FELINE ALPHA-MANNOSIDOSIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。从一个波斯猫家族中发现了一群携带α -甘露甘露病的猫，其中发现了受感染的小猫。育种研究表明，这是一种常染色体隐性性状，伴有进行性神经症状和6个月大时死亡。临床、病理、酶和分子研究表明这种疾病是基因治疗的潜在模式。骨髓移植的研究已经显示出神经系统体征的显著改善和生命的延长，这表明基因转移到骨髓干细胞应该是一种有效的治疗手段。我们与华盛顿州西雅图市华盛顿大学的Janis Abkowitz博士合作开展了一项利用猫模型进行活化的成年猫单核细胞子宫移植和基因治疗的合作研究。这项工作是通过Abkowitz博士的分包合同单独资助的。我们描述了猫的周围和中枢神经系统髓鞘异常与α -甘露糖苷酶，这是必要的计划基因转移研究。腺相关病毒（AAV）载体能够将治疗性基因传递到小鼠大脑，从而导致长期和广泛的蛋白质产生。然而，人类婴儿的大脑比老鼠的大脑大1000多倍，这将使治疗儿童全身神经代谢紊乱变得更加困难。我们评估了三种AAV血清型（1、2和5）在猫脑中转导细胞的能力，作为大型哺乳动物脑模型。人类溶酶体酶-葡糖苷酸酶（GUSB）被用作报告基因，因为它可以通过热稳定性与猫的GUSB区分开来。将载体注入脑锥。X、尾状核、丘脑、辐射冠、内囊和半卵圆体。术后10周，采用原位杂交和酶组织化学方法对脑组织进行基因表达评估。AAV2载体能够在灰质中转导细胞，而aav1载体导致灰质的转导比AAV2和白质的转导更大。AAV5在猫脑中没有可检测到的转导。基于这些数据，我们用携带猫MANB cDNA的AAV I载体治疗了6只患病猫，颅内注射该载体显著改善了临床神经学症状和白质磁共振成像。虽然基因转移仅限于注射轨迹周围的区域，但整个大脑的储存病变都减少了。因此，通过临床可行的注射次数，可以在大型哺乳动物大脑中实现神经病理学的全面改善，并且可以介导神经疾病综合征的临床显着改善。

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.