血友病A病人诱导多能干细胞rDNA区F8基因打靶及其巨核-血小板分化治疗研究

Hemophilia A (HA) is an X chromosome-linked recessive genetic disorder caused by a deficiency of factor Ⅷ that was encoded by the F8 gene and affecting approximately 1:5000 newborn male individuals. FVIII replacement therapy is effective, but it requires frequent infusions, leading to huge expenses and infections. And nearly 35% of patients develop anti-FVIII inhibitors. Recently AAV-mediated gene therapy has clinically potential, but is not much applicable for HA owing to the large size of F8. And viral approaches still have limitations including non-permanent expression, unwanted insertional mutation or side effects resulted from immune responses. In this study, therefore, we try to target the BDD-F8 gene into the non-viral and integration-free HA-iPSCs derived from urine cells by using an rDNA-targeting vector and TALENickases. The gene-targeted HA-iPSCs will be differentiated into megakaryocyte progenitor cells and transplanted to HA model mice. Therapeutic effect will be evaluated by whole blood clotting and tailing test, and the inhibitors also will be tested. This approach would not only address the safety concern and immunogenicity of viral materials, but also increase efficiency and avoid inhibitor of FVIII by taking advantages of vWF-expression and local aggregation of megakaryocyte-platelets. Combining the ex vivo gene therapy with hemostatic mechanism, we are hopeful to break through the limitation of hemophilia A gene therapy at the current stage.

血友病A是由F8基因突变引起的X连锁隐性遗传病。目前采取的蛋白替代疗法因价格高昂、反复输注、且将近35%病人产生FⅧ抑制物而备受限制。近来病毒载体的基因疗法显示出一定临床应用前景，但病毒载体有限的装载量、非持续性表达、免疫原性、甚至插入突变的高风险仍然存在。由此，本研究通过联合自主改造的TALENickase打靶系统与巨核细胞特异性启动子ITGA2B驱动的BDD-F8基因的rDNA区打靶载体，对HA-iPSCs进行基因修复，将修复后的iPSCs定向分化为巨核祖细胞，经骨髓移植至HA模型鼠，检测小鼠凝血功能及生存时间，同时监测FⅧ抑制物产生情况。本研究规避了病毒载体引起的随机插入与免疫原性问题，同时利用巨核-血小板表达vWF和出血局部聚集等特性，既有效提高FVIII的凝血效能，也避免FⅧ抑制物产生。这种将非病毒载体ex vivo方式与凝血机制相结合的策略可望突破现阶段血友病A基因治疗的瓶颈。

A型血友病(HA)是由凝血因子VIII (FVIII)基因(F8)突变引起的X连锁隐性遗传病。目前采取的蛋白替代疗法因价格高昂、需反复输注、且将近35%病人产生FVIII抑制物而备受限制。考虑到血小板可以通过局部释放α颗粒促进凝血，研究人员在血小板中异位表达FVIII，并结合血小板能表达vWF及其在出血部位局部聚集等特性，有效提高FVIII的凝血效能，避免了FVIII抑制物产生，是一种很有希望的HA治疗方法。尽管近年基于病毒载体的血小板策略基因疗法显示出一定临床应用前景，但病毒载体有限的装载量、非持续性表达、免疫原性、甚至插入突变的高风险仍然存在。由此，本研究通过联合自主改造的TALENickase打靶系统与巨核细胞特异性启动子ITGA2B启动子驱动的B区缺失F8（BDD-F8）基因的rDNA区打靶载体，对病人iPSCs（HA-iPSCs）进行基因打靶，将定点整合后的iPSCs (2bF8-iPSCs）分化为造血祖细胞（iHPCs）、巨核细胞（iMKs）和间充质干细胞（iMSCs）。研究结果表明，FVIII在2bF8-iHPCs和2bF8-iMSCs中高表达，提示这种血小板靶向治疗HA方法的潜在临床前景。

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