Transient Expression of Green Fluorescent Protein in Integrase-Defective Lentiviral Vector-Transduced 293T Cell Line.
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.
Cell Therapy Centre, University Kebangsaan Malaysia Medical Centre (UKMMC), 12th Floor, Clinical Block, Jalan Yaccob Latif, Bandar Tun Razak, Cheras, 56000, Kuala Lumpur, Malaysia. nordinf@ppukm.ukm.edu.my.
Fazlina
F
Nordin
Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300, Kuala Lumpur, Malaysia.
Zariyantey Abdul
ZA
Hamid
Department of Haematological Medicine, School of Medicine, King's College London, The Rayne Institute, 123 Coldharbour Lane, SE5 9NU, London, UK.
Lucas
L
Chan
Department of Haematological Medicine, School of Medicine, King's College London, The Rayne Institute, 123 Coldharbour Lane, SE5 9NU, London, UK.
Farzin
F
Farzaneh
Cell Therapy Centre, University Kebangsaan Malaysia Medical Centre (UKMMC), 12th Floor, Clinical Block, Jalan Yaccob Latif, Bandar Tun Razak, Cheras, 56000, Kuala Lumpur, Malaysia.
M K Azaham A
MK
Hamid
D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D019426:Integrases / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics
D049452:Green Fluorescent Proteins; D019426:Integrases
D64 point mutation; GFP reporter gene; Integrase-defective lentiviral vector; Transduction efficiency; U937 cell lines