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-10233454-B2
Publication Date
2019-03-19
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 patent describes engineered transposases derived from Bombyx transposases and Xenopus tropicalis transposases, including transposon ends and ITR sequences configured with piggyBac-like transposon/transposase pairs. The disclosed transposase architecture includes non-naturally occurring proteins at least 90% identical to SEQ ID NO:61 and differing from SEQ ID NO:48 by amino acid substitutions shown in Table 4. The system supports high-expression eukaryotic genome modification and efficient stably integrated reporter expression from synthetic transposons when paired with engineered transposase variants.
A central aspect is that naturally occurring transposases become integration-competent only when fused to a heterologous nuclear localization signal, and the engineered variants are described as hyperactive and/or integration-deficient. The disclosure links these engineered transposase variants to transposons configured with specified piggyBac-like transposon ends and defined target site sequence options. Transposase activity can be delivered via transposase mRNA, and reporter expression increases are observed compared with transposon-alone controls.
The document further identifies variants by selection criteria in which mutations are enriched for transposition and/or excision and by quantitative activity measures showing increased excision or transposition activity. Hyperactive Bombyx transposases are also defined using at least 90% identity thresholds to specified SEQ IDs, and the disclosure includes additional transposase/selection frameworks for screening variant activity in eukaryotic cells.
Claims Coverage
The provided independent claims cover engineered, non-naturally occurring piggyBac-like transposase polynucleotides that are at least 90% identical to SEQ ID NO:61 and differ from a reference SEQ ID NO:48 by at least one amino acid specified in Table 4. The independent claims require fusion or linkage to a heterologous nuclear localization signal and characterize functional performance as increased excision and/or transposition activity relative to SEQ ID NO:48 fused to a heterologous nuclear localization signal. Three inventive features are identified across the independent claims.
Engineered transposase polynucleotide with hyperactive excision/transposition
A polynucleotide encoding a transposase, other than a naturally occurring protein, wherein the transposase is at least 90% identical to SEQ ID NO:61 and differs from SEQ ID NO:48 by at least one amino acid shown in Table 4 column C or D, and wherein the transposase, when fused to a heterologous nuclear localization signal, can excise or transpose a transposon from SEQ ID NO:44 with at least two-fold increased excision or transposition activity respectively relative to the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.
NLS-linked engineered transposase polynucleotide with increased excision activity
A polynucleotide encoding a transposase linked to a heterologous nuclear localization sequence, wherein the transposase is other than a naturally occurring protein, at least 90% identical to SEQ ID NO:61, and differs from SEQ ID NO:48 by at least one amino acid shown in Table 4 column C or D and can excise a transposon from SEQ ID NO:44 with increased activity relative to the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.
Nuclear localization fusion enabling increased excision or transposition
The transposase, when fused to a heterologous nuclear localization signal, can excise or transpose a transposon from SEQ ID NO:44 with at least two-fold increased excision or transposition activity relative to the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.
Overall, the claims require an engineered transposase polynucleotide with at least 90% identity to SEQ ID NO:61 and one or more amino acid differences specified by Table 4, plus fusion or linkage to a heterologous nuclear localization signal. The encoded system is characterized by increased excision and/or transposition activity on a transposon from SEQ ID NO:44 relative to a SEQ ID NO:48 fused comparison.
Stated Advantages
Increased excision or transposition activity from SEQ ID NO:44 compared with the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.
At least two-fold increased excision or transposition activity relative to the transposase of SEQ ID NO:48 fused to a heterologous nuclear localization signal.
Efficient stably integrated reporter expression from synthetic transposons when paired with the engineered transposase variants.
Documented Applications
High-expression eukaryotic genome modification using synthetic piggyBac-like transposon/transposase pairs to obtain efficient stably integrated reporter expression.
Creating transgenic eukaryotic cells by introducing piggyBac-like transposase and a transposon-flanked heterologous polynucleotide with specified transposon end sequences, followed by identifying cells with transposon incorporation into the genome.
Assaying transposition/excision activity using reporter transposons in yeast (Saccharomyces cerevisiae) and CHO cells, with selection and quantitative activity measures.
Transposon utility in yeast (Pichia pastoris) using zeocin selection.
Use of IRES elements for two-polypeptide expression in the disclosed context.
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