Application of a split-Cre system for high-capacity adenoviral vector amplification

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• High-capacity adenoviral vectors (HC-AdVs) offer large DNA payloads and stable gene expression.
• HC-AdV production relies on helper viruses (HVs) for trans-complementation of viral proteins.
• The Cre/loxP system is used to excise HV packaging signals, but chronic Cre exposure is toxic to packaging cells.

Purpose of the Study:

• To develop a safer and more efficient method for HC-AdV production.
• To overcome the limitations of chronic Cre recombinase exposure in packaging cells.
• To apply a dimerizable Cre system for improved HC-AdV amplification.

Main Methods:

• The Cre recombinase was split into two non-functional fragments.
• Two versions of the split-Cre system were created: DiCre (rapamycin-dependent) and oCre.
• Packaging cells and HVs were engineered to express complementary Cre fragments for HC-AdV production.

Main Results:

• Both DiCre and oCre systems prevented toxicity to packaging cells.
• The oCre system demonstrated particular suitability for HC-AdV amplification.
• The split-Cre system did not interfere with packaging cell growth.

Conclusions:

• The split-Cre system significantly improves packaging cell performance for HC-AdV production.
• HC-AdV amplification time and cost can be reduced by up to 30% and 15%, respectively.
• This technology facilitates the standardization of HC-AdV production processes.