CNS WHITE MATTER TRACTS AS A NOVEL AVENUE FOR GENE THERAPY FOR KRABBE DISEASE

这个子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 基因治疗（GT）代表了一种有前途的方法，用于治疗溶酶体储存障碍（LSD）的CNS病理，因为它有可能提供一个永久的来源，缺乏酶。我们的小组一直在开发一种慢病毒（LV）介导的脑内GT来治疗球样细胞脑白质营养不良（GLD），它可以从有限数量的注射部位在CNS中实现最大的转基因分散。我们小组公布的数据（在本提案的初步数据部分）清楚地表明，将单个慢病毒载体（LV）注射到高度互连的白色物质区域中（外/内囊; EC/IC）导致功能性半乳糖苷酶（GALC）在Twitch小鼠的整个脑和脊髓中快速和稳健地表达（Krabbe病的鼠模型），导致酶活性的全面拯救，以及活化的小胶质细胞的显著减少。在受影响的新生小鼠中EC/IC注射后实现的GALC酶的稳定生产和广泛分布表明，这种方法有效地产生了生理水平的缺失酶。这种基因递送技术成熟的合乎逻辑的科学进展是在大型动物模型中评估这种策略，包括非人灵长类动物（NHP）。假设：将表达GALC的慢病毒载体给予受Krabbe影响的灵长类动物脑的EC/IC区域将导致整个CNS的转导和稳健表达。 我们将通过两个具体目标来检验这一假设。 #1.确定LV介导的GALC基因在EC/IC中的传递是否改善了受Krabbe影响的恒河猴的行为和神经运动缺陷。 #2.评估注射NHP（受Krabbe影响的动物和正常动物）CNS组织中GALC酶的基因转移效率、扩散、分布和长期表达、组织病理学结局和与白色物质定向基因递送策略相关的免疫应答。 随着这项研究走向人类临床试验，拟议的研究计划对于在重现疾病的大型动物模型中测试这种新的GT方法是绝对必要的。从这个试点提案产生的数据将是不可或缺的准备未来的赠款提案，并在设计人体临床试验的重要价值。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Gene therapy (GT) represents a promising approach for the treatment of the CNS pathology in Lysosomal Storage Disorders (LSD), as it has the potential to provide a permanent source of the deficient enzyme. Our group has been developing a lentiviral (LV)-mediated intracerebral GT to treat Globoid Cell Leukodystrophy (GLD) that could achieve maximal transgene dispersal in the CNS from a limited number of injection sites. Published data from our group (in Preliminary Data section in this proposal) clearly demonstrate that a single lentiviral vector (LV) injection into a highly interconnected white matter region (external/internal capsule; EC/IC) resulted in rapid and robust expression of functional galactocerebrosidase (GALC) throughout the entire brain and spinal cord in the Twitcher mouse (murine model of Krabbe disease), resulting in global rescue of enzymatic activity, and marked decrease of activated microglia. The stable production and widespread distribution of the GALC enzyme achieved following EC/IC injection in affected neonatal mice suggests that this approach effectively produces physiological levels of the missing enzyme. The logical scientific progression in the maturation of this gene delivery technology is to assess this strategy in large animals models, including nonhuman primates (NHPs). Hypothesis: Administration of lentiviral vector expressing GALC to the EC/IC region of the Krabbe-affected primate brain will result in transduction and robust expression throughout the CNS. We will test this hypothesis through two Specific Aims. #1. Determine if LV-mediated delivery of the GALC gene in to the EC/IC improves the behavioral and neuromotor deficits of Krabbe-affected rhesus macaques. #2. To assess gene transfer efficiency, diffusion, distribution and long-term expression of the GALC enzyme in CNS tissues of injected NHP (Krabbe-affected and normal animals), histopathologic outcomes and immune response associated with the white matter-directed gene delivery strategy. As this research moves toward human clinical trials, the proposed research plan is absolutely essential for testing this novel GT approach in a large animal model recapitulating the disease. The data generated from this pilot proposal will be integral for the preparation of future grant proposals and be of significant value in designing human clinical trials.