Viral Gene Expression to Therapeutic Levels in Models of Heart Failure

描述（由申请人提供）：心血管疾病占美国每年发生的所有死亡的近40%，美国经济的成本为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，治疗方法的微小改进可能会带来巨大的好处。本申请的总体目标是控制全身递送后心脏中的重组腺相关病毒（rAAV）治疗性转基因表达。为了检查衰竭心脏中的表达，我们将采用心脏病的标准模型。LIM敲除将用作我们的肥大模型。将采用两种方法来限制rAAV介导的转基因在心脏的表达：1）最佳亲本和转衣壳之间的比较（将来自不同AAV血清型的衣壳亚基组合成一个病毒体）血清型，将允许在全身注射后检查重组病毒的心脏嗜性;和2）赋予由心力衰竭诱导的基因表达的心脏特异性增强子/启动子构型将用于进一步帮助rAAV递送至靶组织的特异性。AAV血清型6和9在全身尾静脉和直接冠状动脉内递送后在心脏中均表现出显著的转基因表达。在递送等量的病毒颗粒后，心脏中荧光素酶表达和病毒基因组拷贝（vg）数的定量表明，AAV9递送导致尾静脉注射后的vg是AAV6的两倍，而AAV6在冠状动脉内递送后具有更大的vg。AAV6对心脏组织具有更有限的天然向性，胸腔中总光单位的高达15%，而AAV9的不到2%。了解控制AAV6对心脏的天然向性的氨基酸是本申请的一个目的。第二个目的是通过病毒转衣壳作用将有利的表型协同作用到一个病毒体中来组合联合收割机特性。利用AAV6的心脏向性和来自AAV9的稳健表达以及最佳的转衣壳混合物，我们将在全身注射后以剂量依赖性方式评估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/启动子结构，使转基因表达偏向心脏组织。在美国，心力衰竭是死亡的主要原因。此外，住院和误工的费用也是我们经济的一个负担。随着对这种疾病的分子变化的阐明，基因治疗开始在动物模型中显示出效益。心力衰竭动物模型中几种基因的表达提高了心脏泵血能力，而无需心室扩张。然而，转基因和敲除模型不能容易地适应人类心力衰竭，因此，基因递送到心脏的机制变得与这些未来的治疗努力更加相关。本申请的终点是确定由实验基因转移载体递送的治疗基因的治疗剂量。

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.