Development of integrating HSV amplicons for Parkinson's disease

基因转移方法为开发帕金森氏病(PD)等人类神经系统疾病的基因治疗创造了机会。腺相关病毒(AAV)和慢病毒载体都整合到宿主细胞染色体中，已被证明在帕金森病动物模型中提供长期的基因表达和治疗效果。然而，这些载体表现出一些缺点，其中之一是无法携带大的转基因片段。基于单纯疱疹病毒(HSV)的基因治疗载体为帕金森病新疗法的开发提供了许多优点。这些包括广泛的细胞取向，大的DNA包装容量，允许表达多个 基因，转导效率高。我们建议修改HSV来源的扩增载体，以提供稳定的黑质纹状体细胞特异性基因表达。对于这个管道项目，我们将首先构建新的扩增子整合形式，它将指导报告基因产物在纹状体和黑质细胞中的特异性表达。DNA绝缘体元件将被引入，以促进整合转录单位保持在等染状态。这些新载体将与表达睡美人转座酶的扩增子共同注射到未受损大鼠的纹状体内，在那里将通过实验获得基因表达持续时间和细胞类型特异性。然后，这些载体将被用来传递神经胶质 在6-OHDA诱导的大鼠损毁前应用细胞系衍生神经营养因子(GDNF)，以评估新载体系统保护黑质纹状体通路的能力。这些研究将产生一种新的HSV载体系统，提供对转基因在体内表达的详细了解，并评估其在保护PD啮齿动物模型黑质纹状体神经元方面的治疗效果。

Gene transfer methodologies have created the opportunity for developing gene therapy for human neurological diseases such as Parkinson's Disease (PD). Adeno-associated virus (AAV) and lentivirus vectors, both of which integrate into the host cell chromosome, have been shown to provide long-term expression of genes and therapeutic efficacy in animal models of PD. These vectors, however, exhibit a number of disadvantages, among them an inability to harbor large transgene segments. Gene therapy vectors based upon the Herpes Simplex virus (HSV) offer numerous advantages for the development of novel therapeutics for PD. These include broad cellular tropism, large DNA packaging capacity that allows for expression of multiple genes, and high transduction efficiency. We propose to modify HSV-derived amplicon vectors to provide stable nigrostriatal cell-specific gene expression. For this pipeline project, we will initially construct novel integrating forms of the amplicon that will direct expression of a reporter gene product specifically within cells of the striatum and substantia nigra. DNA insulator elements will be introduced to facilitate maintenance of the integrated transcription unit in a euchromatic state. These new vectors will be co-administered with an amplicon expressing the Sleeping Beauty transposase into the striata of unlesioned rats, where gene expression duration and cell-type specificity will be experimentally derived. The vectors will then be used to deliver glial cell line-derived neurotrophic factor (GDNF) prior to 6-OHDA-induced lesioning of rats to assess the capacity of the new vector system to confer protection to the nigrostriatal pathway. The proposed studies will yield a novel HSV vector system, provide a detailed understanding of transgene expression in vivo, and evaluate its therapeutic effectiveness in protecting nigrostriatal neurons in a well-established rodent model of PD.