DEVELOPMENT OF HSV VECTORS FOR TREATMENT OF INHERITED DISEASES

1型单纯疱疹病毒（HSV-1）是一种包膜DNA病毒，在细胞核内复制。虽然HSV通常在感觉神经元中处于潜伏状态，但它可以有效地感染并在大量不同的细胞类型和组织中表达其基因组。HSV基因组和衣壳也可以容纳相对较大的DNA插入物。因此，HSV作为一种基因传递载体，可以有效地将较大或多个转基因载体转移到广泛的细胞和组织类型中。野生型HSV是一种快速生长和极具破坏性的病毒，因为它编码的活动可以增强或扰乱宿主细胞代谢的几乎所有方面。这些功能主要是由病毒的即时早期蛋白提供的。先前资助期的一个主要目标是废除HSV IE蛋白的表达，提供有效传播多种IE突变体的手段，并评估多种IE基因敲除病毒作为基因传递载体的潜力。这一系统的工作最终导致了基于全基因组的载体的衍生（d109）；1. 不表达任何IE基因；对细胞完全无毒。可以获得相对较高的滴度，4。有效地将其基因组传递给细胞核，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，因此转基因表达量相当低，并且部分载体基因组可能整合到细胞DNA中（f=约1%）。本提案的目标是完成HSV复制缺陷载体系统的开发和表征，使其成为通用的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. 将开发更有效地将有希望的转基因和启动子插入载体主干以供评估和使用的方法。2. 将探索在体内和体外增加或调节转基因表达的合理策略。我们还将检验HSV载体株在体内重新激活先前潜伏的野生型HSV的能力。3. 载体与细胞基因组之间的相互作用将在随机插入、生化转化以及HSV作为载体有效传递基因组DNA以促进病毒与细胞之间同源重组的潜在用途方面进行研究。这些后一种实验对利用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启示意义。

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.