Department of Neurology, Massachusetts General Hospital, and Neuroscience Program, Harvard Medical School, Boston, MA, USA.
J Neurooncol. 2010 Feb;96(3):337-47. doi: 10.1007/s11060-009-9972-7. Epub 2009 Jul 19.
Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10(4) genome copies/cell), and at low doses it was 1.45-1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.
多形性胶质母细胞瘤（GBM）是一种严重的脑癌，目前尚无有效的治疗方法。替代策略，如腺相关病毒（AAV）载体介导的脑肿瘤细胞的基因修饰，用编码抗肿瘤蛋白的基因，在 GBM 的临床前模型中显示出有希望的结果，尽管这些肿瘤的转导效率往往较低。由于更高的肿瘤细胞转导效率应该导致增强的治疗效果，因此一种快速工程化具有提高的转导效率的 AAV 载体的方法是有吸引力的策略。在这里，我们通过 DNA shuffling 对六个不同的 AAV 衣壳基因（AAV1、AAV2、AAV5、AAVrh.8、AAV9、AAVrh.10）的新构建嵌合 AAV 衣壳文库进行培养，测试了通过选择鉴定用于人 U87 神经胶质瘤细胞的高效 AAV 载体的可能性。经过七轮选择，我们获得了一种高效转导 U87 细胞的嵌合 AAV 衣壳（在 10（4）基因组拷贝/细胞的剂量下转导效率为 97%），并且在低剂量下比 AAV2 好 1.45-1.6 倍，证明是最有效的亲本衣壳。有趣的是，新的 AAV 衣壳对所有测试的神经胶质瘤细胞（包括原代神经胶质瘤细胞）均具有强大的基因传递特性，相对荧光指数比 AAV2 高 1-14 倍。当需要高效转导几种细胞系时，所选载体应可用于体外神经胶质瘤研究，并提供了一种概念证明，即 AAV 文库可用于产生具有增强的神经胶质瘤细胞转导效率的 AAV 载体。
