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Publication Number
US-11485959-B2
Publication Date
2022-11-01
Expiration Date
Abstract
The present invention provides PiggyBac transposase proteins, nucleic acids encoding the same, compositions comprising the same, kits comprising the same, non-human transgenic animals comprising the same, and methods of using the same.
Core Innovation
The invention provides hyperactive PiggyBac transposase proteins, including proteins defined by at least 90% sequence identity to SEQ ID NO: 2. The hyperactive transposase proteins include an amino acid substitution at position 226, specifically a Phenylalanine for a Methionine (M226F), and are capable of mediating transposon integration at a higher frequency than a wild type transposase having the sequence of SEQ ID NO: 2.
The disclosure further covers hyperactive transposase proteins with additional amino acid substitutions at specified positions, including named substitutions such as M185L, A187G, F200W, Q591P, and other position-defined variants reported to increase transposition readouts relative to wild-type PBo. In particular, additional variants are described as containing substitutions at positions beyond M226F, and are associated with higher transposition performance.
The invention also encompasses nucleic acids encoding the hyperactive transposase proteins, vectors for delivering the components, cells, kits, and non-human transgenic animals. PiggyBac transposons are described as being flanked by inverted repeats, and methods are described that administer a transposon and a hyperactive transposase to excise and stably integrate an exogenous gene into target cell genomes, including generating and mobilizing transgenic animals.
Claims Coverage
The provided claim set includes one independent claim directed to a hyperactive transposase protein, and dependent claims that further narrow or add features by specifying additional amino-acid substitutions, nucleic-acid/genome integration using piggyBac-like inverted repeats, and transposase delivery as RNA or as an expressed protein, as well as vector delivery modalities.
Hyperactive transposase with at least 90% identity to SEQ ID NO: 2 and M226F substitution
A hyperactive transposase protein comprising at least 90% sequence identity to SEQ ID NO: 2, comprising an amino acid substitution at position 226 wherein the substitution at position 226 is a Phenylalanine for a Methionine (M226F), wherein the hyperactive transposase protein is capable of mediating transposon integration at a higher frequency compared to a wild type transposase having the sequence of SEQ ID NO: 2.
Additional amino-acid substitutions in the hyperactive transposase protein
A hyperactive transposase protein further comprising specific amino-acid substitutions at positions 30, 165, and 282, namely I30V, G165S, and an M282X change where X is Leucine, Isoleucine, Valine, or Alanine.
Stably integrating an exogenous nucleic acid using a piggyBac-like transposon with inverted repeats
A method for stably integrating an exogenous nucleic acid into a cell genome by delivering a piggyBac-like transposon flanked by first and second inverted repeats and a hyperactive transposase protein to excise and integrate the nucleic acid.
Introducing the hyperactive transposase as transposase RNA
The method is carried out by having the transposon on a vector and introducing the hyperactive transposase protein as transposase RNA.
Introducing the hyperactive transposase as an expressed protein
The method further specifies that the transposon is carried on a vector and that a hyperactive transposase protein is introduced as an expressed protein.
Selecting the vector from plasmid, liposome, virus, or nanoparticle
The vector is selected from a plasmid, a liposome, a virus, or a nanoparticle.
Overall, the claim coverage centers on a hyperactive transposase protein defined by high sequence identity to SEQ ID NO: 2 together with the M226F substitution, and extends to variants with additional named substitutions. It further covers methods for stable genome integration of an exogenous nucleic acid using piggyBac-like inverted repeats, with narrowing based on whether the hyperactive transposase is introduced as transposase RNA or as an expressed protein, and vector modality selection including plasmid, liposome, virus, or nanoparticle.
Stated Advantages
Mediates transposon integration at a higher frequency compared to a wild type transposase having the sequence of SEQ ID NO: 2.
Documented Applications
Excising and stably integrating an exogenous gene into target cell genomes using a transposon flanked by inverted repeats and a hyperactive transposase.
Generating and mobilizing transgenic animals using piggyBac transposons and hyperactive transposase.
Screening/selection of hyperactive mutations using polyA-trap genetrap transposons with GFP readouts and FACS/drug selection.
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