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，用于保护心肌免受未来缺血/再灌注损伤（I/R），涉及单次给予治疗性基因，并使用能够有效和长期的肌细胞特异性和诱导表达治疗性基因的载体系统。我们将利用重组腺相关病毒载体（rAAV）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，并使用这些元件作为分子开关来实现对血红素氧合酶-1 （HO-1）表达的最佳时间和空间控制，作为治疗性转基因。我们报道了心肌定向过表达HO-1的转基因小鼠对I/ r诱导的心肌损伤产生抗性；此外，在I/ r诱导的心肌损伤发生前8周，rAAV在大鼠心肌内单次递送HO-1基因可显著降低心肌梗死的发生率，表明rAAV载体与HO-1基因结合是一种有效可行的心肌保护治疗策略。因此，在本提案中，我们将(1)首先记录raav介导的心肌内HO-1传递对I/R损伤引起的急性心肌梗死后长期生存的影响，并确定转基因的表达持续时间和治疗效果的可持续性；(2)我们将开发诱导、缺氧和再氧敏感的HO-1表达，作为内源性调节的心肌保护治疗方法；(3)我们将建立心肌细胞特异性的HO-1转基因表达，最后(4)我们将开发细胞特异性和诱导表达相结合的方法，作为具有最大组织特异性和安全性的转基因传递的理想策略。

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.