Perabo Luca, Endell Jan, King Susan, Lux Kerstin, Goldnau Daniela, Hallek Michael, Büning Hildegard
Gene Center, Ludwig-Maximilians-University of Munich, Germany.
J Gene Med. 2006 Feb;8(2):155-62. doi: 10.1002/jgm.849.
Viruses are being exploited as vectors to deliver therapeutic genetic information into target cells. The success of this approach will depend on the ability to overcome current limitations, especially in terms of safety and efficiency, through molecular engineering of the viral particles.
Here we show that in vitro directed evolution can be successfully performed to randomize the viral capsid by error prone PCR and to obtain mutants with improved phenotype.
To demonstrate the potential of this technology we selected several adeno-associated virus (AAV) capsid variants that are less efficiently neutralized by human antibodies. These mutations can be used to generate novel vectors for the treatment of patients with pre-existing immunity to AAV.
Our results demonstrate that combinatorial engineering overcomes the limitations of rational design approaches posed by incomplete understanding of the infectious process and at the same time offers a powerful tool to dissect basic viral biology by reverse genetics.
病毒正被用作载体，将治疗性遗传信息传递到靶细胞中。这种方法的成功将取决于通过对病毒颗粒进行分子工程来克服当前局限性的能力，尤其是在安全性和效率方面。
在此我们表明，可以通过易错PCR成功地进行体外定向进化，以使病毒衣壳随机化，并获得具有改善表型的突变体。
为了证明该技术的潜力，我们选择了几种被人抗体中和效率较低的腺相关病毒（AAV）衣壳变体。这些突变可用于生成新型载体，用于治疗对AAV已有免疫力的患者。
我们的结果表明，组合工程克服了由于对感染过程理解不完整而导致的理性设计方法的局限性，同时提供了一种通过反向遗传学剖析基本病毒生物学的强大工具。
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