P-76: Design and Construction an Integrase Minus Lentiviral Vector for Gene Therapy (Pages: 74-74)

Farazmandfar T *, Khanahmad H , Sharifian R , Taghipour M , Azadeh H ,


Objective: Gene targeting and viral vectors based gene therapy are the most common methods that have been used for correction of genetic disorders. Gene targeting is an ideal method for gene therapy but has low efficiency compare with viral vector methods. In this study we are going to design and construct an integrase minus lentiviral vector. This vector is suitable for transient delivery of gene and gene targeting with viral vector. Materials and Methods: The pLP1 plasmid was isolated from lentiviral helper mixture plasmids, including pLP1, pLP2, pLP/VSVG by digestion with AgeI and MluI restriction enzymes and self ligation of them. A missense mutation was induced in the catalytic domain of pLP1 integrase gene at the position D64 (aspartic acid changed to Valine) by overlap extension PCR method. Then the native segment was replaced by final amplified segment in pLP1 plasmid. The transfer vector plasmid, pLOX-CWgfp, association with native and mutated packaging mix were transfected into 293T cell line by lipofectamine 2000 and recombinant viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess viral titer and GFP expression. Results: Recombinant and wild lentiviruses titer was about 106 transducing unit per mililitre in COS-7 cell line. Amount of GFP-positive cells which were 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 GFP positive cells were observed. Conclusion: Integration of lentiviral genome into a host genome would be overcome. Integrase minus lentiviral vectors can be used for transient gene delivery and gene targeting if a cassette of gene targeting is placed in the backbone of lentiviral construct. This combination method decrease disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.