慢病毒载体介导在造血干细胞红系分化过程中特异性表达高凝血活性FIX蛋白治疗血友病B

Hemophilia B is an X-linked bleeding disorder resulted from mutations in the coagulation factor IX (FIX) gene. A majority of HB patients have less than 1% of normal FIX activity with life-threatening phenotypes. Protein replacement therapy (PRT) only have transient effects with high-expense pressure and infusion-associated complications. Recent advances in gene therapy showed promising effects in treatment of HB yet having limited efficacy as well as safety drawbacks related to the vector design. Here we propose an alternative gene therapy strategy with a self-inactivation lentiviral vector (LV) configuration for ex vivo gene therapy. Upon LV transduction of hematopoietic stem cells, the hyper-functional human hFIX protein carrying the R338L mutation under the transcriptional control of β-globin LCR/promoter is specifically expressed in red blood cells during HSC differentiation. With this strategy, we propose that transplantation of gene-modified HSCs can mediate efficient expression of hFIX protein with hyper activity to a therapeutic level and consequently correct bleeding phenotypes in hemophilia B (HB) mice. More importantly, the efficacy and safety of this vector will be verified in hFIX-immunized HB mice mimicking the patients with existing anti-FIX inhibitors after protein replacement therapy. Our gene therapy strategy is very promising to overcome the host immune response against the delivery vector and transgene product to generate therapeutic effects. This therefore will provide a potential one-off therapeutic approach with life-long effects in treatment of naive and PRT-treated HB patients in the future.

血友病B（HB）是X染色体连锁性凝血因子IX缺陷导致的单基因遗传性疾病。患者中约一半以上属于重型并伴有反复自发性出血及继发关节畸形和功能障碍。最近基因治疗HB在临床试验上有一定进展，但是临床疗效还有待增高。自我失活型慢病毒载体（LV）因其强大的转染效率、可塑性及安全性被广泛应用于基因治疗。在此我们提出了一种基因治疗策略，利用慢病毒载体转染整合造血干细胞（HSC），在HSC向红细胞分化成熟过程中以β珠蛋白LCR/启动子驱动高效特异的高凝血活性FIX蛋白突变体表达。我们将以HB小鼠为模型评估这种策略中基因的传递效率，功能蛋白的浓度和活性以及宿主与载体的相互作用，从而探索为更多的HB患者提供一项终生有效的一次性治疗手段。

迄今为止临床上缺乏对血友病患者长期有效的治疗措施。在本项目中我们成功地构建了慢病毒载体（LV）在红细胞系统表达人凝血因子IX（hFIX），并在小鼠模型中成功证实了应用此基因治疗策略治疗血友病B的长期疗效。通过红细胞特异性元件的转录调控， LV载体携带的hFIX表达框架在小鼠骨髓造血干细胞红系分化的过程中可以有效地表达hFIX蛋白，从而纠正小鼠的出血表型。此外，高凝血活性hFIXR338L蛋白的表达可以在不增加hFIX抗原或LV载体剂量的前提下，有效提高hFIX的凝血活性，甚至可以降低LV载体剂量以减少不良反应。此外，动态监测骨髓移植后的小鼠体内均未发现明显的特异性hFIX抑制物或高凝血状态所致的D二聚体水平增高。以上结果都肯定了这种治疗策略的疗效和安全性。我们的研究为血友病B患者提供了一个可长期有效治愈的临床治疗选择。临床转化后可以为缓解患者的疾病状态，改善患者的生活质量，以及减轻社会经济负担均有重要的意义。