Manipulation of VEGF splicing in angiogenic gene therapy

描述（由申请人提供）： 选择性剪接提供了从单个基因转录物产生相关但不同的蛋白质的机制，从而产生多样性。基于基因序列的选择性剪接的基因治疗应该比使用单个cDNA更接近地模拟全范围的生理效应，可能导致增加的功效和减少的不良反应。在使用血管内皮生长因子（VEGF）的血管生成基因治疗的情况下，所有人类研究都使用单个cDNA。然而，生理性血管生成通过至少三种主要亚型121、165和189的协同作用而发生;这些亚型具有微妙不同的性质，并且在各种组织中以不同的比例产生。该提案的重点是将选择性剪接的概念纳入基因治疗的范例，从而更准确地反映所转移基因的真实表型潜力。该提议的基本假设是，指导同种型混合物产生的VEGF基因将增强VEGF介导的血管生成的有效性，同时减少由使用表达单一同种型的单一cDNA所导致的不良后果。尽管所提出的研究仅集中于VEGF基因，并且仅利用腺病毒（Ad）基因转移载体，但是将产生的基本原理应该是适用的 to any gene基因and any gene基因transfer转移vector载体.在此背景下，该提案的具体目标包括：具体目标1。工程基因组/cDNA杂交基因，表达不同比例的主要VEGF亚型，并在体外和体内证明，这些结构的基因转移导致不同的细胞和组织特异性选择性剪接模式。具体目标2。当靶向在同一器官的不同细胞类型中表达时，评估混合基因组/cDNA VEGF基因的剪接模式。具体目标3。评价在体内转移VEGF基因组/cDNA杂合体（设计用于产生不同的选择性剪接模式）后，基因转移的疗效与不良表型，导致VEGF亚型的不同混合物。

DESCRIPTION (provided by applicant): Alternative splicing provides a mechanism for the production of related yet distinct proteins from a single gene transcript, thus generating diversity. Gene therapy based on alternative splicing of gene sequences should mimic the full range of physiological effects more closely than the use of a single cDNA, likely resulting in increased efficacy and reduced adverse effects. In the case of angiogenic gene therapy using vascular endothelial growth factor (VEGF), all human studies have used single cDNAs. However, physiological angiogenesis occurs by the concerted action of at least three major isoforms, 121, 165 and 189; these isoforms have subtly different properties and are produced in different ratios in various tissues. The focus of this proposal is to incorporate the concept of alternative splicing into the paradigm of gene therapy, thus more accurately reflecting the true phenotypic potential of the transferred gene. The underlying hypothesis of this proposal, is that VEGF genes that direct the production of mixtures of isoforms will enhance the effectiveness of VEGF-mediated angiogenesis while reducing the adverse consequences resulting from the use of a single cDNA that expresses a single isoform. Although the proposed studies focus only on the VEGF gene, and utilize only adenovirus (Ad) gene transfer vectors, the basic principles that will be generated should be applicable to any gene and any gene transfer vector. With this background, the specific aims of the proposal include: Specific Aim 1. Engineer genomic/cDNA hybrid genes that express different ratios of the major VEGF isoforms and demonstrate in vitro and in vivo that gene transfer with these constructs results in different patterns of cell- and tissue-specific alternative splicing. Specific Aim 2. Assess splicing patterns of the hybrid genomic/cDNA VEGF genes when targeted to express in different cell types of the same organ. Specific Aim 3. Evaluate the efficacy vs adverse phenotypes of gene transfer resulting in different mixtures of VEGF isoforms following in vivo transfer of VEGF genomic/cDNA hybrids designed to generate different patterns of alternative splicing.