Small antibody fragments as alternative tools in haemophilia care

凝血因子VIII(FVIII)的功能性缺失与一种称为血友病A的严重出血性疾病有关，每10,000名男性中有1-2人受到影响。临床治疗主要包括使用提纯的FVIII浓缩液进行替代治疗。由于这种方法的一些缺点(频繁静脉输注，产生抑制性抗体，成本高)，医学上有强烈的需求来开发新的治疗策略。本项目的目的是同时开发一种低成本的蛋白质疗法和一种利用单域抗体片段(纳米体)对血友病A进行创新的AAV基因疗法。纳米抗体有几个优点，包括应用于人类时的低免疫原性(如果有的话)。此外，它们的小尺寸(±17 kDa)有利于它们的分子工程和它们的DNA在病毒颗粒中的掺入。将产生以天然抗凝剂为靶标的纳米体。抑制这些抗凝血剂可以恢复血友病小鼠有缺陷的凝血酶生成能力。事实上，我们联盟中的一个合作伙伴的初步研究表明，这种纳米体完全恢复了血友病血浆中凝血酶的生成，并减少了血友病小鼠的失血。在这个研究项目中，我们将探索两种不同的纳米体内输送模式：蛋白质注射和基因转移。第一种方法将侧重于皮下给药的发展，导致改进的低成本治疗方案。第二种方法提供了一种长期治疗方案的潜力，以尽量减少血友病的出血。

The functional absence of coagulation factor VIII (FVIII) is associated with a severe bleeding disorder, known as haemophilia A, affecting 1-2 per 10,000 males. Clinical management mainly involves replacement therapy using purified FVIII concentrates. Due to a number of disadvantages of this approach (frequent intravenous infusions, development of inhibitory antibodies, high costs), there is a strong medical need for the development of novel therapeutic strategies. The aim of the present project is to develop in parallel a low-cost protein therapy and an innovative AAV-based gene therapy approach for haemophilia A using single domain antibody fragments (nanobodies). Nanobodies have several advantages, including a low (if any) immunogenicity when applied to humans. Further, their small size (±17 kDa) facilitates their molecular engineering and the incorporation of their cDNA in viral particles. Nanobodies will be generated that target natural anticoagulants. Inhibition of these anticoagulants restores the defective thrombin generation capacity in haemophilic mice. Indeed, preliminary studies by one of the partners in our consortium have shown that such nanobodies fully restore thrombin generation in haemophilic plasma and reduce blood loss in haemophilic mice. In this research program, we will explore two different modes of nanobody delivery: protein administration and gene transfer. The first approach will focus on the development of subcutaneous delivery, leading to an improved low-cost treatment regimen. The second approach provides the potential of a long-term therapeutic solution to minimize bleeding in haemophilia.