SOMATIC CELL HUMAN HPRT TRANSFER

Lesch-Nyhan综合征是一种毁灭性的，最终是致命的 神经功能完全缺乏所致的神经功能障碍 次黄嘌呤-鸟嘌呤磷酸核糖基转移酶(HPRT)。这 无序已被确定为最初的候选之一 体细胞基因替代疗法。其他几个团体是 发展逆转录病毒载体将人HPRT基因转移到 造血干细胞。然而，有几条证据表明 反对这种方法在以下方面的有效性 中枢神经酶缺乏症的矫正 系统。在我们目前资助的赠款中，我们提议建造 一种来源于1型单纯疱疹病毒的嗜神经载体 (HSV-1)将可表达的人HPRT基因转移到神经元 组织。我们已经构建了一种这样的病毒，它可以表达 培养的大鼠神经细胞中的人HPRT。在这个构造中， 复制HPRT基因，从而表达HPRT 活动，需要病毒基因组的复制。因此， 重组病毒仍然具有毒力，尽管程度较小 比野生型单纯疱疹病毒1型更强。 在本提案中，我们描述了一种替代方法 单纯疱疹病毒1型衍生载体的构建。其目的是构建 一种可转移HPRT基因的复制缺陷病毒 在一个自主复制的单元中。自主性小鼠 复制序列(AR)将链接到HPRT微型基因中 哪个人HPRT基因受HSV-1的控制 胸苷激酶启动子。此构造将插入到 编码ICP4基因的HSV-1突变体 已删除。ICP4是病毒复制所必需的，这个突变体是 因此复制是有缺陷的。重组病毒的传代 通过包装细胞系，它已经被 野生型ICP4基因，允许病毒复制和组装 成熟的病毒颗粒。然而，这些病毒粒子仍然存在 复制有缺陷。HPRT缺陷的神经细胞将是 感染了这些缺陷重组体。尽管病毒式传播 不会发生复制，ARS/HPRT最小化应该 以附体的形式复制。这一创新的方法 DNA病毒载体的构建绕过了某些问题 复制能力强的载体系统所固有的。然而， 将需要解决几个关键的实验问题 在这些载体的发展过程中。出于这些原因，这 建议书符合这项拨款所要求的“高风险”标准。 程序。

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.