Nonviral transposon‑engineered stem cells characterization: dose‑dependency between vector copy number and transgene...

Area of Science:

• Cellular and Molecular Medicine
• Genomic Engineering
• Regenerative Medicine

Background:

• Genomic safety of engineered stem cells is critical for regenerative medicine.
• Vector copy number (VCN) is a key genomic safety concern.
• Current VCN assessment methods are limited for non-viral transposon systems.

Purpose of the Study:

• To establish a robust, quantitative framework for interim VCN determination in transposon-engineered stem cells.
• To align VCN assessment with FDA recommendations for lot release.
• To validate a workflow linking plasmid input, genomic integration, and transgene expression.

Main Methods:

• Extended VCN determination to non-viral, transposon-engineered stem cells.
• Demonstrated dose-dependent VCN estimates based on plasmid input.
• Correlated VCN with EGFP median fluorescence intensity (MFI) and gene-of-interest (GOI) protein expression.

Main Results:

• Established a reliable method for interim VCN estimation at lot release.
• Showed a dose-dependent relationship between plasmid input and VCN.
• Validated VCN accuracy through strong linear correlations with MFI and GOI expression.
• Identified gene-specific differences in expression efficiency.

Conclusions:

• Validated a standardized VCN determination workflow for engineered cells.
• The workflow quantitatively links plasmid dose, genomic integration, and functional transgene expression.
• Provides comprehensive data for risk-based analyses to ensure cell product genomic safety and stability.