Temporospatial psychosine accumulation and targeted adeno-associated virus (AAV) gene therapy in canine Krabbe disease

项目总结/摘要 球样细胞脑白质营养不良（GLD），通常被称为克拉伯病，是一种使人衰弱且总是致命的疾病 由编码水解溶酶体的基因突变引起的儿科神经退行性疾病 半乳糖神经酰胺酶（GALC）。GALC的靶细胞毒性psychosine的积累导致损失 少突胶质细胞和雪旺氏细胞以及广泛的中枢和外周神经系统脱髓鞘。 神经功能障碍在生命的第一年就很明显，死亡通常发生在两岁之前。 值得注意的是，GLD是一种在狗中自然发生的遗传性疾病，为此建立了繁殖群; 博士布拉德伯里和她的导师目前维持这个繁殖殖民地。GLD犬的疾病进展 紧密地概括了人类疾病的临床、病理和生化异常。可预测 疾病进展和寿命（15.7 ± 4.8周龄）允许及时识别病理性 治疗干预的变化和评价。此外，增加的规模和复杂性， 狗的大脑与婴儿的大脑相似，允许实施临床相关的方法， 评估疾病进展和治疗策略。长期目标是在这方面进行治疗试验。 有价值的大型动物模型，为儿科临床试验提供信息。然而，初步的腺相关病毒 (AAV)在GLD狗中的研究提供了对当前基因治疗方案的关键限制的见解。 申请人认为，进一步阐明阻碍治疗的潜在致病机制至关重要 并随后考虑更多新的基因治疗方法来治疗这种疾病。 在此，布拉德伯里博士将使用狗的GLD模型，以进一步确定精神病的神经毒性作用， 犬少突胶质细胞，并使用这些数据来开发通过AAV递送的GALC的时空靶向 在这种自然发生的动物模型中治疗疾病。在K99阶段，她将评估 精神分裂素对犬少突胶质细胞成熟的影响，以及这如何影响基因治疗的拯救。她将 还确定了大脑和脊髓中精神病碱的时空积累以及它如何与 与犬脑中髓鞘成熟的时间有关。在R 00阶段，布拉德伯里博士将专注于开发 改善GALC生物分布和靶向的策略。AAV生物学研究进展 十年来，通过靶向特定组织、细胞和受体，提供了更定制的治疗方法。 K99/R 00资助机制将提供从NRSA博士后研究员到 独立调查员除了获得新的实验室技能，申请人的职业发展将 由一个特殊的咨询/指导委员会的专业知识加强。此外，The University of 宾夕法尼亚州是一个顶级的研究机构，拥有无与伦比的资源和充足的教育和培训 机会出色的指导，无与伦比的资源，以及来自她所在部门的坚定承诺 布拉德伯里的候选人资格，并过渡到一个独立的终身教职职位。 !

Project Summary/Abstract Globoid cell leukodystrophy (GLD), commonly referred to as Krabbe disease, is a debilitating and always fatal pediatric neurodegenerative disease caused by a mutation in the gene encoding for the hydrolytic lysosomal enzyme galactosylceramidase (GALC). Accumulation of cytotoxic psychosine, a target of GALC, results in loss of oligodendrocytes and Schwann cells and widespread central and peripheral nervous system demyelination. Neurologic dysfunction is apparent in the first year of life and death often occurs by two years of age. Notably, GLD is a naturally occurring hereditary disease in dogs for which a breeding colony was established; Dr. Bradbury and her mentors currently maintain this breeding colony. Disease progression in GLD dogs closely recapitulates clinical, pathological, and biochemical abnormalities of human disease. The predictable disease progression and lifespan (15.7 ± 4.8 weeks of age) allow for timely identification of pathological changes and evaluation of therapeutic interventions. Furthermore, the increased size and complexity of the canine brain are similar to that of an infant allowing for implementation of clinically relevant methods to evaluate disease progression and therapeutic strategies. The long-term goal is to conduct therapy trials in this valuable large animal model to inform pediatric clinical trials. However, preliminary adeno-associated virus (AAV) studies in the GLD dog have provided insights to critical limitations of current gene therapy protocols. The applicant believes that it is vital to further elucidate underlying pathogenic mechanisms hindering therapy in this particular model and subsequently consider more novel gene therapy approaches to treat this disease. Herein Dr. Bradbury will use the canine model of GLD to further define the neurotoxic effect of psychosine on canine oligodendrocytes and use these data to develop temporospatial targeting of GALC delivered via AAV vectors to treat disease in this naturally occurring animal model. In the K99 phase she will evaluate the effect of psychosine on canine oligodendrocyte maturation and how this impacts rescue by gene therapy. She will also determine the temporospatial accumulation of psychosine in the brain and spinal cord and how it relates to the timing of myelin maturation in the canine brain. In the R00 phase Dr. Bradbury will focus on developing strategies to improve GALC biodistribution and targeting of pyschosine. Advances in AAV biology over the past decade provide for more tailored therapeutic approaches by targeting specific tissues, cells, and receptors. The K99/R00 funding mechanism will provide a necessary transition from NRSA postdoctoral fellow to an independent investigator. In addition to gaining new laboratory skills, the applicant's career development will be enhanced by the expertise of an exceptional advisory/mentoring committee. Additionally, the University of Pennsylvania is a top tier research institution with unparalleled resources and ample educational and training opportunities. The outstanding mentoring, unmatched resources, and strong commitment from her department will strengthen Dr. Bradbury's candidacy for, and transition to, an independent tenure-track faculty position. !