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Updated: Jun 8, 2026
Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors
Published on: October 20, 2023
Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are
Christina Quirin1, Stanimira Rohmer, Inés Fernández-Ulibarri
1Helmholtz-University Group Oncolytic Adenoviruses @ DKFZ, German Cancer Research Center, Heidelberg University Hospital, 69120 Heidelberg, Germany.
Abstract:
Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase-uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies.
Insights
This study optimized oncolytic adenoviruses for cancer therapy, enhancing tumor-specific transgene expression for potent multimodal treatment. The engineered virus effectively targets melanoma via prodrug activation, increasing cancer cell killing.
Area of Science:
- Oncolytic virotherapy
- Cancer gene therapy
- Molecular virology
Background:
- Developing tumor-specific cancer therapies and multimodal treatment regimens remains a challenge.
- Oncolytic adenoviruses offer potential for tumor-selective replication and therapeutic gene delivery.
- Efficient and tumor-specific transgene expression from oncolytic viruses is crucial for enhanced efficacy.
Purpose of the Study:
- To investigate strategies for achieving tumor-specific transgene expression in oncolytic adenoviruses.
- To optimize the insertion and expression of therapeutic transgenes without compromising viral replication.
- To develop an armed oncolytic adenovirus for targeted prodrug activation therapy in melanoma.
Main Methods:
- Utilized luciferase reporter assays and genetic prodrug activation systems.
- Engineered adenovirus late transcription units with promoter control of E1A for transgene expression.
- Compared transgene insertion methods including internal ribosome entry site (IRES), splice acceptor (SA), and viral 2A sequences.
- Optimized transgene expression cassettes by modifying splice sites and surrounding sequences.
Main Results:
- Promoter control of E1A enabled highly selective transgene expression, requiring multistep optimization.
- IRES and SA insertion facilitated replication-dependent and indirectly targeted transgene expression.
- Splice acceptor (SA) mediated more selective (up to 1,500-fold) but less effective transgene expression than IRES.
- Optimized SA-mediated expression of the FCU1 prodrug convertase led to potent bystander killing and a 10-fold increase in viral cytotoxicity against melanoma.
Conclusions:
- Strategies for targeted transgene expression can significantly improve armed oncolytic viruses.
- Optimized oncolytic adenoviruses can achieve selective, multimodal cancer therapies.
- This engineered adenovirus provides effective oncolysis combined with targeted prodrug activation for melanoma treatment.
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