Schematic representation of fVIII and gammaretroviral vector constructs. (A) Full-length fVIII and bioengineered fVIII molecules with improved secretion and reduced immunogenicity. The sfVIIIΔB variant is a BDD-fVIII transgene that has the HSPA5 binding site double mutation F309S/L303E for enhanced secretion of recombinant fVIII.21 The R484A/R489A/P492A BDD-fVIII variant displays reduced immunogenicity in C57-fVIIIKO mice.29 The F309S/N6 BDD-fVIII variant was secreted approximately 14-fold more efficiently than BDD-fVIII (15- to 25-fold more efficiently than full-length fVIII) from COS-1 cells.18 The efVIII cDNA contains the F309S/L309E, the R484A/R489A/P492A, and the N6 modifications. ??? indicates extent of improvement to be determined. Potential N-linked glycosylation sites are indicated. (B) Schematic representation of the MSGV-sfVIIIΔB-IRES-GFP (abbreviated MSGV-sfVIIIΔB), RMSin-sfVIIIΔB-OFB (referred to as FB.sfVIIIΔB in the text), and RMSin-efVIII-OFB (referred to as FB.efVIII in the text) gammaretroviral vectors. Plasmid forms of the vectors are illustrated. Arrows depict transcription initiation sites. LTR indicates long terminal repeat; RSV, Rous sarcoma virus enhancer-promoter sequences; RMSin, RSV enhancer-promoter–driven self-inactivating MSGV-based retroviral vector backbone; SD, splice donor; SA, splice acceptor; CA, internal promoter consisting of the human cytomegalovirus immediate early region enhancer linked to the chicken β-actin promoter; sfVIIIΔB, secretion-enhanced BDD-fVIII gene; efVIII, glycosylation-facilitated secretion-enhanced fVIII gene with reduced immunogenicity; IRES, internal ribosome entry site; EGFP, enhanced GFP gene; OPRE, safety-optimized woodchuck hepatitis virus posttranscriptional regulatory element; ΔU3, deletion in the U3 region of the 3′ LTR; FB, FII enhancer-blocking component of the chicken β-globin 5′HS4 insulator and the BEAD-A homologous region from the human T-cell receptor α/δ BEAD-I insulator; S, simian virus 40 late poly(A) signal; and B, bovine growth hormone poly(A) signal. Transfer of the deletion in the U3 region of the 3′ LTR to the 5′ LTR after reverse transcription self-inactivates the vector as denoted by the dashed arrow with an X through it.
