FELINE ALPHA-MANNOSIDOSIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。一群阿尔法甘露糖症携带者来自一个波斯猫科动物家庭，在那里发现了受影响的小猫。育种研究表明，这是一种常染色体隐性遗传特征，具有进行性神经系统症状，并在6个月大时死亡。临床、病理、酶和分子研究已将这种疾病描述为一种潜在的基因治疗模式。骨髓移植的研究表明，骨髓移植可以显著改善神经体征，延长生命，提示向骨髓干细胞转移基因应该是一种有效的治疗手段。我们与华盛顿州西雅图华盛顿大学的Janis Abkowitz医学博士开展了一项合作研究，使用CAT模型进行激活的成年猫单核细胞宫内移植和基因治疗。这项工作是通过阿布科维茨博士的分包合同单独提供资金的。我们用α-甘露糖苷酶表征了猫的外周和中枢神经系统髓鞘异常，这是计划基因转移研究所必需的。腺相关病毒(AAV)载体能够将治疗性基因输送到小鼠的大脑中，从而导致长期和广泛的蛋白质生产。然而，人类婴儿的大脑比小鼠的大脑大1000多倍，这将使儿童全球神经代谢障碍的治疗变得更加困难。我们评估了三种AAV血清型(1，2和5)作为大型哺乳动物大脑模型的猫脑细胞转导能力。人溶酶体酶fl-葡萄糖醛酸苷酶(GUSB)因其热稳定性与猫科动物的GUSB不同而被用作报告基因。将载体注射到8周龄猫的大脑锥体、尾状核、丘脑、放射冠、内囊和半卵圆中心。术后10周用原位杂交和酶组织化学方法检测脑组织基因表达。AAV2载体能够转导灰质中的细胞，而AAV1载体对灰质的转导比AAV2更大，白质的转导也更多。AAV5没有在猫脑中产生可检测到的转导。根据这些数据，我们用携带猫科动物MANB基因的AAV I载体治疗了6只猫，脑组织内注射该载体后，临床神经体征和脑白质磁共振成像均有显著改善。尽管基因转移仅限于注射轨迹周围的区域，但整个大脑的储存损伤都减少了。因此，使用临床上可行的注射次数，可以在大型哺乳动物的大脑中实现神经病理的整体改善，并可以在临床上显著改善神经疾病综合征。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A colony of carriers of alpha-mannosidosis was derived from a family of Persian cats in which affected kittens were found. Breeding studies showed this to be an autosomal recessive trait, with progressive neurologic signs and death by 6 months of age. Clinical, pathologic, enzyme, and molecular studies have characterized this disorder as a potential model for gene therapy. Studies of bone marrow transplantation have shown substantial amelioration of neurologic signs and prolongation of life, indicating that gene transfer to bone marrow stem cells should be an effective means of therapy. We have developed a collaborative study using the cat model for in utero transplantation of activated adult cat monocytes and gene therapy with Janis Abkowitz, MD, University of Washington, Seattle, WA. This work is separately funded through a subcontract of Dr. Abkowitz. We have characterized the peripheral and central nervous system myelin abnormalities in cats with alpha-mannosidase which was necessary to plan gene transfer studies. Adeno-associated virus (AAV) vectors are capable of delivering a therapeutic gene to the mouse brain that can result in long-term and widespread protein production. However, the human infant brain is more than 1,000 times larger than the mouse brain, which will make the treatment of global neurometabolic disorders in children more difficult. We evaluated the ability of three AAV serotypes (1,2, and 5) to transduce cells in the cat brain as a model of a large mammalian brain. The human lysosomal enzyme fl-glucuronidase (GUSB) was used as a reporter gene because it can be distinguished from feline GUSB by heat stability. The vectors were injected into the cerebral cone.x, caudate nucleus, thalamus, corona radiata, internal capsule, and centrum semiovale of eight-week-old cats. The brains were evaluated for gene expression using in situ hybridization and enzyme histochemistry 10 weeks after surgery. The AAV2 vector was capable of transducing cells in the gray matter, while the AAV 1 vector resulted in greater transduction of the gray matter than AAV2 as well as transduction of the white matter. AAV5 did not result in detectable transduction in the cat brain. Based on these data, we treated six affected cats with an AAV I vector carrying the feline MANB cDNA, Intracranial injection of this vector resulted in remarkable improvement in clinical neurological signs and in magnetic resonance imaging of white matter. Although gene transfer was limited to the areas surrounding the injection tracks, storage lesions were reduced throughout the brain. Thus global improvement in neuropathology can be achieved in a large mammalian brain using a clinically feasible number of injections, and can mediate clinically significant improvement in the neurological disease syndrome.