Lentiviral Vectors for Position-Independent Expression

描述：(研究人员摘要)以造血干细胞(HSCs)为靶细胞群体的基因治疗具有巨大的改进潜力 治疗多种遗传性和获得性血液病。 复制缺陷逆转录病毒已成为基因研究的首选载体 由于其稳定整合的能力，在HSCs中的传递和表达 进入目标细胞的基因组。十多年来，我们的实验室已经 一直在设计和优化逆转录病毒载体，用于基因转移研究 HSC生物学。特别是，我们的MSCV(小鼠干细胞病毒)逆转录病毒载体 已被证明能高效地将功能基因传递给小鼠 造血系统。出于这个原因，选择MSCV平台用于 两项HSC基因治疗试验目前正在美国进行。到目前为止， 然而，大多数逆转录病毒载体临床试验的结果是 令人失望。这被认为部分是由于表面密度较低的 两性包膜受体和逆转录病毒载体，如 源自肿瘤逆转录病毒的MSCV只能整合到细胞中 正在进行有丝分裂的。因此，有人提出了泛向矢量的发展 来自慢病毒，人类免疫缺陷病毒(HJV)，它很容易 将基因转移到各种类型的静止细胞中，可能更适合 向HSCs的基因传递，几乎完全驻留在G0/G1期 细胞周期。即使在HSCs中基于慢病毒的有效基因转移 已经实现，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，建议开发基于HIV的下一代慢病毒载体 专门用于人类HSC基因转移的应用。我们的假设是 允许表达的转录调控元件的利用 HSCs与染色质绝缘子序列和支架/基质结合 附着区将导致保持高水平的转基因表达 在HSCs及其分化的后代中。为此， 利用MSCV长末端重复序列AS构建新一代慢病毒载体 一种内部启动子，含有鸡b-珠蛋白5‘组分 超敏部位(5‘HS4)绝缘体和/或人干扰素-b支架 将在人类造血系统中评估附着区(干扰素-SAR) 使用非肥胖型糖尿病/重症糖尿病联合替代物重新填充细胞 免疫缺陷(NOD/SCID)异种移植试验和小鼠血友病A模型。

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.