GENETIC AND BIOCHEMICAL ANALYSIS OF AAV TRANSCRIPTIONAL CONTROL

本项目的长期目标是开发基于病毒载体的基因转移策略，用于治疗遗传性和获得性心肺疾病。为此，一组具有不同但互补的跨学科兴趣和专业知识的研究人员建立了一项综合研究努力，这一努力突出了在基因治疗的实际应用方面的共同兴趣。该计划的一个主要重点是开发改进的基因转移方法。其中四个组成部分(子项目3、子项目4、子项目和试点2)主要侧重于开发适合转导终末分化细胞的载体。这些载体包括两个稳定表达载体，腺相关病毒(AAV，子项目3)和慢病毒(子项目4)，以及两个瞬时表达载体，腺病毒(Ad，子项目5)和昆虫痘病毒(EPV，Pilot 2)。在每一种情况下，努力都是为了查明和绕过基因疗法实际应用的障碍，这些障碍大多植根于每个载体系统所基于的单个病毒的基本生物学。除了载体开发部分，还非常重视开发新的动物模型，包括开发单基因心脏(分项目1)和肺(试点1，分项目2)疾病的敲除小鼠模型，以及用于AAV整合的转基因模型(分项目1)。利用已建立的高血压和急性肺损伤模型对于次级项目6和5也同样至关重要。该计划还将研究这些载体系统在三种心肺疾病中的应用：阿尔法-1-抗胰蛋白酶缺乏症(阿尔法1AT，子项目2)、酸性麦芽糖酶缺乏症(GAA，子项目1)和高血压(子项目6)。这将通过将载体定向到肌肉(子项目1和2)和血管内皮细胞(子项目6)来实现。此外，该计划还建立了一个媒介核心实验室(核心B)，该实验室将向所有子项目提供质量统一且可重现的媒介，并研究产生重组AAV(RAAV)和腺病毒(Ad)载体的改进方法。向量核心还将作为一种机制，确保所有次级项目之间迅速交换信息。最后，一个行政核心(核心A)将确保对次级项目和试点/可行性项目进行集中的财政管理和监督。

PARENT ABSTRACT - SUBPROJECT ABSTRACT NOT AVAILABLE The long-range goal of this Program Project is to develop viral vector-based gene transfer strategies for treating genetic and acquired cardiopulmonary disorders. To this end, a group of investigators with diverse, yet complementary interdisciplinary interests and expertise has established an integrated research effort that is underscored by a common interest inpracticalapplications of gene therapy. A major focus of the Programis the development of improved methods of gene transfer. Four of the components (Subproject 3, Subproject 4, Subproject and Pilot 2) are primarily focused onthe development of vectors suitable for transduction of terminally differentiated cells. These include two stable expression vectors, Adeno-associated virus (AAV, Subproject 3) and lentivirus (subproject 4), as wellas two transient expression vectors, adenovirus (Ad, Subproject 5) and the entomopoxvirus (EPV, Pilot 2). In each case, the efforts are directed at identifying and circumventing obstacles to the practical applicationof gene therapy, most of which are rooted in the basic biology of the individual virus upon which each vector system is based. Along with the vector development components, there is a strong emphasis on the development of new animal models, including the development of knock-out mouse models for monogenic cardiac (Subproject 1) and pulmonary (Pilot 1, Subproject 2) disorders, and a transgenic model for AAV integration (Subproject 1). The utilization of established models of hypertension and acute lung injury are likewise pivotal for Subprojects 6 and 5, respectively. The program also will investigate the applications of these vector systems to three cardiopulmonary disorders: alpha-1-antitrypsin deficiency (alpha 1AT, Subproject 2), acid maltase deficiency (GAA, Subproject 1) and hypertension (Subproject 6). This will be done by targeting vectors to muscle (Subprojects 1 and 2) and vascular endothelium (Subprogject 6). Additionally, the Program has established a Vector Core Laboratory (Core B) which will supply vectors of uniform and reproducible quality to all subprojects, and investigate improved methods of generating recombinant AAV (rAAV) and adenovirus (Ad) vectors. The Vector Core will also serve as a mechanism to insure rapid exchange of information among all subprojects. Finally, an Administrative Core (Core A) will insure centralized fiscal management and oversight for the subprojects and pilot/feasibility projects.