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Updated: Jun 1, 2026
A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
Published on: December 12, 2017
Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in
Liane Viru1, Gregory Heller, Taavi Lehto
1Institute of Technology, University of Tartu, Tartu, Estonia.
Background:
The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems.
Methods:
Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s).
Results:
It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s), which corrected the splicing defects.
Conclusions:
Splice-switch technology, originally developed for genetic disease therapy, can also be used to control gene expression of viral vectors. This approach represents a novel, universal and powerful method for controlling gene expression, replication, viral spread and, by extension, virus-induced cytotoxic effects and can be used both for basic studies of virus infection and in virus-based gene- and anti-cancer therapy.
Insights
Introducing aberrantly spliced introns into viral vectors controls gene expression and replication. This splice-switch technology offers a universal method for regulating viral activity in research and therapy.
Area of Science:
- Virology
- Molecular Biology
- Gene Therapy
Background:
- Viral gene expression and replication require precise coordination for infection outcomes.
- Viruses are valuable tools for cancer therapy and immune response induction.
- Current viral vector construction methods lack specificity and risk reversion.
Purpose of the Study:
- To investigate the use of aberrantly spliced introns for controlling viral gene expression and replication.
- To evaluate the therapeutic potential of splice-switch technology in viral vectors.
Main Methods:
- Aberrantly spliced introns were introduced into adenovirus and alphavirus vectors.
- Effects on viral gene expression, replication, and genome release were assessed.
- Splice-switching oligonucleotides were used to correct splicing defects.
Main Results:
- Introduction of aberrantly spliced introns effectively blocked viral gene expression and replication.
- Adenovirus vector gene expression was reduced over fifty-fold.
- Alphavirus vector infectivity was reduced over one hundred-fold, with effects reversible by splice-switching oligonucleotides.
Conclusions:
- Splice-switch technology provides a novel, universal method for controlling viral vectors.
- This approach enables precise regulation of gene expression, replication, and viral spread.
- It holds promise for basic virus research and virus-based gene and anti-cancer therapies.
