6型重组腺相关病毒载体的生物学活性评估及预存免疫对其的影响

Efficient and Durable Gene Delivery of Self Complementary Adeno-associated Virus 6 Vector and Impact of Pre-existing Immunity

重组腺相关病毒(rAAV)载体是一种高效的基因治疗转导载体,但因灵长类动物中存在天然的免疫抗体,使其作用受到很大限制。本研究采用含人源α-1抗胰蛋白酶(hAAT)的rAAV6载体转导C57BL/6小鼠,结果显示hAAT的表达在转导后7d出现,14d达峰值,28d仍持续表达。采用含增强型绿色荧光蛋白(eGFP)的rAAV6转导C57BL/6小鼠,荧光成像结果显示rAAV6具有嗜肝性,但也会导致肝脏出现自限性损伤。此外,我们检测了猴群中AAV6的中和抗体(NAb)的分布情况,发现以1∶5为起始稀释度检测,血清中NAb阳性率高达52.17%,在此基础上,我们进行体内过继免疫实验,评估预存免疫对rAAV6的影响,结果显示NAb可以阻断rAAV6的体内转导,即使低滴度的NAb血清也会产生强大的体内中和效应。

Recombinant adenoassociated viral （rAAV） vectors are promising vectors ~or human gene therapy. How ever, AAVmediated gene transduetion can be hampered because of the preexisting neutralized natural antibodies （NAbs） in primates. We evaluated transduction efficiency of rAAV6 expressing human alphalantitrypsin （hAAT） vectors in murine models, and found that these vectors showed stable and high levels of transgene expression. Fluorescence imaging showed that AAV6 expressing enhanced green luorescent protein （eGFP） by intravenous adminis tration predominantly targeted the liver, but led to selflimited hepatitis. Besides, our study evaluated epidemiology o（ antiAAV6 NAb in nonhuman primates （NHPs） by NAb assay in vitro, The result indicated that 52.17 of NHPs had detectable NAb at 1：5 dilution rate. In vivo passive transfer experiment showed that AAV6 specific neu tralizing antibody, even though with low NAb titer, could significantly inhibit rAAV6 transduction.