Use of Integrase-Minus Lentiviral Vector for Transient
Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector.
Materials and Methods:
In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the
Recombinant and wild lentiviruses titer was about 5~8×106 transducing units/ml in
This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.
Vectors based on retroviruses and lentiviruses have been used to
introduce genes efficiently and stably into cells for long-term expression.
Their ability to stable transduces in cells is due to encode integrase protein
in their genomes. This protein identifies specific sequences called long terminal
The third-generation Lentiviral Expression System is based on vectors developed by
Dull et al. (7). This system has a significant number of safety features, including:
Development of a deletion in the 3′ Reduction of genes to three ( Usage of the Insertion of genes encoding the structural and
packaging components required for making virus into four
separate plasmids (7).
Development of a deletion in the 3′
Reduction of genes to three (
Usage of the
Insertion of genes encoding the structural and packaging components required for making virus into four separate plasmids (7).
In this generation of vectors, none of the HIV structural genes are actually present in the packaged viral genome, thus they are never expressed in the transduced target cell. So, no new replication-competent virus can be produced.
Although the integrated form of lentivirus DNA is classically considered to be responsible for viral gene expression, several studies have suggested that nonintegrated viral DNA can support transcription (10, 11). Themis et al. (12) have reported the oncogenic potential of lentiviral vectors after the transduction of fetal and neonatal tissues. These observations have led to the development of several integrase-defective [Int(-)] lentiviral vectors (13, 14). These vectors were made of specific mutations designed to defuse vector integration without affecting vector entry into cells, providing short-term gene expression. Lentivirus integrase is a 32-kDa protein with a core domain that contains a triad of amino acids essential for its catalytic activity, specifically aspartic acid 64, aspartic acid 116, and glutamic acid 152 (15-17).
The aim of this study was to design nonintegrating lentiviral vector by mutating
aspartic acid 64 to valine (
Materials and Methods
Construction of lentiviral vector
In this expereimental assay, lentiviral particles was produced
Producing lentivirus in 293T cells
The new generated Int(-)
Titering lentiviral stock
Viral vector titers were assayed using
Invitrogen’s ViraPower protocol by infection of
Transduction of COS7 cells
The African green monkey cell line, COS-7 (ATCC No. CRL-1651), was thawed and expanded in DMEM (10% FBS, 100 u/ml penicillin, 100 Ag/ml streptomycin, and 2 mmol/liter L-glutamine) to 1×106 cells/ml in culture flasks. Cells were then treated with vector at an multiplicity of infection (MOI) of 2 in 500 µl for 2 hours in the presence of 80 mg/ml polybrene and expanded to total volume of 2 ml. Cells were analyzed for GFP expression by a fluorescence microscopy.
The Mazandaran University of Medical Sciences Research Ethics Committee approves this study from an ethical point of view.
All integrase-minus vectors gave titers within 48 hours and they were comparable to those of the native vector. It indicated that the mutations made to create integrase-minus vectors did not affect their ability to produce functional virus particles.
We confirmed that GFP expression from integrase-minus vectors did not result from integrated provirus because GFP fluorescence levels decreased during the consecutive passages. Cells were initially transduced with equal transduction unit (TU) amounts of vector (mutant and native), then and cultured and passaged for up to 20 days. The cells were consistently analyzed by a fluorescence microscopy to assure of stability assessment of GFP expression through passages (Fig 1,) . The percentage of GFP-positive cells was stable after transduction with the integrative vectors. Also, its growth rates were 28.42 ± 0.22% and 22.64 ± 1.25% after 3 and 20 days, respectively. In contrast, the percentage of GFP fluorescence cells were rapidly decreased in cells transduced with the mutant integrase-minus vectors, and its growth rates were 31.81 ± 0.36% and 0.7 ± 0.09% after 3 and 20 days, respectively (Fig 2,). Progressive loss of transgene expression in dividing cells observed with the recombinant vector was consistent with a nonintegrating phenotype.
In this study, we constructed nonintegrating lentiviral vectors for high efficiency
gene transfer to primary cells for transient gene expression. We produced
integrase-defective vectors from HIV-1-based lentiviral vectors by introducing
mutations to inactivate the integrase catalytic function in the viral genome.
Applying a packaging plasmid with missense mutations in the integrase gene was highly
effective to limit constant expression. In a standard packaging plasmid, the integrase
gene open-reading frame needs to maintain in order to allow sufficient DNA synthesis,
so only few missense mutations, none of nonsense mutation or large deletion, can be used
(18). The rare integrations created by nonintegrating vectors may also occur with other
transient gene delivery methods, such as adenoviral vectors and nonviral plasmid-mediated
gene delivery. The transduction efficiency of the
These vectors could be used in gene therapy methods inquiring transient expression of transgene, in dividing cells, and long-term gene expression in non-dividing cells.
This Report would not be possible without the essential and gracious support of Alireza Rafiei, Director-General of the Cellular and Molecular Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran. There is no conflict of interest in this study.