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。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 基因治疗(GT)是治疗溶酶体储存障碍(LSD)中枢神经系统病变的一种很有前途的方法，因为它有可能提供一个永久性的缺陷酶来源。我们小组一直在开发一种慢病毒(LV)介导的脑内GT来治疗球状细胞白血病(GLD)，这种方法可以从有限数量的注射部位在中枢神经系统实现最大程度的转基因扩散。我们小组已发表的数据(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)注射到高度相互连接的白质区域(外囊/内囊；EC/IC)可导致功能性半乳糖苷酶(GALC)在Twitcher小鼠(Krabbe病小鼠模型)整个脑和脊髓内快速而强劲的表达，导致酶活性得到全面挽救，并显著减少激活的小胶质细胞。在感染的新生小鼠注射EC/IC后，GALC酶获得了稳定的生产和广泛的分布，这表明这种方法有效地产生了缺失的酶的生理水平。在这种基因传递技术的成熟过程中，合乎逻辑的科学进展是在大型动物模型中评估这一策略，包括非人类灵长类动物(NHP)。假设：将表达GALC的慢病毒载体注射到受Krabbe影响的灵长类动物大脑的EC/IC区域，将导致整个中枢神经系统的转导和强劲表达。 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基因进入EC/IC是否改善了Krabbe感染的恒河猴的行为和神经运动缺陷。 2.研究注射NHP(Krabbe感染动物和正常动物)后GALC酶在中枢神经系统组织中的基因转移效率、扩散、分布和长期表达，以及与白质导向基因传递策略相关的组织病理学结果和免疫反应。 随着这项研究走向人类临床试验，拟议的研究计划对于在再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。从这一试点提案中产生的数据将是未来拨款提案准备过程中不可或缺的一部分，并对设计人类临床试验具有重要价值。

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.