Viral Gene Expression to Therapeutic Levels in Animal Models of Heart Failure

心力衰竭动物模型中病毒基因表达达到治疗水平

• 批准号: 7665572
• 负责人: JOSEPH E RABINOWITZ
• 金额: $ 38.63万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7665572
• 关键词: Absenteeism at work; Accounting; Actins; Adenovirus Vector; American Heart Association; Amino Acid Substitution; Amino Acids; Animal Disease Models; Animal Model; Atrial Natriuretic Factor; Automobile Driving; Biological Assay; Budgets; C-terminal; Ca(2+)-Transporting ATPase; Capsid; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cessation of life; Clinical; Coronary; Coronary artery; Coupled; Cytomegalovirus; Data; Discipline; Disease; Dose; EFRAC; Elongation Factor; Enhancers; Failure; Fetal Reduction; Future; GTP-Binding Proteins; Gene Delivery; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Goals; Green Fluorescent Proteins; Heart; Heart Diseases; Heart failure; Heating; Hospitalization; Human; Hypertrophy; Immunohistochemistry; In Vitro; Infarction; Injection of therapeutic agent; Introns; Kinetics; Knock-out; Ligation; Light; Link; Liver; Luciferases; MM form creatine kinase; Measures; Mediating; Methods; Modeling; Molecular; Mus; Mutation; Myocardium; Myoglobin; Nature; Neonatal; Organ; Pattern; Performance; Phenotype; Phosphotransferases; Property; Pump; Rattus; Recombinant adeno-associated virus (rAAV); Regulation; Regulator Genes; Reporter Genes; Route; S100A1 protein; SERCA2a; Safety; Sarcoplasmic Reticulum; Serotyping; Skeletal Muscle; Specificity; System; Tail; Testing; Therapeutic; Thoracic cavity structure; Time; Tissues; Transgenes; Transgenic Animals; Transgenic Organisms; Translating; Treatment Efficacy; Tropism; United States; United States National Institutes of Health; Veins; Ventricular; Viral; Viral Genes; Viral Genome; Virion; adeno-associated viral vector; base; cost; design; dosage; ds-DNA; gene transfer vector; heart function; improved; in vivo; interest; mortality; particle; promoter; rat atrial natriuretic peptide; recombinant virus; response; targeted delivery; therapeutic gene; therapeutic transgene; transduction efficiency; transgene expression; vector; vector genome

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.

描述（由申请人提供）：心血管疾病占美国每年死亡人数的近 40%，给美国经济造成的损失达 3,940 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。就这个范围而言，治疗方法的微小改进可能会带来巨大的好处。该应用的总体目标是在全身递送后控制重组腺相关病毒（rAAV）治疗性转基因在心脏中的表达。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 介导的转基因表达到心脏： 1) 最佳亲本和转衣壳（来自不同 AAV 血清型的衣壳亚基组合成一种病毒颗粒）血清型之间的比较，将允许在全身注射后检查重组病毒的心脏向性； 2) 赋予心力衰竭诱导的基因表达的心脏特异性增强子/启动子配置将用于进一步帮助rAAV递送至靶组织的特异性。 AAV 血清型 6 和 9 均在全身尾静脉和直接冠状动脉内递送后在心脏中表现出显着的转基因表达。递送同等数量的病毒颗粒后，对心脏中的荧光素酶表达和病毒基因组拷贝 (vg) 数量进行定量表明，AAV9 递送导致尾静脉注射后的 vg 比 AAV6 大两倍，而 AAV6 在冠状动脉内递送后的 vg 更大。 AAV6 对心脏组织的自然趋向性更受限制，胸腔内的总光单位高达 15%，而 AAV9 的这一比例不到 2%。了解控制 AAV6 对心脏的天然趋向性的氨基酸是本应用的目标之一。第二个目标是通过病毒转衣壳结合特性，将有利的表型协同到一种病毒粒子中。利用 AAV6 的心脏向性和 AAV9 的强大表达以及最佳的转壳混合物，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表达，我们设计了增强子/启动子配置，以使转基因表达偏向心脏组织。叙述 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会消耗我们的经济。随着导致该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。心力衰竭动物模型中多种基因的表达可提高心脏泵血能力，而无需心室扩张。然而，转基因和敲除模型不能轻易适应人类心力衰竭，因此，将基因传递到心脏的机制与这些未来的治疗工作变得更加相关。该应用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。