Production of Recombinant Eukaryotic Ferroxidases as Protein Therapeutics

作为蛋白质治疗剂的重组真核铁氧化酶的生产

• 批准号: 7291433
• 负责人: DANIEL J. KOSMAN
• 金额: $ 22.43万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7291433
• 关键词: Address; Alleles; Anesthesia procedures; Animals; Biochemical; Biochemistry; Biological Assay; Biological Models; Blood; Blood - brain barrier anatomy; Blood Circulation; Blood capillaries; Brain; Cells; Ceruloplasmin; Characteristics; Chemistry; Clinical; Condition; Defect; Deposition; Development; Dietary Iron; Disease; Drug Industry; Endothelium; Enterocytes; Enzymes; Equilibrium; Equus caballus; Etiology; Eukaryota; Eukaryotic Cell; Evaluation; Exhibits; Face; Functional disorder; Funding; Gene Duplication; Generations; Genes; Genetic; Glycosylphosphatidylinositols; Goals; Health; Hereditary Disease; Human; Human Genetics; In Vitro; Injection of therapeutic agent; Institutes; Interferons; Ions; Iron; Lead; Lesion; Life; Link; Mammalian Cell; Mammals; Maps; Measures; Metabolism; Metals; Modality; Modeling; Molecular; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Nutrient; Nutritional; Organism; Outcome; Oxidases; Oxidation-Reduction; Parkinson Disease; Pathology; Patients; Pattern; Peripheral; Personal Satisfaction; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Physiology; Plasma; Polyethylene Glycols; Procedures; Production; Proteins; Protocols documentation; Radioactivity; Range; Reactive Oxygen Species; Recombinants; Recycling; Research; Saccharomyces cerevisiae; Serum; Small Intestines; Specificity; Stress; Suspension substance; Suspensions; Symptoms; System; Technology; Testing; Therapeutic; Time; Tissues; Transfection; Transferrin; Transferrin Receptor; United States National Institutes of Health; Vacuole; ViraferonPeg; Withdrawal; Work; Yeasts; base; capillary; cohort; cytotoxic; design; enzyme activity; in vivo; insight; interest; iron metabolism; mouse model; mutant; neuron loss; neuropathology; paralogous gene; prevent; small molecule; therapeutic protein; therapeutic target; tissue/cell culture; uptake; yeast protein

DESCRIPTION (provided by applicant): Managing iron within a eukaryote requires the activity of one or more multicopper oxidases specific towards ferrous iron, FeII, as substrate. These enzymes are called ferroxidases. Their absence leads to phenotypes that range from a decline in the activity of iron-dependent enzymes, as in yeast, to a neurodegeneration in humans and other mammals that is due to the Fe-deposition in the brain that hCp deficiency causes. Two of these ferroxidases have been well characterized: the Fet3 protein from the yeast Saccharomyces cerevisiae, yFet3p, and human ceruloplasmin, hCp. In humans, genetic lesions in the Cp gene lead to aceruloplas- minemia (AC), a lack of hCp activity in the blood and in the central nervous system (CNS). The former deficiency causes a decline in iron transferrin leading to a decline in the delivery of Fe to peripheral tissues. De- spite this latter decline, Fe accumulates in the CNS and likely supports the chemistry that leads to neuronal cell death, a mechanism perhaps involved in other neurodegenerative disorders as well. The objective of this R21 application is two-fold: 1) to develop high throughput, mammalian cell-based recombinant hCp production; and 2) to develop strategies for converting yFet3p and rhCp into pharmacologically efficacious protein therapeutics in the managing of systemic iron. The rational for establishing a high-volume expression system for hCp is two-fold: 1) to provide wild type hCp for its development as a protein therapeutic and 2) to provide AC mutant proteins for the subsequent elucidation of their molecular defects. We believe that this insight will be significant for two reasons: 1) it will lead to a greater understanding of the hCp mechanism in general and 2) in some cases, it will suggest small molecule strategies for suppressing a given defect in vivo. Our high-throughput protein production will use HEK298 EBNA1 cells in suspension under conditions of continuous transient transfection, the system most widely used in the biotech and pharmaceutical industries. Furthermore, we will produce these proteins under animal origin free (AOF) conditions as increasingly required in pharmaceutical development. The rationale for developing ferroxidases into effective protein therapeutics is obvious; they have the potential to suppress the mismanagement of iron that leads to a functional (not nutritional) iron deficiency systemically and to a neuronal cell death specifically. Our protein therapeutic development strategy has two steps: 1) stabilize yFet3p (and subsequently, rhCp) in the circulation by PEGylation (polyethylene glycol conjugation) without compromise of ferroxidase activity; 2) target this protein to the CNS via the transferrin receptor (TfR) found in the capillary endothelia that form the blood brain barrier (BBB). PEGylation is now in use (e.g. the interferon drugs, Pegasus and PegIntron) and the TfR, "Trojan Horse" strategy has been used successfully in model systems with both small molecule and protein therapeutics. We will use the aceruloplasminenic, Cp-/- mouse model of AC to test the efficacy of our protein therapeutics in restoring blood iron balance using a highly ethical protocol involving no radioactivity, anesthesia or post-procedure stress. Iron is an essential yet toxic nutrient; a break-down in its normal metabolism in the brain is linked to many neurodegenerative diseases. A clear example of this connection is found in the genetic disorder, aceruloplasminemia, in which the patient has a deficiency in the activity of the enzyme, ceruloplasmin, an enzyme essential for normal iron metabolism. The long-term goals of this research are to determine what is defective in the patients' ceruloplasmin protein and to develop a protein-based drug that can supplement the ceruloplasmin activity that the patients lack.

描述(申请人提供)：在真核生物中管理铁需要一种或多种针对亚铁的多铜氧化酶的活性，FeII，作为底物。这些酶被称为铁氧合酶。它们的缺失导致了各种表型，从酵母中依赖铁的酶的活性下降，到人类和其他哺乳动物的神经退化，这是由于HCP缺乏导致大脑中的铁沉积。其中的两种铁氧合酶已经被很好地描述：来自酿酒酵母的Fet3蛋白，yFet3p和人铜蓝蛋白，hcp。在人类中，CP基因的遗传损伤会导致无白血球蛋白血症(AC)，即血液和中枢神经系统(CNS)中HCP活性的缺乏。前者的缺乏会导致铁转铁蛋白的减少，从而导致铁向外周组织的输送减少。尽管后一种下降，铁在中枢神经系统积累，并可能支持导致神经细胞死亡的化学物质，这一机制可能也参与了其他神经退行性疾病。这项R21应用的目标有两个：1)开发高通量、基于哺乳动物细胞的重组HCP生产；以及2)开发将yFet3p和RHCP转化为在管理全身铁的药理上有效的蛋白质疗法的策略。建立HCP高效表达系统的理由有两个：1)提供野生型HCP作为蛋白质治疗药物；2)提供AC突变蛋白，用于后续阐明其分子缺陷。我们认为，这一见解具有重要意义，原因有两个：1)它将有助于更好地理解HCP的一般机制；2)在某些情况下，它将建议抑制体内给定缺陷的小分子策略。我们的高通量蛋白质生产将使用HEK298 EBNA1细胞在连续瞬时转染的条件下悬浮，这是生物技术和制药行业最广泛使用的系统。此外，我们将在无动物来源(AOF)的条件下生产这些蛋白质，因为制药开发越来越需要这些蛋白质。将铁氧合酶开发成有效的蛋白质疗法的理由是显而易见的；它们有可能抑制铁的管理不善，这种管理不善会导致系统性的功能性(非营养性)铁缺乏，并特别导致神经细胞死亡。我们的蛋白质治疗开发策略包括两个步骤：1)通过聚乙二醇化(聚乙二醇偶联物)稳定循环中的yFet3p(以及随后的RHCP)，而不影响铁氧合酶的活性；2)通过在形成血脑屏障(BBB)的毛细血管内皮细胞中发现的转铁蛋白受体(TFR)将该蛋白质靶向CNS。目前正在使用聚乙二醇化反应(如干扰素药物Pegasus和PegIntron)和TFR，“特洛伊木马”策略已成功地用于小分子和蛋白质疗法的模型系统。我们将使用AC型纤毛虫肺炎的CP-/-小鼠模型来测试我们的蛋白质疗法在恢复血铁平衡方面的有效性，该疗法采用高度道德的方案，不涉及放射性、麻醉或术后应激。铁是一种基本但有毒的营养物质；它在大脑中正常新陈代谢的障碍与许多神经退行性疾病有关。这种联系的一个明显例子是遗传性疾病--无浆蛋白血症，患者缺乏铜蓝蛋白酶的活性，铜蓝蛋白是正常铁代谢所必需的一种酶。这项研究的长期目标是确定患者铜蓝蛋白中存在哪些缺陷，并开发一种基于蛋白质的药物，以补充患者缺乏的铜蓝蛋白活性。