Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA.

中文翻译：

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修饰因子 VIII 基因的转导提高了 A 型血友病的慢病毒基因治疗效果。

血友病 A (HA) 是一种由凝血因子 VIII (F8) 缺乏引起的出血性疾病。F8 替代是护理标准，而 HA 的基因治疗（F8 基因）是一种有吸引力的研究方法。然而，F8基因的大尺寸和产品的免疫原性给F8基因疗法的开发带来了挑战。为了解决这些问题，我们合成了一个缩短的 F8 基因 (F8-BDD) 并将其克隆到慢病毒载体 (LV) 中。F8-BDD 在 LV 转导的细胞中主要产生短切割的非活性产物。为了提高 F8 的功能，我们设计了两个新的 F8-BDD 基因，一个插入了八个特定的 N-糖基化位点 (F8-N8)，另一个则恢复了 B 域中的所有 N-糖基化位点 (F8-299)。虽然整体蛋白表达降低，但高凝血活性（> 在 LV-F8-N8 和 LV-F8-299 转导细胞的上清液中检测到 100 倍。F8 和促凝血辅助因子 von Willebrand 因子的蛋白质分析显示，在使用 F8-299 恢复 B 域糖基化后，相互作用增强。HA 小鼠造血干细胞移植研究表明，将 LV-F8-N8 或 -299 基因转移到造血干细胞后，出血表型得到纠正。重要的是，F8-299 修饰显着降低了这些 HA 小鼠中 F8 蛋白的免疫原性。总之，修饰的 F8-299 基因可以有效地包装到 LV 中，并且尽管表达降低，但产生了高度稳定和功能性的 F8 蛋白，可纠正出血表型而没有抑制性免疫原性。