DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.
Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160,232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy--in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.
Wang
Gary P
GP
University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.
Garrigue
Alexandrine
A
Ciuffi
Angela
A
Ronen
Keshet
K
Leipzig
Jeremy
J
Berry
Charles
C
Lagresle-Peyrou
Chantal
C
Benjelloun
Fatine
F
Hacein-Bey-Abina
Salima
S
Fischer
Alain
A
Cavazzana-Calvo
Marina
M
Bushman
Frederic D
FD
D000818:Animals; D049109:Cell Proliferation; D004262:DNA Restriction Enzymes; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D016147:Genes, Tumor Suppressor; D015316:Genetic Therapy / Q000009:adverse effects*; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics; D008232:Lymphoproliferative Disorders / Q000235:genetics; D051379:Mice; D011519:Proto-Oncogenes; D017422:Sequence Analysis, DNA / Q000379:methods*
D004262:DNA Restriction Enzymes