IRES Mediated Multicistronic Vectors for Antibody Expression
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Solution Overview
Problem
Current methods for generating stable, high monoclonal antibody-producing CHO cell lines are labor-intensive and time-consuming due to rare simultaneous amplification of both antibody chains, leading to a large number of clones needing to be screened, and existing IRES and 2A peptide approaches have limitations in translation efficiency and product stability.
Innovation Solution
A nucleic acid molecule comprising sequences encoding for a peptide or protein of interest, a selectable marker, and IRES sequences, with the IRES sequence located between the peptide and selectable marker sequences, and optionally mutated for reduced ribosome binding affinity or translation efficiency, to enhance co-expression and stability of antibody chains in CHO cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If two separate vectors are used to express DHFR selection marker and mAb genes, then selection stringency can be improved by weakening the selection marker, but simultaneous amplification of both antibody chains is rare and requires screening of large number of clones
Solution Approach 1:
The patent merges the DHFR selection marker and mAb gene expression cassettes into a single vector construct. This ensures that both genes are co-amplified together during selection, eliminating the need to screen large numbers of clones for simultaneous expression of both antibody chains. The single vector approach guarantees coordinated amplification of both selection marker and mAb genes.
Solution Approach 2:
The patent uses an IRES (internal ribosome entry site) element as an intermediary between the DHFR selection marker and the mAb gene. This IRES element enables translational coupling, where the IRES facilitates ribosome binding and translation initiation at the mAb gene without requiring a separate promoter. This intermediary mechanism ensures that both genes are expressed from a single transcript, improving both selection stringency and clone screening efficiency.
2Productivity
If IRES sequences are used for co-expression of multiple genes, then simultaneous amplification of both antibody chains is ensured, but translation efficiency of the gene driven by IRES is lower than 5'-cap dependent translation
Solution Approach 1:
The patent applies local quality by using different translational mechanisms for different genes within the same transcript. The DHFR selection marker gene uses traditional 5'-cap dependent translation, while the mAb gene uses IRES-mediated translation. This localized differentiation allows each gene to be expressed through the most suitable mechanism, optimizing overall protein production while maintaining co-expression.
Solution Approach 2:
The patent modifies the IRES sequence parameters (nucleotide composition, secondary structure, ribosome binding affinity) to optimize translation efficiency. By adjusting these parameters, the patent enhances the translation capability of the IRES-driven mAb gene while maintaining the benefits of co-expression and translational coupling.
3Reliability
If weak regulatory elements like promoters are used on the selection marker to reduce expression level, then selection stringency is improved, but the selection marker function is impaired
Solution Approach 1:
The patent segments the expression control into two independent parts: the DHFR selection marker is driven by a strong promoter to maintain robust selection function, while the mAb gene expression is controlled by the IRES element. This segmentation allows the selection marker to maintain full functionality while still enabling stringent selection through the IRES-mediated control of mAb expression.
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
This approach minimizes non-expressing clones, enhances productivity, and controls product quality by ensuring balanced expression of antibody light and heavy chains, allowing for the rapid generation of high-producing cell lines with improved stability and consistency.
Implementation Method 1
Internal ribosome entry site (IRES) elements allow expression of multiple genes in one transcript (Mountford and Smith 1995). IRES-based bicistronic or tricistronic vectors, which express the product and selection marker genes in one transcript, can minimize the escape of non-expressing clones from selection
Implementation Method 2
the at least one IRES sequence is located 5′ to a nucleic acid sequence encoding for a selectable marker and is mutated such that it has a reduced ribosome binding affinity compared to the corresponding wildtype IRES sequence
Implementation Method 3
DHFR catalyzes the reduction of dihydrofolate into tetrahydrofolate, an essential co-factor in the synthesis of purines and amino acids
Implementation Method 4
enhanced expression level can be achieved by exposure to methotrexate (MTX), a DHFR inhibitor, leading to the amplification the gene copy numbers
Data Source
AI summary
This invention relates to nucleic acid molecules comprising at least one nucleic acid sequence encoding for a peptide or protein of interest, at least one nucleic acid sequence encoding for a selectable marker, and at least one IRES sequence, wherein the at least one IRES sequence is located between the at least one nucleic acid sequence encoding for the peptide or protein of interest and the at least one nucleic acid sequence encoding for the selectable marker. Furthermore, this invention relates to host cells comprising such nucleic acid molecule and to methods of recombinant protein expression using such host cells.