Comprehensive genomic access to vector integration in clinical gene therapy
Comprehensive genomic access to vector integration in clinical gene therapy
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DOI：
10.1038/nm.2057
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发表时间：
2009-12-01
期刊：
影响因子：
82.9
通讯作者：
Schmidt, Manfred
中科院分区：
文献类型：
--
作者：
Gabriel, Richard;Eckenberg, Ralph;Schmidt, Manfred
逆转录病毒载体在许多基因治疗患者中引起了微妙的克隆偏差，并在其中一些患者中引起了严重的克隆增殖和白血病，这强调需要进行全面的整合位点分析，以评估载体的生物安全性和基因组药代动力学以及体内基因修饰细胞的克隆命运。线性扩增介导的 PCR (LAM-PCR) 等整合位点分析需要限制性消化，生成不均匀的基因组小片段。在这里，我们表明，每个限制性基序仅允许识别所有基因组整合体的一小部分，从而妨碍了对载体插入后生物学后果的理解和预测。我们开发了一个模型来定义病毒整合位点的基因组访问，该模型提供最佳的限制性基序组合并最大限度地减少不可访问的插入位点的百分比。我们引入了一种新的非限制性 LAM-PCR 方法，该方法具有在临床前和临床样本中进行全面、无偏见的整合位点检索的卓越能力，不受限制性基序和扩增效率低下的影响。
Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification-mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.