Physiological levels of HBB transgene expression from S/MAR element-based replicating episomal vectors

Area of Science:

• Molecular Biology
• Gene Therapy
• Epigenetics

Background:

• Replicating episomal vectors (REVs) offer transgene expression without genomic integration.
• Scaffold/matrix attachment regions (S/MARs) confer stable episomal replication and retention.
• Transcription through the S/MAR element is essential for its function.

Purpose of the Study:

• To assess tissue-specific expression from an S/MAR-based plasmid REV using a beta-globin microlocus model.
• To determine the minimal transcription levels required for S/MAR function in episomal vectors.

Main Methods:

• Constructed plasmid vectors containing the IFNB1 S/MAR element and the betaLCR-HBB microlocus.
• Varied transcription levels through the S/MAR element.
• Assessed episomal replication, retention, and transgene expression in mammalian cells.

Main Results:

• The betaLCR-HBB microlocus demonstrated reproducible and stable expression at physiological levels on an episome copy number basis.
• Even low levels of transcription through the S/MAR element were sufficient for efficient episomal replication and retention.
• Confirmed the functionality of the IFNB1 S/MAR in supporting stable episomal vector maintenance.

Conclusions:

• The betaLCR-HBB microlocus can achieve robust, tissue-specific expression from an S/MAR-based REV.
• Transcription through the S/MAR element is a critical determinant for episomal vector stability.
• Established principles for designing flexible S/MAR-based REVs for various applications.