Integrated Strategies for Novel Treatment of Myocardial Ischemia

描述（由申请人提供）：冠状动脉疾病（CAD）是美国发病率和死亡率的头号原因。尽管医学治疗取得了进展，但一些患者继续出现难治性症状，需要采取更积极的措施，如左心室辅助装置（LVAD）或原位心脏移植（OHT）。在过去的十年中，重组DNA技术的显着进步使得CAD的分子和细胞治疗的发展成为可能。特别是心脏基因治疗领域已经从体外研究发展到临床前试验再到多中心试验。然而，最近的II/III期试验已经发现了一些问题，例如难以控制腺病毒载体的免疫原性、靶向表达至心脏组织以及递送后重组基因的体内监测。因此，开发安全且可产生靶向组织递送的新型非病毒载体将是一个重大进展。另一个重大进展将是非侵入性技术的发展，以评估基因表达，为整个研究领域提供新的催化剂。该提案的目的是（1）开发非免疫原性“小环”DNA载体，其将显著提高心脏中的转染效率，（2）使用遗传和分子测定的综合策略理解基于基因的治疗的机制，以及（3）评估临床前大型动物模型中的安全性和有效性。在5年结束时，我们希望将来将这些发现转化为基于微环的基因治疗对CAD患者的治疗。 公共卫生相关性：心脏基因治疗领域的进展因缺乏合适的递送载体而受阻。我们的建议使用定制设计的微环，其是可以包含任何感兴趣的转基因的超螺旋重组DNA分子。这些微环既没有起点或复制，也没有抗生素选择标记（因此使免疫反应最小化），并且尺寸较小（因此有利于基因转移）。我们将通过引入心脏组织特异性启动子来提高其靶向特异性。治疗性血管生成将通过突变型缺氧诱导因子（HIF-1a）转录因子来实现，所述突变型缺氧诱导因子（HIF-1a）转录因子抵抗降解并且具有特别长的半衰期。基因治疗的机制将使用遗传和分子方法相结合的综合方法进行评估。最后，非侵入性成像模式在小型和大型动物模型中的应用将有助于在临床竞技场中验证和翻译基因治疗方案。

DESCRIPTION (provided by applicant): Coronary artery disease (CAD) is the number one cause of morbidity and mortality in the United States. Despite advances in medical therapies, some patients continue to develop refractory symptoms requiring more aggressive measures such as left ventricular assist device (LVAD) or orthotopic heart transplantation (OHT). Over the past decade, remarkable progress in recombinant DNA technology has enabled the development of molecular and cellular treatments for CAD. In particular, the field of cardiac gene therapy has evolved from in vitro studies to pre-clinical testing to multi-center trials. However, recent phase II/III trials have uncovered problems such as difficulties in controlling immunogenicity of adenoviral vectors, targeting expression to cardiac tissues, and in vivo monitoring of recombinant genes post delivery. Thus, the development of novel non-viral vectors that are safe and can yield targeted tissue delivery would be a major advance. Another significant advance will be the development of noninvasive techniques to assess gene expression, providing a new catalyst for the entire field of investigation. The aims of this proposal are to (1) develop non- immunogenic "minicircle" DNA vectors will significantly improve transfection efficiency in the heart, (2) understand the mechanisms of gene based therapy using integrated strategies of genetic and molecular assays, and (3) to evaluate the safety and efficacy in pre-clinical large animal models. At the end of 5 years, we hope to translate these findings to treatment of CAD patients with minicircle- based gene therapy in the future. PUBLIC HEALTH RELEVANCE: Progress in the field of cardiac gene therapy has been hindered by the lack of suitable delivery vectors. Our proposal uses a custom designed minicircles, which are supercoiled recombinant DNA molecules that can contain any transgene of interest. These minicircles have neither an origin or replication nor an antibiotic selection marker (thus minimizing immune response) and are of smaller size (thus facilitating gene transfer). We will improve its target specificity by incorporating a cardiac tissue specific promoter. Therapeutic angiogenesis will be achieved via a mutant hypoxia inducible factor (HIF-1a) transcriptional factor that is resistant to degradation and has exceptionally long half-life. Mechanisms of gene therapy will be evaluated using an integrative approach combining genetic and molecular approaches. Finally, the application of non-invasive imaging modalities in small and large animal models will help validate and translate gene therapy protocols in the clinical arena.