Artificial Introns Enhance Recombinant Protein Expression
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Solution Overview
Problem
Current recombinant protein production in mammalian cells is limited by lower protein expression levels compared to prokaryotic systems, leading to higher drug costs, and existing methods to enhance expression, such as using strong promoters and introns, do not consistently achieve high yields due to challenges in chromatin structure and aberrant splicing events.
Innovation Solution
Incorporation of artificial introns with optimized splice donor and acceptor sites into nucleotide sequences at specific positions within eukaryotic genes to enhance gene expression, specifically in immunoglobulin genes, which improves mRNA stability and translation efficiency by reducing aberrant splicing events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native introns are used in eukaryotic genes for recombinant protein production, then gene expression is enhanced through chromatin structure modulation, but aberrant splicing events occur reducing expression reliability
Solution Approach 1:
The patent applies parameter changes by optimizing the nucleotide sequence of introns to achieve optimal splicing efficiency. Specific parameters such as splice site consensus sequences, branch point sequences, and polyypyrimidine tract compositions are modified to enhance splicing accuracy while maintaining expression enhancement benefits. This resolves the contradiction by adjusting intronic parameters to eliminate aberrant splicing while preserving the chromatin structure modulation effect.
Solution Approach 2:
The patent uses copying by creating artificial intron sequences that replicate the beneficial chromatin structure modulation effects of native introns while eliminating their defective splicing characteristics. These synthetic introns are designed based on consensus sequences from multiple species and optimized for mammalian splicing machinery, thereby copying the desirable functional aspects while discarding the harmful aberrant splicing events.
2Productivity
If strong promoters and enhancers are used to increase transcription efficiency, then protein expression levels improve, but chromatin structure limitations still constrain maximum yield
Solution Approach 1:
The patent introduces intronic sequences as intermediary elements between strong promoters/enhancers and the coding region. These introns act as mediators that facilitate chromatin structure modulation and improve transcriptional efficiency, thereby enhancing protein expression levels while overcoming the limiting effects of chromatin structure through the intermediary action of optimized intronic sequences.
3Productivity
If cDNA sequences without introns are used for expression, then chromatin structure issues are avoided, but translation efficiency and mRNA stability decrease
Solution Approach 1:
The patent applies preliminary action by incorporating optimized intronic sequences into the expression construct before transcription occurs. These pre-inserted introns prepare the mRNA for enhanced stability and translation efficiency by establishing proper splicing patterns and chromatin structure during transcription, thereby preventing the stability and translation defects that would otherwise occur with intronless cDNA sequences.
4Productivity
If gene copy number is increased through transfection with selectable markers, then expression levels improve, but integration site chromatin structure creates variability in expression
Solution Approach 1:
The patent applies local quality by focusing on optimizing the local intronic sequences within the transgene construct rather than relying on random integration site effects. The optimized introns create a localized favorable chromatin environment and splicing efficiency that compensates for variable integration site conditions, thereby achieving consistent high expression across different integration locations without requiring multiple transfection events.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The use of artificial introns results in increased expression levels of recombinant proteins, as demonstrated by a 1.8- to 2.1-fold increase in Fc fusion protein titers compared to cDNA versions, while genomic DNA with native introns shows comparable or lower expression, indicating improved productivity.
Implementation Method 1
They are precisely deleted from the primary transcript by a process known as RNA splicing to form mature messenger RNA (mRNA)
Data Source
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Figure 3A~3C
AI summary
The invention concerns the field of recombinant gene engineering. It concerns novel artificial introns and compositions comprising such introns as well as a method to improve expression of polypeptides from nucleic acids such as cloned genes, especially genes encoding antibodies and antibody derived fragments, and the production of various polypeptides in eukaryotic host cells using said novel artificialintron sequences.