Pre-Clinical Trial of MSC-Based Therapy for CNS Disease

描述（由申请人提供）：本提案旨在通过相关动物模型评估成体干细胞注入中枢神经系统治疗溶酶体贮积性疾病相关神经系统后遗症的安全性、可行性和有效性。具体来说，从骨髓中提取的成体干细胞，称为间充质干细胞（MSCs），将通过MRI确定的立体定位坐标，单侧注射到婴儿恒河猴（Macaca mulatta）的尾状壳核中。在移植之前和移植后1年的不同时期，将使用一系列适合年龄的测试来评估每个移植受体的健康、发育、行为以及认知和运动技能，并将其与假手术动物或规范对照数据进行比较。此外，将使用我们开发的针对猕猴Y染色体序列的实时PCR检测来评估植入雌性移植受体中枢神经系统的雄性间质干细胞的水平和解剖分布。为了确认实时PCR数据，植入的MSCs将通过免疫荧光染色或FISH在脑切片中显示，并使用现代立体技术进行计数。将骨髓间充质干细胞植入大脑与健康、发育、行为和运动功能的长期影响联系起来，将直接评估颅内骨髓间充质干细胞给药的安全性和可行性。随后，MSCs将被注射到患有球状细胞白质营养不良（克拉伯氏病）的幼猴的中枢神经系统中。后者的特征是缺乏半乳糖脑苷酶（GALC）活性，导致储存物质在少突胶质细胞中异常积累，导致神经系统进行性脱髓鞘。在这些研究中，MSC的植入水平和分布将与整个中枢神经系统中GALC mRNA的表达水平和酶活性直接相关。此外，将使用生化和组织学方法以及行为和运动性能测试来评估对疾病进展的影响。因此，这一阶段的研究将直接评估MSC给药的治疗效果。总的来说，这些拟议的研究将在相关动物模型中产生重要的临床前数据，这对于开发基于干细胞的疗法来治疗患有各种溶酶体贮积性疾病的人类婴儿和儿童的神经系统后遗症至关重要。

DESCRIPTION (provided by applicant): This proposal aims to assess in a relevant animal model the safety, feasibility and efficacy of adult stem cell administration to the CNS for treating neurological sequelae associated with lysosomal storage diseases. Specifically, adult stem cells derived from bone marrow, referred to as mesenchymal stem cells (MSCs) will be injected unilaterally into the caudate putamen of infant rhesus macaques (Macaca mulatta) using stereotaxic coordinates determined by MRI. Prior to and at various periods up to 1 year post-transplantation, the health, development, behavior as well as cognitive and motor skills of each transplant recipient will be evaluated using a battery of age appropriate tests and compared to sham-operated animals or normative control data. Additionally, the levels and anatomical distribution of male MSCs engrafted in the CNS of female transplant recipients will be evaluated using a real-time PCR assay we developed that targets sequences in the Macaca sp. Y chromosome. To confirm the real-time PCR data engrafted MSCs will be visualized in brain sections by immunofluorescent staining or FISH and counted using modern stereological techniques. Correlating MSC engraftment in brain with long-term affects on health, development, behavior and motor function will directly assess the safety and feasibility of intracranial MSC administration. Subsequently, MSCs will be injected into the CNS of infant macaques afflicted with globoid cell leukodystrophy (Krabbe's Disease). The latter is characterized by a lack of galactocerebrosidase (GALC) activity, which results in abnormal accumulation of storage material in oligodendrocytes causing progressive demyelination in the nervous system. In these studies MSC engraftment levels and distribution will be directly correlated with levels of expressed GALC mRNA and enzyme activity throughout the CNS. Furthermore, affects on disease progression will be evaluated using biochemical and histological methods as well as behavioral and motor performance tests. Consequently, this phase of the study will directly evaluate the therapeutic efficacy of MSC administration. Collectively, these proposed studies will yield important pre-clinical data in a relevant animal model that will be essential for developing stem cell-based therapies to treat neurological sequelae in human infants and children afflicted with various lysosomal storage diseases.