Non-invasive gene therapy for inherited neurodegenerative disorders (How to cross the BBB?)

非侵入性基因治疗遗传性神经退行性疾病如偏色差性白细胞营养不良（MLD）的可行性进行了研究。甘露糖-6-磷酸受体（M6PR）在溶酶体酶跨血脑屏障的交叉校正和胞吞作用中起重要作用。M6PR是一种多功能细胞表面受体，它也与胰岛素样生长因子II （IGF-II）相互作用，由15个重复结构域组成。我们克隆了7.6kb的M6PR cDNA，并构建了一系列的小基因。4.8kb的包含结构域1至9的迷你基因被证明可以结合M6P而不结合IGF-II。利用含有4.8kb miniigene的慢病毒载体，M6PR可以在多种细胞系和动物模型组织中表达。M6PR表达载体对研究溶酶体酶的亚细胞定位和转运以及提高酶替代治疗溶酶体疾病的疗效具有重要意义。在另一种方法中，我们研究了鞘内注射腺相关病毒载体血清型1 （AAV1）治疗MLD的可行性。将表达芳基磺化酶A （ASA）或绿色荧光蛋白（GFP）的AAV1载体注入ASA敲除MLD模型小鼠鞘内。GFP的表达广泛分布于整个大脑。此外，脊髓背侧的大量神经纤维和背根神经节内的许多神经细胞体都得到了有效的转导。在治疗后的MLD模型小鼠中证实了ASA活性的广泛分布和硫脂含量的显著降低。AAV1载体的IT注射是一种有效且无创的基因广泛传递到大脑和背根神经节的方法。

The feasibility of non-invasive gene therapy for inherited neurodegenerative disorders like metachromatic leuko dystrophy (MLD) has been investigated. Mannose-6-phosphate receptor (M6PR) plays important roles in both corss-correction and transcytosis across the blood brain barrier of lysosomal enzymes. M6PR is a multifunctional cell surface receptor which also interacts with insulin-like growth factor II (IGF-II) and is composed of 15 repeated domains. We have cloned 7.6kb M6PR cDNA and constructed a series of minigenes. The 4.8kb minigene containing domain 1 to 9 was shown to bind M6P but not IGF-II. Using a lentiviral vector containing the 4.8kb minigene, M6PR can be expressed in various cell lines and tissues of animal models. M6PR expression vector should be useful for studying subcellular localization and trafficking of lysosomal enzymes and for enhancing the efficacy of enzyme replacement therapy for lysosomal disorders. In an alternative approach, we examined the feasibility of intrathecal (IT) injection of adeno-associated viral vector serotype 1 (AAV1) to treat MLD. AAV1 vector expressing arylsulfatase A (ASA) or green fluorescence protein (GFP) was intrathecally injected into ASA knockout MLD model mice. Broad distribution of GFP expression was seen throughout the brain. In addition, a large number of nerve fibers in the dorsal spinal cord and many neural cell bodies in the dorsal root ganglia were efficiently transduced. Widespread distribution of ASA activity and significant reduction of sulfatide content were confirmed in treated MLD model mice. IT injection of AAV1 vector is a useful and noninvasive method for widespread gene delivery to the brain and dorsal root ganglia.