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

描述（由申请方提供）：本提案旨在在相关动物模型中评估CNS给予成体干细胞治疗溶酶体贮积病相关神经系统后遗症的安全性、可行性和有效性。具体地，将源自骨髓的成体干细胞（称为间充质干细胞（MSC））使用通过MRI确定的立体定位坐标单侧注射到幼年恒河猴（Macaca mulatta）的尾状壳核中。在移植前和移植后1年内的不同时期，将使用一组年龄适当的测试评价每名移植受体的健康、发育、行为以及认知和运动技能，并与假手术动物或正常对照数据进行比较。此外，将使用我们开发的靶向猕猴属物种Y染色体序列的实时PCR测定来评估植入女性移植受体CNS中的雄性MSC的水平和解剖分布。为了确认实时PCR数据，将通过免疫荧光染色或FISH在脑切片中观察移植的MSC，并使用现代体视学技术计数。将MSC在脑中的植入与对健康、发育、行为和运动功能的长期影响相关联将直接评估颅内MSC施用的安全性和可行性。随后，将MSC注射到患有球状细胞脑白质营养不良（克拉伯病）的幼年猕猴的CNS中。后者的特征在于缺乏半乳糖苷酶（GALC）活性，这导致少突胶质细胞中储存物质的异常积累，从而引起神经系统中的进行性脱髓鞘。在这些研究中，MSC植入水平和分布将与整个CNS中表达的GALC mRNA水平和酶活性直接相关。此外，将使用生物化学和组织学方法以及行为和运动性能试验评价对疾病进展的影响。因此，该研究阶段将直接评估MSC施用的治疗功效。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.