STEREOTAXIC GENE THERAPY TO THE RHESUS CNS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 中枢神经系统(CNS)是基因治疗的主要靶点，因为影响大脑的遗传性和获得性疾病范围广泛。帕金森氏病、阿尔茨海默病和肌萎缩侧索硬化症等神经退行性疾病是基因治疗的主要目标。虽然罕见，但遗传性溶酶体储存障碍和脑白质营养不良，如粘多糖症和克拉贝氏病，也是基因治疗的关键靶点。大多数中枢神经系统疾病的治疗选择仅限于支持性护理，因此提供的治疗选择很少。直接的中枢神经系统基因转移可能是消除疾病和功能障碍的最有效的选择。这些研究的目的是直接比较腺相关病毒(AAV)载体系统的转导与猕猴中枢神经系统中基因转移的水平和模式。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显微镜描绘转导的细胞类型，绘制了各种载体的转导模式。 比较了AAV2/1、AAV2基因组和AAV1衣壳、AAV2/2和AAV2/7、AAV2/8、AAV2/9和AAV2/RH10 6种AAV型重组AAV载体在3~6岁恒河猴中枢神经系统内的作用。猕猴接受了PBS注射，或AAV血清型注射到各自的大脑半球。所有动物在手术后四周进行尸检。注射所有AAV血清型后，通过直接荧光共聚焦显微镜分析可检测到EGFP活性。注射管周围及白质和灰质均可检测到高频率的EGFP阳性细胞。所有载体的尾状核、海马体、壳核、黑质、苍白球、杏仁核、大脑小脚和脊髓小脑束中均可检测到EGFP阳性细胞。一旦检测到EGFP阳性细胞，就用几种神经元和胶质细胞抗体进行免疫组织化学，包括GFAP、S100、IBA-1、Neun、MAP2和NSE。发现EGFP阳性细胞与所有被测抗原共存于整个中枢神经系统的不同结构中。除AAV2/8外，所有的载体血清型均在白质中产生显著水平的EGFP阳性细胞。在距注射道4-6 mm处可检测到表达EGFP的细胞。载体转移到对侧大脑半球可见EGFP蛋白阳性轴突小体，但未检测到载体转导细胞。没有证据表明与外科手术或在大脑或脊髓中注射任何载体有关的炎症或退化。拟议的翻译研究结果将为人类，特别是儿童的中枢神经系统定向基因治疗方案的设计和执行提供宝贵的信息。

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 (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.