# US 11,111,483 B2 · Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems and methods

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- Number: US 11,111,483 B2
- Document ID: US11111483B2
- Status: Granted
- Kind: B2
- Application number: 15/844,363
- Filed: 2017-12-15
- Published: 2018-08-02
- Granted: 2021-09-07
- Earliest claimed: 2016-12-16
- Assignees: B-Mogen Biotechnologies, Inc.
- Applicants: B-MoGen Biotechnologies, Inc.
- Inventors: David Largaespada; Branden Moriarity; Beau Webber; Neil Otto; Sandeep Kumar; Leah Hogdal
- Web page: https://patentgrep.com/patent/US11111483B2

## CPC

- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- A61P 35/00: Antineoplastic agents
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C12N 9/1088: {Glutathione transferase (2.5.1.18)}
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Y 207/07: Nucleotidyltransferases (2.7.7)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/23: containing a GST-tag
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

This disclosure provides various TcBuster transposases and transposons, systems, and methods of use.

## Claims

### Claim 1

A fusion transposase comprising a TcBuster transposase amino acid sequence and a DNA sequence specific binding domain, wherein the TcBuster transposase amino acid sequence has at least 70% identity to full-length SEQ ID NO: 1 and an amino acid substitution of D189A, V377T, E469K, K573E, E578L, or any combination thereof, when numbered in accordance with SEQ ID NO: 1.

### Claim 2 (depends on 1)

The fusion transposase of claim 1, wherein the DNA sequence specific binding domain comprises a TALE domain, zinc finger domain, AAV Rep DNA-binding domain, or any combination thereof.

### Claim 3 (depends on 1)

The fusion transposase of claim 1, wherein the DNA sequence specific binding domain comprises a TALE domain.

### Claim 4 (depends on 1)

The fusion transposase of claim 1, wherein the TcBuster transposase amino acid sequence has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to full-length SEQ ID NO: 1.

### Claim 5 (depends on 1)

The fusion transposase of claim 1, wherein the TcBuster transposase amino acid sequence has increased transposition efficiency in comparison to a wild-type TcBuster transposase having amino acid sequence SEQ ID NO: 1 when measured by an assay that comprises: (i) introducing the fusion transposase or the wild-type TcBuster transposase and a TcBuster transposon containing a reporter cargo cassette into a population of cells and (ii) detecting transposition of the reporter cargo cassette in genome of the population of cells induced by the fusion transposase and the wild-type TcBuster transposase, respectively.

### Claim 6 (depends on 1)

The fusion transposase of claim 1, wherein the TcBuster transposase amino acid sequence and the DNA sequence specific binding domain are separated by a linker.

### Claim 7 (depends on 6)

The fusion transposase of claim 6, wherein the linker comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 50 amino acids.

### Claim 8 (depends on 6)

The fusion transposase of claim 6, wherein the linker comprises SEQ ID NO: 9.

### Claim 9 (depends on 1)

The fusion transposase of claim 1, which comprises amino acid substitutions of D189A, V377T, and E469K, when numbered in accordance with SEQ ID NO: 1.

### Claim 10 (depends on 9)

The fusion transposase of claim 9, which further comprises an amino acid substitution K573E and/or E578L, when numbered in accordance with SEQ ID NO: 1.

### Claim 11 (depends on 10)

The fusion transposase of claim 10, which comprises amino acid substitutions of D189A, V377T, E469K, K573E, and E578L when numbered in accordance with SEQ ID NO: 1.

### Claim 12 (depends on 1)

A polynucleotide that codes for the fusion transposase of claim 1.

### Claim 13 (depends on 12)

The polynucleotide of claim 12, wherein the polynucleotide comprises a messenger RNA (mRNA) that encodes the fusion transposase.

### Claim 14 (depends on 13)

The polynucleotide of claim 13, wherein the mRNA encoding the fusion transposase is chemically modified.

### Claim 15

A system for genome editing comprising: a transposon and a fusion transposase or a polynucleotide coding for the fusion transposase, wherein the fusion transposase comprises a TcBuster transposase amino acid sequence and a DNA sequence specific binding domain, wherein the TcBuster transposase amino acid sequence (i) recognizes the transposon, (ii) has at least 70% identity to full-length SEQ ID NO: 1, and (iii) comprises an amino acid substitution of D189A, V377T, E469K, K573E, E578L, or any combination thereof, when numbered in accordance with SEQ ID NO: 1.

### Claim 16 (depends on 15)

The system of claim 15, wherein the transposon is present in a mini-circle plasmid.

### Claim 17 (depends on 15)

The system of claim 15, wherein the transposon comprises a cargo cassette positioned between two inverted repeats.

### Claim 18 (depends on 17)

The system of claim 17, wherein a left inverted repeat of the two inverted repeats comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3, or a right inverted repeat of the two inverted repeats comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4.

### Claim 19 (depends on 17)

The system of claim 17, wherein the cargo cassette is in a reverse direction.

### Claim 20 (depends on 17)

The system of claim 17, wherein the cargo cassette comprises a transgene coding for a cellular receptor selected from the group consisting of: a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), and any combination thereof.

### Claim 21 (depends on 15)

The system of claim 15, wherein the fusion transposase comprises amino acid substitutions of D189A, V377T, and E469K, when numbered in accordance with SEQ ID NO: 1.

### Claim 22 (depends on 21)

The system of claim 21, wherein the fusion transposase further comprises an amino acid substitution K573E and/or E578L, when numbered in accordance with SEQ ID NO: 1.

### Claim 23 (depends on 22)

The system of claim 22, wherein the fusion transposase comprises amino acid substitutions of D189A, V377T, E469K, K573E, and E578L when numbered in accordance with SEQ ID NO: 1.

### Claim 24

A method of genome editing comprising: introducing into a cell a transposon and a fusion transposase or a polynucleotide encoding the fusion transposase, wherein the fusion transposase comprises a TcBuster transposase amino acid sequence and a DNA sequence specific binding domain, wherein the TcBuster transposase amino acid sequence (i) recognizes the transposon, (ii) has at least 70% identity to full-length SEQ ID NO: 1, and (iii) comprises an amino acid substitution of D189A, V377T, E469K, K573E, E578L, or any combination thereof, when numbered in accordance with SEQ ID NO: 1.