GENE THERAPY FOR LONG-TERM MYOCARDIAL PROTECTION

描述(由申请人提供)：在本申请中，我们建议开发一种预防性基因治疗策略，用于预防未来的缺血/再灌注损伤(I/R)，该策略涉及一种单一的治疗基因给药系统，该载体系统能够有效和长期地对心肌细胞特异性和可诱导的治疗基因进行表达。我们将使用重组腺相关病毒载体(RAAV)来实现转导基因在心肌中的长期稳定表达。我们将选择能够诱导和细胞特异性基因表达的顺式作用启动子元件，并利用这些元件作为分子开关来实现作为治疗性转基因的血红素氧合酶-1(HO-1)表达的最佳时空调控。我们已经报道了心脏导向过表达HO-1的转基因小鼠对I/R诱导的心肌损伤产生了抵抗；此外，在大鼠心肌I/R损伤前8周，通过rAAV单次心肌内注射HO-1基因，显著减少了心肌梗死的发生，证明rAAV载体和HO-1基因的结合是一种有效和可行的心肌保护治疗策略。因此，在这项提案中，我们将(1)首先记录rAAV介导的HO-1心肌内递送对I/R损伤诱导的急性心肌梗死后长期存活的影响，并确定转基因表达的持续时间和治疗效果的可持续性；(2)我们将开发可诱导的、低氧和复氧敏感的HO-1表达，作为一种内源性调控的心肌保护治疗方法；(3)我们将建立HO-1的心肌细胞特异性转基因表达，最后(4)我们将开发一种细胞特异性和诱导性表达相结合的方法，作为一种理想的转基因治疗策略，具有最大的组织特异性和安全性。

DESCRIPTION (provided by applicant): In this application we propose to develop a preventive gene therapy strategy for myocardium protection from future ischemia/reperfusion injury (I/R) involving a single administration of a therapeutic gene with a vector system capable of efficient and long term myocyte specific and inducible expression of the therapeutic gene. We will employ recombinant adeno-associated viral vectors (rAAV) to achieve long-term and stable expression of transduced genes in the myocardium. We will select cis-acting promoter elements that are capable of conferring inducible and cell-specific gene expression and to use these elements as molecular switches to achieve optimal temporal and spatial control of expression of heme oxygenase-1 (HO-1) as the therapeutic transgene. We have reported that transgenic mice with cardiac-directed overexpression of HO-1 develop resistance to I/R-induced myocardial injury; furthermore, a single intramyocardial delivery of HO-1 gene by rAAV in rats, eight weeks in advance of I/R-induced myocardial injury, resulted in dramatic reduction in myocardial infarction, demonstrating that the combination of rAAV vector and HO-1 gene is an efficacious and feasible therapeutic strategy for myocardial protection. Accordingly, in this proposal we will (1) first document the impact of rAAV-mediated intramyocardial delivery of HO-1 on long-term survival following acute myocardial infarction induced by I/R injury and to determine the duration of expression and sustainability of the therapeutic effect of the transgene; (2) we will develop inducible, hypoxia and reoxygenation-sensitive expression of HO-1 as an endogenously regulated therapeutic approach for myocardial protection; (3) We will establish cardiomyocyte specific transgene expression of HO-1, and finally (4) we will develop a combined approach of cell-specific and inducible expression as an ideal strategy for transgene delivery with maximal tissue specificity and safety.