Comprehensive genomic access to vector integration in clinical gene therapy.

Retroviral vectors have induced subtle clonal skewing inmany gene therapy patients and severe clonal proliferationand leukemia in some of them, emphasizing the needfor comprehensive integration site analyses to assess thebiosafety and genomic pharmacokinetics of vectors andclonal fate of gene-modified cells in vivo. Integration siteanalyses such as linear amplification–mediated PCR(LAM-PCR) require a restriction digest generating unevenlysmall fragments of the genome. Here we show that eachrestriction motif allows for identification of only a fractionof all genomic integrants, hampering the understanding andprediction of biological consequences after vector insertion.We developed a model to define genomic access to theviral integration site that provides optimal restriction motifcombinations and minimizes the percentage of nonaccessibleinsertion loci. We introduce a new nonrestrictive LAM-PCRapproach that has superior capabilities for comprehensiveunbiased integration site retrieval in preclinical andclinical samples independent of restriction motifs andamplification inefficienc