STEREOTAXIC GENE THERAPY TO THE RHESUS CNS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。中枢神经系统是基因治疗的主要目标，因为广泛的遗传和获得性疾病会影响大脑。神经退行性疾病，如帕金森病、亨廷顿病、阿尔茨海默病和肌萎缩性侧索硬化症（ALS）是影响老年人的最常见疾病，因此是基因治疗发展的主要目标。尽管罕见，遗传性溶酶体储存疾病和白质营养不良，如Tay-Sach病、粘多糖病和Krabbe病，也是开发新的基因治疗干预措施的主要目标。这些和其他中枢神经系统疾病的治疗选择主要局限于支持性护理，因此提供的治疗选择很少。直接中枢神经系统基因转移可能为消除与这些疾病相关的病理提供了最好的机会。本研究的目的是比较重组慢病毒（LV）和腺相关病毒（AAV）载体系统在非人灵长类动物（特别是恒河猴）中枢神经系统中基因转移水平、（细胞和结构）模式和基因表达持久性方面的转导。本提案的最终目的是确定哪种载体或载体组合在与人类非常密切相关的动物模型中提供高水平的基因转移到最广泛的细胞类型。该建议的主要优点是，两种病媒系统的准备工作将使用一致的生产方案进行准备，并直接向中枢神经系统管理。我们已经进行了一系列的基因转移和生物分布研究，使用直接立体定向注射这些重组病毒制剂来靶向大脑的多个区域，如海马、皮质、尾状壳核/纹状体、丘脑和小脑。在使用载体制剂后，我们目前正在绘制各种载体的转导模式，通过确定基因转移到大脑选定区域的水平，并通过免疫组织化学和共聚焦显微镜描绘转导的特定细胞类型。我们目前的数据表明，不同血清型AAV （AAV 1、2、7、9和10）载体提供的基因转移模式存在显著差异。VSV-G假型慢病毒载体的生物分布表明，基因可以广泛转移到许多细胞类型，但效率低于AAV载体。提出的转化研究的结果将为设计和执行人类，特别是儿童的中枢神经系统导向的基因治疗方案提供宝贵的信息。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The central nervous system is a primary target for gene therapy because the broad spectrum of inherited and acquired disorders that affect the brain. Neurodegenerative diseases such as Parkinson¿s disease, Huntington¿s disease, Alzheimer¿s disease, and amyotrophic lateral sclerosis (ALS) are among the most common disorders that affect older humans and as such are prime targets for the development of gene therapy. Although rare, inherited lysosomal storage disorders and leukodystrophys, such as Tay-Sach¿s, mucopolysaccharidosis and Krabbe¿s disease, are also primary targets for the development of novel gene therapy interventions. Treatment options for these and other CNS disorders are primarily limited to supportive care, thus providing few therapeutic options. Direct CNS gene transfer may provide the best opportunity for eliminating the pathology associated with these conditions. The goal of this proposal is the comparison of the transduction of recombinant lentivirus (LV) and adeno-associated virus (AAV) vector systems with regard to levels of gene transfer, pattern of (cells and structures), and persistence of gene expression in the CNS of nonhuman primates, specifically rhesus macaques. The ultimate aim of this proposal is to determine which vector or combination of vectors provides high levels of gene transfer to the broadest range of cell types in an animal model that is very closely related to humans. The primary advantage of this proposal is that preparations of both vector systems will be prepared using consistent production protocols and administered directly to the CNS. We have performed a series of gene transfer and biodstribution studies using direct stereotaxic injection of these recombinant virus preparations to target multiple regions of the brain, such as the hippocampus, cortex, caudate putamen/striatum, thalamus and cerebellum. Following administration of the vector preparations, we are currently mapping the transduction patterns of the various vectors by determining the levels of gene transfer to selected regions of the brain and delineating the specific cell types transduced by immunohistochemistry and confocal microscopy. Our current data suggests that marked differences in the patterns of gene transfer provided by various serotypes of AAV (AAV 1, 2, 7, 9 and 10) vectors can be detected. The biodistribution of VSV-G pseudotyped lentivirus vectors demonstrated broad gene transfer to many cell types, but at lower efficiency than AAV vectors. . The results of the proposed translational studies will serve as invaluable information on the design and execution of CNS-directed gene therapy protocols in humans, especially children.