New research from Chinese scientists reveals significant advancements in the production of recombinant therapeutic proteins (RTP) by optimizing intron sequences combined with the cytomegalovirus (CMV) promoter. The study highlights the importance of the widely-used CHO (Chinese hamster ovary) cells as the primary vehicle for producing over 89% of approved recombinant monoclonal antibodies.
Published on January 30, 2025, the findings demonstrate how the manipulation of intron sequences from the CMV promoter can substantially increase the efficiency of protein production, addressing the growing demand from the biopharmaceutical industry for improved yield of RTPs. The researchers focused on various synthetic introns—truncated, EF-1α first, chimeric, and β-globin—assessing their effectiveness when combined with the CMV promoter.
"Optimization of the intron sequences combined with the CMV promoter can achieve a higher yield of recombinant proteins in CHO cells," stated the authors of the article. The study involved comparing multiple synthetic intron sequences to identify their impact on stable transgene expression. Results revealed significant improvements, underscoring the potential of this methodology for more efficient protein production.
CHOs are widely favored for RTP manufacturing due to their capability to perform proper protein folding and human-like post-translational modifications. Over the years, researchers have employed several strategies to boost protein yields, including bioprocess engineering and the optimization of gene expression vectors. The focus on the CMV promoter—an entity considered to regulate transcriptional initiation—opens new avenues for enhancing protein yields under different cellular conditions.
To explore the role of introns, the research team implemented a series of experiments using the enhanced green fluorescent protein (EGFP) and secreted alkaline phosphatase (SEAP) as reporter genes. They found significant variations between vectors containing the modified introns and those using the standard sequences. The researchers reported, "The truncated, EF-1α first, chimeric, and β-globin introns can significantly improve stable transgene expression."
Using qPCR analysis, the team assessed the mRNA levels of the genes involved. The analysis confirmed increased expression levels when optimized intron sequences were incorporated, leading to more efficient protein assembly, which is particularly beneficial for producing complex proteins such as monoclonal antibodies. This enhancement is particularly notable since the traditional reliance on basic CMV promoter constructs has shown limitations due to methylation and histone modifications affecting gene transcription.
To demonstrate practical applications, the researchers replaced reporter genes with genes encoding human serum albumin and adalimumab (commonly known as Humira), two important therapeutics. Their findings indicate not just efficacy through transient expression, but also stability and higher yields through optimized stable expressions.
Transcriptomics analysis unveiled 48 differentially expressed genes linked to processes like mRNA processing and cell-cycle regulation, emphasizing the complexity of how intron optimization influences overall gene expression. The report emphasizes the need for increased exploration of other intronic variations to fully exploit this strategy.
Overall, these findings present promising new methods for enhancing RTP yield, which can be beneficial for future industrial applications. The research highlights the synergistic potential of optimizing various genetic elements, paving the way for subsequent protocols and methodologies aimed at improving the productivity of CHO cell lines.
Considering the importance of this work, the authors conclude: "The yields of various genes of interest were increased in CHO cells by optimizing the intron element combined with the CMV promoter, which has the potential for industrial applications." The study encourages the continuation of research surrounding expression vectors and the role of structural elements such as introns to attain optimal functioning in therapeutic protein production.