Regulation of translation of expressed genes
Inventors
Verhaert, Raymond Michael Dimphena • Schut, Pieter Victor • Barends, Sharief • van der Heijden, Maurice Wilhelmus
Assignees
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Publication Number
US-11667921-B2
Publication Date
2023-06-06
Expiration Date
Abstract
The present invention describes mRNA usage improving and/or translation-enhancing nucleic acid sequences, nucleic acid constructs comprising such sequences, and host cells comprising such nucleic acid constructs. The invention further pertains to a method for expressing a protein of interest in a cell or organism using such nucleic acid sequences, as well as their uses for increasing intergration of such nucleic acid construct into a genome, for enhancing mRNA usage and/or translation of a recombinantly expressed polypeptide, and for increasing the number of transformants upon transformation of a cell with such nucleic acid construct.
Core Innovation
The invention relates to nucleic acid molecules that include a first nucleotide sequence extending from a 5′ end to a 3′ end. This first nucleotide sequence contains a TC-rich nucleotide sequence, multiple A-rich nucleotide sequences, and a GT-rich nucleotide sequence, together with an intron sequence that includes splice sites required for splicing of the intron sequence. The first nucleotide sequence has at least 90% sequence identity with SEQ ID NO: 2.
In the disclosed nucleic acid architecture, the TC-rich and A-rich regions and the GT-rich region are arranged within the first nucleotide sequence alongside the intron sequence. The intron sequence includes splice sites required for splicing, including 5′ GT and 3′ AG, and a branch-site motif located on the 5′ side of a TC-rich nucleotide sequence. The document further contemplates variations in intron and UTR configurations while maintaining sequence identity constraints relative to SEQ ID NO: 2.
The document further discloses nucleic acid constructs and expression contexts in which the first nucleotide sequence is operably linked to a protein/polypeptide coding region. A promoter may be included, and the document describes expression in non-human cells/organisms, including transformed cells and transformants obtained by integration into a genome. The overall purpose stated in the disclosure is to improve mRNA usage and/or enhance translation, including cap-dependent translation behavior.
Claims Coverage
The independent claims identified in the partial content include three inventive features: a structured nucleic acid molecule, sequence identity to SEQ ID NO: 2, and an expression method using a translation-increasing first nucleotide sequence.
Structured first nucleotide sequence with TC-rich, A-rich, and GT-rich regions plus intron splice sites
A nucleic acid molecule comprising a first nucleotide sequence from its 5′ end to its 3′ end including a TC-rich nucleotide sequence, 4 A-rich nucleotide sequences, a GT-rich nucleotide sequence, and an intron sequence comprising at least splice sites required for splicing of the intron sequence.
Sequence identity to SEQ ID NO: 2
The nucleic acid molecule where the first nucleotide sequence has at least 90% sequence identity with SEQ ID NO: 2.
Expression method using a nucleic acid construct with a translation-increasing first nucleotide sequence
A method for expressing a polypeptide of interest by providing a nucleic acid construct comprising a first nucleotide sequence that increases translation of a second nucleotide sequence encoding the polypeptide of interest, where the construct also includes a promoter, transforming the cell with the construct to obtain a transformed cell, and expressing the polypeptide in the transformed cell.
The claim coverage centers on a defined TC-rich/A-rich/GT-rich first nucleotide sequence combined with an intron sequence providing required splice sites, constrained by sequence identity to SEQ ID NO: 2, and an expression method in which such a translation-increasing first nucleotide sequence is used within a promoter-containing construct to express a polypeptide of interest in a transformed cell.
Stated Advantages
Improves translation and/or mRNA usage.
Enhances translation in a cap-dependent manner as described for competitive in vitro translation.
Increases production of secreted proteins/polypeptides in multiple systems as described, including monoclonal antibody yield, secreted laccase, hIL-8, luciferase signal, and secreted SeAP and hIL-4.
Improves mRNA levels associated with expression outcomes as described for CHO cell experiments using UN variants.
Documented Applications
Expression and/or production of a protein/polypeptide of interest in non-human cells/organisms using constructs containing the disclosed nucleic acid sequences, including transformed cells and transformants obtained by integration into a genome.
Competitive in vitro translation using constructs with elements including a GAA repeat and UN elements, evaluated in rabbit reticulocyte lysate and/or wheat germ extract contexts as described.
Monoclonal antibody production in CHO cells using modified UN variants.
Secreted laccase production in Aspergillus niger using UN variants and positional adaptations.
hIL-8 production in yeast using UN variants and effects attributed to genomic integration behavior.
Plant protoplast luciferase increase using positional adaptation of an imperfect GAA toward the start codon.
Mammalian Flp-In cell lines showing increased secreted SeAP and hIL-4 with UN2 and tailored intron, and increased hIL-4 via a “free” untranslated UN1 transcript cassette in stably transfected CHO cells.
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