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Therapies that target the multicellular pathology of central nervous system (CNS) disease/injury are urgently required. Modified non-anticoagulant heparins mimic the heparan sulphate (HS) glycan family and have been proposed as therapeutics for CNS repair since they are effective regulators of numerous cellular processes. Our in vitro studies have demonstrated that low-sulphated modified heparan sulphate mimetics (LS-mHeps) drive CNS repair. However, LS-mHeps are derived from pharmaceutical heparin purified from pig intestines, in a supply chain at risk of shortages and contamination. Alternatively, cellular synthesis of heparin and HS can be achieved using mammalian cell multiplex genome engineering, providing an alternative source of recombinant HS mimetics (rHS). TEGA Therapeutics (San Diego) have manufactured rHS reagents with varying degrees of sulphation and we have validated their ability to promote repair in vitro using models that mimic CNS injury, making comparisons to LS-mHep7, a previous lead compound. We have shown that like LS-mHep7, low-sulphated rHS compounds promote remyelination and reduce features of astrocytosis, and in contrast, highly sulphated rHS drive neurite outgrowth. Cellular production of heparin mimetics may, therefore, offer potential clinical benefits for CNS repair. 

Background: Although most biologics are produced using recombinant technologies, heparin persists as a product purified from animal tissues. A cell based system for production of heparin would eliminate risk of supply shortage and contamination. Additionally, genetic engineering could yield heparin with improved qualities such as reduced risk of heparin-induced thrombocytopenia. Aims: This work is focused on engineering mammalian cell lines and bioprocess methods to produce recombinant heparin. Methods: The heparan sulfate biosynthetic pathway of mastocytoma cells was genetically engineered to alter the expression of heparan sulfate sulfotransferases. The resulting cell lines were screened for production of anti-FXa activity. Heparan sulfate production from a candidate cell line was tested in chemically defined medium. The recombinant product was characterized structurally and in clotting, anti-protease and heparin induced thrombocytopenia assays. Results: Engineered cells produced heparan sulfate in chemically defined medium with anti-Xa and anti-IIa activity exceeding the requirement for unfractionated heparin despite having lower sulfate content. Chain length was longer than unfractionated heparin. Additionally, binding to platelet factor 4 was reduced compared to unfractionated heparin, suggesting less risk of heparin-induced thrombocytopenia. Conclusion: These results demonstrate the feasibility of producing a substitute for unfractionated heparin from recombinant cell culture. Additionally, recombinant technology may allow production of heparin substitutes with improved properties such as reduced side effects.