Abstract
Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors.
Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fluorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fluorescence microscope and FACS analysis.
Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells.
Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.
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Meissner A, Wernig M, Jaenisch R (2007) Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol 25:1177–1181
Park IH, Zhao R, West JA et al (2007) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141–146
Wernig M, Meissner A, Foreman R et al (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–324
Zufferey R, Dull T, Mandel RJ et al (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72:9873–9880
Wanisch K, Yanez-Munoz RJ (2009) Integration-defective lentiviral vectors: a slow coming of age. Mol Ther 17:1316–1332
Yamashita M, Emerman M (2006) Retroviral infection of non- dividing cells: old and new perspectives. Virology 344:88–93
Nightingale SJ, Hollis RP, Pepper KA et al (2006) Transient gene expression by nonintegrating lentiviral vectors. Mol Ther 13:1121–1132
Aiuti A, Biasco L, Scaramuzza S et al (2013) Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science 341:1233151
Lewinski MK, Bisgrove D, Shinn P et al (2005) Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription. J Virol 79:6610–6619
Hacein-Bey-Abina S, Von Kalle C, Schmidt M et al (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 348:255–256
Okita K, Matsumura Y, Sato Y et al (2011) A more efficient method to generate integration-free human iPS cells. Nat Methods 8:409–412
Vargas J, Gusella GL, Najfeld V et al (2004) Novel integrase-defective lentiviral episomal vectors for gene transfer. Hum Gene Ther 15:361–372
Peluffo H, Foster E, Ahmed SG et al (2013) Efficient gene expression from integration-defective lentiviral vectors in the spinal cord. Gene Ther 20:645–657
Apolonia L, Waddington SN, Fernandes C et al (2007) Stable gene transfer to muscle using non-integrating lentiviral vectors. Mol Ther 15:1947–1954
Leavitt A, Robles G, Alesandro N et al (1996) Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J Virol 70:721–728
Engelman A (1999) In vivo analysis of retroviral integrase structure and function. Adv Virus Res 52:411–426
Shawand A, Cornetta K (2014) Design and potential of non-integrating lentiviral vectors. Biomedicines 2:14–35
Bayer M, Kantor B, Cockrell A et al (2008) A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector. Mol Ther 16:1968–1976
Lombardo A, Genovese P, Beausejour CM et al (2007) Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 25:1298–1306
Kingston RE, Chen CA, Rose JK (2003) Calcium phosphate transfection. Curr Protoc Mol Biol. Chapter 9, Unit 9.1
Nordin F, Abdul Karim N, Wahid SFA (2014) Transgene expression is transient in non-integrating lentiviral-based transduction system: an alternative approach. Regen Res 3:1–7
Philippe S, Sarkis C, Barkats M et al (2006) Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo. Proc Natl Acad Sci U S A 103:17684–17689
Cornu TI, Cathomen T (2007) Targeted genome modifications using integrase-defective lentiviral vectors. Mol Ther 15:2107–2113
Sloan R, Wainberg M (2011) The role of unintegrated DNA in HIV infection. Retrovirology 8:52
Acknowledgments
This work was supported by the Ministry of Higher Education of Malaysia and Universiti Kebangsaan Malaysia. The work has been carried out at the King’s College London, Department of Haematological Medicine, The Rayne Institute, London, UK.
Conflict of interest: The authors declare no conflict of interest.
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Nordin, F., Hamid, Z.A., Chan, L., Farzaneh, F., Hamid, M.K.A.A. (2016). Transient Expression of Green Fluorescent Protein in Integrase-Defective Lentiviral Vector-Transduced 293T Cell Line. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_12
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DOI: https://doi.org/10.1007/978-1-4939-3753-0_12
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