肌注腺伴随病毒基因治疗血友病B的安全性研究

Safety Assessment of Intramuscular Administration of Recombinant Adeno-associated Virus(AAV)-mFⅨ

研究了肌注腺伴随病毒 (adeno -associatedvirus,AAV)介导凝血Ⅸ因子基因治疗血友病B的安全性。首先采用PCR、反转录PCR(RT -PCR)分别检测rHSV/AAV包装系统产生的重组腺伴随病毒AAV -mFⅨ ,和病毒感染细胞后所得上清再次感染的BHK细胞中的HSV(herpessimplexvirus,HSV)和野生型AAV。同时观察HSV引起的细胞毒作用。结果表明 :纯化后的AA -mFⅨ中HSV≤ 1个病毒基因组 (viralgenome ,v g ) /10 8个病毒基因组AAV -mFⅨ ,且不具备感染活性 ;没有野生型AAV。其次 ,采用PCR、RT -PCR、免疫组化等方法检测了AAV -mFⅨ在体内的分布和表达时间 ;通过抗AAV的抗体检测和病理切片等方法观察了AAV -mFⅨ在体内引起的免疫反应和病理变化。结果表明 :AAV -mFⅨ仅分布在注射点肌肉组织内 ,表达可持续 2 0 0天以上 ;抗体水平低 ,各主要脏器均未发生明显的病理变化。AAV介导的凝血因子Ⅸ系统是安全的。

The safety of intramuscular injection of recombinant adeno-associated virus(AAV)-mFⅨ to rescue hemophilia B was assessed.The helper virus,herpes simplex virus(HSV),and wild-type adeno-associated virus in purified AAV-mFⅨ were assayed by detecting glycoprotein gene D(gD) of HSV and rep gene of wild-type AAV in AAV-mFⅨ with the method of polymerase chain reaction(PCR).The mRNA of gD and rep gene in HBK cells infected by the supernatant of AAV-mFⅨ infecting cells were examined by RT-PCR,and cytopathogenic effect caused by HSV was observed.The results showed that there was no wild-type AAV and there was less than 1 viral genome(v.g.) of HSV per 10 8 v.g. of AAV-mFⅨ, and the HSV had no biological activity.In in vivo study,the results showed that AAV-mFⅨ only existed in muscle around the injection site and the expression of mouse clotting factor IX lasted more than 200 days. Mice injected with AAV-mFⅨ,resulted in little antibody to AAV, and there was no evidence of inflammation or other toxicity. These indicated that intramuscular injection of AAV-mFⅨ was relatively safe.