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: 7455765
• 负责人: DANIEL J. KOSMAN
• 金额: $ 19.02万
• 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 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7455765
• 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（由申请人提供）：在真核生物中管理铁需要一种或多种针对亚铁（FeII）作为底物的多铜氧化酶的活性。这些酶被称为铁氧化酶。它们的缺失导致了各种表型，从铁依赖性酶的活性下降，如酵母，到人类和其他哺乳动物的神经变性，这是由于hCp缺乏导致的铁沉积在大脑中。其中两种氧化铁酶已被很好地表征：来自酵母酿酒酵母的fe3蛋白（yFet3p）和人类铜蓝蛋白（hCp）。在人类中，Cp基因的遗传病变导致急性粒细胞性贫血（AC），血液和中枢神经系统（CNS）中缺乏hCp活性。前者的缺乏导致铁转铁蛋白的下降，导致铁向外周组织的输送下降。尽管后一种下降，铁在中枢神经系统中积累，并可能支持导致神经元细胞死亡的化学反应，这一机制可能也涉及其他神经退行性疾病。该R21应用的目的有两个：1)开发高通量、基于哺乳动物细胞的重组hCp生产；2)制定策略，将yFet3p和rhCp转化为药理学上有效的蛋白质治疗药物，以管理全身铁。建立hCp大容量表达系统的原因有两个：1)为野生型hCp作为蛋白质治疗药物的开发提供依据；2)为后续阐明AC突变蛋白的分子缺陷提供依据。我们认为，这一发现具有重要意义，原因有二：1)它将使我们对hCp的一般机制有更深入的了解；2)在某些情况下，它将提出抑制体内给定缺陷的小分子策略。我们的高通量蛋白生产将使用连续瞬时转染条件下悬浮的HEK298 EBNA1细胞，该系统在生物技术和制药行业中应用最广泛。此外，我们将在无动物源性（AOF）条件下生产这些蛋白质，这在药物开发中越来越需要。将氧化铁酶开发成有效的蛋白质疗法的基本原理是显而易见的；他们有可能抑制铁的管理不善，导致功能性（非营养性）缺铁系统和神经元细胞死亡特异性。我们的蛋白质治疗开发策略有两个步骤：1)在不影响铁氧化酶活性的情况下，通过聚乙二醇偶联（PEGylation）稳定yFet3p（以及随后的rhCp）在循环中；2)通过在形成血脑屏障（BBB）的毛细血管内皮中发现的转铁蛋白受体（TfR）将该蛋白靶向到中枢神经系统。聚乙二醇化现在正在使用（例如干扰素药物Pegasus和PegIntron），而TfR“特洛伊木马”策略已成功地用于小分子和蛋白质治疗的模型系统中。我们将使用aceruloplasminenic， Cp-/-小鼠AC模型来测试我们的蛋白质疗法在恢复血铁平衡方面的功效，使用高度道德的方案，不涉及放射性，麻醉或术后应激。铁是一种必需但有毒的营养素；大脑正常代谢的中断与许多神经退行性疾病有关。这种联系的一个明显的例子是在遗传性疾病——青铜质纤溶酶血症中发现的，在这种疾病中，患者缺乏酶的活性，铜蓝蛋白是正常铁代谢所必需的酶。这项研究的长期目标是确定患者的铜蓝蛋白中有什么缺陷，并开发一种基于蛋白质的药物，可以补充患者缺乏的铜蓝蛋白活性。