# US 2019/0169597 A1 · Genome editing enhancers

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- Number: US 2019/0169597 A1
- Document ID: US20190169597A1
- Status: Published application
- Kind: A1
- Application number: 16/325,013
- Filed: 2017-08-18
- Published: 2019-06-06
- Earliest claimed: 2016-08-19
- Assignees: bluebird bio, Inc.
- Applicants: bluebird bio, Inc.
- Inventors: Sasha ASTRAKHAN; Garrett C. HEFFNER
- Web page: https://patentgrep.com/patent/US20190169597A1

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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

## Abstract

The invention provides improved compositions for gene therapy compositions for treatment, prevention, or amelioration of numerous diseases, disorders, and conditions.

## Claims

### Claim 1

A composition comprising a population of cells, a genome editing enhancer, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 2

A composition comprising a population of cells, a genome editing enhancer, a donor repair template, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 3 (depends on 1, 2)

The composition of claim 1 or claim 2, wherein the population of cells comprises stem cells.

### Claim 4 (depends on 1, 2, 3)

The composition of any one of claims 1 to 3, wherein the the population of cells comprises hematopoietic cells.

### Claim 5 (depends on 1, 2, 3, 4)

The composition of any one of claims 1 to 4, wherein the population of cells comprises CD34+ cells, CD133+ cells, CD34+CD133+ cells, or CD34+CD38LoCD90+CD45RA− cells.

### Claim 6 (depends on 1, 2)

The composition of claim 1 or claim 2, wherein the population of cells comprises immune effector cells.

### Claim 7 (depends on 1, 2)

The composition of claim 1 or claim 2, wherein the population of cells comprises CD3+, CD4+, CD8+ cells, or a combination thereof.

### Claim 8 (depends on 1, 2)

The composition of claim 1 or claim 2, wherein population of cells comprises T cells.

### Claim 9 (depends on 1, 2)

The composition of claim 1 or claim 2, wherein the population of cells comprises cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cells.

### Claim 10 (depends on 1, 2)

The composition of claim 1 or claim 2, wherein the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.

### Claim 11

A composition comprising a genome editing enhancer, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 12

A composition comprising a genome editing enhancer, a donor repair template, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The composition of any one of claims 1 to 12, wherein the genome editing enhancer is a DNA intercalator.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The composition of any one of claims 1 to 13, wherein the genome editing enhancer is selected from the group consisting of: a monofunctional DNA intercalator, a bifunctional DNA intercalator, or a polyfunctional DNA intercalator.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The composition of any one of claims 1 to 14, wherein the genome editing enhancer is selected from the group consisting of: acridines, anthracyclines, alkaloids, coumarins, and phenanthridines.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The composition of any one of claims 1 to 15, wherein the genome editing enhancer is selected from the group consisting of: 1,8-naphthalimide, 4′6-diamidino-α-phenylindole, acridines, acridine orange, acriflavine, acronycine, actinodaphnidine, aminacrine, amsacrine, anthracycline, anthramycin, anthrapyrazole, benzophenanthridine alkaloids, berbamine, berberine, berberrubine, bleomycin, BOBO-1, BOBO-3, boldine, BO-PRO-1, BO-PRO-3, bublocapnine, camptothecin, cassythine, chartreusin, chloroquine, chromomycin, cinchonidine, cinchonine, coptisine, coralyne, coumarin, cryptolepine, dactinomycin, DAPI, daunorubicin, dicentrine, dictamine, distamycin, doxorubicin, ellipticine, emetine, ethacridine, ethidium, evolitrine, fagarine, fagaronine, fluorcoumanin, GelStar, gentamicin, glaucine, harmaline, harmine, harmine, hedamycin, hexidium, Hoechst 33258, Hoechst 33342, homidium, hycanthone, imidazoacridinone, an indazole analog, iodide, isocorydine, isoquinoline alkaloids, jatrorrhizine, JOJO-1, JO-PRO-1, kinetin riboside, kokusainine, lobeline, LOLO-1, LO-PRO-1, lucanthone, masculine, matadine, mepacrine, a metallo-intercalator, mithramycin, mitoxantrone, neocryptolepine, netropsin, nitidine, nitracrine, nogalamycin, norharman, OliGreen, palmatine, phenanthridine, PicoGreen, pirarubicin, polypyridyls, POPO-1, POPO-3, PO-PRO-1, PO-PRO-3, proflavine, propidium, psoralen, quinacrine, quinidine, quinine, quinoxalines, RiboGreen, a rhodium based intercalator, a ruthenium based intercalator, sanguinarine, serpentine, skimmianine, streptomycin, SYBR DX, SYBR Gold, SYBR Green I, SYBR Green II, SYTO-11, SYTO-12, SYTO-13, SYTO-14, SYTO-15, SYTO-16, SYTO-17, SYTO-20, SYTO-21, SYTO-22, SYTO-23, SYTO-24, SYTO-25, SYTO-40, SYTO-41, SYTO-42, SYTO-43, SYTO-44, SYTO-45, SYTO-59, SYTO-60, SYTO-61, SYTO-62, SYTO-63, SYTO-64, SYTO-80, SYTO-81, SYTO-82, SYTO-83, SYTO-84, SYTO-85, SYTOX blue, SYTOX green, SYTOX orange, tacrine, thalidomide, thiazole orange, tilorone, TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-3, usambarensine, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3, and analogs and derivatives thereof.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The composition of any one of claims 1 to 16, wherein the genome editing enhancer is selected from the group consisting of: tilorone, aminacrine, homidium bromide (ethidium bromide), harmine, hycanthone, daunorubicin, sanguinarine sulfate, kinetin riboside, ethacridine lactate, and cyclohexamide.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The composition of any one of claims 1 to 17, wherein the genome editing enhancer is selected from the group consisting of: tilorone, aminacrine, homidium bromide (ethidium bromide), and harmine.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The composition of any one of claims 1 to 18, wherein the genome editing enhancer is an acridine or diacridine.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)

The composition of any one of claims 1 to 19, wherein the genome editing enhancer is aminacrine (9-aminoacridine).

### Claim 21

A composition comprising a cell, an acridine, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 22

A composition comprising a cell, an acridine, a donor repair template, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 23

A composition comprising a cell, 9-aminoacridine, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 24

A composition comprising a cell, 9-aminoacridine, a donor repair template, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 25

A composition comprising an acridine, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 26

A composition comprising an acridine, a donor repair template, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 27

A composition comprising 9-aminoacridine, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 28

A composition comprising 9-aminoacridine, a donor repair template, and an engineered nuclease, and/or optionally, an mRNA encoding the engineered nuclease.

### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)

The composition of any one of claims 1-28, wherein the engineered nuclease is selected from the group consisting of: a meganuclease, a megaTAL, a TALEN, a ZFN, or a CRISPR/Cas nuclease.

### Claim 30 (depends on 29)

The composition of claim 29, wherein the meganuclease is engineered from an LAGLIDADG homing endonuclease (LHE) selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

### Claim 31 (depends on 29, 30)

The composition of claim 29 or claim 30, wherein the meganuclease is engineered from an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI.

### Claim 32 (depends on 29, 30, 31)

The composition of any one of claims 29-31, wherein the meganuclease is engineered from an I-OnuI LHE.

### Claim 33 (depends on 29)

The composition of claim 29, wherein the megaTAL comprises a TALE DNA binding domain and an engineered meganuclease.

### Claim 34 (depends on 33)

The composition of claim 33, wherein the TALE binding domain comprises about 9.5 TALE repeat units to about 11.5 TALE repeat units.

### Claim 35 (depends on 33, 34)

The composition of claim 33 or claim 34, wherein the meganuclease is engineered from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

### Claim 36 (depends on 33, 34, 35)

The composition of any one of claims 33-35, wherein the meganuclease is engineered from an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI.

### Claim 37 (depends on 33, 34, 35, 36)

The composition of any one of claims 33-36, wherein the meganuclease is engineered from an I-OnuI LHE.

### Claim 38 (depends on 29)

The composition of claim 29, wherein the TALEN comprises a TALE DNA binding domain and an endonuclease domain or half-domain.

### Claim 39 (depends on 38)

The composition of claim 38, wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 11.5 TALE repeat units.

### Claim 40 (depends on 38, 39)

The composition of claim 38 or claim 39, wherein the endonuclease domain is isolated from a type-II restriction endonuclease.

### Claim 41 (depends on 38, 39, 40)

The composition of any one of claims 38-40, wherein the endonuclease domain is isolated from FokI.

### Claim 42 (depends on 39)

The composition of claim 39, wherein the ZFN comprises a zinc finger DNA binding domain and an endonuclease domain or half-domain.

### Claim 43 (depends on 42)

The composition of claim 42, wherein the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.

### Claim 44 (depends on 42, 43)

The composition of claim 42 or claim 43, wherein the ZFN comprises a TALE binding domain.

### Claim 45 (depends on 44)

The composition of claim 44, wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 11.5 TALE repeat units.

### Claim 46 (depends on 42, 43, 44, 45)

The composition of any one of claims 42-45, wherein the endonuclease domain is isolated from a type-II restriction endonuclease.

### Claim 47 (depends on 42, 43, 44, 45, 46)

The composition of any one of claims 42-46, wherein the endonuclease domain is isolated from FokI.

### Claim 48 (depends on 39)

The composition of claim 39, wherein the engineered nuclease comprises a CRISPR/Cas nuclease.

### Claim 49 (depends on 48)

The composition of claim 48, wherein the Cas nuclease is Cas9 or Cpf1.

### Claim 50 (depends on 48, 49)

The composition of claim 48 or claim 49, wherein the Cas nuclease further comprises one or more TALE DNA binding domains.

### Claim 51 (depends on 48, 49, 50)

The composition of any one of claims 48-50, wherein the composition further comprises a tracrRNA, and one or more crRNAs that target a protospacer sequence in the genome of the cell.

### Claim 52 (depends on 48, 49, 50)

The composition of any one of claims 48-50, wherein the composition further comprises one or more sgRNAs that target a protospacer sequence in the genome of the cell.

### Claim 53 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)

The composition of any one of claims 1 to 52, wherein the engineered nuclease comprises an end-processing enzymatic activity.

### Claim 54 (depends on 53)

The composition of claim 53, wherein the end-processing enzymatic activity is 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase or template-independent DNA polymerases activity.

### Claim 55 (depends on 54)

The composition of claim 54, wherein the end-processing enzymatic activity is 3-5′exonuclease activity of Trex2 or a biologically active fragment thereof.

### Claim 56 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)

The composition of any one of claims 1 to 52, wherein the composition further comprises an end-processing enzyme, or an mRNA encoding the end-processing enzyme.

### Claim 57 (depends on 56)

The composition of claim 56, wherein the end-processing enzyme exhibits 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase or template-independent DNA polymerases activity.

### Claim 58 (depends on 56, 57)

The composition of claim 56 or claim 57, wherein the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.

### Claim 59 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The composition of any one of claims 1 to 58, comprising a donor repair template that encodes: β globin, δ globin, γ globin, BCL11A, KLF1, CCR5, CXCR4, PPP1R12C (AAVS1), HPRT, albumin, Factor VIII, Factor IX, LRRK2, Htt, SOD1, C9orf72, TARDBP, FUS, RHO, CFTR, SFTPB, TRAC, TRBC, PD1, CTLA-4, HLA A, HLA B, HLA C, HLA-DP, HLA-DQ, HLA-DR, LMP7, TAP 1, TAP2, TAPBP, CIITA, DMD, GR, IL2RG, Rag-1, RFX5, FAD2, FAD3, ZP15, KASII, MDH, EPSPS, or a fragment thereof.

### Claim 60 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The composition of any one of claims 1 to 58, comprising a donor repair template that encodes a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-α), a chemokine (e.g., MIP-1α, MIP-1β, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor), or an engineered antigen receptor.

### Claim 61 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The composition of any one of claims 1 to 58, comprising a donor repair template that encodes an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor.

### Claim 62

A method of increasing genome editing in a population of cells comprising:
(a) introducing an engineered nuclease into a population of cells; and
(b) contacting the population of cells with a genome editing enhancer,
wherein expression of the engineered nuclease in the presence of the genome editing enhancer increases the frequency of genome editing in the population of cells.

### Claim 63

A method of increasing homology directed repair (HDR) in a population of cells comprising:
(a) contacting the population of cells with a genome editing enhancer;
(b) introducing an engineered nuclease to generate a double-strand break (DSB) at a target site; and
(b) introducing a donor repair template into the population of cells;
wherein expression of the engineered nuclease in the presence of the genome editing enhancer and the donor repair template increases the frequency of incorporation of the donor repair template at the target site by homology directed repair (HDR).

### Claim 64

A method of increasing non-homologous end joining (NHEJ) in a population of cells comprising:
(a) contacting the population of cells with a genome editing enhancer;
(b) introducing an engineered nuclease to generate a double-strand break (DSB) at a target site;
(a) introducing an engineered nuclease into a population of cells; and
wherein expression of the engineered nuclease in the presence of the genome editing enhancer increases the frequency of NHEJ at the target site.

### Claim 65 (depends on 62, 63, 64)

The method of any one of claims 62-64, wherein the cell is a hematopoietic cell.

### Claim 66 (depends on 62, 63, 64, 65)

The method of any one of claims 62-65, wherein the cell is an immune effector cell.

### Claim 67 (depends on 62, 63, 64, 65, 66)

The method of any one of claims 62-66, wherein the cell is CD3+, CD4+, CD8+, or a combination thereof.

### Claim 68 (depends on 62, 63, 64, 65, 66, 67)

The method of any one of claims 62-67, wherein the cell is a T cell.

### Claim 69 (depends on 62, 63, 64, 65, 66, 67, 68)

The method of any one of claims 62-68, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

### Claim 70 (depends on 62, 63, 64, 65, 66, 67, 68, 69)

The method of any one of claims 62-69, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.

### Claim 71 (depends on 62, 63, 64, 65)

The method of any one of claims 62-65, wherein the cell is a hematopoietic stem cell or hematopoietic progenitor cell.

### Claim 72 (depends on 62, 63, 64, 65)

The method of any one of claims 62-65, wherein the cell is a CD34+ cell.

### Claim 73 (depends on 62, 63, 64, 65)

The method of any one of claims 62-65, wherein the cell is a CD133+ cell.

### Claim 74 (depends on 62, 63, 64, 65)

The method of any one of claims 62-65, wherein the cell is a CD34+CD38LoCD90+CD45RA− cell.

### Claim 75 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74)

The method of any one of claims 62-74, wherein the genome editing enhancer is a DNA intercalator.

### Claim 76 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)

The method of any one of claims 62-75, wherein the genome editing enhancer is selected from the group consisting of: a monofunctional DNA intercalator, a bifunctional DNA intercalator, or a polyfunctional DNA intercalator.

### Claim 77 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76)

The method of any one of claims 62-76, wherein the genome editing enhancer is selected from the group consisting of: acridines, anthracyclines, alkaloids, coumarins, and phenanthridines.

### Claim 78 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77)

The method of any one of claims 62-77, wherein the genome editing enhancer is selected from the group consisting of: 1,8-naphthalimide, 4′6-diamidino-α-phenylindole, acridines, acridine orange, acriflavine, acronycine, actinodaphnidine, aminacrine, amsacrine, anthracycline, anthramycin, anthrapyrazole, benzophenanthridine alkaloids, berbamine, berberine, berberrubine, bleomycin, BOBO-1, BOBO-3, boldine, BO-PRO-1, BO-PRO-3, bublocapnine, camptothecin, cassythine, chartreusin, chloroquine, chromomycin, cinchonidine, cinchonine, coptisine, coralyne, coumarin, cryptolepine, dactinomycin, DAPI, daunorubicin, dicentrine, dictamine, distamycin, doxorubicin, ellipticine, emetine, ethacridine, ethidium, evolitrine, fagarine, fagaronine, fluorcoumanin, GelStar, gentamicin, glaucine, harmaline, harmine, harmine, hedamycin, hexidium, Hoechst 33258, Hoechst 33342, homidium, hycanthone, imidazoacridinone, an indazole analog, iodide, isocorydine, isoquinoline alkaloids, jatrorrhizine, JOJO-1, JO-PRO-1, kinetin riboside, kokusainine, lobeline, LOLO-1, LO-PRO-1, lucanthone, masculine, matadine, mepacrine, a metallo-intercalator, mithramycin, mitoxantrone, neocryptolepine, netropsin, nitidine, nitracrine, nogalamycin, norharman, OliGreen, palmatine, phenanthridine, PicoGreen, pirarubicin, polypyridyls, POPO-1, POPO-3, PO-PRO-1, PO-PRO-3, proflavine, propidium, psoralen, quinacrine, quinidine, quinine, quinoxalines, RiboGreen, a rhodium based intercalator, a ruthenium based intercalator, sanguinarine, serpentine, skimmianine, streptomycin, SYBR DX, SYBR Gold, SYBR Green I, SYBR Green II, SYTO-11, SYTO-12, SYTO-13, SYTO-14, SYTO-15, SYTO-16, SYTO-17, SYTO-20, SYTO-21, SYTO-22, SYTO-23, SYTO-24, SYTO-25, SYTO-40, SYTO-41, SYTO-42, SYTO-43, SYTO-44, SYTO-45, SYTO-59, SYTO-60, SYTO-61, SYTO-62, SYTO-63, SYTO-64, SYTO-80, SYTO-81, SYTO-82, SYTO-83, SYTO-84, SYTO-85, SYTOX blue, SYTOX green, SYTOX orange, tacrine, thalidomide, thiazole orange, tilorone, TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-3, usambarensine, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3, and analogs and derivatives thereof.

### Claim 79 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78)

The method of any one of claims 62-78, wherein the genome editing enhancer is selected from the group consisting of: tilorone, aminacrine, homidium bromide (ethidium bromide), harmine, hycanthone, daunorubicin, sanguinarine sulfate, kinetin riboside, ethacridine lactate, and cyclohexamide.

### Claim 80 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)

The method of any one of claims 62-79, wherein the genome editing enhancer is selected from the group consisting of: tilorone, aminacrine, homidium bromide (ethidium bromide), and harmine.

### Claim 81 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80)

The method of any one of claims 62-80, wherein the genome editing enhancer is an acridine or diacridine.

### Claim 82 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81)

The method of any one of claims 62-81, wherein the genome editing enhancer is aminacrine (9-aminoacridine).

### Claim 83 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The method of any one of claims 62-82, wherein the engineered nuclease is selected from the group consisting of: a meganuclease, a megaTAL, a TALEN, a ZFN, or a CRISPR/Cas nuclease.

### Claim 84 (depends on 83)

The method of claim 83, wherein the meganuclease is engineered from an LAGLIDADG homing endonuclease (LHE) selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

### Claim 85 (depends on 83, 84)

The method of any one of claim 83 or claim 84, wherein the meganuclease is engineered from an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI.

### Claim 86 (depends on 84, 85)

The method of any one of claims 84-85, wherein the meganuclease is engineered from an I-OnuI LHE.

### Claim 87 (depends on 83)

The method of claim 83, wherein the megaTAL comprises a TALE DNA binding domain and an engineered meganuclease.

### Claim 88 (depends on 87)

The method of claim 87, wherein the TALE binding domain comprises about 9.5 TALE repeat units to about 11.5 TALE repeat units.

### Claim 89 (depends on 87, 88)

The method of claim 87 or claim 88, wherein the meganuclease is engineered from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

### Claim 90 (depends on 87, 88, 89)

The method of any one of claims 87-89, wherein the meganuclease is engineered from an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI.

### Claim 91 (depends on 87, 88, 89, 90)

The method of any one of claims 87-90, wherein the meganuclease is engineered from an I-OnuI LHE.

### Claim 92 (depends on 83)

The method of claim 83, wherein the TALEN comprises a TALE DNA binding domain and an endonuclease domain or half-domain.

### Claim 93 (depends on 92)

The method of claim 92, wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 11.5 TALE repeat units.

### Claim 94 (depends on 92, 93)

The method of claim 92-93, wherein the endonuclease domain is isolated from a type-II restriction endonuclease.

### Claim 95 (depends on 92, 93, 94)

The method of any one of claims 92-94, wherein the endonuclease domain is isolated from FokI.

### Claim 96 (depends on 83)

The method of claim 83, wherein the ZFN comprises a zinc finger DNA binding domain and an endonuclease domain or half-domain.

### Claim 97 (depends on 96)

The method of claim 96, wherein the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.

### Claim 98 (depends on 96, 97)

The method of claim 96 or claim 97, wherein the ZFN comprises a TALE binding domain.

### Claim 99 (depends on 98)

The method of claim 98, wherein the TALE DNA binding domain comprises about 9.5 TALE repeat units to about 11.5 TALE repeat units.

### Claim 100 (depends on 96, 97, 98, 99)

The method of any one of claims 96-99, wherein the endonuclease domain is isolated from a type-II restriction endonuclease.

### Claim 101 (depends on 96, 97, 98, 99, 100)

The method of any one of claims 96-100, wherein the endonuclease domain is isolated from FokI.

### Claim 102 (depends on 83)

The method of claim 83, wherein the engineered nuclease comprises a CRISPR/Cas nuclease.

### Claim 103 (depends on 102)

The method of claim 102, wherein the Cas nuclease is Cas9 or Cpf1.

### Claim 104 (depends on 102, 103)

The method of claim 102 or claim 103, wherein the Cas nuclease further comprises one or more TALE DNA binding domains.

### Claim 105 (depends on 102, 103, 104)

The method of any one of claims 102-104, wherein the composition further comprises a tracrRNA, and one or more crRNAs that target a protospacer sequence in the genome of the cell.

### Claim 106 (depends on 102, 103, 104)

The method of any one of claims 102-104, wherein the composition further comprises one or more sgRNAs that target a protospacer sequence in the genome of the cell.

### Claim 107 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106)

The method of any one of claims 62 to 106, wherein the engineered nuclease comprises an end-processing enzymatic activity.

### Claim 108 (depends on 107)

The method of claim 107, wherein the end-processing enzymatic activity is 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase or template-independent DNA polymerases activity.

### Claim 109 (depends on 108)

The method of claim 108, wherein the end-processing enzymatic activity is 3-5′exonuclease activity of Trex2 or a biologically active fragment thereof.

### Claim 110 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106)

The method of any one of claims 62-106, wherein the composition further comprises an end-processing enzyme, or an mRNA encoding the end-processing enzyme.

### Claim 111 (depends on 110)

The method of claim 110, wherein the end-processing enzyme exhibits 5-3′ exonuclease, 5-3′ alkaline exonuclease, 3-5′exonuclease, 5′ flap endonuclease, helicase or template-independent DNA polymerases activity.

### Claim 112 (depends on 110, 111)

The method of claim 110 or claim 111, wherein the end-processing enzyme comprises Trex2 or a biologically active fragment thereof.

### Claim 113 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112)

The method of any one of claims 62 to 112, comprising a donor repair template that encodes: β globin, δ globin, γ globin, BCL11A, KLF1, CCR5, CXCR4, PPP1R12C (AAVS1), HPRT, albumin, Factor VIII, Factor IX, LRRK2, Htt, SOD1, C9orf72, TARDBP, FUS, RHO, CFTR, SFTPB, TRAC, TRBC, PD1, CTLA-4, HLA A, HLA B, HLA C, HLA-DP, HLA-DQ, HLA-DR, LMP7, TAP 1, TAP2, TAPBP, CIITA, DMD, GR, IL2RG, Rag-1, RFX5, FAD2, FAD3, ZP15, KASII, MDH, EPSPS, or a fragment thereof.

### Claim 114 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112)

The method of any one of claims 62 to 112, comprising a donor repair template that encodes: a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-α), a chemokine (e.g., MIP-1α, MIP-1β, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, and an IL-21 receptor), or an engineered antigen receptor.

### Claim 115 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112)

The method of any one of claims 62 to 112, comprising a donor repair template that encodes: an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor.

### Claim 116 (depends on 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115)

A cell produced by a method of any one of claims 62 to 115.

### Claim 117 (depends on 116)

A composition comprising a cell according to claim 116.

### Claim 118 (depends on 116)

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cell according to claim 116.