IP Library Granted Patent US 12,559,742
Granted Patent B2
US 12,559,742 · App. 18/048,631 · Granted Feb 24, 2026

Donor repair templates multiplex genome editing

Inventors: Baeckseung Lee (Kenmore, WA); Alexander Astrakhan (Seattle, WA)
Assignee: Novo Nordisk A/S
C12N15/102A61K40/11A61K40/31A61K40/4215C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2878C12N9/22C12N15/66C12N15/86A61K38/00C07K2317/622C07K2317/76C07K2319/02C07K2319/03C07K2319/30C07K2319/33C12N2740/15043C12N2750/14143C12N2830/50
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Quick Facts
Patent No.
US 12,559,742
App. No.
18/048,631
Granted
Feb 24, 2026
Kind
B2
Abstract

The present disclosure provides improved multiplex genome editing compositions and methods. The disclosure further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of a hemoglobinopathy, a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.

Claims (25)

1 . A method of editing the genome of a cell at multiple target sites by introducing one or more engineered nucleases and a DNA donor repair template, wherein the DNA donor repair template comprises from 5′ to 3′:

(a) a 5′homology arm homologous to a DNA sequence 5′ of a first target site;

(b) a 5′homology arm homologous to a DNA sequence 5′ of a second target site;

(c) a transgene;

(d) a 3 ‘homology arm homologous to a DNA sequence 3’ of the second target site; and

(e) a 3′homology arm homologous to a DNA sequence 3′ of the first target site.

2 . The method of claim 1 , wherein the lengths of the 5′ and 3′ homology arms to the first target site and/or the second target site are independently selected from about 100 bp to about 500 bp.

3 . The method of claim 1 , wherein the first target site and the second target site are in different genes.

4 . The method of claim 1 , wherein the first target site or the second target site comprises an engineered nuclease cleavage site in a TCRα gene or in an immune system checkpoint gene.

5 . The method of claim 4 , wherein the immune system checkpoint gene is selected from the group consisting of: programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR) genes.

6 . The method of claim 1 , wherein the first target site is a first engineered nuclease cleavage site in a TCRα gene and the second target site is a second engineered nuclease cleavage site in an immune system checkpoint gene.

7 . The method of claim 6 , wherein the immune system checkpoint gene is selected from the group consisting of: programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR) genes.

8 . The method of claim 1 , wherein the first target site is a first engineered nuclease cleavage site in a first immune system checkpoint gene and the second target site is a second engineered nuclease cleavage site in a second immune system checkpoint gene.

9 . The method of claim 8 , wherein the first and second immune system checkpoint genes are independently selected from the group consisting of: programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR) genes.

10 . The method of claim 1 , wherein a viral vector comprises the DNA donor repair template of claim 1 .

11 . The viral vector of claim 10 , wherein the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus.

12 . The method of claim 11 , wherein the rAAV has one or more inverted terminal repeats (ITRs) from AAV2.

13 . The method of claim 1 , wherein the cell is a hematopoietic cell.

14 . The method of claim 1 , wherein the cell is a T cell or immune effector cell.

15 . The method of claim 1 , wherein the first target site is an engineered nuclease cleavage site in a TCRα gene and the second target site is an engineered nuclease cleavage site in an immune system checkpoint gene or a gene that encodes an immunosuppressive signaling component.

16 . The method of claim 1 , wherein the first target site is an engineered nuclease cleavage site in a first immune system checkpoint gene or a gene that encodes an immunosuppressive signaling component and the second target site is an engineered nuclease cleavage site in a second immune system checkpoint gene or a gene that encodes an immunosuppressive signaling component.

17 . The method of claim 15 , wherein the gene that encodes an immunosuppressive signaling component is independently selected from the group consisting of: interleukin receptor 10 alpha, transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), aryl hydrocarbon receptor (AHR), serum and glucocorticoid-regulated kinase 1 (SGK1), tuberous sclerosis complex 2 (TSC2), von Hippel-Lindau tumor suppressor (VHL), adenosine A2a receptor (A2AR), and Cbl proto-oncogene B (CBLB).

18 . The method of claim 16 , wherein the gene that encodes an immunosuppressive signaling component is independently selected from the group consisting of: interleukin receptor 10 alpha, transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), aryl hydrocarbon receptor (AHR), serum and glucocorticoid-regulated kinase 1 (SGK1), tuberous sclerosis complex 2 (TSC2), von Hippel-Lindau tumor suppressor (VHL), adenosine A2a receptor (A2AR), and Cbl proto-oncogene B (CBLB).

19 . The method of claim 1 , wherein the transgene comprises a polynucleotide encoding an engineered antigen receptor.

20 . The method of claim 19 , wherein the transgene comprises a polynucleotide encoding a chimeric antigen receptor (CAR).

Assignments (5)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 11, 2024
From: 2SEVENTY BIO, INC.
To: NOVO NORDISK A/S
Reel/Frame 068553/0482 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 16, 2022
From: ASTRAKHAN, ALEXANDER; LEE, BAECKSEUNG
To: BLUEBIRD BIO, INC.
Reel/Frame 062121/0689 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 15, 2022
From: BLUEBIRD BIO, INC.
To: 2SEVENTY BIO, INC.
Reel/Frame 062137/0536 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 17, 2022
From: ASTRAKHAN, ALEXANDER; LEE, BAECKSEUNG
To: BLUEBIRD BIO, INC.
Reel/Frame 061809/0787 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 17, 2022
From: BLUEBIRD BIO, INC.
To: 2SEVENTY BIO, INC.
Reel/Frame 061959/0527 →
Continuity (3)
Continuation 16485345
Provisional Application 62459203 · Feb 15, 2017
Related Publication 20230174967A1 · Jun 8, 2023
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