Cytotoxic synergy of crispr-cas9 with DNA damage repair inhibitors

Abstract

Provided herein are methods for increasing the cytotoxicity of CRISPR-Cas9 systems for use in treating subjects in need thereof. Also disclosed are methods of treating a subject suffering from cancer by administering to a subject in need of treatment thereof a combination of at least one CRISPR-Cas9 system and at least one inhibitor of DNA double strand repair.

Description

Cytotoxic Synergy of CRISPR-Cas9 with DNA Damage Repair InhibitorsRELATED APPLICATION INFORMATION

[0001] This application claims priority to U.S. Application No. 63 / 637,030, filed on April 22, 2024, the contents of which are herein incorporated by reference.TECHNICAL FIELD

[0002] The present disclosure relates to methods for increasing the cytotoxicity of CRISPR- Cas9 systems for use in treating subjects in need thereof. In some embodiments, the methods described herein relate to treating a subject suffering from cancer by administering to a subject in need of treatment thereof a combination of at least one CRISPR-Cas9 system and at least one inhibitor of DNA damage repair.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] None.BACKGROUND

[0004] Solid tumors arise from multistep carcinogenesis, produced by the accumulation of driver mutations in oncogenes and tumor suppressor genes (2, 3). However, the vast majority of mutations found in cancers are passengers (1, 4). Since cancer is a clonal disease, all malignant cells should contain the mutations present in the cancer initiating cell at the beginning of tumorigenesis.

[0005] Since its discovery, reduction to a two-component system, and demonstration of activity in human cells, the CRISPR-Cas9 system has been rapidly adopted by scientists as the tool of choice for gene editing (5-7). CRISPR-Cas9 works by introducing a double-strand break (DSB) as directed by a complementary single-guide RNA (sgRNA) sequence in the presence of a protospacer adjacent motif (PAM), where the break is then repaired by one of the endogenous DNA repair systems. However, CRISPR-Cas9 has been associated with off-target activity and other toxicities, sometimes resulting in unintentional loss of whole chromosome arms (8, 9).SUMMARY|0006] In one embodiment, the presently disclosed subject matter relates to a method for reducing the number of double stranded breaks required to obtain death of one or more target cells in a tumor in a subject being treated or to be treated with a CRISPR-Cas9 system. The method comprises the step of administering to a subject in need of treatment thereof a therapeutically effective amount of at least one CRISPR-Cas9 system and at least one inhibitor of DNA damage repair.

[0007] In some embodiments, the CRISPR-Cas9 system targets one or more sgRNAs that target a sequence adjacent to one or more target protospacer adjacent motifs (PAMs).

[0008] In some embodiments, the tumor is cancer.

[0009] In some embodiments, the cancer is pancreatic cancer, lung cancer, esophageal cancer, or any combinations thereof.

[0010] In still other embodiments, the inhibitor of DNA damage repair is a phosphoinositide 3-kinase (PI3K) inhibitor.

[0011] In still yet other embodiments, the inhibitor of DNA damage repair is a DNA- dependent protein kinase (DNA-PK) inhibitor.

[0012] In still yet other embodiments, the inhibitor of DNA damage repair is a poly(ADP- ribose) polymerase (PARP) inhibitor.

[0013] In still yet other embodiments, the inhibitor of DNA damage repair is an ataxia telangiectasia and Rad3 -related (ATR) inhibitor.

[0014] In some embodiments, the inhibitor of DNA damage repair is a biologic moleculebased inhibitor, a chemical molecule inhibitor, a small molecule inhibitor or an RNA-based inhibitor.

[0015] In some embodiments, the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair are administered simultaneously or sequentially.

[0016] In other embodiments, the present disclosure relates to a method of treating a subject suffering from pancreatic cancer, lung cancer, esophageal cancer, or any combination thereof. The method comprises administering to a subject a therapeutically effective amount of the CRISPR-Cas9 system and at least one inhibitor of a DNA damage repair.

[0017] In some embodiments, the CRISPR-Cas9 system targets one or more sgRNAs that target a sequence adjacent to one or more target protospacer adjacent motifs (PAMs). In some9other embodiments, the inhibitor of DNA damage repair is a phosphoinositide 3-kinase (PI3K) inhibitor. In still other embodiments, the inhibitor of DNA damage repair protein is a DNA- dependent protein kinase inhibitor. In still yet other embodiments, the inhibitor of DNA damage repair is a poly(ADP-ribose) polymerase (PARP) inhibitor. In still yet other embodiments, the inhibitor of DNA damage repair is an ataxia telangiectasia and Rad3 -related (ATR) inhibitor. |0018] In some embodiments, the inhibitor is a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor, or an RNA-based inhibitor.

[0019] In still other embodiments, the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair are administered simultaneously or sequentially.

[0020] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0023] FIG. 1A-1E show DNA-PK killing curves generated based on the methods described in the Examples.

[0024] FIG. 2A-2D show that DSB-cytotoxicity is enhanced by KU0060648 and nedisertib treatment.

[0025] FIG. 3A-3B show that DSB-cytotoxicity is enhanced by AZD2461 and talazoparib treatment.

[0026] FIG. 4A-4B show that DSB-cytotoxicity is enhanced by ceralasertib and berzosertib treatment.

[0027] FIG. 5A-5C show that synergy with KU0060648 begins at 3-5 targets.

[0028] FIG. 6A-6B show Tag survival with DNA-PK inhibition and increasing numbers ofCRISPR-DSBs.

[0029] FIG. 7A-7B show nedisertib synergizes with CRISPR-DSBs at greater than 3 target sites.DETAILED DESCRIPTION

[0030] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.1. Definitions

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0032] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. The present disclosure contemplates other embodiments "comprising," "consistingof" and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0033] The singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. Following long-standing patent law convention, the terms “a,” '‘an.” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

[0034] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0035] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0036] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0037] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term“about,” when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments + 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments + 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0038] Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0039] As used herein, the term “administering” means the actual physical introduction of a CRISPR-Cas9 system alone, or in combination with one or more other therapeutic or pharmaceutical agents (e.g., a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor or an RNA (e.g., microRNA, shRNA, siRNA or gRNA molecule) -based inhibitor) into or onto (as appropriate) a target cell. Any and all methods of introducing the CRISPR-Cas9 system alone or in combination with one or more therapeutic or pharmaceutical agents into the target cell are contemplated according to the disclosure; the method is not dependent on any particular means of introduction and is not to be so constmed. Means of introduction are well-known to those skilled in the art and are also exemplified herein.

[0040] As used herein, the phrase, “biologic molecule-based”, refers to a compound or molecule that is based in part or in whole on a biologic molecule such as DNA, RNA, peptides, polypeptides and the like. For example, in some embodiments a biologic molecule-based inhibitor of DNA damage repair can be an RNA polynucleotide (e.g., an antisense oligo, short hairpin RNA, siRNA, etc.). In some embodiments, the inhibitor of DNA damage repair is a DNA aptamer. In some embodiments, the inhibitor of DNA damage repair is an antibody or antibody fragment.

[0041] As used herein, the term “cancer” refers to a disease caused by an uncontrolled division of abnormal cells in a part of the body. Examples of cancer include, but are not limitedto, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain tumor and / or cancer, breast cancer, bronchial tumors, Burkitt lymphoma, cardiac tumors, cervical cancer, leukemia, colorectal cancer, uterine cancer, esophageal cancer, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, head and neck cancer, kidney cancer, liver cancer, lip and oral cavity cancer, lung cancer, lymphoma, melanoma, skin cancer, metastatic cancer, mouth cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, salivary gland cancer, throat cancer, thyroid cancer, or any combinations thereof.

[0042] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly, a CRISPR-Cas9 system described herein and at least one additional therapeutic agent or pharmaceutical agent, such as a chemotherapeutic agent and / or one or more inhibitors of a DNA damage repair. More particularly, the term “in combination” refers to the concomitant administration of a CRISPR-Cas9 system and at least one or more additional therapeutic agents or pharmaceutical active agents for the treatment of a single disease state (such, as for example, cancer). As used herein, the CRISPR-Cas9 system and one or more therapeutic or pharmaceutical agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the CRISPR-Cas9 system and the one or more therapeutic or pharmaceutically active agents are combined and administered in a single dosage form. In another embodiment, the CRISPR-Cas9 and one or more therapeutic or pharmaceutical agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other) and such administration can be made simultaneously or sequentially, in any order.

[0043] As used herein, the phrase “CRISPR-Cas9” or “CRISPR-Cas9 system” as used interchangeably herein, is a molecular scissor that can induce a double strand break (DSB) at a specific genomic location as determined by the sgRNA sequence. In one embodiment, DSBs are known to be toxic to cells and lead to cell death, which is the driving mechanism behind many cytotoxic therapies, such as radiation therapies. In one embodiment, the CRISPR-Cas9 is known as a gene-editing technology for modifying, deleting, correcting, or inserting precise regions of DNA. In some embodiments, the CRISPR / Cas9 edits genes by precisely cutting DNA and then letting natural DNA repair processes take over. In some aspects, the CRISPR-Cas9 systemtargets protospacer adjacent motifs (PAMs). Such CRISPR-Cas9 systems that target protospacer adjacent motifs can be prepared or designed using the methods described in WO 2024 / 044304, the contents of which are herein incorporated by reference.

[0044] As used herein, the term “inhibit” or “inhibits” means to decrease, suppress, attenuate, diminish, arrest, or stabilize an activity associated with a disease or a disease-related pathway or the development or progression of a disease, disorder, or condition, e.g. cancer, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% compared to an untreated control subject, cell, biological pathway, or biological activity.

[0045] As used herein, an “inhibitor of DNA damage repair” refers to one or more therapeutic agents or pharmaceutical agents (e.g., a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor, or an RNA-based inhibitor) that inhibit the repair of DNA damage in a target cell by: 1) inhibiting repair of DNA single strand breaks and / or stalls at the DNA replication fork (e.g. in which poly(ADP-ribose) polymerase (PARP) and the ataxiatelangiectasia and Rad3-related (ATR) kinase pathways are involved), that can cause an accumulation of DSBs; or 2) preventing repair of the DSB through the error-prone nonhomologous end joining (NHEJ) pathway or the error-free homologous recombination pathway (e.g., in which phosphoinositide 3-kinase (PI3K)-like kinases, DNA-PK (e.g., the catalytic subunit of DNA-PKs) and ataxia telangiectasia mutated (ATM) kinases are centrally involved). In some embodiments, the inhibitor of DNA damage repair is an inhibitor of phosphoinositide 3-kinase, an inhibitor of a DNA-dependent protein kinase, an inhibitor of an ATM -kinase, an inhibitor of PARP, an inhibitor of ATR, or any combinations thereof.

[0046] As used herein, an “inhibitor of phosphoinositide 3-kinase”, “a phosphoinositide 3- kinase inhibitor” or a “inhibitor of a phosphoinositide 3-kinase pathway” refers to one or more pharmaceutical agents (e.g., a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor, or an RNA-based inhibitor) that inhibit one or more of PI3K enzymes, which are part of PI3K / AKT / mT0R pathway. Examples of inhibitors of PI3K that can be used include (a) dual PI3K inhibitors such as dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotolisib, bimiralisib, paxalisib, voxtalsib and DNA-PKs; (b) Pan-PI3K inhibitors, such as, buparlisib, CH5132799, pilaralisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100- 155, RIDR-PI-103; (c) isoform-specific inhibitors such as alpelisib (alpha), serabelisib (alpha), GSK2636771 (beta), idelalisib (delta), zandelisib (delta), AMG319 (delta), linperlisib (delta),parsaclisib (delta), umbralisib (delta), leniolisib (delta), eganelisib (gamma), tenalisib (delta / gamma), taselisib (alpha / delta / gamma), AZD8186 (beta / delta), AZD8835 (delta / alpha), duvelisib (delta / gamma); and (d) miscellaneous PI3K inhibitors, such as, fimepinostat (HDAC / PI3K) and rigosertib (PLK1 / PI3K).

[0047] In some aspects, the inhibitor of PI3K includes dual inhibitors, such as, for example, pharmaceutical agents that inhibit PI3K as well as DNA-dependent protein kinases (DNA-PKs). Examples of inhibitors of DNA-PKs include KU0060648 (4-Ethyl-N-[4-[2-(4-morpholinyl)-4- oxo-4H-l-benzopyran-8-yl]-l -dibenzothienyl] -1 -piperazineacetamide), nedisertib, CC-115 (1- ethyl-3 ,4-dihydro-7 - [2-methyl-6-( 1 H- 1 ,2,4-triazol-5-yl)-3-pyridinyl] -pyrazino [2,3-b]pyrazin- 2(lH)-one) or any combinations thereof.

[0048] As used herein, an “inhibitor of poly(ADP-ribose) polymerase (PARP)” refers to one or more pharmaceutical agents (e.g., a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor, or an RNA-based inhibitor) that inhibit PARP enzymes (primarily PARP1 and PARP2) that repair DNA single strand breaks through the base excision repair pathway. PARP inhibitors are most effective in cancers with deficiencies in the error-free homologous recombination pathway, e.g. breast cancer (BRCA) mutations. Examples of PARP inhibitors that can be used include AZD2461, talazoparib, olaparib, rucaparib, niraparib, veliparib, or any combination thereof.

[0049] As used herein, an “inhibitor of ataxia-telangiectasia and Rad3-related (ATR)” refers to one or more pharmaceutical agents (e.g., a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor, or an RNA-based inhibitor) that inhibit an ATR kinase, a member of the phosphatidylinositol 3-kinase-related kinase family involved in preventing the collapse of replication forks and repairing DNA single strand breaks. ATR inhibitors are most effective in cancers with high replication stress or defects in DNA damage repair. Examples of ATR inhibitors that can be used include ceralasertib, berzosertib, elimusertib, ART0380, or RP-3500, or any combination thereof.

[0050] As used herein, the term “metastasis cancer” refers to a cancer that spreads from where it started to a distant part of the body is called metastatic cancer. For many types of cancer, it is also called stage IV (4) cancer.

[0051] As used herein, the term “protospacer adjacent motifs (PAMs)” refers to a short DNA sequence (typically 2-6 base pairs in length) that follows the DNA region targeted for cleavageby the CRISPR system, such as CRISPR-Cas9. The PAM is generally required for a Cas nuclease to cut and is typically found 3-4 nucleotides downstream from the cut site.

[0052] As used herein, the term “pancreatic cancer” refers to a type of cancer that starts in the pancreas. Pancreatic cancer types include, but are not limited to, exocrine pancreatic cancer, neuroendocrine pancreatic cancer. The most common type of pancreatic cancer, adenocarcinoma of the pancreas, starts when exocrine cells in the pancreas start to grow out of control.

[0053] As used herein, the term “benign pancreatic disease” and “pancreatic disease” as used herein interchangeably refer to pancreatic disease which is not cancer or has become cancer. Benign pancreatic disease includes pancreatitis, various types of cysts and tumors, pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) lesions, and mucinous cystic neoplasm (MCN).

[0054] As used herein, the term “early-stage pancreatic cancer” as used herein refers to pancreatic cancer which is limited to the pancreas, outside the pancreas or nearby lymph nodes, but has not expanded into nearby major blood vessels or nerves or distant organs. Early-stage pancreatic cancer includes stage 0, stage I and stage II pancreatic cancers. See Yachida et al. (2010) Nature 467:1114-1119; see also National Comprehensive Cancer Network (NCCN) Guidelines Version 2.2012 Pancreatic Adenocarcinoma.

[0055] As used herein, the term “late-stage pancreatic cancer” as used herein refers to pancreatic cancer which has expanded into nearby major blood vessels, nerves or distant organs. Late-stage pancreatic cancer includes stage III or stage IV pancreatic cancer.

[0056] As used herein, the term “stage 0 pancreatic cancer” as used herein refers to pancreatic cancer limited to a single layer of cells in the pancreas. The pancreatic cancer is not visible on imaging tests or to the naked eye. The tumor is confined to the top layers of pancreatic duct cells and has not invaded deeper tissues or spread outside of the pancreas. Stage 0 tumors are sometimes referred to as pancreatic carcinoma in situ or pancreatic intraepithelial neoplasia III (Panin III).

[0057] As used herein, the term “stage I pancreatic cancer” as used herein refers to cancer confined or limited to the pancreas and has not spread to nearby lymph nodes. "Stage IA" refers to a tumor confined to the pancreas and is less than 2 cm in size. "Stage IB" refers to a tumor confined to the pancreas and is greater than 2 cm in size.

[0058] As used herein, the term “stage II pancreatic cancer” as used herein refers to local spread cancer that has grown outside the pancreas or has spread to nearby lymph nodes. "Stage IIA" refers to a tumor growing outside the pancreas but not into large blood vessels, nearby lymph nodes or distant sites. "Stage IIB" refers to a tumor either confined to the pancreas or growing outside the pancreas but has not spread into nearby large blood vessels or major nerves. Stage IIB may spread to nearby lymph nodes but has not spread to distant sites.

[0059] As used herein, the term “stage III pancreatic cancer” as used herein refers to wider spread cancer that has expanded into nearby major blood vessels or nerves but has not metastasized. The tumor grows outside the pancreas into nearby large blood vessels or major nerves and may or may not have spread to nearby lymph nodes. It has not spread to distant sites.

[0060] As used herein, the term “stage IV pancreatic cancer” as used herein refers to confirmed spread cancer that has spread to distant organs or sites. Stage IVA pancreatic cancer is locally confined, but involves adjacent organs or blood vessels, thereby hindering surgical removal. Stage IVA pancreatic cancer is also referred to as localized or locally advanced. Stage IVB pancreatic cancer has spread to distant organs, most commonly the liver. Stage IVB pancreatic cancer is also called metastatic.

[0061] As used herein, the term “PDAC” refers to pancreatic ductal adenocarcinoma cells, cancerous cells that originate in the pancreatic ducts.

[0062] As used herein, the phrase “pharmaceutical agent” or “therapeutic agent” as used interchangeably herein refers to one or more biologic molecules, one or more chemical molecules, one or more small molecules, or one or more RNA (e.g., microRNA, shRNA, siRNA or gRNA molecule) molecules. For example, in some embodiments, the pharmaceutical agent or therapeutic agent is one or more biologic molecule-based inhibitors, chemical molecule inhibitors, small molecule inhibitors, an RNA-based inhibitors or any combination thereof.

[0063] As used herein, the term “phosphoinositide 3-kinases” (PI3Ks) or “phosphatidylinositol 3-kinases”, refers to a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. The PI3K pathway is a crucial intracellular signaling pathway which is mutated or amplified in a wide variety of cancers such as, for example, breast, gastric, ovarian, colorectal, prostate, glioblastoma and endometrial cancers. PI3K signaling plays an important role in cancer cell survival, angiogenesis and metastasis.

[0064] PI3K proteins are classified into three main classes (I, II and III) based on the substrate specificities and structural characteristics. Class I PI3Ks are categorized into two subtypes (A and B) based on the mode of regulation. Class IA PI3Ks form dimers containing a regulatory (p85a, p850, p55a, p55y, p50a) and a catalytic (pl 10a, pl 100, pl 105) subunit. Class I PI3Ks act downstream of both G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In addition, these regulatory subunits play an important role in stabilization of the pl 10 catalytic subunits and in suppression of the basal lipid kinase activity. Class IB PI3Ks which are activated downstream of GPCRs form heterodimers consisting of pl 10y as catalytic subunit with other regulatory subunits such as p 101 , p87 or p84. This class consists of three isoforms (PI3K-C2a, PI3K-C2B and PI3K-C2y). These isoforms have a RAS-binding domain (RBD), a helical domain, and a catalytic domain but lack a regulatory domain. The class III PI3Ks also known as vacuolar protein sorting 34 (VPS34) heterodimerize with membrane-associated VPS 15 regulatory subunit.

[0065] As used herein “poly(ADP-ribose) polymerase (PARP)” refers to a family of enzymatic proteins that play a role in various cellular processes, including DNA repair, genomic stability, and programmed cell death. PARP proteins detect and initiate repair of single strand DNA breaks by binding the damaged DNA and synthesizing poly (ADP-ribose) chains that signal the binding of additional proteins necessary for DNA repair. PARP proteins facilitate the repair of DNA single strand breaks through base excision repair, a DNA repair pathway that removes damaged bases in the DNA that do not cause significant distortion to the DNA helix and replaces them with the correct bases.

[0066] As used herein “ataxia-telangiectasia and Rad3-related (ATR) kinase” refers a serine / threonine kinase that is a member of the phosphatidylinositol 3-kinase-related kinase family that also includes ataxia telangiectasia mutated (ATM) and DNA-protein kinase (DNA- PK). ATR kinases primarily respond to replication stress that result from replication fork stalls and single strand DNA breaks. ATR kinases activate checkpoint kinase 1 and other proteins that halt the cell cycle, allowing DNA repair. ATR kinase can also be recruited by ATM to repair DNA double strand breaks.

[0067] As used herein, the term “sgRNAs” or “sgRNA-guided Cas 9” as used interchangeably herein, refers to a single guide RNA, which is a single RNA molecule that contains the custom-designed short crRNA sequence fused to the scaffold tracrRNA sequences. In some embodiments, sgRNA is synthetically made in vitro or in vivo from a DNA template.

[0068] As used herein, the “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0069] As used herein, the term “target cell” refers to a cell selectively affected, identified by, attacked and / or targeted by a CRISPR-Cas9 system, particularly, the combination of a CRISPR- Cas9 system and one or more inhibitors of DNA damage repair. In some embodiments, the target cells are, but are not limited to, one or more cells having one or more somatic mutations, such as, cancer cells, particularly pancreatic, lung, and esophageal cancer. In some aspects, the one or more somatic mutations produce one or more protospacer adjacent motifs (PAMs) and / or target sites (e.g., sequences).

[0070] As used herein, the phrase “therapeutically effective amount”, “effective amount” or “effective dose” as used interchangeable herein, refers to an amount that provides a therapeutic or aesthetic benefit in the treatment, prevention, or management of a disorder or disease, e.g., an amount that provides a statistically significant decrease in at least one symptom, sign, or marker of the disorder or disease. Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.

[0071] As used herein, the term “treating,” “treat,” or “treatment” can include reversing, alleviating, inhibiting the progression of, preventing or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed combinations of CRISPR-Cas9 systems and one or more inhibitors of DNA damage repair can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.2. Methods Increasing the Cytotoxicity of CRISPR-Cas9 Systems and Reducing the Number of Double Stranded Breaks Required to Obtain the Death of One or More Target Cells

[0072] In some embodiments, the present disclosure relates to methods of increasing the cytotoxicity of a CRISPR-Cas9 system that has been or is to be administered to a subject in need of treatment thereof. In other embodiments, the present disclosure relates to methods for reducing the number of double stranded breaks required to obtain the death of one or more target cells in a tumor in a subject that has been or is to be administered a CRISPR-Cas9 system. In some embodiments, the tumor is cancer. Using the methods described herein, it was surprisingly discovered that the number of DSBs needed for greater than 90% cell death, greater than 91% cell death, greater than 92% cell death, greater than 93% cell death, greater than 94% cell death, or greater than 95% cell death in a target cell can be reduced by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16% percent, 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%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, or at least 40% when compared to when a CRISPR-Cas9 system is administered alone without using the combination described herein. Thus, the use of the combination described herein increases the cytotoxicity of CRISPR- Cas9 when used for treating subjects in need of treatment thereof.

[0073] In some embodiments, the methods described herein involve administering a CRISPR- Cas9 system in combination with at least one inhibitor of DNA damage repair to a subject or patient in need of treatment thereof. In some embodiments, the subject receives a therapeutically effective amount of a CRISPR-Cas9 system and a therapeutically effective amount of at least one inhibitor of DNA damage repair. The CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair can be administered simultaneously or sequentially, in any order. If administered sequentially, the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair can be administered at the same time or at separate times, where the administration is at least an hour apart, at least several hours apart, at least a day apart, at least several days apart or at least a week or more apart.

[0074] In some embodiments, the subject to be administered the combination of the CRISPR- Cas9 system and at least one inhibitor of DNA damage repair has previously received or has been administered a CRISPR-Cas9 system. In other embodiments, the subject to be administered the combination of the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair has not previously received or been administered a CRISPR-Cas9 system.

[0075] In some embodiments, the subject in need of treatment here is suffering from one or more tumors. In some embodiments, the tumor is cancer. In yet further embodiments, the cancer is pancreatic cancer, lung cancer, esophageal cancer, or any combination thereof. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is esophageal cancer.

[0076] In other embodiments, the at least one inhibitor of DNA damage repair is a pho sphoino sitide-3 -kinase inhibitor.

[0077] In some embodiments, the at least one inhibitor of DNA damage repair is a DNA- dependent protein kinase inhibitor. In yet other embodiments, the DNA-dependent protein kinase inhibitor is KU0060648 (4-Ethyl-N-[4-[2-(4-morpholinyl)-4-oxo-4H-l-benzopyran-8-yl]- I -dibenzothienyl] -I -piperazineacetamide), nedisertib, CC-115 (l-ethyl-3,4-dihydro-7-[2-methyl- 6-(lH-l,2,4-triazoT5-yl)-3-pyridinyl]-pyrazino[2,3-b]pyrazin-2(lH)-one), or any combinations thereof.

[0078] In some embodiments, the at least one inhibitor of DNA damage repair is a poly(ADP- ribose) polymerase (PARP) inhibitor. In yet other embodiments, the PARP inhibitor is AZD2461 or talzoparib, or any combination thereof.

[0079] In some embodiments, the at least one inhibitor of DNA damage repair is an ataxiatelangiectasia and Rad3-related (ATR) inhibitor. In yet other embodiments, the ATR inhibitor is ceralasertib or berzosertib, or any combination thereof.

[0080] In some embodiments, the CRISPR-Cas9 system and at least one inhibitor of DNA damage repair are administered to a subject in a single dosage form. In some embodiments, the CRISPR-Cas9 system at least one inhibitor of DNA damage repair are administered in separate dosages forms, either simultaneously or sequentially, in any order. If administered sequentially, the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair can be administered at the same time or at separate times, where the administration is at least an hour apart, at least several hours apart, at least a day apart, at least several days apart or at least a week or more apart.

[0081] In some embodiments, the CRISPR-Cas9 system and at least one inhibitor of DNA damage repair can be administered to a subject by any method including, for example, injection, infusion, deposition, implantation, oral ingestion, or topical administration, or any combination thereof. Injections can be, e.g., intravenous, intramuscular, intradermal, subcutaneous or intraperitoneal. Single or multiple doses can be administered over a given time period.

[0082] The exact amount of CRISPR-Cas9 and at least one inhibitor of DNA damage repair to be administered to a subject will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.3. Methods of Treating Cancer

[0083] In some embodiments, the present disclosure relates to methods of treating a subject suffering from cancer. In some embodiments, the cancer is pancreatic cancer, lung cancer, esophageal cancer, or any combination thereof. Using the methods described herein, it was surprisingly discovered that the number of DSBs needed for greater than 90% cell death, greater than 91% cell death, greater than 92% cell death, greater than 93% cell death, greater than 94% cell death, or greater than 95% cell death can be reduced by at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16% percent, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, atleast 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, or at least 40% when compared to when a CRISPR-Cas9 system is administered alone without using the combination described herein. Thus, the use of the combination described herein increased the cytotoxicity of CRISPR-Cas9 when used for treating subjects suffering from cancer.

[0084] In some embodiments, the methods described herein involve administering a CRISPR- Cas9 system in combination with at least one inhibitor of DNA damage repair to a subject or patient suffering from cancer. In some embodiments, the subject receives a therapeutically effective amount of a CRISPR-Cas9 system and a therapeutically effective amount of at least one inhibitor of a DNA damage repair. The CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair can be administered simultaneously or sequentially, in any order. If administered sequentially, the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair can be administered at the same time or at separate times, wherein the administration is at least an hour apart, at least several hours apart, at least a day apart, at least several days apart or at least a week or more apart.

[0085] In some embodiments, the subject to be administered the combination of the CRISPR- Cas9 system and at least one inhibitor of DNA damage repair has previously received or has been administered a CRISPR-Cas9 system. In other embodiments, the subject to be administered the combination of the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair has not previously received or been administered a CRISPR-Cas9 system.

[0086] In some embodiments, the at least one inhibitor of DNA damage repair is a pho sphoino sitide-3 -kinase inhibitor.

[0087] In other embodiments, the at least one inhibitor of DNA damage repair is a DNA- dependent protein kinase inhibitor. In yet other embodiments, the DNA-dependent protein kinase inhibitor is KU0060648 (4-Ethyl-N-[4-[2-(4-morpholinyl)-4-oxo-4H-l-benzopyran-8-yl]- 1 -dibenzothienyl] -1 -piperazineacetamide), nedisertib, CC-115 (l-ethyl-3,4-dihydro-7-[2-methyl- 6-(lH-l,2,4-triazol-5-yl)-3-pyridinyl]-pyrazino[2,3-b]pyrazin-2(lH)-one) or any combinations thereof.

[0088] In some embodiments, the at least one inhibitor of DNA damage repair is a poly(ADP- ribose) polymerase (PARP) inhibitor. In some other embodiments, the PARP inhibitor is AZD2461 or talazoparib, or any combination thereof.

[0089] In other embodiments, the at least one inhibitor of DNA damage repair is an ataxiatelangiectasia and Rad3-related (ATR) inhibitor. In yet other embodiments, the ATR inhibitor is ceralasertib or berzosertib, or any combination thereof.

[0090] In some embodiments, the CRISPR-Cas9 system and at least one inhibitor of DNA damage repair are administered to a subject in a single dosage form. In some embodiments, the CRISPR-Cas9 system at least one inhibitor of DNA damage repair are administered in separate dosages forms, either simultaneously or sequentially, in any order. If administered sequentially, the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair can be administered at the same time or at separate times, where the administration is at least an hour apart, at least several hours apart, at least a day apart, at least several days apart or at least a week or more apart.

[0091] In some embodiments, the CRISPR-Cas9 system and at least one inhibitor of DNA damage repair can be administered to a subject by any method including, for example, injection, infusion, deposition, implantation, oral ingestion, or topical administration, or any combination thereof. Injections can be, e.g., intravenous, intramuscular, intradermal, subcutaneous or intraperitoneal. Single or multiple doses can be administered over a given time period.

[0092] The exact amount of CRISPR-Cas9 and at least one inhibitor of DNA damage repair to be administered to a subject will depend on the type of cancer being treated and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.EX MPLES

[0093] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of thepresently disclosed subject matter. The descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be constmed as limiting in any manner.

[0094] Example 1 - Synergy with DNA Damage Repair Inhibition

[0095] Killing Curve Experiments

[0096] Killing curve experiments were performed in Pane 10.05, TSO 111, and Panc480 PDAC cell lines to assess baseline toxicity of small molecule DNA-PK inhibitors in the absence of CRISPR-DSBs. Cells were plated at 1000 cells / well in 96- well plates and allowed to adhere overnight. After 24 hours, cells were treated with KU0060648 (Sigma-Aldrich, USA), Nedisertib (Selleckchem, USA), or CC-115 (Selleckchem, USA) at 0,1, 3,10, 30,100, 300, 1000, 3000, or 10,000 nM. When the OnM wells reached confluence, approximately 10 days after plating, growth media and drug were aspirated and changed to fresh media containing 10% alamarBlue Cell Viability Reagent. Fluorescent signal was read and normalized to the OnM samples for each cell line.

[0097] Clonogenicity Assays

[0098] TSOI 11, Panc480, and Pane 10.05 PDAC cell lines were transduced with a lentiviral vector (lentiCRISPRv2_puro, Addgene 98290) simultaneously expressing Cas9 and multitarget sgRNAs or PAM-based, TSOI 11-specific sgRNAs in 24- well plates for 24 hours. The multitarget sgRNA each cut at a known number of loci in the human genome, ranging from 1-20 target sites and were previously described in WO 2024 / 044304, the contents of which are incorporated by reference. Two days after transduction, the cells were expanded into 96-well plates at -100 cells / well in complete media (DMEM supplemented with 10% fetal bovine serum, 1% L- glutamine, and lx Antibiotic-Antimycotic (ThermoFisher Scientific, USA)). Selection was performed on both treatment arms with 1 ug / mL puromycin. For each sgRNA, control plates received puromycin-only media (labeled no drug or -KU0060648 (-KU) (FIG 2B)) and drug treated plates received puromycin and 1 pM KU0060648 (labeled +KU) (Sigma- Aldrich, USA), I pM Nedisertib (Selleckchem, USA), IpM AZD 2461 (Selleckchem, USA), lOOnM Talazoparib (Selleckchem, USA), IpM Ceralasertib (Selleckchem, USA), or lOOnM Berzosertib (Selleckchem, USA) in DMSO. The 96-well plates were grown in a tissue culture incubator with media changes and fresh drug every 1-2 weeks. The endpoint was determined when the NT transduced plates reached confluency in at least one well, a period of 21-30 days posttransduction. At end point, growth media was aspirated and changed to fresh media containing 10% alamarBlue Cell Viability Reagent (ThermoFisher Scientific, USA).

[0099] Tag Survival AssaysTag survival assays were performed in the TSOI 11, Panc480, and Pancl0.05 PDAC cell lines. Cells were plated at a density of 4xl06in T75 flasks. The follow day, the cells were transduced for 20-24 hours at low MOI with a pool of lentiCRISPRv2_puro (Addgene 98290) virus expressing NT sgRNA, multitarget sgRNA, or AGGn and Alu positive control sgRNA. The sgRNA were previously described in WO 2024 / 044304. Two days after transduction, selection was begun with 1 ug / mL puromycin and DNA-PK inhibitors. The drugs and dosages were no drug control (puromycin only), KU0060648 (Sigma-Aldrich, USA), IpM or lOOnM, nedisertib (Selleckchem, USA) IpM, or CC-115 (Selleckchem, USA), 30nM. The flasks were grown for 21 days post transduction with trypsinization and passaging 1-2 times weekly as necessary to prevent confluency. At 21 days, cells were trypsinized and harvested for genomic DNA extraction. DNA extraction was performed with the QIAamp DNA Mini Kit (Qiagen, USA) and quantified with Qubit HS Assay (ThermoFisher Scientific, USA). To assay relative abundance of each guide, -75-100ng DNA (>10,000 genomes) from each treatment group was PCR amplified using NGS-adaptor primers previously designed to amplify the transduced sgRNA sequence (See, Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017; 12(4):828-63). Amplicons were submitted for deep sequencing (>50,000x coverage) by the Illumina AmpliconEZ service at Azenta. Tags were quantified by a previously described Python script, count_spacersl .py (See, Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017; 12(4):828-63)).

[0100] Results

[0101] Killing curve experiments were performed for KU0060648, nedisertib, and CC-115 to determine baseline toxicity of small molecule DNA-PK inhibition to PDAC cells and choose an optimal experimental dose that would allow for detection of synergy with CRISPR-Cas9 with less background toxicity from drug treatment alone. FIG. 1 shows killing curves for the three compounds tested in 3 PDAC cell lines. Based on these curves, further experiments were performed with nedisertib at IpM, KU0060648 at lOOnM, and CC-115 at 30nM.

[0102] Cytotoxic Synergy between CRISPR-Cas9 induced DSBs and DNA-PK inhibition

[0103] Clonogenicity assays with TSOI 11, Panc480, and Pane 10.05 PDAC cells were performed to determine a relationship between the number of CRISPR targets and cytotoxicity, with and without DNA-PK / PI3K inhibition. In TSOI 11 cells, CRISP-Cas9 scission at 10-12 targets was necessary to induce >80% cell death in the absence of KU0060648 (-KU) or nedisertib (see FIG. 2A). This number was reduced to 2-3 targets in the presence of KU0060648 and nedisertib (see FIG. 2A). TSOI 11 cells grown in the presence of KU0060648 demonstrated about 95% cell death beginning at 2 targets and about 70% cell death in the presence of nedisertib (see FIG. 2A). Of note, the 8-cutting sgRNA exhibited low cytotoxicity in the absence of KU0060648 or nedisertib, likely due to decreased cutting efficiency compared to the other sgRNAs (see FIG. 2A).

[0104] FIG. 2B shows that Panc480 cells demonstrated the same cytotoxicity curve as TSOI 11 in the presence of KU0060648. The + KU0060648 (+KU) curve is indistinguishable from the - KU0060648 (-KU) curve in this cell line, however, as Panc480 has a higher baseline sensitivity to CRISPR induced DSBs.

[0105] FIG. 2C shows that in Pancl0.05 cells, CRISPR-Cas9 scission at 10-12 targets was necessary to induce >80% cell death in the absence of nedisertib. As with TSOI 11 cells, this number was reduced to 2-3 targets, with 95% cell death demonstrated in the presence of nedisertib.

[0106] FIG. 2D shows a plot of p-values for each target number for the TSOI 11 dose response curve. Significance (p>0.05) represented by dotted line, r2determined by linear regression.

[0107] Overall, the data show that PDAC cells display a dramatic increase in cytoxicity in the presence of NHEJ inhibition. Data is shown in FIG. 2 for A) TSOI 11 cells, B) Panc480 and C) Pancl0.05 pancreatic cancer cells. N=3 for TSOI 11 and Panc480 cells; N=1 for Pancl0.05. cells, error in SEM.

[0108] Cytotoxic Synergy between CRISPR-Cas9 induced DSBs and PARP inhibition

[0109] Clonogenicity assays were performed on TSOI 11 and Pane 10.05 PDAC cell lines transduced with lentiviral vectors co-expressing Cas9 and multi-target sgRNAs. Results are shown for FIG. 3 for A) TSOI 11 cells only and B) TSOI 11 and Pancl0.05 cells. FIG. 3A shows the synergistic toxicity of the PARP inhibitor AZD2461 and TSOI 11 cells expressing non-targeting sgRNAs (Neg ctrl(2)), two PAM-based, TSOl l l-specific sgRNAs (chrl2 and chrl4), a 3-target sgRNA (52F(3)) or a positive control sgRNA (AGGn (rep)). There was synergy between the PAM-based, TSOl ll-specific chrl2 and chrl4 sgRNAs and the 3-target sgRNA and AZD2461, as shown by a reduction in the percentage of cell survival. There was even greater synergy between the chrl4 PAM-based sgRNA and the 3-target sgRNA and AZD2461 compared to that induced by chrl2 PAM-based sgRNA and AZD2461 (N=l).

[0110] FIG. 3B shows the results of synergistic toxicity of the PARP inhibitor talazoparib on TSOI 11 and Pancl0.05 cell lines expressing non-targeting sgRNAs (Neg cntrl (2)) a 12 cutter sgRNA (230F(I2), or a positive control sgRNA (AGGn(rep)). There was synergy between TSOI 11 and Pancl0.05 cells expressing the 12 cutter sgRNA and talazoparib, with even greater synergy between the 12 cutter sgRNA and talazoparib in TSOI 11 cells than in Pancl0.05 cells (N=l).

[0111] Cytotoxic Synergy between CRISPR-Cas9 induced DSBs and ATR inhibition

[0112] Clonogenicity assays were performed on TSOI 11 and Pancl0.05 PDAC cell lines transduced with lentiviral vectors co-expressing Cas9 and multi-target sgRNAs. Results are shown in FIG. 4 for A) TSOI 11 cells and B) Pane 10.05 cells. FIG. 4A shows the synergistic toxicity of the ATR inhibitors ceralasertib and berzosertib and expression of a 12 target sgRNA (230F(12) in TSOI 11 cells, as shown by a reduction in cell survival (N=l). FIG. 4B shows the synergistic toxicity of the ATR inhibitor berzosertib and Pane 10.05 cells expressing both a 3 cutter sgRNA and the 12 cutter sgRNA, with greater synergy between berzosertib and the 12 cutter sgRNA (N=l).

[0113] Synergy between CRISPR-induced DSBs and DNA-PK inhibition begins at 3 target sites

[0114] Tag survival experiments were performed in the 3 PDAC cell lines. Log2 fold change from day-0 for multitarget sgRNAs was shown (FIG. 5 A- 5C). The cells were grown in the presence or absence of KU0060648 for 21 days and relative abundance of each sgRNA was determined relative to the day of initial lentiviral transduction. Results are shown in FIG. 5 for A) TSOI 11, B) Panc480, and C) Pancl0.05 PDAC cell lines. Synergy between CRISPR-DSBs and KU0060648 is apparent beginning at 3-5 targets in TSOI 11 and Panc480 cells. Pancl0.05 cells demonstrated high sensitivity to KU0060648 in the absence of sgRNA transduction potentially obscuring a synergistic effect. N=3 for TSOI 11, N=2 for Panc480 and Pancl0.05.N=1 for experiments in FIG 5. Error in SEM. Pairwise comparisons by paired t-test with significance (denoted by *) at p > 0.05. Results are shown in FIG. 5 for A) TSOI 11 and B) Panc480.

[0115] Tag survival studies were performed using TSOI 11, Panc480, and Pane 10.05 cells that were transduced with a pool of the multitarget sgRNAs in addition to a negative control (NT) sgRNA and positive control sgRNAs targeting the AGG(n) trinucleotide repeat and the Alu retro transposon (FIG. 6A- 6B). Relative abundance of each guide (tag) was determined after 21 days of growth in the presence or absence of KU0060648, nedisertib, or CC-115. In the TSOI 11 and Panc480 cell lines, synergy is apparent beginning at 3 targets and is most pronounced between 3 and 8 targets (FIG. 6A-6B). Similar to the clonogenicity dose response curves in FIG. 2, KU0060648 and nedisertib treatment exhibits the highest effects with smaller target numbers. This is likely because induction of >8 simultaneous DSBs alone exhibits >90% toxicity as shown in FIG. 2.

[0116] Nedisertib synergizes with CRISPR-DSBs at >3 target sites.

[0117] TS011 and Panc480 PDAC cells were transduced with multitarget sgRNAs as detailed in the tag survival assay and grown in the presence of nedisertib (' 1 uM) for 21 days. Normalized read counts of each sgRNA were shown for day 1 and day 21. There was a positive selection for cells cut at less than 3 targets and a negative selection for 3 or more targets. Alu and AGGn were included as positive controls. N=l.REFERENCES

[0118] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0119] 1. F. Blokzijl et al., Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260-264 (2016).

[0120] 2. P. C. Nowell, The clonal evolution of tumor cell populations. Science 194, 23-28(1976).

[0121] 3. E. R. Fearon, B. Vogelstein, A genetic model for colorectal tumorigenesis. Cell61, 759-767 (1990).

[0122] 4. C. Tomasetti, B. Vogelstein, G. Parmigiani, Half or more of the somatic mutations in cancers of self-renewing tissues originate prior to tumor initiation. Proc Natl Acad Sci U SA 110, 1999-2004 (2013).

[0123] 5. M. Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).

[0124] 6. L. Cong et al., Multiplex genome engineering using CRISPR / Cas systems.Science 339, 819-823 (2013).

[0125] 7. P. Mali et al., RNA-guided human genome engineering via Cas9. Science 339,823-826 (2013).

[0126] 8. Y. Fu et al., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31, 822-826 (2013).

[0127] 9. G. Alanis-Eobato et al., Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos. Proc Natl Acad Sci USA 118, (2021).

[0128] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

What is claimed is:

1. A method for reducing the number of double stranded breaks required to obtain death of one or more target cells in a tumor in a subject being treated or to be treated with a CRISPR-Cas9 system, the method comprising the step of administering to a subject in need of treatment thereof a therapeutically effective amount of at least one CRISPR-Cas9 system and at least one inhibitor of a DNA damage repair.

2. The method of claim 1, wherein the CRISPR-Cas9 system targets one or more sgRNAs that target a sequence adjacent to one or more target protospacer adjacent motifs (PAMs).

3. The method of claim 1 or claim 2, wherein the tumor is cancer.

4. The method of any of claims 1-3, wherein the cancer is pancreatic cancer, lung cancer, esophageal cancer, or any combinations thereof.

5. The method of any of claims 1-4, wherein the inhibitor of DNA damage repair is a phosphoinositide 3-kinase (PI3K) inhibitor.

6. The method of claim 5, wherein the inhibitor of DNA damage repair protein is a DNA-dependent protein kinase inhibitor.

7. The method of claim 5, wherein the inhibitor of DNA damage repair is a poly [ADP-ribose] polymerase (PARP) inhibitor.

8. The method of claim 5, wherein the inhibitor of DNA damage repair is an ataxia telangiectasia and Rad3 -related (ATR) inhibitor.

9. The method of any of claims 1-8, wherein the inhibitor of DNA damage repair is a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor, or an RNA-based inhibitor10. The method of any of claims 1-9, wherein the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair are administered simultaneously or sequentially.

11. A method of treating a subject suffering from pancreatic cancer, lung cancer, esophageal cancer, or any combination thereof, the method comprising administering to the subject a therapeutically effective amount of the CRISPR-Cas9 system and at least one inhibitor of a DNA damage repair.

12. The method of claim 11, wherein the CRISPR-Cas9 system targets one or more sgRNAs that target a sequence adjacent to one or more target protospacer adjacent motifs (PAMs).

13. The method of claim 12, wherein the inhibitor of DNA damage repair is a phosphoinositide 3-kinase (PI3K) inhibitor.

14. The method of claim 12, wherein the inhibitor of DNA damage repair is a DNA- dependent protein kinase inhibitor.

15. The method of claim 12, wherein the inhibitor of DNA damage repair is a poly(ADP-ribose) polymerase (PARP) inhibitor.

16. The method of claim 12, wherein the inhibitor of DNA damage repair is an ataxia telangiectasia and Rad3 -related (ATR) inhibitor.

17. The method of any of claims 11-16, wherein the DNA damage repair inhibitor is a biologic molecule-based inhibitor, a chemical molecule inhibitor, a small molecule inhibitor or an RNA-based inhibitor.

18. The method of any of claims 11-17, wherein the CRISPR-Cas9 system and the at least one inhibitor of DNA damage repair are administered simultaneously or sequentially.

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