Gene Transfer and NMR Studies in Alpha-Mannosidosis Brain

描述（由申请人提供）：溶酶体贮积病（lsd）是一大类遗传性酶缺乏症，可在儿童中产生致命的退行性综合征，其中大多数患有严重的脑部疾病，表现为智力迟钝。神经遗传疾病在整个大脑中都有病变，因此人们认为需要进行全局矫正。载体介导的正常基因cDNA的转移可以纠正有缺陷的细胞，但目前的载体介导的基因在大脑中的传递相对有限。然而，中枢神经系统基因疗法可能对大多数lsd起作用，因为这种酶是从基因校正的细胞分泌的，并被邻近细胞吸收；酶可以通过轴突途径运输到远端部位。为了研究在比啮齿类动物大脑大得多的大脑中进行基因治疗的方法，我们将使用人类α -甘露糖醇病（AMD）的猫模型，这是由溶酶体α -甘露糖醇酶（LAMAN）基因突变引起的。在目前的资助期内，我们发现：1)AAV1血清型载体增加了总基因转移量，并扩大了白质和灰质区域的转导。2)一个单一的外科手术将载体注射到大脑周围的几个轨道中，产生足够的正常LAMAN来逆转大面积的储存病变。在临床上，接受治疗的AMD猫在神经系统综合征方面表现出有希望的改善，但没有完全解决。3)评估了几种磁共振（MR）方式对活体动物大脑疾病病理的无创监测。磁化转移成像（MTI）显示，基因治疗后AMD猫脑白质明显改善。弥散加权成像（Dw-MRI）和光谱学（1H-MRS）能够检测到灰质区域的疾病，在灰质区域可以看到大部分存储。4)病理学研究表明，治疗后的猫在灰质和白质中恢复了正常的脑细胞形态，但一些未纠正的疾病区域仍然存在。为了进一步提高这些进展，我们在继续拨款中提出：1)评估一种利用PET监测活脑中载体基因表达的方法。2)研究替代矢量输送方法，以实现更完整的中枢神经系统校正。3)采用3T临床磁体，研究Dw-MRI和1H-MRS测量治疗后灰质变化的能力。4)研究长期矫正（> - 1年），生命早期开始治疗的效果，并评估神经生理学研究中疾病矫正的功能相关性。总之，这些在哺乳动物大脑中进行的实验比啮齿动物的大脑大得多，为研究aav介导的基因治疗在神经遗传性lsd中纠正全局脑损伤的潜力提供了一个强大的转化模型。这些研究的转化性质涉及来自多个领域的专家团队，包括神经学，MRI和PET成像，基因载体开发，分子生物学，神经生理学和病理学。

DESCRIPTION (provided by applicant): Lysosomal storage diseases (LSDs) are a large group of inherited enzyme deficiencies that produce fatal degenerative syndromes in children, with most having severe brain disease manifest as mental retardation. Neurogenetic diseases have lesions throughout the brain, thus it is thought that global correction will be needed. Vector mediated transfer of a normal cDNA of the gene can correct defective cells, but current vectors mediate relatively limited gene delivery in the brain. Nevertheless, CNS gene therapy may work in most LSDs because the enzyme is secreted from genetically corrected cells and taken up by neighboring cells; and the enzyme may be transported via axonal pathways to distal sites. To study approaches to gene therapy in a brain that is significantly larger than a rodent brain, we will use a cat model of human alpha- mannosidosis (AMD), caused by a mutation in the lysosomal alpha-mannosidase (LAMAN) gene. In the current grant period we found: 1) An AAV1 serotype vector increased the amount of total gene transfer and it expanded transduction to both white and gray matter areas. 2) A single surgical procedure injecting the vector into several tracks spaced around the brain produced enough normal LAMAN to reverse large areas of storage lesions. Clinically, the treated AMD cats showed promising improvements in the neurological syndrome, but not complete resolution. 3) Several magnetic resonance (MR) modalities were evaluated to non-invasively monitor disease pathology in the living animal brain. Magnetization transfer imaging (MTI) showed that significant improvements in the white matter could be measured in AMD cat brains treated by gene therapy. Diffusion-weighted imaging (Dw-MRI) and spectroscopy (1H-MRS) were able to detect disease in the gray matter areas, where most of the storage is seen. 4) Pathology studies demonstrated significant restoration of normal brain cell morphology in treated cats, in both gray and white matter, but some regions of uncorrected disease remained. To improve on these advances, we propose in the continuation grant to: 1) Evaluate a method to monitor vector gene expression in the living brain by PET. 2) Investigate alternate vector delivery approaches to achieve more complete correction of the CNS. 3) Study the ability of Dw-MRI and 1H-MRS to measure changes in the gray matter after treatment, using a 3T clinical magnet. 4) Study long-term correction (>1 year), the effects of starting treatment earlier in life, and evaluate functional correlates of disease correction in neuro-physiological studies. Together, these experiments in a mammalian brain that is significantly larger than a rodent brain provide a robust translational model to study the potential of AAV-mediated gene therapy to correct the global brain lesions in neurogenetic LSDs. The translational nature of these studies involves a team of experts from several fields, including neurology, MRI and PET imaging, gene vector development, molecular biology, neurophysiology, and pathology. PUBLIC HEALTH RELEVANCE: The lysosomal storage diseases are a large group of inherited disorders that have severe disease in the brain. We are studying gene therapy methods to correct the brain disease. The proposed studies are directed towards improving gene and protein delivery sufficiently to make them feasible for human clinical trials.