Lentiviral-mediated Hematopoietic Stem Cell Gene Therapy for Canine Globoid Cell Leukodystrophy

 描述（由申请人提供）：球样细胞脑白质营养不良（GLD），也称为克拉伯病，是一种进行性和致死性神经退行性溶酶体贮积症，由水解酶β-半乳糖苷酶（GALC）活性不足引起，该酶负责降解髓鞘脂质半乳糖神经酰胺（GalCer）和半乳糖鞘氨醇（Psychosine）。在没有GALC的情况下，已知的神经毒素--精神病碱的积累会导致少突胶质细胞的损失和随后的广泛脱髓鞘，患者死亡通常发生在生命的第二年。迄今为止，对儿童唯一可用的治疗干预是来自供体脐带血或骨髓的造血干细胞移植（HSCT）。当早期使用时，HSCT已被证明能够减缓疾病进展;然而，由于神经系统恶化导致认知和运动功能丧失，儿童的生活质量很差。我们假设，移植前基因增强HSC将增加GALC的治疗水平，进一步减缓疾病进展，并延长GLD患者的生存期。为了检验这一假设，我们将利用GLD的犬模型，该模型忠实地再现了在人类患者中观察到的临床疾病进展、神经病理学改变和生化异常。在目标1中，我们将利用编码GFP的载体优化幼犬骨髓收获、HSC分离和正常犬中的慢病毒转导。在目标2中，我们将评估慢病毒HSC基因治疗的安全性和耐受性，包括一种新的预处理方案，在正常犬中使用编码犬GALC的创新慢病毒载体。在目标3中，我们将在GLD犬中建立慢病毒HSC基因疗法的功效，并与用单独的HSCT（无慢病毒转导）治疗的GLD犬和未治疗的GLD对照进行比较。目前在其他脑白质营养不良中进行的临床试验的安全性和有效性数据以及我们的GLD犬模型中令人信服的临床前数据可能导致Krabbe病的慢病毒HSC基因治疗的快速临床实施。针对这种疾病模型的培训将通过与我的赞助商的定期互动进行，我的赞助商是一位委员会认证的兽医神经学家和NIH资助的研究人员，他们具有利用大型动物模型开发多种LSD疗法的丰富研究经验。该培训将通过以下方式得到加强：1）与病毒载体专家共同赞助，2）与犬体外基因治疗专家合作。宾夕法尼亚大学提供足够的资源来完成该项目，并提供充足的补充教育和培训机会。我所选择的申办者和共同申办者的专业知识以及该机构的资源将使我能够发展新的技能，并使我能够成为一名独立的研究者，能够将实验室治疗转化为患者治疗。

 DESCRIPTION (provided by applicant): Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a progressive and fatal neurodegenerative lysosomal storage disorder caused by the deficient activity of the hydrolytic enzyme β- galactocerebrosidase (GALC), which is responsible for degrading the myelin lipids galactosylceramide (GalCer) and galactosylsphingosine (psychosine). In the absence of GALC, accumulation of psychosine, a known neurotoxin, causes loss of oligodendrocytes and subsequent widespread demyelination with patient death typically occurring in the second year of life. To date the only available therapeutic intervention for children is hematopoietic stem cell transplantation (HSCT) from donor umbilical cord blood or bone marrow. When employed early, HSCT has proven capable of slowing disease progression; however, the quality of life in children is poor due to neurologic deterioration resulting in loss of cognitive and motor functions. We hypothesize that genetically enhancing HSCs prior to transplantation will increase therapeutic levels of GALC, further attenuate disease progression, and prolong survival in GLD patients. To test this hypothesis, we will take advantage of a canine model of GLD that faithfully recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. In Aim 1 we will optimize bone marrow harvest from young pups, HSC isolation, and lentiviral transduction in normal dogs utilizing a vector encoding GFP. In Aim 2 we will assess the safety and tolerability of lentiviral HSC gene therapy, including a novel preconditioning regimen, in normal dogs using an innovative lentiviral vector encoding canine GALC. In Aim 3 we will establish the efficacy of lentiviral HSC gene therapy in GLD dogs and compare to GLD dogs treated with HSCT alone (no lentiviral transduction) and untreated GLD controls. Safety and efficacy data from current clinical trials in other infantile-onset leukodystrophies and convincing preclinical data in our canine model of GLD could lead to rapid clinical implementation of lentiviral HSC gene therapy for Krabbe disease. Training specific to this disease model will occur through regular interactions with my sponsor, a board certified veterinary neurologist and NIH-funded investigator with extensive research experience utilizing large animal models to develop therapies for numerous LSD. This training will be augmented by 1) co-sponsorship with a viral vector specialist, and 2) collaboration with an expert in canine ex vivo gene therapy. The University of Pennsylvania provides adequate resources to complete the project and ample supplemental educational and training opportunities. The expertise of my selected sponsor and co-sponsor and resources of this institution will allow development of new skill sets and prepare me to become an independent investigator capable of translating therapies from the bench to use in patients.