通过原位转导造血干细胞建立高效安全的血友病基因治疗方法和机制

Hemophilia B is a hereditary bleeding disorder caused by human factor IX (hFIX) deficiency. In treatment of hemophilia B，gene therapy is no doubt the best choice. However, the therapeutic effect of current clinical trials for hemophilia B are still disappointing. This is mainly because of lack of vector transduction efficiency, low therapeutic gene expression level, as well as inhibition of therapeutic gene expression due to immune responses .In this proposal, we plan to perform hemophilia B gene therapy through intrafemoral transduction of hematopoietic stem cells (HSCs) using lentiviral vector that is capable of erythroid specifically expressing hFIX. To achieve therapeutic level of hFIX gene expression, in situ transduced HSCs will be enriched using drug resistance gene, methylguanine methyltransferase-P140K mediated drug selection. Furthermore, to reduce the risk of insertional mutagenesis and improve integration safety of lentiviral vector transduction, ΦC31 integrase site-specific integration will be studied to achieve erythroid specific hFIX gene expression. So far, we have established a highly efficient erythroid specific gene expression system, as well as a live-imaging mouse model for intrafemoral lentiviral vector transduction of HSCs and erythroid specific gene expression. Although the main focus of this proposal is to develop an effective gene therapy for hemophilia B, the conclusion of our research will have broad impact for developing effective treatment for other genetic disorders that require gene replacement therapies.

B型血友病是人第九凝血因子(hFⅨ)基因缺陷引起的遗传性出血疾病，在治疗这一单基因缺陷遗传疾病方面，基因治疗无疑是最佳选择。然而，由于载体转导效率较低、基因表达水平不高和免疫抑制等因素，目前血友病基因治疗的疗效还有待提高。本项目拟通过股骨内慢病毒载体原位转导途径，将红细胞特异性表达的hFⅨ基因转入小鼠造血干细胞，并通过耐药基因富集使其表达到治疗水平，从而建立高效的血友病基因治疗方法。为进一步提高这一治疗的安全性，我们还将探讨通过ΦC31整合酶系统达到hFⅨ治疗基因位点特异性整合的可行性。作为本项目的前期结果，申请人已建立了高效的红细胞特异性慢病毒载体表达系统，和可供活体监测的小鼠股骨内原位转导动物模型，为采用原位转导造血干细胞基因治疗血友病这一方法提供了可靠的技术基础。本项研究不但对发展高效、安全的血友病基因治疗具有指导意义，还能为治疗其他单基因缺陷遗传病提供新的途径。