title: Comprehensive genomic access to vector integration in clinical gene therapy.
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.
authors: Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany.
authors: Richard
authors: R
authors: Gabriel
authors: Ralph
authors: R
authors: Eckenberg
authors: Anna
authors: A
authors: Paruzynski
authors: Cynthia C
authors: CC
authors: Bartholomae
authors: Ali
authors: A
authors: Nowrouzi
authors: Anne
authors: A
authors: Arens
authors: Steven J
authors: SJ
authors: Howe
authors: Alessandra
authors: A
authors: Recchia
authors: Claudia
authors: C
authors: Cattoglio
authors: Wei
authors: W
authors: Wang
authors: Katrin
authors: K
authors: Faber
authors: Kerstin
authors: K
authors: Schwarzwaelder
authors: Romy
authors: R
authors: Kirsten
authors: Annette
authors: A
authors: Deichmann
authors: Claudia R
authors: CR
authors: Ball
authors: Kamaljit S
authors: KS
authors: Balaggan
authors: Rafael J
authors: RJ
authors: Yáñez-Muñoz
authors: Robin R
authors: RR
authors: Ali
authors: H Bobby
authors: HB
authors: Gaspar
authors: Luca
authors: L
authors: Biasco
authors: Alessandro
authors: A
authors: Aiuti
authors: Daniela
authors: D
authors: Cesana
authors: Eugenio
authors: E
authors: Montini
authors: Luigi
authors: L
authors: Naldini
authors: Odile
authors: O
authors: Cohen-Haguenauer
authors: Fulvio
authors: F
authors: Mavilio
authors: Adrian J
authors: AJ
authors: Thrasher
authors: Hanno
authors: H
authors: Glimm
authors: Christof
authors: C
authors: von Kalle
authors: William
authors: W
authors: Saurin
authors: Manfred
authors: M
authors: Schmidt
mesh_terms: D015316:Genetic Therapy; D005822:Genetic Vectors; D015894:Genome, Human; D023281:Genomics; D006801:Humans; D016133:Polymerase Chain Reaction