Gene Transfer and NMR Studies in Alpha-Mannosidosis Brain

描述(申请人提供)：溶酶体储存疾病(LSD)是一大组遗传性酶缺乏，在儿童中会导致致命的退行性综合征，大多数严重的脑部疾病表现为智力低下。神经遗传性疾病的损害遍及整个大脑，因此被认为需要进行全球纠正。载体介导的正常基因的cDNA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。尽管如此，CNS基因治疗在大多数LSD中可能有效，因为这种酶是从经过基因校正的细胞分泌并被邻近细胞吸收的；而且这种酶可能通过轴突途径运输到远端部位。为了研究在比啮齿动物大脑大得多的大脑中进行基因治疗的方法，我们将使用人类阿尔法甘露糖苷酶(AMD)的猫模型，这是由溶酶体α甘露糖苷酶(LAMAN)基因突变引起的。在当前的资助期间，我们发现：1)AAV1血清型载体增加了总的基因转移量，并将转导扩大到白质和灰质区域。2)一次外科手术将载体注射到大脑周围的几个轨道上，就可以产生足够的正常拉曼来逆转大面积的存储性病变。临床上，接受治疗的AMD猫在神经综合征方面显示出有希望的改善，但并未完全消失。3)评价了几种磁共振(MR)方法对活体动物脑内疾病病理的非侵入性监测。磁化转移成像(MTI)显示，经过基因治疗的AMD猫的脑白质有了显著的改善。弥散加权成像(DW-MRI)和波谱(1H-MRS)能够发现灰质区域的疾病，那里是大部分存储空间的地方。4)病理学研究显示，用药猫脑灰质和白质的正常脑细胞形态明显恢复，但仍有一些未矫正的疾病区域。为了改进这些进展，我们在继续拨款中建议：1)评估一种通过正电子发射计算机断层扫描(PET)监测活脑中载体基因表达的方法。2)研究替代载体传递方法，以实现更完整的CNS矫正。3)使用3T临床磁铁，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(&gt；1年)，在生命早期开始治疗的效果，并在神经生理学研究中评估疾病矫正的功能相关性。总之，这些在显著大于啮齿类动物大脑的哺乳动物大脑中的实验提供了一个强大的翻译模型，以研究AAV介导的基因治疗纠正神经遗传性LSD中的全局脑损伤的潜力。这些研究的翻译性质涉及来自多个领域的专家团队，包括神经学、核磁共振和PET成像、基因载体开发、分子生物学、神经生理学和病理学。 与公共卫生相关：溶酶体储存疾病是一大组遗传性疾病，在大脑中有严重的疾病。我们正在研究基因治疗方法来纠正脑部疾病。拟议的研究旨在充分改善基因和蛋白质的传递，使其在人体临床试验中可行。

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.