Gene Transfer and NMR Studies in Alpha-Mannosidosis Brain

α-甘露糖苷沉积症脑的基因转移和NMR研究我们将调查系统的能力， 递送新型腺相关病毒（AAV）基因转移载体以治疗中枢神经系统 (CNS)在人溶酶体贮积病（LSD）α-甘露糖苷沉积症（AMD）的猫模型中的疾病。在 大多数LSDs对相对少量突变细胞的遗传校正导致正常细胞的分泌， 酶和受体介导的周围细胞的摄取，导致非- 转导的细胞。治疗脑的主要问题是病理学存在于整个CNS中 因为所有细胞都存在代谢缺陷。因此，治疗整个大脑需要全球分布 治疗正常的α-甘露糖苷酶（MANB）酶。这一问题因巨大的 小鼠和人类大脑之间的大小差异（约3，000倍）。家猫的大脑是一种极好的 在大小上是中等的，因为它比老鼠的大脑大100倍，而人类的大脑在出生到10岁之间。 一年（当治疗预计是最有效的）是只有10-30倍大于猫的大脑。的 猫的大脑也有一个脑回皮层，在结构上与人类大脑更相似 比啮齿类动物的多因此，这个翻译项目的基本前提是， 全球正确的AMD猫脑更有可能有效地转化为临床试验。我们已经展示了在 该项目通过多次实质内注射或通过 CSF输注可改善临床和组织学参数，但纠正不完全。 某些AAV可在小鼠中全身血管内递送后进入CNS并介导广泛的免疫应答。 转导，但在大型动物脑中载体分布要有限得多。我们已经开发出一种新颖 用于全身递送的AAV载体，其转导整个大脑皮层和脑内其他区域的神经元。 猫和猴的大脑该新载体还具有将基因有效递送到细胞中的新特性。 具有单链AAV载体基因组的大大脑，而先前的大动物血管内AAV 递送实验已经使用了自身互补（sc）-AAV，其太小而不能容纳所述载体。 MANB cDNA（~3kb）。将新载体与其他AAV进行比较，以确定互补模式是否 在中枢神经系统和其他器官发生转导，以提高治疗效果。我们还开发了 基于MR的成像分析可以非侵入性地测量大脑病理学，初步实验表明， 可以定量监测对AAV治疗的反应。具体目标是优化 递送，最小化剂量，评估临床和寿命改善，以及评估非 通过与组织病理学的相关性来确定侵入性成像参数。虽然取得了重大进展， 到目前为止，该项目的处理仍然不完整，需要进一步改进，以确保 最有效地转化为人类患者的临床方案。

Gene Transfer and NMR Studies in Alpha-Mannosidosis Brain. We will investigate the ability of systemic delivery of a novel adeno-associated virus (AAV) gene transfer vector to treat the central nervous system (CNS) disease in a cat model of the human lysosomal storage disease (LSD) alpha-mannosidosis (AMD). In most LSDs genetic correction of a relatively small number of mutant cells results in secretion of the normal enzyme and receptor-mediated uptake by surrounding cells, resulting in metabolic correction of the non- transduced cells. The major problem for treating the brain is that pathology is present throughout the CNS because the metabolic defect is present in all cells. Thus, treating the whole brain requires global distribution of the therapeutic normal alpha-mannosidase (MANB) enzyme. This problem is exacerbated by the enormous size difference between mouse and human brains (~3,000 fold). The domestic cat brain is an excellent intermediate in size as it is ~100 times larger than a mouse brain, while the human brain between birth and 1 year (when treatment is expected to be the most effective) is only 10-30 times larger than the cat brain. The cat brain also has a gyrencephalic cerebral cortex that is structurally much more similar to the human brain than the rodent's. Thus, the underlying premise of this translational project is that strategies developed to globally correct the AMD cat brain are more likely to translate effectively into clinical trials. We have shown in this project that AAV gene transfer into the AMD cat brain either by multiple intraparenchymal injections or by infusion into the CSF can improve clinical and histological parameters, but the correction is incomplete. Certain AAVs can enter the CNS after systemic intravascular delivery in mice and mediate widespread transduction, but in large animal brains vector distribution is much more limited. We have developed a novel AAV vector for systemic delivery that transduces neurons throughout the cerebral cortex and other regions of both cat and monkey brains. The new vector also has the novel property of efficiently delivering the gene into large brains with a single-stranded AAV vector genome, whereas previous large animal intravascular AAV delivery experiments have used self-complementary (sc)-AAVs, which are too small to accommodate the MANB cDNA (~3kb). The novel vector will be compared to other AAVs to determine if complimentary patterns of transduction occur in the CNS and other organs to improve the therapeutic effect. We also have developed MR-based imaging assays to non-invasively measure brain pathology and preliminary experiments show they can quantitatively monitor the response to AAV treatment. The specific aims are directed towards optimizing delivery, minimizing dose, evaluating clinical and lifespan improvements, and assessing the accuracy of non- invasive imaging parameters by correlation with histopathology. Although significant progress has been made on this project to date, the treatment is still incomplete and further improvements are needed to ensure the most effective translation into a clinical protocol for human patients.