Lentiviral Vectors for Position-Independent Expression

描述：（研究者摘要）使用造血干细胞的基因治疗 作为靶细胞群的造血干细胞（HSC）具有很大的潜力， 治疗广泛的遗传性和获得性血液疾病。 复制缺陷型逆转录病毒一直是基因治疗的首选载体。 因为它们能够稳定地整合 导入靶细胞的基因组中。十多年来，我们的实验室 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（鼠干细胞病毒）逆转录病毒载体 已被证明在将功能性基因传递给小鼠方面是高效的 造血系统因此，选择MSCV平台用于 目前在美国进行的两项HSC基因治疗试验。到目前为止， 然而，大多数逆转录病毒载体临床试验的结果 失望据信这部分是由于所述聚合物的低表面密度。 嗜亲性包膜受体以及逆转录病毒载体如 来源于肿瘤逆转录病毒的MSCV只能整合到细胞中， 进行有丝分裂。因此，有人提出泛热带媒介的发展 从慢病毒，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，可能更适合于 基因递送到HSC，其几乎完全位于G 0/G1期， 细胞周期即使在HSC中有效的基于慢病毒的基因转移是可行的， 获得，累积的数据表明，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，我建议开发下一代基于HIV的慢病毒载体 明确用于人HSC基因转移应用。我们的假设是 利用允许表达的转录调节元件， HSC与染色质绝缘子序列和支架/基质结合 附着区将导致高水平转基因表达的维持 在HSC及其分化的后代中。为此， 利用MSCV长末端重复序列作为 一个内部启动子，含有鸡B-珠蛋白5'组成型 超敏位点（5'HS 4）绝缘子和/或人干扰素-b支架 将在人造血干细胞中评估粘附区（IFN-SAR） 使用替代非肥胖糖尿病/重度糖尿病组合 免疫缺陷（NOD/SCID）异种移植物测定和鼠血友病A模型中。

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.