Related Experiment Videos
A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus
Chunping Qiao1, Bing Wang, Xiaodong Zhu
1Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Abstract:
Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene expression since its promoter is located within the coding sequence of Rep78/68. To tightly regulate all four Rep proteins by using their own promoters, we have developed a novel gene control paradigm termed "dual splicing switch," which disrupts all four Rep genes by inserting into their shared coding region an intron that harbors transcription termination sequences flanked the LoxP sites. As a result, the structure and activities of the Rep gene promoters, both p5 and p19, are not affected; however, all of the Rep transcripts are prematurely terminated and the genes were inactivated. Removal of the terminator by Cre protein reactivates the transcription of all four Rep proteins derived from their own promoters. This switch system was initially tested in the lacZ gene and a 600-fold induction of beta-galactosidase activity was observed. Using the dual splicing switch strategy, we have subsequently established a number of AAV packaging cell lines from 293 cells, which showed a normal growth rate, high stability, and more importantly, high yields of AAV vectors. Such a gene control paradigm is also useful for other viruses, e.g., autonomous parvoviruses. Finally, the high-titer 293-based AAV packaging cell lines should greatly reduce the risk of wild-type adenovirus contamination and provide a scalable AAV vector production method for both preclinical and clinical studies.
Insights
Researchers developed a novel "dual splicing switch" to control adeno-associated virus (AAV) Rep gene expression, enabling the creation of stable, high-titer AAV packaging cell lines for scalable vector production.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Adeno-associated virus (AAV) vector production is hindered by challenges in creating stable, high-titer packaging cell lines.
- Previous attempts failed due to the cytostatic and cytotoxic effects of uncontrolled AAV Rep gene expression in host cells.
Purpose of the Study:
- To develop a novel gene control system for precise regulation of all four AAV Rep proteins.
- To establish stable and high-titer AAV packaging cell lines using this new system.
Main Methods:
- Developed a "dual splicing switch" system to disrupt and conditionally reactivate AAV Rep genes.
- Inserted a LoxP-flanked intron with transcription termination sequences into the Rep coding region.
- Validated the system using the lacZ gene and subsequently generated 293-based AAV packaging cell lines.
Main Results:
- The dual splicing switch effectively inactivated and reactivated all four Rep proteins without affecting promoter activity.
- Demonstrated a 600-fold induction of beta-galactosidase activity in initial tests.
- Established 293-based AAV packaging cell lines exhibiting normal growth, high stability, and high AAV vector yields.
Conclusions:
- The dual splicing switch is a novel and effective gene control paradigm for regulating viral gene expression.
- This strategy enables the development of scalable, high-titer AAV packaging cell lines for research and clinical applications.
- The approach is potentially applicable to other autonomous parvoviruses, improving vector production methods.