Blood Research Institute, Versiti Wisconsin, Milwaukee, WI.
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI.
Blood Adv. 2021 Mar 9;5(5):1224-1238. doi: 10.1182/bloodadvances.2020004071.
Gene therapy may lead to a cure for hemophilia B (HB) if it is successful. Data from clinical trials using adeno-associated virus (AAV)-mediated liver-targeted FIX gene therapy are very encouraging. However, this protocol can be applied only to adults who do not have liver disease or anti-AAV antibodies, which occur in 30% to 50% of individuals. Thus, developing a protocol that can be applied to all HB patients is desired. Our previous studies have demonstrated that lentivirus-mediated platelet-specific FIX (2bF9) gene therapy can rescue bleeding diathesis and induce immune tolerance in FIXnull mice, but FIX expression was only ∼2% to 3% in whole blood. To improve the efficacy, we used a codon-optimized hyperfunctional FIX-Padua (2bCoF9R338L) to replace the 2bF9 cassette, resulting in 70% to 122% (35.08-60.77 mU/108 platelets) activity levels in 2bCoF9R338L-transduced FIXnull mice. Importantly, sustained hyperfunctional platelet-FIX expression was achieved in all 2bCoF9R338L-transduced highly immunized recipients with activity levels of 18.00 ± 9.11 and 9.36 ± 12.23 mU/108 platelets in the groups treated with 11 Gy and 6.6 Gy, respectively. The anti-FIX antibody titers declined with time, and immune tolerance was established after 2bCoF9R338L gene therapy. We found that incorporating the proteasome inhibitor bortezomib into preconditioning can help eliminate anti-FIX antibodies. The bleeding phenotype in 2bCoF9R338L-transduced recipients was completely rescued in a tail bleeding test and a needle-induced knee joint injury model once inhibitors dropped to undetectable. The hemostatic efficacy in 2bCoF9R338L-transduced recipients was further confirmed by ROTEM and thrombin generation assay (TGA). Together, our studies suggest that 2bCoF9R338L gene therapy can be a promising protocol for all HB patients, including patients with inhibitors.
如果基因治疗成功，它可能为乙型血友病 (HB) 带来治愈的希望。使用腺相关病毒 (AAV) 介导的肝靶向 FIX 基因治疗的临床试验数据非常令人鼓舞。然而，该方案仅适用于没有肝病或抗 AAV 抗体的成年人，而这些抗体在 30%至 50%的个体中出现。因此，需要开发一种可以应用于所有 HB 患者的方案。我们之前的研究表明，慢病毒介导的血小板特异性 FIX（2bF9）基因治疗可以挽救出血倾向并诱导 FIXnull 小鼠的免疫耐受，但全血中的 FIX 表达仅为 2%至 3%。为了提高疗效，我们使用了经过密码子优化的超功能 FIX-Padua（2bCoF9R338L）替代 2bF9 盒，导致 2bCoF9R338L 转导的 FIXnull 小鼠中的活性水平达到 70%至 122%（35.08-60.77 mU/108 血小板）。重要的是，所有接受 2bCoF9R338L 转导的高度免疫化受者均实现了持续的超功能血小板-FIX 表达，其中分别接受 11 Gy 和 6.6 Gy 治疗的组中的活性水平为 18.00 ± 9.11 和 9.36 ± 12.23 mU/108 血小板。抗 FIX 抗体滴度随时间下降，2bCoF9R338L 基因治疗后建立了免疫耐受。我们发现，将蛋白酶体抑制剂硼替佐米纳入预处理可以帮助消除抗 FIX 抗体。一旦抑制剂降至不可检测水平，2bCoF9R338L 转导受者的尾部出血试验和针诱导的膝关节损伤模型中的出血表型完全得到纠正。2bCoF9R338L 转导受者的止血效果在 ROTEM 和凝血酶生成试验 (TGA) 中得到进一步证实。总之，我们的研究表明，2bCoF9R338L 基因治疗可能成为所有 HB 患者的一种有前途的方案，包括有抑制剂的患者。
