High-titer helper virus-free HSV-1 vectors

高滴度辅助无病毒 HSV-1 载体

基本信息

批准号：
7277800
负责人：
ALFRED I. GELLER
金额：
$ 24.46万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-01 至 2008-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7277800
关键词：
Adenovirus Vector Adenoviruses Adverse effects Affect Age Aging Appendix Aromatic-L-Amino-Acid Decarboxylases Bacterial Artificial Chromosomes Behavioral Biochemical Biological Assay Brain Brain Neoplasms Cell Count Cell Line Cell Nucleus Cells Complement Convection Corpus striatum structure Cosmids DNA Disease Dopamine Early Promoters Elements Event Fibroblasts Galactosidase Gene Expression Gene Transfer Genes Genetic Genetic Recombination Genetic Transcription Genome Goals Guanosine Triphosphate HSV-1 vector Helper Viruses Herpesvirus 1 Homebound Persons Human Human Herpesvirus 4 Hybrids Immediate-Early Genes Inflammatory Response Injection of therapeutic agent Laboratories Lentivirus Vector Measures Methods Modeling Mutate Neurodegenerative Disorders Neurons Numbers Oxidopamine Parkinson Disease Plasmid Cloning Vector Plasmids Procedures Production Property Prosencephalon Proteins Rattus Recombinants Reporting Research Personnel Retroviridae Safety Sequence Homology Simplexvirus Site Standards of Weights and Measures Subfamily lentivirinae System Technology Testing Transfection Transgenic Mice Tyrosine 3-Monooxygenase United States National Institutes of Health Viral Genes Virus adeno-associated viral vector base design gene therapy gutless adenoviral vector homologous recombination nervous system disorder neurotropic virus promoter recombinase size vector vesicular monoamine transporter

项目摘要

DESCRIPTION (provided by applicant): HSV-1 vectors are attractive for gene therapy of aging disorders that affect the brain because HSV-1 can persist indefinitely in neurons in the latent state and large HSV-1 vectors can coexpress multiple genes. This laboratory has developed a Herpes Simplex Virus (HSV-1) plasmid vector system for gene transfer into neurons. Using this system, we have begun to explore gene therapy approaches to specific aging disorders that affect the brain, such as Parkinson's Disease (PD). We have shown that delivery of a HSV-1 vector that expresses human tyrosine hydroxylase into the partially denervated striatum in the 6-hydroxydopamine rat model of PD results in long-term (1 year) biochemical and behavioral correction. Other investigators have demonstrated the potential of using this vector system for gene therapy for a number of other neurological disorders. We developed a helper virus-free packaging system for these HSV-1 vectors. This improvement substantially reduces the cytopathic effects and the inflammatory response previously associated with gene transfer. Furthermore, we and others have recently identified specific promoters that support long-term expression in rat forebrain neurons. However, the relatively low titers remain one of the primary barriers to the use of this vector system for human gene therapy of aging disorders that affect the brain. The goal of this proposal is to develop a packaging cell line that can produce high-titer helper virus-free HSV-1 vector stocks. High-titer retrovirus, lentivirus, and adenovirus vector stocks have been produced using packaging cell lines, and these vector stocks have been used in human gene therapy. The first specific aim will isolate a cell line that stably maintains an HSV-1 genome that does not express any immediate early (IE) genes and lacks a packaging site. The second specific aim will produce high-titer, helper virus-free HSV-1 vector stocks by using an HSV-1 vector that contains the 3 essential IE genes flanked by lox sites. This packaging system is based upon standard genetic complementation. High-titer vector stocks will be produced by serial passaging. To excise the IE genes from the vector, the final passage will use a cell line that expresses Cre recombinase. The third specific aim will evaluate the safety features of this packaging system. The fourth specific aim will use these high-titer vector stocks to achieve gene transfer to large numbers of cells in the rat striatum.

描述（由申请人提供）：HSV-1载体对于影响大脑的衰老疾病的基因治疗很有吸引力，因为HSV-1可以在潜伏状态下无限期地存在于神经元中，并且大的HSV-1载体可以共表达多个基因。该实验室开发了一种单纯疱疹病毒（HSV-1）质粒载体系统，用于将基因转移到神经元中。利用这个系统，我们已经开始探索针对影响大脑的特定衰老疾病的基因治疗方法，例如帕金森病（PD）。我们已经证明，将表达人酪氨酸羟化酶的 HSV-1 载体递送到 6-羟基多巴胺 PD 大鼠模型中部分去神经的纹状体中，可以实现长期（1 年）的生化和行为纠正。其他研究人员已经证明了使用该载体系统对许多其他神经系统疾病进行基因治疗的潜力。我们为这些 HSV-1 载体开发了辅助无病毒包装系统。这种改进大大减少了先前与基因转移相关的细胞病变效应和炎症反应。此外，我们和其他人最近发现了支持大鼠前脑神经元长期表达的特定启动子。然而，相对较低的滴度仍然是使用该载体系统对影响大脑的衰老疾病进行人类基因治疗的主要障碍之一。该提案的目标是开发一种包装细胞系，能够产生高滴度的无辅助病毒 HSV-1 载体原液。使用包装细胞系生产了高滴度逆转录病毒、慢病毒和腺病毒载体原液，这些载体原液已用于人类基因治疗。第一个具体目标是分离稳定维持 HSV-1 基因组的细胞系，该基因组不表达任何立即早期 (IE) 基因并且缺乏包装位点。第二个具体目标是通过使用包含侧翼为 lox 位点的 3 个必需 IE 基因的 HSV-1 载体来生产高滴度、无辅助病毒的 HSV-1 载体原种。该包装系统基于标准的遗传互补。高滴度载体库存将通过连续传代产生。为了从载体中切除 IE 基因，最后的传代将使用表达 Cre 重组酶的细胞系。第三个具体目标将评估该包装系统的安全特性。第四个具体目标是利用这些高滴度载体库实现基因转移到大鼠纹状体中的大量细胞。