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Recombinant Enzyme Fusion Protein for Lysosomal Storage Disorders

用于治疗溶酶体贮积症的重组酶融合蛋白

基本信息

批准号：
7218543
负责人：
YUN ZHANG
金额：
$ 35.74万
依托单位：
ARMAGEN TECHNOLOGIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-01 至 2009-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7218543
关键词：
Adult Affect Affinity Affinity Chromatography Animal Model Anions Antibodies Beta-glucuronidase Binding Biological Assay Bioreactors Blood - brain barrier anatomy Blood Vessels Blood capillaries Brain Brain Part Bypass COS Cells Canis familiaris Carbohydrates Cations Cells Cephalic Cessation of life Child Chimeric Proteins Chinese Hamster Ovary Cell Chromatography Clinical Trials Cloning Complementary DNA Condition Conditioned Culture Media Contracts DNA Restriction Enzymes DNA Sequence Analysis Dermatan Sulfate Development Dihydrofolate Reductase Disease Dose Drug Kinetics Electroporation Engineering Enzyme-Linked Immunosorbent Assay Enzymes Equus caballus Filtration Future Genes Genetic Engineering Glucuronides Goals Grant Guanosine Monophosphate Heparitin Sulfate Hereditary Disease Histamine Human Hydrolysis Immunoglobulin G Inborn Errors of Metabolism Injection of therapeutic agent Institution Insulin Receptor Isoelectric Focusing Laboratories Legal patent Light Liver Macaca mulatta Mass Spectrum Analysis Measures Mediating Methods Methotrexate Modeling Molecular Sieve Chromatography Molecular Weight Monoclonal Antibodies Mucopolysaccharidosis VII Mus Mutate Neuraxis Organ Orphan Drugs Outsourcing Patients Pharmaceutical Preparations Pharmacologic Substance Pharmacology and Toxicology Phase Phase II Clinical Trials Phase III Clinical Trials Plasmids Polyacrylamide Gel Electrophoresis Polymerase Chain Reaction Preparation Primates Process Production Protein Engineering Proteins Radiolabeled Recombinant Antibody Recombinant Fusion Proteins Recombinant Proteins Recombinants Research Rodent Running Serum Small Business Funding Mechanisms Small Business Innovation Research Grant Sodium Dodecyl Sulfate Solutions Spinal Cord Staging Structure System Technology Testing Therapeutic Therapeutic Effect Transfection United States Food and Drug Administration Western Blotting Work abstracting brain cell capillary cell bank chimeric antibody commercialization dalton design enzyme activity enzyme replacement therapy expression vector fusion gene glucuronide human INSR protein in vivo intravenous administration intravenous injection lysosomal glycosyl hydrolase milligram molecular trojan horse novel novel strategies peptide permease preclinical study progressive neurodegeneration prototype radiotracer receptor receptor binding size small molecule therapeutic enzyme transcytosis uptake vector

项目摘要

DESCRIPTION (provided by applicant): Zhang, Yun Abstract There are over 40 lysosomal storage disorders, and most of these diseases affect adversely the central nervous system (CNS). The mainstay of treatment 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. 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. 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 permanent transfection of cells. These phase II studies will enable production of a master cell bank and development of the purification and downstream processing of the 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. 1 Zhang, Yun Project Narrative Lysosomal storage disorders are serious inborn errors of metabolism, and about 75% of the ~40 lysosomal storage disorders affect the brain. The mainstay of treatment is Enzyme Replacement Therapy (ERT). However, ERT is ineffective in the brain, because the enzymes do not cross the blood-brain barrier (BBB). The present work will produce a novel recombinant fusion protein that is able to both (a) bind a human BBB receptor to trigger transport into the brain, and (b) retain high lysosomal enzyme activity. This novel drug, designated AGT-180, will be a model for the treatment of the brain for multiple genetic diseases. 1

描述(申请人提供)：张云摘要有40多种溶酶体储存障碍，这些疾病大多对中枢神经系统(CNS)造成不利影响。目前主要的治疗方法是酶替代疗法(ERT)。然而，ERT对大脑无效，因为这些酶不会穿过大脑毛细血管壁，而毛细血管壁在体内形成血脑屏障(BBB)。如果不对中枢神经系统进行治疗，这些年轻患者注定会出现进行性神经变性和死亡。在这些疾病的未来治疗中，限制因素是酶通过血脑屏障的运输。直接向脑内注射血脑屏障是无效的，因为只有一小部分大脑通过经颅输送系统进行治疗。相反，几乎所有的脑细胞都可以通过血管输送系统进行治疗，这种系统可以使酶在静脉注射后穿过血脑屏障。一种新的BBB递送大分子如酶的方法是分子特洛伊木马技术。利用基因工程技术生产双功能融合蛋白，将缺失的重组酶与BBB分子木马融合。后者是一种基因工程蛋白，能够通过内源性BBB多肽转运系统上的受体介导的跨细胞作用穿过人的BBB。临床前研究表明，一种分子量为100,000道尔顿的大酶可以通过跨血脑屏障的运输传递到大脑，然后连接到血脑屏障受体特异性特洛伊木马上。目前的工作将产生一种新的融合基因，编码一种溶酶体酶和一种基因工程的分子特洛伊木马，这将允许生产相应的融合蛋白AGT-180。融合基因将被整合到真核表达载体中，然后永久转染细胞。这些第二阶段的研究将使主细胞库的生产和融合蛋白的纯化和下游加工的开发成为可能。由BBB靶向抗体和重组酶组成的融合蛋白可能在治疗人类溶酶体储存障碍的大脑方面具有治疗作用。1张云项目叙述溶酶体储存障碍是一种严重的先天代谢障碍，约75%的~40个溶酶体储存障碍影响大脑。主要的治疗方法是酶替代疗法(ERT)。然而，ERT在大脑中是无效的，因为这些酶不能穿过血脑屏障(BBB)。本工作将产生一种新型的重组融合蛋白，它能够(A)结合人的BBB受体以触发转运到大脑中，(B)保持高溶酶体酶活性。这种名为AGT-180的新药将成为治疗多种遗传性疾病的大脑模型。1