DEVELOPMENT OF HSV VECTORS FOR TREATMENT OF INHERITED DISEASES

单纯疱疹病毒1型(HSV-1)是一种在细胞核内复制的包膜DNA病毒。虽然单纯疱疹病毒在自然界中最常见的是潜伏在感觉神经元中，但它有效地感染并在大量不同类型的细胞和组织中表达其基因组。HSV基因组和衣壳也有相对较大的DNA插入。因此，单纯疱疹病毒作为一种基因载体，可以有效地将相对较大或较多的转基因导入多种细胞和组织类型。野生型单纯疱疹病毒是一种快速增长和极具破坏性的病毒，因为它编码的活动几乎可以增强或扰乱宿主细胞代谢的方方面面。这些功能主要由病毒的即刻早期(IE)蛋白提供。前一个资助阶段的主要目标是消除单纯疱疹病毒IE蛋白的表达，提供高效繁殖多个IE突变株的手段，并评估多个IE基因敲除病毒作为基因载体的潜力。这项系统化的工作最终导致了基于全基因组的载体(D109)的产生，该载体1.不表达任何IE基因，2.对细胞完全无毒，3.可获得相对较高的滴度，4.有效地将其基因组运送到细胞核，以及5.在感染细胞中持续较长时间。我们还发现，由于该载体不表达任何HSV激活蛋白，转基因表达相当低，并且载体基因组的部分可能整合到细胞DNA中(f=约1%)。该方案的目标是完成HSV复制缺陷载体系统的开发和鉴定，以便将其用作通用的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.将开发更有效地将预期的转基因和启动子插入载体骨架的方法，以供评估和使用。2.探索在体内和体外增强或调节转基因表达的合理策略。我们还将检测HSV载体株在体内重新激活先前潜伏的野生型HSV的能力。3.将从随机插入、生化转化以及利用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目的来研究载体与细胞基因组之间的相互作用。这些后面的实验对利用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重要意义。

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.