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SOMATIC CELL HUMAN HPRT TRANSFER

体细胞人体 HPRT 转移

基本信息

批准号：
3426119
负责人：
WILLIAM N KELLEY
金额：
$ 4.74万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-09-01 至 1988-08-31
项目状态：
已结题

项目摘要

The Lesch-Nyhan syndrome is a devastating and ultimately fatal neurological disorder caused by the complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). This disorder has been identified as one of the initial candidates for somatic cell gene replacement therapy. Several other groups are developing retroviral vectors to transfer the human HPRT gene to hematopoietic stem cells. However, several lines of evidence argue against the efficacy of this approach with respect to correction of the enzyme deficiency in the central nervous system. In our currently funded grant, we proposed construction of a neurotropic vector, derived from herpes simplex virus type 1 (HSV-1) to transfer an expressible human HPRT cDNA to neuronal tissue. We have constructed one such virus which expresses human HPRT in cultured rat neuronal cells. In this construct, replication of HPRT cDNA, and hence expression of HPRT activity, requires replication of the viral genome. Thus, the recombinant virus remains virulent, although to a lesser degree than wild-type HSV-1. In this proposal, we describe an alternative approach to the development of an HSV-1 derived vector. The aim is to construct a replication defective virus capable of transferring HPRT cDNA within an autonomously replicating unit. Murine autonomously replicating sequences (ARS) will be linked to an HPRT minigene in which human HPRT cDNA is under the control of the HSV-1 thymidine kinase promoter. This construct will be inserted into an HSV-1 mutant from which the gene encoding ICP4 has been deleted. ICP4 is required for viral replication and this mutant is therefore replication defective. Passage of the recombinant virus through a packaging cell line, which has been transformed by the wild-type ICP4 gene, allows viral replication and assembly of mature virus particles. These virions, however, remain replication defective. HPRT deficient neuronal cells will be infected with these defective recombinants. Although viral replication will not occur, the ARS/HPRT minigenes should replicate as episomes. This innovative approach to the construction of DNA viral vectors circumvents certain problems inherent in replication-competent vector systems. However, several critical experimental questions will need to be addressed during development of such vectors. For these reasons, this proposal meets the standard of "high risk" required by this grant program.

莱希-尼汉综合征是一种毁灭性的，由于完全缺乏神经系统疾病，次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）。这疾病已被确定为最初的候选人之一，体细胞基因替代疗法其他几个团体是开发逆转录病毒载体以转移人HPRT基因，造血干细胞。然而，有几条证据表明，反对这种方法的有效性，纠正中枢神经系统中的酶缺乏系统在我们目前的拨款中，我们提议建造来自单纯疱疹病毒1型的亲神经性载体将可表达的人HPRT cDNA转移至神经元细胞组织. 我们已经构建了一种这样的病毒，人HPRT在培养的大鼠神经元细胞中的表达。在这个结构中，复制HPRT cDNA，从而表达HPRT 活性，需要病毒基因组的复制。因此重组病毒仍然具有毒性，尽管程度较低野生型HSV-1。在本提案中，我们描述了一种替代方法， HSV-1衍生载体的开发。其目的是构建能够转移HPRT cDNA的复制缺陷型病毒在一个自主复制的单元中。小鼠自主复制序列（ARS）将连接到HPRT小基因，人HPRT cDNA受HSV-1的控制，胸苷激酶启动子。该结构将被插入到 HSV-1突变体，其中编码ICP 4的基因已被的页面不存在或 ICP 4是病毒复制所需的，这种突变体是因此复制缺陷。重组病毒传代通过一个包装细胞系，它已经被转化，野生型ICP 4基因，允许病毒复制和组装，成熟病毒颗粒然而，这些病毒体仍然存在，复制缺陷。 HPRT缺陷的神经元细胞将被感染了这些缺陷重组体尽管病毒不会发生复制，ARS/HPRT小基因应该作为游离体复制。这种创新的方法， DNA病毒载体的构建避免了某些问题在复制能力载体系统中固有的。然而，在这方面，需要解决几个关键的实验问题在这些载体的发展过程中。由于这些原因，这建议书符合本补助金所要求的“高风险”标准程序.