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Transgene expression after rep-mediated site-specific integration into chromosome 19.
Nicola J Philpott1, Janette Gomos, Erik Falck-Pedersen
1Department of Microbiology and Immunology, William Randolph Hearst Foundation Molecular Biology Graduate Program, Weill Medical College of Cornell University, New York, NY 10021, USA.
Human Gene Therapy
|February 18, 2004
Summary
Adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) can lead to transgene instability and loss of expression over time. Providing Rep in trans, however, maintains transgene stability and expression.
Area of Science:
- Molecular Biology
- Gene Therapy
- Virology
Background:
- Adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) is a pathway for targeted gene insertion.
- Understanding transgene stability and expression post-integration is crucial for gene therapy applications.
Purpose of the Study:
- To characterize the stability and expression of transgenes integrated via the AAV RMSSI pathway.
- To investigate the role of the AAV Rep protein in transgene DNA and expression stability.
Main Methods:
- Utilized a plasmid-based transfection model in HeLa cells using constructs with the AAV p5 integration efficiency element (p5IEE).
- Constructs varied in the presence of AAV inverted terminal repeats (ITRs) and the Rep gene (in cis or trans).
- Monitored transgene (GFP or CAT) integration, DNA presence, and protein expression over 18 weeks post-transfection.
Main Results:
- All constructs showed site-specific integration (10-40% efficiency) in the absence of drug selection.
- Cell lines with Rep in cis to the transgene exhibited declining transgene expression and DNA loss by 18 weeks.
- Cell lines with Rep provided in trans maintained transgene integration and expression throughout the 18-week study.
Conclusions:
- The AAV Rep protein, when present in cis, mediates transgene instability and loss of expression.
- Rep-mediated pathways contribute to transgene instability.
- Providing Rep in trans offers a strategy for stable transgene integration and sustained expression.