CLK2 inhibition as a radiation countermeasure

Abstract

The disclosure relates to methods of using CLK2 inhibitors for treating subjects exposed to radiation.

Description

[0001] CLK2 INHIBITION AS A RADIATION COUNTERMEASURE

[0002] REEATED APPLICATION

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 619,361, filed January 10, 2024, which is incorporated by reference herein in its entirety.

[0004] GOVERNMENT LICENSE RIGHTS

[0005] This invention was made with government support under 75F40119C10098 awarded by U.S. Food and Drug Administration (FDA). The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (H049870803WO00-SEQ-KVC.xml; Size: 3,298 bytes; and Date of Creation: January 8, 2025) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] In humans, acute radiation syndrome (ARS) caused by exposure to ionizing radiation is associated with injury to the hematopoietic, gastrointestinal, pulmonary, and neurovascular systems. Despite the critical role of the endothelium in mediating radiation damage to other tissues, currently available drugs for radiation medical countermeasures all target the hematopoietic system.

[0010] SUMMARY

[0011] The disclosure relates, at least in part, to the use of Cdc2-like kinase 2 (CLK2) inhibition as a strategy for promoting recovery of the endothelium following exposure to (e.g., treatment with) ionizing radiation. There is a crucial need for safe and effective radiation medical countermeasures (MCMs) for military and civilian biodefense applications, and for mitigation of toxicities associated with medical radiotherapies. The nature and severity of such toxicities depend primarily on the dose of radiation and the exposure time. Exposure to high levels of radiation, even for short periods of time, can cause DNA and protein damage, produce free radicals and reactive oxygen species, and result in cell cycle arrest, loss of epithelial cells, endothelial cell toxicity, mucosal barrier breakdown, and inflammation. In humans, exposure to ionizing radiation can result in acute radiation syndrome (ARS), which is associated with injury to the hematopoietic, gastrointestinal, pulmonary, and / or neurovascular systems.

[0012] Endothelial injury is a major complicating factor in conditions arising from radiation exposure. Microvascular endothelial cells are particularly sensitive to ionizing radiation, and radiation-induced endothelial damage and its associated vascular changes often lead to chronic lesions or organ failure. Currently, only three drugs have been approved as MCMs - NEUPOGEN® (filgrastim; G-CSF), NEULASTA® (pegfilgrastim; PEGylated G-CSF), and LEUKINE® (sargramostim; GM-CSF) - all of which target the hematopoietic system. Given the critical role of the endothelium in mediating radiation damage to other tissues, particularly the pulmonary and gastrointestinal systems, therapies that can retroactively protect against damage to the endothelium are of great importance as MCMs.

[0013] The data described herein show that CLK2 inhibition increases endothelial cell numbers and decreases apoptosis following exposure to ionizing radiation. As toxicity to endothelial cells is known to be responsible for many of the adverse effects of radiation exposure, the methods provided by the disclosure, which leverage CLK2 inhibition, are useful therapeutic strategies to counter such endothelial cell toxicities resulting from exposure to radiation. These findings were surprising and unexpected, particularly in view of reports linking CLK2 inhibition to the inhibition of cell growth (Nam et al. J Biol Chem. 2010 Oct 8;285(41):31157-63) and the induction of cell apoptosis (Araki et al. PLoS One. 2015; 10(1): eOl 16929).

[0014] Accordingly, aspects of the present disclosure relate to a method, comprising administering a CLK2 inhibitor to a subject who has been or will be exposed to radiation.

[0015] In some embodiments, the subject has been exposed to radiation therapy.

[0016] In some embodiments, the CLK2 inhibitor is administered in a therapeutically effective amount, optionally wherein the therapeutically effective amount reduces radiation toxicity in the subject.

[0017] In some embodiments, cell number is increased in the subject, relative to baseline or control. In some embodiments, cell death is decreased, relative to baseline or control.

[0018] In some embodiments, the therapeutically effective amount reduces radiation toxicity to cells of the subject. In some embodiments, the method is used as an adjunctive therapy to chemotherapy or radiotherapy.

[0019] Aspects of the present disclosure relate to a method, comprising administering a therapeutically effective amount of a CLK2 inhibitor to a subject in need thereof, wherein the subject has a condition arising from radiation exposure, and optionally wherein the therapeutically effective amount is sufficient to reduce radiation toxicity in the subject.

[0020] In some embodiments, the condition arising from radiation exposure is acute radiation syndrome (ARS), an acute radiation subsyndrome associated with one or more of the blood (hematopoietic syndrome, H-ARS), the immune system, the gastrointestinal tract (gastrointestinal syndrome, GI-ARS), the skin (cutaneous syndrome), the pulmonary system (pulmonary syndrome), the kidney, or the central nervous system (neurovascular syndrome), cutaneous radiation injury, acute respiratory syndrome, or cancer.

[0021] In some embodiments, the subject has been exposed to at least 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 Gray of radiation. In some embodiments, the subject has been exposed to the radiation over a time frame of less than 1 minute, or up to 5 minutes, 10 minutes, 30 minutes, 60 minutes, 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, or 1 year.

[0022] In some embodiments, the CLK2 inhibitor is administered at least once a day for 1, 2, 3, 4, 5, 6, or 7 days.

[0023] In some embodiments, the subject is exposed to radiation before administration of the CLK2 inhibitor, during administration of the CLK2 inhibitor, after administration of the CLK2 inhibitor, or combinations thereof.

[0024] Aspects of the present disclosure relate to a method, comprising contacting cells exposed to radiation with a CLK2 inhibitor in an amount sufficient to decrease cell death, relative to a control.

[0025] In some embodiments, the radiation is ionizing radiation. In some embodiments, the radiation is whole body radiation or partial radiation. In some embodiments, the radiation is chemotherapy or radiotherapy.

[0026] In some embodiments, the CLK2 inhibitor is selected from Cirtuvivint, Lorecivivint, Silmitasertib, TG003, SM08502, SM04755, SM09419, Cpd-2, Cpd-3, T3, CC-671, T-025, NR9, DB18, ML106, MU1210, KuWall51, CX-4945, acrifoline, CaNDY, leucettine, polyandrocarpamines A (PAC 12), ML315, KH-CB19, ML167, or CTX-712, preferably Cirtutivint, Lorecivivint, or TG003. In some embodiments, the CLK2 inhibitor is administered intravenously or orally.

[0027] Each of the limitations of the compositions and methods described in this disclosure may encompass various described embodiments. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings.

[0028] BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0030] Figures 1A-1E: Design and optimization of a kinome-scale radiation resistance screen in endothelial cells (Figure 1A) Design of improved all-in-one CRISPR / Cas9 vector (Figure IB) Cloning strategy and sgRNA representation frequency in ECv2-GE-kinome library. Shading indicates all sgRNAs between 90th and 10th percentile. (Figures 1C-1D) Radiation dose titration in human umbilical vein endothelial cells (HUVECs) and human small intestinal microvascular endothelial cells (HSIMECs). (Figure IE) Pan-endothelial cell responses to potential therapies.

[0031] Figures 2A-2G: Identification of CEK2 inhibition as a combined anti-cancer and radioprotective therapeutic strategy. (Figure 2A) Recovery effect of CEK inhibition with TG003 (left) and Cirtuvivint (right) at 0, 2, 4, and 8 Gy of ionizing radiation on primary HUVECs. (Figure 2B) Recovery effect of CEK inhibition via TG003 on primary HSIMECs at 0 and 4 Gy of ionizing radiation. (Figure 2C) CEK2 inhibitor TG003 reduces apoptosis 72h days post-irradiation. (Figure 2D) Quantification of endothelial cell size by forward scatter geometric mean fluorescence intensity (FSC-A gMFI). (Figure 2E) Quantification of intracellular reactive oxygen species (ROS) levels by CellROX Deep Red Reagent. (Figure 2F) Foci counts of 53bpl (left) gamma- H2AX (center) and representative images (right) (Figure 2G) Quantification of leukocyte adhesion to endothelial cells 24 hours postirradiation.

[0032] Figures 3A-3D: RNA-sequencing indicates a reversal of radiation-induced gene signatures. (Figure 3 A) Correlation plot (left) and Heatmap (right) showing that radiation- related transcriptional changes are reversed by TG003 treatment across all RNA transcripts. (Figure 3B) Pathway analyses showing irradiated vs. control (left) and TG003-treated and irradiated endothelial cells vs. vehicle-treated and irradiated endothelial cells, (right) (Figure 3C) Correlation plot showing effect sizes of protein phosphorylation across the proteome. (Figure 3D) Model of NF AT phosphorylation driven by CLK2.

[0033] Figures 4A-4F: Multi-organ Chip Assessment of CLK2 inhibition as a therapeutic strategy. (Figure 4A) Schematic showing experimental overview. (Figure 4B) Cell counts from the vascular channel (left), apical (epithelial) channel (center), and villus height measurements (right) of intestine chips treated as indicated. (Figure 4C) Cytokine levels measured by Luminex assay. (Figure 4D) VE-Cadherin average quantification per chip (left) and representative images (right) of lung chips treated as indicated. (Figure 4E) Quantification per chip (left) and representative images (right) of PBMC adhesion to lung chips (right). (Figure 4F) Levels of cytokines detected by Luminex assay.

[0034] DETAILED DESCRIPTION

[0035] The present disclosure relates, at least in part, to methods of using CLK2 inhibitors, such as CLK2 inhibiting small molecules, for reducing toxicity to endothelial cells of a subject exposed to radiation. In some embodiments, CLK2 inhibitors can be used to decrease cell death associated with radiation exposure.

[0036] CLK2 Inhibitors

[0037] CDC-like kinases (CLKs) phosphorylate serine-, threonine-, and tyrosine-containing substrates and regulate serine- and arginine-rich (SR) proteins of the spliceosomal complex, influencing alternative transcript splicing. CLKs are classified as a CMGC kinase (cyclin- dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK-like kinases) group and share an ATP co-factor for phosphorylating downstream protein substrates. The CLK kinase domain contains an “EHLAMMERILG” (SEQ ID NO: 2) motif located at the C-terminus of each family member, which includes CLK1, CLK2, CLK3, and CLK4 encoded by CLK1, CLK2, CLK3, and CLK4, respectively, in humans. CLKs are located in the nucleus and cytoplasm, where they phosphorylate the serine / arginine-rich domain of splicing factors, controlling pre-mRNA splicing and generating various protein isoforms.

[0038] A non-limiting example of a CLK2 enzyme from Homo sapiens is provided by UniProt Accession No. P49760 and SEQ ID NO: 1. MPHPRRYHS SERGSRGSYREHYRSRKHKRRRSRSWS S S SDRTRRRRREDSYHVRSRS SYDDRS SDRRVYDRRYCG SYRRNDYSRDRGDAYYDTDYRHSYEYQRENS SYRSQRS SRRKHRRRRRRSRTFSRS S SQHS SRRAKSVEDDAEGH LI YHVGDWLQERYEIVSTLGEGTFGRWQCVDHRRGGARVALKI IKNVEKYKEAARLEINVLEKINEKDPDNKNL CVQMFDWFDYHGHMCI SFELLGLSTFDFLKDNNYLPYP IHQVRHMAFQLCQAVKFLHDNKLTHTDLKPENILFVN SDYELTYNLEKKRDERSVKSTAVRWDFGSATFDHEHHSTIVSTRHYRAPEVILELGWSQPCDVWS IGCI IFEYY VGFTLFQTHDNREHLAMMERILGP IP SRMIRKTRKQKYFYRGRLDWDENTSAGRYVRENCKPLRRYLTSEAEEHH QLFDLIESMLEYEPAKRLTLGEALQHPFFARLRAEPPNKLWDS SRDI SR (SEQ ID NO: 1)

[0039] As described herein, it has been found that inhibition of CLK2 can reduce toxicity to cells by decreasing cell death (e.g., apoptosis). The present disclosure relates to methods of reducing toxicity to endothelial cells of a subject exposed to radiation, comprising administering to the subject a therapeutically effective amount of CLK2 inhibitor, thereby reducing toxicity to the endothelial cells.

[0040] The term “CLK2 inhibitor”, as used herein, is an agent that reduces a measurable level of CLK2 activity, for example, by inhibiting CLK2 gene expression, mRNA expression, protein expression, and / or protein activity (e.g., serine, threonine, or tyrosine phosphorylation). In some embodiments, the CLK2 inhibitor is an inhibitor of CLK2 gene expression. In some embodiments, the CLK2 inhibitor is an inhibitor of CLK2 mRNA expression. In some embodiments, the CKL2 inhibitor is an inhibitor of CLK2 protein expression. In some embodiments, the CLK2 inhibitor is an inhibitor of CLK2 protein activity (e.g., serine, threonine, or tyrosine phosphorylation). In some embodiments, the CLK2 inhibitor is an inhibitor of any combination of CLK2 gene expression, CLK2 mRNA expression, CLK2 protein expression, and CLK2 protein activity. Non-limiting examples of CLK2 inhibitors are described below, and the development of CLK2 inhibitors is discussed, for example, in Qin et al. J Med Chem. 2021 Sep 23;64(18): 13191-13211.

[0041] In some embodiments, a CLK2 inhibitor inhibits (i.e., reduce or eliminate) CLK2 gene expression. For example, a CLK2 inhibitor may inhibit CLK2 gene expression by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, relative to a control, for example, a baseline level of CLK2 gene expression.

[0042] In some embodiments, a CLK2 inhibitor inhibits (i.e., reduce or eliminate) CLK2 mRNA expression. For example, a CEK2 inhibitor may inhibit CEK2 mRNA expression by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, relative to a control, for example, a baseline level of CEK2 mRNA expression. In some embodiments, a CLK2 inhibitor inhibits (i.e., reduce or eliminate) CLK2 protein expression. For example, a CLK2 inhibitor may inhibit CLK2 protein expression by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, relative to a control, for example, a baseline level of CLK2 protein expression.

[0043] In some embodiments, a CLK2 inhibitor inhibits (i.e., reduce or eliminate) CLK2 protein activity (e.g., serine, threonine, or tyrosine phosphorylation). For example, a CLK2 inhibitor may inhibit CLK2 protein activity (e.g., serine, threonine, or tyrosine phosphorylation) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, relative to a control, for example, a baseline level of CLK2 protein expression.

[0044] Small molecules

[0045] In some embodiments, the CLK2 inhibitor is a small molecule. The term “small molecule(s)”, as used herein, include organic and inorganic compounds (including heterorganic and organometallic compounds) generally having a molecular weight less than about 5,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, e.g., organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. In some embodiments, small molecule inhibitors of CLK2 compete with ATP in the binding pocket of the kinase. In some embodiments, the CLK2 inhibitor is Cirtuvivint, Lorecivivint, Silmitasertib, TG003, SM08502, SM04755, SM09419, Cpd-2, Cpd-3, T3, CC-671, T-025, NR9, DB18, ML106, MU 1210, KuWall51, CX-4945, acrifoline, CaNDY, leucettine, polyandrocarpamines A (PAC 12), ML315, KH-CB19, ML167, or CTX-712. In some embodiments, the CLK2 inhibitor is Cirtuvivint. In some embodiments, the CLK2 inhibitor is Lorecivivint. In some embodiments, the CLK2 inhibitor is TG003. In some embodiments, the CLK2 inhibitor is Silmitasertib.

[0046] In some embodiments, the CLK2 inhibitor is TG003:

[0047] In some embodiments, the CLK2 inhibitor is Lorecivivint:

[0048] In some embodiments, the CLK2 inhibitor is SM08502:

[0049] In some embodiments, the CLK2 inhibitor is SM04755: In some embodiments, the CLK2 inhibitor is Cpd-2:

[0050] I e CLK2 inhibitor is Cpd-3:

[0051] In some embodiments, the CLK2 inhibitor is T3:

[0052] In some embodiments, the CLK2 inhibitor is CC-671:

[0053]

[0054] In some embodiments, the CLK2 inhibitor is T-025:

[0055] I e CLK2 inhibitor is NR9:

[0056] In some embodiments, the CLK2 inhibitor is DB18: , the CLK2 inhibitor is ML106: In some embodiments, the CLK2 inhibitor is MU1210:

[0057] I ts, the CLK2 inhibitor is KuWall51: In some embodiments, the CLK2 inhibitor is CX-4945: In some embodiments, the CLK2 inhibitor is acrifoline:

[0058] In some embodiments, the CLK2 inhibitor is CaNDY : In some embodiments, the CLK2 inhibitor is leucettine:

[0059] In some embodiments, the CLK2 inhibitor is polyandrocarpamines A (PAC12):

[0060] In some embodiments, the CLK2 inhibitor is ML315:

[0061] In some embodiments, the CLK2 inhibitor is KH-CB19:

[0062] In some embodiments, the CLK2 inhibitor is ML167:

[0063] In some embodiments, the CLK2 inhibitor is an indazole derivative, imidazopyridine derivative, a benzothiazoline derivative, a pyrrolopyrimidine derivative, a purine derivative, a pyrazolopyridazine derivative, a quinoline derivative, a quinazoline derivative, a furopyridine derivative, a dihydropyrroloindol-one derivative, a benzonaphthyridine derivative, or a benzofluorene derivative. As is known in the art, a derivative is produced by converting a chemical compound into a product (the reaction's derivate / derivative) of similar chemical structure. Other small molecule CLK2 inhibitors are contemplated.

[0064] Antibodies

[0065] In some embodiments, the CLK2 inhibitor is an antibody or antigen-binding fragment thereof. The term “antibody”, as used herein, refers to a molecule that specifically binds to, or is immunologically reactive with, a particular antigen and includes at least the variable domain of a heavy chain, and normally includes at least the variable domains of a heavy chain and of a light chain of an immunoglobulin. Unless otherwise indicated, the term “antibody” (Ab) is meant to include both intact (whole) molecules as well as antibody fragments (such as, for example, Fab and F(ab’)2 fragments) that are capable of specifically binding to a target protein. Antibodies (including intact antibodies and antigen-binding fragments), variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and quad-specific antibodies, diabodies, triabodies, and tetrabodies), single-domain antibodies (sdAb), epitope-binding fragments, e.g., Fab, Fab’, and F(ab’)2, Fd, Fvs, single-chain Fvs (scFv), rlgG, single-chain antibodies, disulfide- linnked Fvs (sdFv), fragments containing either a Vi.or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. Fab and F(ab’)2 fragments lack the Fc fragment of an intact antibody. Antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecule.

[0066] The term “antigen-binding fragment,” as used herein, refers to one or more fragments of an immunoglobulin that retain the ability to specifically bind to a target antigen. The antigen-binding function of an immunoglobulin can be performed by fragments of a full- length antibody. The antibody fragments can be a Fab, F(ab’)2, scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment that consists of a VH domain; (vii) a dAb that consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Other antibody fragments are described above. These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.

[0067] Nucleic acids

[0068] In some embodiments, the CLK2 inhibitor is a nucleic acid. In some embodiments, the CLK2 inhibitor is an antisense oligonucleotide. Antisense oligonucleotides (ASOs) are small- sized single- stranded nucleic acids that bind to their target RNA or DNA sequence inside cells to cause gene silencing. In some embodiments, the CLK2 inhibitor is an ASO that binds to a nucleic acid encoding CLK2.

[0069] In some embodiments, the CLK2 inhibitor is an RNA interference molecule. Nonlimiting examples of RNA interference molecules include micro RNAs, short interfering RNAs, and short hairpin RNAs. In some embodiments, a CLK2 inhibitor is an RNA interference molecule that binds to a nucleic acid encoding CLK2.

[0070] In some embodiments, the CLK2 inhibitor is a programmable nuclease, for example, an RNA-guided nuclease. Non-limiting examples of programmable nucleases include CRISPR nucleases, zinc finger nucleases, transcription activator- like effector nucleases, and meganucleases.

[0071] Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases.

[0072] Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences, and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.

[0073] The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages and provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR- associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea. These systems have created CRISPR gene editing that commonly utilizes the cas9 gene. For a review of ZFN, TALEN and CRISPR / Cas-based methods for genome engineering see, e.g., Gaj T el al. Trends Biotechnol. 2013 Jul; 31(7): 397-405, incorporated herein by reference. Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double- stranded DNA sequences of 12 to 40 base pairs); as a result, this site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I- Scel meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance (although sequences with a single mismatch occur about three times per human-sized genome). Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes. In some embodiments, a CLK2 inhibitor is a programmable nuclease system designed to target a nucleic acid encoding CLK2.

[0074] Methods of Use

[0075] The methods described herein, in some aspects, are used to reduce radiation toxicity in a subject. Such methods may include, for example, administering to a subject exposed to radiation a CLK2 inhibitor in an amount effective to reduce radiation toxicity to endothelial cells of the subject. This effective amount may also be referred to as a therapeutically effective amount. An effective amount of a CLK2 inhibitor is an amount sufficient to reduce radiation toxicity to endothelial cells of a subject. A reduction in radiation toxicity may be assessed relative to a control or baseline. A control may be, for example, radiation toxicity to endothelial cells of a subject that has not received a CLK2 inhibitor. Baseline is the state of the endothelial cells of the subject prior to radiation exposure. In some embodiments, an effective amount of a CLK2 inhibitor reduces radiation toxicity by at least 20%, at least 30%, at least 40%, or at least 50%, relative to a control or baseline. In some embodiments, a reduction in radiation toxicity is assessed by evaluating endothelial cell numbers. Thus, in some embodiments, an effective amount of a CLK2 inhibitor increases endothelial cell number in a subject relative to a control or baseline. An increase in endothelial cell number may be, for example, an increase by at least 20%, at least 30%, at least 40%, or at least 50%, relative to a control or baseline. In some embodiments, a reduction in radiation toxicity is assessed by evaluating endothelial cell death. Thus, in some embodiments, an effective amount of a CLK2 inhibitor decreases endothelial cell death in a subject relative to a control or baseline. A decrease in endothelial cell death may be, for example, a decrease by at least 20%, at least 30%, at least 40%, or at least 50%, relative to a control or baseline.

[0076] In some embodiments, an effective amount of a CLK2 inhibitor is administered to a subject as a pre-exposure treatment. A pre-exposure treatment refers to the administration of a CLK2 inhibitor to a subject before the subject is exposed to radiation (e.g., within 96 hours of an anticipated exposure to radiation). Thus, in some embodiments, a CLK2 inhibitor is used to protect cells from the harmful effects of radiation (e.g., prevent increased cell death relative to a baseline or control). In some embodiments, an effective amount of a CLK2 inhibitor is administered to a subject as a post-exposure treatment. A post-exposure treatment refers to the administration of a CLK2 inhibitor to a subject after the subject is exposed to radiation. Thus, in some embodiments, a CLK2 inhibitor is used to counteract the effects of radiation exposure - to treat cells damaged from the harmful effects of radiation.

[0077] In some embodiments, an effective amount of a CLK2 inhibitor is administered to a subject to alleviate one or more symptoms associated with exposure to radiation. Alleviating the symptoms include delaying the development or progression of the symptom or reducing symptom severity. Alleviating the symptom does not necessarily require curative results. Symptoms associated with exposure to radiation include, for example, nausea, vomiting, diarrhea, fever, skin redness, itching, blistering, fatigue, hair loss, and internal and external bums. In some embodiments, an effective amount of a CLK2 inhibitor is administered to a subject to alleviate one or more symptoms associated with a disease or condition caused by exposure to radiation (e.g., cancer, thyroid disorders, cataracts, radiation dermatitis, radiation pneumonitis, radiation enteritis, or radiation fibrosis). Symptoms associated with a disease or condition caused by exposure to radiation include, for example, abnormal weight loss, fatigue pain, abnormal bleeding, blurred vision, sensitivity to light, difficulty with night vision, redness, itching, dryness, blistering, skin ulcers, cough, shortness of breath, fever, chest pain, diarrhea, and fibrosis.

[0078] In some embodiments, an effective amount of a CLK2 inhibitor is administered to a subject to reduce toxicity to cells. In some embodiments, increased numbers of cells (e.g., endothelial cells) relative to a baseline or control, indicates reduced toxicity to cells. In some embodiments, decreased cell death (e.g., apoptosis), relative to a baseline or control, indicates reduced toxicity to cells. In some embodiments, toxicity to cells, such as toxicity to endothelial cells, is decreased by at least 5%, relative to a baseline or control. For example, toxicity to cells, such as toxicity to endothelial cells, may be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, relative to a baseline or control. In some embodiments, toxicity to cells, such as toxicity to endothelial cells, is decreased by 5%-100%, 5%-90%, 5%-80%, 5%-70%, 5%- 60%, 5%-50%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%- 40%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 40%-100%, 40%- 90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%- 70%, or 50%-60%, relative to a baseline or control.

[0079] In some embodiments, an effective amount of CLK2 inhibitor is an amount required to increase cell viability (e.g., cell numbers). In some embodiments, an effective amount of CLK2 inhibitor is an amount required to increase endothelial cell numbers. In some embodiments, an agent, or a combination of agents, as disclosed herein, is administered in an amount effective for increasing cell numbers, such as endothelial cell numbers. In some embodiments, cell numbers, such as endothelial cell numbers, are increased by at least 5%, relative to a baseline or control. For example, cell numbers, such as endothelial cell numbers, may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, relative to a baseline or control. In some embodiments, cell numbers, such as endothelial cell numbers, are increased by 5%- 100%, 5%-90%, 5%-80%, 5%-70%, 5%-60%, 5%-50%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, or 50%-60%, relative to a baseline or control.

[0080] In some embodiments, an effective amount of a CLK2 inhibitor is administered to a subject to decrease cell death (e.g., apoptosis). In some embodiments, an effective amount of a CLK2 inhibitor is an amount required to decrease cell death (e.g., endothelial cell death). In some embodiments, an agent, or a combination of agents, as disclosed herein, is administered in an amount effective for reducing cell death, such as reducing endothelial cell death. In some embodiments, cell death, such as endothelial cell apoptosis, is decreased by at least 5%, relative to a baseline or control. For example, cell death, such as endothelial cell apoptosis, may be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, relative to a baseline or control. In some embodiments, cell death, such as endothelial cell apoptosis, is decreased by 5%-100%, 5%-90%, 5%-80%, 5%-70%, 5%-60%, 5%-50%, 20%-100%, 20%-90%, 20%-80%, 20%- 70%, 20%-60%, 20%-50%, 20%-40%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%- 60%, 30%-50%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%- 100%, 50%-90%, 50%-80%, 50%-70%, or 50%-60%, relative to a baseline or control.

[0081] Suitable routes of administration include, without limitation, oral, intravenous, intranasal, intramuscular, subcutaneous, intradermal, intraperitoneal, and intrathecal. In some embodiments, a CLK2 inhibitor is administered orally. In some embodiments, a CLK2 inhibitor is administered intravenously. In some embodiments, a CLK2 inhibitor is administered intranasally. In some embodiments, a CLK2 inhibitor is administered intramuscularly. In some embodiments, a CLK2 inhibitor is administered subcutaneously. In some embodiments, a CLK2 inhibitor is administered intradermally. In some embodiments, a CLK2 inhibitor is administered intraperitoneally. In some embodiments, a CLK2 inhibitor is administered intrathecally. Other routes of administration are contemplated herein.

[0082] Any of the agents disclosed herein may be administered to a subject (e.g., mammalian subject, such as a human, mouse, rabbit, goat, sheep, pig, or non-human primate) to treat exposure to radiation. As used herein, the term “treating” refers to the application or administration of a CLK2 inhibitor to a subject, who has been exposed to radiation, is at risk of exposure to radiation, or has a condition or disease arising from radiation exposure, with the purpose to cure or improve the condition or disease.

[0083] A CLK2 inhibitor may be administered as a single dose or as multiple doses. In some embodiments, a CLK2 inhibitor is administered as a prophylactic to a subject before exposure to radiation. For example, a CLK2 inhibitor may be administered to a subject as a prophylactic within 96 hours of being exposed to radiation (e.g., within 96 hours of an anticipated exposure to radiation). In some embodiments, a CLK2 inhibitor is administered within about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day.

[0084] In some embodiments, a CLK2 inhibitor is administered as a treatment to a subject after exposure to radiation. For example, a CLK2 inhibitor may be administered to a subject as a treatment within about 12 hours, about 24 hours, about 36 hours, or about 48 hours. In some embodiments, a CLK2 inhibitor is administered without about a week or about a month after exposure to radiation.

[0085] CLK2 inhibitors provided herein may include or may be administered in combination with other agents, such as chemotherapeutic or radiotherapeutic agents. In some embodiments, a CLK2 inhibitor, as disclosed herein, is administered in an amount effective for reducing toxicity to cells, such as reducing toxicity to endothelial cells. In some embodiments, an increased number of cells and decreased cell death (e.g., apoptosis), relative to a baseline or control, indicates reduced toxicity to cells. A baseline or control may be, for example, a subject prior to radiation exposure, a subject exposed to radiation prior to receiving an amount of CLK2 inhibitor, or a subject exposed to radiation prior to receiving an additional amount of CLK2 inhibitor. Subjects and Conditions

[0086] A subject is an individual who has been exposed to or is at risk of being exposed to radiation (e.g., ionizing radiation). A subject, in some embodiments, is a mammalian subject. For example, a mammalian subject may be a human. In some embodiments, a subject is a non-human primate, e.g., a laboratory animal such as a rhesus monkey. Other examples of laboratory animals include rodents, such as rats and mice. In some embodiments, a subject treated with a CLK2 inhibitor has already been exposed to radiation, while in other embodiments, a CLK2 inhibitor is administered to a subject in anticipation of radiation exposure.

[0087] A subject has been “exposed” to radiation if the subject comes into contact with a source of radiation or comes within the vicinity of a source of radiation. The vicinity of a source of radiation can be calculated as any distance that would result in a subject being exposure to a dose in excess of 1.25 rem per calendar quarter to the whole body, 18.75 rem per calendar quarter to the hands, forearms, feet, or ankle, or 7.5 rem per calendar quarter to the skin of the whole body. A “rem” is the equivalent dose of radiation, which represents the equivalent biological effect of the deposit of one hundred ergs or one rad of X-rays or gamma rays. This distance can be calculated, for example, by using the inverse square law, to calculate the change in dose rate when a person moves farther or closer to a point source of radiation.

[0088] A subject is considered “at risk of exposure” to radiation, for example, if the subject plans to undergo a medical procedure involving radiation therapy, works or regularly spends time within the vicinity of a source of radiation, or plans to travel to region within the vicinity of a source of radiation. Professionals at risk of radiation exposure include, for example, healthcare professionals (e.g., radiologists and radiologic technologists, nuclear medicine technologists, and radiation therapists), nuclear power plant workers (e.g., engineers and technicians), research scientists (e.g., biologists and medical researchers, aviation industry professionals (e.g., airline pilots and cabin crew), military personnel (e.g., nuclease submarine crew and radiologic technicians), mining and industrial workers (e.g., uranium miner and industrial radiographers), and emergency response teams (e.g., hazmat teams and firefighters).

[0089] There are various forms of radiation to which a subject may be exposed, including ionizing radiation and non-ionizing radiation. Ionizing radiation has enough energy to remove tightly bound electrons from atoms, creating ions in the process. This type of radiation poses a significant risk to living tissues. Examples of ionization radiation include alpha particles, beta particles, gamma rays, x-rays, and neutron radiation. Non-ionizing radiation has less energy and cannot remove electrons from atoms. Although generally considered less harmful, excessive exposure to certain types of non-ionizing radiation can still pose risks. Examples of non-ionizing radiation include radio waves, microwaves, infrared radiation, visible light, ultraviolet radiation, and extremely low frequency (ELF) radiation.

[0090] A unit of ionizing radiation, referred to as a “Gray (Gy),” is the absorption of one joule or radiation energy per kilogram of matter and describes the amount of radiation absorbed by a subject. In some embodiments, a subject has been exposed to at least 0.01 Gy, at least 0.1 Gy, at least 0.25 Gy, at least 0.5 Gy, at least 0.75 Gy, at least 1 Gy, at least 2 Gy, at least 3 Gy, at least 4 Gy, at least 5 Gy, at least 6 Gy, at least 7 Gy, at least 8 Gy, at least 9 Gy, at least 10 Gy, at least 11 Gy, at least 12 Gy, at least 13 Gy, at least 14 Gy, at least 15 Gy, or at least 16 Gy. In some embodiments, a subject has been exposed to at least 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 Gray of radiation over a time frame of less than 1 minute, or up to 5 minutes, 10 minutes, 30 minutes, 60 minutes, 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, or 1 year. In some embodiments, the subject has been exposed to at least 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 Gray of radiation over a time frame of more than 1 month or 1 year.

[0091] In some embodiments, a subject is exposed to or expected to be exposed to wholebody radiation (no portion of the subject is shielded from receiving penetrating radiation). In other embodiments, a subject is exposed to or expected to be exposed to partial-body radiation (shielding of sufficient thickness blocks a portion of the subject from receiving penetrating radiation).

[0092] In some embodiments, a subject has undergone or is expected to undergo radiotherapy. Non-limiting examples of radiotherapies include external beam radiation therapy, such as three-dimensional conformal radiation therapy (3D CRT), intensity modulated radiation therapy (IMRT), image guided radiation therapy (IGRT), stereotactic radiation therapy, intraoperative radiation therapy, proton beam therapy, and neutron beam therapy. In some embodiments, the radiotherapy is a stereotactic radiation therapy. Nonlimiting examples of stereotactic radiation therapies include stereotactic radiosurgery, stereotactic body radiation therapy, and stereotactic ablative radiotherapy, including those that utilize Axesse, CyberKnife, Gamma Knife, Edge, Novalis, Primatom, Synergy, X-Knife, TomoTherapy, Trilogy, Truebeam, Versa HD, and View Ray machines.

[0093] A subject, in some embodiments, has a condition arising, directly or indirectly, from or suspected of arising from radiation exposure. Acute radiation syndrome (ARS) is one example of such a condition. ARS, also known as radiation sickness or radiation poisoning, is an illness that occurs when the body is exposed to a high dose of penetrating ionizing radiation within a short period of time, typically minutes to days. The severity of ARS is directly related to the dose and type of radiation, the duration of exposure, and the specific organs exposed. In some embodiments, acute radiation syndrome is caused by exposure to at least 1 Gray of radiation within less than an hour. The stages of ARS include: (1) the prodromal stage, occurring within minutes to days after exposure with symptoms presenting as nausea, vomiting, diarrhea, and fatigue; (2) the latent stage, which is a period of apparent well-being lasting from hours to several weeks, where the initial symptoms may disappear; (3) the manifest illness stage, where the severe symptoms associated with the specific type of ARS appear, including fever, malaise, infection, bleeding, dehydration, and shock, depending on the specific syndrome (e.g., hematopoietic, gastrointestinal, cardiovascular / central nervous system); and (4) recovery, which may take several weeks to months, or death within days to weeks if the exposure is severe.

[0094] Another example of a condition arising, directly or indirectly, from or suspected of arising from radiation exposure is chronic radiation syndrome. Chronic radiation syndrome is caused by months or years of chronic exposure to ionizing radiation. In some embodiments, a subject with chronic radiation exposure has been exposed to between 0.5 to 2.0 Gray of radiation at a dose rate of at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 1, at least 1.5, or at least 2 Gray per year. In some embodiments, the condition involved what is referred to as Delayed Effects of Acute Radiation Exposure (DEARE). DEARE refers to the health consequences that appear weeks, months, or even years after the initial radiation exposure. While ARS focuses on the immediate impact of high-dose radiation exposure, DEARE considers the long-term health implications. These effects are particularly concerning because they can occur after a period of apparent recovery from the initial exposure. Nonlimiting examples of DEARE include cancer / leukemia, cataracts, infertility and reproductive issues, organ damage, chronic dermatitis, cognitive impairment, weakened immunity, fibrosis, radiation cystitis, mucositis, and proctitis, which may occur months to years after an acute exposure to radiation. Radiation exposure can be measured using various methods and devices, depending on the type of radiation and the specific circumstances, known to those of skill in the art. Nonlimiting methods and devices for measuring radiation exposure include dosimeters, Geiger- Muller counters, scintillation detections, ionization chambers, survey meters, spectrometers, environmental monitoring, biological dosimetry, radiography, and imaging. Those of skill in the art will know that different types of radiation require different measurement techniques, instruments, and that the units of measurement for radiation exposure can vary.

[0095] In some embodiments, a subject has also undergone or is expected to undergo chemotherapy (e.g., curative, adjuvant, neoadjuvant, or palliative chemotherapy).

[0096] Cells

[0097] Aspects of the present disclosure provide a method of reducing toxicity to cells comprising administering a therapeutically effective amount of CLK2 inhibitor to a subject in need thereof. The present disclosure also includes methods of contacting cells with a CLK2- inhibitor in an amount sufficient to decrease cell death (e.g., apoptosis). Contacting refers to the process of exposing cells to a CLK2 inhibitor, for example, by culturing the cells in the presence of the CLK2 inhibitor or by administering the CLK2 inhibitor to a subject in need thereof. In some embodiments, the cells are endothelial cells.

[0098] In some embodiments, the contacting is performed ex vivo. In some embodiments, the ex vivo contacting is performed in cells or cell cultures. In some embodiments, the cells or cell cultures are human, mouse, rabbit, goat, sheep, pig, or non-human primate cells or cell cultures. In some embodiments, the cells or cell cultures are endothelial cells or endothelial cell cultures. In some embodiments, the endothelial cells are primary human umbilical vein (HUVEC), small intestinal microvascular (HSIMEC), or microvascular lung endothelial (HmVEC-L) cells. In some embodiments, the contacting is performed in vivo. In some embodiments, the in vivo contacting is performed in a subject. In some embodiments, the in vivo contacting is performed in a human, mouse, rabbit, goat, sheep, pig, or non-human primate. In some embodiments, the in vivo contacting comprises administering the CLK2 inhibitor to the subject. In some embodiments, the CLK2 inhibitor is administered to the subject intravenously or orally.

[0099] In some embodiments, the present disclosure provides methods of reducing toxicity to endothelial cells of a subject exposed to radiation comprising administering a therapeutically effective amount of CLK2 inhibitor to the subject, thereby reducing toxicity to endothelial cells. Endothelial cells are cells that line the interior surface of blood vessels and play a crucial role in maintaining vascular health and function. In some embodiments, an endothelial cell is a primary dermal microvascular endothelial cell (HDMVEC), a primary pulmonary artery endothelial cell (HPAEC), a primary umbilical vein endothelial cell (HUVEC), a primary aortic endothelial cell (HAEC), a primary coronary artery endothelial cell (HCAEC) a small intestinal microvascular cell (HSIMEC), a primary cardiac microvascular endothelial cell (HMVEC-C), a primary iliac artery endothelial cell (HIAEC), a primary bladder microvascular endothelial cell (HMVEC-Bd), a primary valvular interstitial cell (hVIC), or a microvascular lung endothelial cell (HmVEC-L). In some embodiments, an endothelial cell can be characterized using an endothelial cell marker. Non-limiting examples of endothelial cell markers include CD13, CD29, CD31, CD34, CD36, CD39, CD44, CD47, CD54, CD61, CD62E, CD62P, CD80, CD86, CD93, CD102, CD105, CD106, CD112, CD117, CD121a, CD141, CD142, CD143, CD144, CD146, CD147, CD151, CD160, CD201, CD213a, CD248, CD309, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM 17, ADAM33, ADAMTS-13, ADAMTS-18, CXCL16, DCBLD2, Endomucin, ESAM, FABP, IgG, VLA-4, KLF4, LYVE-1, Notch, Podocalyxin, Podoplanin, RLIP76, Stabilin-1, Stabilin-2, TEM8, THSD1, Tie-1, Tie-2, TNAP, TNF RII, VE-cadherin, VE-statin, VG5Q, and VWF.

[0100] Measuring Cell Toxicity

[0101] Aspects of the present disclosure provide a method of reducing toxicity to cells. In some embodiments, the toxicity to cells is the toxicity to endothelial cells. In some embodiments, endothelial cell toxicity can be measured by endothelial cell viability. In some embodiments, a decrease in endothelial cell viability, relative to a baseline or control, is an indicator of reduced endothelial cell toxicity. In some embodiments, a decrease in endothelial cell death, relative to a baseline or control, is an indicator of reduced endothelial cell toxicity. In some embodiments, the present disclosure provides methods of contacting endothelial cells that have been exposed to radiation with a CLK2 inhibitor in an amount sufficient to decrease endothelial cell death. Cell death can occur through several different mechanisms, such as apoptosis, necrosis, and pyroptosis. A control may be, for example, a subject prior to radiation exposure (baseline), a subject exposed to radiation prior to receiving an amount of CLK2 inhibitor, or a subject exposed to radiation prior to receiving an additional amount of CLK2 inhibitor. Apoptosis is a tightly regulated and highly controlled process of cell self-destruction. It serves several important functions in the body, including removal of unnecessary or damaged cells, tissue remodeling, immune system regulation, prevention of cancer, and homeostasis. Pyroptosis is another form of programmed cell death that plays a critical role in the innate immune response to infections, particularly in response to bacterial and certain viral infections. Unlike apoptosis, which is generally a non-inflammatory and non-disruptive form of cell death, pyroptosis is a highly inflammatory process that results in the release of pro- inflammatory molecules and the formation of pores in the cell membrane, leading to cell lysis and the release of cellular contents. Necroptosis is another form of programmed cell death that triggers an inflammatory response and involves the rupture of the cell membrane, leading to the release of cellular contents into the extracellular space.

[0102] Methods of assessing cell viability include dye exclusion assays (e.g., trypan blue staining) or fluorescence-based viability assays (e.g., propidium iodide staining). In some embodiments, endothelial cell viability is identified using an apoptosis marker (e.g., annexin), a pyroptosis marker (e.g., caspase 1, caspase 4, caspase 5, or caspase 11), a necroptosis marker (e.g., RIPK1, RIPK3), or a marker of DNA double-stranded breaks (e.g., y-H2AX or 53bpl). A “decrease” in endothelial cell death (characterized by an apoptosis marker, e.g., annexin; a pyroptosis marker, e.g., caspase 1, caspase 4, caspase 5, or caspase 11; a necroptosis marker, e.g., RIPK1 or RIPKL2; or a marker of DNA double- stranded breaks, e.g., y-H2AX or 53bpl) can be, for example, a decrease in endothelial cell apoptosis of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40 %, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, relative to a baseline or control. Methods of evaluating endothelial cell death such as immunofluorescent staining (e.g., fluorescent in situ hybridization, FISH) are known in the art.

[0103] Additional Embodiments

[0104] The disclosure also relates to the additional embodiments in the following numbered paragraphs:

[0105] 1. A method, comprising: administering a CLK2 inhibitor to a subject who has been or will be exposed to radiation. 2. The method of paragraph 1, wherein the subject has been exposed to radiation therapy.

[0106] 3. The method of paragraph 1 or 2, wherein the CLK2 inhibitor is administered in a therapeutically effective amount, optionally wherein the therapeutically effective amount reduces radiation toxicity in the subject.

[0107] 4. The method of any one of paragraphs 1-3, wherein endothelial cell number is increased in the subject, relative to baseline or control.

[0108] 5. The method of any one of paragraphs 1-4, wherein endothelial cell death is decreased, relative to baseline or control.

[0109] 6. The method of any one of paragraphs 3-5, wherein the therapeutically effective amount reduces radiation toxicity to endothelial cells, optionally selected from umbilical vein endothelial cells, small intestine microvascular endothelial cells, and lung endothelial cells.

[0110] 7. The method of any one of paragraphs 1-6, wherein the method is used as an adjunctive therapy to chemotherapy or radiotherapy.

[0111] 8. A method, comprising: administering a therapeutically effective amount of a CLK2 inhibitor to a subject in need thereof, wherein the subject has a condition arising from radiation exposure, and optionally wherein the therapeutically effective amount is sufficient to reduce radiation toxicity in the subject.

[0112] 9. The method of paragraph 8, wherein the condition arising from radiation exposure is acute radiation syndrome (ARS), an acute radiation subsyndrome associated with one or more of the blood (hematopoietic syndrome, H-ARS), the immune system, the gastrointestinal tract (gastrointestinal syndrome, GI-ARS), the skin (cutaneous syndrome), the pulmonary system (pulmonary syndrome), the kidney, or the central nervous system (neurovascular syndrome), cutaneous radiation injury, acute respiratory syndrome, or cancer.

[0113] 10. The method of paragraph 8 or 9, wherein the subject has been exposed to at least 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 Gray of radiation.

[0114] 11. The method of any one of paragraphs 8-10, wherein the subject has been exposed to the radiation over a time frame of less than 1 minute, or up to 5 minutes, 10 minutes, 30 minutes, 60 minutes, 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, or 1 year. 12. The method of any one of paragraphs 8-11, wherein the CLK2 inhibitor is administered at least once a day for 1, 2, 3, 4, 5, 6, or 7 days.

[0115] 13. The method of any one of paragraphs 8-12, wherein the subject is exposed to radiation before administration of the CLK2 inhibitor, during administration of the CLK2 inhibitor, after administration of the CLK2 inhibitor, or combinations thereof.

[0116] 14. A method, comprising: contacting endothelial cells exposed to radiation with a CLK2 inhibitor in an amount sufficient to decrease cell death, relative to a control.

[0117] 15. The method of paragraph 14, wherein the endothelial cells are umbilical vein endothelial cells, small intestine microvascular endothelial cells, or lung endothelial cells.

[0118] 16. The method of any one of paragraphs 1-15, wherein the radiation is ionizing radiation.

[0119] 17. The method of any one of paragraphs 1-16, wherein the radiation is whole body radiation or partial radiation.

[0120] 18. The method of any one of paragraphs 1-17, wherein the radiation is chemotherapy or radiotherapy.

[0121] 19. The method of any one of paragraphs 1-19, wherein the CLK2 inhibitor is selected from Cirtuvivint, Lorecivivint, Silmitasertib, TG003, SM08502, SM04755, SM09419, Cpd-2, Cpd-3, T3, CC-671, T-025, NR9, DB18, ML106, MU1210, KuWall51, CX-4945, acrifoline, CaNDY, leucettine, polyandrocarpamines A (PAC 12), ML315, KH- CB19, ML167, or CTX-712, preferably Cirtutivint, Lorecivivint, or TG003.

[0122] 20. The method of any one of paragraphs 1-19, wherein the CLK2 inhibitor is administered intravenously or orally.

[0123] EXAMPLES

[0124] To identify radioprotective therapeutic targets, kinome-wide CRISPR / Cas9 screens were performed in endothelial cells of different organ origins. These screens led to the identification of CDC-like kinase 2 (CLK2) as a shared and druggable target for protection of endothelial cells against acute radiation injury, and CLK2 inhibition using the small molecule inhibitors, TG003 and Cirtuvivint, protected cultured endothelial cells against acute radiation injury. Transcriptomic analysis indicated that these treatments reversed radiation-induced changes in gene expression and pathways associated with cell-cycle arrest, inflammation, and leukocyte-endothelial cell surface interactions. Administration of CLK2 inhibitors to a microfluidic human organ-on-a-chip (Organ Chip) models of acute radiation injury in both intestine and lung lined by organ- specific primary epithelial cells and vascular endothelium resulted in protection of the epithelium against radiation-induced tissue injury. These findings demonstrate the utility of combining kinome-wide CRISPR screens and Organ Chips to identify therapeutic targets and enable drug repurposing.

[0125] Example 1

[0126] Kinome-Scale CRISPR Screening in Primary Human Endothelial Cells

[0127] CRISPR screens in primary cells are often limited by the quantity of cells available and thus, it is desirable to minimize the time in culture required to transduce these cells. All- in-one vectors, which contain both CRISPR / Cas9 and an sgRNA of interest in a single construct, reduce the time and selection pressure required to transduce a cell population of interest, but suffer from poor signal-to-noise expression of reporters in many cell types. To carry out CRISPR screens in primary human endothelial cells, we cloned the recently described mGreenLantern protein in place of GFP in the LentiCRISPRv2-GFP vector (LCv2- GL)(data not shown), which provided superior signal-to-noise as compared to LentiCRISPRv2-GFP (data not shown) and resulted in transduction with high efficiency in three different types of primary human endothelial cells — human small intestine microvascular endothelial cells (HSIMEC), human lung microvascular endothelial cells (HPMEC), and human umbilical vein endothelial cells (HUVEC) (data not shown). To test the ability of this vector to induce a gene knockout in primary endothelial cells, HSIMEC were transduced with lentiviruses containing LCv2-GL with an sgRNA against TP53 (data not shown). At 7 days post- transduction, but not 4 days, we observed nearly complete knockout in transduced cells by Tracking of Indels by Decomposition (TIDE), but not in control transduced cells, confirming that this system is sufficient to induce indel formation in primary endothelial cells.

[0128] To identify possible therapeutic targets in these primary cells, we targeted the human kinome, composed of kinases that are modulated by at least 243 kinase inhibitors previously described in clinical literature. Using sgRNA sequences from the Brunello Human Kinome library, we cloned 3152 sgRNAs targeting 763 kinases, with 4 guides per gene and 100 nontargeting guide sequences into LCv2-GL. The sgRNA sequences in our library were even, with a skew ratio between the 90thand 10thpercentile sgRNAs <1.5 (data not shown). To determine the parameters of our screen, we tested a range of doses of radiation for their effects on human endothelial cell survival (Figure 1A). To be as stringent as possible while allowing sufficient cell coverage after the screen, we selected 4 Gy of radiation, as -15% of two different types of endothelial cells (HSIMEC and HUVEC) survived at this dose (Figure 1A). For HPMEC, 16 Gy was selected based on our lab’s previous work showing this as the optimal radiation dose to produce endothelial injury in human Lung Chips.

[0129] Orthogonal Kinome-Scale CRISPR Screens Identify Potential Novel Radiation Countermeasures

[0130] We next performed kinome-wide screens in HSIMEC, HPMEC, and HUVEC by exposing the cells to 4, 4, and 16 Gy of ionizing radiation for several hours, respectively, 4 days after transduction with lentiviruses, while control cells in similar cultures were not exposed to radiation (Figure IB). Each type of endothelial cell was treated as an independent condition in our design matrix (data not shown), and we identified highly overlapping sets of significantly altered genes that were shared by the different endothelial tissue types (Figure 1C). We also observed a strong correlation between effect sizes and statistical significance across all endothelial subsets tested (Figure IE) both after ionizing radiation and in control cultures; using MAGeCK-MLE, a model for pan-endothelial response to potential therapies was constructed based on this data (Figure IE, bottom graph).

[0131] As endothelial cells of differing tissue origin displayed similar kinome-wide responses, we performed an integrated analysis, stratifying only by irradiation status. To prioritize kinase inhibitors for therapeutic testing, we sought to identify kinases whose loss was enriched in surviving cells following treatment with ionizing radiation, but not in control conditions, because these could represent potential targets for repurposing of existing kinase inhibitors. We also used the MAGeCK Flute pipeline to normalize against previously reported sgRNA effects on cell survival (data not shown). The Angiopoietin- 1 (Ang-1) pathway has previously been described to play an essential role in endothelial cell recovery following acute radiation injury. In line with these reports, our data identified the receptor for Ang-1, TEK, as a dropout in our screen (Figure ID). Interestingly, we identified CLK2 loss as having the largest pan-endothelial enrichment (Figure ID); CLK2 loss showed the most positive effect on endothelial cell survival as compared to all other human kinases. When ranking kinases by radiation- specific effects on endothelial proliferation, CLK2 was among the highest ranked kinases (Figure ID, right), while TEK was found within the lowest ranked kinases. However, this was surprising because past work has shown that CLK2 overexpression, rather than loss, inhibits radiation-induced cell death in cancer-derived HeLa cells. To further confirm the results of our screen, we performed GSEA analysis using the Reactome annotation and found that sgRNAs in pathways necessary for endothelial proliferation, including MAPK- and VEGF-rclatcd pathways, were depleted in our screens (data not shown). Following treatment with acute ionizing radiation, G2 / M cell-cycle related terms were found to be dropouts as well (data not shown). We therefore concluded that the results of our screen warranted follow-up with therapeutic testing in cancer cells versus primary endothelial cells.

[0132] Example 2

[0133] CLK2-targeting Compounds as Selective Radioprotective Therapeutics for Endothelium

[0134] Damage to the vascular endothelium following treatment with ionizing radiation is associated with adverse patient outcomes via a variety of mechanisms. Fortuitously, many small-molecule inhibitors of CLK2 have already been described. Investigation of publicly available expression data indicated that CLK isoforms are well-expressed, particularly in endothelium-rich tissues (data not shown). To investigate whether CLK2 inhibitors could function as effective radiation countermeasures, we tested their ability to protect endothelial cell proliferation following treatment with ionizing radiation. As countermeasures for both military and civilian biodefense applications likely to be administered to patients retroactively, we treated cells with compounds 2 hours after treatment with ionizing radiation. We observed that, at multiple levels of ionizing radiation, CLK2 inhibition using the previously described CLK2 inhibitor TG003 significantly increased HUVEC cell numbers in a dose-dependent fashion (Figure 2A, left). Similar effects were observed with Cirtuvivint, which inhibits CLK2 more selectively than other CLK proteins (Figure 2A, right). Importantly, similar effects were observed in HSIMECs, with TG003 increasing cell numbers as compared to DMSO controls following treatment with ionizing radiation (Figure 2B).

[0135] We then sought to address whether these changes could be due in part to a reduction in ionizing-radiation induced apoptosis. Indeed, we detected a significant reduction in the frequency of endothelial cells positively stained with Annexin V, a dye indicating cellular apoptosis, by flow cytometry (Figure 2C) following treatment with TG003, Cirtuvivint, or Lorecivivint, another small molecule inhibitor of CLK2. Endothelial cells are known to undergo hypertrophy following exposure to ionizing radiation; in line with this, we observed a radiation dose-dependent increase in cell size, as measured by forward scatter area by flow cytometry (Figure 2D). Interestingly, TG003 treatment significantly prevented the radiation exposure induced increase in cell size within 24 hours at all radiation doses tested.

[0136] Finally, as the production of ROS is a key phenomenon in the development of radiation-induced endothelial cell injury, we hypothesized that CLK2 inhibition could promote clearance of ROS. Indeed, staining with CellROX fluorescent dye 24 hours postirradiation reduced ROS to untreated levels (Figure 2E). We therefore conclude that CLK2 inhibition may represent a viable therapeutic strategy to protect the vascular endothelium against radiation injury.

[0137] Another key application of radiation countermeasures is the mitigation of side effects to patients receiving radiotherapy as cancer therapy, and CLK2 overexpression has been previously shown to protect against radiation damage in HeLa cancer cells as described above. This is the opposite what we observed with healthy endothelial cells. However, when we re-analyzed publicly available data from Project Achilles, an atlas of forward genetic screens across the cancer cell line encyclopedia (CCLE), we found that CRISPR knockouts of CLK2 using the same library as we used here resulted in tumor cell killing (data not shown). Additionally, we analyzed available datasets from the PRISM assay, which assesses the effects of compound treatments on a pool of barcoded cell lines. These results showed that treatment with TG003 either inhibited or had no effect on proliferation of cancer cell lines. We confirmed these results experimentally by treating the tumor-derived Caco-2 intestinal epithelial cell line with ionizing radiation in the presence or absence of TG003, where we observed no statistically significant change in cell numbers in irradiated cultures (data not shown).

[0138] Given the observed a reduction in cellular ROS levels, it is possible that CLK2 inhibition could reverse downstream consequences of exposure to ionizing radiation. We therefore assessed the formation of gH2AX and 53bpl foci (a marker of double- stranded DNA breaks) within endothelial cell nuclei, with and without CLK2 inhibition. Interestingly, CLK2 inhibition reduced the numbers of both gH2AX and 53bpl foci per cell, hencing suppressing DNA damage in HSIMECs 6 hours post-irradiation (Figure 2F). To confirm that the effects of TG003 were mediated by CLK2 inhibition, we transduced HUVECs with 4 sgRNAs targeting CLK2 or a non-targeting control. Loss of CLK2 via genetic inhibition also promoted cell survival following exposure to ionizing radiation, while treatment with TG003 showed no additional benefit, confirming inhibition of CLK2 activity as the mechanism of action (data not shown). Importantly, the reduced levels of ROS and double- stranded breaks produced by treatment with TG003 were also accompanied by a reduction in endothelial cellleukocyte adhesion 24 hours post-irradiation (Figure 2G).

[0139] Example 3

[0140] CLK2 Inhibition Reverses Radiation-Induced Changes to the Transcriptome

[0141] To understand the global effects of CLK2 inhibition on endothelial cell function following exposure to ionizing radiation, we performed RNA-sequencing of radiation treated and untreated cells, with and without TG003. We identified 923 differentially regulated genes (485 upregulated and 438 downregulated) when we compared the 0 Gy and 4 Gy controls, while there were 891 upregulated and 1474 downregulated genes when the TG003 and DMSO treatment groups exposed to 4 Gy ionizing radiation were compared. Many of the most upregulated genes between the 4 Gy and 0 Gy conditions were also downregulated in 4 Gy TG003 versus DMSO conditions (data now shown). To quantify this relationship, we plotted the effect sizes of all genes comparing 4 Gy TG003 versus DMSO to 0 Gy versus 4 Gy DMSO. We observed a negative correlation (Pearson’s R = -0.555, p < 2.2* 10'16) in fold changes across all identified transcripts (Figure 3A), indicating a reversal of radiation- associated transcriptomic changes. In agreement with this observation, principal component analysis (PCA) of these data showed that irradiated TG003-treated samples associated more closely with control unirradiated samples than untreated irradiated samples (data now shown).

[0142] We next sought to understand if these global changes in gene expression were associated with relevant biological responses by performing Gene Set Enrichment Analysis (GSEA) using the Reactome annotation. This analysis revealed a negative normalized enrichment score (NES) for terms relating to Cell Cycle driven by many well-characterized proliferation-related genes (CCNA2, CCNB2, CCNB1, CDCA8, CDK1) in irradiated versus non-irradiated cells (Figure 3A-B). In addition, we observed a positive NES of terms relating to an active inflammatory response (Figure 3B, left) driven by many known inflammatory mediators (IRF9, IFIT1, HLA-F, OAS2, HLA-B, IFIT2, JAK1, STAT1, STAT2, ISG15) (Figure 3 A, right), which is consistent with reports that ionizing radiation induces an inflammatory response in endothelial cells. We also observed Reactome terms relevant to extracellular matrix organization, in line with a role of radiation as a driver of tissue remodeling. Importantly, when the same analysis was carried out comparing cells treated with CLK2 inhibitor versus control in the presence of ionizing radiation, we detected a reversal of radiation-associated Reactome terms across the transcriptome (Figure 3B, right) in agreement with the gene level changes we observed (data now shown). Indeed, in line with our experimental data modulating CLK2 activity, we observed reversal of the injury phenotype as indicated by a positive enrichment of Cell Cycle-related terms (Figure 3B, right). In agreement with our observation that CLK2 inhibition reduces endothelial-leukocyte adhesion post-irradiation, we observed a down-regulation of terms related to immune / inflammatory pathways and cell surface interactions, in particular 1CAM1 (Figure 3A-B). Intriguingly, we observed a negative NES score for pathways relating to extracellular matrix organization, indicating that CLK2 inhibition could possibly mitigate this damaging effect of acute radiation exposure as well (Figure 3B, right).

[0143] CLK Inhibition Reverses Radiation-Induced Alterations via NF TC4

[0144] To understand the protein-protein interactions which could mediate the radioprotective effect of CLK2 inhibition, we performed phospho-proteomic analysis of cells treated with and without ionizing radiation, and with and without TG003, 24 hours postirradiation. In agreement with our RNA-seq results, TG003 treatment strikingly reversed (Pearson’s R: -0.935, p < 0.0001) proteome-wide changes in protein phosphorylation induced by exposure to ionizing radiation (Figure 3C). Notably, we observed a radiation-dependent increase in phosphorylation of NFATc4, a member of the NF AT family of transcription factors, which was reversed by treatment with TG003 (Figure 3C). While NFATc4 has not been previously suggested to contribute to radiation sensitivity in any cell type, NF AT family members are known to mediate VEGF-dependent growth signaling in endothelial cells where activation of this pathway results in dephosphorylation of NF AT. We therefore hypothesized that VE47c4-dcpcndcnt VEGF signaling could be disrupted by exposure to ionizing radiation in a CLK2-dependent manner. In agreement with previous results, we observed increased CLK2 phosphorylation after exposure to ionizing radiation, which was inhibited by treatment with TG003 (data not shown). Further, Western blotting revealed a radiation-induced increase in NFATc4 phosphorylation, which was consequently reduced via TG003 treatment in HUVEC exposed to ionizing radiation for 24 hours (data not shown). Our data support a model where ionizing radiation inhibits NF AT phosphorylation and thereby interferes with VEGF-dependent signation of endothelial cell growth and viability (Figure 3D).

[0145] Example 4 CLK Inhibition Protects Against Radiation Injury in Human Intestine and Lung Chips

[0146] We next sought to understand if our observations with cultured endothelial cells were relevant in a human tissue- and organ-relevant context. Our group and others have previously demonstrated a dependency of intestinal epithelial cell radiosensitivity on the presence of microvascular endothelial cells. We utilized our previously described two-channel microfluidic human Intestine Chip that is lined by a highly differentiated villus intestine epithelium composed of primary human ileum organoid-derived cells interfaced with primary HSIMEC across a porous membrane to test if CLK2 inhibition would protect against functional consequences of ionizing radiation exposure in the intestinal epithelium (Figure 4A). When TG003 was flowed through the basal vascular (endothelium- lined) channel 2 hr following exposure to ionizing radiation to simulate intravenous administration of this compound in a therapeutic context, we found that CLK2 inhibition using this compound reduced epithelial and endothelial cell loss (Figure 4B, left and center) and protected against blunting of the intestinal epithelial villi caused by exposure to 4 Gy ionizing radiation 7 days earlier (Figure 4B, right). Immunofluorescence microscopic analysis also confirmed that TG003 treatment reversed radiation-induced loss of ZO-1 containing tight junctions in the intestinal epithelium, which are critical for maintenance of intestinal barrier function (data not shown). Moreover, in accordance with data from our in vitro cultures with cultured endothelial cells alone, TG003 treatment reversed radiation-induced increases in cytokine levels in the effluents the vascular channels of the human Intestine chips (Figure 4C). Thus, CLK2 inhibition of the endothelial response to radiation exposure can protect human intestinal epithelial tissues from injury in a more organ-relevant context.

[0147] To understand if this therapeutic strategy was generalizable to other highly vascularized and radiosensitive tissues, we utilized our previously described Fung Chip model of radiation injury. Similar to results on epithelial cell-cell adhesions observed in the Intestine Chip, TG003 treatment ameliorated radiation-induced loss of VE-Cadherin expression and organization within the endothelium (Figure 4D). Further, TG003 treatment reduced adhesion of PBMCs to the vascular endothelium following exposure to ionizing radiation (Figure 4E). Protection against radiation injury was also evidenced by maintenance of increased numbers of viable pulmonary epithelial cells (data not shown). In addition, we found that TG003 decreases production of radiation-induced cytokines IP-10, 11-6, 11-8, and MCP-1 in the vascular channel of irradiated Fung Chips, similar to our results from the Intestine chip (Figure 4F). Taken together, these results indicate that pharmacologic CLK2 inhibitors potentially could be repurposed as radioprotectants in multiple tissues and organs.

[0148] Methods

[0149] Cell Culture and Radiation Assays

[0150] Human umbilical vein endothelial cells (HUVEC) were cultured in EGM-2 (Lonza), while human small intestine microvascular endothelial cells (HSIMEC) and human pulmonary microvascular endothelial cells (HPMEC) were cultured in EGM-2 MV (Lonza). Cells were cultured for a maximum of 7 passages to maintain cellular phenotypes. For radiation assays in cultured cells, cells were irradiated in a GammaCell 80 Irradiator with a Cesium source. For radiation dose titrations, primary endothelial cells of different tissue origins were trypsinized and placed in a 15 mL conical tube prior to irradiation. Following radiation exposure, 5*104cells were seeded per well in 6 well dishes. Cells were then cultured for 7 days in respective culture medium prior to dissociation by trypsinization and analysis by flow cytometry. For cell counts, 5*103Spherotech counting beads were added per sample prior to assessment. For assays involving treatment of cells with compounds, compounds were diluted in DMSO to 1000X stock concentrations prior to treatment, then diluted in cellular medium to respective working concentrations as indicated at 2 hours postirradiation. To assess intracellular ROS formation, cells were treated for 30 minutes with 2.5 nM CellROX Deep Red 24 hours post-irradiation, then washed with PBS prior to trypsinization and assessment of MFI by flow cytometry. To assess cell swelling, forward scatter area measurements were collected alongside cell counts at 7 days post-irradiation. Similarly, to assess apoptosis, cells were stained with Annexin V (ThermoFisher) according to the manufacturer’s instructions alongside DAPI for live / dead analysis 7 days postirradiation, prior to analysis by flow cytometry.

[0151] Vector Design, Library cloning, and Lenti virus Production

[0152] Human Kinome CRISPR pooled library sgRNA spacer sequences were selected from the previously described Brunello library. The LentiCRISPRv2-mGreenLantem vector was cloned from the previously described LentiCRISPRv2-GFP vector (Addgene #82416) by replacing GFP with the mGreenLantem ORF (Addgene # 161912) via restriction enzyme cloning. Upon confirmation of the new vector sequence, sgRNAs were purchased as pooled oligonucleotides from Twist Bioscience with 40 bp overlap on either side of the sgRNA spacer sequence to the LentiCRISPRv2-mGreenLantem vector. The sgRNAs were then amplified via PCR and inserted into the vector via Gibson cloning, as previously described (Joung et al). To confirm even distribution of sgRNA sequences, spacer sequences were amplified and prepared for next generation sequencing (NGS) by PCR as previously described (Joung et al). Ten different forward primer sequences were used to increase the library diversity for sequencing.

[0153] For CRISPR screens in primary human endothelial cells, lentivirus was produced by transfection of HEK293T cells via Lipofectamine 3000 as previously described. Cells were cultured to 70% confluency in T75 flasks prior to transfection with 13 ug transfer plasmid, 13 ug psPAX2 (Addgene # 12260), and 6.5 ug pCMV-VSVG (Addgene #8454). After 24 hours, medium was changed and further cultured for an additional 48 hours, after which time the supernatant was harvested, filtered, and concentrated with Lenti-X Concentrator (Takara) according to the manufacturer’s instructions. Viral pellets were resuspended in PBS after centrifugation, then aliquoted and frozen at -80 C prior to use for tittering or screening.

[0154] Viral Titer and Kinome-scale CRISPR Screens

[0155] To titer concentrated lentivirus stocks, stocks were thawed from -80 and added to 50,000 HUVEC in 6 well dishes. Virus-treated cells were washed 24 hours after it was added to medium and HUVEC were cultured for another 72 hours, prior to assessment of mGreenLantern positivity by flow cytometry. Once viral MOI was determined, at least 5 * 106cells were transduced with concentrated viral stocks in 6 well plates at an MOI of ~0.3. Prior to irradiation, and four days following transduction, cells were pooled and viral titer and cell counts were confirmed. Cells were then re- seeded at appropriated densities in T75 flasks. This time point was chosen to confirm titer and irradiate cells as the levels of fluorescent protein were stable at subsequent time points, but editing efficiency was still low. Following irradiation at indicated doses, cells were cultured for another 7 days prior to harvesting. Typically, non-irradiated controls were passaged once as they would reach confluency by approximately days 3-4 post-mock irradiation. Due to the effects of ionizing radiation on cell proliferation, irradiated controls did not need to be passaged post-irradiation. Following collection of cells by trypsinization, cells were centrifuged at 300 x g for 5 minutes; supernatant was then aspirated and cells were stored as pellets prior to gDNA isolation.

[0156] Isolation of gDNA, Library Preparation, and Screen Analysis

[0157] Genomic DNA (gDNA) was isolated using the NEB Monarch gDNA Isolation Kit (NEB) according to the manufacturer’s instructions. Genomic DNA was then pooled for each condition prior to addition of Illumina adapters by PCR as previously performed during library cloning. Samples were barcoded using reverse primers (i7 indices) as described by Joung et al. After amplification of adapters was completed, PCR products were purified using the Qiaquick DNA Cleanup and PCR isolation kit (Qiagen). Samples were then run on a 2% E-Gel EX (ThermoFisher) prior to isolation of 250 bp bands using the NEB Monarch Gel Purification Kit (NEB) according to the manufacturer’s instructions. Following library isolation, DNA was quantified using the NEBNext Library Quant Kit for Illumina (NEB) according to the manufacturer’s instructions. Libraries were then diluted to 2 nM and sequenced on the Illumina Miseq platform using the 150 cycle Illumina Miseqv3 kit, such that each sample had at least 1000X reads per sgRNA in the library.

[0158] Screen data were analyzed using Galaxy EU. Read quality was assessed using FastQC, then trimmed using CutAdapt based on the presence of the CACG sequence at the start of each sgRNA spacer. Trimmed reads were then analyzed using the MaGeCK Count program prior to multivariate analysis using the MaGeCK MLE program. In all cases, nontargeting sgRNAs present in the library were used as controls to assess effect sizes. Results from this analysis were then imported into R in Galaxy and normalized using the MaGeCK- VISPR pipeline. In all cases, plasmid read counts were used as a baseline control. Data exported from MaGeCK MLE and MaGeCK VISPR were then imported into Graphpad for plotting. For pathway analyses, gene set enrichment analysis was performed on all genes in the kinome in Galaxy using the FGSEA program with the Reactome annotation.

[0159] Isolation of RNA, RNA-sequencing, and RNA-seq Analysis

[0160] RNA was isolated from cell pellets using the Monarch Total RNA Miniprep Kit (NEB) according to the manufacturer’s instructions. Prior to RNA isolation, cells were treated with compound for 7 days post-irradiation in 6 well dishes, as had been performed previously. Cell counts were assessed via flow cytometry prior to pelleting and isolation of the cells. RNA-sequencing was performed by Azenta. Messenger RNA was isolated by poly(A) selection. A minimum of 25M reads were collected via Illumina sequencing. For analysis of RNA-sequencing data, QC of raw reads was performed via FastQC prior to quantification via Salmon. Differentially expressed genes (DEGs) were then identified using DESeq2, as previously described. DEGs were then used to perform GSEA using the Reactome pathway annotation.

[0161] Leukocyte Adhesion Assays Prior to performing the leukocyte adhesion assay, peripheral blood mononuclear cells (PBMC) were first isolated by Ficoll separation, as previously described (Chou et al, 2020) and frozen in aliquots of 100M cells each. HUVEC were treated with irradiation or mock, then seeded in 6 well plates at a density of 2*105cells per well. Compound was then added as before at doses indicated at least 2 hours post-irradiation. The next day, PBMC were incubated with 2.5 nM CellTracker Deep Red Dye (ThermoFisher) for 30 minutes in PBS at a density of 106cells / mL. Cells were then centrifuged at 300 x g for 5 minutes, resuspended in PBS, and again centrifuged at 300 x g for 5 minutes. Cells were then resuspended to a density of 107cells / mL, such that 106cells could be added to each well. PBMC and HUVEC were then incubated for 30 minutes, prior to washing gently 4X with PBS. Images were then acquired using an ECHO Revolve microscope with a 2X objective using the Cy5 laser filter combination.

[0162] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0163] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0164] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0165] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0166] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

CLAIMSWhat is claimed is:

1. A method, comprising: administering a CLK2 inhibitor to a subject who has been or will be exposed to radiation.

2. The method of claim 1, wherein the subject has been exposed to radiation therapy.

3. The method of claim 1 or 2, wherein the CLK2 inhibitor is administered in a therapeutically effective amount, optionally wherein the therapeutically effective amount reduces radiation toxicity in the subject.

4. The method of any one of claims 1-3, wherein cell number is increased in the subject, relative to baseline or control.

5. The method of any one of claims 1-4, wherein cell death is decreased, relative to baseline or control.

6. The method of any one of claims 3-5, wherein the therapeutically effective amount reduces radiation toxicity to cells of the subject.

7. The method of any one of claims 1-6, wherein the method is used as an adjunctive therapy to chemotherapy or radiotherapy.

8. A method, comprising: administering a therapeutically effective amount of a CLK2 inhibitor to a subject in need thereof, wherein the subject has a condition arising from radiation exposure, and optionally wherein the therapeutically effective amount is sufficient to reduce radiation toxicity in the subject.

9. The method of claim 8, wherein the condition arising from radiation exposure is acute radiation syndrome (ARS), an acute radiation subsyndrome associated with one or more ofthe blood (hematopoietic syndrome, H-ARS), the immune system, the gastrointestinal tract (gastrointestinal syndrome, GI-ARS), the skin (cutaneous syndrome), the pulmonary system (pulmonary syndrome), the kidney, or the central nervous system (neurovascular syndrome), cutaneous radiation injury, acute respiratory syndrome, or cancer.

10. The method of claim 8 or 9, wherein the subject has been exposed to at least 0.01, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 Gray of radiation.

11. The method of any one of claims 8-10, wherein the subject has been exposed to the radiation over a time frame of less than 1 minute, or up to 5 minutes, 10 minutes, 30 minutes, 60 minutes, 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, or 1 year.

12. The method of any one of claims 8-11, wherein the CLK2 inhibitor is administered at least once a day for 1, 2, 3, 4, 5, 6, or 7 days.

13. The method of any one of claims 8-12, wherein the subject is exposed to radiation before administration of the CLK2 inhibitor, during administration of the CLK2 inhibitor, after administration of the CLK2 inhibitor, or combinations thereof.

14. A method, comprising: contacting cells exposed to radiation with a CLK2 inhibitor in an amount sufficient to decrease cell death, relative to a control.

15. The method of any one of claims 1-14, wherein the radiation is ionizing radiation.

16. The method of any one of claims 1-15, wherein the radiation is whole body radiation or partial radiation.

17. The method of any one of claims 1-16, wherein the radiation is chemotherapy or radiotherapy.

18. The method of any one of claims 1-17, wherein the CLK2 inhibitor is selected from Cirtuvivint, Lorecivivint, Silmitasertib, TG003, SM08502, SM04755, SM09419, Cpd-2, Cpd- 3, T3, CC-671, T-025, NR9, DB18, ML106, MU1210, KuWall51, CX-4945, acrifoline, CaNDY, leucettine, polyandrocarpamines A (PAC 12), ML315, KH-CB19, ML167, or CTX- 712, preferably Cirtutivint, Lorecivivint, or TG003.

19. The method of any one of claims 1-18, wherein the CLK2 inhibitor is administered intravenously or orally.

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