DNA vectors, transposons and transposases for eukaryotic genome modification
Inventors
Minshull, Jeremy • Lee, Maggie
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10435696-B2
Publication Date
2019-10-08
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 transposon/transposase-based gene transfer systems that provide stable, high-expression integration into eukaryotic genomes using synthetic transposons and transposases. The systems use transposon ends and a transposase to mediate integration of a heterologous nucleic acid sequence to be expressed, and the constructs are configured for heterologous gene expression while promoting integration performance consistent with stable expression in eukaryotic cells.
A core aspect is the use of piggyBac-like synthetic transposon/transposase systems implemented with Xenopus and Bombyx components. The disclosure includes Xenopus and Bombyx transposases, including hyperactive and integration-deficient variants, and corresponding transposon end sequences defined in the document, and in the Xenopus system the disclosure describes NLS fusion requirements for Xenopus transposases.
The invention further provides engineered target sequence and integration-related design elements intended to enhance integration while preserving expression. The disclosure describes modification of target sequences including switching TTAA to TTAT to enable efficient integration with preserved expression, as well as selectable and counter-selectable markers and chromatin control elements such as insulators, UCOEs, and STARs.
Claims Coverage
The provided independent claims include one polynucleotide claim directed to an EF1 promoter construct, with the inventive features centered on an EF1 promoter defined by SEQ ID NO: 905 and its operable linkage to a heterologous nucleic acid sequence to be expressed. Related claim features further specify selectable marker segments, promoter sequence sets, an intron identity constraint, and a gene-expression vector for mammalian cells.
EF1 promoter defined by SEQ ID NO: 905 for heterologous expression
A polynucleotide comprising an EF1 promoter comprising SEQ ID NO: 905 operably linked to a heterologous nucleic acid sequence to be expressed.
Selectable marker segment in the expression cassette
A polynucleotide comprising a segment encoding a selectable marker, wherein the selectable marker is included as part of the polynucleotide of the EF1 promoter expression construct.
Selectable marker variants defined by specific marker proteins
The polynucleotide further comprises one of the specified selectable markers including glutamine synthase, dihydrofolate reductase, puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acetyl transferase, or a zeocin-binding protein.
Selectable marker promoter driven by SEQ ID NOS: 937-948
A polynucleotide in which a selectable marker segment is operably linked to a promoter containing a sequence selected from SEQ ID NOS: 937-948.
Intron with a defined sequence identity constraint
A polynucleotide comprising an intron containing a sequence that is 95% identical to a sequence selected from SEQ ID NOS: 958-997.
Mammalian gene-expression vector incorporating the EF1 promoter construct
A gene-expression vector for use in a mammalian cell that includes the polynucleotide comprising the EF1 promoter comprising SEQ ID NO: 905 operably linked to a heterologous nucleic acid sequence to be expressed.
Across the independent claim and related limitations, the core claim coverage is directed to an EF1 promoter defined by SEQ ID NO: 905 operably linked to a heterologous nucleic acid sequence to be expressed, with further claim features specifying selectable marker segments, defined selectable marker options, promoter sequence sets for the selectable marker, an intron defined by a 95% identity constraint, and extension to a mammalian gene-expression vector.
Stated Advantages
Reports significantly higher DasherGFP expression with co-transfection using specific hyperactive Bombyx transposases compared with naturally occurring Bombyx transposase SEQ ID NO: 407.
States that promoter/selection stringency and insulator use strongly affect outcomes.
States that construct linearization and transposase expression increase zeocin-resistant colony formation in Pichia pastoris.
Documented Applications
Enhanced DasherGFP expression in mammalian cells using hyperactive Bombyx transposases compared with naturally occurring transposase.
IRES-based dual-polypeptide gene transfer with measured expression ratios in HEK293 and CHO cells.
Genome integration in Pichia pastoris with increased zeocin-resistant colony formation.
Interested in licensing this patent?