GENE TRANSFER AND EXPRESSION IN STEM CELLS

干细胞中的基因转移和表达

• 批准号: 6110745
• 负责人: Maria L. Zapp
• 金额: $ 15.38万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110745
• 关键词: Retroviridae; antiAIDS agent; antisense nucleic acid; antiviral agents; crosslink; gene therapy; genetic transduction; helper T lymphocyte; hematopoietic stem cells; human immunodeficiency virus 1; in situ hybridization; tissue /cell culture; transfection /expression vector; virus protein; virus replication; western blottings

The identification of human immunodeficiency virus type 1 (HIV-1) as the etiological agent of AIDS has led to the proposal of novel intervention strategies to block HIV-1 infection and viral replication. Because HIV-1 infection can be detected early, when the vast majority of the target cells are uninfected, gene therapy is an attractive approach. Our long-ter strategy is to stably transduce pluripotent hematopoietic stem cells with anti-HIV-1 agents that will confer "intracellular immunity" to progeny CD4+ lymphocytes and macrophages, athe permissive host for HIV-1. This approach necessitates; (1) the ability to functionally transduce pluripotent hematopoietic stem cells; and (2) an agent that confers resistance to HIV- 1. We will systematically test the ability of established oncoretroviral and AAV-based gene transfer vectors to functionally transduce human and murine hematopoietic stem cells. We will also examine various well- established methods for gene transfer for their ability to introduce and express transgenes with minimal disruption to cell viability and renewal potential. We will also actively explore the use of HIV-1 pseudotyped vectors. As described in the proposal, these vectors may be advantageous for targetting non-dividing cells., such as pluripotent stem cells. The efficacy of these HIV-based vectors will be assessed in parallel with oncoretroviral and AAV-vectors. Secondly, we will continue to develop the inhibitory potential of two novel peptide inhibitors of the essential HIV-1 regulatory protein, Rev. We have previously identified two novel protein inhibitors of Rev; a new class of dominant-negative Rev mutants and a selected peptide that binds to Rev's effector domain. The potency of these new Rev inhibitors suggest they may be efficacious anti-viral agents. We will perform a series of experiments to directly test their ability to block HIV-1 replication. We will continue to analyze their mechanism of action using biochemical structure- function experiments. These studies will facilitate the development of other potent peptide derivatives.

人类免疫缺陷病毒1型（HIV-1）的鉴定为 艾滋病的病因学家导致了新干预的建议 阻止HIV-1感染和病毒复制的策略。 因为HIV-1 当绝大多数目标细胞时，可以尽早检测到感染 未感染，基因疗法是一种有吸引力的方法。 我们的长期 策略是稳定地转导多能造血干细胞 抗HIV-1代理将赋予后代CD4+的“细胞内免疫” 淋巴细胞和巨噬细胞，HIV-1的宽松宿主。 这种方法 需要 （1）功能传递多能的能力 造血干细胞； （2）赋予对艾滋病毒抗性的药物 1。我们将系统地测试已建立的癌症病毒的能力 和基于AAV的基因转移向量在功能上转导人类和 鼠造血干细胞。 我们还将检查各种好处 建立的基因转移方法，以引入和 对细胞活力和更新的细胞生存力的最小破坏的快速转基因 潜在的。 我们还将积极探索HIV-1假型的使用 向量。 如提案中所述，这些向量可能是有利的 用于靶向非分散细胞，例如多能干细胞。 这 这些基于HIV的载体的功效将与 癌症病毒和AAV向量。 其次，我们将继续发展两个新颖的抑制潜力 基本HIV-1调节蛋白的肽抑制剂，Rev.我们有 先前已经确定了两种新型Rev的蛋白质抑制剂。 一个新的类 主导阴性的REV突变体和与Rev结合的选定肽 效应域。 这些新的Rev抑制剂的效力表明他们可能 有效的抗病毒剂。 我们将执行一系列实验 直接测试其阻止HIV-1复制的能力。 我们将 继续使用生化结构分析其作用机理 - 功能实验。 这些研究将促进 其他有效的肽衍生物。