Metabolic Engineering of Chinese Hamster Ovary Cells for Production of Heparin

肝素是现代医学中应用最广泛的抗凝血药物。为了应对健康危机

Heparin (HP) is the most widely used anticoagulant drug in modem medicine. In response to a health crisis that took place in early 2008, we propose to develop a metabolically engineered HP from a non-animal source. This health crisis involved the adulteration of HP produced from hogs in China with an oversulfated chondroitin sulfate, leading to the death of nearly 100 Americans. HP is a highly sulfated polysaccharide found covalently attached to a core protein as the proteoglycan (PG) serglycin and stored in intracellular granules of mast cells that are found in large numbers in the intestines and lungs of many animals. The heparan sulfate (HS) glycosaminoglycan (GAG) is a less sulfated version of HP that is attached to core proteins, of PGs such as syndecans and glypican, ubiquitously found on the external cell membrane all animal tissues. The proposed 3-year project is a translational and multi-disciplinary research effort aimed at producing HP entirely by the fermentation of metabolically engineered Chinese hamster ovary (CHO) cells. By engineering the CHO cell glycosylation pathway, which normally affords the related polysaccharide HS, a metabolically engineered HP will be prepared with a structure identical to the pharmaceutical HP prepared from animals. Chemical and in vitro bioequivalence studies will provide the necessary pre-clinical data required to carry metabolically engineered HP forward as a generic HP. This is a hypothesis-driven, transformational, and translational research project. We hypothesize that the biosynthetic pathway for HS can be modified to produce HP that will then be secreted outside the CHO cells on an HS core protein and then be shed into the culture medium for collection. There are four specific aims of this proposal. 1. Engineer the CHO cell pathway of HS biosynthesis to afford the more highly sulfated GAG, HP; 2. Engineer the CHO cell so that it makes this highly sulfated GAG attached to a core protein normally carrying HS, export this PG to the external cell surface, and then shed it into the medium; 3. Recover and confirm chemical and in vitro bioequivalence of CHO cell metabolically engineered HP with US Pharmacopeial (USP) HP; and 4. Scale-up the production and recovery of CHO cell metabolically engineered HP while maintaining chemical and bioequivalence. RELEVANCE (See instructions): The broader impacts of the proposed research are to create a metabolically engineered CHO cell heparin that will be safer for patients and can be prepared at costs competitive to heparin obtained from animal tissues, to establish a new paradigm for the use of CHO cells for production of non-protein therapeutics.