Virus-mediated Genetic Surgery: Homologous Recombination With a Little "Helper" From My Friends.
Virus-mediated Genetic Surgery: Homologous Recombination With a Little "Helper" From My Friends.
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病毒介导的基因手术：同源重组与我朋友的小“帮手”。
DOI：
10.1038/mtna.2011.7
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Sargent,RGeoffrey
中科院分区：
文献类型：
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作者：
Gruenert,DieterC;Sargent,RGeoffrey
基因组DNA的序列特异性修饰是基于基因和干细胞的疗法的重要目标。1，2自从证明转基因动物可以通过同源重组靶向特定的基因组位点产生以来，3，4许多研究人员都专注于开发基因靶向策略，以有效和可靠地纠正基因组DNA中的突变。1，5-7最近出现了三篇论文，8-10包括早期在线版的《分子治疗》，描述了一种有效的、基于辅助依赖性腺病毒（HDAV）的系统，用于靶向修饰人胚胎和诱导多能干细胞（分别为hES和hiPS）中的特定序列。这些研究不仅为开发用于基础研究的同基因人类多能干细胞奠定了基础，而且还为产生治疗性、自体、患者来源的干细胞奠定了基础，其中致病突变已被纠正。1998年hES细胞11和2007年hiPS细胞12，13的衍生促使研究人员开发将序列特异性基因组修饰引入这些多能细胞的DNA中的方法。早期的报告显示出一些前景，并使用经典的基因靶向载体，14，15但很少有出版物表明这种策略在多能细胞中足够稳健。到2009年，依靠双链断裂（DSB）增强哺乳动物细胞中的同源重组的观察，16-18几个小组报道了通过将锌指核酸酶（ZFN）的表达与同源质粒的递送19、20或与寡核苷酸/多核苷酸供体DNA相结合，成功靶向hES和hiPS细胞系中的多个基因。7，21 ZFN 5，6和最近的转录激活因子样作用核酸酶（TALEN）22，23为研究人员提供了基于同源重组效率提高在特定基因组位点进行“遗传手术”的工具。19，20这些靶向核酸内切酶系统的主要关注点是它们产生非特异性离位切割的可能性，所述非特异性离位切割可能使靶细胞的基因组不稳定。5，21 Aizawa等人在Molecular Therapy中的报告，8描述了使用HDAV系统来递送与基因组靶标基本上同源的序列，并且克服了同源重组的更经典方法的一些限制以及最小化随机整合到基因组DNA中的可能性。HDAV系统实际上是一种“掏空的”腺病毒，其中所有病毒基因都已缺失，仅保留将DNA载体直接包装到病毒衣壳中所需的那些病毒序列。24通过共转染将HDAV载体包装到具有“辅助”病毒基因组的生产哺乳动物细胞系中，所述“辅助”病毒基因组含有载体复制、衣壳产生和载体包装所必需的所有病毒基因。重组HDAV载体的一个优点是它可以容纳25-35 kb的DNA，而不是例如对腺相关病毒（AAV）系统施加的~ 4.7 kb的限制。这种能力足以容纳基因组DNA和药物选择标记的大同源区域，以刺激同源重组并允许富集已靶向的细胞。由于某些病毒已经进化出将DNA递送到细胞核的精确机制，因此病毒转导往往比化学或物理方法介导的转导更有效。重组病毒载体对细胞活力的负面影响也比非病毒方法小。
The sequence-specific modification of genomic DNA is an important goal of gene-and stem cell-based therapies. 1, 2 Since the demonstration that transgenic animals could be generated by targeting specific genomic loci by homologous recombination, 3, 4 numerous investigators have focused on developing gene-targeting strategies that will efficiently and reliably correct mutations in genomic DNA. 1, 5–7 Three papers have recently appeared, 8–10 including one in the early online edition of Molecular Therapy, that describe an efficient, helper-dependent adenovirus (HDAV)-based system for targeted modification of specific sequences in human embryonic and induced pluripotent stem (hES and hiPS, respectively) cells. These studies lay the foundation not only for the development of isogenic human pluripotent stem cells for basic research, but also for the generation of therapeutic, autologous, patient-derived stem cells in which the diseasecausing mutation has been corrected. The derivation of hES cells in 199811 and hiPS cells in 200712, 13 prompted researchers to develop approaches to introduce sequence-specific genomic modifications into the DNA of these pluripotent cells. Early reports showed some promise and used classical gene-targeting vectors, 14, 15 but few publications indicated that this strategy was sufficiently robust in pluripotent cells. By 2009, relying on the observation that double-strand breaks (DSBs) enhance homologous recombination in mammalian cells, 16–18 several groups reported the successful targeting of multiple genes in hES and hiPS cell lines by combining the expression of zinc-finger nucleases (ZFNs) with the delivery of homologous plasmid19, 20 or with oligo/polynucleotide donor DNA. 7, 21 ZFNs5, 6 and more recently, the transcription activator-like effort nucleases (TALENs) 22, 23 have provided researchers the tools to carryout “genetic surgery” at specific genomic loci based on an increased efficiency of homologous recombination. 19, 20 A primary concern with these targeted endonuclease systems is their potential to generate nonspecific off-site cleavage that could destabilize the genome of the targeted cells. 5, 21 The report in Molecular Therapy by Aizawa et al., 8 describes the use of an HDAV system to deliver sequences essentially homologous to the genomic target and both overcomes some of the limitations of more classical approaches to homologous recombination as well as minimizes the potential for random integration into the genomic DNA. The HDAV system is effectively a “gutted” adenovirus from which all viral genes have been deleted, retaining only those viral sequences required for direct packaging of the DNA vector into the virus capsid. 24 The HDAV vector is packaged by cotransfection into a producer mammalian cell line with a “helper” virus genome that contains all of the viral genes necessary for vector replication, capsid production, and vector packaging. 25 An advantage of the recombinant HDAV vector is that it can accommodate between 25–35 kb of DNA, as opposed to, for example, the~ 4.7 kb limitation imposed on the adeno-associated virus (AAV) system. This capacity is more than sufficient to accommodate large homologous regions of genomic DNA and drug selectable markers to stimulate homologous recombination and to allow enrichment of cells that have been targeted. Because certain viruses have evolved precise mechanisms to deliver DNA to the nucleus, viral transduction tends to be much more efficient than that mediated by chemical or physical methods. Recombinant viral vectors also have less of a negative effect upon cell viability than nonviral methods that …