DEVELOPMENT OF IMPROVED HSV AMPLICON VECTORS

在过去的十年中，基因转移方法取得了迅速的进展， 为人类基因治疗的发展创造了机会 神经系统疾病治疗的一个目标是减缓神经元的损失， 神经保护基因的表达。 基因的发展 慢性病的治疗不仅依赖于长期的 治疗性基因产物的表达， 无毒的基因传递系统。 该项目特别关注 精制单纯疱疹病毒（HSV）扩增子载体，基于质粒 需要辅助病毒进行包装的载体。细胞毒性 与扩增子载体储备物相关的基因表达似乎主要是以下功能的函数： HSV立即早期（IE）基因产物辅助病毒表达。我们 计划利用两种方法来产生扩增子储备， 降低细胞毒性：一个包括使用三组分 利用四环素依赖性的调节包装系统 表达IE基因产物以增加扩增子对辅助物的滴度; 第二种方法包括使用Cre重组酶突变体来衍生 切割含loxP的辅助病毒的包装细胞系， 辅助病毒滴度降低， 扩增子载体储备物。

The rapid advances made in the last decade in gene transfer methods have created the opportunity for developing gene therapy for human neurological disease. One goal of therapy is to slow neuron loss, through the expression of neuroprotective genes. The development of gene therapies for chronic diseases critically depends on not only long-term expression of a therapeutic gene product, but also the development of non-toxic gene delivery systems. This project specifically focuses on refining herpes simplex virus (HSV) amplicon vectors, plasmid-based vectors that require a helper virus for packaging. Cytotoxicity associated with amplicon vector stocks appear to be largely a function of helper virus expression of HSV immediate early (IE) gene products. We plan to utilize two approaches to produce amplicon stocks which exhibit reduced cytotoxicity: One includes the use of a three component regulatory packaging system that utilizes tetracycline-dependent expression of IE gene products to increase amplicon to helper titer; the second approach includes the use of Cre recombinase mutants to derive packaging cell lines that cleave loxP-containing helper virus, resulting in reduced helper virus titers and in effect, decreased cytotoxicity in amplicon vector stocks.