Coagulation factor viii : insertions of unstructured polypeptides ( xten ) result in enhanced expression and extended in vivo half-life

Synthetic biology is revolutionizing how we conceptualize and approach the engineering of biological systems. Recent advances in the field are allowing us to expand beyond the construction and analysis of small gene networks towards the implementation of complex multicellular systems with a variety of applications. In this talk I will describe our integrated computational / experimental approach to engineering complex behavior in mammalian cells. In our research, we appropriate design principles from electrical engineering and other established fields. These principles include abstraction, standardization, modularity, and computer aided design. But we also spend considerable effort towards understanding what makes synthetic biology different from all other existing engineering disciplines and discovering new design and construction rules that are effective for this unique discipline. We will briefly describe the implementation of genetic circuits and modules with finely-tuned digital and analog behavior and the use of artificial cell-cell communication to coordinate the behavior of cell populations. The first system to be presented is a genetic circuit that can detect and destroy specific cancer cells based on the presence or absence or specific biomarkers in the cell. We will also discuss preliminary experimental results for obtaining precise spatiotemporal control over stem cell differentiation for tissue engineering applications. We will conclude by discussing the design and preliminary results for creating an artificial tissue homeostasis system where genetically engineered stem cells maintain indefinitely a desired level of pancreatic beta cells despite attacks by the autoimmune response, relevant for diabetes. Fusion of unstructured polypeptides of defined amino acid composition, known as XTEN, is an effective means of extending the in vivo half-life of biotherapeutics. We are employing the XTEN technology to improve the half-life of human coagulation factor VIII (hFVIII). One or more XTENs were inserted at inter-domain sites, intra-domain sites, and/or the C terminus of hFVIII. The aim of this study was to determine the effect of XTEN insertion(s) on the expression and in vivo half-life of recombinant hFVIII. Potential insertion sites were identified based on comprehensive structure analysis of FVIII and by excluding sites reported implicated in hemophilia A. Expression vector constructs encoding human B domain-deleted FVIII (BDD-FVIII) bearing XTEN modifications were transiently expressed in HEK293 cells, and the activities of the secreted FVIII measured by a chromogenic assay. The pharmacokinetic (PK) properties of hFVIII variants were evaluated in mice models by monitoring plasma FVIII activity over time. Analysis of the effect of 42 and 144 …