Translational Studies of Paracrine CV Gene Transfer

描述(由申请人提供)： 基因转移治疗心血管疾病在概念上是有吸引力的，但在心脏中以一种容易和安全的方式获得高产量的转基因表达一直是进展的主要障碍。目前治疗心脏病的基因转移方法包括肌肉注射或冠状动脉内注射，这些方法通常只能提供有限的表达或应用起来很麻烦。因此，我们考虑了编码旁分泌型转基因的载体的有用性。在这种方法中，转基因充当一种激素，从远处释放到循环中后对心脏产生影响。这种方法将绕过获得高产量心脏基因转移的问题，并使患者能够在办公室就诊时通过全身注射进行治疗。此外，建议的方法将消除静脉注射治疗性多肽的需要，从而避免重复和长时间的住院、高发病率和巨大的经济成本。最适合实现这些目标的载体是腺相关病毒(AAV)，它在静脉注射后在啮齿动物、猪和灵长类动物中提供长期和广泛的表达。最后，通过在AAV载体中调控转基因的表达，可以实现对转基因表达水平的控制。开发和优化这一方法是本提案的重点。胰岛素样生长因子-I(IGFI)是一种具有多种心血管效应(促肌力、促血管生成、抗细胞凋亡)的多肽。我们实验室最近的概念验证研究表明，骨骼肌注射编码IGFI的AAV5载体可以改善衰竭大鼠心脏的功能。在拟议的研究中，IGFI将是一种理想的治疗性转基因。为了发展和完善这样一种方法，我们建议确定：a)IGFI基因转移增强收缩功能的机制；b)静脉注射的最佳AAV载体和启动子，将提供最大限度的转基因表达，同时最小的免疫反应和非靶点效应；c)最佳的调控转基因表达系统，以实现血清转基因水平的微调，并根据需要关闭和开启表达；d)IGFI基因转移的安全性、有效性和生存优势，在充血性心力衰竭大鼠模型中使用这种最佳的旁分泌方法；E)以血清IGFI和免疫反应为终点，在正常猪体内静脉注射该最佳载体后，AAV和转基因表达激活剂的最低有效剂量。这些机械性和概念验证研究的设计范围足以在单独的资助下在患有CHF的猪身上启动一项研究，然后向FDA提交IND申请进行倒数第二项研究：一项针对患有充血性心力衰竭的退伍军人的临床试验。

DESCRIPTION (provided by applicant): Gene transfer for the treatment of cardiovascular diseases is conceptually attractive, but difficulty in obtaining high yield transgene expression in the heart in a manner that can be easily and safely applied has been a chief impediment to progress. Current methods of gene transfer for heart disease include intramuscularinjection into heart muscle or intracoronary delivery, approaches that typically provide limited expression or are cumbersome to apply. Consequently, we have considered the usefulness of a vector encoding a paracrine-type transgene. In this approach, the transgene acts as a hormone, having cardiac effects after being released to the circulation from a distant site. This approach would circumvent the problem of attaining high yield cardiac gene transfer and enable patients to be treated by a systemic injection during an office visit. Furthermore, the approach proposed would eliminate the need for intravenous delivery of therapeutic peptides and thereby circumvent repeated and prolonged hospital stays, high morbidity, and enormous economic costs. The most suited vector to achieve these goals is the adeno-associated virus (AAV), which provides long term and extensive expression after intravenous delivery in rodents, pigs, and primates. Finally, by using regulated expression of the transgene in the AAV vector, one can achieve control of the level of transgene expression. To develop and optimally refine this approach is the focus of this proposal. Insulin-like growth factor-I (IGFI) is a peptide with protean favorable cardiovascular effects (inotrope, angiogenic, anti-apoptotic). Proof-of-concept studies in our laboratory recently showed that skeletal muscle injection of an AAV5 vector encoding IGFI improved function of the failing rat heart. IGFI will be an ideal therapeutic transgene in the proposed studies. To develop and refine such an approach, we propose to determine: a) mechanisms by which IGFI gene transfer increases contractile function; b) the optimal AAV vector and promoter for intravenous delivery that will provide maximal transgene expression with minimal immune response and off-target effects in rats; c) the optimal regulated transgene expression system to enable fine-tuning of serum transgene levels, and turning expression off and on as needed; d) the safety, efficacy, and survival advantage of IGFI gene transfer, using this optimal, paracrine-based approach in a rat model of congestive heart failure; and e) e) the minimally effective doses of AAV and activators of transgene expression following intravenous delivery of this optimal vector in normal pigs, using serum IGFI and immune response as end-points. These mechanistic and proof-of-concept studies are designed to be sufficient in scope to launch, under separate funding, a study in pigs with CHF, and subsequently to file an IND application to the FDA for the penultimate study: a clinical trial in veterans with congestive heart failure.