Single Copy Transgene Integration in a Transcriptionally Active Site for Recombinant Protein Synthesis

Area of Science:

• Biotechnology
• Molecular Biology
• Genomics

Background:

• High transgene transcription in stable cell lines is crucial for efficient biomanufacturing of protein biologics.
• Genome engineering offers precise transgene insertion but requires identifying optimal genomic loci for high expression.
• Current methods for cell line development can be inefficient in achieving consistent high productivity.

Purpose of the Study:

• To identify characteristics of genomic regions that promote high transgene transcription.
• To develop a strategy for streamlining the generation of high-producing mammalian cell lines for biomanufacturing.
• To establish a method for targeted transgene integration into desirable genomic sites.

Main Methods:

• Infection of Chinese hamster ovary (CHO) cells with a lentivirus encoding destabilized Green Fluorescent Protein (dGFP).
• Isolation of high and low GFP-expressing cells, followed by RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq).
• Integration site analysis to correlate genomic features with transcriptional activity and subsequent use of recombinase-mediated cassette exchange for transgene swapping.

Main Results:

• High GFP expression correlated with transgene integration in large regions of high transcriptional activity and chromatin accessibility.
• Desirable integration sites were not necessarily located within highly transcribed genes.
• Successful generation of high Immunoglobulin G (IgG) expressing cell lines with single-copy transgene integration in identified optimal regions.

Conclusions:

• The study presents a strategy to identify and utilize specific genomic regions for enhanced transgene expression in biomanufacturing.
• Targeted integration into transcriptionally active and accessible regions, rather than within genes, is key for high productivity.
• This approach facilitates the development of robust cell lines for producing therapeutic proteins like IgG, erythropoietin, and tumor necrosis factor receptor-Fc.