DNA-PK inhibitors

Abstract

To provide DNA-PK inhibitors.SOLUTION: The present invention relates to compounds useful as inhibitors of DNA-PK. The invention also provides pharmaceutically acceptable compositions comprising the compounds, and methods of using the compositions in treatment of various diseases, conditions or disorders. The present invention relates to compounds useful as inhibitors of DNA-dependent protein kinase (DNA-PK). The invention also provides pharmaceutically acceptable compositions comprising the compounds of the invention, and methods of using the compositions in treatment of cancer, and for increasing genome editing efficiency by administering a DNA-PK inhibitor and a genome editing system to a cell(s).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. US62 / 618,598, filed January 17, 2018, which is incorporated herein by reference in its entirety. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on January 15, 2019 is named 14390-686Sequence Listing_ST25.txt and is 8KB in size.

[0003] The present invention relates to compounds useful as inhibitors of DNA-dependent protein kinase (DNA-PK). The present invention also provides pharmaceutically acceptable compositions comprising the compounds of the present invention, and methods for using the compositions in the treatment of cancer and for increasing genome editing efficiency by administering a DNA-PK inhibitor and a genome editing system to a cell(s). [Background technology]

[0004] Ionizing radiation (IR) induces various DNA lesions, with double-strand breaks (DSBs) being the most cytotoxic. Unless rapidly and completely repaired, these DSBs can lead to cell death via apoptosis and / or mitotic catastrophe. In addition to IR, certain chemotherapeutic agents, including topoisomerase II inhibitors, bleomycin, and doxorubicin, also cause DSBs. These DNA lesions trigger a complex series of signals via the DNA damage response network that function to repair the damaged DNA and maintain cell survival and genomic stability. In mammalian cells, the primary repair pathway for DSBs is non-homologous end joining (NHEJ). This pathway functions regardless of cell cycle phase and does not require a template to religate broken DNA ends. NHEJ requires the coordination of many proteins and signaling pathways. The core NHEJ machinery consists of the Ku70 / 80 heterodimer and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs or DNA-PK), which together comprise the active DNA-PK enzyme complex. DNA-PKcs is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family of serine / threonine protein kinases, which also includes ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), mTOR, and four PI3K isoforms. However, while DNA-PKcs is in the same protein kinase family as ATM and ATR, these latter kinases function to repair DNA damage via the homologous recombination (HR) pathway and are restricted to the S and G2 phases of the cell cycle. ATM is also recruited to sites of DSBs, whereas ATR is recruited to sites of single-strand DNA breaks.

[0005] NHEJ is thought to proceed through three major steps: recognition of the DSB, DNA processing to remove unligatable ends or other forms of end-related damage, and final ligation of the DNA ends. DSB recognition is achieved by binding of the Ku heterodimer to the broken DNA end, followed by recruitment of two DNA-PKcs molecules to the adjacent side of the DSB. This serves to protect the broken end until additional processing enzymes are recruited. Recent data support the hypothesis that DNA-PKcs phosphorylates the processing enzyme, Artemis, as well as itself, to prepare the DNA end for further processing. In some cases, DNA polymerase may be required to synthesize new ends before the ligation step. Autophosphorylation of DNA-PKcs is thought to induce a conformational change that opens the central DNA-binding cavity, releases DNA-PKcs from the DNA, and facilitates eventual religation of the DNA ends.

[0006] DNA-PK - / - It has been known for some time that mice are hypersensitive to the effects of IR and that some nonselective small-molecule inhibitors of DNA-PKcs can radiosensitize various tumor cell types across a broad range of genetic backgrounds. Inhibition of DNA-PK is expected to radiosensitize normal cells to some extent, but to a lesser extent than tumor cells. This is likely due to the fact that tumor cells possess higher basal levels of intrinsic replication stress and that DNA damage (oncogene-induced replication stress) and DNA repair mechanisms are less efficient in tumor cells. Most importantly, combining DNA-PK inhibitors with recent advances in the precise delivery of focused IR, including image-guided RT (IGRT) and intensity-modulated RT (IMRT), offers an improved therapeutic window with greater sparing of normal tissue.

[0007] Inhibition of DNA-PK activity elicits effects in both cycling and non-cycling cells. This is crucial because the majority of cells in solid tumors are not actively replicating at any given time, limiting the effectiveness of many cell cycle-targeting drugs. Equally intriguing is a recent report suggesting a strong relationship between inhibition of the NHEJ pathway and the ability to kill previously radioresistant cancer stem cells (CSCs). In some tumor cells, DSBs in dormant CSCs have been shown to overwhelmingly activate DNA repair via the NHEJ pathway. CSCs are typically thought to be in a quiescent phase of the cell cycle. This may explain why, despite treatment, half of cancer patients may experience local or distant tumor recurrence, as current strategies are unable to effectively target CSCs. DNA-PK inhibitors may have the potential to sensitize these potentially metastatic progenitor cells to the effects of IR and certain DSB-inducing chemotherapeutic agents.

[0008] Considering the involvement of DNA-PK in DNA repair processes, the application of specific DNA-PK inhibitors acts as an agent to enhance the efficacy of both cancer chemotherapy and radiotherapy. Therefore, it is desirable to develop compounds useful as DNA-PK inhibitors.

[0009] In addition, precise genome targeting techniques are required to enable systematic manipulation of genetic variation. The use of genome editing systems, specifically Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-endonuclease-based genome editing techniques, has grown exponentially in the past few years. The type II CRISPR-Cas9 bacterial innate immune system has emerged as an effective genome editing tool for targeted modification of the human genome (Wiedenheft, B. 2012; Hsu, PDeta. 2014). Recently, the CRISPR-Cpf genome editing system has been described. CRISPR-endonuclease-based genome editing relies, in part, on the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways to repair DNA double-strand breaks. Cellular repair mechanisms favor NHEJ over HDR.

[0010] Although achieving insertions or deletions (indels) from NHEJ is up to 70% effective in some reports, the efficiency of HDR remains challenging, with rates of less than 1%.

[0011] Therefore, there is a need to increase genome editing efficiency, particularly HDR efficiency. Another application of certain DNA-PK inhibitory drugs is to act as agents that enhance the efficacy of genome editing systems. Summary of the Invention [Means for solving the problem]

[0012] The compounds of the present invention, and pharmaceutically acceptable compositions thereof, have been found to be effective as inhibitors of DNA-PK. Accordingly, the present invention provides compounds having the general formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , X, ring A, ring B and ring C are as defined elsewhere herein).

[0013] The present invention also provides pharmaceutical compositions comprising a compound of Formula I and a pharmaceutically acceptable carrier, adjuvant, or vehicle. These compounds and pharmaceutical compositions are useful for treating or lessening the severity of cancer.

[0014] The compounds and compositions provided by the present invention are also useful for the study of DNA-PK in biological and pathological phenomena; for the study of intracellular signal transduction pathways mediated by such kinases; and for the comparative evaluation of novel kinase inhibitors.

[0015] The present invention also enables the use of DNA-PK inhibitors to improve HDR efficiency by inhibiting NHEJ enzymes such as DNA-PK.

[0016] In some embodiments, the disclosure provides a method for editing one or more target genomic regions, comprising administering to one or more cells having one or more target genomic regions a genome editing system and a compound represented by Formula I: [ka] or a pharmaceutically acceptable salt thereof (wherein R 1 , R 2 , X, ring A, ring B and ring C are each independently as defined elsewhere herein.

[0017] In some embodiments, the disclosure also provides a method for repairing DNA breaks in one or more target genomic regions via the homology directed repair (HDR) pathway, the method comprising administering to one or more cells harboring the one or more target genomic regions a genome editing system and a compound represented by Formula I or a pharmaceutically acceptable salt thereof.

[0018] The genome editing system interacts with the nucleic acid(s) of the target genomic region, resulting in a DNA break, which is at least partially repaired via the HDR pathway.

[0019] In some embodiments, the disclosure also provides a method for inhibiting or suppressing repair of DNA breaks in one or more target genomic regions via the NHEJ pathway, the method comprising administering to one or more cells harboring the one or more target genomic regions a genome editing system and a compound represented by Formula I or a pharmaceutically acceptable salt thereof.

[0020] The genome editing system interacts with nucleic acid(s) in one or more target genomic regions, resulting in a DNA break, and repair of the DNA break via the NHEJ pathway is inhibited or suppressed.

[0021] In some embodiments, the disclosure also provides a method of modifying expression of one or more genes or proteins, the method comprising administering to one or more cells comprising one or more target genomic regions a genome editing system and a compound represented by Formula I, or a pharmaceutically acceptable salt thereof.

[0022] The genome editing system interacts with nucleic acid(s) of one or more target genomic regions of a target gene(s) to result in editing of the one or more target genomic regions, which edits alter the expression of downstream gene(s) and / or protein(s) associated with the target gene(s).

[0023] In some embodiments, a kit or composition for editing one or more target genomic regions is provided. In some embodiments, the kit or composition comprises a genome editing system and a compound represented by Formula I or a pharmaceutically acceptable salt thereof.

[0024] Other features, objects, and advantages of the present invention will become apparent from the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 depicts the design of the gene editing assay.

[0026] [Figure 2] FIG. 2 is a graph showing gene editing rates in BECs treated with DNA-PK inhibitors.

[0027] [Figure 3] Figures 3A and 3B are graphs showing gene editing rates after treatment with DNA-PK inhibitors in CD34+ cells from two different donors.

[0028] [Figure 4] FIG. 4 is a graph showing gene editing rates in iPSCs treated with DNA-PK inhibitors.

[0029] [Figure 5] FIG. 5 is a graph showing gene editing rates at the ECmax of DNA-PK inhibitors in BECs.

[0030] [Figure 6] FIG. 6 is a graph showing gene editing rates at EC50 of DNA-PK inhibitors in BECs.

[0031] [Figure 7] Figure 7 is a bar graph showing HDR ratios for gene editing constructs delivered by lipid-mediated transfection in BECs.

[0032] [Figure 8] FIG. 8 is a graph showing gene editing rates in CD34+ cells treated with DNA-PK inhibitors.

[0033] [Figure 9] FIG. 9 is a graph showing gene editing rates in CD34+ cells treated with DNA-PK inhibitors.

[0034] [Figure 10] FIG. 10 is a graph showing gene editing rates in CD34+ cells treated with DNA-PK inhibitors.

[0035] [Figure 11] FIG. 11 depicts the design of a gene editing strategy to perform homology-driven repair (HDR) using CRISPR-Cas9 using an AAV donor.

[0036] [Figure 12] FIG. 12 is a graph showing precise gene editing by HDR mediated by an AAV donor, CRISPR-Cas9, and a selective DNA-PK inhibitor. DETAILED DESCRIPTION OF THE INVENTION

[0037] Definitions and General Terms As used herein, the following definitions shall apply unless otherwise indicated: For purposes of this invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75 th Ed. 1994. Additionally, general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito:1999, and "March's Advanced Organic Chemistry," 5 thEd., Smith, MB and March, J. eds., John Wiley & Sons, New York: 2001. Generally, the nomenclature utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this disclosure. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).

[0038] As described herein, the compounds of the present invention may be optionally substituted with one or more substituents, such as those generally exemplified above or those exemplified for specific classes, subclasses, and species of the present invention. It should be recognized that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." In general, the term "substituted," whether preceded by the term "optionally" or not, refers to the replacement of one or more hydrogen radicals in a given structure with a specified substituent group. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structure can be substituted with more than one substituent selected from the specified group, the substituent may be the same or different at each position.

[0039] As used herein, when the term "optionally substituted" precedes a list, said term refers to all of the subsequent substitutable groups in that list. For example, when X is a halogen, optionally substituted C 1~3 When X is alkyl or phenyl, X may be either an optionally substituted alkyl or an optionally substituted phenyl. Similarly, when the term "optionally substituted" follows a list, said term also refers to all of the substitutable groups in the previous list unless otherwise indicated. For example, when X is halogen, C 1~3 alkyl, or phenyl, and X is J X If replaced as necessary by C 1~3 Alkyl and phenyl are both J X As would be apparent to one skilled in the art, groups such as H, halogen, NO, CN, NH, OH, or OCF are not substitutable groups and are therefore not included. As would also be apparent to one skilled in the art, a heteroaryl or heterocyclic ring containing an NH group may be optionally substituted by replacing the hydrogen atom with a substituent. If a substituent group or structure is not specified or defined as "optionally substituted," the substituent group or structure is unsubstituted.

[0040] Combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that do not change substantially when subjected to conditions that allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, stable or chemically feasible compounds are those that do not change substantially when maintained at or below 40°C in the absence of moisture or other chemically reactive conditions for at least one week.

[0041] The term "alkyl" or "alkyl group," as used herein, means a fully saturated, straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain. Unless otherwise specified, alkyl groups contain 1 to 8 carbon atoms. In some embodiments, alkyl groups contain 1 to 6 carbon atoms, and in still other embodiments, alkyl groups contain 1 to 4 carbon atoms ("C 1~4 In other embodiments, the alkyl group contains a covalent bond or a C 1~4 "C" represents either an alkyl chain 0~4 The term "alkyl" is characterized as an alkyl group. Examples of alkyl groups include methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, and tert-butyl. The term "alkylene," as used herein, represents a saturated divalent straight-chain or branched-chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene, and the like. The term "alkylidene," as used herein, represents a divalent straight-chain alkyl linking group. The term "alkenyl," as used herein, represents a monovalent straight-chain or branched-chain hydrocarbon group containing one or more carbon-carbon double bonds. The term "alkynyl," as used herein, represents a monovalent straight-chain or branched-chain hydrocarbon group containing one or more carbon-carbon triple bonds.

[0042] The term "cycloalkyl" (or "carbocycle") refers to a monocyclic C3-C8 hydrocarbon or bicyclic C8-C8 ring that is fully saturated and has a single point of attachment to the rest of the molecule. 12 refers to a hydrocarbon, wherein any individual ring in said bicyclic ring system has from 3 to 7 members. Suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0043] The terms "heterocycle," "heterocyclyl," "heterocycloalkyl," or "heterocyclic," as used herein, refer to a monocyclic, bicyclic, or tricyclic ring system in which at least one ring in the system contains one or more heteroatoms, either the same or different, and which is fully saturated or contains one or more units of unsaturation, but is not aromatic and has a single point of attachment to the rest of the molecule. In some embodiments, a "heterocycle," "heterocyclyl," "heterocycloalkyl," or "heterocyclic" group has 3 to 14 ring members, one or more of which are heteroatoms independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 8 ring members.

[0044] Examples of heterocyclic rings include, but are not limited to, the following monocyclic rings: 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl. nyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl; and the following bicycles: 3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one, indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and 1,3-dihydro-imidazol-2-one.

[0045] The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, or phosphorus, including any oxidized form of nitrogen, sulfur, or phosphorus; the quaternized form of any basic nitrogen; or a substitutable nitrogen of a heterocyclic ring, such as N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (in the case of N-substituted pyrrolidinyl).

[0046] The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0047] The term "alkoxy" or "thioalkyl," as used herein, refers to an alkyl group, as previously defined, attached to the main carbon chain through an oxygen ("alkoxy") or sulfur ("thioalkyl") atom.

[0048] The terms "haloalkyl," "haloalkenyl," and "haloalkoxy" mean alkyl, alkenyl, or alkoxy, as the case may be, substituted with one or more halogen atoms. The term "halogen" means F, Cl, Br, or I.

[0049] The term "aryl," used alone or as part of a larger moiety such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of 6 to 14 ring members, said ring system having a single point of attachment to the rest of the molecule, at least one ring in the system being aromatic, and each ring in the system containing 4 to 7 ring members. The term "aryl" can be used interchangeably with the term "aryl ring." Examples of aryl rings include phenyl, naphthyl, and anthracene.

[0050] The term "heteroaryl," used alone or as part of a larger moiety such as "heteroaralkyl" or "heteroarylalkoxy," refers to monocyclic, bicyclic, and tricyclic ring systems having a total of 5 to 14 ring members, said ring systems having a single point of attachment to the rest of the molecule, at least one ring in the system being aromatic, at least one ring in the system containing one or more heteroatoms independently selected from nitrogen, oxygen, sulfur, or phosphorus, and each ring in the system containing 4 to 7 ring members. The term "heteroaryl" can be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic."

[0051] Further examples of heteroaryl rings include the following monocyclic rings: 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and 5-triazolyl). , 2-thienyl, 3-thienyl, pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyrazinyl, 1,3,5-triazinyl, and the following bicyclic rings: benzimidazolyl, benzofuryl, benzothiophenyl, indolyl (e.g., 2-indolyl), purinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl).

[0052] As described herein, a bond drawn from a substituent to the center of one of the rings in a polycyclic ring system (as shown below) represents substitution of the substituent at any substitutable position on any of the rings in the polycyclic ring system. For example, structure a represents possible substitution at any of the positions shown in structure b. [ka]

[0053] This also applies to polycyclic ring systems (which would be represented by dotted lines) fused to an optional ring system. For example, in structure c, X is an optional substituent for both ring A and ring B. [ka]

[0054] However, when two rings in a polycyclic ring system each have different substituents drawn from the center of each ring, unless otherwise specified, each substituent represents only the substitution on the ring to which it is attached. For example, in structure d, Y is an optional substituent on ring A only, and X is an optional substituent on ring B only. [ka]

[0055] The term "protecting group," as used herein, refers to such groups intended to protect functional groups, such as alcohols, amines, carboxyls, carbonyls, and the like, from adverse reactions during synthetic procedures. Commonly used protecting groups are described in Greene and Wuts, Protective Groups In Organic Synthesis, 3, incorporated herein by reference. rdEdition (John Wiley & Sons, New York, 1999). Examples of nitrogen protecting groups include acyl, aroyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries, such as protected or unprotected D, L, or D,L-amino acids, e.g., alanine, leucine, phenylalanine, etc.; sulfonyl groups, for example, benzenesulfonyl, p-toluenesulfonyl, etc.; carbamate groups, for example, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-di Methoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methyl methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc.; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl, etc.; and silyl groups such as trimethylsilyl, etc.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz). Examples of hydroxyl-protecting groups include ethers such as tetrahydropyranyl, tertbutyl, benzyl, and allyl; silyl ethers such as trimethylsilyl, triethylsilyl, triisopropylsilyl, and tert-butyldiphenylsilyl; esters such as acetyl and trifluoroacetyl; and carbonates. Hydroxyl-protecting groups also include those suitable for protecting phenols.

[0056] Unless otherwise shown or stated, structures recited herein are intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of a structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Generally, the hatched lines ( [ka] ) or bold ( [ka] Compounds drawn with defined stereochemical centers using the (R) bond are stereochemically pure, but the absolute stereochemistry is still not defined. Such compounds can have either the R or S configuration. When the absolute configuration has been determined, the chiral center(s) are labeled in the diagram as (R) or (S).

[0057] Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, a hydrogen is replaced by deuterium or tritium, or a carbon is replaced by 13 C or 14 Compounds having the present structures except for the replacement of C-rich carbons are within the scope of the invention. Such compounds are useful, for example, as analytical tools, probes in biological assays, or as DNA-PK inhibitors with improved therapeutic profiles. Compound Description

[0058] In one aspect, the present invention provides a compound of formula (III-E-1) or (III-E-2): [ka] or a pharmaceutically acceptable salt thereof.

[0059] X is O or NR, and R is H or C1-C4 alkyl.

[0060] Y is O or NR, and R is H or C1-C4 alkyl.

[0061] R 3 is hydrogen, C 1~4 Alkyl, or OC 1~2 It is alkyl.

[0062] R 1 is a 6-membered aromatic heterocycle containing one or two nitrogen atoms, which aromatic heterocycle is selected from the group consisting of halo, CN, C1-C4-alkyl, C1-C4-haloalkyl, C3-C4-cycloalkyl, OR 6 , C(=O)OR 6 , C(=O)NR 7 R 6 , and N.R. 4 R 50, 1, 2 or 3 substituents R independently selected from the group consisting of 2 may be substituted with.

[0063] Each C1-C4-alkyl and C1-C4-haloalkyl may be 0, 1 or 2 OR 6 is substituted with a group.

[0064] Each R 6 and R 7 are independently H, C1-C4 alkyl or C1-C4-haloalkyl.

[0065] Each R 4 and R 5 are independently H, C1-C4 alkyl, or C(=O)C1-C4 alkyl.

[0066] R 4 and R 5 together with the N atom to which they are attached form a heterocyclic ring containing 0 or 1 additional O or N atoms, said heterocyclic ring being C1-C4 alkyl or OR 6 may be substituted with.

[0067] Ring B is [ka] and W is selected from the group consisting of N or CR 7 and Z is O or S, and R 7 is H or C1-C4 alkyl.

[0068] In another embodiment, R 1 is a 6-membered aromatic heterocycle containing one or two nitrogen atoms, and the aromatic heterocycle is selected from the group consisting of C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 6 , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 may be substituted with.

[0069] R 6 is C1-C4-alkyl.

[0070] Each R 4 and R 5 are independently H, C1-C4 alkyl, or C(=O)C1-C4 alkyl.

[0071] R 4 and R 5 together with the N atom to which they are attached form a heterocyclic ring containing 0 or 1 additional N atom, said heterocyclic ring being optionally substituted with C1-C4-alkyl.

[0072] Ring B is [ka]

[0073] and W is selected from the group consisting of N or CR 7 and Z is O or S, and R 7 is H or C1-C4-alkyl.

[0074] In one embodiment, the compound has the formula [ka] It has.

[0075] In one embodiment, X is O.

[0076] In another embodiment, X is NH.

[0077] In one embodiment, Y is O.

[0078] In another embodiment, Y is NH.

[0079] In a further embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen.

[0080] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 2’ , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 is a pyrimidine ring substituted with

[0081] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 2’ , and N.R. 4 R 5 one substituent R selected from the group consisting of 2 is a pyrimidine ring substituted with

[0082] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl and C(=O)NHR 2’ one substituent R selected from the group consisting of 2 is a pyrimidine ring substituted with

[0083] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is one substituent R selected from the group consisting of C1-C2-alkyl and C(=O)NHC1-C2-alkyl 2 is a pyrimidine ring substituted with

[0084] In another embodiment, X is O, Y is NH, and R 3 is hydrogen and R 1 is one substituent R selected from the group consisting of C1-C2-alkyl and C(=O)NHC1-C2-alkyl 2is a pyrimidine ring substituted with

[0085] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 6 , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is selected from the group consisting of N or CR 3 and Z is O or S, and R 3 is H or C1-C4 alkyl.

[0086] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 6 , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is N or CR 3 and Z is O or S, and R 3 is H or C1-C4 alkyl.

[0087] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 6 , and N.R. 4 R 50, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is N and Z is O or S.

[0088] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl and C(=O)NHR 6 0, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is N and Z is O or S.

[0089] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is 0, 1, or 2 substituents R independently selected from the group consisting of C1-C2-alkyl and C(=O)NHC1-C2-alkyl 2 and ring B is a pyrimidine ring substituted with [ka] and W is N and Z is O or S.

[0090] In another embodiment, the compound has the formula [ka] It has.

[0091] In another embodiment, X is NH.

[0092] In one embodiment, Y is O.

[0093] In another embodiment, Y is NH.

[0094] In a further embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen.

[0095] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 6 , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 is a pyrimidine ring substituted with

[0096] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 6 , and N.R. 4 R 5 one substituent R selected from the group consisting of 2 is a pyrimidine ring substituted with

[0097] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl and C(=O)NHR 6 one substituent R selected from the group consisting of 2 is a pyrimidine ring substituted with

[0098] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is one substituent R selected from the group consisting of C1-C2-alkyl and C(=O)NHC1-C2-alkyl 2is a pyrimidine ring substituted with

[0099] In another embodiment, X is O, Y is NH, and R 3 is hydrogen and R 1 is one substituent R selected from the group consisting of C1-C2-alkyl and C(=O)NHC1-C2-alkyl 2 is a pyrimidine ring substituted with

[0100] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 2’ , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is selected from the group consisting of N or CR 3 and Z is O or S, and R 3 is H or C1-C4 alkyl.

[0101] In another embodiment, X is O or NH, Y is O or NH, R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 6 , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is N or CR 3 and Z is O or S, and R 3 is H or C1-C4 alkyl.

[0102] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 2’ , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is N and Z is O or S.

[0103] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is C1-C4-alkyl and C(=O)NHR 6 0, 1, or 2 substituents R independently selected from the group consisting of 2 and ring B is a pyrimidine ring substituted with [ka] and W is N and Z is O or S.

[0104] In another embodiment, X is O or NH, Y is O or NH, and R 3 is hydrogen and R 1 is 0, 1, or 2 substituents R independently selected from the group consisting of C1-C2-alkyl and C(=O)NHC1-C2-alkyl 2 and ring B is a pyrimidine ring substituted with [ka] ; and W is N and Z is O or S.

[0105] In one embodiment of the compound having formula (III-E-1) or (III-E-2), [ka] teeth, [ka] is.

[0106] In another embodiment, [ka] teeth, [ka] is. In another embodiment, the invention features a compound selected from the group of compounds listed in Table 1.

[0107] In another embodiment, the invention features a compound selected from the group of compounds listed in Table 2. Compositions, Formulations, and Administration of Compounds

[0108] In another embodiment, the invention provides a pharmaceutical composition comprising a compound of any of the formulae described herein and a pharmaceutically acceptable excipient. In a further embodiment, the invention provides a pharmaceutical composition comprising a compound of Table 1. In a further embodiment, the composition further comprises an additional therapeutic agent.

[0109] According to another embodiment, the present invention provides a composition comprising a compound of the present invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In one embodiment, the amount of the compound in the composition of the present invention is such that it is effective to moderately inhibit DNA-PK in a biological sample or in a patient. In another embodiment, the amount of the compound in the composition of the present invention is such that it is effective to moderately inhibit DNA-PK. In one embodiment, the composition of the present invention is formulated for administration to a patient in need of such a composition. In a further embodiment, the composition of the present invention is formulated for oral administration to a patient.

[0110] The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0111] The term "agent" is used herein to refer to a chemical compound, a small molecule, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0112] As used herein, "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement or effect on one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, the composition may be administered to a subject who is at risk of developing a particular disease, condition, or symptom, even if the disease, condition, or symptom has not yet manifested, or to a subject who reports one or more physiological symptoms of the disease. These terms also refer to the treatment of disease in a mammal, e.g., a human, where treatment includes: (a) inhibiting the disease, i.e., arresting or preventing its onset; (b) relieving the disease, i.e., causing regression of the disease state; or (c) curing the disease.

[0113] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug sufficient to produce beneficial or desired results. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., but can be easily determined by those skilled in the art. This term also applies to a dose that will obtain an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of the specific drug selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system that carries it.

[0114] As used herein, "administering" refers to contacting, injecting, dispensing, delivering, or applying a genome editing system and / or a DNA-PK inhibitor to a cell or a subject. In some embodiments, administering contacts the genome editing system and / or the DNA-PK inhibitor with a cell(s). In some embodiments, administering delivers the genome editing system and / or the DNA-PK inhibitor to a cell(s). In some embodiments, administering applies the genome editing system and / or the DNA-PK inhibitor to a cell(s). In some embodiments, administering injects the genome editing system and / or the DNA-PK inhibitor into a cell(s). Administering can occur in vivo, ex vivo, or in vitro. Administering the genome editing system and the DNA-PK inhibitor to a cell(s) can occur simultaneously or sequentially.

[0115] As used herein, the term "acquired," in reference to a condition or disease, means a disorder or medical condition that develops after the fetal stage, as opposed to a congenital disorder that is present at birth. A congenital disorder may precede an acquired disorder.

[0116] The term "congenital" or "genetic" condition or disease refers to a genetic disorder found in a subject's genome that is present in the subject at birth. "Genome," as used herein, includes all genetic material in the nucleus and cytoplasm, and further includes the mitochondrial genome and the ribosomal genome. Congenital or genetic conditions can be manifested at any time during a subject's life, for example, at birth or in adulthood.

[0117] The term "genetic disorder" or "genetic disease" includes inherited or acquired mutations in a subject's genome that cause or can cause disease.

[0118] The term "polymorphism" or "genetic variation" refers to different forms of a gene at a genetic locus.

[0119] It should also be appreciated that certain compounds of the present invention can be present in free form for treatment, or, where appropriate, in the form of their pharmaceutically acceptable derivatives. In accordance with the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of such esters, or any other adducts or derivatives, which, upon administration to a patient in need thereof, can directly or indirectly provide a compound as otherwise described herein, or a metabolite or residue thereof. As used herein, the term "inhibitorily active metabolite or residue thereof" means that a metabolite or residue thereof is also an inhibitor of DNA-PK.

[0120] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like.

[0121] Pharmaceutically acceptable salts are well known in the art.For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66, 1-19, 1977, which is incorporated herein by reference.The pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and inorganic and organic bases.The example of pharmaceutically acceptable non-toxic acid addition salts is the salt of amino group formed with inorganic acid, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acid, for example, acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxyethanesulfonate. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N-methyl-N ... + (C 1~4The present invention also contemplates the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Such quaternization may result in water or oil-soluble or dispersible products. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include, where appropriate, counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, C 1~8 Included are salts formed by sulfonate ions and arylsulfonate ions with non-toxic ammonium cations, quaternary ammonium cations, and amine cations.

[0122] As noted above, pharmaceutically acceptable compositions of the present invention further comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, which, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. See Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. DB, the contents of each of which are incorporated herein by reference. Troy, Lippincott Williams & Wilkins, Philadelphia and Encyclopedia of Pharmaceutical Technology, eds, J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, disclose various carriers used in formulating pharmaceutically acceptable compositions and known techniques for their preparation. Except in the case where any conventional carrier medium is incompatible with the compounds of the present invention, for example, by producing any adverse biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is considered to be within the scope of the present invention.

[0123] Some examples of substances that can function as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate, cellulose acetate esters ... Cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer, and other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0124] The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral," as used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraocular, intrahepatic, intralesional, epidural, intraspinal, and intracerebral injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media.

[0125] For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful for preparing injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween®, Span, and other emulsifiers or bioavailability enhancers commonly used in the production of pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0126] The pharmaceutically acceptable composition of the present invention can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or liquids.For tablets for oral use, commonly used carriers include lactose and corn starch.Lubricants, such as magnesium stearate, are also usually added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are required for oral use, active ingredients are combined with emulsifiers and suspending agents.If desired, certain sweeteners, flavorings or colorings can be added.

[0127] Alternatively, the pharmaceutically acceptable composition of the present invention can be administered in the form of suppositories for rectal administration.These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycol.

[0128] The pharmaceutically acceptable compositions of the invention may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0129] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.

[0130] For topical application, pharmaceutically acceptable compositions can be formulated into a suitable ointment, which contains the active ingredient suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of the present invention include but are not limited to mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.Alternatively, pharmaceutically acceptable compositions can also be formulated into a suitable lotion or cream, which contains the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include but are not limited to mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0131] For ophthalmic use, pharmaceutically acceptable compositions can be formulated, for example, as finely divided suspensions in isotonic, pH-adjusted, sterile saline or other aqueous solutions, or, preferably, as solutions in isotonic, pH-adjusted, sterile saline or other aqueous solutions, with or without preservatives such as benzylalkonium chloride. Alternatively, for ophthalmic use, pharmaceutically acceptable compositions can be formulated in an ointment such as petrolatum. The pharmaceutically acceptable compositions of the present invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can also be formulated as solutions in saline, utilizing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0132] Most preferably, the pharmaceutically acceptable compositions of this invention are formulated for oral administration.

[0133] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active compounds, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and their mixtures.In addition to inert diluents, oral compositions can also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

[0134] Injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents.Sterile injectable preparations can also be injectable sterile solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol.Among acceptable vehicles and solvents, water, Ringer's solution, USP, and isotonic sodium chloride solution can be used.In addition, sterile, fixed oils are commonly used as solvents or suspending media.For this purpose, any bland, fixed oil, including synthetic monoglycerides or diglycerides, can be used.In addition, fatty acids such as oleic acid are used in the preparation of injectable preparations.

[0135] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0136] In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility. Thus, the rate of absorption of the compound depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, dissolving or suspending the compound in an oil vehicle achieves delayed absorption of parenterally administered compound forms. Injectable depot forms are produced by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0137] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing a compound of the invention with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

[0138] The solid dosage form for oral administration includes capsules, tablets, pills, powders and granules.In such solid dosage form, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) filler or extender, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) moisturizer, such as glycerol, d) disintegrant, such as agar, calcium carbonate In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0139] Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. These can optionally contain opacifying agents, and can also have compositions that release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0140] The active compound may also be in microencapsulated form with one or more excipients as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. These dosage forms may also contain, as is customary, additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents, and may have compositions that release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0141] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers, as needed. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present invention. The present invention also contemplates the use of transdermal patches, which offer the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the appropriate medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0142] The compounds of the present invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, the term "unit dosage form" refers to a physically discrete unit of drug appropriate for the patient to be treated. However, it should be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific composition being used; the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific compound being used; the duration of treatment; drugs used in combination or simultaneously with the specific compound being used, and similar factors well known in the medical arts.

[0143] The amount of the compounds of the invention that can be combined with a carrier material to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration, etc. Preferably, the compositions should be formulated so that a dosage of between 0.01-100 mg / kg body weight / day of the inhibitor can be administered to a patient receiving these compositions.

[0144] Depending on the particular proliferative condition or cancer to be treated, additional therapeutic agents that are normally administered to treat or prevent that condition may also be present in the compositions of the invention. As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular proliferative condition or cancer are known as "appropriate for the disease or condition being treated." Examples of additional therapeutic agents are provided below.

[0145] The amount of additional therapeutic agent present in the compositions of the invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably, the amount of additional therapeutic agent in the compositions disclosed herein will be in the range of about 50% to 100% of the amount that would normally be present in a composition comprising that agent as the only therapeutically active agent. Uses of the Compounds and Compositions

[0146] In some embodiments, provided herein are methods of sensitizing cells to therapeutic agents or disease conditions that induce DNA damage, comprising contacting cells with one or more DNA-PK inhibitors disclosed herein, e.g., a DNA-PK inhibitor of formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof.

[0147] In some embodiments, provided herein is a method for enhancing a therapeutic regimen for the treatment of cancer, comprising administering to an individual in need thereof an effective amount of a DNA-PK inhibitor disclosed herein, e.g., a DNA-PK inhibitor of formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof. In one aspect, the therapeutic regimen for the treatment of cancer includes radiation therapy.

[0148] The DNA-PK inhibitors disclosed herein are useful when radiation therapy is used to enhance the therapeutic benefit of such treatment.In addition, radiation therapy is often used as an adjuvant to surgery in the treatment of cancer.In the adjuvant setting, the goal of radiation therapy is to reduce the risk of recurrence and enhance disease-free survival when the primary tumor is controlled.Adjuvant radiation therapy is used for several diseases, including colon, rectum, lung, gastroesophageal, and breast cancer, as described below.

[0149] In some embodiments, the DNA-PK inhibitor disclosed herein and other anti-cancer chemotherapy agents are used in a therapeutic regimen for cancer treatment with or without radiation therapy.The combination of the DNA-PK inhibitor disclosed herein with such other agents can enhance chemotherapy protocols.For example, the DNA-PK inhibitor disclosed herein can be administered with etoposide or bleomycin, which are known agents that cause DNA strand breaks.

[0150] In some embodiments, further disclosed are methods for radiosensitizing tumor cells using the DNA-PK inhibitor compounds disclosed herein. As used herein, a DNA-PK inhibitor capable of "radiosensitizing" a cell is defined as a molecule, preferably a low-molecular-weight molecule, that is administered to an animal in a therapeutically effective amount to increase the sensitivity of the cell to electromagnetic radiation and / or facilitate the treatment of diseases treatable with electromagnetic radiation (e.g., X-rays). Diseases treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancerous cells.

[0151] Also provided herein, in some embodiments, is a method for treating cancer in an animal, comprising administering to the animal an effective amount of a DNA-PK disclosed herein. Further provided herein, in some embodiments, is a method for inhibiting cancer cell growth, including the processes of cell proliferation, invasion, and metastasis, in a biological system. Such a method includes the use of a DNA-PK inhibitor disclosed herein as an inhibitor of cancer cell growth. In certain embodiments, the method is used to inhibit or reduce cancer cell growth, invasion, metastasis, or tumor incidence in a living animal, such as a mammal. The DNA-PK inhibitors disclosed herein can be used alone or in combination with IR or one or more chemotherapeutic agents in treating cancer or inhibiting cancer cell growth. In some embodiments, such methods are adaptable for use in assay systems, for example, for assaying cancer cell growth and its characteristics, and for identifying compounds that affect cancer cell growth.

[0152] A tumor or neoplasm comprises the growth of tissue cells, where the proliferation of the cells is uncontrolled and progressive. Some such growths are benign, while others are termed "malignant" and can result in the death of the organism. Malignant neoplasms or "cancers" are distinguished from benign growths in that, in addition to exhibiting aggressive cell proliferation, they are capable of invading and metastasizing into surrounding tissues. Furthermore, malignant neoplasms are characterized by exhibiting greater differentiation and loss of tissue (greater "dedifferentiation") relative to each other and to their surrounding tissues. This property is also referred to as "anaplasia."

[0153] Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas. Carcinomas include malignant neoplasms derived from epithelial cells that infiltrate (invade) surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue or tissues that form recognizable glandular structures. Another broad category of cancer includes sarcomas, which are tumors whose cells are embedded in a homogeneous substance such as fibrillar or embryonic connective tissue. The DNA-PK inhibitors disclosed herein can also enable the treatment of myeloid or lymphatic cancers, including leukemia, lymphoma, and other cancers that do not usually present as a tumor mass but are distributed within the vascular or lymphoreticular tissue systems.

[0154] DNA-PK activity is recognized in, for example, adult and pediatric oncology, solid tumor / malignant tumor growth, mucinous and round cell carcinoma, locally advanced tumors, metastatic cancer, human soft tissue sarcomas including Ewing's sarcoma, cancer metastasis including lymphoid metastasis, squamous cell carcinoma, especially of the head and neck, esophageal squamous cell carcinoma, oral cancer, blood cell malignancies including multiple myeloma, acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia. Leukemias, including chronic myeloid leukemia, and hairy cell leukemia; effusion lymphoma (cavity-based lymphoma); thymic lymphoma, including small cell lung cancer; cutaneous T-cell lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; cancer of the adrenal cortex; ACTH-producing tumors; breast cancer, including non-small cell carcinoma, small cell carcinoma, and ductal carcinoma; gastrointestinal cancers, including stomach cancer, colon cancer, and colorectal cancer; polyps associated with colorectal neoplasia; pancreatic cancer , liver cancer, primary superficial bladder tumors, invasive transitional cell carcinoma of the bladder, and muscle-invasive bladder cancer, urinary tract cancer including bladder cancer including prostate cancer, ovarian cancer, primary peritoneal epithelial neoplasms, malignancies of the female reproductive tract including cervical cancer, endometrial cancer, vaginal cancer, cancer of the vulva, uterine cancer and solid tumors within the ovary, malignancies of the male reproductive tract including testicular cancer and penile cancer, kidney cancer including renal cell carcinoma, brain cancer including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, glioma, metastatic tumor cell invasion in the central nervous system, bone cancer including osteoma and osteosarcoma, malignant melanoma, skin cancer including tumor progression of human skin keratinocytes, squamous cell carcinoma, thyroid cancer, retinoblastoma, neuroblastoma, ascites, malignant pleural effusion, mesothelioma, Wilms' tumor, gallbladder cancer, trophoblastic tumor, hemangiopericytoma, and Kaposi's sarcoma. Methods for enhancing the treatment of these and other forms of cancer are also disclosed herein.

[0155] Also provided herein is a method for inhibiting DNA-PK activity in a biological sample, comprising contacting the biological sample with a compound or composition of the present invention. The term "biological sample," as used herein, refers to a sample outside of a living organism, including, without limitation, cell cultures or extracts thereof; biopsy material or extracts thereof obtained from mammals; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof. Inhibition of kinase activity, particularly DNA-PK activity, in a biological sample is useful for various purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, biological specimen storage and biological assays. In one embodiment, the method for inhibiting DNA-PK activity in a biological sample is limited to non-therapeutic methods.

[0156] In some embodiments, the present disclosure provides methods, compositions and kits for editing target genomes, for example, by correcting mutations. Such methods, compositions and kits can increase genome editing efficiency by using DNA-PK inhibitors.

[0157] Genome editing systems can, for example, stimulate or induce DNA break(s) at DSB(s) at a desired locus (or target genomic region) within the genome. The generation of the DNA break prompts cellular enzymes to repair the break site via either the error-prone NHEJ pathway or the error-free HDR pathway. In NHEJ, the DNA lesion is repaired by fusing the two ends of the DNA break in a series of enzymatic processes involving the Ku70 / 80 heterodimer and the DNA-dependent protein kinase (DNA-PK) enzyme. The repair mechanism involves tethering and alignment of the two DNA ends, excision, extension, and ligation (Rouet et al.; Dexheimer T. DNA repair pathways and mechanisms. In: Mathews L, Cabarcas S, Hurt E, editors. DNA repair of cancer stem cells. Dordrecht: Springer; 2013. pp. 19-32.), which forms small insertion or deletion mutations (indels) at the break site. Indels introduced into the coding sequence of a gene can cause either premature stop codons or frame-shift mutations, resulting in the production of nonfunctional, truncated proteins. The mechanism of the HDR pathway is poorly understood and involves a different set of repair proteins, such as Rad51, that stimulate strand invasion by donor repair templates for base insertion or gene replacement. Therefore, HDR allows the introduction of exogenous DNA templates and the desired DNA editing results within the genome, making it a powerful strategy for translational disease modeling and therapeutic genome editing to restore gene function.

[0158] Of the two DNA repair pathways, NHEJ occurs much more frequently and has been reported to be able to achieve efficiencies greater than 70%, even in neurons (Swiech et al., "In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9”, Nat Biotechnol. 2015 Jan; 33(1):102-6 (2014). However, gene correction by HDR occurs at very low frequency during the S and G2 phases, when DNA replication is complete and sister chromatids are available to serve as repair templates (Heyer et al., Regulation of homologous recombination in eukaryotes. Annual Review of Genetics 44:113-139, 2010). Because NHEJ occurs throughout the cell cycle and competes with and is favored over HDR during the S and G2 phases, targeted insertion via the HDR pathway remains challenging and is the focus of ongoing research.

[0159] DNA protein kinase (DNA-PK) plays a role in various DNA repair processes. DNA-PK is involved in DNA double-strand break repair through activation of the non-homologous end joining (NHEJ) pathway. NHEJ is thought to proceed through three steps: recognition of the DSB, DNA processing to remove unligatable ends or other forms of end damage, and finally ligation of the DNA ends. DSB recognition is achieved by binding of a Ku heterodimer to the broken DNA end, followed by recruitment of two molecules of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the adjacent side of the DSB; this serves to protect the broken end until additional processing enzymes are recruited. Recent data support the hypothesis that DNA-PKcs phosphorylates the processing enzyme, Artemis, as well as itself, to prepare the DNA end for further processing. In some cases, DNA polymerase may be required to synthesize new ends before the ligation step. Autophosphorylation of DNA-PKcs is thought to induce a conformational change that opens the central DNA-binding cavity, releases DNA-PKcs from DNA, and facilitates eventual religation of the DNA ends.

[0160] In some embodiments, the present disclosure provides methods, compositions and kits for enhancing gene editing in genome editing systems, including repair by CRISPR-based HDR in cells, particularly for increasing the repair efficiency of DNA break(s) via HDR pathway, or the efficiency of inhibiting or suppressing the repair of DNA break(s) via NHEJ pathway.Without being bound by any particular theory, it is believed that the genome editing system administered to cell(s) interacts with the nucleic acid(s) of target gene, causing or causing DNA break; such DNA breaks are repaired by some repair pathways, for example, HDR, and the DNA-PK inhibitor administered to cell(s) can inhibit, block or suppress NHEJ repair pathway, and increase or promote the frequency or efficiency of the DNA repair pathway by HDR.

[0161] The interaction between the genome editing system and the nucleic acid(s) of the target gene can be hybridization of at least a portion of the genome editing system with the nucleic acid(s) of the target gene, or any other recognition of the nucleic acid(s) of the target gene by the genome editing system. In some embodiments, such an interaction is a protein-DNA interaction or hybridization between base pairs.

[0162] In some embodiments, the present disclosure provides a method for editing one or more target genomic regions in a cell(s) by administering a genome editing system and a DNA-PK inhibitor to the cell(s). Editing can be performed simultaneously or sequentially. Editing of one or more target genomic regions includes any type of genetic manipulation or manipulation of the cellular genome. In some embodiments, editing of one or more target genomic regions can include insertion, deletion, or replacement of a genomic region in a cell(s). A genomic region includes genetic material in a cell(s), such as DNA, RNA, polynucleotides, and oligonucleotides. A genomic region in a cell(s) also includes the genomes of mitochondria or chloroplasts contained in the cell(s).

[0163] In some embodiments, the insertion, deletion, or substitution may occur within a coding or non-coding genomic region, within an intron or exon region, or any combination thereof, including overlapping or non-overlapping segments thereof. As used herein, "non-coding region" refers to a genomic region that does not encode an amino acid sequence. For example, a non-coding region includes introns. A coding region refers to a genomic region that encodes an amino acid sequence. For example, a coding region includes exons.

[0164] In some embodiments, the editing of one or more target genomic regions can occur within any one or more target regions within the genome of a cell(s). In some embodiments, the editing of one or more target genomic regions can occur in, for example, exons, introns, transcription start sites, promoter regions, enhancer regions, silencer regions, insulator regions, anti-repressors, post-translational regulatory elements, polyadenylation signals (e.g., minimal polyA), conserved regions, transcription factor binding sites, or any combination thereof.

[0165] In some embodiments, administering a DNA-PK inhibitor and a genome editing system to a cell(s) results in an increase in targeted genome editing efficiency compared to when a DNA-PK inhibitor and a genome editing system are not administered to a cell(s). In some embodiments, the increase in editing efficiency is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or 100-fold compared to when a DNA-PK inhibitor and a genome editing system are not administered to a cell(s), or when only a genome editing system is administered to a cell(s) without a DNA-PK inhibitor. Genome editing efficiency can be measured by any method known in the art, for example, by determining the frequency of targeted polynucleotide integration or by measuring the frequency of targeted mutation introduction. Targeted polynucleotide integration can also result in sequence alteration or replacement within a region of interest in a genome, chromosome, or cellular chromatin. Targeted polynucleotide integration can result in targeted mutations including, but not limited to, point mutations (i.e., conversion of a single base pair to a different base pair), substitutions (i.e., conversion of multiple base pairs to different sequences of the same length), insertions of one or more base pairs, deletions of one or more base pairs, and any combination of the above sequence changes.

[0166] In some embodiments, the method of editing one or more target genomic regions in a cell(s) comprises administering to the cell(s) a genome editing system and a DNA-PK inhibitor. In some embodiments, the cell(s) are synchronized to the S or G2 cell cycle phase. Synchronization of a cell(s) in the S or G2 cell cycle phase can be achieved by any method known in the art. By way of non-limiting example, agents that can be used to synchronize a cell(s) to the S or G2 cell cycle phase include aphidicolin, hydroxyurea, lovastatin, mimosine, nocodazole, thymidine, or any combination thereof. (Lin et al. "Enhanced homology-directed human genome engineering by timing of CRISPR / Cas9 delivery,” Elife. 2014 Dec 15;3). In some embodiments, the agent for cell synchronization can be administered at any time during the gene editing process. In some embodiments, the cell(s) can be synchronized to the S or G2 phase of the cell cycle before, during, or after administering the genome editing system and / or DNA-PK inhibitor to the cell(s).

[0167] In some embodiments, the method of editing one or more target genomic regions in a cell(s) by administering to the cell(s) a genome editing system and a DNA-PK inhibitor results in increased cell survival compared to conditions where the genome editing system and DNA-PK inhibitor were not administered to the cell(s), or compared to conditions where only the gene editing system is contacted or administered to the cell(s) but not the DNA-PK inhibitor.

[0168] In some embodiments, provided herein are methods for repairing DNA breaks in one or more target genomic regions via HDR pathways. Administering a genome editing system and a DNA-PK inhibitor to a cell(s) results in DNA breaks in the targeted region of the genome, and the DNA breaks are then at least partially repaired by HDR pathways. These methods result in an increased amount of HDR-mediated repair (e.g., HDR pathway) in one or more target genomic regions, resulting in HDR-mediated repair at a higher efficiency compared to when a DNA-PK inhibitor and a genome editing system are not administered to a cell(s). In some embodiments, the efficiency of HDR pathway-mediated repair of DNA breaks is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or 100-fold higher than when a DNA-PK inhibitor and a genome editing system are not administered to a cell(s), or when only a genome editing system is administered to a cell(s) without a DNA-PK inhibitor. The efficiency of HDR pathway-mediated repair can be measured by any method known in the art, for example, by determining the frequency of targeted polynucleotide integration or by measuring the frequency of targeted mutagenesis.

[0169] In some embodiments, the methods herein provide for repair of DNA breaks by increasing the efficiency of the HDR pathway.

[0170] HDR pathways can be "canonical" or "alternative." "HDR" (homologous recombination repair) refers to a specialized form of DNA repair that occurs, for example, during repair of double-strand breaks or DNA nicks in a cell(s). HDR of double-strand breaks is generally based on nucleotide sequence homology and uses a "donor" molecule as a template for repair of a "target" molecule (e.g., one that has suffered a double-strand break), which can result in the transfer of genetic information from the donor to the target. Canonical HDR of double-strand breaks is generally based on BRCA2 and RAD51 and typically utilizes a dsDNA donor molecule. Non-canonical, or "alternative," HDR is an HDR mechanism that is suppressed by BRCA2, RAD51, and / or functionally related genes. Alternative HDR can also use ssDNA or nicked dsDNA donor molecules. See, for example, WO2014172458.

[0171] In some embodiments, the method of repairing DNA breaks in one or more target genomic regions via the HDR pathway by administering to a cell(s) a genome editing system and a DNA-PK inhibitor results in increased cell survival compared to a condition in which the genome editing system and the DNA-PK inhibitor are not administered to the cell(s), or compared to a condition in which only the gene editing system and not the DNA-PK inhibitor is administered to the cell(s).

[0172] In some embodiments, provided herein is a method for inhibiting or suppressing NHEJ-mediated repair of DNA breaks in one or more target genomic regions in a cell(s). In some embodiments, inhibiting or suppressing NHEJ-mediated repair of DNA breaks is carried out by inhibiting or suppressing the NHEJ pathway. The NHEJ pathway can be either classical ("standard") or alternative NHEJ pathway (alt-NHEJ, or microhomology-mediated end joining (MMEJ)). The NHEJ pathway or alt-NHEJ pathway is suppressed in a cell(s) by administering a genome editing system and a DNA-PK inhibitor to the cell(s).

[0173] The classical NHEJ repair pathway is a DNA double-strand break repair pathway in which the ends of the double-strand break are ligated without extensive homology. Classical NHEJ repair utilizes several factors, including the KU70 / 80 heterodimer (KU), XRCC4, ligase IV, and DNA protein kinase catalytic subunit (DNA-PKcs). Alt-NHEJ is an alternative pathway for repairing double-strand breaks. Alt-NHEJ utilizes microhomologous sequences of 5–25 base pairs during alignment of the break ends before joining them. Alt-NHEJ is largely independent of the KU70 / 80 heterodimer (KU), XRCC4, ligase IV, DNA protein kinase catalytic subunit (DNA-PKcs), RAD52, and ERCC1. See Bennardo et al., "Alternative-NHEJ is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair," PLOS Genetics, June 27, 2008.

[0174] In some embodiments, the method of inhibiting or suppressing NHEJ-mediated repair of a DNA break via the NHEJ pathway in one or more target genomic regions in a cell(s) by inhibiting or suppressing the NHEJ pathway via administration of a genome editing system and a DNA-PK inhibitor to a cell(s) results in increased efficiency of inhibition or suppression of NHEJ-mediated repair of the DNA break compared to a cell(s) that has not received the genome editing system and the DNA-PK inhibitor, or compared to a condition in which the cell(s) receive the genome editing system but not the DNA-PK inhibitor. In some embodiments, the increase in the efficiency of inhibiting or suppressing repair of DNA breaks via the NHEJ pathway by contacting a cell(s) with a DNA-PK inhibitor and a genome editing system is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or 100-fold compared to the condition in which a DNA-PK inhibitor and a genome editing system are not administered to a cell(s), or compared to the condition in which only a genome editing system is administered to a cell(s) without a DNA-PK inhibitor. The efficiency of inhibiting or suppressing repair of DNA breaks via the NHEJ pathway can be measured by any method known in the art, for example, by determining the frequency of targeted polynucleotide integration or by measuring the frequency of targeted mutation introduction.

[0175] In some embodiments, the method of inhibiting or suppressing NHEJ-mediated repair of DNA breaks in one or more target genomic regions in a cell(s) by inhibiting or suppressing the NHEJ pathway via administration of a genome editing system and a DNA-PK inhibitor to a cell(s) results in increased cell viability compared to conditions in which the genome editing system and the DNA-PK inhibitor are not contacted or administered to the cell(s), or compared to conditions in which only the gene editing system is contacted or administered to the cell(s) and not the DNA-PK inhibitor.

[0176] The DNA break can be a double-strand break (DSB) or two single-strand breaks (e.g., two DNA nicks). The DSB can be blunt-ended or have either a 5' or 3' overhang; if the strands are broken far apart, the overhangs continue to anneal to each other and present as two nicks rather than a single DSB.

[0177] In some embodiments, provided herein are methods of modifying the expression of one or more genes (target gene(s)) and / or corresponding or downstream proteins by administering to a cell(s) a genome editing system and a DNA-PK inhibitor. In some embodiments, the genome editing system creates, for example, an insertion, deletion, replacement, modification, or disruption in a target genomic region(s) of the target gene(s) in the cell(s), resulting in modified expression of the target gene(s). In some embodiments, the insertion, deletion, replacement, modification, or disruption can result in targeted expression of a particular protein, or group of proteins, or downstream protein. In some embodiments, the genome editing system can create an insertion, deletion, or replacement within a non-coding region or a coding region. In some embodiments, the genome editing system can create insertions, deletions, replacements, modifications, or disruptions in promoter regions, enhancer regions, and / or any other gene regulatory elements, including exons, introns, transcription start sites, silencer regions, insulator regions, anti-repressors, post-translational regulatory elements, polyadenylation signals (e.g., minimal polyA), conserved regions, transcription factor binding sites, or any combination thereof. In some embodiments, the genome editing system can create insertions, deletions, replacements, modifications, or disruptions in more than one target region simultaneously or sequentially. In some embodiments, administering a genome editing system and a DNA-PK inhibitor to a cell(s) can enable targeted modification of gene expression in a cell(s). Such targeted modification of gene expression can result in the expression of a specific protein and its downstream proteins.

[0178] In some embodiments, expression of the downstream gene and / or protein is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or 10-fold compared to conditions in which a DNA-PK inhibitor and a genome editing system are not administered to a cell(s), or compared to conditions in which only a genome editing system but not a DNA-PK inhibitor is administered to a cell(s).

[0179] In some embodiments, gene expression of the downstream gene and / or protein is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% compared to a condition in which a DNA-PK inhibitor and a genome editing system are not administered to a cell(s), or compared to a condition in which only a genome editing system but not a DNA-PK inhibitor is administered to a cell(s).

[0180] The cell of the methods herein can be any cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the vertebrate cell is a mammalian cell. In some embodiments, the vertebrate cell is a human cell.

[0181] The cell can be any type of cell at any developmental stage. In some embodiments, the cell can be a differentiated cell, a totipotent stem cell, a pluripotent stem cell, an embryonic stem cell, an embryonic germ cell, an adult stem cell, a progenitor cell, an induced pluripotent stem cell, or any combination thereof. Differentiated cells are specialized cells that perform specific functions within a tissue. Totipotent stem cells are undifferentiated cells derived from an embryo, fetus, or adult that can divide over a long period of time and have the potential to differentiate into any cell type in any of the organism's three germ layers. Pluripotent stem cells are undifferentiated cells derived from an embryo, fetus, or adult that can divide over a long period of time and have the potential to differentiate into any cell type in the organism except extraembryonic tissues or the placenta. Embryonic stem cells are undifferentiated stem cells found in the inner cell mass of an embryo and have the potential to differentiate into any cell type in any of the three germ layers. Embryonic germ cells are embryonic cells that can give rise to germ cells such as sperm cells or egg cells. Adult stem cells are undifferentiated cells found in differentiated tissues, capable of self-renewal and differentiation into any of the cells of the tissue in which they reside. Precursor or progenitor cells are partially differentiated cells that can usually only differentiate into one type of cell (e.g., unipotent cells). Induced pluripotent stem cells are a type of pluripotent stem cell that can be generated from differentiated or partially differentiated cells of adults. See, for example, WO / 2010 / 017562.

[0182] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof. For example, "one or more cells" and "cell(s)" are used interchangeably herein. Similarly, "one or more target genomic regions" and "target genomic region(s)" are used interchangeably herein.

[0183] The terms "approximately" and "about" are used interchangeably herein. When applied to one or more values ​​of interest, the term "approximately" or "about" refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value (greater or less), unless otherwise stated or otherwise clear from the context (except when such number exceeds 100% of possible values).

[0184] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. These terms refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can also be further modified after polymerization, such as by conjugation with a labeling component. The term "ssDNA" refers to a single-stranded DNA molecule. The term "ssODN" refers to a single-stranded oligodeoxynucleotide.

[0185] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides having modified or substituted sugars, etc. The term "oligonucleotide linkage" referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroseleroate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoronmidate, etc. If desired, the oligonucleotide can include a detectable label.

[0186] The term "synthetic RNA" refers to RNA that is engineered or does not occur in nature.

[0187] As used herein, the term "wild-type" is a term of art understood by those skilled in the art and means the typical form of an organism, strain, gene or characteristic as found in nature, as distinguished from mutant or variant forms.

[0188] The terms "non-naturally occurring" and "engineered" are used interchangeably and indicate the involvement of the hand of man. When referring to a nucleic acid molecule or polypeptide, this term means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is naturally associated and found in nature.

[0189] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid, either by conventional Watson-Crick or other non-conventional methods. The percentage of complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all adjacent residues of a nucleic acid sequence will hydrogen bond with the same number of adjacent residues in a second nucleic acid sequence. "Substantially complementary," as used herein, refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0190] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0191] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling moiety. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both D- and L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0192] A "viral vector" is defined as a recombinantly produced virus or viral particle containing a polynucleotide that is delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors, adenoviral vectors, lentiviral vectors, herpes simplex virus vectors, and chimeric virus vectors. In some embodiments where gene transfer is mediated by a retroviral vector, the vector construct refers to a polynucleotide that includes the genome of a retrovirus or a portion thereof.

[0193] Some embodiments of the present disclosure relate to a vector system comprising one or more vectors, or to the vector itself.Vector can be designed for the expression of CRISPR transcript (for example, nucleic acid transcript, protein or enzyme) in prokaryotic or eukaryotic cells.For example, CRISPR transcript can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vector), yeast cells or mammalian cells.

[0194] Cells may be primary cells, induced pluripotent stem cells (iPSCs), embryonic stem cells (hESCs), adult stem cells, progenitor cells, or cell lines. "Primary cells" are cells obtained directly from living tissue and placed and grown in vitro. Primary cells rarely undergo population doublings and have a finite lifespan relative to population doublings in vitro. "Stem cells," "embryonic stem cells," and "induced pluripotent stem cells" are unspecialized, undifferentiated cells that are capable of self-renewal and have the potential to differentiate into different cell types with specialized functions. "Cell lines" include cell cultures derived from one cell type or a set of cells of the same type that can proliferate indefinitely. Non-limiting examples of mammalian cell lines include CD34 cells, 293 cells, HEK cells, CHO cells, BHK cells, CV-1 cells, Jurkat cells, HeLa cells, or any variants thereof.

[0195] In some embodiments, the vector can be used to drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 and pMT2PC. When used in mammalian cells, the control functions of the expression vector are usually provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. Other promoters can include, for example, the EF1 promoter or the EF1 alpha promoter. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, See Chapters 16 and 17 of Cold Spring Harbor, NY, 1989.

[0196] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment of a biotinyl moiety to a polypeptide that is detectable by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected optically or calorimetrically). In certain circumstances, the label or marker may be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "drug or drug," as used herein, refers to a chemical compound or composition capable of eliciting a desired therapeutic effect when appropriately administered to a patient.

[0197] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., on a molar basis, more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition in which the target species constitutes at least about 50 percent (on a molar basis) of all macromolecular species present.

[0198] Generally, a substantially pure composition comprises greater than about 80 percent of all macromolecular species present in the composition. In some embodiments, a substantially pure composition comprises greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity (contaminating species are not detectable in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species. Genome editing system

[0199] Various types of genome manipulation systems can be used. Terms such as "genome editing system," "gene editing system," and the like are used interchangeably herein to refer to systems or technologies for editing target genes or their function or expression. A genome editing system includes: at least one endonuclease component that enables cleavage of target genomic region(s) (or target sequence(s)); and at least one genome targeting element that delivers or targets the endonuclease component to the target genomic region(s). Examples of genome targeting elements include DNA-binding domains (e.g., zinc finger DNA-binding proteins or TALE DNA-binding domains), guide RNA elements (e.g., CRISPR guide RNAs), and guide DNA elements (e.g., NgAgo guide DNAs). Programmable genome targeting and endonuclease elements enable precise genome editing by introducing DNA breaks, such as double-strand breaks (DSBs), at specific genomic loci. The DSB subsequently recruits endogenous repair mechanisms for either non-homologous end joining (NHEJ) or homology-directed repair (HDR) to the DSB site to mediate genome editing. An "endonuclease component" includes an endonuclease or a nucleic acid containing a nucleotide sequence(s) encoding such an endonuclease.

[0200] The term "endonuclease" refers to any wild-type, mutant, variant, or engineered enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within DNA or RNA molecules. Endonucleases can recognize and cleave DNA or RNA molecules at their target genomic region. Examples of endonucleases include homing endonucleases; restriction enzymes, such as FokI; chimeric zinc finger nucleases (ZFNs) generated by fusing an engineered zinc finger domain with the catalytic domain of a restriction enzyme, such as FokI; Cas enzymes, and Cpf enzymes. Chemical endonucleases in which a chemical or peptide cleaving agent is conjugated to either a polymer of nucleic acid that recognizes a specific target sequence or to another DNA, thereby targeting the cleavage activity to a specific sequence, are included in the term "endonuclease." Examples of chemical endonucleases include synthetic nucleases such as conjugates of orthophenanthroline, DNA cleaving molecules, and triplex-forming oligonucleotides (TFOs).

[0201] By "variant" is intended a recombinant protein obtained by replacing at least one residue in the amino acid sequence of a parent protein with a different amino acid.

[0202] In some embodiments, the endonuclease, for example, ZFN, TALEN and / or meganuclease, comprises a cleavage domain and / or a cleavage half-domain.The cleavage domain can be homologous or heterologous to the DNA binding domain.For example, a zinc finger DNA binding domain and a cleavage domain derived from a nuclease, or a meganuclease DNA binding domain and a cleavage domain derived from a different nuclease, can be used.A heterologous cleavage domain can be obtained from any endonuclease or exonuclease.Exemplary endonucleases from which the cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases.See, for example, WO2013 / 130824. Additional enzymes that cleave DNA are known (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

[0203] The cleavage half-domains can be derived from any nuclease or portion thereof, as described above, that requires dimerization for cleavage activity. In some embodiments, when a fusion protein contains a cleavage half-domain, two fusion proteins are required for cleavage. In some embodiments, a single protein containing two cleavage half-domains can be used. In some embodiments, the two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof). In some embodiments, each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof). In addition, the target sites for the two fusion proteins are preferably positioned relative to each other such that binding of the two fusion proteins to their respective target sites positions the cleavage half-domains in a spatial orientation relative to each other that allows for the formation of a functional cleavage domain, e.g., by dimerization. Thus, in certain embodiments, the proximal ends of the target sites are separated by 5-50 nucleotides, 5-8 nucleotides, or 15-18 nucleotides. It is noted that any integral number of nucleotides or nucleotide pairs can intervene between the two target sites (e.g., 2 to 50 nucleotide pairs or more). In some embodiments, the cleavage site is located between the target sites.

[0204] Restriction endonucleases (restriction enzymes) exist in many species and are capable of sequence-specific binding (at recognition sites) to DNA and cleaving the DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at sites excluded from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme FokI catalyzes double-stranded cleavage of DNA. See, e.g., U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31, 978-31, 982.

[0205] In some embodiments, the endonuclease component comprises a fusion protein(s) containing a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains (engineered or unengineered). An exemplary type IIS restriction enzyme whose cleavage domain is separable from the binding domain is FokI. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575. The portion of the FokI enzyme used in such fusion proteins is considered the cleavage half-domain. Thus, two fusion proteins, each containing a FokI cleavage half-domain, can be used to reconstitute a catalytically active cleavage domain for targeted double-strand cleavage and / or targeted replacement of cellular sequences using zinc finger fusions or TALE-FokI fusions. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can be used.

[0206] Exemplary type IIS restriction enzymes are described in International Publication No. WO 07 / 014275, which is incorporated herein in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and these are contemplated in this disclosure. See, e.g., Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

[0207] In certain embodiments, the cleavage domain comprises one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in U.S. Patent Publication Nos. 20050064474 and 20060188987 and WO2013 / 130824. Exemplary engineered cleavage half-domains of FokI that form essential heterodimers include pairs in which the first cleavage half-domain comprises mutations of amino acid residues at positions 490 and 538 of FokI, and the second cleavage half-domain comprises mutations at amino acid residues 486 and 499. See, e.g., U.S. Patent Application Publication Nos. 2008 / 0131962 and 2011 / 0201055. The engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis (FokI) of the wild-type cleavage half-domain as described in U.S. Patent Publication Nos. 20050064474 and 20080131962.

[0208] The terms "edit", "edits", "editing" and the like refer to any kind of manipulation, alteration, modification or modulation (in each case including but not limited to by means of gene knockout, gene tagging, gene disruption, gene mutation, gene insertion, gene deletion, gene activation, gene silencing or gene knock-in).

[0209] As used herein, "genetic modification," "genome editing," "genome modification," "genetic modification," and "gene editing" refer to the addition, deletion, knockout, knockin, tagging, mutation, activation, silencing, modification, and / or disruption of any gene to the nucleotides of a cell. In this context, the cell may be in vitro, in vivo, or ex vivo.

[0210] By "target genomic region," "target gene," "DNA target," "DNA target sequence," "target sequence," "target nucleotide sequence," "target site," "target," "site of interest," "recognition site," "polynucleotide recognition site," "recognition sequence," and "cleavage site" is intended a polynucleotide sequence that is recognized and cleaved by a genome editing system. These terms refer to a distinct DNA location, preferably a genomic location, at which a DNA break (cleavage) will be induced by the genome editing system.

[0211] The above-mentioned editing, including manipulation, modification, alteration and modulation, can occur simultaneously or sequentially.Can use any genome editing system known in the art.In some embodiments, genome editing system is meganuclease-based system, zinc finger nuclease (ZFN)-based system, transcription activator-like effector-based nuclease (TALEN)-based system, CRISPR-based system or NgAgo-based system.

[0212] Meganuclease-based, ZFN-based, and TALEN-based systems each include a nucleic acid containing at least one DNA-binding domain or a nucleic acid sequence(s) encoding the DNA-binding domain, and achieve specific targeting or recognition of a target genomic region(s) through protein-DNA interactions. CRISPR-based systems include a nucleic acid containing at least one guide RNA element or a nucleic acid sequence(s) encoding the guide RNA element, and achieve specific targeting or recognition of a target genomic region(s) through direct base pairing with the DNA of the target genomic region(s). NgAgo-based systems include a nucleic acid containing at least one guide DNA element or a nucleic acid sequence(s) encoding the guide DNA element, and achieve specific targeting or recognition of a target genomic region(s) through direct base pairing with the DNA of the target genomic region(s).

[0213] In some embodiments, the genome editing system is a meganuclease-based system. Meganuclease-based systems utilize meganucleases, which are endonucleases with large (>14 bp) recognition sites, whose DNA-binding domains are also responsible for cleaving the target sequence. The meganuclease DNA-binding domain can have a double-stranded DNA target sequence of 12-45 bp. In some embodiments, meganucleases are either dimeric enzymes in which each meganuclease domain is on a monomer, or monomeric enzymes containing the two domains on a single polypeptide. In addition to wild-type meganucleases, various meganuclease variants have been generated by protein engineering to encompass countless unique sequence combinations. In some embodiments, chimeric meganucleases with recognition sites composed of half sites from meganuclease A and half sites from protein B can also be used. Specific examples of such chimeric meganucleases include the protein domains of I-DmoI and I-CreI. Examples of meganucleases include homing endonucleases from the LAGLIDADG family.

[0214] LAGLIDADG meganucleases include I-SceI, I-ChuI, I-CreI, I-CsmI, PI-SceI, PI-TliI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, PI-CivI, PI-C trI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI, PI-MflI, PI-MgaI, PI-MgoI, PI-MinI, PI-MkaI, PI-MleI, PI- The LAGLIDADG meganuclease may be MmaI, PI-MshI, PI-MsmI, PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI, PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, or I-MsoI, or a functional mutant or variant thereof, whether homodimeric, heterodimeric, or monomeric. In some embodiments, the LAGLIDADG meganuclease is an I-CreI derivative. In some embodiments, the LAGLIDADG meganuclease shares at least 80% similarity with the naturally occurring I-CreI LAGLIDADG meganuclease. In some embodiments, the LAGLIDADG meganuclease shares at least 80% similarity with residues 1-152 of naturally occurring I-CreI LAGLIDADG meganuclease. In some embodiments, the LAGLIDADG meganuclease may consist of two monomers that share at least 80% similarity with residues 1-152 of naturally occurring I-CreI LAGLIDADG meganuclease and are linked together with or without a linker peptide.

[0215]

[0216] "LAGLIDADG meganuclease" refers to a homing endonuclease from the LAGLIDADG family, as defined in Stoddard et al. (Stoddard, 2005), or an engineered variant comprising a polypeptide that shares at least 80%, 85%, 90%, 95%, 97.5%, 99% or more identity or similarity with said naturally occurring homing endonuclease. Such engineered LAGLIDADG meganucleases can be derived from monomeric or dimeric meganucleases. When derived from dimeric meganucleases, such engineered LAGLIDADG meganucleases can be single-chain or dimeric endonucleases.

[0217] "I-CreI" refers to the naturally occurring wild-type I-CreI meganuclease having the sequence pdb accession code 1g9y.

[0218] The DNA recognition and cleavage functions of meganucleases are generally intertwined within a single domain. Unlike meganucleases, the DNA binding domain of ZFN-based and TALEN-based systems is distinct from that of endonucleases in terms of cleavage function. ZFN-based systems include: at least one zinc finger protein or variant thereof, or a nucleic acid comprising a nucleotide sequence(s) encoding a zinc finger protein or variant thereof as its DNA binding domain; and an endonuclease element, such as a zinc finger nuclease (ZFN) or Fok1 cleavage domain. Zinc finger proteins (ZFPs) are not naturally occurring in that they are engineered to bind to a selected target site. For example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct Biol. 10:411-416; U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061.

[0219] Engineered zinc finger binding domains can have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design, for example, includes the use of a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a specific triplet or quadruplet sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261, the entire contents of which are incorporated herein by reference.

[0220] Various kinds of selection methods can be used together with the method herein.Exemplary selection methods include phage display and two-hybrid system are disclosed in United States Patent No. 5,789,538;5,925,523;6,007,988;6,013,453;6,410,248;6,140,466;6,200,759;and 6,242,568;and WO98 / 37186;WO98 / 53057;WO00 / 27878;WO01 / 88197 and GB2,338,237.In addition, the binding specificity enhancement for zinc finger binding domain is described, for example, in WO02 / 077227. Additionally, as disclosed in these and other references, zinc finger domains and / or multi-fingered zinc finger proteins may be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. See also U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences of 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. Methods for selecting target sites, ZFPs, and designing and constructing fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and are described in U.S. Patent Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200, 759; WO95 / 19431; WO96 / 06166; WO98 / 53057; WO98 / 54311; WO00 / 27878; WO01 / 60970; WO01 / 88197; WO02 / 099084; WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536 and WO03 / 016496.

[0221] Additionally, as disclosed in these and other references, zinc finger domains and / or multi-fingered zinc finger proteins may be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. See also U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences of 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[0222] The transcription activator-like effector-based nuclease (TALEN) system refers to a genome editing system that utilizes one or more transcription activator-like effector (TALE)-DNA binding domains and an endonuclease element, such as a Fok1 cleavage domain. The TALE-DNA binding domains contain one or more TALE repeat units, each having a length of 30 to 38 (e.g., 31, 32, 33, 34, 35, or 36) amino acids. The TALE-DNA binding domain may utilize a full-length TALE protein, a fragment thereof, or a variant thereof. The TALE-DNA binding domain can be fused or linked to the endonuclease domain via a linker.

[0223] The terms "CRISPR-based system," "CRISPR-based gene editing system," "CRISPR genome editing," "CRISPR gene editing," "CRISPR endonuclease-based genome editing," and the like are used interchangeably herein to collectively refer to a genome editing system comprising one or more guide RNA elements and one or more RNA-guided endonuclease elements. The guide RNA element comprises a targeter RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence at one or more target genomic regions, or a nucleic acid comprising a nucleotide sequence(s) encoding the targeter RNA. The RNA-guided endonuclease element comprises an endonuclease that is guided or delivered to the target genomic region(s) by the guide RNA element; or a nucleic acid comprising a nucleotide sequence(s) encoding such an endonuclease. Examples of such CRISPR-based gene editing systems, including CRISPR-based systems, are the CRISPR-Cas system or the CRISPR-Cpf system.

[0224] As used herein, the terms "guide RNA element," "guide RNA," "gRNA," "gRNA molecule," and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence that includes a targeter RNA that hybridizes to a target nucleic acid sequence or a nucleic acid that includes a nucleotide sequence(s) encoding the targeter RNA. The targeter RNA of a gRNA includes a targeting domain that includes a nucleotide sequence that is substantially complementary to a nucleotide sequence in a target genomic region. The phrase "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0225] The guide RNA element can further include an activator RNA capable of hybridizing with the targeter RNA, or a nucleic acid containing a nucleotide sequence(s) encoding the activator RNA. The activator RNA and the targeter RNA can be separate or fused as a single nucleic acid via a linker loop sequence to form a single gRNA molecule. The gRNA molecule can include several domains. For example, such a gRNA can include, for example, from 5' to 3': a targeting domain (complementary to the target nucleic acid); a first complementarity domain; a linking domain; a second complementarity domain (complementary to the first complementarity domain); a proximal domain; and, optionally, a tail domain. See WO2015048557.

[0226] A "first complementarity domain" has substantial complementarity with a second complementarity domain and is capable of forming a duplex region under at least some physiological conditions.

[0227] The "linking domain" serves to link the first complementarity domain to the second complementarity domain of the unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently.

[0228] A "proximal domain" can be 3 to 25 nucleotides in length, or 5 to 20 nucleotides in length. A proximal domain can share homology with or be derived from a naturally occurring proximal domain.

[0229] A "tail domain" can be absent or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Tail domains can include sequences that are complementary to each other and form a duplex region under at least some physiological conditions.

[0230] The guide RNA element can form a complex with the endonuclease of the RNA-guided endonuclease element, such as, for example, Cas endonuclease (" gRNA / nuclease complex "). An example of a gRNA / nuclease complex is a CRISPR complex, as described below with respect to the CRISR-based system. In some embodiments, the CRISPR complex comprises the endonuclease of the RNA-guided endonuclease system, which forms a complex with targeter RNA. In some embodiments, the CRISPR complex comprises the endonuclease of the RNA-guided endonuclease system, which forms a complex with targeter RNA and activator RNA.

[0231] The targeting domain of the targeter RNA facilitates specific targeting or homing of the gRNA / nuclease complex to the target nucleotide sequence. In some embodiments, the targeting domain can be 10-30 bp, e.g., 15-25 bp, 18-22 bp, or 20 bp.

[0232] Methods for designing gRNAs, including methods for selecting, designing, and validating target domains, are known in the art. For example, see WO2015048577, Mali et al., 2013 SCIENCE, 339(6121): 823-826; Hsu et al., 2013 NATBIOTECHNOL, 31(9):827-32; Fu et al., 2014 NATBTOTECHNOL, doi: 10.1038 / nbt.2808. PubMed PMID: 24463574; Heigwer et al., 2014, NAT METHODS 11(2):122-3. doi: l 0.1038 / nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 BIOTNFORMATICS PubMed PMID: 24463181; Xiao A et al., 2014 BIOINFORMATICS Pub Med PMID: 24389662.

[0233] In some embodiments, an RNA-guided endonuclease, such as a Cas enzyme or protein (e.g., a type II Cas9 protein) or a Cpf enzyme or protein (e.g., a Cpfl protein), can be used. In some embodiments, modified versions of such Cas or Cpf enzymes or proteins can also be used.

[0234] In some embodiments, the CRISPR-based system is a CRISPR-Cas system. The CRISPR-Cas system comprises: (a) at least one guide RNA element or a nucleic acid comprising a nucleotide sequence(s) encoding this guide RNA element, wherein this guide RNA element comprises a targeter RNA comprising a nucleotide sequence substantially complementary to the nucleotide sequence in one or more target genome regions, and an activator RNA comprising a nucleotide sequence capable of hybridizing with the targeter RNA; and (b) a Cas protein element comprising a Cas protein or a nucleic acid comprising a nucleotide sequence encoding this Cas protein. The targeter RNA and the activator RNA may be separate or may be fused together in a single RNA.

[0235] In some embodiments, the CRISPR-based system includes class 1 CRISPR and / or class 2 CRISPR systems. Class 1 systems utilize several Cas proteins together with CRISPR RNA (crRNA) as the targeter RNA to construct a functional endonuclease. Class 2 CRISPR systems utilize a single Cas protein and crRNA as the targeter RNA. Class 2 CRISPR systems, including type II Cas9-based systems, contain a single Cas protein to mediate cleavage, rather than the multi-subunit complex utilized by class 1 systems. CRISPR-based systems also include class II and type V CRISPR systems, which utilize Cpf1 protein and crRNA as the targeter RNA.

[0236] The Cas protein is a CRISPR-associated (Cas) double-stranded nuclease. In some embodiments, the CRISPR-Cas system includes a Cas9 protein. In some embodiments, the Cas9 protein is SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or D10A nickase. The term "Cas protein," e.g., Cas9 protein, includes wild-type Cas proteins or functional derivatives thereof (e.g., truncated versions or variants of wild-type Cas proteins that have nuclease activity).

[0237] In some embodiments, Cas9 proteins from species other than S. pyogenes and S. thermophiles can be used. Additional Cas9 protein species can be obtained and used herein, including: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus; Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfingens, Corynebacterium accolens, Corynebacterium dolichum, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemoplzilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, llyobacter polytropus, Kingella kingae, lactobacillus crispatus, listeria ivanovii, listeria monocytogenes, listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutells, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

[0238] In some embodiments, one or more elements of the CRISPR-based system are derived from a type I, type II, or type III CRISPR system.

[0239] In some embodiments, one or more elements of a CRISPR-based system are expressed in a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes, Staphylococcus aureus, Francisella tularensis, Prevotella sp., Acidaminococcus sp., and Lachnospiraceae sp. Generally, CRISPR-based systems are characterized by an element (also called a protospacer in the context of endogenous CRISPR systems) that promotes the formation of a CRISPR complex at the site of a target genomic region or target sequence. In the context of CRISPR complex formation, the term "target sequence" refers to a sequence to which a guide sequence is designed to have substantial complementarity, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence can comprise any polynucleotide, for example, a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell(s). In some embodiments, the target sequence may be within an organelle of a eukaryotic cell(s), for example, a mitochondrion or chloroplast.

[0240] The sequence or template that can be used for recombination into the targeted locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." An exogenous template polynucleotide can be referred to as an editing template or donor template. In some embodiments, single-stranded DNA and double-stranded DNA of synthetic or biological origin can be used. Non-limiting examples of suitable editing templates include ssODN, dsODN, PCR products, plasmids, and viruses, including AAV, adenovirus, retrovirus, lentivirus, etc. Additional editing templates are also possible. In some embodiments, the recombination is homologous recombination.

[0241] In some embodiments, the CRISPR-based system is a CRISPR-Cas9 system. The targeter RNA of the CRISPR-Cas9 system comprises a CRISPR targeting RNA (crRNA), and the activator RNA of the CRISPR-Cas9 system comprises a trans-activating CRISPR RNA (tracRNA). The Cas protein element of the CRISPR-Cas9 system utilizes a Cas9 protein. The crRNA and tracrRNA can be separate or combined into a single RNA construct via a linker loop sequence. This combined RNA construct is called a single guide RNA (sgRNA; or guide RNA).

[0242] General information regarding CRISPR-Cas systems, their components, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, and making and using the same, including with respect to amounts and formulations, can be found in: U.S. Patent Nos. 8,999,641; 8,993,233; 8,945,839; 8,932,814; Nos. 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, and 8,697,359; U.S. Patent Publications US2014-0310830, US2014-0287938A1, US2014-0273234A1, US2014-0273232A1 , US2014-0273231, US2014-0256046A1, US2014-0248702A1, US2014-0242700A1, US2014-0242699A1, US2014 -0242664A1, US2014-0234972A1, US2014-0227787A1, US2014-0189896A1, US2014-0186958, US2014-018691 9A1, US2014-0186843A1, US2014-0179770A1 and US2014-0179006A1, US2014-0170753; European patents EP2784162B1 and EP2771468B1; European patent applications EP2771468 (EP13818570.7), EP2764103 (EP13824232.6), and EP2784162 (EP14170383.5); and PCT patent application publications WO2014 / 093661, WO2014 / 093694, WO2014 / 093595, WO2014 / 093718, WO2014 / 093709, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093 712, WO2014 / 093701, WO2014 / 018423, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, and WO2016 / 028682.

[0243] In some embodiments, the CRISPR-based system is a CRISPR-Cpf system. A "CRISPR-Cpf system" includes (a) at least one guide RNA element or a nucleic acid comprising a nucleotide sequence(s) encoding the guide RNA element, wherein the guide RNA comprises a targeter RNA having a nucleotide sequence complementary to a nucleotide sequence at a locus of a target nucleic acid; and (b) a Cpf protein element or a nucleic acid comprising a nucleotide sequence encoding the Cpf protein element.

[0244] Examples of Cpf protein elements include Cpf1 nucleases, such as Francisella Cpf1 (FnCpf1) and any variants thereof. See, for example, Zetsche et al., "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system," Cell, 163(3): pages 759-71; and Fonfara et al., "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA," Nature, 532(7600): pages 517-21. The preferred PAM of Cpf1 is 5'-TTN, which differs from that of Cas9 (3'-NGG) in both genomic location and GC content. The CRISPR-Cpf system may not utilize an activator RNA (tracrRNA). Both Cpf1 and the guide RNA are generally smaller than their SpCas9 counterparts. The Cpf1 locus contains a mixed alpha / beta domain, RuvC-I, followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have the HNH endonuclease domain, and the N-terminus of Cpf1 lacks the alpha-helical recognition lobe of Cas9. The Cpf1 locus encodes Cas1, Cas2, and Cas4 proteins, which are more similar to type I and III systems than type II systems. Cpf1-family proteins can be found in many bacterial species.

[0245] Without being bound by any particular theory, the CRISPR-Cpf system utilizes the Cpf1-crRNA complex to cleave target DNA or RNA by recognizing the protospacer adjacent motif 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3, as opposed to the G-rich PAM targeted by Cas9. After PAM recognition, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0246] In some embodiments, the genome editing system is an NgAgo-based system. The NgAgo-based system includes a nucleic acid comprising at least one guide DNA element or a nucleic acid sequence(s) encoding the guide DNA element; and a DNA-guided endonuclease. The NgAgo-based system uses DNA as the guide element. Its operating principle is similar to that of the CRISPR-Cas9 technology, but the guide element is a segment of guide DNA (dDNA) rather than the gRNA of the CRISPR-Cas9 technology. An example of a DNA-guided endonuclease is the Argonaute endonuclease (NgAgo) from Natronobacterium gregoryi. See, for example, Feng Gao et al., "DNA-guided genome editing using the Natronobacterium gregoryi Argonaute", Nature Biotechnology, (2016):doi:10.1038 / nbt.3547.

[0247] By "linker," "peptide linker," "peptidic linker," or "peptide spacer" is intended to mean a peptide sequence that allows the connection of different monomers in a fusion protein and the adoption of the correct conformation for said fusion protein activity, without altering the activity of any of the monomers. Peptide linkers may be of various sizes, with non-limiting indicative ranges being 1, 2, 3, 4, 5, 10, 15, 20, 30, 40-50 amino acids, or any intermediate value within this range.

[0248] DNA-PK inhibitors for increasing genome editing efficiency

[0249] The efficiency of targeted genome editing can be increased by administering one or more compounds (e.g., DNA-PK inhibitors) and a genome editing system described herein to a cell(s). Genome editing systems suitable for use include, for example, meganuclease-based systems, zinc finger nuclease (ZFN)-based systems, transcription activator-like effector-based nuclease (TALEN) systems, CRISPR-based systems, or NgAgo-based systems. The methods, compositions, and kits disclosed herein provide DNA-PK inhibitors and / or genome editing systems for increasing genome editing efficiency. In some embodiments, HDR genome editing efficiency is increased after administering a DNA-PK inhibitor to a cell(s).

[0250] In some embodiments, the genome editing system is a CRISPR-based genome editing system.The CRISPR-based genome editing system can be a CRISPR-Cas system or its variant.The CRISPR-Cas system can use any Cas endonuclease, for example, Cas9 endonuclease and its variant.Examples of Cas9 endonuclease include Cas9 endonuclease and its variant, for example, SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or CasD10A nickase.The Cas endonuclease can be wild-type, engineered, or nickase mutant, or any variant thereof.

[0251] In some embodiments, the CRISPR-based genome editing system comprises a CRISPR sequence, a trans-activating cr (tracr) sequence, a guide sequence, and a Cas endonuclease, or any combination thereof.

[0252] In some embodiments, the CRISPR-based genome editing system comprises an RNA containing a CRISPR sequence (crRNA), an RNA containing a trans-activating cr (tracr) sequence (tracrRNA), and a Cas endonuclease, or any combination thereof.

[0253] In some embodiments, the CRISPR-based genome editing system comprises a CRISPR sequence, a guide sequence, and a Cas endonuclease or a Cpf endonuclease, or any combination thereof.

[0254] In some embodiments, the CRISPR-based genome editing system is a CRISPR-Cpf system. Cpf nuclease is a Class 2 CRISPR-Cas system endonuclease. Cpf is a single RNA-guided endonuclease. Cpf nuclease can be wild-type, engineered, or a nickase mutant, or any variant thereof. For example, Zetsche et al., "CPF1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system." 2 CRISPR-Cas System”, Cell, 163(3):759-71. In some embodiments, the Cpf nuclease is a Cpf1 endonuclease.

[0255] In some embodiments, the genome editing system is a meganuclease-based system. Meganuclease-based genome editing uses sequence-specific endonucleases that recognize large DNA target sites (e.g., usually about >12 bp). See, e.g., US9,365,964. Meganucleases can cleave unique chromosomal sequences without affecting the overall genome integrity. In some embodiments, the meganuclease can be a homing endonuclease. In some embodiments, the meganuclease can be an intron endonuclease or an intein endonuclease. The homing endonuclease can belong to the LAGLIDADG family. The meganuclease can be wild-type, engineered, or a nickase mutant.

[0256] In some embodiments, the gene editing system is based on zinc finger nuclease (ZFN) system.ZFN is an artificial restriction enzyme that is engineered based on the fusion between zinc finger DNA binding domain and DNA cleavage domain.See, for example, US9,145,565.

[0257] In some embodiments, gene editing system is transcription activator-like effector-based nuclease (TALEN).TALEN is an engineered restriction enzyme that is produced by fusing TAL effector DNA binding domain with DNA cutting domain.See, for example, US9,181,535.

[0258] In some embodiments, the gene editing system is an Argonaute-based system. The Argonaute-based gene editing system includes an endonuclease derived from Argonaute and 5' phosphorylated ssDNA. In some embodiments, the phosphorylated ssDNA can be 10-40 nucleotides, 15-30 nucleotides, or 18-30 nucleotides (e.g., about 24 nucleotides) in length. In some embodiments, the Argonaute endonuclease can be any endonuclease. In some embodiments, the Argonaute endonuclease is derived from Thermus thermophiles (TtAgo), Pyrococcus furiosus (PfAgo), or Natronobacterium gregoryi (NgAgo). In some embodiments, the Natronobacterium gregoryi (NgAgo) is strain 2 (i.e., N. gregoryi SP2). In some embodiments, the Argonaute endonuclease is NgAgo. See, for example, Gao et al., "DNA-guided See “Genome editing using the Natronobacterium gregoryi Argonaute,” Nature Biotechnology, May 2016.

[0259] The DNA-PK inhibitor can be any DNA-PK inhibitor. The DNA-PK inhibitor can be any compound or substance that causes inhibition of DNA-PK. The DNA-PK inhibitor can be a compound, a small molecule, an antibody, or a nucleotide sequence. In some embodiments, the DNA-PK inhibitor is a compound represented by formula (III-E-1) or (III-E-2).

[0260] In some embodiments, the disclosure provides a method for editing one or more target genomic regions, comprising administering to one or more cells having one or more target genomic regions a genome editing system and a compound represented by formula (III-E-1) or (III-E-2):

[0261] [ka]

[0262] [In the formula,

[0263] X is O or NR, R is H or C1-C4 alkyl,

[0264]

[0265] Y is O or NR, R is H or C1-C4 alkyl,

[0266]

[0267] R 3 is hydrogen, C 1~4 Alkyl, or OC 1~2 is alkyl,

[0268] R 1 is a 6-membered aromatic heterocycle containing one or two nitrogen atoms, which aromatic heterocycle is selected from the group consisting of C1-C4-alkyl, C1-C4-haloalkyl, C(=O)NHR 2’ , and N.R. 4 R 5 0, 1, or 2 substituents R independently selected from the group consisting of 2 may be substituted with

[0269] R 2’ is a C1-C4 alkyl;

[0270] Each R 4 and R 5 are independently H, C1-C4 alkyl, or C(=O)C1-C4 alkyl; or

[0271] R 4 and R 5 together with the N atom to which they are attached form a heterocyclic ring containing 0 or 1 additional N atom, said heterocyclic ring being optionally substituted with C1-C4 alkyl;

[0272] Ring B is

[0273] [ka]

[0274] (Wherein, W is N or CR 3 and Z is O or S, and R 3 is H or C1-C4 alkyl] The method includes administering

[0275] In some embodiments, the disclosure provides a method of editing one or more target genomic regions, the method comprising administering to one or more cells harboring the one or more target genomic regions a genome editing system and a compound represented by Formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof.

[0276] In some embodiments, the disclosure also provides a method for repairing DNA breaks in one or more target genomic regions via the homology directed repair (HDR) pathway, the method comprising administering to one or more cells harboring the one or more target genomic regions a genome editing system and a compound represented by Formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof.

[0277] The genome editing system interacts with the nucleic acid(s) of the target genomic region, resulting in a DNA break, which is at least partially repaired via the HDR pathway.

[0278] In some embodiments, the disclosure also provides a method for inhibiting or suppressing repair of DNA breaks in one or more target genomic regions via the NHEJ pathway, the method comprising administering to one or more cells having the one or more target genomic regions a genome editing system and a compound represented by Formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof.

[0279] The genome editing system interacts with nucleic acid(s) in one or more target genomic regions, resulting in a DNA break, and repair of the DNA break via the NHEJ pathway is inhibited or suppressed.

[0280] In some embodiments, the disclosure also provides a method of modifying expression of one or more genes or proteins, the method comprising administering to one or more cells comprising one or more target genomic regions a genome editing system and a compound represented by Formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof.

[0281] The genome editing system interacts with nucleic acid(s) of one or more target genomic regions of a target gene(s) to result in editing of the one or more target genomic regions, which edits alter the expression of downstream gene(s) and / or protein(s) associated with the target gene(s).

[0282] In some embodiments, the DNA break comprises a DNA double-strand break (DSB).

[0283] In some embodiments, the efficiency of repair of a DNA break in a target genomic region in one or more cells via the HDR pathway is increased compared to an otherwise identical cell(s) in the absence of the compound.

[0284] In some embodiments, the efficiency of inhibiting or suppressing repair of a DNA break in a target genomic region in one or more cells via the NHEJ pathway is increased compared to an otherwise identical cell(s) in the absence of the compound.

[0285] In some embodiments, the efficiency is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or 100-fold compared to an otherwise identical cell(s) in the absence of the compound.

[0286] In some embodiments, efficiency is measured by the frequency of targeted polynucleotide integration. In some embodiments, efficiency is measured by the frequency of targeted mutagenesis. In some embodiments, targeted mutagenesis comprises point mutations, deletions, and / or insertions.

[0287] In some embodiments, expression of downstream gene(s) and / or protein(s) associated with the target gene(s) is increased compared to baseline expression levels in the one or more cells prior to administration, e.g., the expression is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or 10-fold compared to baseline expression levels in the one or more cells prior to administration.

[0288] In some embodiments, expression of downstream gene(s) and / or protein(s) associated with the target gene(s) is reduced compared to baseline expression levels in the one or more cells prior to administration, e.g., gene expression is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% compared to baseline expression levels in the one or more cells prior to administration.

[0289] In some embodiments, expression of downstream gene(s) and / or protein(s) associated with the target gene(s) is substantially abolished in one or more cells.

[0290] In some embodiments, the cells are synchronized to the S or G2 cell cycle phase.

[0291] In some embodiments, one or more cells administered or contacted with the compound have increased survival time compared to one or more cells not administered or contacted with the compound.

[0292] In some embodiments, the genome editing system and the compound are administered simultaneously to one or more cells. In some embodiments, the genome editing system and the compound are administered sequentially to one or more cells. In some embodiments, the genome editing system is administered to one or more cells before the compound. In some embodiments, the compound is administered to one or more cells before the genome editing system.

[0293] In some embodiments, the one or more cells are cultured cells. In some embodiments, the one or more cells are in vivo cells within an organism. In some embodiments, the one or more cells are ex vivo cells from an organism.

[0294] In some embodiments, the organism is a mammal, hi some embodiments, the organism is a human.

[0295] In some embodiments, the genome editing system and the compound are administered via the same route. In some embodiments, the genome editing system and the compound are administered via different routes. In some embodiments, the genome editing system is administered intravenously and the compound is administered orally.

[0296] In some embodiments, the genome editing system is selected from a meganuclease-based system, a zinc finger nuclease (ZFN)-based system, a transcription activator-like effector-based nuclease (TALEN) system, a CRISPR-based system, or an NgAgo-based system.

[0297] In some embodiments, the genome editing system is a CRISPR-based system. In some embodiments, the CRISPR-based system is a CRISPR-Cas system or a CRISPR-Cpf system.

[0298] In some embodiments, the CRISPR-based system is a CRISPR-Cas system, which includes (a) at least one guide RNA element comprising: (i) a targeter RNA or a nucleic acid comprising nucleotide sequence(s) encoding the targeter RNA, the targeter RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence in one or more target genomic regions; (ii) and an activator RNA or a nucleic acid comprising nucleotide sequence(s) encoding the activator RNA, the nucleotide sequence(s) comprising a nucleotide sequence capable of hybridizing to the targeter RNA; and (b) a Cas protein element comprising a Cas protein or a nucleic acid comprising nucleotide sequence(s) encoding the Cas protein.

[0299] In some embodiments, the targeter RNA and the activator RNA are fused as a single molecule.

[0300] In some embodiments, the Cas protein is a type II Cas9 protein. In some embodiments, the Cas9 protein is SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or D10A nickase, or any combination thereof.

[0301] In some embodiments, the CRISPR-based system is a CRISPR-Cpf system comprising: (a) at least one guide RNA element or a nucleic acid comprising nucleotide sequence(s) encoding the guide RNA element, wherein the guide RNA comprises a targeter RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence in one or more target genomic regions; and (b) a Cpf protein element comprising a Cpf protein or a nucleic acid comprising a nucleotide sequence encoding the Cpf protein.

[0302] In some embodiments, the genome editing system is delivered by one or more vectors.

[0303] In some embodiments, the one or more vectors are selected from a viral vector, a plasmid, or ssDNA.

[0304] In some embodiments, the viral vector is selected from retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex viral vectors.

[0305] In some embodiments, the genome editing system is delivered by synthetic RNA.

[0306] In some embodiments, the genome editing system is delivered in a nanoformulation.

[0307] In some embodiments, a kit or composition is provided for editing one or more target genomic regions. In some embodiments, the kit or composition includes a genome editing system; and a compound represented by formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof.

[0308] In some embodiments, the genome editing system of kit or composition is meganuclease-based system, zinc finger nuclease (ZFN)-based system, transcription activator-like effector-based nuclease (TALEN) system, CRISPR-based system or NgAgo-based system.In some embodiments, the genome editing system of kit or composition is CRISPR-based system.In some embodiments, the CRISPR-based system of kit or composition is CRISPR-Cas system or CRISPR-Cpf system.

[0309] In some embodiments, the CRISPR-based system of the kit or composition is a CRISPR-Cas system, which includes (a) at least one guide RNA element comprising: (i) a targeter RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence in one or more target genomic regions, or a nucleic acid comprising nucleotide sequence(s) encoding the targeter RNA; (ii) and an activator RNA comprising a nucleotide sequence capable of hybridizing to the targeter RNA, or a nucleic acid comprising nucleotide sequence(s) encoding the activator RNA; and (b) a Cas protein element comprising a Cas protein, or a nucleic acid comprising nucleotide sequence(s) encoding the Cas protein.

[0310] In some embodiments, the Cas protein of the kit or composition is a type II Cas9 protein, hi some embodiments, the Cas9 protein of the kit or composition is SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or D10A nickase, or any combination thereof.

[0311] In some embodiments, the CRISPR-based system of the kit or composition is a CRISPR-Cpf system, which includes: (a) a targeter RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence in one or more target genomic regions, or a nucleic acid comprising nucleotide sequence(s) encoding the targeter RNA; and (b) a Cpf protein element comprising a Cpf protein, or a nucleic acid comprising nucleotide sequence(s) encoding the Cpf protein.

[0312] In some embodiments, the genome editing system of the kit or composition is contained in or packaged in one or more vectors. In some embodiments, the one or more vectors are selected from a viral vector, a plasmid, or a ssDNA. In some embodiments, the viral vector is selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated, and herpes simplex virus vectors.

[0313] In some embodiments, the increase in genome editing efficiency is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, or 100-fold compared to a condition in which a DNA-PK inhibitor and a genome editing system are not administered to a cell(s), or compared to a condition in which only a genome editing system but not a DNA-PK inhibitor is administered to a cell(s).

[0314] Uses of DNA-PK inhibitors and genome editing systems, kits, and compositions thereof

[0315] Genome editing, in which specific genomic regions are precisely altered, holds great therapeutic potential.

[0316] In some embodiments, provided herein are methods for editing one or more target genomic regions to repair DNA breaks in one or more target genomic regions via the HDR pathway, to inhibit or suppress NHEJ-mediated repair of DNA breaks in one or more target genomes, and to alter expression of one or more genes or proteins, via administering to a cell(s) a genome editing system and a DNA-PK inhibitor.

[0317] In some embodiments, provided herein are methods of modifying expression of one or more genes or proteins, the methods comprising administering to one or more cells comprising one or more target genomic regions a genome editing system as described herein and a DNA-PK inhibitor, wherein the genome editing system interacts with nucleic acid(s) of the one or more target genomic regions of the target gene(s), resulting in editing of the one or more target genomic regions, which editing modifies expression of downstream gene(s) and / or protein(s) associated with the target gene(s).

[0318] The genome editing system can be any genome editing system that can edit a target genome region in a cell (or cells). Exemplary genome editing systems are described in detail above and can include, for example, meganuclease-based systems, zinc finger nuclease (ZFN)-based systems, transcription activator-like effector-based nuclease (TALEN) systems, CRISPR-based systems, or NgAgo-based systems.

[0319] The editing of one or more target genomic regions includes any type of genetic manipulation or manipulation of the genome of a cell. The editing of one or more target genomic regions can include the insertion, deletion, or replacement of a genomic region in a cell(s) performed by one or more endonucleases. A genomic region includes genetic material in a cell(s), such as DNA, RNA, polynucleotides, and oligonucleotides. A genomic region in a cell(s) also includes the genomes of mitochondria or chloroplasts contained in the cell(s).

[0320] In some embodiments, provided herein are methods of treating a subject having a disease or condition requiring editing of one or more target genomic regions in a cell(s) of the subject, the method comprising administering to the one or more cells a genome editing system and a DNA-PK inhibitor.

[0321] In some embodiments, the methods provided herein are used to modify the expression of genes, RNA molecules, proteins, groups of proteins, or downstream proteins in a pathway. Such modifications can be used to treat diseases, dysfunctions, abnormal organism homeostasis, whether acquired or inherited, or due to the aging process. As used herein, the term "modify" or "modifying" includes modulating, promoting, reducing, increasing, inserting, deleting, knocking out, knocking in, etc.

[0322] Those skilled in the art will understand that the disease, whether acquired, genetic, or otherwise acquired, involves dysregulation of homeostatic mechanisms, including the involvement of gene or protein function. To this end, those skilled in the art can use the methods provided herein to modulate, alter, enhance, reduce, or otherwise provide gene function in a subject.

[0323] Altering the expression of a gene and the resulting protein expression in a cell(s) can be achieved by the methods provided herein, for example, by specifically editing (e.g., by substitution, insertion, or deletion, any combination thereof) nucleic acid sequences in any of the following: exons, introns, transcription start sites, promoter regions, enhancer regions, silencer regions, insulator regions, anti-repressors, post-translational regulatory elements, polyadenylation signals (e.g., minimal polyA), conserved regions, transcription factor binding sites, or any combination thereof.

[0324] In some embodiments, the methods, kits, and compositions provided herein are used to treat a subject with cancer. The method for treating a subject with cancer or a cancer-related condition comprises administering a DNA-PK inhibitor and a genome editing system to a cell(s) of the subject. The administration of the DNA-PK inhibitor and the genome editing system is It can be in vivo or ex vivo.

[0325] Cancer can be any type of cancer.Cancer includes solid tumors, such as breast, ovary, prostate, lung, kidney, stomach, colon, testicle, head and neck, pancreas, brain, melanoma, and other tissue and organ tumors, and blood cell cancers, such as lymphomas and leukemias, including acute myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.Cancer can include melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, chronic lymphocytic leukemia, and cancers of the pancreas, breast, thyroid, ovary, uterus, testicle, pituitary gland, kidney, stomach, esophagus, and rectum.

[0326] In some embodiments, the methods, kits, and compositions provided herein are used to treat a subject with any one or more of the following cancers: acute lymphocytic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, childhood cerebellar or cerebral astrocytoma, basal cell carcinoma, extrahepatic bile duct cancer (see cholangiocarcinoma), bladder cancer, bone tumor, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain cancer, brain tumor (cerebellar astrocytoma), brain tumor (cerebral astrocytoma / malignant glioma), brain tumor (ependymoma), brain tumor (medulloblastoma). ), Brain tumors (supratentorial primitive neuroectodermal tumors), Brain tumors (visual pathway and hypothalamic gliomas), Breast cancer, Bronchial adenoma / carcinoid, Burkitt lymphoma, Carcinoid tumor, Pediatric carcinoid tumor, Gastrointestinal carcinoid tumor, Carcinoma of unknown primary, Primary central nervous system lymphoma, Pediatric cerebellar astrocytoma, Pediatric brain astrocytoma / malignant glioma, Cervical cancer, Childhood cancer, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myeloid leukemia, Chronic myeloproliferative disorder, Colon cancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Epithelioid hemangioendothelioma Hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma of the Ewing family of tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (intraocular melanoma), eye cancer (retinoblastoma), gallbladder cancer, stomach cancer (gastric cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), extracranial, extragonadal, or ovarian germ cell tumor, gestational trophoblastic tumor, brainstem glioma, glioma (pediatric cerebral astrocytoma) cysts), childhood visual pathway and hypothalamic gliomas, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, childhood hypothalamic and visual pathway gliomas, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemias, acute lymphocytic leukemia (also called acute lymphocytic leukemia), acute myeloid leukemia (also called acute myelogenous leukemia),Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), Leukemia, chronic myelogenous (also called chronic myeloid leukemia), hairy cell leukemia, lip and oral cavity cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin (an older classification of all lymphomas except Hodgkin) lymphoma, primary central nervous system Waldenstrom macroglobulinemia, male breast cancer, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, childhood melanoma, intraocular (Ocular) melanoma, Merkel cell carcinoma, mesothelioma, adult malignant mesothelioma, unknown primary, metastatic squamous neck cancer of childhood, cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myeloid leukemia, chronic myeloid leukemia, acute myeloid leukemia of adults, acute myeloma of childhood, multiple (bone marrow cancer), myeloproliferative disorder, chronic myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer , oligodendroglioma, cancer of the mouth, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, ovarian low-grade malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, Rectal cancer, renal cell carcinoma (kidney cancer), renal pelvic and ureteral cancer (caner), transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing family tumor, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma - see skin cancer (non-melanoma), primary unknown, metastatic cervical squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor,Cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome), testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroid carcinoma, transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, carcinoma of unknown primary site in adults, carcinoma of unknown primary site in children, transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, or Wilms' tumor (kidney cancer).

[0327] One of the active ingredients is that it is specifically the active ingredient of ABL1 and AB L2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31. AKAP9, AT1, AKT2, ALDH2, AL, AL017, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT, ASPSCR 1, ASXL1, ATF1, ATIC, ATM, ATRX, AXIN1, BAP1, BCL10, BCL11A, BCL11B, BCL2, BCL3, BCL5 BCL6, BCL7A, BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPRIA, BRAF, BRCAl, BRCA2, BRD3, BRD 4. BRIPI, BTG1, BUB1B, C12orf9, C15orf21, C15orf55, C16orf75, C2orf44, CAMTA1, CAN T1, CARD11, CARS, CBFA2T1, CBFA2T3, C.BFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1. CCND2, CCND3, CCNE1, CD273, CD274, CD74, CD79A, CD79B, CDH1, CDH11, CDK12, CDK4, CDK 6, CDN2A, CDN2a(pl4), CDN2C, CDX2, CEBPA, CEPl, CHCHD7, CHEK2, CHIC2, CHNl, CIC, Cin A. CLTC, CLTCL1, CMKOR1, CNOT3, COL1Al, COPEB, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRTC3, CTNNB1, CYLD, D10S17 0, DAXX, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK, D1CER1, DNM2, DNM T3A, DUX4, EBFI, ECT2L, EGFR, E1F4A2, ELF4, ELK4, ELKS, ELL, EL N、EML4、EP300、EPS15、ERBB2、ERCC2、ERCC3、ERCC4、ERCC5、ERG ETVl, ETV4, ETV5, ETV6, EVIl, EWSR1, EXTl, EXT2, EZH2, EZR, FA CL6, FAM22A, FAM22B, FAM46C, 1ANCA, EANCC, FANCD2, FANCE, FAN CF, FANCG, FBXOl1, FBXW7, FCGR2B, FEV, FGFR1, FGFRIOP, FGFR2<h2 style=";text-align:left;direction:ltr">FGFR3、FTI、FIIIT、FIP1L1、FLU、FLJ27352、FLT3、FNBP1、FOXL2、FOXOIA、FOX03A、FOXP1、 FSTL3、FUBP1、FUS、FVT1、GAS7、GATA1、GATA2、GATA3、GMPS、GNA11、GNAQ、GNAS、GOLGA5、G OPC、GPC3、GPHN、GRAF、H3F3A、IICMOGT-1、IIEAB、HERPUD1、IIEY1、IIIPl、HIST1IT3B、II IST1II4I、IILF、HLXB9、HMGA1、HMGA2、HNRNPA2BI、HOOK3、HOXA11、HOXA13、HOXA9、HOXC11 HOXC13, HOXD11, HOXD13, HRAS, IIRPT2, HSPCA, HSPCB, IDHl, IDH2, IGH, IGK, IGL, IKZFl, IL2, TL21R, IL6ST, IL7R, IRF4, IRTA1, ITK, JAK1, JAK2, JAK3, JAZF1, JUN, KCNJ5, KDM5A KDM5C、KDM6A、KDR、KIAA1549、KIF5B、KIT、KLF4、KLK2、KRAS、KTN1、LAF4、LASPl、LCK、LCP 1、LCX、LHFP、LIFR、LMOl、LM02、LPP、LRIG3、LYL1、MADH4、MAF、MAFB、MALT1、MAML2、MAP2KL MAP2K2、ΜλΡ2Κ4、MAX、MDM2、MDM4、MDS1、MDS2、MECTl、MED12、M EN1、MET、MITF、MKL1、MLF1、MLIIl、MLL、MLL2、MLL3、MLLT1、ML LT10、MLLT2、MLLT3、MLLT4、MLLT6、MLLT7、MN1、MPL、MSF、MSH2 、MSH6、MSI2、MSN、MTCP1、MUC1、MUTYH、MYB、MYC、MYCL1、MYCN、M YD88, MYH11, MYH9, MYST4, NACA, NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2, NFE2L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCHI, NOTCH2, NPMl, NR4A3, NRAS, NSDl, NT5C2, NTRKl, NTRK3, NUMAL, NUP214, NUP98, OLIG2, OMD, P2RY8, PFAH1B2, PALB2, PAX3,PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7, PDE4DIP, PDGFB, PDGFRA, PDG FRB, PERI, PIIF6, PHOX2B, PICALM, PIK3CA, PIK3R1, PIM1, PLAG1, PML, PMS 1, PMS2, PMX1, PNUTL1, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRD M1, PRDM16, PRF1, PRKAR1A, PRO1073, PSIP2, PTCH, PTEN, PTPN11, RAB5EP ACl, RAD51L1, RAFl, RALGDS, RANBP17, RAPIGDSI, RARA, RBI, RBM15, RECQL 4. REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RUNDC2A, RUNX1, RUNXB P2, SBDS, SDC4, SDH5, SDHB, SDHC, SDHD, SEPT6, SET, SETBP1, SETD2, SF3B1 SFPQ, SFRS3, SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMARCA4, SMARCB1, S.K MARCE1, SMO, SOCS1, SOX2, SRGAP3, SRSF2, SSI8, SS18L1, SSH3BP1, SSX1, S SX2, SSX4, STAT3, STK11, STL, SUFU, SIJZ12, SYK, TAF15, TALI, TAL2, and TCEA 1, TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG, T FPT, TFRC, THRAP3, TIF1, TLX1, TLX3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF 17 TNFRSF6, TOPI, TP53, TPM3, TPM4, TPR, TRA, TRAF7, TRB, TRD, TRIM27, T RIM33, TRIP11, TSC1, TSC2, TSHR, TTL, U2AF1, USP6, VHL, VTUA, WAS, WHSC1 WHSC1L1, WIF1, WRN, WT1, WTX, WWTR1, XPA, XPC, XPOl, YWHAE, ZNF145, ZNF1 98 ZNF278, ZNF331, ZNF384, ZNF521, ZNF9, and ZRSR2.

[0328] In some embodiments, the methods provided herein are used to treat a subject with a genetic disorder. The method for treating a subject with a genetic disease or condition or a genetic disorder comprises administering a DNA-PK inhibitor and a genome editing system to a cell(s) of the subject. The administration of the DNA-PK inhibitor and the genome editing system is It can be in vivo or ex vivo.

[0329] The genetic disorder can result from a mutation or duplication within a chromosomal region (e.g., from a point mutation, deletion, insertion, frameshift, chromosomal duplication or deletion). The genetic disorder can be any genetic disorder.

[0330] In some embodiments, the genetic disorder is 22q11.2 deletion syndrome, Angelman syndrome, Canavan disease, Charcot-Marie-Tooth disease, color blindness, cri-a-cat syndrome, Down syndrome, Duchenne muscular dystrophy, hemochromatosis, hemophilia, Klinefelter syndrome, neurofibromatosis, phenylketonuria, polycystic kidney disease, Prader-Willi syndrome, sickle cell disease, spinal muscular atrophy, spinal muscular atrophy, Tay-Sachs disease, Turner syndrome, hemoglobinopathies, or any combination thereof.

[0331] In some embodiments, the genetic disorder is 1p36 deletion syndrome, 18p deletion syndrome, 21-hydroxylase deficiency, 47XXX (Triple X syndrome), 47XXY (Klinefelter's syndrome), 5-ALA dehydratase deficiency porphyria (ALA dehydratase deficiency), 5-aminolevulinic acid dehydratase deficiency porphyria, 5p deletion syndrome (cri-c-cat syndrome) (also known as 5p-syndrome), ataxia telangiectasia (also known as AT), alpha 1-antitrypsin deficiency (AAT), ceruloplasmin deficiency, achondrogenesis type II (ACG2), achondroplasia chondroplasia (ACH). , acid beta-glucosidase deficiency, Gaucher disease (any type, e.g., type 1, 2, 3), brachycephalic syndactyly (Apert) (Apert syndrome), brachycephalic syndactyly (any type, e.g., type 1, 2, 3, 5), Pfeiffer syndrome, acrocephaly, acute cerebral Gaucher disease, acute intermittent porphyria, (AIP)ACY2 deficiency, Alzheimer's disease (AD), Adelaide craniosynostosis, Muenck syndrome, adenomatous polyposis, familial adenomatous polyposis, adenomatous polyposis of the colon, familial adenomatous polyposis (ADP), adenylosuccinate lyase deficiency, adrenal disorders, adrenogenital syndrome, adrenal cerebral white matter Dystrophy, androgen insensitivity syndrome (AIS), alkaptonuria (AKU), ALA-dehydratase deficiency porphyria, ALA-D porphyria, ALA-dehydratase deficiency, Alagille syndrome, albinism, alkaptonuria, Alexander disease, alkaptonuria, alkaptonuric histochromia, alkaptonuria (alpha-1 protease inhibitor disease), alpha-1 related emphysema, alpha-galactosidase A deficiency, Fabry disease, Alstroem syndrome, Alexander disease (ALX), hereditary amelogenesis imperfecta syndrome, aminolevulinic acid dehydratase deficiency, aminoacylase 2 deficiency, Canavan disease, Anderson-Fabry disease, androgen insensitivity syndrome, hereditary sideroblastic anemia, X-linked sideroblastic splenic anemia and / or familial anemia, diffuse truncal angiokeratoma, diffuse angiokeratoma corporis, retinal angiomatosis, von Hippel-Lindau disease, APC resistance, Leiden type, factor V Leiden thrombophilia, Apert syndrome, AR deficiency, androgen insensitivity syndrome, Charcot-Marie-Tooth disease (any type, e.g., CMT1, CMTX, CMT2, CMT4, severe early-onset CMT),Arachnodactyly, Marfan syndrome, ARNSHL, non-syndromic hearing loss (autosomal recessive, autosomal dominant, x-linked, or mitochondrial), hereditary progressive arthro-ophthalmopathy, Stickler syndrome (e.g., COL2A1, COL11A1, COL11A2, COL9A1), congenital multiple arthrochalasia, Ehlers-Danlos syndrome (e.g., hypermobile, multiple arthrochalasia, classic, vascular, kyphoscoliotic, skin fragility), Asp deficiency, Aspa deficiency, aspartoacylase deficiency, ataxia-telangiectasia, autism-dementia-ataxia-purposeless hand movement syndrome, Rett syndrome, autosomal dominant juvenile onset ALS, autosomal dominant opitz G / BBB syndrome, autosomal recessive juvenile ALS type 3, amyotrophic lateral sclerosis (any type; e.g., ALS1, ALS2, ALS3, ALS4, ALS5, ALS6, ALS7, ALS8, ALS9, ALS10, ALS11, ALS12, ALS13, ALS14, ALS15, ALS16, ALS17, ALS18, ALS19, ALS20, ALS21, ALS22, FTDALS1 , FTDALS2, FTDALS3, FTDALS4, FTDALS4, IBMPFD2), autosomal recessive nonsyndromic hearing loss, autosomal recessive sensorineural hearing impairment and goiter, Pendred syndrome, Alexander disease (AxD), Ayer's syndrome, familial pulmonary arterial hypertension, B variant of hexosaminidase GM2 gangliosidosis, Sandhoff disease, BANF-related disorders, neurofibromatosis (any type, e.g., NF1, NF2, schwannoma), Behre-Stevenson gyrate scalp syndrome, benign paroxysmal peritonitis, Benjamin syndrome, beta-thalassemia, BH4 deficiency, tetrahydrobiopterin deficiency, bilateral acoustic neurofibromatosis, biotinidase deficiency, bladder cancer, bleeding disorders, factor V Leiden thrombophilia, Bloch-Sulzberger syndrome, incontinentia pigmenti, Bloom syndrome , bone diseases, Brutaville disease, tuberous sclerosis, brain diseases, prion diseases, breast cancer, Birt-Hogg-Dubé syndrome, osteoporosis, osteogenesis imperfecta, broad finger-toe syndrome, Rubinstein-Taybi syndrome, bronze diabetes, hemochromatosis, bronze cirrhosis, spinal-bulbar muscular atrophy, X-linked spinal-bulbar muscular atrophy, Bürger-Grütz syndrome, lipoprotein lipase deficiency, familial CADASIL syndrome, chronic granulomatous disorder (CGD),Ankle dysplasia, familial cancer syndromes, hereditary nonpolyposis colorectal cancer, breast cancer, bladder cancer, carboxylase deficiency, multiple late-onset biotinidase deficiency, cri-a-cat syndrome, Köhler cardio-facial syndrome, ceramide trihexosidase deficiency, cerebellar retinal angiomatosis, familial von Hippel-Lindau disease, cerebral arteriopathy, CADASIL syndrome, cerebral autosomal dominant arteriopathy (ateriopathy), CADASIL syndrome, cerebral atrophic hyperammonemia, Rett syndrome, cerebroside lipidosis syndrome, Charcot disease, CHARGE syndrome, chondrodysplasia, cartilage formation Dysmorphic syndrome, chondrodysplasia with sensorineural hearing loss, otospondylomegaly epiphyseal dysplasia, achondroplasia, choreoathetosis (self-injurious behavior), hyperuricemia syndrome (Lesch-Nyhan syndrome), classic galactosemia, galactosemia, cleft lip and palate, Stickler syndrome, cloverleaf cephalic dysplasia with lethal dwarfism, lethal osteodysplasia (e.g., type 1 or type 2), Coffin-Lowry syndrome (CLS), Cockayne syndrome, Coffin-Lowry syndrome, collagen dysplasia types II and XI, familial nonpolyposis colorectal cancer, hereditary nonpolyposis colorectal cancer, familial ... Intestinal cancer, familial adenomatous polyposis, colorectal cancer, complete HPRT deficiency (Lesch-Nyhan syndrome), complete hypoxanthine-guanine phosphoribosyltransferase deficiency, compression neuropathy, hereditary neuropathy with liability to pressure palsies, connective tissue disease, conotruncal dysfacies syndrome, Cooley anemia, beta-thalassemia, copper storage disease, Wilson's disease, copper transport disease, Menkes disease, coproporphyria, hereditary coproporphyria, coproporphyrinogen oxidase deficiency, Cowden syndrome, CPX deficiency, craniofacial joint abnormalities, Crouzon syndrome, craniofacial dysostosis, Crohn's syndrome, Crohn's disease, fibrostenosis, Crouzon syndrome, Crouzon syndrome with acanthosis nigricans, Crouzon cutaneous skeletal syndrome, Crouzon cutaneous skeletal syndrome, Cockayne syndrome (CS), Cowden syndrome, Kurschmann-Batten-Steinert syndrome, Behle-Stevenson gyral scalp syndrome, Behle-Stevenson gyral scalp syndrome, D-glycerate dehydrogenase deficiency, hyperoxaluria, primary mottled metaphyseal syndrome, Strudwick spondyloepiphyseal dysplasia, dementia of the Alzheimer type (DAT), hereditary hypercalciuria, Dent's disease,Muscular dystrophies (e.g., Duchenne and Becker), hearing loss associated with goiter, Pendred syndrome, deafness-retinitis pigmentosa syndrome, Usher syndrome, phenylalanine hydroxylase deficiency disorders, neurodegenerative disorders, de Grouchy syndrome 1, De Grouchy syndrome, Dejerine-Sottas syndrome, delta-aminolevulinic acid dehydratase deficiency porphyria, dementia (CADASIL syndrome), demyelinating leukodystrophy, Alexander disease, cutaneous Ehlers-Danlos syndrome (fragile skin), hereditary developmental disorders, distal hereditary motor neuropathy (dHMN), distal hereditary motor neuropathy (e.g., DHMN-V), DHTR deficiency, androgen insensitivity syndrome, diffuse globoid body sclerosis, Krabbe disease, DeJones syndrome Zy's syndrome, dihydrotestosterone receptor deficiency, androgen insensitivity syndrome, distal hereditary motor neuropathy, myotonic dystrophy (type 1 or 2), distal spinal muscular atrophy (any type, e.g., types 1, 2, 3, 4, 5, and 6), Duchenne / Becker muscular dystrophy, dwarfism (any type, e.g., achondroplasia, achondroplasia, lethal skeletal dysplasia), dwarfism-retinal atrophy-deafness syndrome, Cockayne syndrome, demyelinating leukodystrophy (Alexander disease), myotonic dystrophy, dystrophic retinitis pigmentosa (Usher syndrome), early-onset familial Alzheimer's disease (EOFAD), Alzheimer's disease (including, for example, types 1, 2, 3, or 4), Ekman-Lobstein disease, osteogenesis imperfecta, entrapment neuropathy, hereditary neuropathy with liability to pressure palsies, erythropoietic protoporphyria (EPP), erythroblastic anemia, beta-thalassemia, erythroblastic hepatic protoporphyria, erythrocyte 5-aminolevulinic acid synthase deficiency, X Linked sideroblastic anemia, Eye cancer, Retinoblastoma FA - Friedreich's ataxia, Friedreich's ataxia, FA, Fanconi anemia, Facial injuries and disorders, Factor V Leiden thrombophilia, FALS, Amyotrophic lateral sclerosis, Familial acoustic neuroma, Familial adenomatous polyposis, Familial Alzheimer's disease (FAD), Familial amyotrophic lateral sclerosis, Amyotrophic lateral sclerosis, Familial dysautonomia, Familial lipoprotein lipase deficiency, Familial hemochromatosis, Hemochromatosis,Familial LPL deficiency (familial lipoprotein lipase deficiency), familial nonpolyposis colon cancer, hereditary nonpolyposis colorectal cancer, familial paroxysmal polyserositis, familial PCT, porphyria cutanea tarda, familial pressure-sensitive neuropathy, hereditary neuropathy with liability to pressure palsy, familial primary pulmonary hypertension (FPPH), familial vasculo-leukoencephalopathy, CADASIL syndrome, FAP (familial adenomatous polyposis), FD (familial dysautonomia), ferrochelatase deficiency, ferroportin disease, hemochromatosis (any type, e.g., type 1, type 2A, type 2B) , type 3, type 4, neonatal hemochromatosis, aceruloplasmin deficiency, congenital atransferrinemia, Gracile syndrome, periodic fever syndrome, familial Mediterranean fever (FMF), FG syndrome, FGFR3-related coronal synostosis, fibrinoid degeneration of astrocytic cells (Alexander disease), fibrocystic disease of the pancreas, Folling disease, fra(X) syndrome, fragile X syndrome, fragile bone disease, osteogenesis imperfecta, FRAXA syndrome, Friedreich's ataxia (FRDA), G6PD deficiency, galactokinase deficiency Galactosylceramidase deficiency (galactosemia), galactose-1-phosphate uridyltransferase deficiency (galactosemia), galactosylceramidase deficiency (Krabbe disease), galactosylceramide lipidosis (Krabbe disease), galactosylcerebrosidase deficiency, galactosylsphingosine lipidosis, GALC deficiency, GALT deficiency (galactosemia), Gaucher disease-like disease, pseudo-Gaucher disease, GBA deficiency, hereditary encephalopathy, hereditary pulmonary emphysema, hereditary hemochromatosis, hemochromatosis, giant cell hepatitis (neonatal), neonatal hemochromatosis, GLA deficiency, retinal Glioblastoma (retinoblastoma), retinal glioma (retinoblastoma), globoid cell leukodystrophy (GCL, GLD), (Krabbe disease), globoid cell leukoencephalopathy, glucocerebrosidase deficiency, glucocerebrosidosis, glucosylcerebroside lipidosis, glucosylceramidase deficiency, glucosylceramide beta-glucosidase deficiency, glucosylceramide lipidosis, glyceric aciduria, primary hyperoxaluria, glycine encephalopathy, nonketotic hyperglycinemia, glycolic aciduria, primary hyperoxaluria, GM2 gangliosidosis,Tay-Sachs disease, goiter-deafness syndrome (Pendred syndrome), Graef-Usher syndrome (Usher syndrome), Glenn-Blood-Strandberry syndrome, pseudoxanthoma elasticum, hemochromatosis, Hallgren syndrome, Usher syndrome, harlequin ichthyosis, Hb S disease, chondrohyperplasia, Hemoglobin M disease (HCH), hereditary coproporphyria (HCP), head and brain malformations, hearing impairment and deafness, hearing problems in children, HEF2A, HEF2B, hematoporphyria (porphyria), heme synthesis enzyme deficiency, hemochromatosis, hemoglobin M disease, beta-globin methemoglobinemia, hemoglobin S disease, hemophilia, myeloid hepatic porphyria (HEP), hepatic AGT deficiency, primary hyperoxaluria, hepatolenticular degeneration syndrome, Wilson's disease, hereditary Articular-ocular diseases, Stickler syndrome, Hereditary ectopic lipidosis, Hereditary hemochromatosis (HHC) (hemochromatosis), Hereditary hemorrhagic telangiectasia (HHT), Hereditary inclusion body myositis, Skeletal muscle regeneration, Hereditary iron-addition anemia, X-linked sideroblastic anemia, Hereditary motor and sensory neuropathies, Hereditary motor neuropathy, type V, Distal hereditary motor neuropathy, Hereditary multiple exostoses, Hereditary nonpolyposis colorectal cancer, Hereditary periodic fever syndromes, Hereditary polyposis coli, Familial adenomatous polyposis Possibility of pulmonary emphysema, hereditary resistance to activated protein C, factor V Leiden thrombophilia, hereditary sensory and autonomic neuropathy type III, familial dysautonomia, hereditary spastic paraplegia, infantile-onset ascending hereditary spastic motor paralysis, hereditary spinal ataxia, Friedreich's ataxia, hereditary spinal sclerosis (Friedreich's ataxia), Herrick's anemia, heterozygous OSMED (Weisenbacher-Zweimüller syndrome), heterozygous otospinal macrobiontosis Acral dysplasia (Weissenbacher-Zweimüller syndrome), HexA deficiency (Tay-Sachs disease), Hexosaminidase A deficiency (Tay-Sachs disease), Hexosaminidase alpha-subunit deficiency (any variant, e.g., variant A, variant B) (Tay-Sachs disease), HFE-associated hemochromatosis (hemochromatosis), HGPS (progeria), Hippel-Lindau disease (von Hippel-Lindau disease), Hemochromatosis (HLAH), Distal hereditary motor neuropathy (HMN V), hereditary nonpolyposis colorectal cancer (HNPCC), hereditary neuropathy with liability to pressure palsies (HNPP), homocystinuria, homogentisic acid oxidase deficiency (alkaptonuria), homogentisic aciduria (alkaptonuria), homozygous porphyria cutanea tarda (myelohepatic porphyria), primary hyperoxaluria (HP1),Hyperoxaluria (HP2), hyperphenylalaninemia (HPA), HPRT-hypoxanthine-guanine phosphoribosyltransferase deficiency (Lesch-Nyhan syndrome), HSAN Type III, familial dysautonomia, familial dysautonomia (HSAN3), hereditary sensory neuropathy (any type, e.g., HSN-1, HSN-II, HSN-III), familial dysautonomia, human fragile skin, Huntington's disease, Hutchinson-Gilford progeria syndrome (progeria), non-classic hyperandrogenism due to 21-hydroxylase deficiency, hyperchylomicronemia, familial lipoprotein lipase deficiency, familial hyperglycinemia (propionic acidemia) with ketoacidosis and leukopenia, hyperlipoproteinemia type I, lipoprotein lipase deficiency, familial hyperoxaluria, primary hyperphenylalaninaemia, hyperphenylalanineemia, chondrodysplastic hypochondroplasia, hypochondroplasia, hypochromic anemia, X-linked sideroblastic anemia, hypoxanthine phosphatase Hormone transferase (HPRT) deficiency, Lesch-Nyhan syndrome, infantile-onset ascending hereditary spastic paralysis (IAHSP), ICF syndrome, immunodeficiency, centromere instability and facial malformation syndrome, idiopathic hemochromatosis, hemochromatosis type 3, idiopathic neonatal hemochromatosis (hemochromatosis), neonatal idiopathic pulmonary hypertension, immune system disorders, X-linked severe combined immunodeficiency, incontinentia pigmenti, infantile cerebral Gaucher disease, infantile Gaucher disease, infantile-onset ascending hereditary spastic paralysis Paralysis, infertility, hereditary emphysema, hereditary tendency to pressure palsies, hereditary neuropathy with liability to pressure palsies, Insley-Astley syndrome (otospondylomegaly epiphyseal dysplasia), intermittent acute porphyria syndrome (acute intermittent porphyria), intestinal polyposis-cutaneous pigmentation syndrome (Peutz-Jeghers syndrome), incontinentia pigmenti (IP), iron storage disorder (hemochromatosis), isodicentric 15, isodicentric 15, nonsyndromic deafness, Jackson-Weiss syndrome, Joubert syndrome, juvenile primary lateral sclerosis (JPLS), juvenile amyotrophic lateral sclerosis, juvenile gout, choreoathetosis, mental retardation syndrome, Lesch-Nyhan syndrome, juvenile hyperuricemia syndromeLesch-Nyhan syndrome, Jackson-Weiss syndrome (JWS), spinal-bulbar muscular atrophy, Kennedy disease, spinal-bulbar muscular atrophy, Kennedy spinal-bulbar muscular atrophy, spinal-bulbar muscular atrophy, keratin histiocytosis, keratin lipoidosis, keratin storage disease, ketotic glycinemia (propionic acidemia), ketotic hyperglycinemia (propionic acidemia), kidney disease, primary hyperoxaluria, Kniest dysplasia, Krabbe disease, Kugelberg-Welander disease, spinal muscular atrophy, lacunar dementia, CADASIL syndrome, Langer-Saldino achondrogenesis , Langer-Saldino dysplasia, late-onset Alzheimer's disease, late-onset Krabbe disease (LOKD) (Krabbe disease), learning disability, learning disability, perioral lentigines, Peutz-Jeghers syndrome, Lesch-Nyhan syndrome, leukodystrophy, leukodystrophy with Rosenthal fibers (Alexander disease), spongiform leukodystrophy, Li-Fraumeni syndrome (LFS), Li-Fraumeni syndrome lipase D deficiency, lipoprotein lipase deficiency, familial LIPD deficiency, lipoprotein lipase deficiency, familial cerebroside lipidosis, breast Childhood ganglioside lipidosis, Tay-Sachs disease, Lipoid histiocytosis (kerasin type), Lipoprotein lipase deficiency, Familial liver disease (galactosemia), Lou Gehrig's disease, Louis-Barr syndrome, Ataxia-telangiectasia, Lynch syndrome (hereditary nonpolyposis colorectal cancer), Lysyl hydroxylase deficiency, Machado-Joseph disease, Spinocerebellar degeneration (any type, e.g., SCA1, SCA2, SCA3, SCA18, SCA20, SCA21, SCA23, SCA26, SCA28, SCA29), Male breast cancer (breast cancer), Male reproductive disorders, Malignant neoplasms of the breast ( Breast cancer), malignant tumor of the breast (breast cancer), malignant tumor of the bladder (bladder cancer), cancer of the breast (breast cancer), Marfan syndrome, Marfan X syndrome, Fragile X syndrome, Martin-Bell syndrome, Fragile X syndrome, McCune-Albright syndrome, McLeod syndrome, MEDNIK syndrome, Mediterranean anemia, beta-thalassemia, gigantic epiphyseal dwarfism, otospondylomegaly epiphyseal dysplasia, Menkea's disease (Menkes disease), Menkes disease, mental retardation with osteochondral abnormalities, Coffin-Lowry syndrome, metabolic disorders, morphologic dwarfism type II, Kniest dysplasia, morphologic dysplasia type II, Kniest dysplasia,Methemoglobinemia (any type, e.g., congenital, beta-globin, congenital methemoglobinemia type II), methylmalonic acidemia, Marfan syndrome (MFS), MHAM, Cowden syndrome, Micro syndrome, microcephaly, MMA, methylmalonic acidemia, Menkes disease (also known as MK or MNK), monosomy 1p36 syndrome, motor neuron disease, amyotrophic lateral sclerosis, movement disorder, Mowat-Wilson syndrome, mucopolysaccharidosis (MPS) I), pancreatic fibrosis, multiple sclerosis dementia, CADASIL syndrome, multiple carboxylase deficiency, late-onset biotinidase deficiency, multiple hamartoma syndrome (Cowden syndrome), multiple neurofibromatosis, muscular dystrophy (any type, including Duchenne and Becker), atrophic myotonia, myotonic dystrophy, myotonic dystrophy, Nance-Insley syndrome (otospondylomegaly epiphyseal dysplasia), Nance-Sweene syndrome ychondrodysplasia (otospondylomegalyphyseal dysplasia), NBIA1, pantothenate kinase-associated neurodegeneration, Neil-Dingwall syndrome, Cockayne syndrome, retinal neuroblastoma, retinoblastoma, neurodegeneration with brain iron accumulation type 1, pantothenate kinase-associated neurodegeneration, neurological disorders, neuromuscular disorders, distal hereditary motor neuropathy, Niemann-Pick (Niemann-Pick disease), Noack syndrome, nonketotic hyperglycinemia, glycine encephalopathy, non-neuropathic Gaucher disease, Non-phenylketonuric hyperphenylalaninemia, tetrahydrobiopterin deficiency, non-syndromic hearing loss, Noonan syndrome, Norbotton-type Gaucher disease, histochromia, alkaptonuria, alkaptonuric arthritis, Ogden syndrome, osteogenesis imperfecta (OI), Osler-Weber-Rendu disease, hereditary hemorrhagic telangiectasia, OSMED (otospondylomegaphyseal dysplasia), osteogenesis imperfecta, osteofragility (osteogenesis imperfecta), congenital osteosclerosis (otospondylomegaphyseal dysplasia) dysplasia), otospondylomegaly epiphyseal dysplasia (otospondylomegaly epiphyseal dysplasia), oxalosis, primary hyperoxaluria, primary oxaluria, primary hyperoxaluria, pantothenate kinase-associated neurodegeneration, Patau syndrome (trisomy 13), PBGD deficiency (acute intermittent porphyria), PCC deficiency, propionic acidemia, porphyria cutanea tarda (PCT), PDM disease, Pendred syndrome, periodic diseases (Mediterranean fever, familial periodic peritonitis),Perioral multiple lentigines syndrome (Peutz-Jeghers syndrome), peripheral neuropathy (familial dysautonomia), peripheral neurofibromatosis, peroneal muscular atrophy, peroxisomal alanine:glyoxylate aminotransferase deficiency, hyperoxaluria, primary Peutz-Jeghers syndrome, phenylalanine hydroxylase deficiency disease, pheochromocytoma, von Hippel-Lindau disease, Pierre-Robin syndrome with fetal chondrodysplasia, Weissenbacher-Zweimüller syndrome, pigmentary cirrhosis (hemochromatosis), Peutz-Jeghers syndrome (PJS), pantothenate kinase-associated neurodegeneration (PKAN), PKU, phenylketonuria, pulmopovolphys dyslipidemia, ALA deficiency porphyria, PMA, polycystic kidney disease, polyostotic fibrous dysplasia (McCune-Albright syndrome), familial adenomatous polyposis, hamartomatous polyposis coli, polyps and pigmented spots syndrome (Peutz-Jeghers syndrome), porphobilinogen synthase deficiency, ALA deficiency porphyria, porphyrin disorders, PPOX deficiency, variegate porphyria, Prader-Larbhardt-Willi syndrome, Prader-Willi syndrome, presenile and senile dementia, primary ciliary dyskinesia (PCD), primary hemochromatosis (hemochromatosis), primary hyperuricemia syndrome, Lesch-Nyhan syndrome, primary senile degenerative dementia, procollagen-type EDS VII, Mutant Progeroid Hutchinson-Gilford Progeria Syndrome, Progeria-like Syndrome (Cockayne Syndrome), Progeria Dwarfism (Cockayne Syndrome), Progressive Chorea, Chronic Hereditary (Huntington's), Huntington's Disease, Osteogenesis Imperfecta Progressiva with Normal Sclera, Osteogenesis Imperfecta (Any Type, e.g., Type I, II, III, IV, V, VI, VII, VIII), Proximal Myotonic Dystrophy (PROMM), Propionic Acidemia, Propionyl-CoA Carboxylase Deficiency deficiency, protein C deficiency, protein S deficiency, protoporphyria, protoporphyrinogen oxidase deficiency, variegate porphyria, proximal myotonic dystrophy, myotonic dystrophy type 2, proximal myotonic myopathy, pseudo-Gaucher disease, pseudoxanthoma elasticum, psychosine lipidosis (Krabbe disease), pulmonary arterial hypertension, pulmonary hypertension, pseudoxanthoma elasticum (PXE), pseudoxanthoma elasticum, retinoblastoma (Rb), Recklinghausen's disease, recurrent polyserositis,Retinopathy, Retinitis Pigmentosa-Hearing Loss Syndrome, Usher Syndrome, Retinoblastoma, Rett Syndrome, RFALS Type 3, Ricker Syndrome, Riley-Day Syndrome, Familial Dysautonomia, Lucy-Levi Syndrome, Rubinstein-Taybi Syndrome (RSTS), Rett Syndrome (RTS), Rubinstein-Taybi Syndrome, Rubinstein-Taybi Syndrome, N-Taybi syndrome, Sac-Barabas syndrome, SADDAN disease, Li and Fraumeni familial sarcoma syndrome (Lie-Fraumeni syndrome), SBLA syndrome (Sarcoma, Breast, Leukemia, and Adrenal Syndrome) (Li-Fraumeni syndrome), Spinal-bulbar muscular atrophy (SBMA), Schwannoma, bilateral, Acoustic neurofibromatosis type II, Schwartz-Jamper syndrome, X-linked severe combined immunodeficiency (SCIDX1), Congenital SED, Spondyloepiphyseal dysplasia congenita, Strudwick type SED, Strudwick type spondyloepiphyseal dysplasia congenita (SEDc), Spondyloepiphyseal dysplasia (SEMD), Strudwick type SEMD, Severe achondroplasia with senile dementia, developmental delay and acanthosis nigricans, SADDAN disease, Shprintzen syndrome, Siderius caused by mutations in the PHF8 gene X-linked mental retardation syndrome, Achondroplasia, Disorders of skin pigmentation, Spinal muscular atrophy (SMA), Spondyloepiphyseal dysplasia (SMED) (any type, e.g., Studwick, type 1), Smith-Lemli-Opitz syndrome, Smith-Magenis syndrome, South African hereditary porphyria, Infantile-onset ascending spastic motor paralysis, Infantile-onset ascending hereditary spastic motor paralysis, Speech and communication disorders, Sphingolipidosis, Tay-Sachs (Tay-Sachs disease), Spinal-bulbar muscular atrophy, Spinal muscular atrophy, Spinal muscular atrophy (distal type V), Distal hereditary motor neuropathy, Distal spinal muscular atrophy with upper limb dominance, Distal hereditary motor neuropathy, Spinocerebellar degeneration, Congenital spondyloepiphyseal dysplasia, Spondyloepiphyseal dysplasia, Collagen anomalies (any type, e.g., types II and XI), spondyloepiphyseal dysplasia, spondyloepiphyseal dysplasia (SMD) (spondyloepiphyseal dysplasia), spondyloepiphyseal degeneration of the central nervous system, spondyloepiphyseal degeneration of the brain, spondyloepiphyseal degeneration of the white matter of infancy, sporadic primary pulmonary hypertension, SSB syndrome, Setar syndrome (Menkes disease), Steinert disease (myotonic dystrophy), Steinert myotonic dystrophy syndrome (myotonic dystrophy), Stickler syndrome, stroke, CADASIL syndrome, Strudwick syndrome, subacute neuropathic Gaucher disease, Swedish hereditary porphyria, acute intermittent porphyria, acute intermittent porphyria, Swiss cheese chondrodysplasia, Kniest dysplasia, Tay-Sachs disease, TD-lethal dwarfism, lethal skeletal dysplasia,TD with straight femurs and cloverleaf skull, Thoracthal skeletal dysplasia type 2, Telangiectasia, Ataxia oculocutaneous telangiectasia, Testicular feminization syndrome (androgen insensitivity syndrome), Tetrahydrobiopterin deficiency, Testicular feminization syndrome (TFM) (androgen insensitivity syndrome), Thalassemia intermedia (beta-thalassemia), Thalassemia major (beta-thalassemia), Thoracthal skeletal dysplasia, Activated protein C cofactor deficiency, Leiden thrombophilia (Factor V Leiden thrombophilia), Thyroid disease, Sausage neuropathy, Hereditary neuropathy with liability to pressure palsies, Total HPRT deficiency, Lesch-Nyhan syndrome, Total hypoxanthine-guanine phosphoribosyltransferase deficiency, Lesch-Nyhan syndrome, Trehar-Collins syndrome, Trisigmoid osteomyelitis, Triple X syndrome, Triplo X syndrome, Trisomy 1 syndrome M1 21, Trisomy X, Troageau-Hanot-Schauffard syndrome (hemochromatosis), Tay-Sachs disease (TSD), Tuberous sclerosis syndrome (TSC), Tuberous sclerosis complex, Turner-like syndrome, Noonan syndrome, UDP-galactose-4-epimerase deficiency disease, galactosemia, UDP-glucose 4-epimerase deficiency disease, galactosemia, UDP-glucose hexose-1-phosphate uridylyltransferase deficiency, galactosemia, undifferentiated hearing loss, nonsyndromic hearing loss, UPS deficiency, acute intermittent porphyria, bladder cancer, bladder cancer, UROD deficiency, uroporphyrinogen decarboxylase deficiency, uroporphyrinogen synthase deficiency, acute intermittent porphyria, Usher syndrome, UTP-hexose-1-phosphate uridylyltransferase deficiency (galactosemia), Van Bogaert-Bertrand syndrome, van der Havel syndrome, palatocardiofacial syndrome, VHL syndrome, von Hippel-Lindau disease, visual dysfunction and blindness, Alström syndrome, Von Bogaert-Bertrand disease, von Hippel-Lindau disease, Von Recklenhausen-Applebaum disease (hemochromatosis), von Recklinghausen disease, neurofibromatosis type I, Frolik disease, osteogenesis imperfecta, Waardenburg syndrome, Warburg-Sjo-Fledelius syndrome, Micro syndrome, Wilson disease (WD),Weissenbacher-Zweimüller syndrome, Werdnig-Hoffmann disease, spinal muscular atrophy, Williams syndrome, Wilson's disease, Wilson's disease, Wilson's disease, Wolf-Hirschhorn syndrome, Wolff periodic disease, Weissenbacher-Zweimüller syndrome (WZS), xeroderma pigmentosum, X-linked mental retardation and macroorchidism, fragile X syndrome, X-linked primary hyperuricemia (Lesch-Nyhan syndrome), X-linked severe combined immunodeficiency, X-linked sideroblastic anemia, X-linked spinal-bulbar muscular atrophy, spinal-bulbar muscular atrophy atrophy, X-linked uric acid uric enzyme deficiency, (Lesch-Nyhan syndrome), X-SCID, X-linked severe combined immunodeficiency, X-linked sideroblastic anemia (XLSA), X-SCID, X-linked severe combined immunodeficiency, X-linked sideroblastic anemia (XLSA), XSCID, X-linked severe combined immunodeficiency, X-linked sideroblastic anemia (XLSA), XSCID, X-linked severe combined immunodeficiency, XXX syndrome, triple X syndrome, XXXX syndrome, XXXXX syndrome, XXXXX, XXY syndrome, XXY trisomy, Klinefelter syndrome, XYY syndrome, triple repeat disorder, or any combination thereof.

[0332] In certain embodiments, specific post-transcriptional regulatory modulators are targeted for modulation, alteration, enhancement, or reduction of activity by administering a DNA-PK inhibitor and a genome editing system. For example, post-transcriptional regulatory modulators include PARN, PAN, CPSF, CstF, PAP, PABP, PAB2, CFI, CFII, RNA triphosphatase, RNA glutamyltransferase, RNA methyltransferase, SAM synthase, ubiquitin-conjugating enzyme E2R, SR proteins SFRS1 to SFR11, hnRNP proteins (e.g., HNRNPA0, HNRNPA1, HNRNPA1L1, HNRNPA1L2, HNRNPA2, HNRNPA2B1, HNRNPAB, HNRNPB1, HNRNPC, HNRNPCL1, HNRNPD, HNRPDL, HNRNPF, HNRNHP1, HNRNPH2, HNRNPH3, HNRNPK, HNRNPL, HNR Examples of such proteins include NPLL, HNRNPM, HNRNPR, HNRNPU, HNRNPUL1, HNRNPUL2, HNRNPUL3, ADAR, Mex67, Mtr2, Nab2, Dead-box helicase, eIF4A, eIF4B, eIF4E, eIF4G, GEF, GCN2, PKR, HRI, PERK, eEF1, eEF2, GCN, eRF3, ARE-specific binding proteins, EXRN1, DCP1, DCP2, RCK / p54, CPEB, eIF4E, microRNAs and siRNAs, DICER, Ago proteins, nonsense-mediated mRNA decay proteins, UPF3A, UPF3BeIF4A3, MLN51, Y14 / MAGOH, MG-1, SMG-5, SMG-6, SMG-7, or any combination thereof.

[0333] In some embodiments, the activity of gene pathways related to cell cycle is modulated, enhanced or reduced by administering a DNA-PK inhibitor and a genome editing system. Exemplary pathways and genes related to cell cycle include ATM, PMS2, FAS-L, MRE11, MLH1, FasR, NBS1, MSH6, Trail-L, RAD50, MSH2, Trail-R, 53BP1, RFC, TNF-Ct, P53, PCNA, TNF-R1, CHKE, MSH3, FADD, E2F1, MutS homolog, TRADD, PML, MutL homolog, R1P1, FANCD2, exonuclease, MyD88, SMC1, DNA, polymerase delta, IRAK, BLM1, (POLD1, POLD2, POLD3, NIL, BRCA1, and POLD4 - genes encoding IKK and H2AX subunits), NFκβ, ATR, topoisomerase 1, IκBα, RPA, topoisomerase 2, IAP, ATRIP, RNAseHl, caspase 3, RAD9, ligase 1, caspase 6, RAD1, DNA polymerase 1, caspase 7, HUS, DNA polymerase 3, caspase 8, RAD17, primase, caspase 10, RFC, helicase, HDAC1 , CHK1, single-stranded, binding, HDAC2, TLK1 protein, cytochrome C, CDC25, Bxl-xL, STAT3, STAT5, DFF45, Vcl-2, ENDO-G, PI3K, Akt, calpain, Bad, Bax, ubiquitin-mediated proteolysis, hypoxia, cell proliferation, HIF-loc, MAPK, El, HERC1, TRAF6, HIF-Ιβ, MAPKK, E2, UBE2Q, MEKK1, Refl, MAPKKK, E3, UBE2R, COP!, HSP90, c-Met, UBLE1A, UBE 2S, PIFH2, VEGF, HGF, UBLE1B, UBE2U, cIAP, PAS, ER, S1 / 2, UBLEIC, UBE2W, PIAS, ARNT, ATK, UBE2A, UBE2Z, SYVN, VHL, PKC, UBE2B, AFC, LLC, N , NHLRC1, HLF, Paxillin, UBE2C, UBE1, AIRE, EPF, FAK, UBE2A, E6AP, MGRN1, VDU2, Adducin, UBE2E, UBE3B, BRCA1, SUMORESUME, PYK1, UBE2F, Smurf,FANCL, SENP1, RB, UBE2G1, Itch, MIDI, Calcineurin A, RBI, UBE2G2, HERC2, Cdc20, RACK1, Raf-1, UBE2I, HERC3, Cdhl, PTB, A-Raf, UBE2J1, HERC4, Apcl, Hur, B-raf, UBE2J2, UBE4A, Apc2, PHD2, MEK1 / 2, UBE2L3, UBE4B, Apc3, SSAT2, ERK1 / 2, UBE2L6, CHIP, Apc4, SSAT1, Ets, UBE2M, CYC4, Apc5, GSK3, Elkl, UBE2N, PPR19, Apc6, C BP, SAP1, UBE20, UIP5, Apc7, FOX04, cPLA2, WWPI, Mdm2, Apc8, FlH-1, WWP2, Parkin, Apc9, TRIP, 12, Trim32, Ape10, NEED4, Trim37, Ape11, ARF-BP1, SIAH-1, Ape12, EDD1, PML, cell viability, cell cycle arrest, SMADI, P21, SMAD5, BAX, SAMD8, MDR, LEF1, DRAIL, IGFBP3, TCF3, GADD45, TCF4, P300, HAT1, PI3, Akt, GF1, or any combination thereof.

[0334] In some embodiments, the activity of genes associated with angiogenesis is modulated, enhanced, or reduced by administering a DNA-PK inhibitor and a genome editing system to a cell (or cells).Exemplary genes and gene pathways associated with angiogenesis and angiogenesis-related conditions include VEGF, VEGFR2, SHC, E2F7, VEGFB, VEGFR3, PI3, VEGFC, Nrp1, PIP3, EGFDIP3, DAG, GRB2, SOS, Akt, PB, PKC, Ras, RAF1, DAG, eNOS, NO, ERK1, ER2, cPLA2, ME1, MEK2, or any combination thereof.

[0335] In some embodiments, the activity of gene pathways and / or genes associated with mitochondrial function is modulated, enhanced, or reduced by administering to a cell(s) a DNA-PK inhibitor and a genome editing system. Exemplary genes and gene pathways associated with mitochondrial function include malate dehydrogenase, aminotransferase, hydratase, deacylase, dehydrogenase, carboxylase, mutase, fatty acid oxidation, leucine oxidation, isoleucine disorder (enzyme pathway oxidative pathway deficiency), aminotransferase, aminotransferase, OCTN2, branched chain aminotransferase, FATP1-6, aminotransferase 2, CPT-1, mitochondrial mitochondrial, CACT, isobutyl-CoA, 2-methylbutyl-CoA, CPT-II, dehydrogenase, SCAD, branched chain dehydrogenase, MCAD, ketoacid, VLCAD, dehydrogenase, ETF-DH complex, alpha-ETF hydratase, beta-ETF Examples include HMG-CoA lyase 2-methyl-3-OH-SCHAD butyryl-CoA, LCHAD dehydrogenase, MTP3-oxothiolase, LKAT, DECR1, HMGCS2, HMGCL, or any combination thereof.

[0336] In some embodiments, the activity of gene pathways and / or genes associated with DNA damage or genomic instability is modulated, enhanced, or reduced. Exemplary genes and gene pathways associated with pathways and / or genes related to DNA damage and genomic instability include 53BP1, BLM, MBD2, DNA ligase 4, MDC1, H2AX, XLF, SMC1, 53BP1, Rad50, P53, P53, Artemis, Rad27, TdT, APE1, PMS2, APE2, UvrA, RecA, MLH1, NEIL1, UvrB, SSB, MSH6, NEIL2, UvrC, Mrell, MSH2, NEIL3, XPC, Rad50, RFC, XRCC1, Rad23B, Nbsl, PCNA, PNKP, CEN2, CtIP, MSH 3, Tdpl, DDB1, RPA, MutS, APTX, XPE, Rad51, MutL, DNA polymerase β, CSA, Rad52, DNA polymerase δ, CSB, Rad54, Topoisomerase 1, DNA, TFT1H, BRCA1, Topoisomerase 2, PCNA, XPB, BRCA2, RNAseHl, FEN1, XPD, Exol, Ligase 1, RFC, XPA, BLM, DNA, Polymerase 1, PAR, 1, RPA, Topllla, DNA, Ligl, XPG, GEN1, Primase, Lig3, ERCC1Yenl helicase, UNG, XPF, Slxl, SSB, MUTY DNA polymerase δ, Slx4, SMUG DNA polymerase ε, Mus8, MBD4, Emel, Dssl, ASH1L, SETD4, DQT1L, SETD5, EHMT1, SETD6, EHMT2, SETD7, EZH1, SETD8, EZH2, SETD9, MLL, SETDB1, MLL2, SETDB2, MLL3, SETMAR, MLL4, SMYD,1, MLL5, SMYD2, NSD,1, SMYD3, PRDM2, SMYD4, SET, SMYD5, SETBP1, SUV39H1, SETD1A, SUV39H2, SETD1B, SUV420H1, SETD2, SUV420H2, SETD3, or any combination thereof.

[0337] In some embodiments, a gene encoding a mammalian transcription factor is modulated, enhanced, reduced, or provided to a cell. Exemplary human transcription factors include AFF4, AFF3, AFF2, AFF1, AR, TFAP2B, TFAP2D, TFAP2C, TFAP2E, TFAP2A, JARID2, KDM5D, ARID4A, ARID4B, KDM5A, ARID3A, KDM5B, KDM5C, ARID5B, ARID3B, ARID2, ARID5A, ARID3C, ARID1A, ARID1B, HIF1A, NPAS1, NPAS3, NPAS4, MLXIPL, ARNTL2, MXD1, AHRR, TFE3, HES2, MNT, TC F3, SREBF1, TFAP4, TCFL5, LYL1, USF2, TFEC, AHR, MLX, MYF6, MYF5, SIM1, TFEB, HAND1, HES1, ID2, MYCL1, ID3, TCF21, MXI1, SOHLH2, MYOG, TWIST 1, NEUROG3, BHLHE41, NEUROD4, MXD4, BHLHE23, TCF15, MAX, ID1, MYOD1, ARNTL, BHLHE40, MYCN, CLOCK, HEY2, MYC, ASCL1, TCF12, ARNT, HES6, FER D3L, MSGN1, USF1, TAL1, NEUROD1, TCF23, HEYL, HAND2, NEUROD6, HEY1, SOHLH1, MESP1, PTF1A, ATOH8, NPAS2, NEUROD2, NHLH1, ID4, ATOH1, ARNT2 , HES3, MLXIP, ASCL3, KIAA2018, OLIG3, NHLH2, NEUROG2, MSC, HES7, ATOH7, BHLHA15, BHLHE22, NEUROG1, FIGLA, ASCL2, OLIG1, TAL2, MITF, SCXB , HELT, ASCL4, MESP2, HES4, SCXA, TCF4, HES5, SREBF2, BHLHA9, OLIG2, MXD3, TWIST2, LOC388553, C13orf38-SOHLH2, CEBPE, XBP1, BATF3, CREB5 , CEBPG, ATF3, ATF7, CEBPB, CEBPD, CEBPA, CBFB, CAMTA2, CAMTA1, EBF4, EBF3, EBF1, EBF2, NR2F6, NR2F1, NR2F2, GRHL2, TFCP2L1, GRHL1, TFCP2,UBP1、GRHL3、YBX2、CSDE1、CSDA、YBX1、LIN28A、CARHSP1、CSDC2、LIN28B、NFIX、NFIC、NFIB、NFIA、CUX2、ONECUT2、CUX1、ONECUT1、SATB1、ONECUT3、SATB2、DMRT3、DMRT1、DMRTC2、DMRTA2、DMRTB1、DMRT2、DMRTA1、E2F2、E2F1、E2F3、TFDP2、E2F8、E2F5、E2F7、E2F6、TFDP3、TFDP1、E2F4、NR1H3、NR1H2、ETV1、ETV7、SPI1、ELF4、ETV2、ERF、ELF2、ELK3、ETV3、ELF1、SPDEF、ELK1、ETS1、EHF、ELF5、ETV6、SPIB、FLI1、GABPA、ERG、ETS2、ELK4、ELF3、FEV、SPIC、ETV4、ETV5、FOXN3、FOXC1、FOXJ2、FOXF1、FOXN1、FOXM1、FOXP1、FOXO3、FOXA2、FOXP2、FOXJ1、FOXP4、FOXF2、FOXN4、FOXK2、FOXO1、FOXH1、FOXQ1、FOXK1、FOXI1、FOXD4、FOXA3、FOXN2、FOXB1、FOXG1、FOXR1、FOXL1、FOXC2、FOXE1、FOXS1、FOXL2、FOXO4、FOXD4L1、FOXD4L4、FOXD2、FOXI2、FOXE3、FOXD3、FOXD4L3、FOXR2、FOXJ3、FOXO6、FOXB2、FOXD4L5、FOXD4L6、FOXD4L2、KIAA0415、FOXA1、FOXP3、GCM2、GCM1、NR3C1、GTF2IRD1、GTF2I、GTF2IRD2B、GTF2IRD2、SOX8、SOX30、PMS1、CIC、TCF7、TOX4、SOX10、HMGXB4、HBP1、TFAM、UBTF、WHSC1、SOX6、HMGXB3、BBX、TOX2、SOX4、SOX21、SOX9、SOX15、SOX5、SOX3、LEF1、HMG20A、SOX13、TCF7L2、SSRP1、TCF7L1、SOX17、SOX14、PINX1、SOX7、SOX11、SOX12、SOX2、SOX1、SRY、SOX18、UBTFL1、UBTFL2、TOX、HMGB1、HMGB2、PBRM1、TOX3、SMARCE1、HMG20B、HMGB3、HMGA2、HMGA1、ARX、HOXA11、MEOX1、DLX6、ISL1、HOXC8、BARX2、ALX4、GSC2、DLX3、PITX1、HOXA9、HOXA10、LHX5、LASS4、ZFHX4、SIX4、VSX1、ADNP、RHOXF1、MEIS3、PBX4、DLX5、HOXA1、HOXA2、HOXA3、HOXA5、HOXA6、HOXA13、EVX1、NOBOX、MEOX2、LHX2、LHX6、LHX3、TLX1、PITX3、HOXB6、HNF1B、DLX4、SEBOX、VTN、PHOX2B、NKX3-2、DBX1、NANOG、IRX4、CDX1、TLX2、DLX2、VAX2、PRRX1、TGIF2、VSX2、NKX2-3、HOXB8、HOXB5、HOXB7、HOXB3、HOXB1、MSX2、LHX4、HOXA7、HOXC13、HOXC11、HOXC12、ESX1、BARHL1、NKX2-4、NKX2-2、SIX1、HOXD1、HOXD3、HOXD9、HOXD10、HOXD11、HOXD13、MNX1、CDX4、BARX1、RHOXF2、LHX1、GSC、MEIS2、RAX、EMX1、NKX2-8、NKX2-1、HLX、LMX1B、SIX3、LBX1、PDX1、LASS5、ZFHX3、BARHL2、LHX9、LASS2、MEIS1、DLX1、HMBOX1、ZEB1、VAX1、NKX6-2、VENTX、HHEX、TGIF2LX、LASS3、ALX3、HOXB13、IRX6、ISL2、PKNOX1、LHX8、LMX1A、EN1、MSX1、NKX6-1、HESX1、PITX2、TLX3、EN2、UNCX、GBX1、NKX6-3、ZHX1、HDX、PHOX2A、PKNOX2、CDX2、DRGX、NKX3-1、PBX3、PRRX2、GBX2、SHOX2、GSX1、HOXD4、HOXD12、EMX2、IRX1、IRX2、SIX2、HOXB9、HOPX、OTP、LASS6、HOXC5、HOXB2、RAX2、EVX2、ZHX3、PROP1、ISX、HOXD8、TGIF2LY、IRX5、SIX5、TGIF1、IRX3、ZHX2、LBX2、NKX2-6、ALX1、GSX2、HOXC9、HOXC10、HOXB4、NKX2-5、SIX6、MIXL1、DBX2、PBX1、<h2 style=";text-align:left;direction:ltr">SHOX、ARGFX、HMX3、HMX2、BSX、HOXA4、DMBX1、HOXC6、HOXC4、RHOXF2B、PBX2 、DUXA、DPRX、LEUTX、NOTO、HOMEZ、HMX1、DUX4L5、DUX4L2、DUX4L3、DUX4L6、N KX1-1、HNF1A、HSF4、HSFY2、HSFX1、HSFX2、HSFY1、HSF1、LCORL、LCOR、IRF6、 IRF1、IRF3、IRF5、IRF4、IRF8、IRF2、IRF7、IRF9、MBD3、BAZ2B、MBD4、SETDB2 、MBD1、MECP2、SETDB1、MBD2、BAZ2A、SMAD7、SMAD5、SMAD9、SMAD6、SMAD4、S MAD3、SMAD1、SMAD2、ZZZ3、RCOR1、CDC5L、MYBL2、DNAJC2、TADA2A、RCOR3、MY B、TERF2、DMTF1、DNAJC1、NCOR1、TERF1、MIER3、MYSM1、SNAPC4、RCOR2、TADA 2B、MYBL1、TERF1P2、NCOR2、CCDC79、SMARCC1、SMARCC2、TTF1、C11orf9、NFY A、NFYC、NFYB、NRF1、NR4A3、NR4A1、NR4A2、ESR1、NR0B2、NR0B1、PREB、EAF2 、SPZ1、TP63、TP73、TP53、PAX6、PAX7、PAX2、PAX4、PAX8、PAX1、PAX3、PAX5、P AX9、SUB1、POU2F2、POU1F1、POU4F3、POU6F2、POU2F3、POU2F1、POU4F2、POU4 F1、POU6F1、POU3F2、POU3F1、POU3F4、POU3F3、POU5F1、POU5F1B、PPARD、PPA RG、PPARA、PGR、PROX1、PROX2、NR2E1、NR5A2、NR2C1、NR5A1、NR6A1、ESRRA、N R2C2、RFX3、RFX2、RFX4、RFX1、RFX5、RFX7、RFX6、RFX8、NFATC3、NFKB2、NFAT C4、NFATC2、NFAT5、RELB、NFKB1、NFATC1、REL、RELA、RORA、RORC、NR1D2、ROR B、RUNX3、RUNX1、SP100、SP140、GMEB2、SP110、AIRE、GMEB1、DEAF1、SP140L、<h2 style=";text-align:left;direction:ltr">LOC729991-MEF2B、MEF2A、SRF、MEF2D、MEF2B、STAT1、STAT5A、STAT4、STAT6 、STAT3、STAT2、STAT5B、TBX21、TBX5、TBX15、TBX18、TBX2、TBX4、TBX22、TBX 3、TBR1、TBX19、TBX6、EOMES、T、TBX20、TBX10、MGA、TBX1、TEAD3、TEAD2、TEA D1、TEAD4、CREBL2、NFE2L3、CREB3L3、FOSL2、NFE2L1、CREM、DBP、CREB3、HLF、 BACH2、ATF2、NFE2L2、ATF6、CREB1、ATF1、NFE2、FOSB、ATF4、NRL、JUND、JDP2 、CREB3L4、BATF、BACH1、CREB3L1、NFIL3、TEF、BATF2、ATF5、FOS、JUNB、DDIT3 、FOSL1、JUN、MAF、CREB3L2、MAFA、MAFF、MAFG、MAFK、MAFB、ATF6B、CRX、OTX1 、OTX2、THAP3、THAP10、THAP1、PRKRIR、THAP8、THAP9、THAP11、THAP2、THAP6、 THAP4, THAP5, THAP7, NR1H4, NR2E3, RARB, HNF4A, VDR, ESRRB, THRA, NR1D1, RARA, ESR2, NR1I3, NR1I2, THRB, NR3C2, HNF4G, RARG, RXRA, ESRG, RXRB, TSC 22D1、TSC22D3、TSC22D4、TSC22D2、TULP3、TULP2、TULP1、TULP4、TUB、ZBTB3 3、ZBTB32、ZBTB11、MYNN、ZBTB25、PATZ1、ZBTB16、ZBTB24、BCL6、ZBTB47、ZBT B17、ZBTB45、GZF1、ZBTB1、ZBTB46、ZBTB8A、ZBTB7B、BCL6B、ZBTB49、ZBTB43 、HIC2、ZBTB26、ZNF131、ZNF295、ZBTB4、ZBTB34、ZBTB38、HIC1、ZBTB41、ZBTB 7A, ZNF238, ZBTB42, ZBTB2, ZBTB20, ZBTB40, ZBTB7C, ZBTB37, ZBTB3, ZBTB6, ZBTB44, ZFP161, ZBTB12, ZBTB48, ZBTB10, ZBED4, ZBED3, ZBED2, C11orf95,ZBED1、IKZF5、ZNF821、ZNF451、ZNF195、ZFX、ZNF263、ZNF200、HIVEP2、WIZ、ZNF582、SNAI2、ZFP64、IKZF2、ZIC2、ZNF800、PRDM1、PRDM6、ZFP112、ZNF275、ZNF76、 ZFAT、KLF6、ZFY、ZXDC、GLI2、ZNF532、ZNF37A、ZNF510、ZNF506、ZNF324、ZNF671、ZNF416、ZNF586、ZNF446、ZNF8、ZNF264、REST、MECOM、ZNF213、ZNF343、ZNF302、ZNF268、ZNF10、HIVEP1、ZNF184、MZF1、SALL4、ZNF516、KLF8、KLF5、ZNF629、ZNF423、CTCF、ZNF500、ZNF174、SALL1、MAZ、ZNF419、OVOL3、ZNF175、ZNF14、ZNF574、ZNF85、SP4、ZKSCAN1、GLI3、GLIS3、KLF3、PRDM4、GLI1、PRDM13、ZNF142、PRDM2、ZNF684、ZNF541、KLF7、PLAGL1、ZNF430、KLF12、KLF9、ZNF410、BCL11A、EGR1、ZFP30、TSHZ3、ZNF549、ZSCAN18、ZNF211、ZNF639、ZSCAN20、GTF3A、ZNF205、ZNF644、EGR2、IKZF4、CTCFL、ZNF831、SNAI1、ZNF576、ZNF45、TRERF1、ZNF391、RREB1、ZNF133、OVOL2、ZNF436、PLAGL2、GLIS2、ZNF384、ZNF484、HIVEP3、BCL11B、KLF2、ZNF780B、FEZF1、KLF16、ZSCAN10、ZNF557、ZNF337、PRDM12、ZNF317、ZNF426、ZNF331、ZNF236、ZNF341、ZNF227、ZNF141、ZNF304、ZSCAN5A、ZNF132、ZNF20、EGR4、ZNF670、VEZF1、KLF4、ZFP37、ZNF189、ZNF193、ZNF280D、PRDM5、ZNF740、ZIC5、ZSCAN29、ZNF710、ZNF434、ZNF287、ZIM3、PRDM15、ZFP14、ZNF787、ZNF473、ZNF614、PRDM16、ZNF697、ZNF687、OSR1、ZNF514、ZNF660、ZNF300、RBAK、ZNF92、ZNF157、ZNF182、ZNF41、ZNF711、PRDM14、ZNF7、ZNF214、ZNF215、SALL3、ZNF827、ZNF547、ZNF773、ZNF776、ZNF256、ZSCAN1、ZNF837、PRDM8、ZNF117、ZIC1、FEZF2、ZNF599、ZNF18、KLF10、ZKSCAN2、ZNF689、ZIC3、ZNF19、ZSCAN12、ZNF276、ZNF283、ZNF221、ZNF225 、ZNF230、ZNF222、ZNF234、ZNF233、ZNF235、ZNF362、ZNF208、ZNF714、ZNF394、ZNF333、ZNF382、IKZF3、ZNF577、ZNF653、ZNF75A、GFI1、ZNF281、ZNF496、ZNF496 NF2、ZNF513、ZNF148、KLF15、ZNF691、ZNF589、PRDM9、ZNF12、SP8、OSR2、ZNF367、ZNF22、GFI1B、ZNF219、SALL2、ZNF319、ZNF202、ZNF143、ZNF3、ZSCAN21 ZNF606、SP2、ZNF91、ZNF23、ZNF226、ZNF229、ZNF180、ZNF668、ZNF646、ZNF641、ZNF610、ZNF528、ZNF701、ZNF526、ZNF146、ZNF444、ZNF83、ZNF558、ZNF23 2、E4F1、ZNF597、INSM2、ZNF30、ZNF507、ZNF354A、ZEB2、ZNF32、KLF13、ZFPM2、ZNF764、ZNF768、ZNF35、ZNF778、ZNF212、ZNF282、PRDM10、SP7、SCRT1、ZNF 16、ZNF296、ZNF160、ZNF415、ZNF672、ZNF692、ZNF439、ZNF440、ZNF581、ZNF524、ZNF562、ZNF561、ZNF584、ZNF274、ZIK1、ZNF540、ZNF570、KLF17、ZNF217 ,ZNF57,ZNF556,ZNF554,KLF11,HINFP,ZNF24,ZNF596,OVOL1,SP3,ZNF621,ZNF680,BNC2,ZNF483,ZNF449,INSM1,ZNF417,ZNF791,ZNF80,GLIS1,ZNF497,KLF14,ZNF266,ZIC4,ZNF408,ZNF519,ZNF25,ZNF77,ZNF169,ZNF613,ZNF683,ZNF135,ZSCAN2,ZNF575,ZNF491,ZNF620,ZNF619,ZNF354C,ZNF114,ZNF366、ZNF454、ZNF543、ZNF354B、ZNF223、ZNF713、ZNF852、ZNF552、ZFP42、ZNF664、EGR3、ZFPM1、ZNF784、ZNF648、FIZ1、ZNF771、TSHZ1、ZNF48、ZNF816、ZNF571、ZSCAN4、ZNF594、ZFP3、ZNF443、ZNF792、ZNF572、ZNF707、ZNF746、ZNF322A、ZNF467、ZNF678、ZFP41、HKR1、PLAG1、ZNF329、ZNF101、ZNF716、ZNF708、ZSCAN22、ZNF662、ZNF320、ZNF623、ZNF530、ZNF285、ZFP1、WT1、ZFP90、ZNF479、ZNF445、ZNF74、SP1、SNAI3、ZNF696、IKZF1、ZNF267、ZNF566、ZNF224、ZNF529、ZNF284、ZNF749、ZNF17、ZNF555、ZNF75D、ZNF501、ZNF197、ZNF396、ZFP91、ZNF732、ZNF397、ZSCAN30、ZNF546、ZNF286A、ZKSCAN4、ZNF70、ZNF643、ZNF642、ZSCAN23、ZNF490、ZNF626、ZNF793、ZNF383、ZNF669、ZNF559、ZNF177、ZNF548、MTF1、ZNF322B、ZNF563、ZNF292、ZNF567、SP6、ZNF573、ZNF527、ZNF33A、ZNF600、ZKSCAN3、ZNF676、ZNF699、ZNF250、ZNF79、ZNF681、ZNF766、ZNF107、ZNF471、ZNF836、ZNF493、ZNF167、ZNF565、ZNF34、ZNF781、ZNF140、ZNF774、ZNF658、ZNF765、ZNF124、ZNF569、ZNF777、ZNF775、ZNF799、ZNF782、ZNF846、ZNF136、ZKSCAN5、ZNF502、ZFP62、ZNF33B、ZNF512B、ZNF431、ZNF418、ZNF700、ZNF239、ZSCAN16、ZFP28、ZNF705A、ZNF585A、ZNF138、ZNF429、ZNF470、ZNF100、ZNF398、ZNF498、ZNF441、ZNF420、ZNF763、ZNF679、ZNF682、<h2 style=";text-align:left;direction:ltr">ZNF772、ZNF257、ZNF785、ZSCAN5B、ZNF165、ZNF655、ZNF98、ZNF786、ZNF517 、ZNF675、ZNF860、ZNF628、ZNF665、ZNF624、ZNF841、ZNF615、ZNF350、ZNF432 、ZNF433、ZNF460、ZNF81、ZNF780A、ZNF461、ZNF181、LOC100287841、ZNF44、 ZNF790、ZNF677、ZNF823、ZNF311、ZNF347、ZNF71、ZNF121、ZNF335、ZNF560、Z NF273、ZNF84、ZNF667、ZNF649、ZNF248、ZNF544、ZNF770、ZNF737、ZNF251、Z NF607、ZNF334、ZXDA、ZNF485、ZIM2、PEG3、ZNF192、ZNF442、ZNF813、ZNF26、Z NF69、ZNF583、ZNF568、ZXDB、ZNF480、ZNF587、ZNF808、ZNF43、ZNF28、ZNF62 7、ZNF789、ZNF536、ZNF534、ZNF652、ZNF521、ZNF358、ZFP2、SP5、ZNF814、ZNF 551、ZNF805、ZSCAN5C、ZNF468、ZNF616、ZFP57、ZNF155、ZNF783、ZNF425、ZN F580、ZNF611、ZNF254、ZNF625、ZNF134、ZNF845、ZNF99、ZNF253、ZNF90、ZNF9 3、ZNF486、REPIN1、LOC100131539、ZNF705D、LOC100132396、ZNF705G、SCRT 2、ZNF407、SP9、ZNF579、ZNF880、ZNF630、ZNF844、ZNF469、ZNF717、ZNF865、Z NF492、ZNF688、YY2、ZNF878、ZNF879、ZNF736、ZNF323、ZNF709、ZNF512、ZNF 585B、ZNF154、ZNF324B、ZNF564、ZFP82、GLI4、ZNF674、ZNF345、ZNF550、KLF1 YY1, MYST2, ST18, L3MBTL4, MYT1L, MYT1, L3MBTL1, MTA3, GATA1, TRPS1, GATA3, GATA5, GATA4, GATA6, GATAD2B, GATAD1, GATA2, MTA1, ZGLP1, MTA2, RERE,C16orf5, LITAF, PIAS1, PIAS2, PIAS4, ZMIZ1, ZMIZ2, PIAS3, RNF138, NFX1, NFXL1, or any combination thereof.

[0338] In some embodiments, cells are engineered (e.g., converted or differentiated) from one cell type to another. In some embodiments, pancreatic cells are engineered into beta pancreatic islet cells. In some embodiments, fibroblasts are engineered into iPS cells. In some embodiments, preadipocytes are engineered into brown adipocytes. Other exemplary cells include, for example, muscle cells, nerve cells, white blood cells, and lymphocytes.

[0339] In some embodiments, the cell is disease cell or mutation-carrying cell.Such cell can be engineered to treat disease, for example, correct mutation, or change cell phenotype, for example, inhibit the growth of cancer cell.For example, the cell is associated with one or more diseases or conditions described herein.

[0340] In some embodiments, the engineered cells are normal cells.

[0341] In some embodiments, the engineered cells are stem or progenitor cells (e.g., iPS, embryonic, hematopoietic, adipose, germ line, lung, or neural stem or progenitor cells). In some embodiments, the engineered cells can be cells from any of the three germ layers (i.e., mesoderm, endoderm, or ectoderm). In some embodiments, the engineered cells can be derived from extraembryonic tissue, for example, from the placenta.

[0342] In some embodiments, the engineered cells are selected from fibroblasts, monocyte precursors, B cells, exocrine cells, pancreatic precursors, endocrine precursors, hepatoblasts, myoblasts, or preadipocytes. In some embodiments, the cells are engineered (e.g., converted or differentiated) into muscle cells, erythroid-megakaryocyte cells, eosinophils, iPS cells, macrophages, T cells, pancreatic islet beta-cells, neurons, cardiomyocytes, blood cells, endocrine precursors, exocrine precursors, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, cholangiocytes, hemangioblasts, mesoangioblasts, or brown adipocytes.

[0343] In some embodiments, the cells are muscle cells, erythroid-megakaryocyte cells,

[0344] Eosinophils, iPS cells, macrophages, T cells, pancreatic islet beta-cells, neurons, cardiomyocytes, blood cells, endocrine precursors, exocrine precursors, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, bile duct cells, or white or brown adipocytes.

[0345] In some embodiments, the cell is a progenitor cell, a pluripotent cell, a totipotent cell, an adult stem cell, an inner cell mass cell, an embryonic stem cell, or an iPS cell.

[0346] In some embodiments, the engineered cells are cancer cells, which may be lung cancer cells, breast cancer cells, skin cancer cells, brain cancer cells, pancreatic cancer cells, hematopoietic cancer cells, liver cancer cells, kidney cancer cells, ovarian cancer cells, prostate cancer cells, or skin cancer cells.

[0347] In some embodiments, the cell is a muscle cell, an erythroid-megakaryocyte cell, an eosinophil, an iPS cell, a macrophage, a T cell, a pancreatic islet beta-cell, a neuron, a cardiomyocyte, a blood cell, an endocrine precursor, an exocrine precursor, a ductal cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a hepatocyte, a bile duct cell, or a white or brown adipocyte.

[0348] Administration of a DNA-PK inhibitor and a gene-editing system to a cell(s)

[0349] Administration of the genome editing system and the DNA-PK inhibitor to a cell(s) can be carried out by any method known in the art. Administration can be in vitro, ex vivo, or in vivo. Administration of the genome editing system and the DNA-PK inhibitor to a cell(s) can be simultaneous or sequential. In some embodiments, administration results in entry of the DNA-PK inhibitor and genome editing system components into the cell membrane. In some embodiments, administration results in entry of the DNA-PK inhibitor and genome editing system components into the cell nucleus. In some embodiments, administration includes incubating the cells in the presence of the DNA-PK inhibitor and the genome editing system.

[0350] The gene editing system can be administered to a cell(s) by any method known in the art. For example, any nucleic acid or protein delivery method known in the art can be used. The gene editing system is administered (e.g., delivered) to a cell as a nucleic acid encoding the components of the gene editing system. The gene editing system can be administered to a cell by either a viral vector or a non-viral vector. In some embodiments, a viral vector is used. The viral vector can be a retrovirus (e.g., murine leukemia, HIV, or lentivirus) or a DNA virus (e.g., adenovirus, herpes simplex, and adeno-associated). In some embodiments, the genome editing system is introduced into a cell using a transfection method (e.g., a non-viral delivery method). Transfection methods include contacting the cell with DEAE-dextran, calcium phosphate, liposomes, or electroporating a plasmid into the cell. Additional methods of non-viral delivery include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and drug-enhanced DNA uptake. Sonoporation, for example, using the Sonitron 2000 system (Rich-Mar), can also be used to deliver nucleic acids. In some embodiments, one or more nucleic acids are delivered as mRNA. In some embodiments, capped mRNA is used to increase translation efficiency and / or mRNA stability. In some embodiments, an ARCA (anti-reverse cap analog) cap or a variant thereof is used. See U.S. Patents US7074596 and US8153773.

[0351] In one embodiment, the endonuclease (e.g., Cas, Cpf1, etc.) and gRNA are transcribed from DNA.

[0352] In one embodiment, the endonuclease (e.g., Cas, Cpf1, etc.) is transcribed from DNA and the gRNA is provided as RNA.

[0353] In one embodiment, the endonuclease (e.g., Cas, Cpf1, etc.) and gRNA are provided as RNA.

[0354] In one embodiment, the endonuclease (e.g., Cas, Cpf1, etc.) is provided as a protein and the gRNA is provided as DNA.

[0355] In one embodiment, the endonuclease (e.g., Cas, Cpf1, etc.) is provided as a protein and the gRNA is provided as RNA.

[0356] Additional nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA) and Copernicus Therapeutics Inc. (see, for example, US6008336). Lipofection is described, for example, in U.S. Patent Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™, Lipofectin™, and Lipofectamine™ RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in Feigner, WO91 / 17424, WO91 / 16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).

[0357] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy, 2:710-722 (1995)).

[0358] An additional delivery method involves packaging the nucleic acid to be delivered into an EnGeneIC delivery vehicle (EDV). These EDVs are specifically delivered to target tissues using bispecific antibodies, one arm of which has specificity for the target tissue and the other arm for the EDV. The antibody delivers the EDV to the surface of the target cell, which then delivers the EDV into the cell by endocytosis. Once inside the cell, the contents are released (MacDiarmid et al (2009) Nature Biotechnology 27(7):643; Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0359] In some embodiments, transfection can be transient, in which case the transfected genome editing system containing the plasmid enters the nucleus but does not integrate into the cell's genome during replication. Transfection can be stable, in which case the transfected plasmid integrates into the cell's genomic region.

[0360] In some embodiments where transient expression is used, an adenovirus-based system can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. This vector can be mass-produced using a relatively simple system. Adeno-associated virus ("AAV") vectors have also been used, for example, to transduce cells with target nucleic acids in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, for example, West et al., Virology, 160:38-47 (1987); U.S. Patent No. 4,797,368; WO93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in several publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol., 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol 63:03822-3828 (1989).

[0361] In some embodiments, administration of the DNA-PK inhibitor to a cell(s) is carried out by culturing the isolated cell(s) in the presence of the DNA-PK inhibitor and any suitable medium that allows the DNA-PK inhibitor to enter the cell membrane and / or the cell nucleus.

[0362] In some embodiments, the DNA-PK inhibitor is administered to a cell(s) in vitro, in vivo, or ex vivo. In some embodiments, the DNA-PK inhibitor is contacted with a cell(s) for about 5 hours, 10 hours, 15 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 85 hours, 90 hours, 100 hours, 125 hours, 150 hours, 200 hours, or any period therebetween. In some embodiments, the DNA-PK inhibitor is contacted with a cell(s) for about 1.5 weeks, 2.0 weeks, 2.5 weeks, 3.0 weeks, 3.5 weeks, 4 weeks, or any period therebetween. The DNA-PK inhibitor may be re-administered together with a change of cell culture medium. The DNA-PK inhibitor may be contacted with a cell before, during, or after the introduction of a genome editing system component.

[0363] In some embodiments, the DNA-PK inhibitor is administered to a cell(s) at a concentration of about 0.1 μM, 0.25 μM, 0.5 μM, 0.75 μM, 1.0 μM, 1.25 μM, 1.50 μM, 1.75 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM, 5.5 μM, 6.0 μM, 6.5 μM, 7.0 μM, 7.5 μM, 8.0 μM, 8.5 μM, 9.0 μM, 9.5 μM, 10 μM, 10.5 μM, 11.0 μM, 11.5 μM, 12 μM, or any concentration therebetween. The DNA-PK inhibitor concentration can be modified during the course of administration.

[0364] In some embodiments, the gene editing components are delivered to cell(s) by one or more vectors, or in the form of RNA, mRNA, or in the case of an endonuclease component, as purified protein or mRNA (e.g., Cas9 protein). The one or more vectors can include a viral vector, a plasmid, or ssDNA. The viral vector can include a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex viral vector, or any combination thereof. In some embodiments, the gene editing components are delivered via RNA or synthetic RNA.

[0365] In some embodiments, administering a DNA-PK inhibitor to a cell together with a gene editing system increases the amount of gene editing results from homologous recombination repair compared to baseline conditions in which the DNA-PK inhibitor is not administered to the cell. In some embodiments, administering a DNA-PK inhibitor to a cell(s) together with a gene editing system results in the suppression of on-target and off-target indels (derived from NHEJ). In some embodiments, administering a DNA-PK inhibitor to a cell(s) together with a gene editing system results in increased or decreased expression of a gene of interest. Administering a DNA-PK inhibitor to a cell(s) together with a gene editing system can result in the expression of a gene that is not endogenous to the cell. In some embodiments, administering a DNA-PK inhibitor to a cell(s) together with a gene editing system results in the complete or partial removal or modification of a gene from the cell(s). In some embodiments, administering a DNA-PK inhibitor to a cell(s) together with a gene editing system results in the complete or partial removal or modification of an intron and / or exon in the cell(s). In some embodiments, administering a DNA-PK inhibitor together with a gene editing system to a cell(s) results in the complete or partial removal or modification of non-coding regions in the cell(s). In some embodiments, administering a DNA-PK inhibitor together with a gene editing system to a cell(s) results in the simultaneous or sequential complete or partial removal or modification of coding and / or non-coding genetic regions in the cell(s). In some embodiments, administering a DNA-PK inhibitor together with a gene editing system to a cell(s) results in the simultaneous or sequential complete or partial removal or modification of coding and / or non-coding genetic regions in the cell(s), including extrachromosomal DNA or RNA. The extrachromosomal DNA may be mitochondrial DNA, chloroplast DNA, extrachromosomal circular DNA, or viral extrachromosomal DNA.

[0366] In some embodiments, administering a DNA-PK inhibitor to cells together with a genome editing system results in increased or decreased expression of a gene of interest. In some embodiments, the increase or decrease in expression of the gene of interest can be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or any combination thereof, compared to a baseline condition in which a DNA-PK inhibitor is not administered to cells. In some embodiments, the increase or decrease in expression of the gene of interest can be about 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or 10-fold, or any combination thereof, compared to a baseline expression level in which a DNA-PK inhibitor is not administered to cells.

[0367] In some embodiments, administering a DNA-PK inhibitor to cells together with a genome editing system results in increased genome editing. In some embodiments, the increase in genome editing can be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or any combination thereof, compared to a baseline condition in which a DNA-PK inhibitor is not administered to cells. In some embodiments, the increase in genome editing can be about 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or 10-fold, or any combination thereof, compared to a baseline expression level in which a DNA-PK inhibitor is not administered to cells.

[0368] In some embodiments, administering a DNA-PK inhibitor and a gene editing system to a cell population results in greater cell survival compared to a baseline condition in which the cell population is administered only the gene editing system, but not the DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor that results in greater cell survival is a compound of Formula (III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof.

[0369] In some embodiments, cells are synchronized to the S or G2 cell cycle phase either before, after, or during administration of a DNA-PK inhibitor. In some embodiments, cells are synchronized to the S or G2 cell cycle phase either before, after, or during introduction of a gene editing component. Synchronization of cells in the S or G2 cell cycle phase can be achieved by any method known in the art. By way of non-limiting example, agents that can be used to synchronize cells to the S or G2 cell cycle phase include aphidicolin, hydroxyurea, lovastatin, mimosine, nocodazole, thymidine, or any combination thereof. (Lin et al., Elife. 2014 Dec 15;32014). In some embodiments, the agent for cell synchronization can be administered at any time during the gene editing process.

[0370] In some embodiments, the DNA-PK inhibitor agent and / or genome editing system can be included in a container, pack, or dispenser together with instructions for use. In some embodiments, the DNA-PK inhibitor agent and / or genome editing system included in a container, pack, or dispenser together with instructions for use is a kit.

[0371] In some embodiments, the DNA-PK inhibitor and / or genome editing system is included in a kit together with instructions for use. The kit can contain any genome editing system and / or DNA-PK inhibitor and instructions for use. In some embodiments, the DNA-PK inhibitor is a compound represented by structural formula (III-E-1) or (III-E-2). In some embodiments, the genome editing system is selected from a meganuclease-based system, a zinc finger nuclease (ZFN)-based system, a transcription activator-like effector-based nuclease (TALEN) system, a CRISPR-based system, or an NgAgo-based system. The genome editing system can be provided in the kit in any form, for example, as a plasmid, vector, DNA, or RNA construct.

[0372] In some embodiments, the DNA-PK inhibitor and / or the genome editing system are administered in vivo.The DNA-PK inhibitor and the gene editing system are formulated to be compatible with their intended administration route.Examples of administration routes are described above.

[0373] In some embodiments, the formulation may also contain more than one active compound, as needed for the particular indication being treated, for example, those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0374] In some embodiments, the DNA-PK inhibitor drug and / or genome editing system is administered in combination therapy, i.e., in combination with other drugs, such as therapeutic agents, that are useful for treating pathological conditions or disorders, such as various forms of cancer and inflammatory diseases.The term "in combination" in this context means that the drugs are administered simultaneously or sequentially at substantially the same time.When administered sequentially, it is preferred that at the start of administration of the second compound, the first compound of the two compounds is still detectable at an effective concentration at the treatment site. Genome editing screening method

[0375] Any method known in the art can be used to screen cells for genome editing efficiency, including the efficiency of NHEJ and / or HDR.For example, screening method can include PCR-based amplification of target region to confirm genome editing, followed by sequencing or deep sequencing of the amplified region.PCR genotyping allows quantification and ranking of compounds in HDR stimulation.Another screening method can include next-generation sequencing.See, for example, Bell et al., "A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing," BMC Genomics, 15:1002 (2014).

[0376] PCR primers can be engineered to selectively amplify both unmodified and modified gene regions, resulting in amplicons of different lengths depending on the genetic modification status. The amplicons can then be resolved on a gel, and HDR efficiency can be predicted by densitometry using a Bio-Imager. Alternatively, a novel PCR technique, rapid digital droplet PCR (DDPCR), can be used to simultaneously measure HDR and NHEJ events in genome-edited samples. For example, Miyaoka et al. See, e.g., et al., "Systematic quantification of HDR and NHEJ reveals effectors of locus, nuclease, and cell type on genome-editing," Scientific Reports, 6, 2016. Other methods can be used to screen cells for genome modifications, including Sanger sequencing, deep sequencing, and RT-PCR. Preparation of Compounds of the Invention

[0377] As used herein, all abbreviations, symbols, and conventions are consistent with those used in the contemporary scientific literature, e.g., Janet S. Dodd, ed., The See ACS Style Guide: A Manual for Authors and Editors, 2nd Ed., Washington, DC: American Chemical Society, 1997. The following definitions describe terms and abbreviations used herein: BPin Pinacolboronic acid ester Brine NaCl saturated aqueous solution DCM dichloromethane DIAD Diisopropyl azodicarboxylate DIEA Diisopropylethylamine DMA Dimethylacetamide DMF Dimethylformamide DMSO dimethyl sulfoxide DTT Dithiothreitol ESMS Electrospray Mass Spectrometry Et2O ethyl ether EtOAc ethyl acetate EtOH Ethyl alcohol HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High Performance Liquid Chromatography IPA Isopropanol LAH Lithium Aluminum Hydride LC-MS Liquid Chromatography-Mass Spectrometry LDA Lithium diisopropylethylamide Me methyl MeOH Methanol MsCl methanesulfonyl chloride MTBE Methyl t-butyl ether NMP N-methylpyrrolidine Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0) Pd(dppf)Cl2 1,1'bis(diphenylphosphino)-ferrocenedichloro-palladium PG protecting group Ph Phenyl (rac)-BINAP Racemic 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl RockPhos Di-tert-butyl(2',4',6'-triisopropyl-3,6-dimethoxy-[1,1'-biphenyl]-2-yl)phosphine RT or rt Room temperature SFC Supercritical Fluid Chromatography SPhos 2-Dicyclohexylphosphino-2',6'-dimethoxybiphenyl TBAI Tetrabutylammonium iodide tBu tertiary butyl THF tetrahydrofuran TEA Triethylamine TMEDA Tetramethylethylenediamine VPhos [3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxyphenyl]sulfonyloxysodium General synthetic procedure

[0378] In general, the compounds of this invention can be prepared by the methods described herein or by other methods known to those skilled in the art. [Example]

[0379] Example 1 General Preparation of Compounds of Formula G [ka] Compounds of formula (III-E-1) or (III-E-2), where X is NH, can be prepared as outlined in Scheme 1 below. Thus, as shown in Scheme 1, step 1-i, a heteroaryl compound of formula A can be reacted with morpholine or a morpholine analog by heating the mixture in a polar, aprotic solvent to produce a compound of formula B. As shown in Scheme 1, step 1-ii, a palladium-catalyzed, phosphine ligand-assisted Buchwald / Hartwig-type coupling can be utilized to react a compound of formula B with an aminocyclohexane of formula C to produce a compound of formula D, where R 1 and R 2 (wherein R is as described elsewhere herein). In one example, when a monoprotected mesocyclohexane-1,4-diamine of formula E is prepared, removal of the protecting group forms a compound of formula F, as shown in step 1-iii of Scheme 1. The resulting free amine can then be reacted with various moieties bearing groups reactive towards amines (e.g., R 1a -L, where L is a leaving group such as chloro, bromo, iodo, toluenesulfonate, methanesulfonate, or trifluoromethanesulfonate, or L is a reactive carbonyl-containing moiety such as an activated ester or isocyanato group, to produce a compound of formula G, as shown in Scheme 1, step 1-iv. Example 2 General Preparation of Compounds of Formulas M, N, R, and S [ka]

[0380] Compounds of formula (III-E-1) or (III-E-2), where X is O, can be prepared as outlined in Schemes 2a and 2b below. Thus, as shown in step 2-i of Scheme 2a, the hydroxyl group of a heteroaryl compound of formula H can be protected to produce a compound of formula J, which can then be reacted with morpholine or a morpholine analog by heating the mixture in a polar, aprotic solvent to produce, after removal of the protecting group, a compound of formula K, as shown in steps 2-ii and 2-iii of Scheme 2a. Subsequently, as shown in step 2-iv of Scheme 2a, the compound of formula K is reacted with a compound of formula L under conditions sufficient to affect SN2 displacement of its leaving group (e.g., L is a leaving group, e.g., chloro, bromo, iodo, toluenesulfonate, methanesulfonate, or trifluoromethanesulfonate), to produce R 1 or R 2 Depending on whether R is hydrogen, compounds of formula M or M can be produced. 1 or R 2 When is protected at the nitrogen or oxygen moiety, the compounds of the invention can be generated by removal of the protecting group and subsequent synthetic manipulation of the resulting free amine / alcohol.

[0381] Alternatively, as shown in Scheme 2b, the hydroxyl group of a compound of formula O can be reacted with a compound of formula L to produce a fused bicycloheteroaryl bromide of formula P, which can then be reacted with morpholine or a morpholine analog to produce a compound of formula M or formula N. [ka]

[0382] Alternatively, as shown in Scheme 2c, compounds of the invention in which ring B is a dihydropyran ring can be prepared by reacting a compound of formula Q with a dialkyl(3,6-dihydro-2H-pyran-4-yl)boronate to produce a compound of formula R. The compound of formula R can then be subsequently reduced to form a compound of formula S. [ka] Example 3 Ethyl (4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate (Compound 6) and N 1 -(7-morpholinoquinoxalin-5-yl)-N 4 Preparation of -(pyrimidin-2-yl)cyclohexane-1,4-diamine (compound 18) [ka]

[0383] As shown in Scheme 3, step 3-i, to a solution of 3-bromo-5-fluoro-benzene-1,2-diamine (compound 1001, 1.11 g, 5.41 mmol) in methanol (11 mL) was added oxaldehyde (1.57 mL of 40% w / v, 10.8 mmol). The reaction mixture was stirred under nitrogen at room temperature. After 2 hours, a yellow solid precipitated. The reaction mixture was diluted with water (20 mL), stirred for an additional 5 minutes, filtered, and the collected solid was dried under high vacuum to produce 5-bromo-7-fluoroquinoxaline (compound 1002, 868 mg, 70.6% yield): [ka] ESMS(M+H + )=227.14.

[0384] As shown in Scheme 3, Step 3-ii, to a solution of 5-bromo-7-fluoroquinoxaline (4.5 g, 19.8 mmol) in NMP (67.5 mL) was added morpholine (3.1 mL, 35.6 mmol). The reaction mixture was heated to 140 °C and stirred for 15 h. After cooling, the mixture was poured into water (200 mL), extracted with ethyl acetate (2 × 100 mL), dried over magnesium sulfate, filtered, evaporated under reduced pressure, and purified by medium-pressure silica gel chromatography (10-80% EtOAc / hexane gradient) to give 4-(8-bromoquinoxalin-6-yl)morpholine (compound 1003, 3.86 g, 66% yield) as a yellow solid: [ka] ESMS(M+H + )=227.14.

[0385] As shown in Scheme 3, Step 3-iii, a mixture of 4-(8-bromoquinoxalin-6-yl)morpholine (1.57 g, 5.34 mmol), tert-butyl-N-(4-aminocyclohexyl)carbamate (1.37 g, 6.40 mmol), (rac)-BINAP (664 mg, 1.07 mmol), cesium carbonate (5.22 g, 16.0 mmol), and Pd(dba) (489 mg, 0.534 mmol) in toluene (50 mL) was heated at 100° C. for 12 hours. After cooling, the mixture was diluted with ethyl acetate (150 mL) and water (25 mL) and then filtered through diatomaceous earth, followed by an ethyl acetate wash. The combined organics were washed with brine, dried over sodium sulfate, concentrated under reduced pressure, and purified by medium pressure silica gel chromatography (0-60% EtOAc / hexanes gradient) to give tert-butyl (-4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate (compound 1004, 1.83 g, 83.2% yield): [ka]

[0386] As shown in Scheme 3, step 3-iv, to a solution of tert-butyl (-4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate (900 mg, 2.00 mmol) in dichloromethane (16 mL) was added trifluoroacetic acid (3 mL, 38.9 mmol). The resulting black reaction mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. Saturated aqueous sodium bicarbonate (150 mL) was added slowly until the color changed from black to orange. The mixture was extracted with dichloromethane (2 x 100 mL), and the combined organics were washed with brine (50 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford N 1 -(7-morpholinoquinoxalin-5-yl)cyclohexane-1,4-diamine, trifluoroacetate (compound 1005): [ka] [ka] ESMS(M+H + )=328.34. This compound was used as is without further purification.

[0387] As shown in Scheme 3, Step 3-v, N 1 To a solution of 4-(7-morpholinoquinoxalin-5-yl)cyclohexane-1,4-diamine (25 mg, 0.07 mmol) and diisopropylethylamine (18.0 mg, 24.3 μL, 0.14 mmol) in dichloromethane (750 μL) was added ethyl chloroformate (11.4 mg, 10.0 μL, 0.105 mmol). The reaction mixture was stirred for 12 h, diluted with dichloromethane (10 mL), washed with saturated aqueous sodium bicarbonate (5 mL), dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by HPLC preparative chromatography using a 10–90% acetonitrile / water (0.1% TFA) gradient as the eluent to give ethyl (4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate (compound 6, 14 mg, 50% yield): [ka] ESMS(M+H + )=400.17.

[0388] As shown in Step 3-vi of Scheme 3, N 1 A mixture of -(7-morpholinoquinoxalin-5-yl)cyclohexane-1,4-diamine (185 mg, 0.56 mmol), 2-bromopyrimidine (93 mg, 0.58 mmol), and triethylamine (143 mg, 197 μL, 1.41 mmol) in 1-methylpyrrolidin-2-one (3 mL) was heated to 130 °C and stirred for 15 h. After cooling to room temperature, the mixture was diluted with ethyl acetate (70 mL) and methyl tert-butyl ether (20 mL), washed with water (3 × 20 mL), brine (15 mL), dried over sodium sulfate, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (10–100% EtOAc / hexane gradient) to give N 1 -(7-morpholinoquinoxalin-5-yl)-N 4 -(pyrimidin-2-yl)cyclohexane-1,4-diamine was obtained (compound 18, 102 mg, 45% yield): [ka] ESMS(M+H + )=328.34. Example 4 Preparation of 1-(4-((7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)amino)cyclohexyl)-3-ethylurea (compound 22) [ka]

[0389] As shown in Scheme 4, Step 4-i, to a solution of 5-bromo-7-fluoroquinoxaline (compound 1002, 150 mg, 0.66 mmol) in NMP (2.3 mL) was added 8-oxa-3-azabicyclo[3.2.1]octane (178 mg, 1.2 mmol) at RT. The reaction mixture was sealed in a microwave vial and heated at 180° C. for 20 min. After cooling to RT and pouring into water, the aqueous phase was extracted with EtOAc (3×). The combined extracts were dried over MgSO4, filtered, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0-100% EtOAc / hexanes gradient) to give 3-(8-bromoquinoxalin-6-yl)-8-oxa-3-azabicyclo[3.2.1]octane (compound 1006, 87 mg, 41% yield) as a dark orange oil: ESMS (M+H) + )=320.07.

[0390] As shown in Scheme 4, Step 4-ii, a degassed solution of 3-(8-bromoquinoxalin-6-yl)-8-oxa-3-azabicyclo[3.2.1]octane (261 mg, 0.815 mmol), tert-butyl N-(4-aminocyclohexyl)carbamate (210 mg, 0.98 mmol), rac-BINAP (102 mg, 0.163 mmol), CsCO (797 mg, 2.45 mmol), and Pd(dba) (75 mg, 0.0815 mmol) in toluene (10.5 mL) was heated in a sealed microwave tube at 100 °C (oil bath temperature) for 15 h. After cooling, the mixture was directly loaded onto a chromatography column and purified by medium-pressure silica gel chromatography (0-100% EtOAc / hexanes gradient) to yield tert-butyl (4-((7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)amino)cyclohexyl)carbamate (compound 1007, 141 mg, 36% yield) as a white solid: [ka] ESMS(M+H + )=453.96.

[0391] As shown in Scheme 4, step 4-iii, to a solution of compound 1007 (141 mg, 0.295 mmol) in CHCl (2.5 mL) was added TFA (656 mg, 443 μL, 5.75 mmol) at RT. The resulting black solution was stirred for 2 h, and then the reaction was quenched by the addition of saturated NaHCO until the black color gradually turned orange. The reaction mixture was extracted with CHCl (3×), and the combined organic extracts were dried over NaSO, evaporated to dryness, and purified with N. 1 -(7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)cyclohexane-1,4-diamine, trifluoroacetate (compound 1008): ESMS (M+H + )=354.20. This material was used in subsequent reactions without any further purification.

[0392] As shown in Scheme 4, step 4-iv, to a solution of compound 1008 (45 mg, 0.071 mmol) and DIEA (36.5 mg, 49.0 μL, 0.28 mmol) in CHCl (1.4 mL) was added ethyl isocyanate (20 mg, 0.28 mmol) at RT. The solution was stirred at this temperature for 15 h and then directly loaded onto a chromatography column and purified by medium-pressure silica gel chromatography (0–100% EtOAc / hexane gradient) to produce 1-(4-((7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)amino)cyclohexyl)-3-ethylurea as a white solid (compound 22, 8 mg, 27% yield): [ka] ESMS(M+H + )=425.35. Example 5 N 1 -(6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)-N 4 Preparation of -(pyrimidin-2-yl)cyclohexane-1,4-diamine (compound 23) [ka]

[0393] As shown in Scheme 5, step 5-i, a mixture of tert-butyl ((cis)-4-aminocyclohexyl)carbamate (compound 1009, 490 mg, 2.3 mmol), 2-chloropyrimidine (262 mg, 2.3 mmol), and TEA (463 mg, 637 μL, 4.6 mmol) in DMF (10 mL) was subjected to microwave irradiation at 150 °C for 20 min. The reaction mixture was diluted with EtOAc, washed with HO, dried over NaSO, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0-50% EtOAc / hexanes gradient) to give tert-butyl ((cis)-4-(pyrimidin-2-ylamino)cyclohexyl)carbamate (compound 1010) as a white solid: [ka]

[0394] As shown in Scheme 5, step 5-ii, HCl (3 mL, 4 M in THF, 12 mmol) was added to compound 1010. The mixture was stirred for 30 min and concentrated under reduced pressure to give (cis)-N 1 -(pyrimidin-2-yl)cyclohexane-1,4-diamine hydrochloride was produced (compound 1011). This material was used directly in the subsequent reaction without further purification.

[0395] As shown in Scheme 5, Step 5-iii, 4-bromo-6-morpholinobenzo[c][1,2,5]oxadiazole (compound 1012, 147 mg, 0.5 mmol), (cis)-N 1A mixture of 1,4-(pyrimidin-2-yl)cyclohexane-1,4-diamine hydrochloride (120 mg, 0.6 mmol), (rac)-BINAP (32 mg, 0.05 mmol), Pd(dba) (24 mg, 0.026 mmol), and cesium carbonate (506 mg, 1.55 mmol) in toluene (5 mL) was flushed with nitrogen gas and stirred overnight at 90 °C under a nitrogen atmosphere. The mixture was filtered through a layer of diatomaceous earth, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0–80% EtOAc / hexane gradient) to give (cis)-N as an orange solid. 1 -(6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)-N 4 -(pyrimidin-2-yl)cyclohexane-1,4-diamine was obtained (compound 23): [ka] Example 6 Preparation of 5-methoxy-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (Compound 134) [ka]

[0396] As shown in Scheme 6, Step 6-i, to a mixture of 5-bromo-2-fluoro-pyrimidine (1 g, 5.651 mmol) in iPrOH (10 mL) was added TEA (1.143 g, 1.574 mL, 11.30 mmol) and trans-4-aminocyclohexan-1-ol (650.8 mg, 5.651 mmol). The mixture was microwaved at 150 °C for 20 min, concentrated under reduced pressure, diluted with EtOAc, washed with water, and dried over Na SO . After removing the volatiles under reduced pressure, the residue was purified by medium-pressure silica gel chromatography (0–80% EtOAc / hexane gradient) to give (trans)-4-((5-bromopyrimidin-2-yl)amino)cyclohexanol (compound 1013, 1.2 g): [ka] [ka]

[0397] As shown in Scheme 6, step 6-ii, to compound 1013 (1.2 g, 4.41 mmol) in DCM (20 mL) was added TEA (1.134 g, 1.84 mL, 13.2 mmol) and MsCl (505 mg, 341 μL, 4.41 mmol). The reaction mixture was stirred for 1 h, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0-80% EtOAc / hexanes gradient) to give trans-4-((5-bromopyrimidin-2-yl)amino)cyclohexyl methanesulfonate (compound 1014): [ka]

[0398] As shown in Scheme 6, Step 6-iii, to a solution of 2-amino-3-nitrophenol (5.00 g, 32.4 mmol) in dioxane (50 mL) was added bromine (6.22 g, 2.01 mL, 38.9 mmol). The mixture was stirred for 2 hours, and a precipitate formed, which was collected and washed with dioxane and ether. The resulting yellow solid was treated with saturated NaHCO solution and extracted with EtOAc (3x). The combined organics were dried over NaSO, filtered, and concentrated under reduced pressure to produce 2-amino-5-bromo-3-nitrophenol (compound 1015) as a brown solid. This material was used directly in subsequent reactions without further purification.

[0399] As shown in Scheme 6, step 6-iv, to a solution of 2-amino-5-bromo-3-nitrophenol (7.5 g, 31.8 mmol) in ethyl acetate (60 mL) was added Raney Nickel™ (1.90 g, 214 μL, 32.4 mmol), and the reaction mixture was shaken under an atmosphere of H at 30 psi for 2 hours. After filtration and drying over NaSO, the mixture was concentrated under reduced pressure to give 2,3-diamino-5-bromophenol (compound 1016), which was used directly in the subsequent reaction without further purification.

[0400] As shown in Scheme 6, Step 6-v, 2,3-diamino-5-bromophenol (6.0 g, 29.5 mmol) was dissolved in methanol, and to this solution was added glyoxal (3.77 g, 2.98 mL, 64.9 mmol) and stirred overnight. The reaction mixture was concentrated under reduced pressure to a minimum volume, and the resulting tan solid was collected by filtration and dried under high vacuum to yield 7-bromoquinoxalin-5-ol (compound 1017), which was used directly in the subsequent reaction without further purification.

[0401] As shown in Scheme 6, Step 6-vi, to a solution of 7-bromoquinoxalin-5-ol (2.0 g, 8.89 mmol) in DCM (20 mL) was added imidazole (1.82 g, 26.7 mmol) and tert-butyldimethylsilyl chloride (1.34 g, 1.65 mL, 8.89 mmol). The reaction mixture was stirred at RT overnight, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0-20% EtOAc / hexanes gradient) to give 7-bromo-5-((tert-butyldimethylsilyl)oxy)quinoxaline (compound 1018) as a colorless oil. [ka]

[0402] As shown in Scheme 6, Step 6-vii, a mixture of 7-bromo-5-((tert-butyldimethylsilyl)oxy)quinoxaline (700 mg, 2.06 mmol), morpholine (270 mg, 270 μL, 3.09 mmol), Pd(dba) (94.50 mg, 0.1032 mmol), (rac)-BINAP (129 mg, 0.206 mmol), and cesium carbonate (2.02 g, 6.19 mmol) in toluene (7 mL) was flushed with nitrogen for 10 minutes. The mixture was then heated at 100 °C overnight. After cooling, the reaction mixture was diluted with EtOAc, filtered through a layer of diatomaceous earth, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0–30% EtOAc / hexane gradient) to give 7-morpholinoquinoxalin-5-ol. This compound (450 mg, 1.3 mmol) was dissolved in THF (20 mL) and tetra-n-butylammonium fluoride (539 mg, 2.06 mmol) was added. The reaction mixture was stirred for 0.5 h, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0-100% EtOAc / hexanes gradient) to give 7-morpholinoquinoxalin-5-ol (compound 1019) as a yellow solid: [ka]

[0403] As shown in Scheme 6, step 6-viii, a solution of 7-morpholinoquinoxalin-5-ol (100 mg, 0.432 mmol), (trans)-4-((5-bromopyrimidin-2-yl)amino)cyclohexyl methanesulfonate (compound 1014, 303 mg, 0.865 mmol), and CsCO (282 mg, 0.865 mmol) in dioxane (1.0 mL) was stirred at 105 °C for 16 h. After cooling, the reaction mixture was diluted with EtOAc, filtered through diatomaceous earth, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0–5% MeOH / DCM gradient) to yield 5-bromo-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (compound 1020, 110 mg) as a yellow foam: [ka] [ka]

[0404] As shown in Scheme 6, Step 6-ix, a mixture of 5-bromo-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (75 mg, 0.155 mmol), cesium carbonate (101 mg, 0.309 mmol), allylpalladium(II) chloride dimer (0.28 mg, 0.0015 mmol), RockPhos (2.17 mg, 0.0046 mmol), and MeOH (9.9 mg, 12.5 μL, 0.31 mmol) in toluene (2 mL) was flushed with nitrogen gas and heated at 100° C. for 18 h. The reaction mixture was diluted with EtOAc, filtered through a layer of diatomaceous earth, and concentrated under reduced pressure. Purification by medium pressure silica gel chromatography (0-8% MeOH / DCM gradient) gave 5-methoxy-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (compound 134, 43 mg): [ka] Example 7 Preparation of 4-(8-(((trans)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)quinoxalin-6-yl)morpholine (Compound 34) and 4-(8-(((cis)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)-quinoxalin-6-yl)morpholine (Compound 42) [ka]

[0405] As shown in Scheme 7, step 7-i, to a solution of 1,4-dioxaspiro[4.5]decan-8-ol (compound 1021, 1.0 g, 6.32 mmol) in DMF (10 mL) was added NaH (370 mg, 9.25 mmol). The reaction mixture was stirred for 20 min, and then 2-chloropyrimidine (869 mg, 7.59 mmol) was added. The mixture was stirred at RT for 30 min and then heated to 100 °C for 9 h. After cooling, the mixture was diluted with EtOAc, washed with HO, dried over NaSO, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0–40% EtOAc / hexanes) to yield 2-(1,4-dioxaspiro[4.5]decan-8-yloxy)pyrimidine (compound 1022) as a colorless oil: [ka] ESMS(M+H + )=237.12.

[0406] As shown in Scheme 7, Step 7-ii, 2-(1,4-dioxaspiro[4.5]decan-8-yloxy)pyrimidine (620 mg, 2.624 mmol) was added with HCl (4.0 mL of 6 M, 8.86 mmol), and the reaction mixture was stirred for 2 h. The pH of the mixture was neutralized with saturated NaHCO3 (aqueous), and the mixture was concentrated under reduced pressure as a methanol azeotrope. DCM (30 mL) was added to the residue to form a precipitate, followed by stirring for an additional 20 min. The solid was filtered off, and the mother liquor was concentrated under reduced pressure. The resulting residue was dissolved in methanol, and sodium borohydride (151 mg, 3.99 mmol) was added as a solid. The mixture was stirred for 1 h, and the reaction was quenched with HCl (6 M, 0.70 mL). Stirring was continued until gas evolution ceased. The pH of the mixture was adjusted to approximately 8 with 1 N sodium hydroxide, and the mixture was extracted with EtOAc (20 mL). The organics were dried over sodium sulfate and concentrated under reduced pressure to yield 4-(pyrimidin-2-yloxy)cyclohexanol (compound 1023, 248 mg, 64% yield) as a mixture of (cis)- and (trans)-isomers. A 12 mg aliquot of the sample was purified via HPLC preparative reverse-phase chromatography (10-90% CHCN / water gradient containing 0.1% TFA) to separate the isomers: (trans)-4-pyrimidin-2-yloxycyclohexanol- [ka] ESMS(M+H + ) = 195.07, (cis)-4-pyrimidin-2-yloxycyclohexanol- [ka] ESMS(M+H + )=195.07. The remaining material was used in subsequent reactions as a cis / trans mixture.

[0407] As shown in Scheme 7, Step 7-iii, to a solution of a cis / trans mixture of 4-pyrimidin-2-yloxycyclohexanol (244 mg, 1.256 mmol) and triethylamine (350 μL, 2.51 mmol) in dichloromethane (5 mL) was added methanesulfonyl chloride (145 μL, 1.87 mmol). The reaction mixture was stirred for 2 hours, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0-20% EtOAc / dichloromethane gradient) to give 4-pyrimidin-2-yloxycyclohexyl methanesulfonate as a mixture of cis / trans isomers (compound 1024, 239 mg, 70% yield): [ka] ESMS(M+H + )=273.52.

[0408] As shown in Scheme 7, Step 7-iv, a mixture of (4-pyrimidin-2-yloxycyclohexyl)methanesulfonate (105 mg, 0.386 mmol), 7-morpholinoquinoxalin-5-ol (178.3 mg, 0.7712 mmol), and CsCO (125.6 mg, 0.3856 mmol) in dioxane (1.5 mL) was sealed in a 5 mL microwave tube and heated to 110 °C using an oil bath for 14 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, and filtered through diatomaceous earth, followed by an ethyl acetate wash. The filtrate was concentrated under reduced pressure, and the residue was purified via preparative reverse-phase HPLC (10–90% CHCN / water gradient containing 0.1% TFA). The fractions containing a mixture of cis and trans isomers were further purified via SFC using a chiral OJ column and eluting with 40% MeOH in CO to give 21 mg of 4-(8-(((trans)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)quinoxalin-6-yl)morpholine (compound 34): [ka] ESMS(M+H +) = 408.56, and 22 mg of 4-(8-(((cis)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)-quinoxalin-6-yl)morpholine (compound 42): [ka] ESMS(M+H + )=408.56. Example 8 N-[(cis)-4-[7-(3,6-dihydro-2H-pyran-4-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine (Compound 36) [ka]

[0409] As shown in Scheme 8, Step 8-i, to a mixture of 7-bromoquinoxalin-5-ol (compound 1018, 200 mg, 0.89 mmol) and cesium carbonate (579 mg, 1.78 mmol) in NMP (4.0 mL) was added (trans)-4-(pyrimidin-2-ylamino)cyclohexyl methanesulfonate (compound 1014, 241.1 mg, 0.8887 mmol). The mixture was stirred at 90° C. for 18 hours, at which point an additional 0.5 equivalents of compound 1014 (241 mg, 0.89 mmol) was added. After stirring at 90 °C for an additional 6 h, the reaction mixture was diluted with EtOAc, washed with HO, dried over NaSO, concentrated, and purified by medium pressure silica gel chromatography (0-5% MeOH / DCM) to give N-((cis)-4-((7-bromoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (compound 1025): [ka]

[0410] As shown in Step 8-ii of Scheme 8, a mixture of N-((cis)-4-((7-bromoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (Compound 1025, 52 mg, 0.1299 mmol), Pd(dppf)Cl (10.61 mg, 0.01299 mmol), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Compound 1026, 27.3 mg, 0.13 mmol), NaCO (195 μL of a 2 M (aqueous) solution, 0.39 mmol) in DMF (1 mL) was flushed with nitrogen gas for 10 minutes. The mixture was subjected to microwave irradiation at 150 °C for 20 minutes. After cooling, the mixture was diluted with EtOAc, washed with HO, dried over NaSO, concentrated under reduced pressure, and purified by medium-pressure silica gel chromatography (0-5% MeOH / DCM) to give N-[(cis)-4-[7-(3,6-dihydro-2H-pyran-4-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine (compound 36) as an off-white solid. [ka] ESMS(M+H + )=404.2. Example 9 N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (Compound 28) [ka]

[0411] As shown in Scheme 9, Step 9-i, 7-bromoquinoxalin-5-ol (Compound 1017, 5.4 g, 24.0 mmol), 2-((trans)-4-hydroxycyclohexyl)isoindoline-1,3-dione (5.607 g, 22.86 mmol), and triphenylphosphine (8.994 g, 7.945 mL, 34.29 mmol) were dissolved in anhydrous THF and the flask was cooled in an ice bath. DIAD (6.93 g, 6.64 mL, 34.3 mmol) was added dropwise, and the reaction was stirred at 0° C. for 5 minutes, then warmed to room temperature and stirred for 18 hours. The reaction mixture was concentrated under reduced pressure, the residue was treated with EtO and stirred at RT for 0.5 h, the precipitate was filtered off, the filtrate was concentrated under reduced pressure, and the residue was purified by medium-pressure silica gel chromatography (0–50% EtOAc / hexanes gradient) to give 2-[(cis)-4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (compound 1028, 6.2 g, 60% yield): [ka]

[0412] As shown in Step 9-ii of Scheme 9, a mixture of 2-[4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (6.2 g, 12.34 mmol), morpholine (1.61 g, 1.62 mL, 18.5 mmol), and CsCO (12.06 g, 37.0 mmol) in anhydrous toluene (73 mL) was treated with rac-BINAP (768.4 mg, 1.234 mmol) and Pd(dba) (565 mg, 0.617 mmol) in a round-bottom flask equipped with a condenser. The reaction mixture was heated at 110 °C for 18 h. After cooling to room temperature, the mixture was filtered through diatomaceous earth and concentrated under reduced pressure. The residue was triturated with EtO, and the solid was collected by filtration and washed with EtO to yield 2-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)isoindoline-1,3-dione (compound 1029, 4.2 g) as a yellow solid. The filtrate was concentrated under reduced pressure and purified by medium-pressure silica gel chromatography (0-100% EtOAc / hexanes gradient) to yield an additional 300 mg of compound 1029: [ka]

[0413] As shown in Scheme 9, Step 9-iii, to a suspension of 2-[(cis)-4-(7-morpholinoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (2.3 g, 5.02 mmol) in MeOH (25 mL) was added hydrazine (321 mg, 315 μL, 10.0 mmol), and the reaction mixture was stirred at RT for 18 h, during which time the initial suspension became homogeneous, followed by the appearance of a precipitate. EtO (30 mL) was added, and the reaction mixture was stirred for an additional 30 min. The precipitate was filtered off, the filtrate was concentrated under reduced pressure, and the residue was treated with DCM (30 mL), and any remaining solids were removed by filtration. The filtrate was concentrated under reduced pressure to give (cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexanamine (compound 1030), which was used directly in the subsequent reaction: [ka]

[0414] As shown in Scheme 9, Step 9-iv, to a solution of (cis)4-(7-morpholinoquinoxalin-5-yl)oxycyclohexanamine (415 mg, 1.264 mmol) and 2-methylsulfonylpyrimidine (400 mg, 2.53 mmol) was added DIEA (490 mg, 661 μL, 3.79 mmol), and the reaction mixture was sealed in a vessel and heated to 100 °C for 16 h. After this time, the volatiles were removed under a stream of nitrogen gas, and the crude residue was dissolved in a minimal amount of DCM. Purification by medium-pressure silica gel chromatography (0–10% MeOH / DCM, 1% EtN) afforded N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine, which contained triethylamine hydrochloride as an impurity. The product was dissolved in DCM and stirred with silica-supported amine (Silabond amine® 40-63 μm). The scavenger mixture was filtered, concentrated under reduced pressure, and dried under high vacuum to give N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine (compound 28, 435 mg): [ka] Example 10 Preparation of N-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine (Compound 291) [ka]

[0415] To a mixture of 7-bromoquinoxalin-5-ol (47.53 g, 211.2 mmol), 2-(4-hydroxycyclohexyl)isoindoline-1,3-dione (52.41 g, 213.7 mmol), and PPh3 (87.31 g, 332.9 mmol) in THF (740 mL) was added tert-butyl (NZ)-N-tert-butoxycarbonyliminocarbamate (DTBAD) (79.51 g, 328.0 mmol) in portions at 21 °C over 40 min, maintaining the temperature below 30 °C, and the resulting reaction mixture was stirred at room temperature for an additional 20 h.

[0416] The reaction was evaporated under reduced pressure. The remaining reddish-brown viscous oil was dissolved in CHCl and filtered through a silica plug (made of 1 L of dry silica suspended in CHCl) in a glass column under air pressure. The plug was eluted with CHCl, and the fractions were combined and evaporated under reduced pressure to produce a reddish-brown viscous oil / foam, which was then dissolved in 700 mL of MeOH and subsequently precipitated. The mixture was stirred at room temperature for 1 h, filtered, washed with cold MeOH (500 mL) and EtO (100 mL), and then dried under reduced pressure to produce a tan solid, which was suspended in 300 mL of MeOH and refluxed for 10 min. The suspension was cooled to room temperature, filtered, washed with more MeOH and Et2O (4:1), and dried under reduced pressure to give 2-[4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (58.43 g, 126.6 mmol, 59.94%). [ka] ESI-MS m / z calculated 451.05316, found 452.19 (M+1)+; retention time: 0.92 min. [ka]

[0417] A mixture of 2-[4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (1 g, 2.211 mmol), 6-oxa-3-azabicyclo[3.1.1]heptaneHCl (180 mg, 1.328 mmol), cesium carbonate (2.161 g, 6.633 mmol), Pd(dba) (202.5 mg, 0.2211 mmol), and rac-BINAP (275.3 mg, 0.4422 mmol) in dioxane (5 mL) was stirred at 70 °C overnight and then heated in a microwave reactor at 150 °C for 15 min. The reaction was then diluted with methylene chloride, filtered through Celite, and concentrated. Silica gel flash column chromatography (0–5% MeOH / DCM) afforded 2-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]isoindoline-1,3-dione (750 mg, 72.1%) as a yellow solid, which was used in the next reaction. [ka]

[0418] To a solution of 2-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]isoindoline-1,3-dione (800 mg, 1.700 mmol) in EtOH (10 mL) was added hydrazine monohydrate (85.10 mg, 83.35 μL, 1.700 mmol), and the reaction was stirred at reflux overnight, then concentrated, diluted with DCM, and filtered. The filtrate was concentrated and purified on a 40 g silica gel cartridge using 0–50% (20% NH3 / MeOH) to afford 4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexanamine as a yellow solid (450 mg, 77.8%). [ka] [ka]

[0419] A mixture of 4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexanamine (190 mg, 0.5581 mmol), 2-fluoropyrimidine (60 mg, 0.6118 mmol), and DIEA (200 μL, 1.148 mmol) in 2-propanol (2 mL) was heated in a microwave reactor at 150 °C for 20 min. The reaction mixture was concentrated and then purified through a 12 g silica gel cartridge using 0-6% MeOH / DCM to yield N-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine (120.2 mg, 48.9%) as a yellow solid. Mass +1: 419.23; Retention time: 0.72; NMR notes: [ka] Example 11 Preparation of 4-methyl-N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]pyrimidin-2-amine (Compound 665) [ka]

[0420] A mixture of 4,6-dibromo-2,1,3-benzoxadiazole (3 g, 10.80 mmol) and tert-butyl N-(4-hydroxycyclohexyl)carbamate (2.5 g, 11.61 mmol) was dissolved in THF (60 mL) and cooled to -30 °C. To the mixture, sodium bis(trimethylsilyl)amide (NaHMDS, 23 mL of 1 M, 23.00 mmol) was added dropwise over 10 min, turning the solution deep blue. The mixture was stirred at -20 to 0 °C for 30 min and at rt for 30 min. LC-MS showed only the desired product peak and no starting material. The mixture was cooled to 0 °C, quenched with 10% citric acid solution, extracted with EtOAc, dried over Na2SO4, and concentrated. Purification was performed with 0 to 40% EtOAc / heptane. Recovered 1.9 g (42%) of tert-butyl ((1s,4s)-4-((6-bromobenzo[c][1,2,5]oxadiazol-4-yl)oxy)cyclohexyl)carbamate (C17H22BrN3O4).

[0421] A mixture of tert-butyl N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate (2.6 g, 6.306 mmol), cesium carbonate (4 g, 12.28 mmol), diacetoxypalladium (140 mg, 0.6236 mmol), RuPhos (580 mg, 1.243 mmol), and morpholine (900 μL, 10.32 mmol) in dioxane (30 mL) was purged with N for 5 minutes. The mixture was stirred at 90 °C for 3 hours. LC-MS showed the desired product. The mixture was diluted with DCM, filtered through a layer of Celite, concentrated, and purified using an 80 g silica gel cartridge eluting with 0–80% EtOAc / heptane to recover tert-butyl N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate (2.1 g, 80%). [ka]

[0422] tert-Butyl N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate

[0423] To a solution of tert-butyl N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate (3.5 g, 8.363 mmol) in DCM (30 mL) was added TFA (approximately 953.6 mg, 644.3 μL, 8.363 mmol) and stirred for 1 h. The mixture was concentrated and purified using a 40 g silica gel cartridge eluting with 0-10% (7N NH3 / MeOH / DCM). 4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexanamine (2.5 g, 94%) was recovered. ESI-MS m / z calculated 318.1692, found 319.2 (M+1). + ;Retention time: 0.55 minutes.

[0424] A microwave vial was charged with 4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexanamine (1 g, 3.141 mmol), 2-chloro-4-methyl-pyrimidine (600 mg, 4.667 mmol), RockPhosG3 (80 mg, 0.3175 mmol), and dioxane (10 mL). NaOtBu (4 mL of 2 M, 8.000 mmol) was added to the mixture. The mixture was stirred at 70 °C for 15 min. LC-MS showed conversion, yielding 4-methyl-N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]pyrimidin-2-amine. The mixture was diluted with DCM, filtered through a layer of Celite, concentrated, and purified using a 40 g silica gel cartridge eluting with 0–10% MeOH / DCM. The product was collected, triturated with EtO and dried under vacuum, 4-methyl-N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]pyrimidin-2-amine (795.3 mg, 59%), [ka] ESI-MS m / z measured value 410.20663, actual value 411.3 (M+1) + ;Retention time: 0.63 min.

[0425] Preparation of N-methyl-2-((4-((6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)oxy)cyclohexyl)amino)pyrimidine-4-carboxamide (compound (666)) [ka]

[0426] To a mixture of 4,6-dibromo-2,1,3-benzoxadiazole (1 g, 3.598 mmol), (4-methoxyphenyl)methanol (500 μL, 4.010 mmol) in THF (20 mL) was added dropwise NaHMDS (4 mL of 1 M, 4.000 mmol) at −78 °C. The solution turned blue. The mixture was stirred at −78 °C for 30 min. The mixture was warmed to RT and stirred at RT for 1 h. LC-MS showed a product peak and no starting material. TLC showed a spot slightly less polar than the starting material. The reaction was quenched with saturated NH 4 Cl, extracted with EtOAc, washed with HO, dried over Na 2 SO 4 , and concentrated. The product was purified using a 40 g silica gel cartridge eluting with 0-30% EtOAc to recover 6-bromo-4-[(4-methoxyphenyl)methoxy]-2,1,3-benzoxadiazole (850 mg, 70%) as a slightly yellow solid. [ka]

[0427] A mixture of 6-bromo-4-[(4-methoxyphenyl)methoxy]-2,1,3-benzoxadiazole (830 mg, 2.476 mmol), cesium carbonate (2.4 g, 7.366 mmol), Pd(OAc) (50 mg, 0.2227 mmol), rac-BINAP (300 mg, 0.4818 mmol), and morpholine (300 μL, 3.440 mmol) in dioxane (10 mL) was bubbled with N for 10 min. The mixture was heated to 80 °C. The mixture was stirred at 80 °C for 4.5 h. LC-MS showed clean product. The mixture was cooled to rt, diluted with DCM, filtered through a pad of Celite, and concentrated. The product was purified using a 40 g silica gel cartridge with 0-70% EtOAc / DCM to recover 4-[(4-methoxyphenyl)methoxy]-6-morpholino-2,1,3-benzoxadiazole (750 mg, 89%). [ka]

[0428] To a solution of 4-[(4-methoxyphenyl)methoxy]-6-morpholino-2,1,3-benzoxadiazole (750 mg, 2.197 mmol) in DCM (10 mL) was added TFA (2 mL, 25.96 mmol) and stirred for 1 h. The mixture was concentrated, and the product was purified using a 12 g silica gel cartridge eluting with 0-10% MeOH / DCM. 6-morpholino-2,1,3-benzoxadiazol-4-ol (400 mg, 82%) was recovered as a yellow foam.

[0429] A mixture of 6-morpholino-2,1,3-benzoxadiazol-4-ol (50 mg, 0.2260 mmol), [4-[[4-(methylcarbamoyl)pyrimidin-2-yl]amino]DMF,hexyl]methanesulfonate (240 mg, 0.7308 mmol), and cesium carbonate (250 mg, 0.7673 mmol) in DMF (1 mL) was stirred at 80 °C overnight. LC-MS showed the desired product. The mixture was diluted with DCM and HO, extracted with DCM, dried over NaSO, and concentrated. The product was purified using a 4 g silica gel cartridge eluting with 0-5% MeOH / DCM to recover the desired product, N-methyl-2-((4-((6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)oxy)cyclohexyl)amino)pyrimidine-4-carboxamide (41.8 mg, 39%). [ka] ESI-MS m / z calculated 453.21246, found 454.39 (M+1) + ;Retention time: 0.72 minutes.

[0430] Tables 1 and 2 provide analytical characterization data for certain compounds of formula (III-E-1 and III-E-2) (blank cells indicate that no testing was performed). Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

[0431] Compounds were screened for their ability to inhibit DNA-PK kinase using a standard radiometric assay. Briefly, in this kinase assay: 33 P-ATP terminal33 The transfer of P-phosphate to a peptide substrate was investigated. Assays were performed in 384-well plates containing approximately 6 nM DNA-PK, 50 mM HEPES (pH 7.5), 10 mM MgCl, 25 mM NaCl, 0.01% BSA, 1 mM DTT, 10 μg / mL sheared double-stranded DNA (obtained from Sigma), 0.8 mg / mL DNA-PK peptide (Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp-Leu-Trp-Lys-Lys-Lys, obtained from American Peptide), and 100 μM ATP in a final volume of up to 50 μL per well. Thus, compounds of the present invention were dissolved in DMSO to generate an initial 10 mM stock solution. Then, serial dilutions were made in DMSO to obtain the final assay solution. A 0.75 μL aliquot of DMSO or inhibitor in DMSO was added to each well, followed by 33 ATP substrate solution containing P-ATP (obtained from Perkin Elmer) was added. The reaction was initiated by the addition of DNA-PK, peptide, and ds-DNA. After 45 minutes, the reaction was quenched with 25 μL of 5% phosphoric acid. The reaction mixture was transferred to a MultiScreen HTS 384-well PH plate (obtained from Millipore), allowed to bind for 1 hour, and washed three times with 1% phosphoric acid. After the addition of 50 μL of Ultima Gold™ high-performance scintillant (obtained from Perkin Elmer), samples were counted in a Packard TopCount NXT Microplate Scintillation and Luminescence Counter (Packard BioScience). For competitive and tight binding inhibition, data were fitted to a kinetic model using Microsoft Excel. Use the Solver macro to find K i values ​​were calculated.

[0432] Compounds 18, 23, 24, 27~30, 32~34, 36~42, 44~46, 49~55, 59~61, 63~67, 69, 71, 72, 84, 87, 93, 94, 104, 108, 109, 134 , 135, 139, 140, 142~146, 152, 155, 158, 160~162, 164, 187, 189~191, 193~210, 215, 219, 223~227, 233~237, 2 41-243, 245-247, 254, 256, 257, 260, 263-265, 267-269, 272, 273, 275-277, 282, 283, 285, 286, 287, 291, 295, 297, 298, 308, 309, 312, 314, 315-319, 321, 323, 324, 333-336, 340, 341, 351-354, 366, 369, 373-377, 380, 3 82, 386-388, 393-397, 399, 401, 402, 404-409, 417-422, 424, 427-430, 436, 438-451, 453-457, 463, 465, 467, 469, 471, 472, 474, 476-478, 481, 482, 485, 486, 489-492, 494, 499, 511, 512, 525, 531-535, 537, 539, 547, 5 Each of 48, 553, 558, 565, 567, 572–577, 579, 582–584, 586–595, 597, 598, 600–605, 607–609, 612, 614–616, 618, 622, 623, 625–627, 629, 630, 633, 634, 636, 642–644, 650–653, and 658–666 had a K of less than 1.0 micromolar for inhibition of DNA-PK. iCompounds 18, 23, 24, 27-30, 32-34, 36-37, 39-41, 44-46, 49-55, 59-61, 63-67, 69, 71, 72, 84, 87, 93, 94, 104, 108, 109, 134, 135, 139, 140, 142-146, 152, 155, 158, 160-162, 164, 187, 189-191, 193-200, 202-210, 215, 219, 223-227, 233-237, 241-243, 245-247, 254, 256, 257, 260, 263-265, 267-269, 272, 273, 275-277, 282, 283, 285, 286, 287, 291, 295, 297, 298, 308, 309, 312, 314, 315-319, 321, 323, 324, 333-336, 340, 341, 351-354, 366, 369, 373-374, 3 76-377, 380, 382, ​​386-388, 393-397, 399, 401, 402, 404-409, 417-422, 424, 427-430, 436, 438-451, 453-457, 463, 465, 467, 469, 471, 472, 474, 476-478, 481, 482, 485, 486, 489-492, 494, 499, 511, 512, 531-535, 537, 539, 54 Each of 7, 548, 553, 558, 565, 567, 572–577, 579, 582–584, 586–595, 597, 598, 600–605, 607–609, 612, 614–616, 618, 622, 623, 625–627, 629, 630, 633, 634, 636, 642–644, 650–653, and 658–666 had a K of less than 0.10 micromolar for inhibition of DNA-PK. i For example, for inhibition of DNA-PK, compounds 661, 665, and 666 have a K of 0.001 micromolar. i and compound 663 has a K of 0.008 micromolar i It has.

[0433] Gene editing examples

[0434] Example 12 material and method

[0435] method:

[0436] Cells and cultures

[0437] Bronchial epithelial cells (BECs) were derived from a human donor diagnosed with cystic fibrosis who had the CFTRdF508 / dF508 genotype.

[0438] Induced pluripotent stem cells (iPSCs) were derived from human skin fibroblasts after viral transduction with the Yamanaka reprogramming factors Oct4, Sox2, KLF4, and c-Myc. The derived iPSCs were capable of differentiating into three germ layers and contained a normal karyotype with 23 pairs of chromosomes.

[0439] Primary human mobilized peripheral blood (mPB) CD34 + Hematopoietic stem and progenitor cells (HSPCs) were purchased from Hemacare or AllCells. Cells were thawed, washed, and cultured at 1–3 × 10 cells per mL in serum-free medium consisting of CellGro SCGM (CellGenix) and a cytokine mix (300 ng / mL SCF, 300 ng / mL Flt3L, 100 ng / mL TPO, and 60 ng / mL IL-3). 5 The cells were resuspended in supplemented complete medium at a cell density of 1000 and incubated in a 37°C / 5% CO2 incubator for 48 hours before electroporation.

[0440] DNA-PK inhibitors:

[0441] DNA-PK inhibitor compounds 661, 663, 665, and 666 were used in the gene editing examples. 10 mM stock solutions were made using anhydrous DMSO and stored at -80 °C.

[0442] Electroporation:

[0443] The synthetic sgRNA used was HPLC (high performance liquid chromatography) purified and purchased from Synthego, and contained chemically modified nucleotides (2'-O-methyl 3'-phosphorothioate) at three terminal positions on both the 5' and 3' ends. The sequence of the sgRNA, with the modified nucleotides underlined, is as follows:

[0444] AAVS1 sgRNA: [ka]

[0445] NAV1.7 sgRNA:

[0446] [ka]

[0447] Cas9 mRNA was purchased from TriLink Biotechnologies (L-7206). The Cas9 mRNA expresses a version of the Streptococcus pyogenes SF370 Cas9 protein (CRISPR Associated Protein 9) with a nuclear localization signal. spCas9 mRNA also contains a CAP1 structure, a polyadenylation signal, and modified uridines to ensure optimal expression levels in mammalian cells.

[0448] The donor ssODN was purchased from IDT. The ssODN contains a 10-nucleotide insert sequence flanked by 40 nucleotides of homology arms, allowing HDR events to be measured by the TIDE assay. The ssODN contains a total of 90 nucleotides and phosphorothioate-modified nucleotides at the three terminal positions of both the 5' and 3' ends. The sequence of the donor ssODN, with the phosphorothioate-modified nucleotides underlined, is shown as follows:

[0449] AAVS1 PAM:

[0450] [ka]

[0451] AAVS1 non-PAM:

[0452] [ka]

[0453] NAV1.7 PAM:

[0454] [ka]

[0455] NAV1.7 Non-PAM:

[0456] [ka]

[0457] All electroporations were performed in a Lonza 4D-Nucleofector™ system.

[0458] The following electroporation conditions were used for BEC: 1.8xE5 cells, 250ng Cas9 mRNA, 500ng sgRNA, and 0.66uM ssODN in 20ul P4 electroporation buffer using program CM-138. Electroporated cells were transferred to 96-well plates containing 100ul BEC medium supplemented with DNA-PK inhibitor or left untreated. Cells were incubated at 37°C in a 5% CO2 incubator.

[0459] For iPSCs, the following conditions were used: 2.0xE5 cells, 250ng CAS9 mRNA, 500ng sgRNA, and 0.66uM ssODN in 20ul P3 electroporation buffer using program CA-137. Electroporated cells were transferred to a 96-well plate containing 100ul mTEsR1 medium (Stem Cell Technologies) supplemented with 10uM ROCK inhibitor Y-27632 (Stem Cell Technologies), with or without DNA-PK inhibitor, and then incubated at 37°C in a 5% CO2 incubator.

[0460] CD34 + Cells were electroporated two days after thawing. The following conditions were used for electroporation: 2.0xE6 cells, 15µg Cas9 protein (Feldan), 15µg sgRNA, 1µM ssODN in 100µl P3 electroporation buffer, using program CA-137. Electroporated cells were equally divided and transferred to eight wells of a 24-well plate, each well containing various concentrations of DNA-PK inhibitor. Cells were incubated at 37°C in a 5% CO2 incubator for two days, and cell viability and gene editing were evaluated.

[0461] Lipid-mediated cell transfection:

[0462] One day before transfection, BECs were seeded in BEC medium at a cell density of 1x E4 cells per well in a 96-well plate. First, 0.15 ul of MessengerMax (ThermoFisher, LMRNA 003) was diluted into 5 ul of Opti-MEM and incubated at room temperature for 10 minutes. Meanwhile, 80 ng of Cas9 mRNA (Trilink, L-7206), 20 ng of sgRNA (Synthego), and 1 pmole of ssODN were added to 5 ul of Opti-MEM and then mixed with the MessengerMAx solution. The mixture was incubated for 5 minutes before being added to the cells. The entire solution was added to the cells in the wells of a 96-well plate containing 100 ul of medium with or without DNA-PK inhibitor. The cells were incubated at 37°C in a 5% CO2 incubator.

[0463] Measurement of cell viability:

[0464] Cells were incubated in culture medium with 5 μg / ml Hoechst 33342 (Life Technologies: H3570) and 0.5 μg / ml Propidium Iodide (PI; Life Technologies: P3566) at 37°C for 1 hour. Cells were imaged and measured for Hoescht-positive events (live and dead cells) and PI-positive events (dead cells) using a High-Content Imaging system (Molecular devices). Relative cell viability was calculated as follows: [(Hoescht of sample) + Event-PI + Events)] / Control (Hoescht + Event-PI + Events)]*100. Controls were mock-transfected cells, and cell viability was arbitrarily set to 100%.

[0465] CD34 was isolated using Cell Titer Glo (CTG) reagent (Promega). +The cell viability of HSPCs was measured. 100 μl of cell suspension was mixed with 100 μl of complete CTG reagent. The chemiluminescent signal was measured using a luminometer, and the percentage of viable cells was calculated compared to control cells (cells not treated with DNA-PK inhibitors).

[0466] Measuring gene editing rates:

[0467] Genomic DNA was isolated by incubating cells with 50 ul of DNAQuickextract solution (Epicentre) per well of a 96-well plate for 30 minutes at 37°C. The cell extracts were mixed and transferred to a PCR plate, which was then incubated at 65°C for 6 minutes and 98°C for 2 minutes. AccuPrime™ Pfx PCR reaction was performed on 1 ul of genomic DNA-containing solution using DNA Polymerase (Thermofisher, 12344024). The PCR conditions were 94°C (1x) for 4 minutes, followed by 94°C for 15 seconds, 60°C for 15 seconds, and 60°C for 1 minute (40x). The PCR product was purified and then Sanger sequenced by GENEWIZ. The following primer pairs spanning the target site were used for PCR (FW, forward; RV, reverse). Primers used by Sanger sequencing are indicated with an asterisk (*):

[0468] [ka]

[0469] [ka]

[0470] [ka]

[0471] [ka]

[0472] Each sequence chromatogram was analyzed using TIDE (Tracking of Indels by Decomposition) software (http: / / tide.nki.nl) (see also Brinkman et al., Nucleic Acids Research, Volume 42, Issue 22, 16 December 2014, Pages e168). Using a mock-electroporated sample as the reference sequence, parameters were set for an indel size of 30 nt and a decomposition window covering the largest window possible with a high-quality trace. The total indel (insertion and deletion) rate was obtained directly from the TIDE plot. The HDR rate was the percentage of events with a 10-nucleotide insertion. The NHEJ rate was calculated as the total indel rate minus the HDR rate. Graphs were generated and all statistics were calculated using GraphPad Prism7 software.

[0473] Example 13 DNA-PK inhibitors improve HDR gene editing rates in BECs

[0474] Figure 1 illustrates the design of the gene editing assay used in the following examples. To investigate the effect of DNA-PK inhibitors on HDR gene editing rates, BECs were electroporated with spCAS9 mRNA, NAV1.7 sgRNA, and NAV1.7 non-PAM ssODN, and then incubated with different concentrations of Compound 665 or left untreated (control). 72 hours after electroporation, the gene editing rates were determined using the TIDE assay. The gene editing rates were expressed as a percentage and categorized into HDR and NHEJ. Cell viability was expressed as a percentage, with control cells set at 100%.

[0475] As shown in Figure 2, compound 665, a DNA-PK inhibitor, improves gene editing rates in BECs. For compound 665, the NHEJ IC50 was 0.4163 μM, the HDR EC50 was 0.4834 μM, and the HDR TOP% was 76.03.

[0476] Example 14 DNA-PK inhibitors are CD34 + Improving HDR gene editing rates in cells

[0477] To investigate the effect of DNA-PK inhibitors on HDR gene editing rates, we used mPB CD34 + Cells were electroporated with RNP (spCAS9 protein + NAV1.7 sgRNA) and NAV1.7 non-PAM ssODN. Cells were then incubated with various concentrations of Compound 665. Forty-eight hours after electroporation, gene editing rates were determined using the TIDE assay. Gene editing rates were expressed as a percentage and categorized into HDR and NHEJ as shown in Figure 3A (donor B) and Figure 3B (donor C). Cell viability was expressed as a percentage; in this case, control cells were set as 100%.

[0478] As shown in Figures 3A and 3B, the DNA-PK inhibitor compound 665 inhibits CD34 + Improved gene editing rates in cells. The EC50 values ​​for HDR and indel formation for donor B were 0.28 μM and 0.36 μM, respectively.

[0479] Example 15 DNA-PK inhibitor improves HDR gene editing rates in iPSCs

[0480] To investigate the effect of DNA-PK inhibitors on HDR gene editing rates, iPSCs were electroporated with spCAS9 mRNA, AAVS1 sgRNA, and AAVS1 PAM ssODN, then incubated with different concentrations of Compound 665 or left untreated (control). Seventy-two hours after electroporation, gene editing rates were determined using the TIDE assay. Gene editing rates were expressed as a percentage and categorized into HDR and NHEJ. Cell viability was shown as a percentage; in this case, control cells were set as 100%.

[0481] Compound 665, a DNA-PK inhibitor, improves gene editing rates in iPSCs, as shown in Figure 4. For compound 665, the NHEJ IC50 was 1.274 μM, the HDR EC50 was 0.9337 μM, and the HDR TOP% was 26.27.

[0482] Example 16 Determining gene editing rates at ECmax

[0483] To investigate the gene editing rate at ECmax, BECs were electroporated with spCAS9 mRNA, AAVS1 sgRNA, and AAVS1 PAM ssODN, and then incubated with 10 μM Compound 665 for different times or left untreated (control). Gene editing rates were determined using the TIDE assay and expressed as percentages of HDR and NHEJ. 10 μM is the maximum enhancing concentration (ECmax) of Compound 665.

[0484] Figure 5 shows that a strict inverse correlation exists between HDR and NHEJ events.

[0485] Example 17 Determining gene editing rates at EC50

[0486] BECs were electroporated with spCAS9 mRNA, AAVS1 sgRNA, and AAVS1 PAM ssODN, then incubated with 0.7 μM Compound 665 for different periods of time or left untreated (control). Gene editing rates were determined using the TIDE assay and expressed as percentages of HDR and NHEJ. 0.7 μM is the enhancing concentration 50 (EC50) of Compound 665. Figure 6 illustrates the time course of DNA-PK inhibition on HDR and NHEJ in BECs.

[0487] Example 18 DNA-PK inhibitors improve HDR rates in BECs when gene-editing components are delivered by lipid-mediated transfection

[0488] To investigate the effect of lipid-mediated transfection, BECs were transfected with spCAS9 mRNA, AAVS1 sgRNA, and AAVS1 PAM ssODN, then incubated with different concentrations of Compound 665 or left untreated (control). Seventy-two hours after transfection, gene editing rates were determined using the TIDE assay. Figure 7 shows that HDR efficiency increases with increasing concentrations of Compound 665 delivered via lipid-based transfection.

[0489] Overview: Compound 665, a DNA-PK inhibitor, improves HDR-driven gene editing [Table A-1] [Table A-2]

[0490] (Example 19):

[0491] mPB CD34 +Cells were electroporated with RNP (spCAS9 protein + NAV1.7 sgRNA) and NAV1.7 non-PAM ssODN. Cells were then incubated with various concentrations of compounds 661, 663, and 666. The experiment was performed as described in the methods above, except that lower concentrations of sgRNA (5 μg) and ssODN (0.2 μM) were used. Forty-eight hours after electroporation, gene editing rates were determined using the TIDE assay. Gene editing rates were expressed as a percentage and categorized into HDR and NHEJ. Results for each compound tested in two separate donors are shown below.

[0492] compound 661 [Table B]

[0493] compound 663 [Table C]

[0494] compound 666 [Table D-1] [Table D-2]

[0495] Summary table for compounds 661, 663, and 666 [Table E]

[0496] Example 20 CD34 + DNA-PK inhibitors improve HDR gene editing rates in cells

[0497] To investigate the effect of DNA-PK inhibitors on HDR gene editing rates, we used spCAS9 CD34 mRNA, AAVS1 sgRNA, and AAVS1 non-PAM ssODN + Cells were electroporated and then incubated with different concentrations of compounds 666, 661, and 663, or left untreated (control). 72 hours after electroporation, gene editing rates were determined using the TIDE assay. Gene editing rates were expressed as a percentage and categorized into HDR and NHEJ. Cell viability was expressed as a percentage, with control cells set as 100%.

[0498] As indicated, CD34 + In cells, the DNA-PK inhibitors compounds 666 (Figure 8), 661 (Figure 9), and 663 (Figure 10) improved HDR rates.

[0499] Example 21 Precise gene editing by HDR mediated by AAV donors, CRISPR-Cas9, and selective DNA-PK inhibitors.

[0500] Figure 11 illustrates the design of the gene editing assay used in this example.

[0501] cell

[0502] Lung progenitor cells (LPCs) were derived from human lung donors diagnosed with cystic fibrosis. LPC donor IDs 14071 and 14335 contain the CFTR genotypes dF508 / dF508 and dF508 / G542X, respectively.

[0503] CRISPR-Cas9 gene editing reagents

[0504] Synthetic sgRNA was purchased from Synthego. The gRNA was HPLC (high-performance liquid chromatography) purified and contains chemically modified nucleotides (2'-O-methyl 3'-phosphorothioate) at the three terminal positions of both the 5' and 3' ends. The gRNA contains a 22-nucleotide long spacer sequence that promotes specific binding to the target site and an 80-nucleotide long scaffold sequence that enables binding to the saCAS9 protein. The complete gRNA sequence is shown in Table 3.

[0505] saCas9 mRNA was synthesized at TriLink Biotechnologies. saCas9 mRNA expresses Staphylococcus aureus Cas9 with SV40 and Nucleoplasmine nuclear localization signals (Uniprot entry code J7RUA5). saCas9 mRNA also contains a CAP1 structure and polyadenylation signal, allowing optimal expression levels in mammalian cells. saCAS9 mRNA was HPLC purified.

[0506] AAV donor construct design and AAV transduction.

[0507] The AAV donor construct contains 500-nucleotide-long sequences of the left and right homology arms relative to the gRNA cleavage site and a 10-nucleotide unique HDR footprint insert. AAV donor preparations were generated, purified, and titrated by Vector Biolabs using the AAV6 serotype. AAV titrations were reported as viral genomes per ml. AAV transduction was performed by adding the AAV6 vector to cells at the specified vector genome copy number per cell for 36 hours at 37°C.

[0508] Electroporation

[0509] Electroporation was performed using the Lonza 4D-Nucleofector™ system connected to a 96-well shuttle system. 1.8xE5 LPC cells were resuspended in 20 μl of P4 electroporation buffer Lonza (V4SP-4096). 20 μl of the cell mixture was combined with 2 μl of CRISPR-Cas9 reagent mix containing 1 μg of saCAS9 mRNA and 1 μg of gRNA. 20 μl of the cell and CRISPR-Cas9 mixture was transferred to one well of a 96-well electroporation plate. Cells were electroporated using program CM-138. A portion of the electroporated LPC cells was transferred to one well of a 384-well plate. Cells were transduced with AAV and DNA-PK inhibitor or left untreated (control) for 36 hours in a 5% CO2 incubator. After 72 hours, genomic DNA was isolated.

[0510] Genomic DNA isolation.

[0511] Genomic DNA was isolated by incubating cells with 50 ul and 15 ul of DNA Quickextract solution (Epicentre) per well of 96-well and 384-well plates, respectively, for 30 minutes at 37°C. The cell extracts were mixed and transferred to a 96-well PCR plate, which was then incubated at 65°C for 6 minutes and 98°C for 2 minutes. Genomic DNA was either used immediately for downstream applications or stored at 4°C.

[0512] Indel rate measurement

[0513] DNA fragments corresponding to the target genes were amplified using Phire Green Hot Start II PCR Master Mix (F126L, Thermo Scientific). PCR reactions were performed according to the manufacturer's instructions. Briefly, 1.25 μl of genomic DNA solution was combined with 23.5 μl of Phire Green Hot Start II PCR Master Mix and the corresponding target gene forward an...

Claims

【Request 1】 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof. 【Request 2】 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof. 【Request 3】 【Chemical 1】 or a compound of the formula 【Chemistry 2】 A composition comprising a compound selected from the group consisting of compounds of the formula:

4. A combination comprising the composition described in claim 3 and a genome editing system.

5. The combination described in claim 4 for use in a method for repairing DNA breaks in one or more target genomic regions via the homology directed repair (HDR) pathway.

6. A combination as described in claim 4 for use in a method for inhibiting or suppressing repair of DNA breaks in one or more target genomic regions via the non-homologous end joining (NHEJ) pathway.

7. A combination as described in claim 4 for use in a method for modifying the expression of one or more genes or proteins.

8. The combination described in any one of claims 4 to 7, wherein the genome editing system is selected from a meganuclease-based system, a zinc finger nuclease (ZFN)-based system, a transcription activator-like effector-based nuclease (TALEN)-based system, a CRISPR-based system, or a NgAgo-based system.

9. A combination described in any one of claims 4 to 8, wherein the genome editing system is a CRISPR-based system.

10. The combination described in claim 9, wherein the CRISPR-based system is a CRISPR-Cas system or a CRISPR-Cpf system.

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