Recombinant Enzyme Fusion Protein for Lysosomal Storage

描述（由申请人提供）：有40多种溶酶体贮积性疾病，这些疾病大多对中枢神经系统（CNS）产生不利影响。溶酶体贮积症的主要治疗方法是酶替代疗法（ERT）。然而，ERT对大脑没有效果，因为酶不能穿过脑毛细血管壁，这在体内形成血脑屏障（BBB）。如果不治疗中枢神经系统，年轻的患者注定会发生进行性神经变性和死亡，即使有ERT。未来治疗这些疾病的限制因素是酶在血脑屏障上的转运。直接注射到大脑的血脑屏障旁路是无效的，因为只有一小部分大脑被经颅输送系统治疗，几乎所有的大脑细胞都受到溶酶体储存障碍的影响。相反，几乎所有的大脑细胞都可以通过静脉给药的跨血管输送系统来治疗，该系统使酶能够穿过血脑屏障。分子特洛伊木马技术是一种将诸如酶之类的大分子输送到血脑屏障的新方法。通过基因工程，将缺失的重组酶与血脑屏障分子特洛伊木马融合，产生了一种双功能融合蛋白。后者是一种基因工程蛋白，能够通过受体介导的内源性血脑屏障肽转运系统的胞吞作用穿过人血脑屏障。临床前研究表明，一种分子量为10万道尔顿的大酶，可以通过血脑屏障的运输，附着在血脑屏障受体特异性的特洛伊木马上，进入大脑。目前的工作将产生一种新的融合基因编码溶酶体酶和一个基因工程分子特洛伊木马，这将允许生产相应的融合蛋白AGT-180。融合基因将被纳入真核表达载体，然后转染COS细胞。分泌融合蛋白的双功能将通过ELISA、血脑屏障受体结合试验和酶活性试验来证明。这些I期研究将为未来的II期工作和永久转染CHO细胞系的开发提供支持，生产重组AGT-180融合蛋白。由血脑屏障靶向抗体和重组酶组成的融合蛋白可用于人脑溶酶体贮积症的治疗。

DESCRIPTION (provided by applicant): There are over 40 lysosomal storage disorders, and most of these diseases affect adversely the central nervous system (CNS). The mainstay of treatment of the lysosomal storage disorders is enzyme replacement therapy (ERT). However, ERT is not effective for the brain, because the enzymes do not cross the brain capillary wall, which forms the blood-brain barrier (BBB) in vivo. Without treatment of the CNS, the young patients are destined to progressive neurodegeneration and death, even with ERT. The limiting factor in the future treatment of these diseases is the transport of the enzyme across the BBB. Bypass of the BBB with direct injection into the brain is not effective, because only a small part of the brain is treated with a trans-cranial delivery system, and nearly all cells of the brain are affected in lysosomal storage disorders. Conversely, virtually all cells of the brain can be treated with a trans-vascular delivery system that enables the enzyme to cross the BBB following intravenous administration. A new approach to the BBB delivery of large molecules such as enzymes is the molecular Trojan horse technology. A bi-functional fusion protein is produced with genetic engineering, wherein the missing recombinant enzyme is fused to a BBB molecular Trojan horse. The latter is a genetically engineered protein that is able to cross the human BBB by receptor-mediated transcytosis on endogenous BBB peptide transport systems. Pre-clinical studies show that a large enzyme with a molecular weight >100,000 Daltons, can be delivered to brain via transport across the BBB, following attachment to a BBB receptor-specific Trojan horse. The present work will produce a novel fusion gene encoding a lysosomal enzyme and a genetically engineered molecular Trojan horse, which will allow the production of the corresponding fusion protein, AGT-180. The fusion gene will be incorporated in a eukaryotic expression vector followed by transfection of COS cells. The bi-functionality of the secreted fusion protein will be demonstrated by ELISA, BBB receptor binding assays, and enzyme activity assays. These phase I studies will enable future phase II work and the development of a permanently transfected CHO cell line producing the recombinant AGT-180 fusion protein. Fusion proteins comprised of BBB targeting antibodies and recombinant enzymes could be therapeutic in the treatment of the brain in human lysosomal storage disorders.