WO2024112945A1

WO2024112945A1 - Methods for enhancing editing efficiency - ii

Info

Publication number: WO2024112945A1
Application number: PCT/US2023/081011
Authority: WO
Inventors: Mihee KIM; Brodie MILES; Roslyn RAY; Kaira Caille SAPKOS; David Rawlings; Cole PUGLIANO; Mason BERGER
Original assignee: Seattle Childrens Hospital; CSL Behring LLC
Current assignee: Seattle Childrens Hospital; CSL Behring LLC
Priority date: 2022-11-23
Filing date: 2023-11-22
Publication date: 2024-05-30
Anticipated expiration: 2025-05-23

Abstract

The present disclosure relates generally to methods and compositions for modifying a target sequence in a cell comprising contacting the cell with a gene editing system, a donor template and a second-generation DNA-PK inhibitor and a cellular modulator. In other embodiments contemplated herein, the present disclosure relates to methods and compositions for modifying a target sequence in a cell, resulting in improved engraftment following in vivo transplantation.

Description

METHODS FOR ENHANCING EDITING EFFICIENCY - II

Related Applications

[0001] The present application claims priority from United States Provisional Patent Application No. 63/384,858 filed on November 23, 2022, the entire content of which is incorporated by reference in its entirety.

Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequence Listing.xml, created November 20, 2023, which is 41,185 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

Field

[0003] The present disclosure relates generally to methods and compositions for modifying a target sequence in a cell (e.g., a population of cells) comprising contacting the cell with a gene editing system, a donor template and a second-generation DNA-PK inhibitor (e.g., AZD7648) and a cellular modulator. In other embodiments contemplated herein, the present disclosure relates to methods and compositions for modifying a target sequence in a cell (e.g., a population of cells), resulting in improved engraftment following in vivo transplantation.

Background

[0004] Programmable gene editing is typically mediated by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is an error-prone DNA repair pathway, while HDR results in perfect repair. More recently, a third DNA repair pathway has been identified, which mediates the repair of double stranded breaks (DSBs) by the recombination of 5-25 base pair (bp) homologous regions flanking a DSB, also referred to as "microhomologies" or "pHs". Beneficially, microhomology-mediated end joining (MMEJ) has been shown to generate more predictable editing outcomes, and has the potential to produce 100% dual allele knockouts and improved editing reproducibility at a given locus (Martinez-Galvez el al. 2021, Nucleic Acids Research, 49(1): 67-78). Due to the specificity and reproducibility of HDR and MMEJ, these DNA pathways are preferred for applications that require high fidelity editing or increased predictability of editing outcomes, such as clinical uses. However, HDR is predominantly only active during the late S/G2 phase of the cell cycle, whereas MMEJ is elevated when cells enter the S/G2 phase. By contrast, NHEJ is active throughout the cell cycle, which limits the efficiency of HDR or MMEJ relative to NHEJ.

[0005] NHEJ dominance in gene editing approaches represents a significant limitation to the use of such techniques in somatic applications (e.g., gene therapy), largely due to the unpredictable outcomes generated by NHEJ at the site of a DSB, including at the target sequence and off-target sequences. The lack of predictability of the sequence specific changes that are made at a given locus results in significant molecular heterogeneity in edited cell populations, which often requires complicated selection processes to identify the cells comprising the gene edits of interest. For example, in a diploid genome, the expected frequency of generating dual allele knockouts by NHEJ is < 50%, assuming the repair of both copies at the target site are independent events (Martinez-Galvez et al. 2021, supra). This highlights the need to develop methods of gene editing that reduce the dominance of NHEJ in favor of HDR or MMEJ, which is more suitable for applications that require high fidelity gene editing and/or more reproducible editing outcomes, such as for clinical use.

Summary

[0006] The present disclosure is predicated, at least in part, on the finding that certain DNA-PK inhibitors that reduce NHEJ dominance and enhance alternative DNA repair pathways, including HDR and MMEJ can be delivered concomitant with cellular modulators (e.g., mRNA cellular modulators) in enhanced methods for gene editing, which are suitable for applications that require high-fidelity gene editing and/or more reproducible insertional editing outcomes. In particular, second-generation DNA-PK inhibitors (e.g., AZD7648) have been surprisingly shown to have a synergistic effect when used in combination with cellular modulators (e.g., mRNA cellular modulators) that modulate various cellular processes, leading to improved gene editing outcomes. Moreover, cells modified in accordance with the methods of the present disclosure have unexpectedly improved engraftment following in vivo transplantation. Accordingly, provided herein are composition and methods for enhanced gene editing. [0007] Thus, in an aspect of the present disclosure, there is provided a method for modifying a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; c. AZD7648; and d. an mRNA cellular modulator.

[0008] In an embodiment, the cell is provided with AZD7648 at a concentration of at least about 0.001 pM, e.g., about 0.3 pM.

[0009] In an embodiment, the donor template is provided to the cell by non-viral delivery means, e.g., electroporation, sonication, peptide-based delivery, lipid-based delivery, inorganic delivery or polymeric delivery.

[0010] In an embodiment, the donor template is provided to the cell within a vector, e.g., an adeno-associated virus (AAV) vector.

[0011] In an embodiment, the donor template is selected from the group consisting of double-stranded DNA (dsDNA), a single-stranded DNA (ssDNA), a single- stranded oligodeoxynucleotide (ssODN) and a long single-stranded DNA (IssDNA). In another embodiment, the donor template is an ssODN. In an embodiment, the donor template is a linear template or a plasmid donor template, e.g., a minicircle.

[0012] In an embodiment, the nuclease is a nuclease capable of producing a double stranded break (DSB) in the target sequence, including CRISPR-associated protein (Cas) endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, variants, fragments and combinations thereof.

[0013] In an embodiment, the nuclease is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease may be any suitable RNA-guided nuclease capable of producing a double stranded break (DSB) in the target sequence, including CRISPR- associated protein (Cas) endonucleases, and variants and fragments thereof. In an embodiment, the RNA-guided nuclease is CRISPR-associated endonuclease 9 (Cas9).

[0014] In an embodiment, the gene editing system further comprises a guide RNA (gRNA) that is complementary to a target sequence in a cell. [0015] In an embodiment, the gRNA is a single guide RNA (sgRNA) comprising a sequence that is complementary to the target sequence. In some examples, the sgRNA comprises a sequence of at least 10 contiguous nucleotides that are complementary to the target sequence.

[0016] In an embodiment, the gene editing system comprises the RNA-guided nuclease and the gRNA complexed as a ribonucleoprotein (RNP). Delivery of a RNP may be performed using any methods known in the art. In some examples, the RNP is delivered to the cell by electroporation (z.e., nucleofection).

[0017] In an embodiment, the mRNA cellular modulator is selected from the group consisting of a p53 inhibitor, a cell cycle modulator, a DNA repair modulator and combinations of the foregoing.

[0018] In an embodiment, the mRNA cellular modulator comprises a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof.

[0019] In an embodiment, the mRNA cellular modulator comprises a sequence of any one of SEQ ID NO: 1-6, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0020] In an embodiment, the mRNA cellular modulator consists of a sequence of any one of SEQ ID NO: 1-6.

[0021] In an embodiment, the cell is provided with from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator, e.g., about 0.8 pg/500,000 cells of the mRNA cellular modulator.

[0022] In an embodiment, the cell is a hematopoietic stem cell (HSC), e.g., an allogeneic or autologous HSC.

[0023] Also provided herein is a cell (e.g., a population of cells) modified according to the method of the present disclosure. [0024] Also provided herein is a pharmaceutical composition comprising the cell (e.g., a population of cells) modified according to the method of the present disclosure. Also provided are uses of the cell (e.g., a population of cells) of the present disclosure for the preparation of a medicament.

[0025] Also provided herein are methods of treating a disease or disorder, comprising administering an effective amount of the cell (e.g., a population of cells) or pharmaceutical composition disclosed herein to a subject in need thereof.

[0026] In another aspect of the present disclosure, there is provided AZD7648 and an mRNA cellular modulator for use in a method of modifying of a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; c. AZD7648; and d. an mRNA cellular modulator.

[0027] In an embodiment, the cell is provided from about 0.001 pM to about 5 pM AZD7648, e.g., about 0.3 pM AZD7648.

[0028] In an embodiment, the donor template is provided to the cell by non-viral delivery means, e.g., electroporation, sonication, peptide-based delivery, lipid-based delivery, inorganic delivery or polymeric delivery.

[0029] In an embodiment, the donor template is provided to the cell within a vector, e.g., an AAV vector.

[0030] In an embodiment, the donor template is selected from the group consisting of a dsDNA, a ssDNA, a ssODN and a IssDNA.

[0031] In an embodiment, the nuclease is an RNA-guided nuclease, e.g., Cas9.

[0032] In an embodiment, the gene editing system further comprises a gRNA that is complementary to the target sequence.

[0033] In an embodiment, the gRNA is a sgRNA. [0034] In an embodiment, the gene editing system comprises an RNA-guided nuclease and a gRNA, wherein the RNA-guided nuclease and the gRNA complexed as a RNP.

[0035] In an embodiment, the mRNA cellular modulator is selected from the group consisting of a p53 inhibitor, a cell cycle modulator, a DNA repair modulator and combinations of the foregoing.

[0036] In an embodiment, the mRNA cellular modulator comprises a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof.

[0037] In an embodiment, the mRNA cellular modulator comprises a sequence of any one of SEQ ID NO: 1-6, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0038] In an embodiment, the mRNA cellular modulator consists of a sequence of any one of SEQ ID NO: 1-6.

[0039] In an embodiment, the cell is provided with from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator, e.g., about 0.8 pg/500,000 cells of the mRNA cellular modulator.

[0040] In an embodiment, the cell is a HSC, e.g., an allogeneic or autologous HSC.

[0041] In another aspect of the present disclosure, there is provided a composition comprising AZD7648 and an mRNA cellular modulator.

[0042] In an embodiment, the composition comprises from about 0.001 pM to about 5 pM AZD7648, e.g., about 0.3 pM AZD7648.

[0043] In an embodiment, the mRNA cellular modulator is selected from the group consisting of a p53 inhibitor, a cell cycle modulator, a DNA repair modulator and combinations of the foregoing. [0044] In an embodiment, the mRNA cellular modulator comprises a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof.

[0045] In an embodiment, the mRNA cellular modulator comprises a sequence of any one of SEQ ID NO: 1-6, or sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0046] In an embodiment, the mRNA cellular modulator consists of a sequence of any one of SEQ ID NO: 1-6.

[0047] In an embodiment, the composition comprises the mRNA cellular modulator in an amount sufficient to deliver from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator, e.g., about 0.8 pg/500,000 cells of the mRNA cellular modulator.

[0048] In an embodiment, the composition further comprises one or more of: a. a gene editing system comprising a nuclease; and b. a donor template.

[0049] In an embodiment, the donor template is selected from the group consisting of a dsDNA, a ssDNA, a ssODN and a IssDNA.

[0050] In an embodiment, the nuclease is an RNA-guided nuclease, e.g., Cas9.

[0051] In an embodiment, the gene editing system further comprises a gRNA that is complementary to the target sequence.

[0052] In an embodiment, the gRNA is a sgRNA.

[0053] In an embodiment, the gene editing system comprises an RNA-guided nuclease and a gRNA, wherein the RNA-guided nuclease and the gRNA complexed as a RNP.

[0054] Also provided herein are methods for modifying a target sequence in a cell (e.g., a population of cells), comprising contacting the cell with the composition disclosed herein. In some embodiments, the method further comprises screening the resulting cell population to identify the modified cells, and selecting the modified cells for use, e.g., in methods for the treatment of a disease or disorder.

Brief Description of the Drawings

[0055] Embodiments of the invention are described herein, by way of non-limiting example only, with reference to the accompanying drawings.

[0056] Figure 1 is a representative analysis of the inferred sequences present in an edited population of K562 cells and their relative representation in the edited pool using Inference of CRISPR Edits (ICE), with the contribution (%) of indels and template integration enabling the inference of DNA repair pathway activity.

[0057] Figure 2A is a representative analysis of indel distributions following editing of K562 cells by ICE showing (left panel) the inferred distribution of indel sizes (y-axis) in the entire edited population of genomes (percentage of this indel in mixture; x-axis); and (right panel) a discordance plot with the level of discordance (y-axis) between the nonedited control and the edited sample in the interference window (z.e., the region around the cut site) according to the Sanger coordinates (BP; x-axis).

[0058] Figure 2B is a representative analysis of indel distributions following editing of K562 cells in the presence of AZD7648 using ICE showing (left panel) the inferred distribution of indel sizes (y-axis) in the entire edited population of genomes (percentage of this indel in mixture; x-axis); and (right panel) a discordance plot with the level of discordance (y-axis) between the non-edited control and the edited sample in the interference window (z.e., the region around the cut site) according to the Sanger coordinates (BP; x- axis).

[0059] Figure 3 shows a comparative analysis of the activity of DNA-PK inhibitors on indel frequency following editing using the non-homologous end joining (NHEJ) dominant B2M guide 1. A graphical representation of indel frequency (%; y-axis) with various concentrations (pM; x-axis) of AZD7648 or NU7026 following editing with guide RNA (gRNA) targeting (A) B2M and (B) BST2. [0060] Figure 4 shows that AZD7648 more potently decreases NHEJ efficiency in K562 cells relative to NU7026. A series of graphical representations of indel frequency (%; y-axis) with various concentrations (pM; x-axis) of AZD7648 or NU7026 following editing with gRNA targeting (A-B) B2M (B2M4 gRNA), (C-D) BST2 (TetN4 gRNA), and (E-F) B2M (B2M9 gRNA). Indels counted and presented as WT (0), NHEJ (+/-1, +/-2), or microhomology-mediated end joining (MMEJ) (-3<; >3).

[0061] Figure 5 shows that AZD7648 decreases NHEJ efficiency in K562 cells. A graphical representation of indel distribution (%; y-axis) and AZD7648 concentration (pM; x-axis) using B2M guide 4. Indels counted and presented as WT (0), NHEJ (+/- 1, +/-2), or MMEJ (-3<; >3).

[0062] Figure 6 shows that AZD7648 decreases NHEJ efficiency in K562 cells. A graphical representation of indel distribution (%; y-axis) and AZD7648 concentration (pM; x-axis) using B2M guide 1,3 4 and BST2. A graphical representation of indel frequency (%; y-axis) with various concentrations (pM; x-axis) of AZD7648

[0063] Figure 7 shows that MMEJ rates in mobilized peripheral blood CD34+ cells were increased at increasing AZD7648 concentrations. A graphical representation of indel distribution (%; y-axis) and AZD7648 concentration (pM; x-axis) using gRNAs targeting (A)-(C) B2M and (D) BST2.

[0064] Figure 8 shows that AZD7648 has minimal impact on mPB CD34 cell viability. A graphical representation of viability (%; y-axis) and AZD7648 concentration (pM; x-axis) using gRNAs targeting B2M.

[0065] Figure 9 shows that AZD7648 decreases NHEJ efficiency in CD34+ HSCs. A series of graphical representations of editing efficiency (indel frequency %; y-axis) and AZD7648 concentration (pM; x-axis) in CD34+ HSCs from (A) Donor 1, (B) Donor 2, (C) Donor 3, (D) Donor 4, (E) Donor 5 and (F) Donor 6.

[0066] Figure 10 shows that AZD7648 increases HDR efficiency in CD34+ HSCs. A series of graphical representations of editing efficiency (template integration frequency %; y-axis) and AZD7648 concentration (pM; x-axis) in CD34+ HSCs from (A) Donor 1 RNP + ssODN, (B) Donor 2 RNP + ssODN, (C) Donor 3 RNP + ssODN, (D) Donor 4 RNP + ssODN, (E) Donor 5 RNP + ssODN and (F) Donor 6 RNP + ssODN. [0067] Figure 11 shows that AZD7648 decreases NHEJ efficiency, thereby increasing HDR efficiency in CD34+ HSCs in a dose-dependent manner. A 4-parameter logistic regression model was used to determine (A) NHEJ inhibition (NHEJ percentage, %; y-axis) as a function of AZD7648 concentration (LogM; x-axis) and (B) HDR dominance (HDR percentage, %; y-axis) as a function of AZD7648 concentration (LogM; x-axis) in B2M4 gRNA + ssODN edited CD34+ HSCs from six donors.

[0068] Figure 12 shows that the GSE_Ad5 mRNA cellular modulator increases in vivo engraftment of CD34+ HSCs. (A) A graphical representation of GFP positive CD34+ HSCs (% GFP High; y-axis) and GSE_Ad5 mRNA cellular modulator concentration (pg; x-axis) in peripheral blood of recipient mice at day 5 post-transplantation. (B) A graphical representation of live cells (%; y-axis) and GSE_Ad5 mRNA cellular modulator concentration (pg; x-axis) in peripheral blood of recipient mice at day 5 post-transplantation.

[0069] Figure 13 shows that the GSE_i53 mRNA cellular modulator increases in vivo engraftment of CD34+ HSCs. (A) A graphical representation of GFP positive CD34+ HSCs (% GFP High; y-axis) and GSE_i53 or i53_GSE mRNA cellular modulator concentration (pg; x-axis) in peripheral blood of recipient mice at day 5 post-transplantation. (B) A graphical representation of GFP positive CD34+ HSCs (% GFP High; y-axis) and GSE, i53 and GSE_i53 mRNA cellular modulator concentration (pg; x-axis) in peripheral blood of recipient mice at day 5 post-transplantation.

[0070] Figure 14 shows a comparative analysis of the combination of AZD7648 and the GSE_Ad5 mRNA cellular modulator to increase in vitro HDR efficiency and in vivo engraftment of CD34+ HSCs. (A) A graphical representation of GFP positive CD34+ HSCs (% GFP High; y-axis) treated with either AZD7648 or a combination of GSE_Ad5 and AZD7648 at day 5 post-nucleofection. (B) A graphical representation of GFP positive CD34+ HSCs (% GFP High; y-axis) treated with AZD6748, GSE_Ad5 mRNA or a combination of both in peripheral blood of recipient mice at day 5 post-transplantation.

[0071] Figure 15 shows that AZD7648 and the GSE_Ad5 mRNA cellular modulator increase HDR efficiency relative to commercially available HDR enhancers. A graphical representation of GFP positive CD34+ HSCs (% GFP High; y-axis) with AZD7648, GSE_Ad5 mRNA cellular modulator, a combination of AZD7648 and GSE_Ad5 mRNA cellular modulator or a commercially available HDR enhancer. [0072] Figure 16 shows that AZD7648 and the GSE_Ad5 mRNA cellular modulator increase in vivo engraftment of edited CD34+ HSCs. (A) A graphical representation of peripheral blood hCD45+ cells (%; y-axis) in samples taken at week 10 post-transplantation (x-axis). (B) A graphical representation of peripheral blood GFP positive hCD45+ cells (%; y-axis) in samples taken at week 10 post-transplantation. (C) A graphical representation of peripheral blood edited cells (%GFP x %hCD45; y-axis) in samples taken at week 10 posttransplantation (x-axis).

[0073] Figure 17 is a series of schematic representations of mRNA cellular modulators exemplified herein. (A) GSE56. (B) GSE56-Ad5E4orf6/7 (GSE_Ad5). (C) GSE56-i53 (GSEJ53) and i53-GSE56 (i53_GSE).

Brief Description of the Sequences

[0074] Nucleic acid and amino acid sequences are referred to by sequence identifier (SEQ ID NO) with reference to the accompanying Sequence Listing, in which:

[0075] SEQ ID NO: 1 is the nucleotide sequence of the GSE56 mRNA cellular modulator.

[0076] SEQ ID NO: 2 is the nucleotide sequence of the Ad5E4orf6/7 mRNA cellular modulator.

[0077] SEQ ID NO: 3 is the nucleotide sequence of the i53 mRNA cellular modulator.

[0078] SEQ ID NO: 4 is the nucleotide sequence of the GSE56 and Ad5E4orf6/7 mRNA cellular modulator (z.e., GSE56_Ad5).

[0079] SEQ ID NO: 5 is the nucleotide sequence of the GSE56 and i53 mRNA cellular modulator (z.e., GSE56_i53).

[0080] SEQ ID NO: 6 is the nucleotide sequence of the i53 and GSE56 mRNA cellular modulator (z.e., i53_GSE56).

[0081] SEQ ID NO: 7 is the nucleotide sequence of the B2M4 guide RNA (gRNA).

[0082] SEQ ID NO: 8 is the nucleotide sequence of the B2M9 gRNA. [0083] SEQ ID NO: 9 is the nucleotide sequence of the B2M17 gRNA.

[0084] SEQ ID NO: 10 is the nucleotide sequence of the BST2 / TetN4 gRNA.

[0085] SEQ ID NO: 11 is the nucleotide sequence of the forward primer for amplification of B2M.

[0086] SEQ ID NO: 12 is the nucleotide sequence of the reverse primer for amplification of B2M.

[0087] SEQ ID NO: 13 is the nucleotide sequence of the sequencing primer for amplification of B2M.

[0088] SEQ ID NO: 14 is the nucleotide sequence of the forward primer for amplification of BST2.

[0089] SEQ ID NO: 15 is the nucleotide sequence of the reverse primer for amplification of BST2.

[0090] SEQ ID NO: 16 is the nucleotide sequence of the sequencing primer for amplification of BST2.

[0091] SEQ ID NO: 17 is the nucleotide sequence of the BST2 single stranded oligodeoxynucleotide (ssODN) donor template.

[0092] SEQ ID NO: 18 is the nucleotide sequence of the B2M1 ssODN donor template.

[0093] SEQ ID NO: 19 is the amino acid sequence of the GSE56 dominant negative peptide.

[0094] SEQ ID NO: 20 is the amino acid sequence of Ad5E4ofr6/7.

[0095] SEQ ID NO: 21 is the amino acid sequence of the 53BP1 inhibitor, i53.

[0096] SEQ ID NO: 22 is the amino acid sequence of the P2A linker.

[0097] SEQ ID NO: 23 is the amino acid sequence of the T2A linker. [0098] SEQ ID NO: 24 is the nucleotide sequence of the pU57 plasmid comprising a polynucleotide encoding the GSE56 mRNA cellular modulator.

[0099] SEQ ID NO: 25 is the nucleotide sequence of the pU57 plasmid comprising a polynucleotide encoding the i53 mRNA cellular modulator.

[0100] SEQ ID NO: 26 is the nucleotide sequence of the pU57 plasmid comprising a polynucleotide encoding the Ad5 E4ofr6/7 mRNA cellular modulator.

[0101] SEQ ID NO: 27 is the nucleotide sequence of the pU57 plasmid comprising a polynucleotide encoding the GSE56_Ad5 mRNA cellular modulator.

Detailed Description

[0102] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0103] The articles "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an allele" includes a single allele, as well as two or more alleles; reference to "a cell" includes a single cell, as well as two or more cells, e.g., a population of cells, and so forth.

[0104] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or). [0105] The term “about”, as used herein, means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.

[0106] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment, unless expressly stated otherwise.

[0107] Nucleotide and amino acid sequences are referred to by a sequence identifier number (z.e., SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l (SEQ ID NO: 1), <400>2 (SEQ ID NO: 2), etc. A sequence listing is provided after the claims. A list describing the SEQ ID NOs in the sequence listing is provided above under the section "Brief Description of the Sequences".

[0108] All sequence identifiers (e.g., GenBank ID, EMBL-Bank ID, DNA Data Bank of Japan (DDBJ) ID, etc.) provided herein were current at the filing date.

[0109] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0110] The term “optionally” is used herein to mean that the subsequent described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiment in which the event or circumstance occurs as well as embodiments in which it does not.

Methods for modifying a target sequence

[0111] In an aspect disclosed herein, there is provided a method for modifying a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; and c. a second-generation DNA-PK inhibitor.

[0112] The term "modifying" as used herein refers to any change to the target sequence, including the insertion of one or more nucleotides (e.g., a donor template) into the target sequence.

[0113] In an embodiment, the cell is derived from a mammalian donor. In another embodiment, the cell is derived from a human donor.

[0114] In an embodiment, the cell is a hematopoietic stem cell (HSC).

[0115] As used herein the terms "hematopoietic stem cell" or "HSC" refer to multipotent cells capable of differentiating into all of the cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, lymphocytes, dendritic cells; and self-renewal activity,

the ability to divide and generate at least one daughter cell with the identical (e.g., self-renewing) characteristics of the parent cell.

[0116] The HSCs contemplated herein (e.g., CD4+ T lymphocytes, CD8+ lymphocytes and/or monocytes/macrophages) can be isolated from the bone marrow or peripheral blood of a subject. The term “isolated” as used herein refers to a cell, which is substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment, e.g., whole blood. Methods for the isolation of HSCs from whole blood would be known to persons skilled in the art, illustrative examples of which include flow cytometry based on the expression, or lack of expression of cell surface markers (e.g., CD117⁺, CD34^neg/low, Thy^low, Flk2“).

Gene editing

[0117] The term "gene editing" as used herein refers to a type of genetic alteration in which a donor template is inserted into a target sequence in the genome of a cell, i.e., in the genome of a HSC, induced by a nuclease, e.g., an RNA-guided nuclease. Any suitable nuclease can be introduced into a cell to induce genome editing of a target sequence, illustrative examples include CRISPR-associated protein (Cas) endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, variants, fragments and combinations thereof. Naturally-occurring and synthetic nucleases are contemplated herein.

[0118] In an embodiment, the gene editing system uses a non-RNA-guided nuclease, for example zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, megaTALs, and variants and combinations thereof.

[0119] In an embodiment, the gene editing system uses an RNA-guided nuclease, e.g., Cas endonucleases.

[0120] In an embodiment, the gene editing system is a CRISPR-Cas gene editing system.

[0121] The “clustered regularly interspaced short palindromic repeat” (CRISPR) / “CRISPR-associated protein” (Cas) system (CRISPR/Cas system) evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. The mechanisms of CRISPR-mediated gene editing would be known to persons skilled in the art and have been described, for example, by Doudna et al., (2014, Methods in Enzymology, 546). Briefly, upon exposure to a virus, short segments of viral DNA are integrated in the clustered regularly interspaced short palindromic repeats (z.e., CRISPR) locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complementarity to the viral genome, mediates targeting of a Cas endonuclease to the sequence in the viral genome. The Cas endonuclease cleaves the viral target sequence to prevent integration or expression of the viral sequence. [0122] CRISPR-Cas genome editing systems may be used to generate a site-specific double strand break (DSB) within a double-stranded DNA (dsDNA) target sequence. Once a DSB is detected in a cell, the DNA repair machinery will repair the break by "non- homologous end-joining" or "NHEJ", "homology-directed repair" or "HDR" or "microhomology-mediated end joining" or "MMEJ".

[0123] NHEJ is triggered to repair double-stranded breaks in which the break ends are directly ligated without the need for a homologous template. Due to the error-prone nature of this repair pathway, small insertions or deletions (INDELs) may be introduced at the target locus near the site of the initial cleavage, and such INDELs can cause frameshift mutations, promote internal ribosomal entry, convert pseudo-mRNAs into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (see, e.g., Tuladhar et al., 2019, Nature Communications, 10: 4056). Unpredicted large genome modifications can also be introduced, which can be more than several kilobases (see, e.g., Kosicki et al., 2018 Nature Biotechnology, 36: 765-771). By contrast, HDR accurately and precisely repairs DNA breaks using a homologous template to guide repair. The most common form of HDR is homologous recombination (HR), by which nucleotide sequences are exchanged between two similar or identical molecules of DNA. MMEJ mediates the repair of double stranded breaks (DSBs) by the recombination of 5-25 base pair (bp) homologous regions flanking a DSB, also referred to as "microhomologies" or "pHs". MMEJ has been shown to generate more predictable editing outcomes, resulting in improved editing reproducibility at a given locus.

[0124] As used herein the terms “polynucleotide”, "nucleotide sequence", “nucleic acid” or “nucleic acid molecule” mean a single- or double- stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof, and can include molecules comprising coding and non-coding sequences of a gene, sense and antisense sequences and complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

[0125] In the context of CRISPR/Cas gene editing systems, the term "target sequence" as used herein refers to a sequence within the genome of a cell to which a gRNA is designed to have complementarity, where hybridization between the target nucleic acid sequence and the gRNA promotes the formation of a complex comprising the RNA-guided nuclease, the gRNA and the target nucleic acid sequence (z.e., a gene editing complex). In the context of non-RNA-guided gene editing, such as TALENs, ZFNs, meganucleases, and megaTALs, the term “target sequence” as used herein refers to a sequence within the genome of a cell to which a DNA-binding domain of the nuclease binds.

[0126] By "complementary" or "substantially complementary" it is meant that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non- covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, "anneal", or "hybridize" to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson- Crick base-pairing includes: adenine/adenosine (A) pairing with thymidine/thymidine (T), A pairing with uracil/ uridine (U), and guanine/guanosine (G) pairing with cytosine/cytidine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a target nucleic acid sequence base pairs with a gRNA) G can also base pair with U. For example, G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base pairing with codons in rnRNA. Thus, in the context of this disclosure, a G (e.g., of a target nucleic acid sequence base pairing with a gRNA) is considered complementary to both a U and to C. For example, when a G/U base-pair can be made at a given nucleotide position of a protein binding segment of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0127] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (T_m) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0128] By ‘ ‘gene” it is meant a unit of inheritance that, when present in its endogenous state, occupies a specific locus on a genome and comprises transcriptional and/or translational regulatory sequences and/or a coding region and/or non-tran slated sequences (z.e., introns, 5’ and 3’ untranslated sequences).

[0129] As used herein, the terms “encode”, “encoding” and the like refer to the capacity of a polynucleotide to provide for another nucleic acid or a polypeptide. For example, a polynucleotide is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a polynucleotide may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode," "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0130] The terms “protein”, “peptide” and “polypeptide” are used interchangeably herein to refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure or function.

[0131] In an embodiment, the gene editing system further comprises a gRNA that is complementary to a target sequence in a cell.

[0132] The terms “guide RNA” or “gRNA” refer to a RNA sequence that is complementary to a target nucleic acid sequence and directs a RNA-guided nuclease to the target nucleic acid sequence. gRNA typically comprises CRISPR RNA (crRNA) and a tracr RNA (tracrRNA). "crRNA" is a 17-20 nucleotide sequence that is complementary to the target nucleic acid sequence, while the "tracrRNA" provides a binding scaffold for the RNA- guided nuclease. crRNA and tracrRNA exist in nature a two separate RNA molecules, which has been adapted for molecular biology techniques using, for example, 2-piece gRNAs such as CRISPR tracer RNAs (cr:tracrRNAs).

[0133] In an embodiment, the gRNA is a single guide RNA (sgRNA).

[0134] The terms “single-guide RNA” or “sgRNA” refer to a single RNA sequence that comprises the crRNA fused to the tracrRNA.

[0135] The sgRNA contemplated herein are complementary to a target sequence within the genome of a cell, e.g. , an HSC.

[0136] In an embodiment, the sgRNA comprises a sequence of at least 10 contiguous nucleotides that are complementary to the target sequence. Accordingly, the sgRNA comprises a sequence of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28 , at least 29, or at least 30 nucleotides that are complementary to the target sequence.

[0137] In an embodiment, the sgRNA comprises a sequence of at least 20 contiguous nucleotides that are complementary to the target sequence.

[0138] Methods and tools for the design of sgRNA would be known to persons skilled in the art, illustrative examples of which include CHOPCHOP, CRISPR Design, sgRNA Designer, Synthego and GT-Scan.

[0139] Methods and tools for the design of TALENs and ZFNs would be known to persons skilled in the art, illustrative examples of which include CHOPCHOP, TALE-NT (Doyle, et al., 2012, Nucleic Acids Research, 4O(W1): W117-W122) and ZF Tools (Mandell and Barbas, 2006, Nucleic Acids Research, 34(W1): W516-W523).

[0140] In an embodiment, the target sequence is within the B2M or BST2 genes. Suitable sgRNAs complementary to a target sequence within the B2M or BST2 genes could be designed and produced by persons skilled in the art, illustrative examples of which include the sgRNAs described elsewhere herein, such as the sgRNAs targeting B2M (SEQ ID NOs: 1-3) and BST2 (SEQ ID NO: 4), as shown in Table 1. [0141] In an embodiment, the target sequence is within a gene associated with a disease or disorder. Suitable diseases or disorders would be known to persons skilled in the art, illustrative examples of which include cancer, cardiovascular diseases (e.g., heart failure, hypertension and atherosclerosis), respiratory diseases, renal diseases, gastrointestinal diseases (e.g., inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, hepatic, gallbladder and bile duct diseases, including hepatitis and cirrhosis), hematologic diseases, metabolic diseases, endocrine and reproductive diseases (e.g., diabetes, bone and bone mineral metabolism diseases), hereditary eye diseases (e.g., congenital cataract, congenital glaucoma, retinitis pigmentosa, congenital corneal dystrophy, Leber congenital amaurosis, retinoblastoma and Usher syndrome), immune system diseases (e.g., autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, and other autoimmune diseases), musculoskeletal and connective tissue diseases (e.g., arthritis, achondroplasia), infectious diseases and neurological diseases (e.g., Alzheimer's disease, Huntington's disease and Parkinson's disease). The target sequence / gene suitable for use in the treatment of such diseases or disorders may include, e.g., HBB, KLF1, LDLR, PAH, DUX4, DMD, CFTR, OTC, AGXT, COL7A1, BCL11A, TCRa, TCR/i, PDCD1, CCR5, B2M, CEP290, HPV16, HPV18, CD7, HPK1, CD40L, CALM2 and EGFR.

[0142] In another embodiment, the target sequence is within a safe harbor locus, e.g., CCR5, CXCR4, PPPIRI2c, an albumin gene or a Rosa gene.

[0143] In an embodiment, the nuclease is an RNA-guided nuclease.

[0144] In an embodiment, the RNA-guided nuclease is a CRISPR-associated (Cas) endonuclease. Suitable Cas endonucleases would be known to persons skilled in the art, illustrative examples of which include Cas9 and Cas 12 (e.g., Cas 12a, Cas 12b, Cas 12c, Casl2d, Casl2e).

[0145] In an embodiment, the RNA-guided nuclease is Cas9.

[0146] In an embodiment, the gene editing system is provided to the cell within a vector. The vector can be an episomal vector (z.e., that does not integrate into the genome of the cell), or can be a vector that integrates into the cell genome. Vectors may be replication competent or replication-deficient. Exemplary vectors include, but are not limited to, plasmids, cosmids, and viral vectors, such as adeno-associated virus (AAV) vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. Preferably, however, the vector is suitable for use in biotechnology.

[0147] Vectors suitable for use in biotechnology would be known to persons skilled in the art, illustrative examples of which include viral vectors derived from adenovirus, adeno- associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, self-amplifying single-strand RNA (ssRNA) viruses such as alphavirus (e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis, Ml), and flavivirus (e.g., Kunjin virus, West Nile virus, Dengue virus), rhabdovirus (e.g., rabies, vesicular stomatitis virus), measles virus, Newcastle Disease virus (NDV) and poxivirus as described by, for example, Lundstrom (2019, Diseases, 6: 42).

[0148] In an embodiment, the vector is an adeno-associated virus (AAV) vector. Exemplary AAV vectors include, without limitation, those derived from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, or using synthetic or modified AAV capsid proteins such as those optimized for efficient in vivo transduction. A recombinant AAV vector describes replication-defective virus that includes an AAV capsid shell encapsidating an AAV genome. Typically, one or more of the wild-type AAV genes have been deleted from the genome in whole or part, preferably the rep and/or cap genes.

[0149] In an embodiment, the AAV vector is an AAV6 vector.

[0150] In an embodiment, the vector is a plasmid.

[0151] Where the gene editing system is provided to the cell within a vector, the vector may comprise polynucleotides encoding the nuclease and a gRNA in one or two vectors. When multiple polynucleotides are combined within the same vector, the expression of each polynucleotide may be controlled by the same promoter or different promoters according to the optimal stoichiometry of the different components of the gene editing system. Thus, in some examples, a polynucleotide encoding the will be operably linked to a first promoter and the polynucleotide encoding the gRNA linked to a second promoter.

[0152] The term "promoter" as used herein refers to an array of control sequences that direct the transcription of a polynucleotide. Suitable promoters would be known to persons skilled in the art, illustrative examples of which include retroviral LTR elements, constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, or P-actin, inducible promoters, such as those containing Tet-operator elements, and/or tissue specific promoters.

[0153] The vector may comprise other additional regulatory elements or sequences. Suitable regulatory sequences would be known to persons skilled in the art, illustrative examples of which include leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, and enhancer or activator sequences. It is also contemplated herein that the vector comprises elements and sequences associated with protein localization and interactions. For example, the polynucleotides encoding the polypeptide tag may comprise sequences encoding a nucleus localization sequence (NLS).

[0154] The present disclosure also provides non-viral delivery vehicles of the gene editing systems, and components thereof. Suitable non-viral delivery vehicles will be known to persons skilled in the art, illustrative examples of which include biological methods (e.g., virus-like particle, cell penetrating peptides), chemical methods (e.g., lipids, lipid-like materials or polymeric materials, as described by, e.g., Rui et al. (2019, Trends in Biotechnology, 37(3): 281-293, and nanoparticles/nanocarriers, as described by, e.g., Nguyen et al. (2020, Nature Biotechnology, 38: 44-49)) and physical methods (e.g., electroporation, sonoporation and microinjection).

[0155] In an embodiment, the gene editing system comprises an RNA-guided nuclease and a gRNA, wherein the RNA-guided nuclease and the gRNA are complexed as a ribonucleoprotein (RNP).

[0156] The terms "ribonucleoproteins", "RNPs" and "Cas-gRNA ribonucleoproteins" are used interchangeably herein to refer to a complex comprising a RNA-guided nuclease and gRNA that can be used to directly deliver the gene editing system to the cell.

[0157] Suitable methods for the direct delivery of RNPs to cells (e.g., HSCs) would be known to persons skilled in the art, illustrative examples of which include microinjection and electroporation. In an embodiment, the RNP is delivered to cells by electroporation (z.e., nucleofection). Donor template

[0158] The term "donor template" as used herein refers to any polynucleotide capable of being utilized as a repair template for HDR, e.g., single-stranded DNA (ssDNA), doublestranded DNA (dsDNA), synthetic polynucleotides and oligonucleotides.

[0159] The structure and arrangement of the donor template will be determined by reference to the desired position of the alteration to be made to the target sequence. Typically, a donor template comprises a pair of homology arms corresponding to the target sequence, wherein each pair of homology arms flanks one or more transgenes. In a particular example, the pair of homology arms comprises a 5' homology arm and a 3' homology arm. A “5' homology arm” refers to a polynucleotide sequence that is identical, or nearly identical, or homologous to a DNA sequence 5' of a target sequence. A “3' homology arm” refers to a polynucleotide sequence that is identical, or nearly identical, or homologous to a DNA sequence 3' of the target sequence.

[0160] The donor templates contemplated herein may further comprise one or more additional modifications to, e.g., improve nuclear delivery and HDR efficiency. Suitable modifications would been known to persons skilled in the art, illustrative examples of which include 5'-modifications (e.g., a simple triethylene glycol (TEG) moiety, a 2'-O-methyl (2'0Me) RNA:TEG modification and a peptide nucleic acid (PNA) comprising the SV40 nuclear localization signal), phosphorothioate (PS) linkages, the addition of a biotin moiety, polyethylene glycol (PEG) linkages, etc.

[0161] In an embodiment, the donor template is selected from the group consisting of a double-stranded DNA (dsDNA), a single-stranded DNA (ssDNA), a single- stranded oligodeoxynucleotide (ssODN) and a long single- stranded DNA (IssDNA).

[0162] In an embodiment, the donor template is a linear donor template or a plasmid donor template.

[0163] In an embodiment, the donor template is a double-stranded DNA (dsDNA).

[0164] In an embodiment, the donor template is a double- stranded plasmid. Suitable plasmids would be known to persons skilled in the art, illustrative examples of which include bacterial plasmids, synthetic plasmids, episomes and minicircles. [0165] In another embodiment, the donor template is a linear dsDNA.

[0166] In an embodiment, the donor template is a single- stranded DNA (ssDNA).

Suitable ssDNA would be known to persons skilled in the art, illustrative examples of which include single- stranded oligodeoxynucleotides (ssODN), long single-stranded DNA (IssDNA) and single- stranded AAV genomic DNA.

[0167] In an embodiment, the donor template is an ssODN.

[0168] In an embodiment, the donor template is provided to the cell by a non-viral delivery method. As described elsewhere herein, suitable non-viral delivery methods include biological methods, chemical methods and physical methods. In an embodiment, the non- viral delivery means is selected from the group consisting of electroporation, sonication, peptide-based delivery, lipid-based delivery, inorganic delivery and polymeric delivery.

[0169] In an embodiment, the donor template is provided to the cell within a vector.

[0170] In an embodiment, the vector is an episomal vector (z.e., that does not integrate into the genome of the cell), or a vector that integrates into the cell genome. Vectors may be replication competent or replication-deficient. Suitable vectors would be known to persons skilled in the art, illustrative examples of which include, but are not limited to, plasmids, cosmids, and viral vectors, such as adeno-associated virus (AAV) vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors.

[0171] In an embodiment, the vector is an adeno-associated virus (AAV) vector. Exemplary AAV vectors include, without limitation, those derived from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, or using synthetic or modified AAV capsid proteins such as those optimized for efficient in vivo transduction. A recombinant AAV vector describes replication-defective virus that includes an AAV capsid shell encapsidating an AAV genome. Typically, one or more of the wild-type AAV genes have been deleted from the genome in whole or part, preferably the rep and/or cap genes.

[0172] In an embodiment, the AAV vector is an AAV6 vector. DNA-PK inhibitors

[0173] The repair of DSBs is mediated, at least in part, by DNA-dependent protein kinase (DNA-PK), a complex that consists of the KU heterodimers (KU70 and KU80) and the DNA-dependent protein kinase catalytic subunit DNA-PKcs. DNA-PK is also associated with cellular processes, such as modulation of chromatin structure, telomere maintenance and transcriptional regulation. In view of the importance of DNA-PK activity in the repair of DSB, small molecule inhibitors have been developed for the treatment of, e.g., cancer. First-generation DNA-PK inhibitors, such as NU7441, NU7026 and KU-0060648, while effective for inhibiting DNA-PK, have limited selectivity against PI3K and PIKK members, e.g., mTOR and PI3Ky. Second-generation DNA-PK inhibitors, such as VX-984, M3814 and AZD7648 have improved selectivity against secondary targets, such as ATM, ATR, mTOR, and PI3K isoforms (i.e., PI3Ka, PI3KP and PI3K5), see, e.g., Fok et al., 2019, Nature Communications, 10: 5065. Second-generation DNA-PK inhibitors have a 50-fold or greater selectivity for DNA-PKcs against six or more of the kinases selected from: ATM, ATR, mTOR, PI3Ka, PI3KP, PI3K5, and PI3Ky.

[0174] In an embodiment, the second-generation DNA-PK inhibitor is selected from the group consisting of VX-984, M3814 and AZD7648.

[0175] VX-984 is an orally bioavailable, selective DNA-PK inhibitor with the structure of formula (I). VX-984 has been shown to inhibit NHEJ (see, e.g., Khan et al., 2018, Oncotarget, 9(40): 25833-25841).

[0176] M3814 is an orally bioavailable, selective DNA-PK inhibitor with the structure of formula (II). M3814 has been shown to inhibit NHEJ and enhance the anti-tumor activity of ionizing radiation and double stranded break- inducing chemotherapies (see, e.g., Zenke et al., 2016, Cancer Research, 76(Suppl.): 1658; and Sun et al., 2019, Molecular Cancer Research, 17: 2457-2458).

[0177] AZD7648 is an orally bioavailable, selective DNA-PK inhibitor with the structure of formula (III). AZD7648 is an ATP-competitive inhibitor of DNA-PK that interferes with NHEJ to prevent the repair of DSBs caused by, e.g., ionizing radiation or chemotherapeutic treatment.

[0178] In a particular embodiment, the second-generation DNA-PK inhibitor is AZD7648.

[0179] In an embodiment, the cell is provided with AZD7648 at a concentration of at least about 0.001 pM. In an embodiment, the cell is provided with AZD7648 at a concentration of at least about 0.002 pM, about 0.003 pM, about 0.004 pM, about 0.005 pM, about 0.006 pM, about 0.007 pM, about 0.008 pM, or about 0.009 pM AZD7648. In an embodiment, the cell is provided with AZD7648 at a concentration of at least about 0.01 pM, about 0.02 pM, about 0.03 pM, about 0.04 pM, about 0.05 pM, about 0.06 pM, about 0.07 pM, about 0.08 pM, or about 0.09 pM AZD7648. In an embodiment, the cell is provided with AZD7648 at a concentration of at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, or about 0.9 pM AZD7648. In an embodiment, the cell is provided with AZD7648 at a concentration of at least about 1.0 pM, about 2.0 pM, about 3.0 pM, about 4.0 pM, about 5.0 pM, about 6.0 pM, about 7.0 pM, about 8.0 pM, about 9.0 pM, or about 10.0 pM AZD7648.

[0180] In an embodiment, the cell is provided with AZD7648 at a concentration of from about 0.001 pM to about 3 pM. Accordingly, the cell may be provided with AZD7648 at a concentration of about 0.001 pM, about 0.002 pM, about 0.003 pM, about 0.004 pM, about 0.005 pM, about 0.006 pM, about 0.007 pM, about 0.008 pM, about 0.009 pM, about 0.01 pM, about 0.02 pM, about 0.03 pM, about 0.04 pM, about 0.05 pM, about 0.06 pM, about 0.07 pM, about 0.08 pM, about 0.09 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1.0 pM, about 2.0 pM, or about 3.0 pM AZD7648.

[0181] In an embodiment, the cell is provided with AZD7648 at a concentration of from about 0.001 pM to about 0.5 pM.

[0182] In an embodiment, the cell is provided with AZD7648 at a concentration of about

0.3 pM.

[0183] In an embodiment, the method comprises incubating the cell with AZD7648 at a temperature between about 35°C and 40°C. In an embodiment, the method comprises incubating the cell with AZD7648 at a temperature of about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C.

[0184] In an embodiment, the method comprises incubating the cell with AZD7648 at a temperature of about 37°C.

[0185] In an embodiment, the method comprises incubating the cell with AZD7648 for at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours.

[0186] In an embodiment, the method comprises incubating the cell with AZD7648 for about 24 hours.

[0187] In an embodiment, the cell is provided the AZD7648 after the gene editing system. In another embodiment, the cell is provided the AZD7648 after the gene editing system and the donor template.

[0188] In an embodiment, the cell is provided the AZD7648 simultaneous with the gene editing system. In another embodiment, the cell is provided the AZD7648 simultaneous with the gene editing system and the donor template.

[0189] In an embodiment, the AZD7648 is provided to cells cultured at a concentration of about 1 x 10² cells/mL, about 1 x 10³ cells/mL, about 1 x 10⁴ cells/mL, about 1 x 10⁵ cells/mL, about 1 x 10⁶ cells/mL, about 1 x 10⁷ cells/mL, or about 1 x 10⁸ cells/mL. In an embodiment, the AZD7648 is provided to cells cultured at a concentration of about 1 x 10⁵ cells/mL, about 2 x 10⁵ cells/mL, about 3 x 10⁵ cells/mL, about 4 x 10⁵ cells/mL, about 5 x 10⁵ cells/mL, about 6 x 10⁵ cells/mL, about 7 x 10⁵ cells/mL, about 8 x 10⁵ cells/mL, or about 9 x 10⁵ cells/mL.

[0190] In an embodiment, the AZD7648 is provided to cells cultured at a concentration of about 2 x 10⁵ cells/mL.

[0191] In an embodiment, the AZD7648 is provided at a concentration of between about

0.001 pM and about 3 pM to cells cultured at a concentration of between about 1 x 10⁵ cells/mL and about 5 x 10⁵ cells/mL. In an embodiment, the AZD7648 is provided at a concentration of between about 0.1 pM and about 1.0 pM to cells cultured at a concentration of about 2 x 10⁵ cells/mL. In an embodiment, the AZD7648 is provided at a concentration of about 0.3 pM to cells cultured at a concentration of about 2 x 10⁵ cells/mL. Cellular modulators

[0192] In certain embodiments, the method further comprises providing to the cell one or more cellular modulators, or a polynucleotide encoding the one or more cellular modulators.

[0193] Accordingly, in an aspect of the present disclosure there is provided a method for modifying a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; c. a second-generation DNA-PK inhibitor; and d. a cellular modulator.

[0194] The term "cellular modulator" as used herein refers to a molecule that is capable of modulating one or more cellular functions, including DNA repair, cell cycle, p53 response, expansion, differentiation, viability and engraftment.

[0195] In an embodiment, the cellular modulator is selected from the group consisting of DNA repair modulators, cell cycle modulators, p53 response modulators, stem cell expansion / viability enhancers and engraftment enhancers.

[0196] The term "DNA repair modulator" as used herein refers to a class of molecules that are capable of enhancing or inhibiting (z.e., "modulating") DNA repair pathways. Suitable examples of DNA repair modulators would be known to persons skilled in the art, illustrative examples of which include i53 (see, e.g., US 10,808,017), UNC2170 and RS-1 (Song et al., 2016, Nature Communications, 1 10548; and Jayathilaka et al., 2008, Proceedings of the National Academy of Sciences U.S.A., 105(41): 15848-15853).

[0197] In an embodiment, the DNA repair modulator is i53, or a polynucleotide encoding i53.

[0198] i53 is a polypeptide (e.g. , as shown in SEQ ID NO: 9) that modulates DNA repair by inhibiting the binding of 53BP1 to DSBs, thereby inhibiting NHEJ. Functional fragments of i53 encompassed by the present disclosure may comprise any portion of the i53 polypeptide that retain at least about 60% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) binding affinity to 53BP1. For example, a functional fragment of i53 may have at least 1, at least 2, at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 fewer amino acid residues or nucleic acid bases relative to the full-length i53 polypeptide of SEQ ID NO: 9 or the polynucleotide encoding i53 of SEQ ID NO: 3.

[0199] UNC2170, also known as 3-bromo-N-[3-[(l,l-dimethylethyl)amino]propyl]- benzamide or 2Z-butenedioate, is a small molecule that modulates DNA repair by functioning as a 53BP1 antagonist, thereby inhibiting NHEJ.

[0200] The term "cell cycle modulator" as used herein refers to a class of molecules that are capable of inhibiting or progressing (z.e., modulating) the cell cycle. Suitable cell cycle modulators would be known to persons skilled in the art, illustrative examples of which include CDK inhibitors (e.g., small molecule inhibitors such as staurosporine, flavopiridol, butyrolactone I, olomucine and roscovitine), CDC25 inhibitors (e.g., anti-CDC25 anitibodies), proteasome modulators (e.g., cyclins) and Adenoviral proteins / peptides (e.g., adenovirus 5 E4orf6/7 protein).

[0201] In an embodiment, the cell cycle modulator is the adenovirus 5 E4orf6/7 protein or "Ad5E4orf6/7", or a polynucleotide encoding Ad5E4orf6/7.

[0202] Ad5E4orf6/7 is a polypeptide (e.g., as shown in SEQ ID NO: 8), which recruits the cell-cycle controller E2F on to its target genes. Functional fragments of Ad5E4orf6/7 encompassed by the present disclosure may comprise any portion of the Ad5E4orf6/7 polypeptide that retain at least about 60% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) recruitment activity of E2Fto its target genes. For example, a functional fragment of Ad5E4orf6/7 may have at least 1, at least 2, at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 fewer amino acid residues or nucleic acid bases relative to the full-length Ad5E4orf6/7 polypeptide of SEQ ID NO: 8 or the polynucleotide encoding Ad5E4orf6/7 of SEQ ID NO: 2.

[0203] The term "p53 response modulator" as used herein refers to a class of molecules that are capable of inhibiting or activating (z.e., modulating) p53-dependent transcription. Suitable p53 response modulators would be known to persons skilled in the art, illustrative examples of which include small molecule activators / restorers of p53 (e.g., pifithrin-alpha, CP-31398, PRIMA1 and Nutlins), genetic suppressor elements (GSEs) from p53 (e.g., GSE56) and modulators of p53 transactivation (e.g., 5CHQ).

[0204] In an embodiment, the p53 response modulator is GSE56.

[0205] GSE56 is a dominant negative p53 truncated form peptide (e.g., as shown in

SEQ ID NO: 7), which inhibits p53. Functional fragments of GSE56 encompassed by the present disclosure may comprise any portion of the GSE56 polypeptide that retain at least about 60% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) inhibition of p53- dependent transcriptional response measured by, e.g., transcription of p53 target genes, such as p21. For example, a functional fragment of GSE56 may have at least 1, at least 2, at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 fewer amino acid residues or nucleic acid bases relative to the full-length GSE65 polypeptide of SEQ ID NO: 7 or the polynucleotide encoding GSE56 of SEQ ID NO: 1.

[0206] The term "stem cell expansion / viability enhancer" as used herein refers to a class of molecules that are capable of improving the expansion and/or viability of stem cells in vitro, ex vivo and/or in vivo. Suitable stem cell expansion / viability enhancers would be known to persons skilled in the art, illustrative examples of which include small molecule stimulators of hematopoiesis (e.g., UM171 and UM729), aryl hydrocarbon receptor (AHR) antagonists (e.g., StemRegenin 1 (SR-1)), histone deacetylase inhibitors (e.g., valproic acid (VPA), CAY10433), caspase inhibitors, eltrombopag and nicotinamide.

[0207] The term "engraftment enhancer" as used herein refers to a class of molecules that are capable of improving engraftment of cells in vivo. Suitable engraftment enhancers would be known to persons skilled in the art, illustrative examples of which include preconditioning or systematic administration of chemotherapy (e.g., busulfan, radiation and monoclonal antibodies), proinflammatory cytokines (e.g., IL-6 and IL-6R) and pleiotrophin.

[0208] In an embodiment, the cellular modulator is an mRNA cellular modulator. [0209] The term "mRNA cellular modulators" as used herein refers to an RNA polynucleotide comprising one or more component nucleotide sequences that encode, e.g., a p53 inhibitor, a cell cycle modulator and/or a DNA repair modulator.

[0210] Where the mRNA cellular modulator comprises two heterologous component nucleotide sequences, the mRNA cellular modulator may be variously referred to as a "heterologous mRNA" or "chimeric RNA". Such mRNA cellular modulators can optionally include linker or spacer nucleotides, which are fused together to form the chimeric mRNA.

[0211] The term "linker" refers to any nucleotide or group of nucleotides that joins or connects two components of the heterologous nucleotide sequence. Suitable linkers would be known to persons skilled in the art, illustrative examples of which include a P2A linker (e.g., SEQ ID NO: 10) and a T2A linker (e.g., SEQ ID NO: 11).

[0212] In an embodiment, the linker is selected from a P2A linker and a T2A linker. P2A and T2A are self-cleaving peptide linkers which can create two functional peptides from a single transcript.

[0213] In an embodiment, the mRNA cellular modulator further comprises regulatory elements.

[0214] The term "regulatory elements" as used herein refers to the nucleotide sequences required for the expression of a gene. In certain embodiments, the regulatory elements an enhancer and/or other regulatory elements that are required for the expression of a gene.

[0215] In an embodiment, the mRNA cellular modulator comprises a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE). In an embodiment, the chimeric RNA comprises an WPRE at the 3' end of the mRNA cellular modulator.

[0216] In an embodiment, the mRNA cellular modulator comprises a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof. [0217] In an embodiment, the mRNA cellular modulator comprises a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof.

[0218] In an embodiment, the mRNA cellular modulator comprises a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0219] In an embodiment, the mRNA cellular modulator comprises a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0220] The term "functional variant" refers to a variant of the mRNA cellular modulator that comprises suitable nucleotide substitutions or deletions that do not eliminate the functional properties of the mRNA cellular modulator. In some examples, functional variant of the mRNA cellular modulator will possess at least about 80% identity to the sequence of which it is a variant. Accordingly, the sequence may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of which it is a variant.

[0221] In an embodiment, the mRNA cellular modulator comprises a sequence of any one of SEQ ID NOs: 1-6, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0222] In an embodiment, the mRNA cellular modulator consists of a sequence of any one of SEQ ID NOs: 1-6.

[0223] In an embodiment, the composition comprises the mRNA cellular modulator in an amount sufficient to deliver from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator. Accordingly, in an embodiment, the composition comprises the mRNA cellular modulator in an amount sufficient to deliver about 0.01 pg/500,000 cells, about 0.02 pg/500,000 cells, about 0.03 pg/500,000 cells, about 0.04 pg/500,000 cells, about 0.05 pg/500,000 cells, about 0.06 pg/500,000 cells, about 0.07 pg/500,000 cells, about 0.08 pg/500,000 cells, about 0.09 pg/500,000 cells, about 0.1 pg/500,000 cells, about 0.2 pg/500,000 cells, about 0.3 pg/500,000 cells, about 0.4 pg/500,000 cells, about 0.5 pg/500,000 cells, about 0.6 pg/500,000 cells, about 0.7 pg/500,000 cells, about 0.8 pg/500,000 cells, about 0.9 pg/500,000 cells, about 1.0 pg/500,000 cells, about 2.0 pg/500,000 cells, about 3.0 pg/500,000 cells, about 4.0 pg/500,000 cells, or about 5.0 pg/500,000 cells of the mRNA cellular modulator. [0224] In an embodiment, the composition comprises the mRNA cellular modulator in an amount sufficient to deliver about 0.8 pg/500,000 cells of the mRNA cellular modulator.

[0225] In an embodiment, the mRNA cellular modulator is provided as a polynucleotide encoding the mRNA cellular modulator. In another embodiment, the polynucleotide encoding the mRNA cellular modulator is within a vector.

[0226] In an embodiment, the vector is an episomal vector (z.e., that does not integrate into the genome of the cell), or a vector that integrates into the cell genome. Vectors may be replication competent or replication-deficient. Suitable vectors would be known to persons skilled in the art, illustrative examples of which include, but are not limited to, plasmids, cosmids, and viral vectors, such as adeno-associated virus (AAV) vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors.

[0227] In an embodiment, the polynucleotide encoding the mRNA cellular modulator is within a vector, e.g. , a plasmid. In another embodiment, the plasmid comprises a sequence selected from SEQ ID NOs: 24-27.

[0228] In an embodiment, the gene editing system, donor template and second- generation DNA-PK inhibitor are provided to the cell concurrently.

[0229] In an embodiment, the cellular modulator is provided to the cell concurrently with the gene editing system, donor template and second-generation DNA-PK inhibitor.

[0230] The terms “provided concurrently” or “providing concurrently” and the like refer to the provision of a single composition containing each agent (e.g., the gene editing system, donor template and second-generation DNA-PK inhibitor; or the gene editing system, donor template, second-generation DNA-PK inhibitor and cellular modulator), or the administration of each agent (e.g., the gene editing system, donor template and second- generation DNA-PK inhibitor; or the gene editing system, donor template, second- generation DNA-PK inhibitor and cellular modulator) as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such agents are administered as a single composition. [0231] In an embodiment, the gene editing system, donor template and second- generation DNA-PK inhibitor are provided to the cell simultaneously.

[0232] In an embodiment, the cellular modulator is provided to the cell simultaneously with the gene editing system, donor template and second-generation DNA-PK inhibitor.

[0233] By “simultaneously” is meant that the agents (e.g., the gene editing system, donor template and second-generation DNA-PK inhibitor; or the gene editing system, donor template, second-generation DNA-PK inhibitor and cellular modulator) are administered at substantially the same time, and desirably together in the same composition. By “contemporaneously” it is meant that the agents (e.g., the gene editing system, donor template and second-generation DNA-PK inhibitor; or the gene editing system, donor template, second-generation DNA-PK inhibitor and cellular modulator) are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful.

[0234] In an embodiment, the gene editing system, donor template and second- generation DNA-PK inhibitor are provided to the cell sequentially.

[0235] In an embodiment, the cellular modulator is provided to the cell sequentially with the gene editing system, donor template and second-generation DNA-PK inhibitor.

[0236] The term “separately” as used herein means that the agents (e.g., the gene editing system, donor template and second-generation DNA-PK inhibitor; or the gene editing system, donor template, second-generation DNA-PK inhibitor and cellular modulator) are provided to the cell at an interval, e.g., at an interval of about a minute to several minutes or hours. The agents may be administered in any order. The term “sequentially” as used herein means that the agents (e.g., the gene editing system, donor template and second-generation DNA-PK inhibitor; or the gene editing system, donor template, second-generation DNA-PK inhibitor and cellular modulator) are administered in sequence, e.g. , at an interval or intervals of minutes, hours or days. If appropriate the agents may be administered in a regular repeating cycle.

[0237] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof. [0238] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator and the mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0239] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0240] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0241] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0242] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0243] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0244] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator and the mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof. [0245] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0246] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0247] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0248] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0249] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0250] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 1, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0251] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 2, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto). [0252] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 3, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0253] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 2, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0254] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0255] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1, 2 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0256] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of any one of SEQ ID NO: 1-6, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0257] In an embodiment, the method comprises providing to the cell AZD7648, the gene editing system, the donor template and the mRNA cellular modulator consisting of the sequence of any one of SEQ ID NOs: 1-6.

[0258] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof.

[0259] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator and the mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0260] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0261] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0262] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0263] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0264] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0265] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator and the mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0266] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof. [0267] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0268] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0269] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0270] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0271] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 1, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0272] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 2, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0273] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 3, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto). [0274] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 2, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0275] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0276] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1, 2 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0277] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of any one of SEQ ID NO: 1-6, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0278] In an embodiment, the method comprises providing to the cell VX-984, the gene editing system, the donor template and the mRNA cellular modulator consisting of the sequence of any one of SEQ ID NOs: 1-6.

[0279] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof.

[0280] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator and the mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0281] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof. [0282] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0283] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0284] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0285] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0286] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator and the mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0287] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0288] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof. [0289] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0290] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0291] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0292] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 1, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0293] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 2, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0294] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequence of SEQ ID NO: 3, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0295] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 2, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto). [0296] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0297] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of SEQ ID NO: 1, 2 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0298] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator comprising the sequences of any one of SEQ ID NO: 1-6, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0299] In an embodiment, the method comprises providing to the cell M3814, the gene editing system, the donor template and the mRNA cellular modulator consisting of the sequence of any one of SEQ ID NOs: 1-6.

[0300] In an aspect disclosed herein, there is provided a second-generation DNA-PK inhibitor for use in a method of modifying of a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; and c. the second-generation DNA-PK inhibitor.

[0301] In another aspect disclosed herein, there is provided a second-generation DNA- PK inhibitor and cellular modulator for use in a method of modifying of a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; c. the second-generation DNA-PK inhibitor; and d. a cellular modulator. [0302] In an embodiment, the second-generation DNA-PK inhibitor is selected from the group consisting of VX-984, M3814 and AZD7648. In another embodiment, the second- generation DNA-PK inhibitor is AZD7648.

[0303] In an embodiment, the cellular modulator is an mRNA cellular modulator.

Cells and uses thereof

[0304] In an aspect of the present disclosure, there is provided a cell (e.g., a population of cells) modified by the method disclosed herein.

[0305] Cells according to the present disclosure include any cell into which the gene editing systems, donor templates, vectors and/or RNPs described elsewhere herein may be introduced and expressed.

[0306] The cell (e.g., the population of cells) contemplated herein may be derived from any species, particularly a vertebrate, and even more particularly a mammal. Suitable vertebrates that fall within the scope of the disclosure include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis , and/or rhesus monkeys (Macaca initial la)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In a preferred embodiment, the cell is a mammalian cell. Suitable mammalian cells would be known to persons skilled in the art, illustrative examples of which include human cells, murine cells, non-human primate cells (e.g., rhesus monkey cells), human progenitor cells or stem cells, 293 cells, K562 cells, HeLa cells, D17 cells, MDCK cells, BHK cells, and Cf2Th cells.

[0307] In an embodiment, the cell (e.g., the population of cells) is a hematopoietic stem cell. [0308] As used herein, the term "hematopoietic stem cells" or "HSCs" refer to multipotent cells capable of differentiating into all the cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, lymphocytes, dendritic cells; and self-renewal activity, i.e., the ability to divide and generate at least one daughter cell with the identical (e.g., self-renewing) characteristics of the parent cell. The hematopoietic cells (e.g., CD4+ T lymphocytes, CD8+ T lymphocytes, and/or monocyte/macrophages) to be modified according to the method of the disclosure can be allogeneic, autologous, or from a matched sibling. The hematopoietic cells are, in some embodiments, CD34-positive and can be isolated from a subject’ s bone marrow or peripheral blood. The isolated CD34-positive hematopoietic cells (and/or other hematopoietic cells) are, in some embodiments, modified according to the method as disclosed herein.

[0309] In an aspect, the present disclosure provides a pharmaceutical composition comprising the cell (e.g., the population of cells) disclosed herein.

[0310] The term "pharmaceutical composition" as used herein refers to a composition that is in a form that allows the biological activity of the active ingredient (e.g., the cell disclosed herein) to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered.

[0311] In an embodiment, the pharmaceutical composition comprises the cell (e.g., a population of cells) in a number sufficient to administer a dosage of 10⁴ to 10⁹ cells/kg body weight per dose. Accordingly, the pharmaceutical composition may comprise the cell (e.g., a population of cells) in a number sufficient to administer a dosage of 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹ cells/kg body weight per dose.

[0312] In an embodiment, the pharmaceutical composition comprises the cell (e.g., a population of cells) in a number sufficient to administer a dosage of 10⁵ to 10⁶ cells/kg body weight per dose, including all integer values within those ranges.

[0313] In some embodiments, periodic re-administration of the pharmaceutical composition may be required to achieve a desirable therapeutic effect. The exact amounts and rates of administration of the pharmaceutical composition will depend on a number of factors, examples of which are described elsewhere herein, such as the subject’s age, body weight, general health, sex and dietary requirements, as well as any drugs or agents used in combination or coincidental with the administration of the composition. Where multiple divided doses are required, these may be administered hourly, daily, weekly, monthly or at other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. Alternatively, a continuous infusion strategy can be employed.

[0314] In an embodiment, the pharmaceutical composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.

[0315] The pharmaceutical compositions disclosed herein may be prepared according to conventional methods well known in the pharmaceutical industries, such as those described in Remington’s Pharmaceutical Handbook (Mack Publishing Co., NY, USA), comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.

[0316] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes to render the composition isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, anti-fungal and anti-bacterial agents, isotonic and absorption agents, and the like.

[0317] In an aspect, the present disclosure provides a use of the cell (e.g., a population of cells) disclosed herein in the manufacture of a medicament. In another aspect, the present disclosure provides a use of the cell (e.g., the population of cells) disclosed herein in the manufacture of a medicament for the treatment or prevention of a disease or disorder.

[0318] In an aspect, the present disclosure provides a method for the treatment or prevention of a disease or disorder comprising the administration of a therapeutically effective amount of the cell (e.g., the population of cells) or the pharmaceutical composition disclosed herein to a subject in need thereof.

[0319] In yet another aspect, the cell (e.g., the population of cells) disclosed herein is for use in the treatment or prevention of a disease or disorder. [0320] In an embodiment, the cells (e.g., population of cells) or pharmaceutical compositions disclosed herein are formulated with PLASMA-LYTE A (e.g., a sterile, non- pyrogenic isotonic solution for intravenous administration; where one liter of PLASMA- LYTE A has an ionic concentration of 140 mEq sodium, 5 mEq potassium, 3 mEq magnesium, 98 mEq chloride, 27 mEq acetate, and 23 mEq gluconate). In another embodiment, the cells (e.g., population of cells) are formulated in a solution of PLASMA- LYTE A, the solution comprising between about 8% and about 10% dimethyl sulfoxide (DMSO). In an embodiment, the less than about 2 xlO⁷ cells are present per mL of a formulation including PLASMA-LYTE A and DMSO.

[0321] The therapeutic regimen for the treatment or prevention of a disease or disorder can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the type, stage and molecular characteristics of the disease or disorder in addition to the age, weight and general health of the subject. Another determinative factor may be the risk of developing recurrent disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent disease. Similarly, for a subject identified as having a more advanced stage of disease or disorder, a more aggressive therapeutic regimen may be prescribed as compared to a subject that has a less advanced stage of the disease or disorder.

[0322] The term “subject” as used herein refers to any mammal, including livestock and other farm animals (e.g., cattle, goats, sheep, horses, pigs and chickens), performance animals (e.g., racehorses), companion animals (e.g., cats and dogs), laboratory test animals and humans. In an embodiment, the subject is a human. In an embodiment, the subject is an adult. In another embodiment, the subject is a child.

[0323] As used herein, the term “effective amount” typically refers to an amount of the cell (e.g., the population of cells) disclosed herein that is sufficient to affect one or more beneficial or desired therapeutic outcomes. Said beneficial or desired therapeutic outcomes may be measured using clinical techniques known in the art, illustrative examples of which include the measurement of imaging biomarkers, quantification of the presence of pathogenic inflammatory mediators (e.g., Interleukin- 1, TNF, TGF-P, etc.). An “effective amount” can be provided in one or more administrations. The exact amount required may vary depending on factors such as the nature and severity of the disease or disorder to be treated, and the age and general health of the subject.

[0324] The terms “treat”, "treating", “treatment” and the like are used interchangeably herein to mean relieving, reducing, alleviating, ameliorating or otherwise inhibiting the severity and/or progression of a disease or disorder, or a symptom thereof, in a subject. It is to be understood that the terms “treat”, "treating", “treatment” and the like, as used herein, do not imply that a subject is treated until clinical symptoms of the disease or disorder have been eliminated or are no longer evident. Said treatment may also reduce the severity of the disease or disorder by preventing progression or alleviating the symptoms associated with the disease or disorder.

[0325] The terms “prevent”, “preventing”, “prevention” and the like are used interchangeably herein to mean inhibit, hinder, retard, reduce or otherwise delay the development of a disease or disorder and/or progression of the disease or disorder, or a symptom thereof, in a subject. In the context of the present disclosure, the term “prevent” and variations thereof does not necessarily imply the complete prevention of the specified event. Rather, the prevention may be to an extent, and/or for a time, sufficient to produce the desired effect. Prevention may be inhibition, retardation, reduction or otherwise hindrance of the event, activity or function. Such preventative effects may be in magnitude and/or be temporal in nature.

[0326] In an embodiment, the cell is an autologous cell.

[0327] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later to be administered into the subject in accordance with the methods disclosed herein. Accordingly, in certain embodiments, cells isolated from the subject may be modified according to the method disclosed herein and cultured ex vivo for a time and under conditions suitable for the integration of the donor template, before being reinfused back into the subject in accordance with the method of treatment disclosed herein.

[0328] In another embodiment, the cell is an allogenic cell.

[0329] The term "allogenic" as used herein refers to any material derived from a different animal of the same species as the subject to whom the material is administered. Compositions

[0330] In an aspect of the present disclosure, there is provided a composition comprising a second-generation DNA-PK inhibitor and a cellular modulator.

[0331] In an embodiment, the second-generation DNA-PK inhibitor is selected from the group consisting of VX-984, M3814 and AZD7648.

[0332] In an embodiment, the second-generation DNA-PK inhibitor is AZD7648.

[0333] In an embodiment, the composition comprises at least about 0.001 pM AZD7648. In an embodiment, the composition comprises at least about 0.002 pM, about 0.003 pM, about 0.004 pM, about 0.005 pM, about 0.006 pM, about 0.007 pM, about 0.008 pM, or about 0.009 pM AZD7648. In an embodiment, the composition at least about 0.01 pM, about 0.02 pM, about 0.03 pM, about 0.04 pM, about 0.05 pM, about 0.06 pM, about 0.07 pM, about 0.08 pM, or about 0.09 pM AZD7648. In an embodiment, the composition comprises at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, or about 0.9 pM AZD7648. In an embodiment, the composition comprises at least about 1.0 pM, about 2.0 pM, about 3.0 pM, about 4.0 pM, about 5.0 pM, about 6.0 pM, about 7.0 pM, about 8.0 pM, about 9.0 pM, or about 10.0 pM AZD7648.

[0334] In an embodiment, the composition comprises from about 0.001 pM to about 3 pM AZD7648. Accordingly, in an embodiment, the composition comprises about 0.001 pM, about 0.002 pM, about 0.003 pM, about 0.004 pM, about 0.005 pM, about 0.006 pM, about 0.007 pM, about 0.008 pM, about 0.009 pM, about 0.01 pM, about 0.02 pM, about 0.03 pM, about 0.04 pM, about 0.05 pM, about 0.06 pM, about 0.07 pM, about 0.08 pM, about 0.09 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1.0 pM, about 2.0 pM, or about 3.0 pM AZD7648.

[0335] In an embodiment, the composition comprises from about 0.001 pM to about 0.5 pM AZD7648.

[0336] In an embodiment, the composition comprises about 0.3 pM AZD7648. [0337] In an embodiment, the cellular modulator is an mRNA cellular modulator.

[0338] In an embodiment, the composition comprises an mRNA cellular modulator selected from the group consisting of a p53 inhibitor, a cell cycle modulator, a DNA repair modulator and combinations of the foregoing.

[0339] In an embodiment, the composition comprises an mRNA cellular modulator comprising a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof.

[0340] In an embodiment, the composition comprises an mRNA cellular modulator in an amount sufficient to deliver from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator. Accordingly, in an embodiment, the composition comprises the mRNA cellular modulator in an amount sufficient to deliver about 0.01 pg/500,000 cells, about 0.02 pg/500,000 cells, about 0.03 pg/500,000 cells, about 0.04 pg/500,000 cells, about 0.05 pg/500,000 cells, about 0.06 pg/500,000 cells, about 0.07 pg/500,000 cells, about 0.08 pg/500,000 cells, about 0.09 pg/500,000 cells, about 0.1 pg/500,000 cells, about 0.2 pg/500,000 cells, about 0.3 pg/500,000 cells, about 0.4 pg/500,000 cells, about 0.5 pg/500,000 cells, about 0.6 pg/500,000 cells, about 0.7 pg/500,000 cells, about 0.8 pg/500,000 cells, about 0.9 pg/500,000 cells, about 1.0 pg/500,000 cells, about 2.0 pg/500,000 cells, about 3.0 pg/500,000 cells, about 4.0 pg/500,000 cells, or about 5.0 pg/500,000 cells of the mRNA cellular modulator.

[0341] In an embodiment, the composition comprises the mRNA cellular modulator in an amount sufficient to deliver about 0.8 pg/500,000 cells of the mRNA cellular modulator.

[0342] In an embodiment, the mRNA cellular modulator is provided as a polynucleotide encoding the mRNA cellular modulator. In another embodiment, the polynucleotide encoding the mRNA cellular modulator is within a vector.

[0343] In an embodiment, the vector is an episomal vector (z.e., that does not integrate into the genome of the cell), or a vector that integrates into the cell genome. Vectors may be replication competent or replication-deficient. Suitable vectors would be known to persons skilled in the art, illustrative examples of which include, but are not limited to, plasmids, cosmids, and viral vectors, such as AAV vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors.

[0344] In an embodiment, the polynucleotide encoding the mRNA cellular modulator is within a vector, e.g. , a plasmid. In another embodiment, the plasmid comprises a sequence selected from SEQ ID NOs: 24-27.

[0345] In an embodiment, the composition does not comprise an inhibitor of MMEJ.

[0346] In an embodiment, the inhibitor of MMEJ is selected from novobiocin and a PolQ inhibitor (e.g., ART558, PolQl, PolQ2, PolQ4, PolQ5, PolQ6 and PolQ7).

[0347] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof.

[0348] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0349] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0350] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0351] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0352] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0353] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0354] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequence of any one of SEQ ID NOs: 1-6, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0355] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 1, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0356] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 2, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0357] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 3, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0358] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 2, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0359] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto). [0360] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1, 2 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0361] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator comprising the sequences of any one of SEQ ID NO: 1-6, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0362] In an embodiment, the composition comprises AZD7648 and an mRNA cellular modulator consisting of the sequence of any one of SEQ ID NOs: 1-6.

[0363] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof.

[0364] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0365] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0366] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0367] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof. [0368] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0369] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0370] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequence of any one of SEQ ID NOs: 1-6, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0371] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 1, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0372] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 2, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0373] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 3, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0374] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 2, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0375] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0376] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1, 2 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0377] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator comprising the sequences of any one of SEQ ID NO: 1-6, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0378] In an embodiment, the composition comprises VX-984 and an mRNA cellular modulator consisting of the sequence of any one of SEQ ID NOs: 1-6.

[0379] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof.

[0380] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0381] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0382] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof.

[0383] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0384] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising (i) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (ii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0385] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising (i) a sequence encoding GSE56 (SEQ ID NO: 19), or a functional variant thereof; (ii) a sequence encoding Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and (iii) a sequence encoding i53 (SEQ ID NO: 21), or a functional variant thereof.

[0386] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequence of any one of SEQ ID NOs: 1-6, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0387] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 1, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0388] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 2, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0389] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequence of SEQ ID NO: 3, or a sequence having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0390] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 2, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto). [0391] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0392] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequences of SEQ ID NO: 1, 2 and 3, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0393] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator comprising the sequences of any one of SEQ ID NO: 1-6, or sequences having 90% identity thereto (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto).

[0394] In an embodiment, the composition comprises M3814 and an mRNA cellular modulator consisting of the sequence of any one of SEQ ID NOs: 1-6.

[0395] The compositions disclosed herein may be prepared according to conventional methods well known in the pharmaceutical industries, such as those described in Remington’s Pharmaceutical Handbook (Mack Publishing Co., NY, USA), comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.

[0396] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes to render the composition isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, anti-fungal and anti-bacterial agents, isotonic and absorption agents, and the like.

[0397] The composition may be combined with gene editing system and a donor template to modify a target sequence. Such combinations may be provided to a cell (e.g., a population of cells) simultaneous with the composition or concurrently with the composition. Accordingly, in an embodiment, the composition further comprises: a. a gene editing system comprising a nuclease; and b. a donor template.

[0398] In an embodiment, the donor template is selected from the group consisting of a dsDNA, a ssDNA, a ssODN and a IssDNA.

[0399] In an embodiment, the nuclease is an RNA-guided nuclease.

[0400] In an embodiment, the RNA-guided nuclease is Cas9.

[0401] In an embodiment, the gene editing system further comprises a gRNA that is complementary to a target sequence in a cell.

[0402] In an embodiment the gRNA is a sgRNA.

[0403] In an embodiment, the gene editing system comprises an RNA-guided nuclease and a gRNA, wherein the RNA-guided nuclease and the gRNA are complexed as a RNP.

[0404] In an embodiment, the composition disclosed herein is for use in a method of modifying a target sequence in a cell.

[0405] Those skilled in the art will appreciate that the invention disclosed herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0406] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0407] All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entireties.

[0408] The various embodiments enabled herein are further described by the following non-limiting examples. Examples

Example 1 - Materials and methods

Cells

[0409] The K562 cell line was cultured in RPMI 1640 supplemented with 10% HI FBS, penicillin (100 U/mL) and streptomycin (100 pg/mL). Primary CD34+ hematopoietic stem cells were derived from donors (CD34+ HSCs) or frozen aliquots of mobilized human peripheral blood (mPB CD34 cells) and cultured in vitro with StemSpan SFEM II hematopoietic cell culture medium supplemented with stem cell factor (100 ng/mL), Fms- like Tyrosine Kinase 3 (Flt3) ligand (100 ng/mL), thrombopoietin (TPO) (100 ng/mL), interleukin- 6 (100 ng/mL), UM 171 (35 nM) and StemRegenin 1 (SRI) (1 pM). sgRNA

[0410] The sgRNAs listed in Table 1 were obtained from IDT. sgRNA were resuspended to a final concentration of 2 pg/pL using lx Tris-EDTA (TE) buffer.

Ribonucleoprotein (RNP) complexing

[0411] 3 pg TrueCut V2 Cas9 (ThermoFisher Scientific, #A36497) and 2 pg sgRNA were mixed and pre-incubated for 10-15 minutes at room temperature to form Cas9-sgRNA RNP complexes. For the HDR studies, BST2 ssODN (SEQ ID NO: 17) or B2M1 ssODN (SEQ ID NO: 18) was also included in the complexing mixture to form Cas9-sgRNA-ssODN RNP complexes.

Chimeric RNA polynucleotides

[0412] GSE56-Ad5E4orf6/7 cDNA was cloned into a pUC57 plasmid for the production of mRNA cellular modulators (SEQ ID NO: 27).

Recombinant adeno-associated virus serotype 6 (rAAV6)-MND-GFP donor template

[0413] MND-GFP cDNA was cloned into a pUC57 plasmid containing AAV vector genome inverted terminal repeats for production of rAAV6-MND-GFP donor template for HDR. Electroporation

[0414] K562 cells were re-suspended in TheraPEAK P3 Primary Cell Nucleofector

Solution (Lonza) to a concentration of 2 x 10⁵ cells/mL and electroporated with Cas9-RNP complexes targeting B2M or BST2 using the K562 electroporation protocol on the Amaxa Cell Line Nucleofector (Lonza), in accordance with the manufacturer's instructions. Immediately after electroporation, media was added to the cells before incubating at 37°C for 5 minutes to recover the cells. Following the initial incubation, 2 x 10⁵ cells/mL for each condition were transferred to a 24-well plate containing pre-warmed media comprising the appropriate concentration of the DNA-PK inhibitor (e.g., 0, 0.1, 0.5, 1, 2, 3 pM) and incubated at 37°C for 48 hours.

[0415] mPB CD34 cells or donor CD34+ HSCs were resuspended in TheraPEAK P3 Primary Cell Nucleofector Solution (Lonza) to a concentration of 2 x 10⁵ cells/mL and electroporated with Cas9-sgRNA RNP or Cas9-RNP-ssODN complexes targeting B2M or BST2 using the DZ100 electroporation protocol on the Amaxa Cell Line Nucleofector (Lonza), in accordance with the manufacturer's instructions. Immediately after electroporation, was added to the cells before incubating at 37°C for 5 minutes. Following the initial incubation, 2 x 10⁵ cells/mL for each condition were transferred to a 24-well plate containing pre- warmed media comprising the appropriate concentration of AZD7648 (e.g., 0, 0.01, 0.1, 1 pM) and incubated at 37°C for 48 hours.

[0416] For the gene editing efficiency studies, mPB CD34 cells or donor CD34+ HSCs were electroporated in the same manner described above, however, following electroporation, media was added to the cells before incubating at 37°C for 5 minutes. Following the initial incubation, 2 x 10⁵ cells/mL for each condition were transferred to a 24-well plate containing pre-warmed media comprising the appropriate concentration of AZD7648 concentrations (z.e., 0, 0.0014, 0.004, 0.012, 0.037, 0.11, 0.33, 1 and 3 pM) and incubated at 37°C for 48 hours.

Flow cytometry

[0417] Cell count and viability was assessed by flow cytometry using the MACSQuant Analyzer (Mitenyi Biotec) and a viability dye containing 7-AAD (BD Sciences). DNA extraction and amplification

[0418] Sample genomic DNA (gDNA) was extracted from pelleted cells using QuickExtract DNA Extraction Solution (Biosearch Technologies), in accordance with the manufacturer's instructions. Briefly, pelleted cells were re-suspended in QuickExtract DNA Extraction Solution and heated at 65°C for 15 minutes and 98°C for 15 minutes. The concentration of the resulting gDNA sample was measured using the Qubit 3.0 Fluorometer (Invitrogen), in accordance with the manufacturer's instructions.

[0419] PCR amplification of edited regions was performed using primers (Table 2) flanking the target region. After amplification, the PCR product was visualized by electrophoresis on the Invitrogen E-Gel Power Snap System (ThermoFisher Scientific) in accordance with the manufacturer's instructions to confirm the presence of the expected ~ 500 bp bands.

Sequencing analysis

[0420] gDNA samples with detectable bands were subjected to Sanger sequencing. Sequencing files were retrieved and analyzed by ICE (Synthego), in accordance with the manufacturer's instructions for "Difficult Templates".

[0421] Editing efficiency rates (z.e., percentage of the pool with non- wild type sequence) were determined by comparing the edited trace to a control trace (see, e.g., Figure 1). Indels were counted and graphed as WT (0), NHEJ (+/- 1 , +/-2) or MMEJ (-3 <; >3).

[0422] Analysis of indels and integration by ICE was also used to infer the dominant DNA repair pathway (Figure 2). HDR was inferred from template integration, MMEJ dominance inferred from large deletions (z.e., > 3 bp deletion) and NHEJ dominance inferred from small indels (z.e., < 3 bp indel).

Mice

[0423] NOD.Cg-KilW-4IJ Tyr+ Prkdcsicd Il2rgtmlWjl/ThomJ (NBSGW) mice were obtained from The Jackson Laboratory, or bred and maintained in-house under pathogen- free conditions. Transplantation and tissue isolation

[0424] Human CD34+ HSCs were pre- stimulated for 48 hours in media (StemSpan SFEM) containing 100 ng/mL SCF, Flt-3, TPO and IE-6, with 35 nM UM171 and 1 pM SR- 1. Cells were washed, counted and plated at 1 x 10⁶ cells/mL for gene editing by electroporation of any one or more or all of: (i) adeno-associated virus (AAV) engineered to express a MND-GFP HDR template, (ii) Cas9-sgRNA RNP complexes (1.2 : 1 molar ratio, sgRNA : Cas9) and (iii) GSE_Ad5 mRNA cellular modulator (SEQ ID NO: 4) using the CM 149 electroporation protocol on the Amaxa Cell Line Nucleofector (Lonza), in accordance with the manufacturer's instructions.

[0425] Immediately after electroporation, media comprising 100 nM of AZD7648 was added to the cells before incubating at 37°C for 24 hours.

[0426] 2 x 10⁶ edited, or mock transduced, CD34+ HSCs were transplanted into busulfan conditioned 8- or 9-week old mice via retro-orbital injection. Recipient mice were evaluated by serial peripheral blood analyses beginning at day 5 post-transplantation, and again at 10, 12 and 14 weeks post-transplantation. Cohorts were sacrificed at 16 weeks for detailed analysis of the thymus, bone marrow and spleen.

[0427] Single-cell suspensions prepared from peripheral blood, bone marrow or spleen were assessed for GFP and human CD34+ cells by flow cytometry.

Example 2 - Comparison of NU7026 and AZD7648 for inhibiting NHEJ

[0428] As described elsewhere herein, DNA-PK inhibitors, such as NU7026, NU7441 and CC115, are effective for the targeting of the ATP-binding site of DNA-PKs but have different limited selectivity against PI3K and PIKK members (e.g., mTOR and PI3Ky). Certain DNA-PK inhibitors, such as AZD7648, VX-984 and M3814, are proposed to have higher function and better selectivity for DNA-PKs as compared to other DNA-PK inhibitors (see, e.g., Fok etal., 2019, supra). To assess whether DNA-PK inhibitors have an equivalent effect on the inhibition of NHEJ, various concentrations of NU7026 or AZD7648 was added to K562 cells immediately after electroporation with Cas9-RNP complexes targeting B2M or BST2 using NHEJ-dominant gRNA with high editing efficiency and > 50% NHEJ indel pattern.

[0429] Figure 3 shows the editing efficiency rates of K562 cells electroporated with Cas9-RNP complexes targeting B2M and BST2, respectively, following incubation with AZD7648 or NU7026. The editing efficiency rate was reduced in a dose-dependent manner by AZD7648 when targeting B2M with B2M4 gRNA (Figure 3A). A reduction in the editing efficiency rate was also observed with 0.1 pM NU7026, however, editing efficiency rates recovered at the higher concentrations of NU7026 tested. By contrast, Figure 3B shows that editing efficiency rates were increased in a dose-dependent manner by AZD7648 when targeting BST2 with TetN4 gRNA.

[0430] Despite differences in the editing efficiency rate observed between the different guides, AZD7648 was more potent in enhancing MMEJ over NHEJ across all guides and concentrations tested (Figures 4 A and 4C), whereas NU7026 had little to no effect on reducing NHEJ dominance (Figures 4B and 4D). The enhancement of MMEJ was also observed in two additional NHEJ-dominant guides targeting B2M and BST2 where AZD7648 was again more potent in enhancing the rate of MMEJ as compared to NU7026 (Figures 4E-4H).

[0431] Taken together, these data demonstrate that AZD7648 mediates more potent inhibition of NHEJ and enhancement of MMEJ as compared to NU7026.

Example 3 - Assessment of editing efficiency in human hematopoietic stem cells following inhibition of NHEJ by AZD7648

[0432] To determine the editing efficiency rate of mPB CD34 cells electroporated with Cas9-RNP complexes comprising four different NHEJ-dominant guides targeting B2M or BST2, indel frequency and distribution was assessed following incubation with AZD7648 across a range of concentrations. Figure 6 shows that overall editing efficiency rates were maintained at increasing AZD7648 concentrations across all guides. Further, AZD7648 had a dose-dependent effect on the inhibition of NHEJ and corresponding enhancement of MMEJ (Figure 7). In fact, 1 pM AZD7648 was sufficient to completely inhibit NHEJ in favor of MMEJ for each of the guides tested. [0433] In addition to editing efficiency and indel distribution, cell viability was also assessed in edited mPB CD34 cells following incubation with AZD7648 across a range of concentrations. As shown in Figure 8, cell viability was maintained across increasing AZD7648 concentrations.

Example 4 - Assessment of HDR template integration in human hematopoietic stem cells following inhibition of NHEJ by AZD7648

[0434] CD34+ HSCs derived from six donors were electroporated with Cas9-sgRNA- ssODN RNP complexes, as described elsewhere herein. In all CD34+ HSC donors, AZD7648 inhibited NHEJ in favor of MMEJ in a dose-dependent manner (Figure 9).

[0435] As shown in Figure 10, the increased rate of MMEJ mediated by AZD7648 was associated with a corresponding increase in HDR efficiency. The consolidated data of the proportion of NHEJ in the absence of a HDR donor (Figure 11 A) as compared to the proportion of HDR in the presence of the ssODN template (Figure 11B) shows that AZD7648 increases the rate of HDR in a dose-dependent manner. Table 4 shows the EC 50 for HDR and the IC50 for NHEJ in each of the six CD34+ donors, as well as the mean EC 50 and IC50. These data show that the average concentration of AZD7648 that achieves a 50% increase in HDR in a donor is about 0.34 pM.

Example 5 - Engraftment of edited human hematopoietic stem cells following DNA repair modulation by AZD7648 and/or mRNA cellular modulators

[0436] CD34+ HSCs derived from three donors were transduced with adeno-associated virus (AAV6) engineered to express an MND-GFP HDR template and electroporated with Cas9-sgRNA RNP complexes and the GSE56-Ad5E4orf6/7 mRNA cellular modulator (GSE_Ad5) at various concentrations (e.g., 0.1, 0.2. 0.8, 1.5 pg) before transplantation into mice. As shown in Figure 12A, the GSE56_Ad5 mRNA cellular modulator increased the proportion of GFP positive cells (z.e., the edited cell population) at day 5 post-transplantation by up to 8.6% using mRNA produced in-house and by TriLink, without significantly reducing cell viability (as compared with an AAV-only control and a Mock control with no gene editing machinery, Figure 12B). [0437] In another example, the GSE56_i53 mRNA cellular modulator (GSE_i53) increased the proportion of GFP positive cells (z.e., the edited cell population) at day 5 posttransplantation by up to 7.8% in a dose dependent manner (Figure 13).

[0438] To assess the combined effect of AZD7648 and mRNA cellular modulators in improving HDR template integration, CD34+ HSCs derived from a donor were transduced with AAV6 engineered to express an MND-GFP HDR template and electroporated with Cas9-sgRNA RNP complexes and the GSE_Ad5 mRNA cellular modulator, followed by incubation with 100 nM AZD7648. As shown in Figure 14, the combination of AZD7648 and the GSE_Ad5 mRNA cellular modulator increased the efficiency of HDR template integration, relative to CD34+ HSCs that were not exposed to any mRNA cellular modulator or AZD7648 alone. This surprising improvement in efficiency of HDR template integration resulting from the combination of AZD7648 and GSE56 mRNA cellular modulators has not previously been observed in other studies that have considered the effect of mRNA cellular modulators, such as the p53 inhibitor GSE-CS56 described in WO 2021/232014. Beneficially, the methods and compositions contemplated herein can improve HDR template integration with AZD7648 alone, or in combination with a single mRNA cellular modulator, rather than delivering an additional mRNA cellular modulator to the cell, as is required in WO 2021/232014. Further, the effect observed with the combination of AZD7648 and GSE_Ad5 was surprisingly increased relative to other small molecules for improving the rate of HDR (Figure 15).

[0439] The in vitro results shown in Figure 14A were reproduced in vivo, following transplantation of edited CD34+ HSCs into recipient mice. In particular, the proportion of GFP positive CD40E cells was increased by up to 14.6% relative to edited CD34+ cells that were not exposed to AZD7648 or a mRNA cellular modulator at day 5 post-transplantation (Figure 14B). Similarly, at week 10, the proportion of edited cells in total peripheral blood (z.e., %GFP x %hCD45) was increased by the inclusion of AZD7648 and the GSE_Ad5 mRNA cellular modulator (Figure 16).

[0440] Taken together, these data demonstrated that use of AZD7648 in combination with a gene editing system and a donor template enhances gene editing by the modulation DNA repair to inhibit NHEJ in favor of HDR or MMEJ, the effects observed with AZD7648 were further enhanced by the addition of a mRNA cellular modulator, such as GSE56, GSE56_Ad5, GSE56_i53 and i53_GSE56 (e.g., as shown in Figure 17).

[0441] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0442] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0443] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Table 1. Exemplary sgRNA

Table 2. Primer sequences

Table 3. ssODN sequences

Table 4. AZD7648 increases the rate of HDR in mPB CD34 cells in a dose-dependent manner

Claims

Claims A method of modifying a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; c. AZD7648; and d. an mRNA cellular modulator. The method according to claim 1, wherein the cell is provided with AZD7648 at a concentration of at least about 0.001 pM. The method according to claim 2, wherein the cell is provided with AZD7648 at a concentration of at least about 0.1 pM. The method according to claim 3, wherein the cell is provided with AZD7648 at a concentration of about 0.3 pM. The method according to any one of claims 1 to 4, wherein the donor template is provided to the cell within a vector. The method according to claim 5, wherein the vector is an adeno-associated virus (AAV) vector. The method according to any one of claims 1 to 6, where in the donor template is selected from the group consisting of a double-stranded DNA (dsDNA), a singlestranded DNA (ssDNA), a single- stranded oligodeoxynucleotide (ssODN) and a long single-stranded DNA (IssDNA). The method according to any one of claims 1 to 7, wherein the nuclease is an RNA- guided nuclease. The method according to claim 8, wherein the RNA-guided nuclease is CRISPR- associated endonuclease 9 (Cas9). The method according to any one of claims 1 to 9, wherein the gene editing system further comprises a guide RNA (gRNA) that is complementary to the target sequence. The method according to claim 10, wherein the gRNA is a single guide RNA (sgRNA). The method according to claim 10 and claim 11, wherein the gene editing system comprises an RNA-guided nuclease and a gRNA, wherein the RNA-guided nuclease and the gRNA are complexed as a ribonucleoprotein (RNP). The method according to any one of claims 1 to 12, wherein the mRNA cellular modulator is selected from the group consisting of a p53 inhibitor, a cell cycle modulator, a DNA repair modulator and combinations of the foregoing. The method according to any one of claims 1 to 13, wherein the mRNA cellular modulator comprises a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof. The method according to claim 14, wherein the mRNA cellular modulator comprises a sequence of any one of SEQ ID NOs: 1-6, or a sequence having 90% identity thereto. The method according to claim 15, wherein the mRNA cellular modulator consists of a sequence of any one of SEQ ID NOs: 1-6. The method according to any one of claims 1 to 16, wherein the cell is provided with from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator. The method according to claim 17, wherein the cell is provided with about 0.8 pg/500,000 cells of the mRNA cellular modulator. The method according to any one of claims 1 to 18, wherein the cell is a hematopoietic stem cell (HSC). A cell modified by the method according to any one of claims 1 to 19. A pharmaceutical composition comprising the cell according to claim 20. A method of treating a disease or disorder, the method comprising administering an effective amount of the cell according to claim 20, or the pharmaceutical composition according to claim 21 to a subject in need thereof. AZD7648 and an mRNA cellular modulator for use in a method of modifying of a target sequence in a cell, the method comprising providing to the cell: a. a gene editing system comprising a nuclease; b. a donor template; c. AZD7648; and d. an mRNA cellular modulator. The AZD7648 and an mRNA cellular modulator for use according to claim 23, wherein the cell is provided at least about 0.001 pM AZD7648. The AZD7648 and an mRNA cellular modulator for use according to claim 24, wherein the cell is provided at least about 0.1 pM AZD7648. The AZD7648 and an mRNA cellular modulator for use according to claim 25, wherein the cell is provided about 0.3 pM AZD7648. The AZD7648 and an mRNA cellular modulator for use according to claims 23 to 26, wherein the donor template is provided to the cell within a vector. The AZD7648 and an mRNA cellular modulator for use according to claim 27, wherein the vector is an AAV vector. The AZD7648 and an mRNA cellular modulator for use according to any one of claims 23 to 28, where in the donor template is selected from the group consisting of a dsDNA, a ssDNA, a ssODN and a IssDNA. The AZD7648 and an mRNA cellular modulator for use according to any one of claims 23 to 29, wherein the nuclease is an RNA-guided nuclease. The AZD7648 and an mRNA cellular modulator for use according to claim 30, wherein the RNA-guided nuclease is Cas9. The AZD7648 and an mRNA cellular modulator for use according to any one of claims 23 to 31, wherein the gene editing system further comprises a gRNA that is complementary to the target sequence. The AZD7648 and an mRNA cellular modulator for use according to claim 32, wherein the gRNA is a sgRNA. The AZD7648 and an mRNA cellular modulator according to claim 32 or claim 33, wherein the gene editing system comprises an RNA-guided nuclease and a gRNA, wherein the RNA-guided nuclease and the gRNA are complexed as a RNP. The AZD7648 and an mRNA cellular modulator for use according to any one of claims 23 to 34, wherein the mRNA cellular modulator is selected from the group consisting of a p53 inhibitor, a cell cycle modulator, a DNA repair modulator and combinations of the foregoing. The AZD7648 and an mRNA cellular modulator according to any one of claims 23 to 34, wherein the mRNA cellular modulator comprises a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof. The AZD7648 and an mRNA cellular modulator for use according to claim 36, wherein the mRNA cellular modulator comprises a sequence of any one of SEQ ID NOs: 1-6, or a sequence having 90% identity thereto. The AZD7648 and an mRNA cellular modulator for use according to claim 37, wherein the mRNA cellular modulator consists of a sequence of any one of SEQ ID NOs: 1-6. The AZD7648 and an mRNA cellular modulator for use according to any one of claims 23 to 38, wherein the cell is provided with from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator. The AZD7648 and an mRNA cellular modulator for use according to claim 39, wherein the cell is provided with about 0.8 pg/500,000 cells of the mRNA cellular modulator. The AZD7648 and an mRNA cellular modulator for use according to any one of claims 23 to 40, wherein the cell is an HSC. A composition comprising AZD7648 and an mRNA cellular modulator. The composition according to claim 42, comprising at least about 0.001 pM AZD7648. The composition according to claim 43, comprising at least about 0.1 pM AZD7648. The composition according to claim 44, comprising about 0.3 pM AZD7648. The composition according to any one of claims 42 to 45, wherein the mRNA cellular modulator is selected from the group consisting of a p53 inhibitor, a cell cycle modulator, a DNA repair modulator and combinations of the foregoing. The composition according to any one of claims 42 to 46, wherein the mRNA cellular modulator comprises a sequence encoding one or more or all of: a. GSE56 (SEQ ID NO: 19), or a functional variant thereof; b. Ad5E4orf6/7 (SEQ ID NO: 20), or a functional variant thereof; and c. i53 (SEQ ID NO: 21), or a functional variant thereof. The composition according to claim 47, wherein the mRNA cellular modulator comprises a sequence of any one of SEQ ID NOs: 1-6, or a sequence having 90% identity thereto. The composition according to claim 48, wherein the mRNA cellular modulator consists of a sequence of any one of SEQ ID NOs: 1-6. The composition according to any one of claims 42 to 49, comprising the mRNA cellular modulator in an amount sufficient to deliver from about 0.01 pg/500,000 cells to about 5 pg/500,000 cells of the mRNA cellular modulator. The composition according to claim 50, comprising the mRNA cellular modulator in an amount sufficient to deliver about 0.8 pg/500,000 cells of the mRNA cellular modulator. The composition according to any one of claims 42 to 51, further comprising: a. a gene editing system comprising a nuclease; and b. a donor template. The composition according to claim 52, wherein the donor template is selected from the group consisting of a dsDNA, a ssDNA, a ssODN and a IssDNA. The composition according to claim 52 or claim 53, wherein the nuclease is an RNA- guided nuclease. The composition according to claim 54, wherein the RNA-guided nuclease is Cas9. The composition according to any one of claims 42 to 55, wherein the gene editing system further comprises a gRNA that is complementary to a target sequence in a cell. The composition according to claim 56, wherein the gRNA is a sgRNA. The composition according to claim 56 or claim 57, wherein the gene editing system comprises an RNA-guided nuclease and a gRNA, wherein the RNA-guided nuclease and the gRNA are complexed as a RNP. The composition according to any one of claims 42 to 58 for use in a method of modifying of a target sequence in a cell.