Journal Article

Comprehensive genomic access to vector integration in clinical gene therapy

Saurin, William, Naldini, Luigi, Thrasher, Adrian J, Wang, Wei, Balaggan, Kamaljit S, Paruzynski, Anna, Faber, Katrin, Biasco, Luca, Ball, Claudia R, Ali, Robin R, Bartholomae, Cynthia C, Cohen-Haguenauer, Odile, Yáñez-Muñoz, Rafael J, Arens, Anne, Kirsten, Romy, Schmidt, Manfred, Cattoglio, Claudia, Recchia, Alessandra, Howe, Steven J, Deichmann, Annette, Eckenberg, Ralph, Glimm, Hanno, Cesana, Daniela, Nowrouzi, Ali, Schwarzwaelder, Kerstin, Gaspar, H Bobby, Aiuti, Alessandro, Mavilio, Fulvio, Gabriel, Richard, Montini, Eugenio, von Kalle, Christof

2009

Overview

Adverse events stemming from the use of retroviral vectors in humans has prompted the search for methods predicting the fate and biological consequences of gene-modified cells after vector insertion. Methods of integration site analysis, such as linear amplification-mediated PCR (LAM-PCR), rely on use of restriction enzymes and identify only a fraction of all genomic integrants. This report describes a non–restriction enzyme–based LAM-PCR technique that provides comprehensive, unbiased integration site analysis. 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.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: Biomedical and Life Sciences / Biomedicine / Biosafety / Cancer Research / Care and treatment / Gene therapy / Genetic aspects / Genetic Therapy / Genetic Vectors / Genome, Human / Genomics / Health aspects / Humans / Infectious Diseases / Leukemia / Metabolic Diseases / Molecular Medicine / Neurosciences / Pharmacokinetics / Pharmacology / Polymerase Chain Reaction / technical-report