Producing Integrase Minus Lentivirus for Transient Expression of the Desired Protein and Reduced Side Effects of the Virus in Gene Therapy

Document Type : Original Article (s)

Authors

1 PhD Student, Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

2 MSc Student, Department of Genetics and Molecular Biology, School of Medicine AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 PhD Student, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

5 Associate Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

6 Professor, Department of Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

7 Assistant Professor, Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable Disease AND Department of Genetics and Molecular Biology, School of Medicine, Isfahan University Of Medical Sciences, Isfahan, Iran

Abstract

Background: Lentiviral vectors are very efficient tools for gene therapy. But, proviral integration make their use dangerous; therefore, the safer integration deficient lentiviral vectors (IDLVs) can be produced through the use of integrase gene mutations that specifically prevent proviral integration. This study was launched to design and construct a second generation integration deficient lentiviral vector suitable for transient gene targeting with viral vector.Methods: Applying the site directed mutagenesis strategy through the overlap polymerase chain reaction technique, a missense mutation (D64V) was induced in the catalytic domain of the integrase gene in the psPAX2 (packaging) plasmid and was verified using DNA sequencing. The HEK293T cell line was transfected using the psPAX2 plasmid (native and integrase minus), pLOX (transfer plasmid) and PMD2G (envelope plasmid). The viruses were harvested and the HEK293T cell line was transuded. The levels of expression of the green fluorescent protein (GFP) reporter gene were monitored in the cells transduced with either native or defective virus for ten days.Findings: We observed a slight slope of decrease in the number of GFP-positive cells transduced with native viruses during the period. In contrast, in the case of defective viruses, a significant decrease in the number of GFP positive cells was noted.Conclusion: In this study, the integrase-minus psPAX2 was constructed and confirmed. The results demonstrate that the IDLV can provide a useful tool for efficient transient gene expression and can help to avoid disadvantages of gene targeting using the native virus.

Keywords

References

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Journal of Isfahan Medical School
Volume 33, Issue 326 - Serial Number 326
January and February 2015
Pages 305-315

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History

  • Receive Date: 08 December 2014
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

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