Lentiviral Vectors for Position-Independent Expression

用于位置无关表达的慢病毒载体

• 批准号: 6537868
• 负责人: Robert G. Hawley
• 金额: $ 34.7万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-15 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537868
• 关键词: Lentivirus; NOD mouse; SCID mouse; chromatin; disease /disorder model; flow cytometry; gene expression; gene therapy; genetic manipulation; genetic promoter element; genetic transduction; green fluorescent proteins; hematopoietic stem cells; hemophilia As; human immunodeficiency virus 1; human tissue; interferon beta; polymerase chain reaction; technology /technique development; tissue /cell culture; transfection /expression vector; virus genetics

DESCRIPTION: (Investigator's abstract) Gene therapy using hematopoietic stem cells (HSCs) as the target cell population has great potential to improve treatment of a wide range of inherited and acquired blood diseases. Replication-defective retroviruses have been the vehicles of choice for gene delivery and expression in HSCs because of their ability to stably integrate into the genome of target cells. For more than a decade, our laboratory has been designing and optimizing retroviral vectors for gene transfer studies of HSC biology. In particular, our MSCV (murine stem cell virus) retroviral vector has proven to be highly efficient at delivering functional genes to the murine hematopoietic system. For this reason, the MSCV platform was chosen for use in two HSC gene therapy trials currently underway in the United States. To date, however, the outcomes of most clinical trials with retroviral vectors have been disappointing. This is believed to be due in part to low surface density of the amphotropic envelope receptor and the fact that retroviral vectors such as MSCV, which are derived from oncoretroviruses, can only integrate into cells undergoing mitosis. Thus it has been proposed that pantropic vectors developed from the lentivirus, human immunodeficiency virus (HJV), which can readily transfer genes into various types of stationary cells, may be more suitable for gene delivery to HSCs, which reside almost exclusively in the G0/G1 phase of the cell cycle. Even if efficient lentivirus-based gene transfer in HSCs is achieved, accumulated data indicate that in vivo transgene expression is frequently subject to transcriptional silencing and position effects. We propose therefore to develop next-generation HIV-based lentiviral vectors expressly for human HSC gene transfer applications. Our hypothesis is that utilization of transcriptional regulatory elements permissive for expression in HSCs in conjunction with chromatin insulator sequences and scaffold/matrix attachment regions will lead to maintenance of high-level transgene expression in HSCs and their differentiated progeny. To this end, the performance of next-generation lentiviral vectors utilizing the MSCV long terminal repeat as an internal promoter and harboring the chicken b-globin 5' constitutive hypersensitive site (5' HS4) insulator and/or the human interferon-b scaffold attachment region (IFN-SAR) will be assessed in human hematopoietic repopulating cells using a surrogate non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenograft assay and in a murine hemophilia A model.

描述：（研究者摘要）以造血干细胞（HSC）为靶细胞群的基因治疗具有巨大的改善潜力 治疗多种遗传性和获得性血液疾病。 复制缺陷型逆转录病毒已成为基因选择的载体 由于 HSC 具有稳定整合的能力，因此能够在 HSC 中进行递送和表达 进入靶细胞的基因组。十多年来，我们的实验室 一直在设计和优化用于基因转移研究的逆转录病毒载体 HSC 生物学。特别是，我们的 MSCV（鼠干细胞病毒）逆转录病毒载体 已被证明能够高效地将功能基因传递给小鼠 造血系统。因此，选择 MSCV 平台用于 目前美国正在进行两项 HSC 基因治疗试验。迄今为止， 然而，大多数逆转录病毒载体临床试验的结果都是 令人失望。据信，这部分是由于表面密度低 双嗜性包膜受体和逆转录病毒载体如 MSCV源自致癌逆转录病毒，只能整合到细胞中 正在进行有丝分裂。因此有人提出泛向性载体的发展 来自慢病毒，人类免疫缺陷病毒（HJV），它可以很容易地 将基因转移到各种类型的静止细胞中，可能更适合 基因递送至 HSC，几乎完全存在于 G0/G1 期 细胞周期。即使 HSC 中基于慢病毒的高效基因转移是 已实现，积累的数据表明体内转基因表达是 经常受到转录沉默和位置效应的影响。我们 因此建议开发下一代基于HIV的慢病毒载体 专门用于人类 HSC 基因转移应用。我们的假设是 利用转录调控元件允许表达 HSC 与染色质绝缘体序列和支架/基质结合 附着区域将导致维持高水平转基因表达 在 HSC 及其分化后代中。为此，性能 利用 MSCV 长末端重复序列作为下一代慢病毒载体 内部启动子并带有鸡 b-珠蛋白 5' 组成型 超敏感位点 (5' HS4) 绝缘体和/或人干扰素-b 支架 附着区（IFN-SAR）将在人类造血系统中进行评估 使用非肥胖糖尿病/严重联合替代品重新填充细胞 免疫缺陷 (NOD/SCID) 异种移植测定和小鼠 A 型血友病模型。