# US 11,993,784 B2 · Transposition-based therapies

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- Number: US 11,993,784 B2
- Document ID: US11993784B2
- Status: Granted
- Kind: B2
- Application number: 18/192,382
- Filed: 2023-03-29
- Published: 2023-08-10
- Granted: 2024-05-28
- Earliest claimed: 2020-05-04
- Assignees: Saliogen Therapeutics, Inc.
- Applicants: Saliogen Therapeutics, Inc.
- Inventors: Joseph J. Higgins; Scott McMillan; Ray Tabibiazar
- Web page: https://patentgrep.com/patent/US11993784B2

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/907: {in mammalian cells}
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- 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)
- 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)

## Abstract

Gene therapy compositions and methods are provided that make use of novel transposases and/or chimeric transposases for targeted transposition.

## Claims

### Claim 1

A method for inserting a gene into the genome of a cell, comprising contacting the cell with a composition comprising a nucleic acid encoding a transposase enzyme, wherein the transposase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 and has a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 2,
wherein the transposase enzyme further comprises a substitution C13X2, wherein X2 is selected from lysine (K), arginine (R), and histidine (H).

### Claim 2 (depends on 1)

The method of claim 1, wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

### Claim 3 (depends on 2)

The method of claim 2, wherein the non-polar aliphatic amino acid is alanine (A).

### Claim 4 (depends on 1)

The method of claim 1, wherein the transposase enzyme further comprises a substitution S8X1, wherein X1 is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

### Claim 5 (depends on 4)

The method of claim 4, wherein X1 is proline (P).

### Claim 6 (depends on 1)

The method of claim 1, wherein X2 is arginine (R).

### Claim 7 (depends on 1)

The method of claim 1, wherein the transposase enzyme does not have additional amino acid residues at the C terminus relative to SEQ ID NO: 2.

### Claim 8 (depends on 1)

The method of claim 1, wherein the transposase enzyme comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 2.

### Claim 9 (depends on 1)

The method of claim 1, wherein the nucleic acid further encodes a transcription activator-like effector (TALE) DNA binding domain (DBD), or a nuclease-deficient Cas9 (dCas9) connected to the transposase enzyme.

### Claim 10 (depends on 9)

The method of claim 9, wherein the transposase enzyme is capable of inserting a transposon at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS) of the genome.

### Claim 11 (depends on 10)

The method of claim 10, wherein the TALE DBD or dCas9 is suitable for directing the transposase enzyme to the GSHS sequence.

### Claim 12 (depends on 11)

The method of claim 11, wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, and human Rosa26 locus.

### Claim 13 (depends on 1)

The method of claim 1, wherein the nucleic acid is co-formulated with a nucleic acid encoding a transposon comprising the gene.

### Claim 14 (depends on 13)

The method of claim 13, wherein the composition is in the form of a lipid nanoparticle (LNP).

### Claim 15

A method for inserting a gene into the genome of a cell, comprising contacting the cell with a composition comprising a nucleic acid encoding a transposase enzyme,
wherein the transposase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 and has a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 2, wherein the transposase enzyme further comprises a substitution N125X3, wherein X3 is selected from is selected from lysine (K), arginine (R), and histidine (H).

### Claim 16 (depends on 15)

The method of claim 15, wherein X3 is lysine (K).

### Claim 17 (depends on 15)

The method of claim 15, wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

### Claim 18 (depends on 15)

The method of claim 15, wherein the nucleic acid further encodes a transcription activator-like effector (TALE) DNA binding domain (DBD), or a nuclease-deficient Cas9 (dCas9) connected to the transposase enzyme.

### Claim 19

A method for inserting a gene into the genome of a cell, comprising contacting the cell with a composition comprising a nucleic acid encoding a transposase enzyme,
wherein the transposase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 and has a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 2, wherein the transposase enzyme further comprises two or more of substitutions S8P, C13R, and N125K.

### Claim 20 (depends on 19)

The method of claim 19, wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

### Claim 21 (depends on 20)

The method of claim 20, wherein the nucleic acid further encodes a transcription activator-like effector (TALE) DNA binding domain (DBD), or a nuclease-deficient Cas9 (dCas9) connected to the transposase enzyme.

### Claim 22

A method for inserting a gene into the genome of a cell, comprising contacting the cell with a composition comprising a first nucleic acid encoding a transposase enzyme and a DNA binding domain and a second nucleic acid encoding a transposon,
wherein the transposase enzyme comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 2 and comprises two or more of substitutions of S8P, C13R, and N125K,
wherein the transposase enzyme is operatively linked to the DNA binding domain, and
wherein the first nucleic acid and second nucleic acid are contained in a LNP.

### Claim 23 (depends on 22)

The method of claim 22, wherein the DNA binding domain is a TALE DBD.

### Claim 24 (depends on 22)

The method of claim 22, wherein the DNA binding domain is a dCas9.

### Claim 25 (depends on 23)

The method of claim 23, wherein the transposase enzyme does not have additional amino acid residues at the C terminus relative to SEQ ID NO: 2.

### Claim 26 (depends on 25)

The method of claim 25, wherein the substitutions are S8P and C13R.

### Claim 27 (depends on 24)

The method of claim 24, wherein the transposase enzyme does not have additional amino acid residues at the C terminus relative to SEQ ID NO: 2.

### Claim 28 (depends on 27)

The method of claim 27, wherein the substitutions are S8P and C13R.