VASCULAR SMOOTH MUSCLE SPECIFIC GENE THERAPY VECTORS

血管平滑肌特异性基因治疗载体

• 批准号: 6367914
• 负责人: David A Dean
• 金额: $ 16.48万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-10-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6367914
• 关键词: actins; binding proteins; cell proliferation; cytoplasm; electrophoresis; gel mobility shift assay; gene expression; gene therapy; genetic promoter element; in situ hybridization; nucleic acid sequence; plasmids; tissue /cell culture; transcription factor; transfection; transfection /expression vector; vascular smooth muscle

DESCRIPTION (Adapted from Investigator's Abstract): It is presently quite difficult to transfer genes to non-dividing vascular smooth muscle cells (SMC) and this has limited the development of gene therapy approaches for the treatment of human vascular diseases such as atherosclerosis and restonosis. The applicant has developed a novel strategy that may overcome this problem and has focused upon mechanisms of DNA fate in cytosol. They have demonstrated that plasmid DNA nuclear import in non-dividing eukaryotic cells occurs through the nuclear pore complex and this DNA nuclear traffick is sequence specific. Moreover, they have identified a DNA sequence that mediates nuclear import of plasmid DNA in only smooth muscle cells. The DNA is the proximal portion of the smooth muscle H-actin (SMGA) promoter which contains binding sites for several SMC-specific transcription of this promoter and it is hypothesized that they also mediate the nuclear import of DNA. The hypothesis predicts that transfactors containing nuclear localization signals (NLS) for their nuclear import bind to specific SMGA DNA sequences thereby coating the DNA with NLSs and allowing the DNA to utilize the NLS-mediated import machinery for nuclear entry. If the transfactors are expressed uniquely in SMCs, import should occur only in those cells. The experiments in this application will utilize four specific aims to examine this hypothesis. The specific aims are: 1) To identify DNA sequences in the SMGA promoter needed for smooth muscle cell-specific DNA import. This will be done by studying plasmid containing portions of this promoter, using microinjection and in situ hybridization to identify sequences that mediate import in SMCs only. To define the transcription factors which bind to imported SMGA sequences and potentially mediate cell selective import. This will employ electrophoretic mobility shift assays to identify the respective binding proteins interacting with the sequences identified in Aim 1. Their role will be tested by transfecting their genes into non- muscle cells to reconstitute cell-specific nuclear import. 3) To identify the cellular factors involved in DNA nuclear import. Here they will test the hypothesis that transcription factors identified in Aim 2 and which are unique to smooth muscle cells can mediate SMGA DNA nuclear import using a permeabilized cell assay. The smooth muscle specificity will be confirmed by also studying non-muscle cells as well. 4) To test the efficacy and cell-specificity of SMGA promoter constructs in an in vitro vascular smooth muscle cell proliferation model. Here they will test the ability of the imported sequences to increase cell-specific gene expression by transfection, and apply this to an in vitro model and prevent SMC proliferation.

描述（改编自研究者摘要）：目前相当 难以将基因转移到非分裂的血管平滑肌细胞 (SMC)这限制了基因治疗方法的发展， 治疗人血管疾病如动脉粥样硬化， 复张申请人开发了一种新的策略，可以克服 这个问题，并集中在细胞质中的DNA命运的机制。他们 已经证明，质粒DNA核输入在非分裂 真核细胞通过核孔复合体发生， 核武器是有序列特异性的此外，他们还确定了一个 仅在平滑的细胞中介导质粒DNA的核输入的DNA序列 肌肉细胞DNA是平滑肌H-肌动蛋白的近端部分 （SMGA）启动子，其含有几个SMC特异性结合位点。 该启动子的转录，并且假设它们也 介导DNA的核输入。该假说预测， 含有核定位信号（NLS）的转因子， 核输入结合到特定的SMGA DNA序列，从而包被DNA 并允许DNA利用NLS介导的导入 核武器进入。如果转因子在 SMC，导入应仅发生在这些单元格中。这个实验 本申请将利用四个具体目标来检验这一假设。 具体目的是：1）鉴定SMGA启动子中的DNA序列 平滑肌细胞特异性DNA输入所需的。会来做这项工作 研究含有该启动子部分的质粒，使用 显微注射和原位杂交来鉴定 仅在SMC中介导导入。为了定义转录因子， 与导入的SMGA序列结合，并可能介导细胞选择性 导入.这将采用电泳迁移率变动分析，以确定 与鉴定的序列相互作用的相应结合蛋白 目标1。他们的作用将通过将他们的基因转移到非- 肌细胞来重建细胞特异性核输入。3)以识别 参与DNA核输入的细胞因子。在这里他们将测试 在Aim 2中鉴定的转录因子， 是平滑肌细胞所特有的，可以介导SMGA DNA核输入 使用透化细胞测定。平滑肌特异性将是 也通过研究非肌肉细胞得到证实。4)测试 SMGA启动子构建体在体外试验中功效和细胞特异性 血管平滑肌细胞增殖模型。在这里，他们将测试 导入序列增加细胞特异性基因的能力 表达，并将其应用于体外模型， 防止SMC增殖。