Fig. 1. Strategy for dissection of 3-O-sulfation structure-function and engineering of a cell-based HS anticoagulant.

(A) Chemical synthesis is used to generate 3-O-sulfated tetrasaccharides, which are subsequently digested to the 3-O-sulfated disaccharide standards required for complete analysis of HS and heparin/LMWHs. (B) A comprehensive HPLC method is developed for the analysis of HS and heparin/LMWH disaccharide composition including 3-O-sulfation. (C) To generate a cell-based anticoagulant, functional assays for anticoagulant activity are used for comparing HS from genetically engineered cells with clinical heparin/LMWHs. A major side effect of heparin is HIT due to PF4 binding to heparin chains; therefore, PF4 binding of HS/heparin/LMWHs is measured to identify low-binding variants that would not generate this side effect. (D) CHO cells produce CS/dermatan sulfate (DS) GAGs as well as HS, which necessitates laborious and problematic purification for isolation of HS chains. To avoid this, CRISPR-Cas9 can be used to KO genes to ablate CS/DS biosynthesis. Targeted stable KI of genes can be achieved using ZFNs, producing CHO cell lines individually expressing the seven HS3STs, resulting in a cell-based HS3ST library. (E) Heparin/LMWHs are widely used anticoagulant drugs, yet are complex, heterogeneous, and poorly characterized animal products derived predominantly from porcine intestinal mucosa. Cell-based production could offer reduced complexity, and genetic engineering of the heparin/HS biosynthetic pathway in cells can generate a more homogeneous population of HS/heparin chains with desired properties.