High-Throughput Genomic Mapping of Vector Integration Sites in Gene Therapy Studies
High-Throughput Genomic Mapping of Vector Integration Sites in Gene Therapy Studies
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DOI：
10.1007/978-1-4939-1133-2_22
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发表时间：
2014-01-01
期刊：
影响因子：
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通讯作者：
Kiem, Hans-Peter
中科院分区：
文献类型：
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作者：
Beard, Brian C.;Adair, Jennifer E.;Kiem, Hans-Peter
基因治疗在治疗各种传染病和遗传性疾病方面具有巨大的潜力。迄今为止，全世界数以百计的患者接受了携带治疗性转基因逆转录病毒载体的基因修饰的造血细胞产品，许多患者因此被治愈或显示出疾病稳定(Adair等人，Sci Transl Med 4：133ra57,2012年；Biffi等人，Science 341：1233158,2013年；Aiuti等人，Science 341：1233151,2013年；Fischer等人，Gene525：170-173,2013年)。不幸的是，对于一些患者来说，前病毒整合失调了附近基因的表达，导致了克隆性生长，在某些情况下，还会导致癌症。因此，插入突变的副作用已成为逆转录病毒基因治疗的一个主要问题。仔细研究逆转录病毒整合位点(RIS)和单个基因修饰克隆对造血细胞再繁殖的贡献对于所有基因治疗研究都是至关重要的。为了支持这一点，美国食品和药物管理局(FDA)要求在所有基因治疗的临床试验中仔细监测RIS。发展了一种非常有价值的方法：线性扩增介导的聚合酶链式反应(LAM-PCR)，能够分析体外和复杂的体内样本，直接获取有价值的基因组信息，位于前病毒整合部位的两侧。将这种方法和类似的方法与高通量测序相结合，现在可以前所未有地了解各种逆转录病毒载体的整合情况，并允许对其安全性进行灵敏的监测。它还允许对改进的安全增强型基因治疗载体进行详细比较。安全性的一个重要读数是单个基因修饰的重新繁殖克隆的相对贡献。LAM-PCR的一个局限性是，由于所有当前方法共同的初始线性PCR，捕获单个克隆的相对贡献的能力受到影响。在这里，我们描述了一种改进的方案，用于有效地捕获、测序和分析RI，该方案保留了基因修改的克隆贡献信息。我们还讨论了在临床前和临床模型中评估显性/过度表达的基因修饰克隆的方法。
Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed:linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods. Here, we describe an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. We also discuss methods to assess dominant/overrepresented gene-modified clones in preclinical and clinical models.