The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter β-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases.

Background: Emicizumab is an alternative non-factor approach for treating patients with hemophilia A. However, there is a potential risk of thrombotic events when emicizumab is concomitantly administered with pro-hemostatic therapies.Objectives: To assess the hemostatic effect in vitro when a plasma-derived factor VIII concentrate containing von Willebrand factor (pdFVIII/VWF) was added to hemophilia A plasma (HAp) in combination with emicizumab.Methods: HAp and HAp with FVIII inhibitors (HAp-i) samples with different concentrations of emicizumab (50 and 100 μg/mL) were combined with activated prothrombin complex concentrate at 0.5 to 1 U/mL, recombinant activated factor VII (rFVIIa) at 0.5 to 7 μg/mL, and pdFVIII/VWF at 0.1 to 4.5 IU/mL. Thrombin generation (TG; thrombin peak and endogenous thrombin potential) was determined using a Calibrated Automated Thrombogram assay.Results: When activated prothrombin complex concentrate was added to HAp and HAp-i with emicizumab, TG dramatically increased (multiplier effect > 4.5×). Addition of rFVIIa to HAp or HAp-i with emicizumab moderately increased TG in a concentration-related manner compared with rFVIIa alone. Addition of pdFVIII/VWF to HAp or HAp-i with emicizumab induced a TG response equivalent to those samples without emicizumab. In an in vitro model of immune tolerance induction with bleeds (HAp-i 15 Bethesda units), combination of pdFVIII/VWF, emicizumab, and rFVIIa did not trigger a multiplying effect on TG.Conclusions: pdFVIII/VWF showed a non-additive effect on TG when combined in vitro with emicizumab. This finding suggests that emicizumab has limited ability to promote factor X activation in the presence of pdFVIII/VWF, thus reducing the risk of thrombotic events.

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