Manipulation of VEGF splicing in angiogenic gene therapy

描述(由申请人提供)： 选择性剪接提供了一种机制，可以从单个基因转录本中产生相关但不同的蛋白质，从而产生多样性。基于基因序列选择性剪接的基因治疗应该比使用单一的cDNA更接近于模仿所有的生理效应，从而可能导致更高的疗效和更少的不良反应。在使用血管内皮生长因子(VEGF)进行血管生成基因治疗的情况下，所有的人类研究都使用了单一的cDNA。然而，生理性血管生成是由至少三种主要的异构体121、165和189协同作用发生的；这些异构体具有微妙的不同性质，在不同的组织中以不同的比例产生。这一建议的重点是将选择性剪接的概念纳入基因治疗的范式，从而更准确地反映所转移基因的真实表型潜力。这一建议的基本假设是，指导混合异构体产生的血管内皮生长因子基因将增强血管生成血管生成的有效性，同时减少使用表达单一异构体的单一cDNA所产生的不良后果。虽然拟议的研究只关注血管内皮生长因子基因，并且仅使用腺病毒(Ad)基因转移载体，但所产生的基本原理应该是适用的 任何基因和任何基因转移载体。在此背景下，该提议的具体目的包括：具体目的1.设计表达不同比例的主要血管内皮生长因子亚型的基因组/cDNA杂合基因，并在体内外证明用这些构建体进行基因转移会导致不同的细胞和组织特异性选择性剪接模式。具体目的2.评估在同一器官的不同细胞类型中表达的杂交基因组/cDNAVEGF基因的剪接模式。具体目的3.评价体内转移旨在产生不同剪接模式的血管内皮生长因子基因组/cDNA杂交体后，导致不同混合血管内皮生长因子异构体的基因转移的有效性和不利表型。

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.