AnVWF biosynthetic efficiency correlates with ancestral FVIII. (A) Phylogenetic tree depicting ancestral sequence reconstruction of 59 extant VWF sequences. VWF sequences selected for de novo synthesis are labeled as closed circles. (B) Production rates of AnVWF protein following transient transfection of HEK116 (red bars) and HEK293T (blue bars) cells. Error bars represent SD (n = 4). *P < .05 (Dunnett’s one-way ANOVA). (C) Stable, monoclonal producer cell lines were produced from HEK116 cells, and VWF biosynthesis was measured via ELISA. Horizontal bars represent median values; n = 13, 7, 23, 29, 19, and 14 for cohVWF, An101, An84, An63, An70, and An88, respectively. No significant differences were detected compared with cohVWF (Dunn’s one-way ANOVA). (D) Transcript analysis of monoclonal producer cell lines at steady state was performed by one-step reverse transcription polymerase chain reaction. VWF antigen was normalized to transcript levels to determine biosynthetic efficiency. Box plot reveals range, 25th and 75th percentiles, and median values; n = 6 for An70 and An88 and n = 5 for others. All AnVWF constructs displayed significantly reduced (*P < .05) biosynthetic efficiency compared with cohVWF (Dunnett’s one-way ANOVA). (E) Correlation of VWF and FVIII biosynthetic efficiency was compared as fold change relative to human. FVIII biosynthetic efficiency was reported previously.14 Linear regression equation (not shown) is y = −1.002x + 6.4 × 10−5 with r2 = 1.