Urocortin-2 Gene Transfer for CHF: a Paracrine Approach Using Intravenous AAV8

描述（由申请人提供）：用于治疗心血管疾病的基因转移由于不能安全且容易地获得足够的心脏转基因表达而受到困扰。目前用于心脏病的基因转移方法包括心肌肌内注射或冠状动脉内递送，这些方法应用起来很麻烦。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，转基因作为一种激素，从远处释放到循环中后具有心脏效应。这种方法将避免获得高产量心脏基因转移的问题，并使患者能够在门诊期间通过全身注射进行治疗。此外，所提出的方法将消除对治疗性肽的静脉内递送的需要，从而避免重复和延长的住院时间、高发病率和巨大的经济成本。实现这些目标的最合适的载体是腺相关病毒8型（AAV8），其在啮齿动物、猪和灵长类动物中静脉内递送后提供长期和广泛的表达。尿皮质素-2是最近发现的促肾上腺皮质激素释放因子家族血管活性肽，通过促肾上腺皮质激素释放因子2型受体发挥作用，该受体在心脏和血管系统中强烈表达。在动物和充血性心力衰竭患者中的研究表明，尿皮质素-2肽输注具有良好的血液动力学效应，包括独立于负荷的收缩功能增加，表明直接的心脏效应。Urocortin-2是一种理想的转基因治疗药物。我们已经确定，使用鸡β-肌动蛋白启动子静脉内递送AAV8提供了持续的高血清UCn 2水平，并增加了正常和衰竭小鼠心脏的功能。为了开发和完善这种方法，我们建议在小鼠和猪的连续研究中确定：a）提供调节的转基因表达以能够微调血浆转基因水平并允许根据需要关闭和打开表达的最佳系统;和B）在CHF小鼠模型中使用这种最佳的基于旁分泌的方法进行尿皮质素-2基因转移的安全性和有效性。随后，在正常猪中，我们将确定：a）增加血清尿皮质素-2所需的最低有效载体剂量; B）载体和转基因的生物分布;和c）毒性。假设：静脉内注射具有调节的尿皮质素-2表达的AAV8载体将通过旁分泌介导的作用对衰竭的心脏具有有利的作用。目标1。确定最佳调节表达系统，以实现血浆中尿皮质素-2的长期和剂量响应性表达，同时具有最小的免疫应答和毒性。测试提供尿皮质素-2（在目标1中鉴定）的调节表达的最佳AAV8载体，并在患有和不患有心力衰竭的小鼠中确定：a）12个月内的血浆水平; B）增加衰竭心脏功能和降低死亡率的功效; c）有益作用的机制;和d）载体和转基因的生物分布和毒性。为了在正常猪中测试编码尿皮质素-2的最佳AAV载体（在目的2中确认）以确定：a）尿皮质素-2的血浆水平; B）载体和转基因的生物分布; c）毒性。这些机制和概念验证研究被设计为在范围上足以为在猪的CHF模型中进行的未来研究奠定基础，并随后提交IND以启动临床试验。

DESCRIPTION (provided by applicant): Gene transfer for the treatment of cardiovascular diseases is bedeviled by inability to obtain, safely and easily, sufficient cardiac transgene expression. Current methods of gene transfer for heart disease include intramuscular injection into heart muscle or intracoronary delivery, approaches that 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 type 8 (AAV8), which provides long term and extensive expression after intravenous delivery in rodents, pigs, and primates. Urocortin-2, a recently discovered corticotrophin releasing factor family vasoactive peptide, acts via corticotropin-releasing factor type 2 receptors, which are robustly expressed in the heart and vasculature. Studies in animals and patients with congestive heart failure have shown favorable hemodynamic effects of urocortin-2 peptide infusions, including increased contractile function independent of loading, indicating direct cardiac effects. Urocortin-2 is an ideal selection as a therapeutic transgene in the proposed studies. We have established that intravenous delivery of AAV8 using the chicken beta-actin promoter provides sustained high serum levels of UCn2 and increases function of the normal and failing mouse heart. To develop and refine such an approach we propose to determine, in sequential studies in mice and pigs: a) the optimal system to provide regulated transgene expression to enable fine-tuning of plasma transgene levels, and allow turning expression off and on as needed; and b) the safety and efficacy of urocortin-2 gene transfer using this optimal, paracrine-based approach in a mouse model of CHF. Subsequently, in normal pigs we will determine: a) the minimally effective vector dose required to increase serum urocortin-2; b) biodistribution of the vector and transgene; and c) toxicity. HYPOTHESIS: Intravenous injection of an AAV8 vector with regulated expression of urocortin-2 will, through paracrine-mediated actions, have favorable effects on the failing heart. Aim 1. To determine the optimal regulated expression system to achieve long term and dose-responsive urocortin-2 expression in plasma, with minimal immune response and toxicity Aim 2. To test the optimal AAV8 vector providing regulated expression of urocortin-2 (identified in Aim 1) and determine in mice with and without heart failure: a) plasma levels over 12 months; b) efficacy for increasing function of the failing heart and reducing mortality; c) mechanisms for beneficial effects; and d) biodistribution and toxicity of the vector and transgene Aim 3. To test the optimal AAV vector encoding urocortin-2 (confirmed in Aim 2) in normal pigs to determine: a) plasma levels of urocortin-2; b) biodistribution of the vector and transgene; c) toxicity These mechanistic and proof-of-concept studies are designed to be sufficient in scope to lay the groundwork for future studies to be conducted in a CHF model in pigs and subsequently file an IND to initiate a clinical trial.