; gene therapy; genetic promoter element; genetic transduction; heart failure; nerve /myelin protein; recombinant virus; sarcoplasmic reticulum; swine; therapy design /development; transfection /expression vector

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）： 该提案的目的是开发一种针对分子障碍的新型疗法，具体是，病毒介导的钙调节蛋白SERCA 2A（肌质质网ca 2 pospase）的心肌递送到携带心力衰竭的大型动物，以尝试恢复功能并提高生存能量而无需担心能量能量参数，而无需担心生存。充血性心力衰竭（CHF）代表了要求有效治疗方法的巨大临床问题。尽管传统的治疗方法取得了进步，包括药物管理，心肌血运重建，机械辅助设备和移植，但CHF仍然是全球死亡的主要原因。因此，一种旨在降低CHF的发病率和死亡率并改善数百万患者的生活质量的新型疗法特别有吸引力。心脏基因疗法在早期动物研究中表现出了希望，并为心力衰竭的治疗提供了希望。众所周知，细胞内钙处理的缺陷在人类和实验性CHF中都是关键异常。在人类失败的心脏和动物模型中，肌浆网（SR）的吸收不足与SR Ca2+ATPase（SERCA2A）的表达和活性降低有关。在过去五年中，我们的初步工作表明，1）基因转移是在体外和体内引入SERCA2A基因在肌细胞和2）2）中的有效手段，它增加了SERCA2A的表达可恢复收缩力并在CHF的啮齿动物模型中恢复了细胞内钙循环。现在，我们将实验从啮齿动物扩展到猪模型。我们的目的是1）确定从猪心分离的心肌细胞中病毒载体转移的效率； 2）确定在猪体内基因转移后，AAV载体和EL-E4删除的重组腺病毒的转移效率； 3）测试近端人脑发努力（HBNP）启动子（相对于转录起始位点，-408至+100），以通过压力超负荷与缺血能力诱导的能力。