Enhanced nucleic acid constructs for eukaryotic gene expression
Inventors
Minshull, Jeremy • Welch, Mark • GOVINDRAJAN, SRIDHAR • Caves, Kate
Assignees
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Publication Number
US-9428767-B2
Publication Date
2016-08-30
Expiration Date
Abstract
The present invention provides polynucleotide vectors for high expression of heterologous genes, and methods for constructing such vectors. 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, but not limited to, gene expression, gene therapy, insertional mutagenesis, or gene discovery.
Core Innovation
The invention relates to an improved non-viral eukaryotic gene expression framework that combines systematic optimization of DNA vector elements with piggyBac-like transposon/transposase systems for stable genomic integration. The framework focuses on heterologous polynucleotide expression by using designed DNA element architectures that include promoters, enhancers, introns, UTRs, polyadenylation signals, RNA export elements, and modulating elements, and it also includes regulatory element options such as insulators to influence expression behavior from the integrated construct.
The invention uses piggyBac-like transposons with inverted repeats flanking a heterologous polynucleotide and copies of a defined target site at each end. The disclosed systems target the 5′-TTAT-3′ architecture, and in described embodiments also 5′-TTAA-3′ from Xenopus, where excision leaves a single TTAT target-site copy in place of the transposon and a defined “scar” target-site motif after excision.
For multi-polypeptide control, the patent further describes translational coupling concepts using IRES and CHYSEL to coordinate expression ratios of multiple heterologous coding sequences, including antibody heavy and light chains. The document places the framework in broader gene transfer and expression contexts and describes corresponding vector and kit concepts associated with the transposon and transposase components.
Claims Coverage
The provided independent claims define a piggyBac-like transposon architecture for excisable integration into a defined 5′-TTAT-3′ target site, including specific sequence identity and length constraints, and for one independent claim, identity criteria for inverted terminal repeats. Across the independent claims, the core scaffold is a transposon with inverted repeats flanking a heterologous polynucleotide and leaving a single 5′-TTAT-3′ copy in place upon excision, with additional specificity in the transposon sequence definitions.
Excisable piggyBac-like transposon flanked by 5′-TTAT-3′ target-site copies leaving a TTAT scar
A polynucleotide comprising a transposon comprising inverted repeats of a piggyBac-like transposon flanking a heterologous polynucleotide, wherein the inverted repeats are flanked by copies of the target site 5′-TTAT-3′ at each end, such that the transposon can be excised leaving a single copy of the 5′-TTAT-3′ target site in place of the transposon in the polynucleotide.
Minimum contiguous nucleotides from SEQ ID NO: 1 and SEQ ID NO: 2 to define TTAT target-site copies and inverted repeats
The transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 1 to provide a copy of the 5′TTAT-3′ target site and one inverted repeat and at least 16 contiguous nucleotides from SEQ ID NO: 2 to provide the other copy of the 5′-TTAT-3′ target site and the other inverted repeat.
Inverted terminal repeats at least 90% identical to SEQ ID NO: 32 in an excisable TTAT architecture
A polynucleotide comprising a transposon comprising inverted repeats of a piggyBac-like transposon flanking a heterologous polynucleotide, wherein the inverted repeats are flanked by copies of the target site 5′-TTAT-3′ at each end, such that the transposon can be excised leaving a single copy of the 5′-TTAT-3′ target site in place of the transposon in the polynucleotide, wherein the transposon comprises two inverted terminal repeat (ITR) sequences, each of which is at least 90% identical to SEQ ID NO: 32.
SEQ ID identity thresholds for transposon sequences relative to SEQ ID NO: 1 and SEQ ID NO: 2
A polynucleotide comprising a transposon comprising inverted repeats of a piggyBac-like transposon flanking a heterologous polynucleotide, wherein the inverted repeats are flanked by copies of the target site 5′-TTAT-3′ at each end, such that the transposon can be excised leaving a single copy of the 5′-TTAT-3′ target site in place of the transposon in the polynucleotide, wherein the transposon comprises a sequence that is at least 90% identical to SEQ ID NO: 1 and a sequence that is at least 90% identical to SEQ ID NO: 2.
The independent claims collectively cover a piggyBac-like transposon architecture designed for excision-driven genomic integration at a defined 5′-TTAT-3′ target site. Claim scope is anchored by leaving a single TTAT target-site copy in place of the transposon after excision and by specifying transposon sequence and identity requirements, including contiguous-nucleotide inclusion from SEQ ID NO: 1 and SEQ ID NO: 2, and for one independent claim, inverted terminal repeat identity relative to SEQ ID NO: 32.
Stated Advantages
Improved expression upon co-transfection for stable integration (reported 3–10 fold for stable integration with transposase).
Excision and integration leave a defined “scar” target-site motif after excision.
Transposase co-delivery improves stable integration/expression.
HS4 insulators enhance expression.
Nuclear localization signal fusion to transposases improves performance.
Documented Applications
Protein or antibody production, including multi-polypeptide expression such as antibody heavy and light chains.
Gene therapy.
Insertional mutagenesis.
Transgenic animals.
Pharmaceutical compositions.
Kits related to the vector/transposon and transposase concepts.
Controlled co-expression of two proteins, including antibody heavy/light chains, via translational coupling elements.
Gene discovery/gene tagging via insertional mutagenesis.
CAR T-cell gene therapy.
Gene transfer systems for which non-viral DNA gene transfer vectors/transposon systems are evaluated in CHO and HEK cells.
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