Gene Therapy for the Recovery from Left Ventricular Assist System

近年来，人们对心脏疾病的分子机制进行了研究，并在分子水平上了解了心脏疾病的发病机制和病理生理。关键基因的遗传调控可能在心脏疾病的治疗干预中发挥重要作用，特别是那些对传统疗法有抗性的心脏疾病。在这方面，我们计划建立可行的系统来控制心肌体内感兴趣的基因。作为一种功能获得的方法，我们尝试用非病毒传递的方法将报告基因或治疗基因转移到跳动的心脏中。将β 2-肾上腺素能受体（bAR）基因导入以ebv为基础的质粒载体中，并将其作为裸DNA注射到心肌病仓鼠心肌中。治疗后的动物FS和心输出量显著升高。全身给药异丙肾上腺素进一步增强心脏收缩，提示衰竭心脏的儿茶酚胺反应因基因转移而升高。心率无明显升高。RT-PCR分析表明，转基因在心脏注射区域的表达受到严格限制。在另一种方法中，我们也成功地通过颗粒介导的转移系统将ebv质粒裸载体送入大鼠心肌。最近，我们还将质粒载体转移到大型动物（即狗）的跳动心脏中，表明可以获得显著的标记基因表达。我们还执行了功能丧失策略来控制体内的基因表达。作为第一个实验，我们在体内通过电穿孔的方式将短干扰RNA （siRNA）转移到骨骼肌细胞中调节外源性或内源性基因的功能。siRNA转染能有效抑制与siRNA同时转导的荧光素酶标记基因的表达。在测试携带EGFP标记基因的转基因小鼠时，GFP特异性siRNA的转导导致GFP荧光显著降低。总的来说，我们成功地建立了利用非病毒或裸DNA系统在体内转导或调节基因的策略。这些方法在严重心脏疾病的基因治疗和分子靶向治疗中可能非常有用。该系统也可能适用于心脏功能基因组学研究。少

Recently, molecular mechanisms of heart diseases have been investigated and pathogenesis as well as pathophysiology is understood at molecular levels. Genetic regulation of critical genes may greatly contribute in the therapeutic intervention of cardiac disorders, particularly those that are resistant for the conventional therapies. In this concern, we planned to establish systems feasible for controlling genes of interest in vivo in the myocardium. As a gain-of-function approach, we tried nonviral delivery method to transfer reporter or therapeutic genes into the beating heart. Beta2-adrenergic receptor (bAR) gene was introduced into an EBV-based plasmid vector, which we have previously shown to exert extremely powerful expression in various cells and tissues, and the resultant plasmid was injected into the myocardium of cardiomyopathic hamsters as naked DNA. The treated animals showed significant elevation in the FS and cardiac output. Systemic administration of isoprotelenol further … More augmented the cardiac contraction, suggesting that the catecholamine response of the failing heart was elevated by the genetic transfer. The hear rate was not significantly increased. RT-PCR analyses demonstrated that the expression of the transgene was strictly restricted in the injected region of the heart. In another approach we also succeeded in delivering naked EBV-plasmid vector into the rat myocardium through the particle-mediated transfer system. More recently we also transferred plasmid vector-based transfer into beating heart of a larger animal, i.e., dogs, showing that significant expression of marker genes can be obtained.We also performed loss-of-function strategies to control genetic expression in vivo. As a first trial, we transferred the short interfering RNA (siRNA) to regulate exogenous or endogenous gene function in skeletal muscle cells by means of the electroporation in vivo. The siRNA transfer quite effectively suppressed expression of luciferase marker gene that had been transduced simultaneosly with the siRNA. In case transgenic mice carring the EGFP marker gene was tested, transduction of siRNA specific for GFP resulted in the significant reduction in the GFP fluorescence.Collectively, we succeeded in establishing strategies to transduce or regulate gene in vivo using non-viral or naked DNA systems. These procedures may become quite useful in the gene therapy as well as the therapeutic molecular targeting for severe cardiac disorders. The system may also be applicable to functional genomics investigations for the heart. Less