Directed Evolution of Adeno-associated Virus for Enhanced Gene Delivery and Gene Targeting in Human Pluripotent Stem Cells

被引:92
作者
Asuri, Prashanth [1 ,2 ,3 ]
Bartel, Melissa A. [1 ,2 ,3 ]
Vazin, Tandis [1 ,2 ,3 ]
Jang, Jae-Hyung [1 ,2 ,3 ,5 ]
Wong, Tiffany B. [4 ]
Schaffer, David V. [1 ,2 ,3 ]
机构
[1] Univ Calif Berkeley, Dept Chem & Biomol Engn, Berkeley, CA 94720 USA
[2] Univ Calif Berkeley, Dept Bioengn, Berkeley, CA 94720 USA
[3] Univ Calif Berkeley, Helen Wills Neurosci Inst, Berkeley, CA 94720 USA
[4] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA
[5] Yonsei Univ, Dept Chem & Biomol Engn, Seoul 120749, South Korea
关键词
ZINC-FINGER NUCLEASES; HEPARAN-SULFATE PROTEOGLYCAN; DOUBLE-STRAND BREAKS; IN-VIVO; HOMOLOGOUS RECOMBINATION; VIRAL VARIANT; VECTORS; THERAPY; EFFICIENT; TYPE-2;
D O I
10.1038/mt.2011.255
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stern cell research. Adeno-associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (similar to 50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double-stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZEN-mediated double-stranded DNA breaks.
引用
收藏
页码:329 / 338
页数:10
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