Engineering of a new therapeutic FVIII chimera BIVV001. BIVV001, or rFVIIIFc-VWF-XTEN, results from the fusion of BDD-FVIII with the D′D3 region of VWF, the Fc fragment of human IgG1 and 2 XTEN polypeptides. The engineered molecule lacks the capacity to bind endogenous VWF and has a prolonged half-life. To achieve this, several modifications were implemented: (1) 2 mutations in C1099A and C1142A were introduced in the D′D3 domain of VWF to prevent its dimerization; (2) fusion of the VWF propeptide D1D2 to the D′D3 domain to secure D′D3 optimal folding and to increase its affinity for FVIII (not depicted on the figure); (3) insertion of a 288-amino-acid (aa)-long XTEN between the A2 and A3 domains of FVIII and of a 144-aa-long XTEN between D′D3 and Fc domains to extend the half-life of FVIII; (4) insertion of the FVIII a2 thrombin site to allow optimal FVIII dissociation from D′D3 at the time of activation by thrombin; (5) removal of 3 aa residues (GAP) from the FVIII/XTEN junction and of 9 aa (PPVLKRHQR) from the N-terminal end of the FVIII B domain linker to (i) eliminate predicted major histocompatibility complex II–binding T-cell epitopes and (ii) remove the furin cleavage site between the heavy and light chains of FVIII to ensure its production as a single polypeptide; (6) expression of the FVIII-XTEN-Fc portion as a first chain and of the D′D3-XTEN-Fc portion as a second chain, both held together by disulfide bonds in the Fc region. The goal here is to stabilize the binding of the D′D3 domain to FVIII, to prevent binding of FVIII to the endogenous VWF, and to allow binding of the chimeric molecule to the FcRn. Binding to the FcRn prevents the complex from lysosomal degradation and fosters recycling toward the circulation. BIVV001 is secreted as a single-chain FVIII rather than as a heterodimer. It has a prolonged half-life, is hemostatically functional, and corrects bleeding time in hemophilia A mice.