Production of Recombinant Eukaryotic Ferroxidases as Protein Therapeutics

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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.