DNA vectors, transposons and transposases for eukaryotic genome modification
Inventors
Minshull, Jeremy • Welch, Mark • GOVINDRAJAN, SRIDHAR • Lee, Maggie • Caves, Kate • Ness, Jon
Assignees
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Publication Number
US-10344285-B2
Publication Date
2019-07-09
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 relates to piggyBac-like transposon/transposon-end systems for eukaryotic genome modification, including stable genomic integration of a heterologous polynucleotide. It discloses isolated eukaryotic cells whose genomes comprise piggyBac-like transposon ends flanking a heterologous polynucleotide not associated with the transposon ends in nature, with a defined inverted orientation between the two transposon ends.
A key feature is a piggyBac-like transposon-end architecture in which each transposon end comprises 5-CCYTTTBMCTGCCA-3 (SEQ ID NO: 19) inverted in orientation in the two transposon ends. The ambiguity-code positions within the transposon-end repeats can be selected independently between the two repeats, providing selectable diversity in the transposon ends while preserving the overall piggyBac-like end structure.
The disclosed systems further support improved heterologous gene expression through integration-dependent expression, including configurations that incorporate nuclear localization signal (NLS) requirements for transposase function. It discloses piggyBac-like systems derived from Xenopus and Bombyx transposases, including characterization of hyperactive and integration-deficient transposase variants, and delivery options for transposase as protein or mRNA.
Claims Coverage
Independent claim coverage centers on an isolated eukaryotic cell with piggyBac-like transposon-end sequence architecture flanking a heterologous polynucleotide, with independently selectable ambiguity-code nucleotides in the two inverted repeats. The coverage is narrowed in dependent claims by optional regulatory elements and by quantitative sequence-identity constraints for specific components.
Isolated eukaryotic cell with piggyBac-like transposon ends flanking a heterologous polynucleotide
An isolated eukaryotic cell whose genome comprises a polynucleotide comprising piggyBac-like transposon ends flanking a heterologous polynucleotide not associated with the transposon ends in nature.
Inverted SEQ ID NO: 19 transposon-end repeats with independently selectable ambiguity codes
Each transposon end comprises 5-CCYTTTBMCTGCCA-3 (SEQ ID NO: 19) inverted in orientation in the two transposon ends, wherein the identity of nucleotides of an ambiguity code can be selected independently in the two repeats.
Overall, the claim set centers on a genome engineering construct with piggyBac-like transposon ends flanking a heterologous polynucleotide, using inverted SEQ ID NO: 19 repeats and independently selectable ambiguity-code nucleotides, with further narrowing in dependent claims via optional regulatory components and sequence-identity thresholds.
Stated Advantages
Improved heterologous gene expression via stable eukaryotic genome modification and integration-dependent expression.
Documented Applications
Use in isolated eukaryotic cells for stable genomic integration of a heterologous polynucleotide using piggyBac-like transposon ends.
Use in high mammalian expression vector/gene-transfer configurations using promoters/enhancers, insulators, and IRES/CHYSEL coupling, together with selectable/counter-selectable marker enrichment approaches.
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