# US 2019/0376091 A1 · Primary cell gene editing

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- Number: US 2019/0376091 A1
- Document ID: US20190376091A1
- Status: Published application
- Kind: A1
- Application number: 16/552,714
- Filed: 2019-08-27
- Published: 2019-12-12
- Earliest claimed: 2017-10-30
- Assignees: Pact Pharma, Inc.
- Applicants: Pact Pharma, Inc.
- Inventors: Kyle Jacoby; Alex Franzusoff
- Web page: https://patentgrep.com/patent/US20190376091A1

## CPC

- C12N 15/90: Stable introduction of foreign DNA into chromosome
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/32: T-cell receptors [TCR]
- A61K 40/4201: {Neoantigens}
- A61K 40/4272: {Melan-A/MART}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C12N 5/0636: {T lymphocytes}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/85: for animal cells
- C12N 15/907: {in mammalian cells}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2319/02: containing a signal sequence
- C07K 2319/50: containing protease site
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/315: Phosphorothioates
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- 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)
- 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

Methods and compositions are provided for nuclease-mediated gene editing of primary cells without the use of viral mediated delivery. Methods of treatments using edited primary cells are also provided.

## Claims

### Claim at position 1

-332. (canceled)

### Claim 333

A method of modifying a patient-derived T cell, comprising:
a. introducing into the cell a homologous recombination (HR) template nucleic acid sequence comprising:
i. first and second homology arms homologous to first and second endogenous sequences of the cell;
ii. a T cell receptor (TCR) gene sequence positioned between the first and second homology arms; and
iii. a first 2A-coding sequence positioned upstream of the TCR gene sequence and a second 2A-coding sequence positioned downstream of the TCR gene sequence, wherein the first and second 2A-coding sequences code for the same amino acid sequence but are codon-diverged relative to each other;
b. recombining the HR template nucleic acid into an endogenous locus of the cell comprising the first and second endogenous sequences homologous to the first and second homology arms of the HR template nucleic acid.

### Claim 334 (depends on 333)

The method of claim 333, wherein said recombining comprises:
a. cleavage of the endogenous locus by a nuclease; and
b. recombination of the HR template nucleic acid sequence into the endogenous locus by homology directed repair.

### Claim 335 (depends on 334)

The method of claim 334, wherein the first and second homology arms are each from about 300 bases to about 2,000 bases in length.

### Claim 336 (depends on 334)

The method of claim 334, wherein the HR template nucleic acid sequence further comprises a second TCR gene sequence positioned between the second 2A-coding sequence and the second homology arm.

### Claim 337 (depends on 334)

The method of claim 334, wherein the HR template nucleic acid sequence further comprises a signal sequence positioned between the first 2A-coding sequence and the TCR gene sequence.

### Claim 338 (depends on 336)

The method of claim 336, wherein the HR template nucleic acid sequence further comprises:
a. a first signal sequence positioned between the first 2A-coding sequence and the first TCR gene sequence;
b. a second signal sequence positioned between the second 2A-coding sequence and the second TCR gene sequence;
wherein the first and second signal sequences are codon-diverged relative to each other.

### Claim 339 (depends on 338)

The method of claim 338, wherein the first, the second, or both 2A-coding sequences are immediately downstream of a sequence coding for the amino acid sequence Gly Ser Gly.

### Claim 340 (depends on 339)

The method of claim 339, wherein a nucleic acid sequence coding for a Furin cleavage site is positioned between the TCR gene sequence the second 2A-coding sequence.

### Claim 341 (depends on 334)

The method of claim 334, wherein the HR template nucleic acid sequence further comprises an exogenous sequence of interest.

### Claim 342 (depends on 341)

The method of claim 341, wherein the exogenous sequence of interest codes for a protein useful in autologous cell therapy.

### Claim 343 (depends on 334)

The method of claim 334, wherein the 2A-coding sequence codes for the P2A amino acid sequence.

### Claim 344 (depends on 337)

The method of claim 337, wherein the signal sequence is a human growth hormone signal sequence.

### Claim 345

A modified cell comprising an exogenous nucleic acid sequence incorporated into an endogenous locus comprising:
a. a TCR gene sequence; and
b. a first 2A-coding sequence positioned upstream of the TCR gene sequence and a second 2A-coding sequence positioned downstream of the TCR gene sequence, wherein the first and second 2A-coding sequences code for the same amino acid sequence but are codon-diverged relative to each other.

### Claim 346 (depends on 345)

The modified cell of claim 345, wherein the exogenous nucleic acid sequence further comprises a second TCR gene sequence positioned downstream of the second 2A-coding sequence.

### Claim 347 (depends on 345)

The modified cell of claim 345, wherein the exogenous nucleic acid sequence further comprises a signal sequence position between the first 2A-coding sequence and the TCR gene sequence.

### Claim 348 (depends on 346)

The modified cell of claim 346, wherein the exogenous nucleic acid sequence further comprises:
a. a first signal sequence positioned between the first 2A-coding sequence and the first TCR gene sequence;
b. a second signal sequence positioned between the second 2A-coding sequence and the second TCR gene sequence;
wherein the first and second signal sequences are codon-diverged relative to each other.

### Claim 349 (depends on 345)

The modified cell of claim 345, wherein the first, the second, or both 2A-coding sequences are immediately downstream of a sequence coding for the amino acid sequence Gly Ser Gly.

### Claim 350 (depends on 345)

The modified cell of claim 345, wherein a nucleic acid sequence coding for a Furin cleavage site is positioned between the TCR gene sequence the second 2A-coding sequence.

### Claim 351 (depends on 345)

The modified cell of claim 345, wherein the exogenous nucleic acid sequence further comprises an exogenous sequence of interest coding for a protein useful in autologous cell therapy.

### Claim 352

A method of treating cancer in a human patient comprising:
a. administering to the human patient a therapeutically effective population of modified cells, wherein the cells comprise an exogenous nucleic acid sequence incorporated into an endogenous locus, the exogenous nucleic acid comprising:
i. a TCR gene sequence coding for at least a portion of a TCR capable of binding an antigen present on a cancerous cell, and
ii. a first 2A-coding sequence positioned upstream of the TCR gene sequence and a second 2A-coding sequence positioned downstream of the TCR gene sequence, wherein the first and second 2A-coding sequences are codon-diverged relative to each other, and
b. thereby treating the cancer in the human patient.