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