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人数的近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。在这个范围内，治疗方法的微小改进可能反映出巨大的好处。本应用的总体目标是控制重组腺相关病毒（rAAV）治疗性转基因在全身递送后在心脏中的表达。为了检查衰竭心脏的表达，我们将采用标准的心脏病模型。LIM淘汰赛将作为我们的肥厚模型。将采用两种方法来限制rAAV介导的转基因在心脏中的表达：1)比较最佳亲本和转囊（将来自不同AAV血清型的衣壳亚基组合成一个病毒粒子）血清型，以便在全身注射后检查重组病毒的心脏倾向；2)心脏特异性增强子/启动子配置赋予心力衰竭诱导的基因表达，将用于进一步帮助rAAV递送到靶组织的特异性。AAV血清型6和9在全身尾静脉和直接冠状动脉内输送后的心脏中均有显著的转基因表达。等量病毒颗粒递送后心脏荧光素酶表达和病毒基因组拷贝（vg）数的定量分析表明，尾静脉注射AAV9递送后的vg是AAV6递送后的两倍，而冠状动脉内递送后AAV6的vg更大。AAV6对心脏组织的自然趋向性更有限，在胸腔中占总光单位的15%，而AAV9的这一比例不到2%。了解控制AAV6s对心脏的自然趋向性的氨基酸是该应用的一个目标。第二个目标是通过病毒转壳结合特性，将有利的表型协同到一个病毒粒子中。利用AAV6的心脏倾向性和AAV9的强表达，以及最佳转乘混合物，我们将以剂量依赖的方式评估2ARKct和S100A1转基因在全身注射后的治疗功能。最后，为了进一步限制心脏基因表达，我们设计了增强子/启动子配置，使转基因表达偏向于心脏组织。在美国，心力衰竭是导致死亡的主要原因。此外，住院治疗和错过工作的费用也在消耗我们的经济。随着支持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益处。心衰动物模型中几种基因的表达可提高心脏泵血能力而不引起心室扩张。然而，转基因和基因敲除模型不容易适用于人类心力衰竭，因此，基因传递到心脏的机制与这些未来的治疗工作更加相关。本申请的终点是确定由实验性基因转移载体递送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.