Expression cassette for production of high-expression and high-functionality target protein and use thereof
Inventors
KIM, Man Su • Kim, Min Soo • Cho, Jong Moon • Chang, Shin Jae
Assignees
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Publication Number
US-12188036-B2
Publication Date
2025-01-07
Expiration Date
Abstract
The present invention relates to an expression cassette for a target protein, comprising a promoter, a polynucleotide coding for the target protein, an intron sequence, and a poly A sequence, an expression vector, and a transformant. The expression cassette for a target protein according to the present invention can simultaneously perform the expression of the intron sequence and the target protein through one transduction and exhibits the effect of inducing the high expression and high functionality of the target protein by regulating the expression of an endogenous gene.
Core Innovation
The invention relates to an expression cassette and associated expression vector and transformant system for production of a target protein in which a single promoter transcriptionally links a polynucleotide coding for the target protein with a polynucleotide sequence encoding an intronic RNA sequence. The intronic RNA sequence comprises a splicing donor, a branch, and a splicing acceptor, and is configured to function as a target gene expression regulation RNA sequence. The target protein is an antibody or a fragment thereof.
The intronic RNA is configured to generate intronic shRNA sequences and/or intronic miRNA sequences that regulate endogenous genes selected from FUT8, HDAC5, LDHA, DHFR, CXCR4, and PDK4, thereby enhancing production and functionality of the antibody. The disclosed system is directed to high-expression and high-functionality target protein production, including reduced fucose on antibody Fc.
The approach includes selection of effective intronic shRNA and miR483 based on endogenous mRNA regulation, including mRNA knockdown and/or upregulation, and reports increased antibody production in transient and stable contexts. The document further reports that stable high producers can be identified using fewer cell lines and quantifies fucosylation reduction using Bio-LC together with RT-PCR of endogenous gene regulation in stable lines, with improved selection efficiency associated with a DHFR/methotrexate selection scheme.
Claims Coverage
The claims include one independent claim and multiple dependent claims. The independent claim defines one inventive combination of a single promoter-controlled target protein coding sequence and an intronic RNA sequence containing splicing elements and a target gene expression regulation RNA sequence comprising specified intronic shRNAs. Dependent claims refine the intronic splicing elements, the specific intronic shRNA sequence IDs, an antibody expression level range, and a vector embodiment.
Single-promoter linked target protein and intronic RNA sequence
The single promoter transcriptionally links the polynucleotide coding for the target protein and the polynucleotide sequence encoding the intronic RNA; the intronic RNA sequence comprises a splicing donor, a branch, and a splicing acceptor.
Intronic gene expression regulation RNA sequence comprising specified intronic shRNA targets
The target gene expression regulation RNA sequence comprises one or more shRNA sequences selected from intronic FUT8 shRNA, intronic HDAC5 shRNA, intronic LDHA shRNA, and intronic DHFR shRNA.
Target protein as antibody or fragment
The target protein is an antibody or a fragment thereof.
Specified intronic splicing donor, branch, and splicing acceptor sequence IDs
The splicing donor, branch, and splicing acceptor include specified sequence IDs.
Quantified antibody expression level range in an expression cell line
In an expression cell line containing the cassette, the antibody expression level is within 198.5 to 374.0 μg/ml.
Vector including the expression cassette
A vector includes an expression cassette for producing a target protein as defined in the independent claim.
Overall, the claim set covers a single-promoter cassette that couples antibody or antibody-fragment coding with an intronic RNA containing splicing elements and a targeted intronic shRNA module. Dependent claims further specify particular intronic splicing elements and particular intronic shRNA sequence IDs, and include additional refinement such as a quantified antibody expression level range and a vector embodiment.
Stated Advantages
Enhanced antibody productivity and functionality associated with endogenous gene regulation by intronic shRNA/miRNA.
Reduced fucose on antibody Fc.
Improved selection efficiency associated with DHFR/methotrexate selection.
Ability to identify stable high producers using fewer cell lines.
Documented Applications
Producing antibody and antibody fragments with high expression and high functionality using an expression cassette system.
Regulating endogenous genes via intronic shRNA/miRNA to improve antibody production characteristics, including reduced Fc fucosylation.
Selection of stable high producers using a DHFR/methotrexate selection scheme.
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