结肠灌注重组腺相关病毒2/人凝血因子Ⅸ基因载体治疗血友病B的安全性研究

Safety of Factor Ⅸ Gene Transfer to Colon Cell in Murine Hemophilia B by Recombinant Adeno-associated Viral Vector

目的分析结肠灌注重组腺相关病毒2/人凝血因子Ⅸ(rAAV2/CMV-hFⅨ)基因载体治疗血友病B小鼠的安全性。方法将rAAV2/CMV-hFⅨ(剂量为1×1013 v.g/kg)结肠灌注血友病B模型小鼠,分别检测抗重组腺相关病毒2(rAAV2)抗体和抗人凝血因子Ⅸ(hFⅨ)抗体,并通过组织病理学、免疫组化和PCR技术分析病毒和hFⅨ的组织分布。结果灌注后,在小鼠体内能够检测到抗rAAV2抗体和抗hFⅨ抗体;组织病理学、免疫组化和PCR技术分析结果显示灌注部位肠组织无明显病理改变,rAAV2以及hFⅨ基因仅仅在灌注的肠上皮。结论结肠灌注重组腺相关病毒基因治疗血友病B小鼠,在所观测时间内是安全的。

Objective To analyze, the safety of factor Ⅸ gene transfer to colon cell in murine hemophilia B by recombinant adeno-associated viral vector serotype 2（rAAV2/CMV-hFⅨ ）. Methods Hemophilia B mice were given an enema with recombinant adeno-associated viral vector serotype 2 encoding hF. Ⅸ under the control of the CMV promoter （rAAV2/CMV-hFⅨ） at dose of 1×10^13 v.g/kg. Antibody to rAAV2 and antibody to hFⅨ were measured in plasma samples collated every 7 days following vector administration. Mice were killed at day 21 after vector administration, and colon tissue was harvested and fixed and stained by hematoxylin and eosin and histochemical analysis. Genomic DNA extracted from murine colon tissue transferring with rAAV vectors was analysed. Results Both antibody to rAAV2 and antibody to hFⅨ were detected in plasma samples. Histochemical analysis, histological analysis and DNA analysis all reveal that factor Ⅸ gene only transfer to colon cell by rAAV2 vector, and sections showed no evidence of inflammatory infiltrate or destruction of colon architecture. Conclusion The experiment of faclor Ⅸ gene transfering to colon cell in routine hemophilia B by recombinant adeno-associated viral vector semtype 2 was safe within the inspected time.