# US 2013/0160152 A1 · Hyperactive Piggybac Transposases

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- Number: US 2013/0160152 A1
- Document ID: US20130160152A1
- Status: Published application
- Kind: A1
- Application number: 13/774,718
- Filed: 2013-02-22
- Published: 2013-06-20
- Earliest claimed: 2009-02-26
- Assignees: Transposagen Biopharmaceuticals, Inc.
- Inventors: Eric Ostertag; Blair Madison
- Web page: https://patentgrep.com/patent/US20130160152A1

## CPC

- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A61D 19/04: for embryo transplantation
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- C12Y 207/07: Nucleotidyltransferases (2.7.7)
- A01K 67/61: Genetically modified invertebrates, e.g. transgenic or polyploid
- A01K 2217/052: inducing gain of function
- A01K 2227/00: Animals characterised by species
- C12N 15/907: {in mammalian cells}
- C12N 2799/027: where the vector is derived from a retrovirus
- C12N 2800/90: Vectors containing a transposable element
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61D: VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12Y: ENZYMES

## 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.

## Claims

### Claim at position 1

-20. (canceled)

### Claim 21

A non-human, transgenic animal comprising a nucleic acid molecule encoding a protein comprising at least 90% sequence identity to SEQ ID NO:2, and comprising at least one of the following amino acid substitutions in SEQ ID NO:2: a serine for the alanine at position 46; a threonine for the alanine at position 46; a proline for the arginine at position 119; an alanine for the cysteine at position 125; a leucine for the cysteine at position 125; a lysine for the tyrosine at position 177; a histidine for the tyrosine at position 177; a leucine for the phenylalanine at position 180; an isoleucine for the phenylalanine at position 180; a valine for the phenylalanine at position 180; a leucine for the methionine at position 185; a glycine for the alanine at position 187; a tryptophan for the phenylalanine at position 200; a proline for the valine at position 207; a phenylalanine for the valine at position 209; a phenylalanine for the methionine at position 226; an arginine for the leucine at position 235; a lysine for the valine at position 240; a leucine for the phenylalanine at position 241; a lysine for the proline at position 243; a glutamine for the methionine at position 282; a tryptophan for the leucine at position 296; a tyrosine for the leucine at position 296; a phenylalanine for the leucine at position 296; a leucine for the methionine at position 298; an alanine for the methionine at position 298; a valine for the methionine at position 298; an isoleucine for the proline at position 311; a valine for the proline at position 311; a lysine for the arginine at position 315; a glycine for the threonine at position 319; an arginine for the tyrosine at position 327; a valine for the tyrosine at position 328; a glycine for the cysteine at position 340; a leucine for the cysteine at position 340; a histidine for the aspartic acid at position 421; an isoleucine for the valine at position 436; a tyrosine for the methionine at position 456; a phenylalanine for the leucine at position 470; a lysine for the serine at position 486; a leucine for the methionine at position 503; an isoleucine for the methionine at position 503; a lysine for the valine at position 552; or a threonine for the alanine at position 570.

### Claim 22 (depends on 21)

The non-human, transgenic animal according to claim 21 further comprising an insertion site for an exogenous nucleic acid, wherein the insertion site is flanked by a first inverted repeat sequence comprising a sequence at least about 90% sequence identity to SEQ ID NO:91 and/or a second inverted repeat sequence comprising a sequence at least about 90% sequence identity to SEQ ID NO:92.

### Claim 23

A method of integrating an exogenous nucleic acid into the genome of at least one cell of a multicellular or unicellular organism comprising administering directly to the multicellular or unicellular organism:
a) a transposon comprising the exogenous nucleic acid, wherein the exogenous nucleic acid is flanked by a first inverted repeat sequence comprising a sequence at least about 90% sequence identity to SEQ ID NO:91 and/or a second inverted repeat sequence comprising a sequence at least about 90% sequence identity to SEQ ID NO:92; and
b) a protein, or RNA or DNA encoding the protein, to excise the exogenous nucleic acid from a plasmid, episome, or transgene and integrate the exogenous nucleic acid into the genome, wherein the protein comprises at least 90% sequence identity to SEQ ID NO:2, and comprises at least one of the following amino acid substitutions in SEQ ID NO:2: a serine for the alanine at position 46; a threonine for the alanine at position 46; a proline for the arginine at position 119; an alanine for the cysteine at position 125; a leucine for the cysteine at position 125; a lysine for the tyrosine at position 177; a histidine for the tyrosine at position 177; a leucine for the phenylalanine at position 180; an isoleucine for the phenylalanine at position 180; a valine for the phenylalanine at position 180; a leucine for the methionine at position 185; a glycine for the alanine at position 187; a tryptophan for the phenylalanine at position 200; a proline for the valine at position 207; a phenylalanine for the valine at position 209; a phenylalanine for the methionine at position 226; an arginine for the leucine at position 235; a lysine for the valine at position 240; a leucine for the phenylalanine at position 241; a lysine for the proline at position 243; a glutamine for the methionine at position 282; a tryptophan for the leucine at position 296; a tyrosine for the leucine at position 296; a phenylalanine for the leucine at position 296; a leucine for the methionine at position 298; an alanine for the methionine at position 298; a valine for the methionine at position 298; an isoleucine for the proline at position 311; a valine for the proline at position 311; a lysine for the arginine at position 315; a glycine for the threonine at position 319; an arginine for the tyrosine at position 327; a valine for the tyrosine at position 328; a glycine for the cysteine at position 340; a leucine for the cysteine at position 340; a histidine for the aspartic acid at position 421; an isoleucine for the valine at position 436; a tyrosine for the methionine at position 456; a phenylalanine for the leucine at position 470; a lysine for the serine at position 486; a leucine for the methionine at position 503; an isoleucine for the methionine at position 503; a lysine for the valine at position 552; or a threonine for the alanine at position 570.

### Claim 24 (depends on 23)

The method according to claim 23 wherein the protein of b) is administered as a nucleic acid encoding the protein.

### Claim 25 (depends on 24)

The method according to claim 24 wherein the transposon and nucleic acid encoding the protein of b) are present on separate vectors.

### Claim 26 (depends on 24)

The method according to claim 24 wherein the transposon and nucleic acid encoding the protein of b) are present on the same vector.

### Claim 27 (depends on 23)

The method according to claim 23 wherein the multicellular or unicellular organism is a vertebrate.

### Claim 28 (depends on 27)

The method according to claim 27 wherein the vertebrate animal is a mammal.

### Claim 29 (depends on 23)

The method according to claim 23 wherein the administering is administering systemically.

### Claim 30 (depends on 23)

The method according to claim 23 wherein the exogenous nucleic acid comprises a gene.

### Claim 31

A method of generating a non-human, transgenic animal comprising a germline mutation comprising:
breeding a first non-human, transgenic animal comprising a transposon with a second non-human, transgenic animal comprising a vector comprising a nucleotide sequence encoding a protein comprising at least 90% sequence identity to SEQ ID NO:2, and comprising at least one of the following amino acid substitutions in SEQ ID NO:2: a serine for the alanine at position 46; a threonine for the alanine at position 46; a proline for the arginine at position 119; an alanine for the cysteine at position 125; a leucine for the cysteine at position 125; a lysine for the tyrosine at position 177; a histidine for the tyrosine at position 177; a leucine for the phenylalanine at position 180; an isoleucine for the phenylalanine at position 180; a valine for the phenylalanine at position 180; a leucine for the methionine at position 185; a glycine for the alanine at position 187; a tryptophan for the phenylalanine at position 200; a proline for the valine at position 207; a phenylalanine for the valine at position 209; a phenylalanine for the methionine at position 226; an arginine for the leucine at position 235; a lysine for the valine at position 240; a leucine for the phenylalanine at position 241; a lysine for the proline at position 243; a glutamine for the methionine at position 282; a tryptophan for the leucine at position 296; a tyrosine for the leucine at position 296; a phenylalanine for the leucine at position 296; a leucine for the methionine at position 298; an alanine for the methionine at position 298; a valine for the methionine at position 298; an isoleucine for the proline at position 311; a valine for the proline at position 311; a lysine for the arginine at position 315; a glycine for the threonine at position 319; an arginine for the tyrosine at position 327; a valine for the tyrosine at position 328; a glycine for the cysteine at position 340; a leucine for the cysteine at position 340; a histidine for the aspartic acid at position 421; an isoleucine for the valine at position 436; a tyrosine for the methionine at position 456; a phenylalanine for the leucine at position 470; a lysine for the serine at position 486; a leucine for the methionine at position 503; an isoleucine for the methionine at position 503; a lysine for the valine at position 552; or a threonine for the alanine at position 570.

### Claim 32

A method of generating a non-human, transgenic animal comprising: introducing a nucleic acid molecule encoding a protein into a cell under conditions sufficient to generate a non-human, transgenic animal; wherein the protein comprises at least 90% sequence identity to SEQ ID NO:2, and comprises at least one of the following amino acid substitutions in SEQ ID NO:2: a serine for the alanine at position 46; a threonine for the alanine at position 46; a proline for the arginine at position 119; an alanine for the cysteine at position 125; a leucine for the cysteine at position 125; a lysine for the tyrosine at position 177; a histidine for the tyrosine at position 177; a leucine for the phenylalanine at position 180; an isoleucine for the phenylalanine at position 180; a valine for the phenylalanine at position 180; a leucine for the methionine at position 185; a glycine for the alanine at position 187; a tryptophan for the phenylalanine at position 200; a proline for the valine at position 207; a phenylalanine for the valine at position 209; a phenylalanine for the methionine at position 226; an arginine for the leucine at position 235; a lysine for the valine at position 240; a leucine for the phenylalanine at position 241; a lysine for the proline at position 243; a glutamine for the methionine at position 282; a tryptophan for the leucine at position 296; a tyrosine for the leucine at position 296; a phenylalanine for the leucine at position 296; a leucine for the methionine at position 298; an alanine for the methionine at position 298; a valine for the methionine at position 298; an isoleucine for the proline at position 311; a valine for the proline at position 311; a lysine for the arginine at position 315; a glycine for the threonine at position 319; an arginine for the tyrosine at position 327; a valine for the tyrosine at position 328; a glycine for the cysteine at position 340; a leucine for the cysteine at position 340; a histidine for the aspartic acid at position 421; an isoleucine for the valine at position 436; a tyrosine for the methionine at position 456; a phenylalanine for the leucine at position 470; a lysine for the serine at position 486; a leucine for the methionine at position 503; an isoleucine for the methionine at position 503; a lysine for the valine at position 552; or a threonine for the alanine at position 570.

### Claim 33 (depends on 32)

The method according to claim 32, wherein the cell is a somatic cell.

### Claim 34 (depends on 32)

The method according to claim 32, wherein the cell is a germ cell.

### Claim 35 (depends on 32)

The method according to claim 32, wherein the step of introducing a nucleic acid molecule encoding a protein into a cell is accomplished by pronuclear injection.

### Claim 36 (depends on 32)

The method according to claim 32, wherein
the cell is an oocyte, embryonic cell, or stem cell; and
wherein the step of introducing a nucleic acid molecule encoding a protein into a cell is accomplished by pronuclear injection.)

### Claim 37

A method of establishing germline transmission of a mutation in a non-human, transgenic animal comprising:
introducing a first and second nucleic acid sequence into an oocyte or fertilized embryo of a non-human, transgenic animal under conditions sufficient to mutate a gene in the oocyte or fertilized embryo; and
transplanting the fertilized embryo into a female non-human animal under conditions sufficient to favor development of at least one offspring carrying the gene;
wherein the first nucleic acid sequence encodes a protein comprising at least 90% sequence identity to SEQ ID NO:2, and comprising at least one of the following amino acid substitutions in SEQ ID NO:2: a serine for the alanine at position 46; a threonine for the alanine at position 46; a proline for the arginine at position 119; an alanine for the cysteine at position 125; a leucine for the cysteine at position 125; a lysine for the tyrosine at position 177; a histidine for the tyrosine at position 177; a leucine for the phenylalanine at position 180; an isoleucine for the phenylalanine at position 180; a valine for the phenylalanine at position 180; a leucine for the methionine at position 185; a glycine for the alanine at position 187; a tryptophan for the phenylalanine at position 200; a proline for the valine at position 207; a phenylalanine for the valine at position 209; a phenylalanine for the methionine at position 226; an arginine for the leucine at position 235; a lysine for the valine at position 240; a leucine for the phenylalanine at position 241; a lysine for the proline at position 243; a glutamine for the methionine at position 282; a tryptophan for the leucine at position 296; a tyrosine for the leucine at position 296; a phenylalanine for the leucine at position 296; a leucine for the methionine at position 298; an alanine for the methionine at position 298; a valine for the methionine at position 298; an isoleucine for the proline at position 311; a valine for the proline at position 311; a lysine for the arginine at position 315; a glycine for the threonine at position 319; an arginine for the tyrosine at position 327; a valine for the tyrosine at position 328; a glycine for the cysteine at position 340; a leucine for the cysteine at position 340; a histidine for the aspartic acid at position 421; an isoleucine for the valine at position 436; a tyrosine for the methionine at position 456; a phenylalanine for the leucine at position 470; a lysine for the serine at position 486; a leucine for the methionine at position 503; an isoleucine for the methionine at position 503; a lysine for the valine at position 552; or a threonine for the alanine at position 570; and
wherein the second nucleic acid sequences encodes a transposon comprising an exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence is flanked by a first inverted repeat sequence comprising a sequence at least about 90% sequence identity to SEQ ID NO:91 and/or a second inverted repeat sequence comprising a sequence at least about 90% sequence identity to SEQ ID NO:92; and
wherein, if the cell is an oocyte, the method further comprises fertilizing the oocyte in vitro prior to the step of transplanting the fertilized embryo into a female non-human animal.

### Claim 38 (depends on 37)

The method of claim 37, wherein the first nucleic acid sequence is an RNA.

### Claim 39 (depends on 37)

The method of claim 37, wherein the first nucleic acid sequence is a DNA.

### Claim 40 (depends on 37)

The method of claim 37, wherein the step of introducing a first and second nucleic acid sequence into an oocyte or fertilized embryo of a non-human, transgenic animal comprises pronuclear injection or transfection.

### Claim 41 (depends on 37)

The method of claim 37, wherein the first nucleic acid sequence encodes a protein comprising at least 99% sequence identity to SEQ ID NO:2.

### Claim 42 (depends on 23)

The method of claim 23, wherein the protein comprises at least 99% sequence identity to SEQ ID NO:2.

### Claim 43 (depends on 31)

The method of claim 31, wherein the protein comprises at least 99% sequence identity to SEQ ID NO:2.

### Claim 44 (depends on 32)

The method of claim 32, wherein the protein comprises at least 99% sequence identity to SEQ ID NO:2.