Correcting Rare Blood Disorders Using Coagulation Factors Produced In Vivo By Shielded Living TherapeuticsTM Products

Hemophilia A arises from mutations in the F8 gene, affecting ~ 1/5000 males. Treatment options include frequent intravenous factor and subcutaneous non-factor therapies. While these approaches have been widely used, they have significant limitations, such as breakthrough bleeds and joint disease due to suboptimal adherence, non-ideal factor kinetics, inhibitor generation, (Weyand, Blood 2018) as well as risk of thrombotic events and coagulation test interference with newer non-factor therapies. (Peters, Nat Rev Drug Discov 2018) Alternative modalities such as cell therapies with genetically modified, ready-made human cells are being investigated. To avoid a cytotoxic immune response by the host, allogeneic cells either need to be physically shielded and/or the host immunosuppressed. Various biomaterials, e.g. hydrogels, could serve as the physical barrier that prevents host immune cells from accessing the allogeneic cells, avoiding the need for immunosuppression altogether. However, the host can still activate a foreign body response (FBR), targeting the biomaterial, which significantly limits cell survival and durability of cell therapies. (Anderson, Semin Immunol 2008) We have successfully identified a library of proprietary small molecules, which when conjugated to alginate used to create encapsulating spheres, limit the FBR (Bochenek, Nat Biomed Eng 2018). In addition, we further reduced the FBR using two-compartment design, 1.5 mm diameter spheres, in which the cells are encapsulated in an inner compartment surrounded by an outer, acellular compartment. Using this innovative technology, we aimed to create a novel product that will deliver long-term, sustained human coagulation factor VIII (hFVIII) in vivo. First, we selected a human epithelial cell line with optimal properties for encapsulation within the spheres; considerations included safety, contact inhibition and longevity. We genetically modified this cell line using a non-viral vector and an optimized the coding sequence for a B-domain deleted hFVIII to create a proprietary engineered cell line that constitutively expresses high levels of this protein. Second, we optimized the inner compartment matrix by modulating cell density/sphere and by the addition of a novel modified alginate; these changes maximized cell viability and protein production in vivo. Finally, we further optimized the acellular outer compartment with a proprietary mixture of small-molecule-modified and unmodified alginates. T