Rational Design of Single Copy Expression Cassettes in Defined Chromosomal Sites Overcomes Intraclonal Cell-to-Cell...

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Gene expression in cloned cell populations exhibits significant cell-to-cell heterogeneity.
• Regulatory elements and epigenetic modifications critically influence both endogenous and transgene expression.
• Understanding and controlling expression variability is crucial for biopharmaceutical production.

Purpose of the Study:

• To investigate and mitigate heterogeneity in recombinant protein expression within Chinese hamster ovary (CHO) cells.
• To evaluate the impact of epigenetic modifiers and targeted gene modulation on expression variability.
• To enhance the predictability and titer of transgene expression for industrial applications.

Main Methods:

• Targeting expression cassettes into preselected chromosomal 'hot spots' in CHO cells.
• Utilizing ubiquitous chromatin opening elements (UCOEs) to influence chromatin accessibility.
• Assessing the efficacy of epigenetic modifiers and dCas9-VPR in reducing expression variability.
• Applying the developed methods to recombinant antibody expression systems.

Main Results:

• Significant expression variability was observed, dependent on gene and expression cassette design.
• Epigenetic modifiers, particularly UCOEs, effectively reduced cell-to-cell heterogeneity.
• UCOEs also led to a concomitant increase in recombinant protein expression levels.
• Site-specific recruitment of dCas9-VPR did not significantly reduce expression variability.
• The approach demonstrated success in improving recombinant antibody production.

Conclusions:

• Rational design of expression cassettes, incorporating elements like UCOEs, can significantly reduce heterogeneity in CHO cells.
• This strategy enhances the predictability and increases the yield of transgene expression.
• The findings support the development of robust cell lines for high-titer biopharmaceutical production.