Regulation of translation of expressed genes

Inventors

Verhaert, Raymond Michael Dimphena • Schut, Pieter Victor • Barends, Sharief • van der Heijden, Maurice Wilhelmus

Assignees

Proteonic Biotechnology IP BV

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 organismusing such nucleic acid sequences, as well as their uses for increasing integration 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 regulatory sequences derived from Nicotiana tabacum ntp303 5′UTR that enhance mRNA usage and/or translation. The sequences include GAA repeat nucleotide sequence elements, TC-rich nucleotide sequence elements, multiple A-rich nucleotide sequences, and a GT-rich nucleotide sequence comprising only G or T nucleotides. Variants of these elements are described as UN variants, including UN1/UN2a/UN2b and other UN variants.

In some embodiments, the nucleic-acid molecule further comprises an intron sequence at the 3′ end of the first nucleotide sequence, with required splice-site motifs for splicing. Splice-site requirements include a GT splice site at the 5′ end of the intron and an AG splice site at the 3′ end of the intron nucleotide sequence, and a branch site sequence such as CTGAC. The document also describes sequence identity constraints relative to SEQ ID NOs.

The disclosed nucleic acids are used in nucleic acid constructs and host cells/organisms (non-human), including plants, yeast, fungi, mammals/CHO, and insects, for expression of a protein or polypeptide of interest. The document states outcomes including increased genomic integration (higher copy integration) and increased translation of recombinantly expressed polypeptides, including increased monoclonal antibody production and increased mRNA levels without changing integrated copy number in mammalian stable pools.

Claims Coverage

Independent claim clm-00001 covers a nucleic acid molecule defined by a first nucleotide sequence containing specific TC-rich, A-rich, GT-rich, and GAA repeat motifs with an integer n (n≥6) and requiring at least 90% sequence identity with SEQ ID NO:5. Dependent claims further refine sequence identity constraints, add optional intron/splicing features at the 3′ end, and specify protein-expression use in non-human cells/organisms with defined construct-to-expression steps. The main inventive features across the independent claim set are four motif classes with identity and structural constraints, plus optional intron/splice-site elements and non-human expression implementation.

Overall, the claim coverage is anchored in a motif-defined first nucleotide sequence containing TC-rich, A-rich, GT-rich, and (GAA)n repeat elements with n≥6 and at least 90% sequence identity to SEQ ID NO:5. Dependent claims further constrain identity, add an intron at the 3′ end with defined splice-site and branch-site sequence features, and include non-human expression of a protein of interest using a nucleic acid construct to produce transformed cells that express the protein.

Stated Advantages

Documented Applications