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Updated: Oct 10, 2026
Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors
Published on: October 20, 2023
Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene
1Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Freie Universität, Berlin, Germany.
Abstract:
Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, co-infection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by back-recombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, alpha(v)beta5- and alpha(v)beta3-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdV-mediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was 'trans-complementation' of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in cancers almost refractory to standard adenovector-mediated gene transfer. Whereas RCAs need to be strictly avoided in gene therapy of non-malignant diseases for safety reasons, the magnitude of helper virus-induced therapeutic transgene expression could possibly warrant application of this principle to overcome the resistance of highly malignant cancers against gene therapy.
Insights
To improve cancer gene therapy, researchers explored enhancing adenovector (AdV) transgene expression. Overexpressing the CAR receptor improved uptake, while co-infecting with replication competent adenovirus (RCA) significantly boosted expression, especially in resistant colorectal cancers.
Area of Science:
- Oncology
- Gene Therapy
- Virology
Background:
- Gene therapy for cancer necessitates high transgene expression in target cells.
- Adenovector (AdV)-mediated gene transfer is limited by poor transgene expression in some cancer cells.
- Colorectal cancer cell lines exhibit particularly low AdV-mediated transgene expression.
Purpose of the Study:
- To investigate methods for improving adenovector-mediated transgene expression in poorly permissive cancer cells.
- To evaluate the efficacy of CAR receptor overexpression and replication competent adenovirus (RCA) co-infection in enhancing gene transfer.
- To compare the effectiveness of these two strategies in overcoming cancer cell resistance to gene therapy.
Main Methods:
- Assessed AdV receptor expression (CAR, integrins) in gastrointestinal cancer cell lines.
- Overexpressed the coxsackie and adenovirus receptor (CAR) on tumor cells to enhance AdV uptake.
- Co-infected cancer cells with a therapeutic AdV vector and traces of RCA to induce vector replication.
Main Results:
- AdV uptake correlated with CAR levels, not integrins.
- CAR overexpression enhanced transgene expression by 1.8- to 6.7-fold.
- Co-infection with RCA significantly amplified transgene expression (up to 46-fold), outperforming CAR overexpression, particularly in resistant colorectal cancer cells.
Conclusions:
- CAR overexpression can improve AdV uptake and transgene expression.
- Co-infection with RCA offers a potent strategy to dramatically enhance transgene expression by inducing therapeutic vector replication.
- RCA-mediated enhancement of transgene expression may be a viable approach to overcome resistance in highly malignant cancers, despite safety concerns in non-malignant applications.
