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

描述(由申请人提供)：心血管疾病的基因转移治疗因无法安全和容易地获得足够的心脏转基因表达而受到困扰。目前治疗心脏病的基因转移方法包括心肌内注射或冠状动脉内注射，这些方法应用起来都很麻烦。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8型腺相关病毒(AAV8)，它在啮齿动物、猪和灵长类动物体内静脉注射后提供长期和广泛的表达。Urocortin-2是新近发现的促肾上腺皮质激素释放因子家族血管活性多肽，它通过促肾上腺皮质激素释放因子2型受体发挥作用，而促肾上腺皮质激素释放因子2型受体广泛表达于心脏和血管系统。在动物和充血性心力衰竭患者身上的研究表明，注射urocortin-2肽具有良好的血液动力学效应，包括增加不依赖负荷的收缩功能，表明直接的心脏效应。在拟议的研究中，urocortin-2是一种理想的治疗性转基因选择。我们已经证实，使用鸡β-肌动蛋白启动子静脉注射AAV8可以提供持续高水平的血清UCn2，并增加正常和衰竭小鼠心脏的功能。为了开发和完善这样的方法，我们建议在小鼠和猪的连续研究中确定：a)提供受调控的转基因表达的最佳系统，以实现血浆转基因水平的微调，并允许根据需要关闭和开启表达；以及b)在CHF小鼠模型中使用这种基于旁分泌的最佳方法进行urocortin-2基因转移的安全性和有效性。随后，在正常猪身上，我们将确定：a)增加血清Urocortin-2所需的最低有效载体剂量；b)载体和转基因的生物分布；以及c)毒性。假设：静脉注射调控表达urocortin-2的AAV8载体，通过旁分泌介导的作用，将对衰竭的心脏有良好的效果。目的1.确定最佳的调控表达系统，以获得长期和剂量反应的血浆中urocortin-2的表达，免疫反应和毒性目标最小。2.测试提供urocortin-2调控表达的最佳AAV8载体(在Aim 1中确定)，并在心力衰竭和非心力衰竭小鼠中测定：a)12个月以上的血药浓度；b)增加衰竭心脏功能和降低死亡率的疗效；c)有益作用的机制；以及d)载体和转基因的生物分布和毒性目的3.测试编码urocortin-2(在Aim 2中得到确认)的最佳AAV载体在正常猪体内的水平，以确定：a)载体和转基因的生物分布；c)毒性这些机制和验证性研究的范围足以为未来在猪CHF模型中进行的研究奠定基础，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.