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| The Establishment of the Methodology used for In Situ Detecting Lentiviral Vector Transcriptional Read-through |
| GAO Yue1, TAN Shuo1,2, REN Zhao-rui1,3, ZHANG Jing-zhi1,2 |
1. Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai.Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200040, China;
2. Key Laboratory of Embryo Molecular Biology, Ministry of Health & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China;
3. Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China |
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Abstract As a gene delivery system, Lentiviral vector (LVV) has been being increasingly used in gene therapy trials, and drawn large attention in recent years. Yet, transcriptional read-through (TRT) is one of potential risks in its application. For the purpose of improving the detection methods of TRT, and making the results intuitive and accurate, a codon optimized reporter gene, LacZ, was placed downstream to the 3'- LTR of the proviral vector to simulate its state in chromosome. Upon the transfection followed with LacZ staining, the cells with TRT phenomenon would be stained blue, and the TRT rate would therefore be visualized at the translational level. Correlating well with the outcomes of qRT-PCR, the established methodology could reflect the feature of TRT, which paves a way for improving LVV biosafety for clinical applications.
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Received: 09 February 2015
Published: 25 May 2015
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[1] Buchschacher G L Jr, Wong-Staal F. Development of lentiviral vectors for gene therapy for human diseases. Blood, 2000, 95(8): 2499-2504.
[2] Zufferey R, Dull T, Mandel R J, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol, 1998, 72(12): 9873-9880.
[3] Zaiss A K, Son S, Chang L J. RNA 3' readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy. Journal of Virology, 2002, 76(14): 7209-7219.
[4] Higashimoto T, Urbinati F, Perumbeti A, et al. The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors. Gene Ther, 2007, 14(17): 1298-1304.
[5] Schambach A, Galla M, Maetzig T, et al. Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors. Mol Ther, 2007, 15(6): 1167-1173.
[6] Fang Y, Gong X, Xu M, et al. A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation. J Gene Med, 2013, 15(2): 102-112.
[7] 何佳平, 方彧聃, 张帆, 等. 慢病毒载体转录通读率检测方法的建立. 生物工程学报, 2013, 29(007): 1006-1015. He J P, Fang Y D, Zhang F, et al. Establishment of the methodology for quantifying lentiviral vector transcriptional read-through rate. Chinese Journal of Biotechnology, 2013, 29(007): 1006-1015.
[8] Galy A, Thrasher A J. Gene therapy for the Wiskott-Aldrich syndrome. Curr Opin Allergy Clin Immunol, 2011, 11(6): 545-550.
[9] Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med, 2003, 348(3): 255-256.
[10] Wu X, Li Y, Crise B, et al. Transcription start regions in the human genome are favored targets for MLV integration. Science, 2003, 300(5626): 1749-1751.
[11] Wiley J. Vectors Used in Gene Therapy Clinical Trials.
[2015-02-06]. http://www.wiley.com//legacy/wileychi/genmed/clinical/.
[12] Biffi A, Montini E, Lorioli L, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science, 2013: 341(6148):1233158.
[13] Cavazzana-Calvo M, Payen E, Negre O, et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature, 2010, 467(7313): 318-322.
[14] Aiuti A, Biasco L, Scaramuzza S, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science, 2013: 341(6148):1233151.
[15] Schambach A, Baum C. Clinical application of lentiviral vectors - concepts and practice. Curr Gene Ther, 2008, 8(6): 474-482.
[16] Escors D and Breckpot K. Lentiviral vectors in gene therapy: their current status and future potential. Arch Immunol Ther Exp(Warsz), 2010, 58(2): 107-119.
[17] Dropulic B. Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research. Hum Gene Ther, 2011, 22(6): 649-657.
[18] Pauwels K, Gijsbers R, Toelen J, et al. State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr Gene Ther, 2009, 9(6): 459-474.
[19] Yang Q, Lucas A, Son S, et al. Overlapping enhancer/promoter and transcriptional termination signals in the lentiviral long terminal repeat. Retrovirology, 2007, 4: 4.
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