血小板FVIII基因治疗诱导血友病A小鼠FVIII免疫耐受研究

Currently, there is no curative approach available for hemophilia A. Exogenous factor Ⅷ (FⅧ) infusion is the standard treatment to control bleeding. However, the economic burden of this therapy is remarkable. In addition, around 30% of these patients have been found the development of neutralizing antibody to replacement FⅧ protein. And it is considered as a severe and high mortality complication in hemophilia A patients. With the support from NIH RO1 foundation and NHF JGP fellowship, our previous studies have demonstrated that therapeutic FⅧ levels can be only ectopically expressed in platelet α-granules and restore the hemostasis in murine hemophilia A without inhibitory antibody development after platelet-derived FⅧ gene therapy. The same protocol also works in the recipients with high-titers of anti-FⅧ antibodies, indicating it is a promising approach for the treatment in hemophilia A. So the current investigation should be further expanded upon the strategies of fundamental clinical relevance. Thus, we will fully evaluate the cellular and humoral response after continuous administration of large amounts of FVIII protein in hemophilia A mice based on the FⅧ transgenic platelet infusion technology and platelet-derived FⅧ gene therapy approach. In addition, vector control and plasma-derived FⅧ treatment will be set up as parallel controls. The findings in this study may provide us further insights about the roles of immune tolerance with the specific therapy. Moreover, the immunological assessment may document another critical step toward progressing ultimately to human trials for hemophilia A, especially for those with severe hemophilia A.

血友病A目前尚无法根治，反复输注外源性凝血因子Ⅷ（FⅧ）成本昂贵，且30%左右患者易产生FⅧ抑制性抗体，替代疗法难以奏效，患者因严重出血危及生命。前期在美国NIH RO1 (项目号：HL10203501)等基金资助下，我们开展了血小板FⅧ基因治疗血友病A小鼠研究，率先发现功能性FⅧ仅在治疗鼠血小板α-颗粒持续稳定表达，不但恢复模型鼠止血功能，而且不会诱导产生抗FⅧ抗体，即使在高滴度抗体模型仍然显示疗效，提示该基因疗法具有独特应用前景。为此，本项目将前期研究延续和深入，通过FⅧ转基因血小板直接多次输注血友病A及其抗体模型鼠，以外源FⅧ重复免疫再刺激基因治疗鼠，探讨体内细胞免疫和体液免疫功能改变，并与空载组对照，血浆源FⅧ治疗组比较，阐明血小板源FⅧ基因治疗诱导血友病A小鼠FⅧ免疫耐受及机制。从免疫学角度全新认识该疗法的又一独特疗效，对将来血友病A特别是伴抑制性抗体重症患者的治疗具有重要价值。

血友病A目前尚无法根治，反复输注外源性凝血因子Ⅷ（FⅧ）成本昂贵，且30%左右患者易产生FⅧ抑制性抗体，使替代疗法难以奏效，患者常常因严重出血危及生命。前期，我们开展了血小板FⅧ基因治疗血友病A小鼠的研究，率先发现功能性FⅧ仅在治疗鼠血小板α-颗粒持续稳定表达，不但恢复模型鼠止血功能，而且不会诱导产生抗FⅧ抗体，即使在高滴度抗体模型仍然显示疗效。为此，本项目将前期研究延续和深入，通过FⅧ转基因(2bF8Tg)血小板直接多次输注血友病A及其抗体模型鼠，以外源重组人FⅧ（rhF8）重复免疫再刺激2bF8慢病毒(2bF8LV)介导的基因治疗鼠，旨在阐明血小板源FⅧ基因治疗诱导血友病A小鼠FⅧ免疫耐受及相关作用机制。研究结果显示：① 2bF8Tg血小板在血友病A小鼠体内不具有免疫原性，可重复多次输注，不会激发模型鼠产生FⅧ初始及记忆性免疫应答，受者鼠体内不会产生抗FⅧ抗体，甚至FⅧ抗体阳性模型鼠随着输注次数的增多体内抗体滴度反而下降。② 血小板源FⅧ体外培养不具有免疫原性，不会刺激rhF8预刺激后的CD4+ T细胞发生增殖。③ 2bF8LV基因治疗后的血友病A小鼠表达血小板FⅧ具有功能性，显著缩短模型鼠断尾出血试验中的凝血时间，并且可以诱导基因治疗鼠产生FⅧ特异性免疫耐受，该免疫耐受作用可通过脾脏细胞移植过继到其它血友病A小鼠。④ 调节性T细胞（Treg）在2bF8LV基因治疗诱导FⅧ免疫耐受中发挥重要作用。基因治疗组模型鼠Treg细胞比例和总数均显著高于对照组。⑤ 2bF8LV基因治疗诱导血友病A小鼠FⅧ免疫耐受是由CD4+ T细胞介导的。基因治疗鼠CD4+ T细胞在体外与rhF8共培养后不会发生显著增殖，治疗鼠记忆性B细胞可分化为分泌抗FⅧ抗体的浆细胞。以上研究结果从免疫学角度全新认识该基因疗法的又一独特疗效，对将来血友病A特别是伴有抑制性抗体的重症患者治疗具有重要价值。

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