DEVELOPMENT OF HSV VECTORS FOR TREATMENT OF INHERITED DISEASES

单纯疱疹病毒1型（HSV-1）是一种在细胞核中复制的包膜DNA病毒。虽然HSV在自然界中最常以潜伏状态存在于感觉神经元中，但它在大量不同的细胞类型和组织中有效感染并表达其基因组。还已知HSV基因组和衣壳容纳相对大的DNA插入物。因此，作为基因递送载体，HSV可用于将相对大的或多个转基因有效地载体到广泛的细胞和组织类型中。野生型HSV是一种生长迅速且极具破坏性的病毒，这是由于其编码的活性增强或扰乱宿主细胞代谢的几乎每个方面。这些功能主要由病毒的立即早期（IE）蛋白提供。上一个资助期的主要目标是废除HSV IE蛋白的表达，提供有效繁殖多种IE突变体的方法，并评估多种IE基因敲除病毒作为基因递送载体的潜力。这种系统性的工作最终导致了基于全基因组的载体（d109）的衍生，其：1.不表达任何IE基因，2.对细胞完全无毒，3.可以以相对高的滴度获得，4.有效地将其基因组递送到细胞核，以及5.在受感染的细胞中持续很长时间。我们还发现，由于该载体不表达任何HSV激活蛋白，因此转基因表达相当低，并且载体基因组的部分可以整合到细胞DNA中（f=约1%）。本提案的目的是完成HSV复制缺陷型载体系统的开发和表征，因为它可用作通用的DNA载体和表达系统。作为模型转基因，我们将使用GFP，neo/I，tk和HPRT，并提出3个具体目标：1。将开发方法以更有效地将预期的转基因和启动子插入载体骨架中用于评估和使用。2.将探讨在体内和体外增强或调节转基因表达的合理策略。我们还将检查HSV载体株在体内重新激活先前潜伏的野生型HSV的能力。3.将研究载体和细胞基因组之间的相互作用，包括随机插入、生物化学转化以及HSV作为载体有效递送基因组DNA以促进病毒和细胞之间同源重组的潜在用途。这些后面的实验对使用HSV以可遗传的方式改变细胞，和/或精确敲除或修复细胞基因具有意义。

Herpes Simplex Virus, type 1 (HSV-1) is an enveloped DNA virus that replicates in the nucleus. While HSV is most often found in nature in a latent state in sensory neurons, it efficiently infects and expresses its genome in a very large number of different cell types and tissues. The HSV genome and capsid are also known to accommodate relatively large DNA inserts. Therefore, as a gene delivery vehicle HSV can be used to efficiently vector relatively large or multiple transgenes into a wide range of cell and tissue type. Wild-type HSV is a rapidly growing and very destructive virus, due to activities it encodes that augment or perturb almost every aspect of host cell metabolism. These functions are largely provided by the immediate early (IE) proteins of the virus. A major goal of the previous funding period was to abrogate the expression of HSV IE proteins, provide the means to efficient propagate multiple IE mutants, and evaluate the potential of multiple IE gene knock out viruses as gene delivery vehicles. This systematical undertaking ultimately resulted in the derivation of a whole genome based vector (d109) that; 1. does not express any IE genes, 2. is completely non-toxic to cells, 3. can be obtained in relatively high titers, 4. efficiently delivers its genome to the nucleus, and 5. persists in infected cells for prolonged periods of time. We also found that because this vector does not express any of the HSV activator proteins, transgene expression is fairly low, and that parts of vector genome may integrate into cellular DNA (f= approximately 1%). The goal of this proposal is to complete the development and characterization of the HSV replication-defective vector system for it uses as a general DNA vectoring and expression system. As model transgenes we will use GFP, neo/I, tk and HPRT, and propose 3 specific aims; 1. Methods will be developed to more efficiently insert prospective transgenes and promoters into the vector backbone for evaluation and use. 2. Rational strategies for augmenting or regulating transgene expression in vivo and in vitro will be explored. We will also examine the ability of vector strains of HSV to reactivate previous latent wild type HSV in vivo. 3. The interaction between the vector and cellular genome will be investigated with respect to random insertion, biochemical transformation, and the potential use of HSV as a vector to efficiently deliver genomic DNA for the purpose of promoting homologous recombination between the virus and cell. These latter experiments have implications for the use of HSV to alter cells in an inheritable way, and/or to precisely knock out or repair cellular genes.