Use of integrase-minus lentiviral vector for transient expression

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. In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein [GFP] gene expression by a fluorescence microscopy. Recombinant and wild lentiviruses titer was about 58×10[6] transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. 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

Comparison of several maturation including factors in dendritic cell differentiation

Dendritic cells [DCs] are professional antigen presenting cells that have an important role in the initiation of immune response. The use of maturation factors in dendritic cell differentiation provides a promising approach in immunotherapy. In this study, we compared tumor necrosis factor-alpha, polyribocytidylic acid, lipopolysacharide and CpG oligonucleotides in inducing dendritic cell maturation. We generated immature dendritic cells with GM-CSF in combination with IL-4 from peripheral blood mononuclear adherent cells and used tumor necrosis factor-alpha, polyribocytidylic acid, lipoplysacharide and CpG for the induction of dendritic cell maturation. CD83 maturation marker on the dendritic cells was analyzed by flowcytometry after 7 days. In addition, mixed leukocyte reaction between dendritic cells and T cells was performed by MTT proliferation assay. Flow cytometry results demonstrated a comparable high level of CD83 expression on the mature dendritic cells generated by TNF-alpha, CpG, Poly I:C, and LPS treatment of the immature dendritic cells. However, a significantly poorer proliferation of lymphocytes cocultured with the Poly I:C-treated DCs was observed compared to the CpG-treated DCs in mixed leukocyte reaction [p=0.025]. Our results indicated that all of studied maturation inducing factors can be used in DC maturation but TNF-alpha and CpG were the preferred in vitro maturation factors. It is concluded that maturation of dendritic cells by CpG motif and TNF-alpha can be used to regulate immune responses