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Nat Med. 2009 Dec;15(12):1431-6. doi: 10.1038/nm.2057. Epub 2009 Nov 22.

Comprehensive genomic access to vector integration in clinical gene therapy.

Author information

1
Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany.

Abstract

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.

PMID:
19966782
DOI:
10.1038/nm.2057
[Indexed for MEDLINE]

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