Novel therapies for hemophilia, including non-factor replacement and in vivo gene therapy, are showing promising results in the clinic, including for patients having a history of inhibitor development. Here, we propose a novel therapeutic approach for hemophilia based on llama-derived single-domain antibody fragments (sdAbs) able to restore hemostasis by inhibiting the antithrombin (AT) anticoagulant pathway. We demonstrated that sdAbs engineered in multivalent conformations were able to block efficiently AT activity in vitro, restoring the thrombin generation potential in FVIII-deficient plasma. When delivered as a protein to hemophilia A mice, a selected bi-paratopic sdAb significantly reduced the blood loss in a model of acute bleeding injury. We then packaged this sdAb in a hepatotropic AAV8 vector and tested its safety and efficacy profile in hemophilic mouse models. We show that the long-term expression of the bi-paratopic sdAb in the liver is safe and poorly immunogenic, and results in sustained correction of the bleeding phenotype in hemophilia A and B mice, even in the presence of inhibitory antibodies to the therapeutic clotting factor.

中文翻译：

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靶向抗凝血酶的单域抗体可减少使用或不使用抑制剂的血友病小鼠的出血。


血友病的新疗法，包括非因子替代和体内基因疗法，在临床上显示出有希望的结果，包括对于有抑制剂开发史的患者。在这里，我们提出了一种基于美洲驼衍生的单域抗体片段（sdAb）的血友病新治疗方法，能够通过抑制抗凝血酶（AT）抗凝途径来恢复止血。我们证明，以多价构象设计的 sdAb 能够在体外有效阻断 AT 活性，恢复 FVIII 缺陷血浆中的凝血酶生成潜力。当将一种选定的双互补位 sdAb 作为蛋白质递送给 A 型血友病小鼠时，可显着减少急性出血损伤模型中的失血量。然后，我们将这种 sdAb 包装在亲肝 AAV8 载体中，并在血友病小鼠模型中测试其安全性和有效性。我们证明双互补位 sdAb 在肝脏中的长期表达是安全的且免疫原性差，并且即使存在治疗性凝血的抑制性抗体，也能持续纠正血友病 A 和 B 小鼠的出血表型因素。