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Published on: August 13, 2013
DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors
D W Russell1, I E Alexander, A D Miller
1Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.
Abstract:
Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.
Insights
Researchers found that inhibiting DNA synthesis or topoisomerase can significantly boost adeno-associated virus (AAV) vector gene transfer into non-dividing cells without harming them. This discovery enhances AAV gene therapy potential.
Area of Science:
- Molecular Biology
- Gene Therapy
- Virology
Background:
- Adeno-associated virus (AAV) vectors are crucial for gene therapy but primarily infect cells in the S phase of the cell cycle.
- Previous studies indicated DNA-damaging agents enhance AAV transduction in non-dividing cells, but optimal concentrations proved cytotoxic.
- Improving AAV transduction efficiency in non-dividing cells is critical for effective gene therapy applications.
Purpose of the Study:
- To identify non-toxic methods for enhancing adeno-associated virus (AAV) vector transduction in normal human fibroblasts.
- To investigate the effect of DNA synthesis and topoisomerase inhibitors on AAV vector gene transfer efficiency.
- To explore potential mechanisms underlying enhanced transduction beyond replicative DNA synthesis.
Main Methods:
- Normal human fibroblasts were pre-exposed to DNA synthesis inhibitors (aphidicolin, hydroxyurea) or topoisomerase inhibitors (etoposide, camptothecin).
- Transduction efficiency of adeno-associated virus (AAV) vectors was measured in treated and untreated stationary cultures.
- Cell viability and proliferative potential were assessed to determine non-toxic concentrations of inhibitors.
Main Results:
- Pre-exposure to specific DNA synthesis or topoisomerase inhibitors increased AAV vector transduction efficiency by over 300-fold in stationary cultures.
- Effective concentrations of these inhibitors did not compromise cell viability or the cells' potential to proliferate.
- Enhanced transduction was observed in both S-phase and non-S-phase cells, suggesting broader cellular mechanisms are involved.
Conclusions:
- Non-toxic pre-treatment with DNA synthesis or topoisomerase inhibitors significantly enhances adeno-associated virus (AAV) vector gene transfer into human fibroblasts.
- These findings offer a promising strategy to improve gene transfer protocols for AAV-based gene therapy.
- The observed effect on both S-phase and non-S-phase cells warrants further investigation into the underlying cellular pathways.
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