STEREOTAXIC GENE THERAPY TO THE RHESUS CNS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 中枢神经系统（CNS）是基因治疗的主要靶点，因为影响大脑的广泛的遗传性和获得性疾病。神经退行性疾病如帕金森氏病、阿尔茨海默氏病和肌萎缩侧索硬化症是基因治疗的主要目标。虽然罕见，但遗传性溶酶体贮积症和脑白质营养不良，如粘多糖样变性和克拉伯病，也是基因治疗的关键靶点。大多数CNS疾病的治疗选择仅限于支持性治疗，因此提供的治疗选择很少。直接CNS基因转移可能是消除疾病和功能障碍的最有效选择。这些研究的目的是直接比较腺相关病毒（AAV）载体系统的转导水平和恒河猴中枢神经系统中的基因转移模式。通过确定基因转移到脑的选定区域的水平并通过免疫组织化学和显微镜描绘转导的细胞类型来绘制各种载体的转导模式。 我们比较了6种血清型的重组AAV载体（AAV 2/1、AAV 2基因组和AAV 1衣壳、AAV 2/2和AAV 2/7、AAV 2/8、AAV 2/9和AAV 2/rh 10）通过立体定向注射在成年恒河猴（3-6岁）中枢神经系统中的能力。恒河猴经历PBS或AAV血清型注射到脑的各个半球中。术后4周对所有动物进行尸检。通过直接荧光共聚焦显微镜分析，所有AAV血清型的注射导致可检测的EGFP活性。在注射道周围以及白色和灰质中检测到高频率的EGFP阳性细胞。EGFP阳性细胞容易在以下结构中检测到：尾状核、海马、壳核、黑质、苍白球、杏仁核、小脑和脊髓小脑束。一旦检测到EGFP阳性细胞，使用几种神经元和神经胶质细胞抗体进行免疫组织化学，包括GFAP、S100、IBA-1、NeuN、Map 2和NSE。发现EGFP阳性细胞与所有测试的抗原共定位在整个CNS的各种结构中。除AAV 2/8外，所有载体血清型在白色物质中产生显著水平的EGFP阳性细胞。在距离注射道4-6 mm处检测到表达EGFP的细胞。将载体转移到对侧半球显示EGFP蛋白阳性轴突体，但未检测到载体转导的细胞。没有与外科手术或在脑或脊髓中注射任何载体相关的炎症或变性证据。拟议的翻译研究的结果将作为设计和执行CNS指导的基因治疗方案在人类，特别是儿童的宝贵信息。

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 (CNS) 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, Alzheimer's disease, and amyotrophic lateral sclerosis are prime targets for gene therapy. Although rare, inherited lysosomal storage disorders and leukodystrophies, such as mucopolysaccharidosis and Krabbe's disease, are also key targets for gene therapy. Treatment options for most CNS disorders are limited to supportive care, thus providing few therapeutic options. Direct CNS gene transfer may be the most effective option for eliminating the disease and dysfunction. The goal of these studies was the direct comparison of the transduction of adeno-associated virus (AAV) vector systems with regard to levels of and pattern of gene transfer in the CNS of rhesus macaques. The transduction patterns of the various vectors were mapped by determination of the levels of gene transfer to selected regions of the brain and delineating the cell types transduced by immunohistochemistry and microscopy. We compared the ability of six AAV serotypes of recombinant AAV vectors (AAV2/1, AAV2 genome and AAV1 capsid, AAV2/2 and AAV2/7, AAV2/8, AAV2/9 and AAV2/rh10) in the CNS of adult rhesus monkeys (3-6 years old) by sterotaxic-guided injection. Rhesus macaques underwent injections of PBS, or an AAV serotype into individual hemispheres of the brain. All animals were necropsied four weeks after surgery. The injection of all of the AAV serotypes resulted in detectable EGFP activity by direct fluorescent confocal microscopic analysis. A high frequency of EGFP positive cells were detected immediately surrounding the injection tract and in both the white and gray matter. EGFP positive cells were readily detected in the following structures: caudate nucleus, hippocampus, putamen, substantia nigra, globus pallidus, amygdala, crus cerebri, and spinocerebellar tract for all of the vectors tested. Once EGFP positive cells were detected, immunohistochemistry was performed using several neuronal and glial cell antibodies, including GFAP, S100, IBA-1, NeuN, Map2, and NSE. EGFP-positive cells were found to colocalize with all of the tested antigens in various structures throughout the CNS. All of the vector serotypes, except AAV2/8, produced significant levels of EGFP positive cells in the white matter. Cells expressing EGFP were detected as far as 4-6 mm from the injection tract. The transfer of vector to the contralateral hemisphere revealed EGFP protein positive axonal bodies, but no detectable vector transduced cells. There was no evidence of inflammation or degeneration related to the surgical procedures or injection of any of the vectors in the brain or spinal cord. 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.