Viral Gene Expression to Therapeutic Levels in Models of Heart Failure

简介(申请人提供)：心血管疾病占美国每年所有死亡人数的近40%，给美国经济造成的损失为3940亿美元(美国心脏协会；2005年)，几乎是NIH年度总预算(2007财年284亿美元)的14倍。就这一范围而言，治疗学的微小改进可能反映出巨大的好处。这项应用的总体目标是控制重组腺相关病毒(RAAV)系统递送后在心脏中的治疗性转基因表达。为了检查心脏衰竭的表现，我们将使用心脏病的标准模型。LIM基因敲除将被用作我们的肥厚模型。将使用两种方法来限制rAAV介导的转基因在心脏中的表达：1)最佳亲本和转衣壳(来自不同AAV血清型的衣壳亚单位组合成一个病毒粒子)血清型之间的比较，将允许检查全身注射后重组病毒的心脏趋向性；以及2)心脏特异的增强子/启动子配置，提供心力衰竭诱导的基因表达，将被用于进一步帮助rAAV递送到靶组织的特异性。AAV血清型6型和9型在全身尾静脉和直接冠脉内递送后，在心脏中都显示出显著的转基因表达。对相同数量的病毒颗粒在心脏中的荧光素酶表达和病毒基因组拷贝(VG)数量的定量检测表明，AAV9在尾静脉注射后的VG是AAV6的两倍，而AAV6在冠脉内注射后的VG更大。AAV6对心脏组织有更有限的自然取向，胸腔内总光单位的15%，而AAV9不到2%。了解支配AAV6对心脏的自然取向的氨基酸是这一应用的目标之一。第二个目标是通过病毒转衣壳结合特性，将有利的表型协同作用成一个病毒粒子。利用AAV6的心脏趋向性和AAV9的强势表达，以及最佳的转帽蛋白混合物，我们将在全身注射后以剂量依赖的方式评估2ARKct和S100A1转基因的治疗功能。最后，为了进一步限制心脏基因的表达，我们设计了增强子/启动子配置，将转基因表达定向到心脏组织。在美国，心力衰竭是导致死亡的主要原因。此外，住院和错过工作的费用是对我们经济的一种消耗。随着支撑疾病的分子变化被阐明，基因治疗开始在动物模型中显示出好处。在心力衰竭的动物模型中，几个基因的表达改善了心脏的泵血能力，而不需要扩大心室。然而，转基因和基因敲除模型不能很容易地适应人类心力衰竭，因此，基因输送到心脏的机制与这些未来的治疗努力变得更加相关。这一应用的终点是确定由实验性基因转移载体传递的治疗性基因的治疗剂量。

DESCRIPTION (provided by applicant): Cardiovascular disease accounts for nearly 40% of all deaths that occur in the United States each year, and the cost to the US economy, $394 billion (American Heart Association; 2005), is almost 14 times greater than the NIH total annual budget (FY 2007 $28.4 billion). With respect to that scope, small improvements in therapeutics may reflect enormous benefits. The overall goal of this application is to control recombinant Adeno-associated virus (rAAV) therapeutic transgene expression in the heart after systemic delivery. To examine expression in failing hearts we will employ a standard model of heart disease. The LIM knockouts will be used as our model of hypertrophy. Two methods will be employed to restrict rAAV mediated transgene expression to the heart: 1) Comparisons between the best parental and transcapsidation (combination of capsid subunits from different AAV serotypes into one virion) serotypes, will permit examination of cardiac tropism of the recombinant virus after systemic injection; and 2) Heart specific enhancer/promoter configurations that confer gene expression induced by heart failure will be used to further aid in the specificity of rAAV delivery to the target tissue. Both AAV serotypes 6 and 9 demonstrate significant transgene expression in the heart after systemic tail vein and direct intra-coronary delivery. Quantification of luciferase expression and viral genome copy (vg) number in the heart after delivery of equivalent numbers of viral particles demonstrates that AAV9 delivery results in two-fold greater vg after tail vein injection than AAV6, while AAV6 has greater vg after intra-coronary delivery. AAV6 has a more restricted natural tropism for cardiac tissue, with up to 15% of total light units in the thoracic cavity compared with less than 2% for AAV9. Understanding the amino acids that govern AAV6s' natural tropism for the heart is one objective of this application. A second objective is to combine properties through viral transcapsidation to synergize advantageous phenotypes into one virion. Taking advantage of the cardiac tropism of AAV6 and robust expression from AAV9 as well as the best transcapsidation mixtures we will assess the therapeutic function of 2ARKct and S100A1 transgenes, after systemic injection, in a dose dependent manner. Finally, with the goal of further limiting cardiac gene expression, we have designed enhancer/promoter configurations to bias transgene expression to cardiac tissue. NARRATIVE In the United States heart failure is a major cause of mortality. In addition, the cost of hospitalization and missed work is a drain on our economy. As the molecular changes that underpin the disease are elucidated genetic treatments are beginning to show benefit in animal models. The expression of several genes in animal models of Heart Failure improves the hearts ability to pump without ventricular dilation. However, transgenic and knockout models cannot be easily adapted to human heart failure, therefore, mechanism for gene delivery to the heart become more relevant to these future therapeutic efforts. An end point for this application is to determine the therapeutic dose of a therapeutic gene delivered by an experimental gene transfer vector.