DNA vectors, transposons and transposases for eukaryotic genome modification
Inventors
Minshull, Jeremy • Lee, Maggie
Assignees
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Publication Number
US-10041077-B2
Publication Date
2018-08-07
Expiration Date
Abstract
The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.
Core Innovation
The invention provides a polynucleotide segment that is at least 99% identical to SEQ ID NO: 866, where the segment comprises SEQ ID NO: 869 and either SEQ ID NO: 867 or 868. The segment is effective to enhance expression of a gene when incorporated between a coding sequence and a 3′ polyA sequence, and the disclosed systems include defined sequence elements and sequence-identity constraints that maintain functional expression enhancement when engineered into gene constructs.
The document further describes engineered expression-enhancing integration contexts using piggyBac-like, DNA transposon/transposase gene-transfer systems for stable, high-expression integration into eukaryotic genomes. It includes synthetic transposons defined by transposon-end/ITR sequence requirements and transposase activities, including hyperactive and integration-deficient variants.
The disclosed constructs also incorporate expression-enhancing chromatin and control elements, including insulators, SAR/UCOE/STAR, promoters/introns, and WPRE/HPRE. It further describes configurations supporting dual-gene expression using IRES/CHYSEL, with engineered placement of sequence elements and measured expression readouts across cell types.
Claims Coverage
The provided independent claim is directed to a specific expression-enhancing polynucleotide segment defined by at least 99% sequence identity to SEQ ID NO: 866 and by required internal sequence inclusion, positioned between a coding sequence and a 3′ polyA sequence. The dependently claimed coverage further refines the polynucleotide with additional regulatory elements and imposes explicit identity thresholds for those added components, plus an isolated-cell context with operable linkage.
Polynucleotide segment with defined SEQ ID composition for expression enhancement
A polynucleotide comprising a segment having a sequence at least 99% identical to SEQ ID NO: 866, wherein the segment comprises SEQ ID NO: 869 and either SEQ ID NO: 867 or 868, the segment effective to enhance expression of a gene incorporating the segment between a coding sequence and 3′ polyA sequence.
Promoter with a heterologous intron to support gene expression
The polynucleotide further includes a promoter and an intron that is heterologous to the promoter.
IRES sequence identity threshold using defined sequence set
The polynucleotide has an IRES sequence that is at least 95% identical to a sequence selected from SEQ ID NO: 1050-1094.
Additional sequence element constrained by SEQ ID NO: 998-1049 identity threshold
The polynucleotide includes an additional sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 998-1049.
Additional sequence element constrained by SEQ ID NO: 751-819 identity threshold
The polynucleotide further includes a sequence that is at least 90% identical to a sequence selected from SEQ ID NO: 751-819.
Isolated cell with operable linkage to the expression polynucleotide
An isolated cell containing a genome where the polynucleotide is operably linked to a sequence to be expressed.
Across the independent claim and its dependents, the scope centers on a specifically defined, identity-constrained polynucleotide segment that enhances gene expression when placed between a coding sequence and a 3′ polyA sequence, with additional coverage for regulatory architectures, defined sequence-identity thresholds for added components, and an isolated cell context in which the polynucleotide is operably linked to an expressed sequence.
Stated Advantages
Enhances expression of a gene incorporating the segment between a coding sequence and a 3′ polyA sequence.
Documented Applications
No documented applications found
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