A method to sequence and quantify DNA integration for monitoring outcome in gene therapy

被引：61

作者：

Brady, Troy ^{[1
]}

Roth, Shoshannah L. ^{[1
]}

Malani, Nirav ^{[1
]}

Wang, Gary P. ^{[1
]}

Berry, Charles C. ^{[2
]}

Leboulch, Philippe ^{[3
,4
,5
,6
,7
]}

Hacein-Bey-Abina, Salima ^{[8
]}

Cavazzana-Calvo, Marina ^{[8
]}

Papapetrou, Eirini P. ^{[9
,10
]}

Sadelain, Michel ^{[9
,10
]}

Savilahti, Harri ^{[11
]}

Bushman, Frederic D. ^{[1
]}

机构：

[1] Univ Penn, Sch Med, Dept Microbiol, Philadelphia, PA 19104 USA

[2] Univ Calif San Diego, Sch Med, Dept Family Prevent Med, San Diego, CA 92093 USA

[3] Inst Emerging Dis & Innovat Therapies iMETI, Commissariat Energie Atom & Energies Alternat, F-92265 Fontenay Aux Roses, France

[4] INSERM, U962, F-92265 Fontenay Aux Roses, France

[5] Univ Paris 11, F-92265 Fontenay Aux Roses, France

[6] Brigham & Womens Hosp, Div Genet, Boston, MA 02115 USA

[7] Harvard Univ, Sch Med, Boston, MA 02115 USA

[8] Univ Paris 05, Grp Hosp Univ Ouest, Hop Necker Enfants Malad,Dept Biotherapy, AP HP,Ctr Invest Clin Integre Biotherapies,INSERM, Paris, France

[9] Mem Sloan Kettering Canc Ctr, Ctr Cell Engn, New York, NY 10065 USA

[10] Mem Sloan Kettering Canc Ctr, Mol Pharmacol & Chem Program, New York, NY 10065 USA

[11] Univ Turku, Dept Biol, Div Genet & Physiol, Turku 20014, Finland

来源：

NUCLEIC ACIDS RESEARCH | 2011年 / 39卷 / 11期

关键词：

SEVERE COMBINED IMMUNODEFICIENCY; TARGET SITE SELECTION; HUMAN GENOME; VECTOR INTEGRATION; BACTERIOPHAGE MU; IN-VITRO; SCID-X1; CELLS; TRANSPOSITION; METHYLATIONS;

D O I：

10.1093/nar/gkr140

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.

引用

页数：8

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