CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/019,637, filed May 4, 2020, and U.S. Provisional Patent Application No. 63/007,025, filed April 8, 2020, each of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Liver cancer is a type of cancer for which treatment may be difficult. Systemic treatments such as chemotherapies may be toxic and may have negative side effects on patients. Moreover, the lack of specific biomarkers can complicate development or use of targeted treatments.

[0003] One approach for treating cancer cells includes identifying target genes, or biomarkers, which identify which cancer cells may be sensitive to treatment with a given agent.

SUMMARY

[0004] As recognized herein, identifying synthetic lethal gene pairs, in which an inhibition or deletion of both genes leads to cell death, may be useful therapeutically in killing cancer cells while maintaining viability of non-cancer cells.

[0005] Recognized herein is a need for therapeutic agents for targeted treatments and therapies for treating a subject having or suspected of having a cancer, e.g., liver cancer. The subject having or suspected of having a liver cancer may have, in one or more cancer cells of the liver cancer, mutations or deletions, an increased or decreased expression level, or increased, decreased or otherwise altered activity level of a first gene compared to a healthy or non-cancer control. Disclosed herein are methods and compositions for treating a liver cancer or liver cancer cell or tissue having such an increased or decreased expression or increased, decreased or otherwise altered activity level of the first gene by causing a decrease in expression or activity of a second gene in the liver cancer or liver cancer cell, thereby treating the subject. The first gene and the second gene may form a synthetic lethal pair. The first gene may encode a protein important for the functioning of the tumor cell, e.g., one that regulates transcription, and the second gene may encode a gene target e.g., a kinase protein. [0006] In an aspect, disclosed herein is a method for treating a subject having or suspected of having a liver cancer, comprising administering to said subject a therapeutically effective amount of one or more therapeutic agents that causes a decrease in expression or activity of Aurora kinase A (AURKA) in a liver of said subject, thereby treating said subject for said liver cancer, wherein said liver comprises a cell that has a decreased expression or activity level of AT-rich interactive domain lA (ARIDlA).

[0007] In some embodiments, said one or more therapeutic agents comprise one or more members selected from the group consisting of a small molecule, a protein, a peptide, a ribonucleic acid (RNA) molecule, and, an endonuclease complex and a deoxyribonucleic acid (DNA) construct. In some embodiments, said DNA construct comprises an endonuclease gene. In some embodiments, said endonuclease gene encodes a CRISPR associated (Cas) protein. In some embodiments, said Cas is Cas9. In some embodiments, said DNA construct comprises a guide RNA targeting AURKA gene. In some embodiments, said endonuclease complex comprises an endonuclease. In some embodiments, said endonuclease is a CRISPR associated (Cas) protein. In some embodiments, said small molecule comprises an AURKA inhibitor.

[0008] In some embodiments, said AURKA inhibitor comprises 2-[3-[[7-[3-[ethyl(2- hydroxyethyl)amino]propoxy]quinazobn-4-yl]amino]-lH-pyrazol- 5-yl]-N-(3- fluorophenyljacetamide (Barasertib), 4- { [9-Chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4- d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid (Absertib), N-[5-[(2R)-2-methoxy-2- phenylacetyl]-4,6-dihydro-lH-pyrrolo[3,4-c]pyrazol-3-yl]-4-( 4-methylpiperazin-l-yl)benzamide (Danusertib), l-Cyclopropyl-3-(3-(5-(morpholinomethyl)-lH-benzo[d]imidazol -2-yl)-lH-pyrazol-4- yljurea (AT9283), N-[2-[(l S,4R)-6-[[4-(Cyclobutylamino)-5-(trifluoromethyl)-2- pyrimidinyl]amino]-l ,2,3,4-tetrahydronaphthalen-l ,4-imin-9-yl]-2-oxoethyl] -acetamide (PF 03814735), or N-(4-((3-(2-aminopyrimidin-4-yl)pyridin-2-yl)oxy)phenyl)-4-( 4-methylthiophen-2- yl)phthalazin-l -amine (AMG 900).

[0009] In some embodiments, the method further comprises administering to said subject a therapeutically effective amount of one or more therapeutic agents that causes a decrease in expression or activity of ARID 1 A. In some embodiments, the method further comprises administering to said subject a therapeutically effective amount of one or more therapeutic agents that causes a decrease in expression or activity of MYC. [0010] In another aspect, disclosed herein is a composition for treating a subject having or suspected of having a liver cancer, comprising a formulation comprising (i) at least one therapeutic agent and (ii) an excipient, wherein said at least one therapeutic agent is present in an amount that is effective to cause a decrease in expression or activity of Aurora kinase A (AURKA) following exposure to a liver of said subject, wherein said excipient is configured to (1) stabilize said at least one therapeutic agent or (2) provide therapeutic enhancement of said at least one therapeutic agent following exposure to said liver of said subject as compared to said at least one therapeutic agent being exposed to said liver of said subject in absence of said excipient, and wherein said liver cancer comprises a cell that has a decreased expression or activity level of AT-rich interactive domain 1 A (ARID 1 A).

[0011] In some embodiments, said at least one therapeutic agent comprise one or more members selected from the group consisting of a small molecule, a protein, a peptide, a ribonucleic acid (RNA) molecule, and, an endonuclease complex and a deoxyribonucleic acid (DNA) construct. In some embodiments, said DNA construct comprises an endonuclease gene. In some embodiments, said endonuclease gene encodes a CRISPR associated (Cas) protein. In some embodiments, said Cas is Cas9. In some embodiments, said DNA construct comprises a guide RNA directed to AURKA gene. In some embodiments, said endonuclease complex comprises an endonuclease. In some embodiments, said endonuclease is a CRISPR associated (Cas) protein. In some embodiments, said small molecule comprises an AURKA inhibitor.

[0012] In some embodiments, said AURKA inhibitor comprises 2-[3-[[7-[3-[ethyl(2- hydroxyethyl)amino]propoxy]quinazolin-4-yl]amino]-lH-pyrazol -5-yl]-N-(3- fluorophenyljacetamide (Barasertib), 4- { [9-Chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4- d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid (Absertib), N-[5-[(2R)-2-methoxy-2- phenylacetyl]-4,6-dihydro-lH-pyrrolo[3,4-c]pyrazol-3-yl]-4-( 4-methylpiperazin-l-yl)benzamide (Danusertib), l-Cyclopropyl-3-(3-(5-(morpholinomethyl)-lH-benzo[d]imidazol -2-yl)-lH-pyrazol-4- yljurea (AT9283), N-[2-[(l S,4R)-6-[[4-(Cyclobutylamino)-5-(trifluoromethyl)-2- pyrimidinyl]amino]-l ,2,3,4-tetrahydronaphthalen-l ,4-imin-9-yl]-2-oxoethyl] -acetamide (PF 03814735), or N-(4-((3-(2-aminopyrimidin-4-yl)pyridin-2-yl)oxy)phenyl)-4-( 4-methylthiophen-2- yl)phthalazin-l -amine (AMG 900). [0013] In some embodiments, said composition further comprises a formulation comprising at least one therapeutic agent present in an amount that is effective to cause a decrease in expression or activity of ARID 1 A.

[0014] In some embodiments, said composition further comprises a formulation comprising at least one therapeutic agent present in an amount that is effective to cause a decrease in expression or activity of MYC.

[0015] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0018] FIGS. 1A-C schematically show example signaling pathways for Aurora kinase A (AURKA).

[0019] FIG. 2 shows a table of percentages of mutations in AT-rich interactive domain 1A (ARID 1 A) in various cancer types. [0020] FIG. 3 schematically shows an example workflow for determining the effect of treatment of a population of cultured liver cancer cells with a nucleic acid molecule.

[0021] FIG. 4 schematically shows another example workflow for determining the effect of treatment of a population of cultured liver cancer cells that are deficient in a gene using a single guide RNA that can induce a mutation in a specific gene.

[0022] FIG. 5 shows a plot of frequency of ARID 1 A inactivation or deficiency in different cancer types.

[0023] FIG. 6 shows a plot of expression of AURKA in different cancer types.

[0024] FIG. 7 shows a plot of the expression level of AURKA in cancer versus normal cells in various cancer types.

[0025] FIG. 8 shows a scatterplot of expression level of AURKA in cancer (tumor) cells compared to normal cells.

[0026] FIG. 9 shows a scatterplot of expression level of AURKA in two populations of cells, having deficient ARID 1 A or having wild-type ARID 1 A.

[0027] FIG. 10 shows a plot of expression level of mutation occurrences of ARID 1 A compared to the expression level of AURKA in various cancer types.

[0028] FIGS. 11A-B show example of end-point growth data of a CRISPR-based approach to knock out AURKA and ARID 1 A in tumor cells, both individually and simultaneously.

[0029] FIGS. 12A-B show additional example of growth data of a CRISPR-based approach to knock out AURKA and ARID 1 A in tumor cells, both individually and simultaneously

DETAILED DESCRIPTION

[0030] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0031] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3. [0032] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0033] The term “subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human), reptile, or avian (e.g., bird), or other organism, such as a plant. For example, the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian or a human. The subject can be a healthy individual, an individual that is asymptomatic with respect to a disease (e.g., cancer, such as liver cancer), an individual that has or is suspected of having the disease (e.g., cancer, such as liver cancer) or a pre-disposition to the disease, or an individual that is symptomatic with respect to the disease. The subject may be in need of therapy. The subject can be a patient.

[0034] The term “genome,” as used herein, generally refers to genomic information from a subject, which may be, for example, at least a portion or an entirety of a subject’s hereditary information. A genome can be encoded in a deoxyribonucleic acid (DNA) molecule (s) and may be expressed in a ribonucleic acid (RNA) molecule(s). A genome can comprise coding regions (e.g., that code for proteins) as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome ordinarily has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome.

Methods for treating liver cancer

[0035] In an aspect, provided herein are methods and compositions for the treatment of cancer, such as liver cancer. A method for treating a subject having or suspected of having a liver cancer can comprise administering to the subject a therapeutically effective amount of one or more therapeutic agents that cause a decrease in expression or activity of one or more gene or gene products, thereby treating the subject for the liver cancer. The liver cancer may comprise a cell that has a mutation or deletion, a decreased or increased expression level, or decreased, increased or altered activity level of a first gene, and administration of or exposure to the one or more therapeutic agents that cause a decrease in expression or activity of a second gene may result in the inhibition or death of the cell, particularly in the presence of the altered expression of the first gene. In some instances, the first gene and the second gene form a synthetic lethal gene pair. In some cases, the first gene is AT-rich interactive domain 1 A (ARID 1 A), and the second gene encodes a kinase, e.g., Aurora kinase A (AURKA).

[0036] In some instances, the first gene is a biomarker for a cancer cell. The first gene may be deficient (e.g., under-expressed, mutated, over-expressed) in the cancer (e.g., liver cancer) cell, and the second gene may comprise a gene target to be knocked down or knocked out, thereby decreasing the expression or activity level of the second gene. In some instances, the first gene has a decreased expression or activity level in the cancer cell, and administration of or exposure to a therapeutically effective amount of one or more therapeutic agents that cause a decrease in expression or activity of the second gene in the liver, liver cancer, or cancer cell causes inhibition or death of the cell. In some instances, the first gene encodes a protein that regulates gene expression (e.g., transcription), e.g., ARID1A, and the second gene encodes a kinase, e.g., AURKA.

[0037] The first gene (e.g., ARIDl A) and the second gene (e.g., AURKA) may form a synthetic lethal pair, such that inhibition or decreased expression or activity level in both the first gene and the second gene is lethal to the cell (e.g., results in apoptosis, necrosis, inhibition of proliferation, etc.), but the inhibition or decreased activity of the first gene alone or the second gene alone is not sufficient to kill the cell. In some cases, inhibition or decreased expression or activity of the first gene (e.g., ARIDl A) or the second gene (e.g., AURKA) alone result in a reduction in viability of a cell or cell population, but the decreased expression or activity of both genes (e.g., knockdown or knockout of ARIDl A and AURKA) results in a greater reduction in viability of the cell or cell population. For example, the decrease of expression or activity of ARIDl A and AURKA may act synergistically, with a greater reduction in viability than the sum of the reductions of viability from decreased expression or activity of each member of the gene pair.

[0038] In cases where a cell (e.g., a cancerous liver cell) has a deficiency in the first gene (e.g., ARIDIA), the forced decreased expression or activity level (e.g., via knock down or knock out) of the second gene (also herein “target gene,” e.g., AURKA) may be lethal to the cell having the deficiency in the first gene, but non-toxic or non-lethal in cells that do not have the deficiency in the first gene. Such a method of treating a subject having a cancer (e.g., liver cancer), which cancer comprises a cell having the deficiency in the first gene (e.g., ARIDIA), using a single inhibitor (e.g., a therapeutically effective amount of a therapeutic agent that causes a decrease in expression or activity of AURKA) may be beneficial in reducing toxicity in normal cells of the subject and thereby reducing toxicity or side effects of cancer treatment. [0039] In some cases, the first gene may be or encode a protein that is an upstream agonist or antagonist of the second gene, or the second gene may be or encode a protein that is an upstream agonist or antagonist of the first gene. By way of example, the first gene may be ARID 1 A and the second gene may be AURKA. ARID 1 A, when expressed in a normal (e.g., non- mutated) cell, can inhibit AURKA, which is a kinase that is an upstream agonist of various proteins within a protein signaling cascade or signal transduction pathway (see, FIGS. 1A-C). For instance, AURKA can interact with or regulate PUK1, which can interact or regulate CDC25 and thereby affect CDC2 and/or other cell cycle proteins. In normal or non-cancerous cells, expression of ARID 1 A can promote cell cycle arrest. However, in cells where ARID1 A is deficient (e.g., a cell that has a mutated ARID 1 A), AURKA may not be inhibited, thereby causing increased expression of the downstream proteins PUK1 and CDC25.

[0040] Although ARID 1 A and AURKA are shown as examples, other gene interactions can be possible. In one such example, the first gene may be an agonist or antagonist of another gene (or encoded protein) that regulates the second gene, or the second gene may be an agonist or antagonist of another gene or encoded protein that regulates the first gene. Similarly, the first gene may be an agonist or antagonist of another gene (or encoded protein) that regulates yet another gene (or encoded protein) that may regulate the second gene, or the second gene may be an agonist or antagonist of another gene (or encoded protein) that regulates yet another gene (or encoded protein) that may regulate the first gene. In some cases, the first or second gene may regulate another gene or protein that is at least 1, 2, 3, 4, 5, 6, 7, 8, or more components (e.g., nodes or other genes, proteins, or signal transducers) upstream of the second or first gene, respectively.

[0041] In some instances, the first gene and the second gene may regulate a subset of the same genes downstream. For example, the first gene may regulate a plurality of downstream genes, a subset of which are also regulated by the second gene. In cancer cells, such as liver cancer cells, the downstream genes may comprise genes important in cancer-related processes, e.g., HIPPO pathway, epithelial -to-mesenchymal transition, P13K pathway, DNA replication, cell migration, cell metastasis, etc. Alternatively or in addition to, the first gene and the second gene may be regulated by a subset of the same genes.

[0042] FIG. IB schematically shows a signaling pathway of AURKA. AURKA, when overexpressed, can lead to oncogenic activity. For instance, AURKA may stabilize MYC, which may be kinase-independent. AURKA may inhibit degradation of N-Myc and may interact with c- Myc, which has downstream effectors that can lead to cell proliferation or lymphomagenesis.

[0043] In some cases, the first gene or the second gene may also be a biomarker for a liver cancer. For instance, the first gene may be ARID 1 A. In some cases, in a cancer cell (e.g., liver cancer cell), ARID1A may be lowly expressed, mutated, or otherwise deficient in a cancer cell, e.g., liver cancer cell, when compared to a control cell or population of cells. For instance, FIG. 2 shows a table of the percentages of certain cancer types that contain a mutation in the ARID 1 A gene. In such examples, a range of approximately 6-57% of cancers of the ovaries, liver, breast, colon, and lung, demonstrate a mutation in the ARID 1 A gene. In cancers comprising the ARID 1 A mutation, administration of or exposure to a therapeutically effective amount of one or more therapeutic agents that causes the decrease in expression or activity of AURKA may result in synthetic lethality of the ARIDlA-mutated cells.

[0044] In some cases, the one or more therapeutic agents used to cause a decrease in expression or activity of the second gene (e.g., AURKA) may comprise a small molecule, a protein, a peptide, a ribonucleic acid (RNA) molecule, a deoxyribonucleic acid (DNA) construct, or a combination thereof (e.g., a protein-nucleic acid complex). In an example, the one or more therapeutic agents may comprise a protein-nucleic acid complex, e.g., an endonuclease complex and a DNA construct. In some cases, the endonuclease complex comprises a clustered regularly interspaced short palindromic repeat (CRISPR) associated (Cas) protein or variant thereof (e.g., an engineered variant). In such cases, the DNA construct may be co-administered with the endonuclease complex. Alternatively or in addition to, the DNA construct may comprise an endonuclease gene. In such instances, the DNA construct may comprise a gene encoding for a Cas protein or variant thereof (e.g., an engineered variant). After the DNA construct is introduced or delivered to a cell (e.g., liver cancer cell), the DNA construct may be transcribed and translated by the cell using the cell’s own machinery (e.g., polymerases, ribosomes, etc.).

[0045] In some instances, the one or more therapeutic agents used to cause a decrease in expression or activity of a target gene (e.g., AURKA) comprises a small molecule inhibitor. The small molecule may be configured to decrease the expression level or activity level of the target gene alone, or the small molecule may be configured to decrease the expression level or activity level of the target gene in combination with the deficient or mutated gene (e.g., ARID 1 A in a liver cancer cell). In some cases, the small molecule may directly interact with both the first gene and the second gene. For example, the small molecule may inhibit the protein or proteins encoded by one or both of the first gene and the second gene, respectively. Alternatively or in addition to, the small molecule may inhibit an upstream effector or downstream protein in a signaling pathway in which one or both of the genes interact.

[0046] In some cases, the small molecule inhibitor may comprise an AURKA inhibitor. The AURKA inhibitor may be, for example, 2-[3-[[7-[3-[ethyl(2- hydroxyethyl)amino]propoxy]quinazolin-4-yl]amino]-lH-pyrazol -5-yl]-N-(3- fluorophenyl)acetamide (Barasertib), 4- { [9-Chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4- d][2]benzazepin-2-yl]amino}-2-methoxybenzoic acid (Alisertib), N-[5-[(2R)-2-methoxy-2- phenylacetyl]-4,6-dihydro-lH-pyrrolo[3,4-c]pyrazol-3-yl]-4-( 4-methylpiperazin-l-yl)benzamide (Danusertib), l-Cyclopropyl-3-(3-(5-(morpholinomethyl)-lH-benzo[d]imidazol -2-yl)-lH-pyrazol-4- yl)urea (AT9283), N-[2-[(l S,4R)-6-[[4-(Cyclobutylamino)-5-(trifluoromethyl)-2- pyrimidinyl]amino]-l ,2,3,4-tetrahydronaphthalen-l ,4-imin-9-yl]-2-oxoethyl] -acetamide (PF 03814735), or N-(4-((3-(2-aminopyrimidin-4-yl)pyridin-2-yl)oxy)phenyl)-4-( 4-methylthiophen-2- yl)phthalazin-l -amine (AMG 900). The small molecule inhibitor may be configured to inhibit or decrease the expression of AURKA (the gene) or the activity of Aurora kinase A (a protein derived from the AURKA gene), either directly or indirectly. For instance, the small molecule inhibitor may inhibit the Aurora kinase A protein or another protein that may be upstream or downstream of Aurora kinase A in a signaling pathway, such as, but not limited to, those shown in FIGS. 1A-1C. For example, the small molecule inhibitor may inhibit or otherwise decrease the expression or activity level of c-Myc, Aurora kinase B (AURKB), cyclin A, cyclin B, cyclin-D, cyclin-E, cdk2, cdk4, p27, N-myc, FBXW7, Plkl, cdc25, Sox3, SP1, E2F1, etc.

[0047] In some cases, the small molecule inhibitor may comprise a combination of small molecule inhibitors or derivatives thereof. For example, a small molecule inhibitor may be engineered or modified for dual specificity and may decrease expression or activity of both the first gene and the second gene (e.g., AURKA and ARID 1 A). Alternatively or in addition to, a combination of small molecule inhibitors (e.g., a small molecule “cocktail”) may be used to decrease expression or activity of the target gene (e.g., AURKA) alone or both the first gene and the second gene. In some cases, a small molecule inhibitor may be administered with another agent type (e.g., protein, RNA molecule, DNA molecule, etc.). [0048] The small molecule inhibitor may be administered in any useful concentration. For example, a small molecule may be administered at a concentration of about 0.5 nanomolar (nM), about 1 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 micromolar (mM), about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM. A small molecule may be administered at a concentration of at least about 0.5 nanomolar (nM), at least about 1 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 micromolar (mM), at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM. A small molecule may be administered at a concentration of at most about 10 mM, at most about 9 mM, at most about 8 mM, at most about 7 mM, at most about 6 mM, at most about 5 mM, at most about 4 mM, at most about 3 mM, at most about 2 mM, at most about 1 mM, at most about 900 nM, at most about 800 nM, at most about 700 nM, at most about 600 nM, at most about 500 nM, at most about 400 nM, at most about 300 nM, at most about 200 nM, at most about 100 nM, at most about 90 nM, at most about 80 nM, at most about 70 nM, at most about 60 nM, at most about 50 nM, at most about 40 nM, at most about 30 nM, at most about 20 nM, at most about 10 nM, at most about 1 nM, at most about 0.5 nM, etc.

A range of concentrations may be used, e.g., between 22 nM-1 mM. Where more than one small molecule is used, the concentrations may be the same of different for each small molecule used. [0049] In some cases, the small molecule inhibitor may be configured to have higher selectivity for AURKA over a similar gene (e.g., Aurora Kinase B, Aurora kinase C, etc.). The small molecule inhibitor may have a higher selectivity for AURKA over a similar gene by about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, or more. The small molecule inhibitor may have a higher selectivity for AURKA over a similar gene by at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, or more.

[0050] In cancers (e.g., liver cancers) comprising a deficiency in the first gene (e.g., ARID 1 A), one or more therapeutic agents used to cause a decrease in expression or activity of the target gene (e.g., AURKA) may require a lower concentration or dosage to be delivered to a subject for therapeutic efficacy. For instance, ARID1A and AURKA may be synthetic lethal, and administration of an AURKA inhibitor to a subject having a cancer cell that has a deficiency in ARID 1 A may be therapeutically effective. In such an example, a lower dosage of AURKA inhibitor may be sufficient to kill the ARID1 A-deficient cancer cells, compared to cells (e.g., non-cancer cells) that do not have the ARID 1 A deficiency. As higher dosages or concentrations of AURKA inhibition in a subject may increase toxicity, administration of a lower concentration or dosage of AURKA inhibitor in selected or pre-screened cancer types (e.g., cancers comprising the ARID1 A mutation) may be advantageous to reduce toxicity and side effects to the subject.

[0051] In some cases, the method for treating the subject having a cancer (e.g., liver cancer) further comprises administering to the subject a therapeutically effective amount of one or more therapeutic agents that causes a decrease in expression or activity of ARID 1 A. In some cases, the method for treating the subject having a cancer (e.g., liver cancer) further comprises administering to the subject a therapeutically effective amount of one or more therapeutic agents that causes a decrease in expression or activity of MYC.

[0052] In some cases, the one or more therapeutic agent used to cause a decrease in expression or activity of the target gene comprises a DNA construct. By way of example, the target gene may be AURKA. The DNA construct may comprise a guide RNA (gRNA) sequence, which may be used to direct a protein (e.g., Cas protein) to the target gene (e.g., AURKA). The DNA construct may comprise a gRNA sequence, which may direct the protein (e.g., Cas protein) to a target gene (e.g., AURKA). The DNA construct may comprise an RNA sequence, a DNA sequence, or a combination thereof. In some cases, the DNA construct comprises: (i) a first gRNA sequence, which may be used to direct an endonuclease (e.g., Cas protein) to a targeted location or gene locus for a target gene (e.g., AURKA) and (ii) a first sequence (e.g., a DNA sequence) corresponding to the gene (e.g., a gene replacement for AURKA). It will be appreciated that different combinations of RNA sequences and DNA sequences may be used in the DNA construct. Moreover, other functional sequences may be included in the DNA sequence, including, but not limited to, a barcode sequence, a tag, or other identifying sequence, a primer sequence, a restriction site, a transposition site, etc.

[0053] The endonuclease complex may comprise an endonuclease, e.g., a Cas protein, or other nucleic acid-interacting enzyme (e.g., ligase, helicase, reverse transcriptase, transcriptase, polymerase, etc.). The Cas protein may comprise any Cas type (e.g., Cas I, Cas IA, Cas IB, Cas IC, Cas ID, Cas IE, Cas IF, Cas IU, Cas III, Cas IIIA, Cas IIIB, Cas IIIC, Cas HID, Cas IV, Cas IVA, Cas IVB, Cas II, Cas IIA, Cas IIB, Cas IIC, Cas V, Cas VI). In some instances, the Cas protein may comprise other proteins (e.g., a fusion protein) and may comprise an additional enzyme that may associate with a nucleic acid molecule (e.g., ligase, transcriptase, transposase, nuclease, endonuclease, reverse transcriptase, polymerase, helicase, etc.). The endonuclease complex may be delivered exogenously or may be encoded in the DNA construct for transcription and translation within the cell.

[0054] In some cases, the one or more therapeutic agents used to cause a decrease in expression in the target gene (e.g., AURKA) may comprise a protein or peptide. For example, the one or more therapeutic agents may comprise an antibody, an antibody fragment, a hormone, a ligand, or an immunoglobulin. The protein or peptide may be naturally occurring or may be synthetic. The protein may be an engineered variant of a protein (e.g., recombinant protein), or fragment thereof. The protein may be subjected to other modifications, e.g., post-translational modifications, including but not limited to: glycosylation, acylation, prenylation, lipoylation, alkylation, amidation, acetylation, methylation, formylation, butyrylation, carboxylation, phosphorylation, malonylation, hydroxylation, iodination, propionylation, S-nitrosylation, S-glutationylation, succinylation, sulfation, glycation, carbamylation, carbonylation, biotinylation, carbamylation, oxidation, pegylation, sumoylation, ubiquitination, ubiquitylation, racemization, etc. One or more modifications may be made to the protein or peptide.

[0055] In some cases, the one or more therapeutic agents used to cause a decrease in expression or activity of the target gene (e.g., AURKA) may comprise a nucleic acid molecule, e.g., an RNA molecule. The RNA molecule can comprise any suitable RNA molecule and size sufficient to decrease the expression level or activity of the target gene (e.g., AURKA). The RNA molecule may comprise a small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA), or other useful RNA molecule. In some examples, the RNA molecule may comprise a messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNAs (rRNA), small nuclear RNA (snRNA), piwi-interacting (piRNA), non-coding RNA (ncRNA), long non-coding RNA, (IncRNA), and fragments of any of the foregoing. The RNA molecule may be single-stranded, double-stranded, or partially single- or double-stranded.

[0056] It will be appreciated that one or more therapeutic agents (e.g., peptides, RNA molecules, protein-nucleic acid complexes) are listed as examples and that a combination of therapeutic agent types may be used to treat the subject. For instance, administering one or more different types of therapeutic agents may be used to decrease the expression or activity of the target gene (e.g., AURKA). For example, a protein or peptide may be co-administered with a small molecule, an RNA molecule, a DNA molecule, or a complexed molecule (e.g., protein-nucleic acid molecule).

Similarly, an RNA molecule may be administered with a small molecule, a DNA molecule, or a complexed molecule. In another example, a small molecule may be co-administered with a DNA molecule or a complexed molecule. Any of these combinations may be used to decrease the expression or activity of the target gene (AURKA) in a cell comprising a mutation in the first gene (e.g., ARIDIA). These combinations are non-limiting examples of different combinations of agents that may be used to treat the subject having or suspected of having cancer (e.g., liver cancer).

[0057] For example, FIG. 3 schematically illustrates an example workflow for determining the effect of treatment of a population of cultured liver cancer cells with a protein and nucleic acid molecule. In such an example, the treatment may comprise administration of a nucleic acid molecule to decrease the activity or expression of the first gene (e.g., ARIDIA) and the second gene (e.g., AURKA). The nucleic acid molecule can comprise a DNA construct, which may comprise a first gRNA sequence (sgRNA-A), a second gRNA sequence (sgRNA-B), a first DNA sequence (BC-B) and a second DNA sequence (BC-A). The first DNA sequence or the second DNA sequence, or both the first and the second DNA sequences may comprise a barcode sequence. The first guide sequence may have sequence homology to the first gene (e.g., ARIDIA) and thus may target the first gene for mutagenesis by a protein (e.g., an endonuclease, e.g., Cas9), and the second guide sequence may have sequence homology to the second gene (e.g., AURKA) and thus may target the second gene for mutagenesis by a protein (e.g., an endonuclease, e.g., Cas9). Cells (e.g., liver cancer cells) may be treated with a therapeutically effective amount of the DNA construct and a protein (e.g., Cas9). In some cases, the DNA construct may be introduced via transfection (e.g., using a liposome or other nanoparticle) or transduction (e.g., using a virus). The protein may be administered using a nanoparticle or other vesicle, or by adding the protein to the cell culture media. The protein (e.g., Cas9) may use the sgRNA-A and sgRNA-B to direct the protein to a specific locus or location in the cell genome (e.g., at a locus of ARID1 A and AURKA). Next, the protein may excise and/or replace the endogenous genes (e.g., ARID1 A and AURKA). If replacing the endogenous genes, the protein (e.g., Cas9) may replace the endogenous genes with the first DNA sequence (BC-B) and the second DNA sequence (BC-A). Cells may then be cultivated for a duration of time (e.g., 7 days, 14 days, 20 days, etc.). The proliferation or viability of the cells may be measured, and in some instances, compared to a control population of cells. Such a comparison may be useful, for example, in determining if the pair of gRNAs cause synthetic lethality in the population of cells. In some instances, the genome of a cell or a population of cells may be sequenced to determine if a cell or population of cells were mutated (e.g., by identification of the presence of a barcode comprised in the replacement genes, e.g., via a polymerase chain reaction (PCR) or sequencing approach). In some instances, the presence or quantity of the gRNAs may be analyzed.

[0058] In some cases, only the target gene may be knocked out in a cell or population of cells, which cell comprises a deficient gene that is synthetic lethal with the target gene. FIG. 4 schematically illustrates an example workflow for determining the effect of treatment of a population of cultured liver cancer cells that are deficient in a gene (e.g., ARIDIA). In such an example, the treatment may comprise administration of a nucleic acid molecule to decrease the activity or expression of the target gene (e.g., AURKA). The nucleic acid molecule can comprise a DNA construct, which may comprise a gRNA sequence (sgRNA-A) and a DNA sequence (BC-A). The DNA sequence may comprise a barcode sequence, and the guide sequence may have sequence homology to the target gene (e.g., AURKA) and thus may target the target gene for mutagenesis by a protein (e.g., an endonuclease, e.g., Cas9). A population of cells (e.g., liver cancer cells) comprising the mutation (e.g., ARIDIA mutation) may be treated with a therapeutically effective amount of the DNA construct and a protein (e.g., Cas9). In some cases, the DNA construct may be introduced via transfection (e.g., using a liposome or other nanoparticle) or transduction (e.g., using a virus). The protein may be administered using a nanoparticle or other vesicle, or by adding the protein to the cell culture media. The protein (e.g., Cas9) may use the sgRNA-A to direct the protein to a specific locus or location in the cell genome (e.g., at a locus of AURKA). Next, the protein may excise and/or replace the endogenous genes (e.g., AURKA). If replacing the endogenous genes, the protein (e.g., Cas9) may replace the endogenous genes with the DNA sequence (BC-A). Cells may then be cultivated for a duration of time (e.g., 7 days, 14 days, 20 days, etc.). The proliferation or viability of the cells may be measured, and in some instances, compared to a control population of cells (e.g., non-mutant ARID1A cells). In such an example, the treated mutated cells (e.g., ARIDlA-deficient liver cancer cells) may have reduced viability or proliferation compared to the treated control population (e.g., non-mutant ARID1 A cells). In some instances, the genome of a cell or a population of cells may be sequenced to determine if a cell or population of cells were mutated (e.g., by identification of the presence of a barcode comprised in the replacement genes, e.g., via a polymerase chain reaction (PCR) or sequencing approach).

[0059] In another aspect, disclosed herein is a composition for treating a liver cancer, comprising a formulation comprising (i) at least one therapeutic agent and (ii) an excipient, wherein the at least one therapeutic agent is present in an amount that is effective to cause a decrease in expression or activity of Aurora kinase A (AURKA) following administration or exposure to a liver of the subject, wherein the excipient stabilizes the at least one therapeutic agent or provides therapeutic enhancement of the at least one therapeutic agent following administration or exposure to the liver of the subject as compared to the at least one therapeutic agent being administered to the liver of the subject in absence of the excipient, and wherein the liver cancer comprises a cell that has a decreased expression or activity level of AT-rich interactive domain 1A (ARID 1 A).

[0060] In some cases, the at least one therapeutic agent used to cause a decrease in expression or activity of AURKA may comprise a small molecule, a protein, a peptide, a ribonucleic acid (RNA) molecule, a deoxyribonucleic acid (DNA) construct, or a combination thereof (e.g., a protein-nucleic acid complex). In an example, the at least one therapeutic agent may comprise a protein-nucleic acid complex, e.g., an endonuclease complex and a DNA construct. In some cases, the endonuclease complex comprises a clustered regularly interspaced short palindromic repeat (CRISPR) associated (Cas) protein or variant thereof (e.g., an engineered variant). In such cases, the DNA construct may be co-administered with the endonuclease complex. Alternatively or in addition to, the DNA construct may comprise an endonuclease gene. In such instances, the DNA construct may comprise a gene encoding for a Cas protein or variant thereof (e.g., an engineered variant). After the DNA construct is introduced or delivered to a cell (e.g., liver cancer cell), the DNA construct may be transcribed and translated by the cell using the cell’s own machinery (e.g., polymerases, ribosomes, etc.).

[0061] In some instances, the at least one therapeutic agent used to cause a decrease in expression or activity of AURKA comprises a small molecule inhibitor. The small molecule may be configured to decrease the expression level or activity level of the target gene alone, or the small molecule may be configured to decrease the expression level or activity level of the AURKA and ARID 1 A. In some cases, the small molecule may directly interact with AURKA, or AURKA and ARID 1 A. For example, the small molecule may inhibit the protein or proteins encoded by AURKA alone, or the combination of AURKA and ARID 1 A, respectively. Alternatively or in addition to, the small molecule may inhibit an upstream effector or downstream protein in a signaling pathway in which AURKA or ARID 1 A interact.

[0062] In some cases, the small molecule inhibitor may comprise an AURKA inhibitor. The AURKA inhibitor may be, for example, Barasertib, Danusertib, AT9283, PF 03814735, or AMG 900. The small molecule inhibitor may be configured to inhibit or decrease the expression of AURKA (the gene) or the activity of Aurora kinase A (a protein derived from the AURKA gene) directly or indirectly. For instance, the small molecule inhibitor may inhibit the Aurora kinase A protein or another protein that may be upstream or downstream of Aurora kinase A in a signaling pathway, such as, but not limited to, those shown in FIGS. 1A-1C.

[0063] In some cases, the small molecule inhibitor may comprise a combination of small molecule inhibitors or derivatives thereof. For example, a small molecule inhibitor may be engineered or modified for dual specificity and may decrease expression or activity of both AURKA and ARID1 A. Alternatively or in addition to, a combination of small molecule inhibitors (e.g., a small molecule “cocktail”) may be used to decrease expression of AURKA, or the combination of AURKA and ARID 1 A. In some cases, a small molecule inhibitor may be administered with another therapeutic agent type (e.g., protein, RNA molecule, DNA molecule, etc.).

[0064] The small molecule inhibitor may be administered in any useful concentration. For example, a small molecule may be administered at a concentration of about 0.5 nanomolar (nM), about 1 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 micromolar (mM), about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM. A small molecule may be administered at a concentration of at least about 0.5 nanomolar (nM), at least about 1 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 micromolar (mM), at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM. A small molecule may be administered at a concentration of at most about 10 mM, at most about 9 mM, at most about 8 mM, at most about 7 mM, at most about 6 mM, at most about 5 mM, at most about 4 mM, at most about 3 mM, at most about 2 mM, at most about 1 mM, at most about 900 nM, at most about 800 nM, at most about 700 nM, at most about 600 nM, at most about 500 nM, at most about 400 nM, at most about 300 nM, at most about 200 nM, at most about 100 nM, at most about 90 nM, at most about 80 nM, at most about 70 nM, at most about 60 nM, at most about 50 nM, at most about 40 nM, at most about 30 nM, at most about 20 nM, at most about 10 nM, at most about 1 nM, at most about 0.5 nM, etc.

A range of concentrations may be used, e.g., between 22 nM-1 mM. Where more than one small molecule is used, the concentrations may be the same of different for each small molecule used. As described elsewhere herein, a lower concentration or dosage of the one or more therapeutic agents to inhibit AURKA may be therapeutically effective in cancers that comprise a cell having an ARID 1 A deficiency, as compared to non-deficient cancer cells.

[0065] In some cases, the composition may further comprise at least one therapeutic agent present in an amount that is effective in causing a decrease in expression or activity of ARID 1 A. In some cases, the method for treating the subject having a cancer (e.g., liver cancer) further comprises administering to the subject a therapeutically effective amount of one or more therapeutic agents that causes a decrease in expression or activity of MYC.

[0066] In some cases, the one or more therapeutic agent used to cause a decrease in expression AURKA comprises a DNA construct. The DNA construct may comprise a guide RNA (gRNA) sequence, which may be used to direct a protein (e.g., Cas protein) to the AURKA. The DNA construct may comprise a gRNA sequence, which may direct the protein (e.g., Cas protein) to AURKA. The DNA construct may comprise an RNA sequence, a DNA sequence, or a combination thereof. In some cases, the DNA construct comprises: (i) a first gRNA sequence, which may be used to direct an endonuclease (e.g., Cas protein) to a targeted location or gene locus for AURKA and (ii) a sequence (e.g., a DNA sequence) corresponding to the AURKA gene (e.g., a gene replacement for AURKA). It will be appreciated, that different combinations of RNA sequences and DNA sequences may be used in the DNA construct. Moreover, other functional sequences may be included in the DNA sequence, including, but not limited to, a barcode sequence, a tag, or other identifying sequence, a primer sequence, a restriction site, a transposition site, etc.

[0067] The endonuclease complex may comprise an endonuclease, e.g., a Cas protein, or other nucleic acid-interacting enzyme (e.g., ligase, helicase, reverse transcriptase, transcriptase, polymerase, etc.). The Cas protein may comprise any Cas type (e.g., Cas I, Cas IA, Cas IB, Cas IC, Cas ID, Cas IE, Cas IF, Cas IU, Cas III, Cas IIIA, Cas IIIB, Cas IIIC, Cas HID, Cas IV, Cas IVA, Cas IVB, Cas II, Cas IIA, Cas IIB, Cas IIC, Cas V, Cas VI). In some instances, the Cas protein may comprise other proteins (e.g., a fusion protein) and may comprise an additional enzyme that may associate with a nucleic acid molecule (e.g., ligase, transcriptase, transposase, nuclease, endonuclease, reverse transcriptase, polymerase, helicase, etc.). The endonuclease complex may be delivered exogenously or may be encoded in the DNA construct for transcription and translation within the cell.

[0068] In some cases, the at least one therapeutic agent used to cause a decrease in expression in AURKA may comprise a protein or peptide. For example, the one or more therapeutic agent may comprise an antibody, an antibody fragment, a hormone, a ligand, or an immunoglobulin. The protein or peptide may be naturally occurring or may be synthetic. The protein may be an engineered variant of a protein (e.g., recombinant protein), or fragment thereof. The protein may be subjected to other modifications, e.g., post-translational modifications, including but not limited to: glycosylation, acylation, prenylation, lipoylation, alkylation, amidation, acetylation, methylation, formylation, butyrylation, carboxylation, phosphorylation, malonylation, hydroxylation, iodination, propionylation, S-nitrosylation, S-glutationylation, succinylation, sulfation, glycation, carbamylation, carbonylation, biotinylation, carbamylation, oxidation, pegylation, sumoylation, ubiquitination, ubiquitylation, racemization, etc. One or more modifications may be made to the protein or peptide.

[0069] In some cases, the at least one therapeutic agent used to cause a decrease in expression or activity of AURKA may comprise a nucleic acid molecule, e.g., an RNA molecule. The RNA molecule can comprise any suitable RNA molecule and size sufficient to decrease the expression level or activity of AURKA, and, in some instances, ARID 1 A. The RNA molecule may comprise a small hairpin RNA (shRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA), or other useful RNA molecule. In some examples, the RNA molecule may comprise a messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNAs (rRNA), small nuclear RNA (snRNA), piwi- interacting (piRNA), non-coding RNA (ncRNA), long non-coding RNA, (IncRNA), and fragments of any of the foregoing. The RNA molecule may be single-stranded, double-stranded, or partially single- or double-stranded.

[0070] It will be appreciated that the therapeutic agents (e.g., peptides, RNA molecules, protein- nucleic acid complexes) are listed as examples and that a combination of therapeutic agent types may be used to treat the subject. For instance, the composition may comprise one or more different types of therapeutic agents that may be used to decrease the expression or activity of AURKA. For example, a protein or peptide may be co-administered with a small molecule, an RNA molecule, a DNA molecule, or a complexed molecule (e.g., protein-nucleic acid molecule). Similarly, an RNA molecule may be administered with a small molecule, a DNA molecule, or a complexed molecule. In another example, a small molecule may be co-administered with a DNA molecule or a complexed molecule. These combinations are non-limiting examples of different combinations of agents that may be used to treat the subject having or suspected of having liver cancer.

[0071] The composition may also comprise an excipient. The excipient may comprise a substance, which substance may be used to confer a property to the therapeutic agent or agents used to decrease the expression or activity level of AURKA. For instance, the excipient may comprise a substance for stabilization of the therapeutic agent. The excipient may comprise a substance for bulking up a solid, liquid, or gel formulation of the therapeutic agent. In some cases, the substance may confer a therapeutic enhancement to the therapeutic agent (e.g., by enhancing solubility). The substance may be used to change a property of the composition, such as the viscosity. The substance may be used to change a property of the therapeutic agent, e.g., bioavailability, absorption, hydrophilicity, hydrophobicity, pharmacokinetics, etc. The excipient may comprise a binding agent, anti-adherent agent, a coating, a disintegrant, a glidant (e.g., silica gel, talc, magnesium carbonate), a lubricant, a preservative, a sorbent, a sweetener, a vehicle, or a combination thereof. For instance, the excipient may comprise a powder, a mineral, a metal, a sugar (e.g. saccharide or polysaccharide), a sugar alcohol, a naturally occurring polymer (e.g., cellulose, methylcellulose) synthetic polymer (e.g., polyethylene glycol or polyvinylpyrrolidone), an alcohol, a thickening agent, a starch, a macromolecule (e.g., lipid, protein, carbohydrate, nucleic acid molecule), etc.

Delivery or administration of one or more therapeutic agents [0072] The present disclosure provides methods and compositions for delivery, administration of, or exposure to one or more therapeutic agents described herein. One or more therapeutic agents may be delivered to a subject (e.g., in vivo), or to a cell or population of cells from a subject (e.g., ex vivo or in vitro). In some cases, the one or more therapeutic agents may be delivered to a subject in one or more delivery vesicles, such as a nanoparticle. The nanoparticle may be any suitable nanoparticle and may be a solid, semi-solid, semi-liquid or a gel. The nanoparticle may be a lipophilic and amphiphilic particle. For example, a nanoparticle may comprise a micelle, liposome, exosome, or other lipid-containing vesicle. In some cases, the nanoparticle may be configured for targeted delivery to a certain cell or cell type (e.g., cancer cell). In such cases, the nanoparticle may be decorated with any number of ligands, e.g., antibodies, nucleic acid molecules (e.g., ribonucleic acid (RNA) molecules or deoxyribonucleic acid (DNA) molecules), proteins, peptides, which may specifically bind to a certain cell or cell type (e.g., cancer cell).

[0073] The one or more therapeutic agents may be delivered using viral approaches. For example, the one or more therapeutic agents may be administered using a viral vector. In such cases, the one or more therapeutic agents may be encapsulated in a virus for delivery to a cell, population of cells, or the subject. The virus can be an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or other useful virus. The virus may be engineered or may be naturally occurring.

[0074] The one or more therapeutic agents may be delivered to a subject (e.g., human patient) or a body of the subject (e.g., at the tumor site) using a single or variety of approaches. For example, the one or more therapeutic agents may be delivered or administered orally, intravenously, intraperitoneally, intratumorally, subcutaneously, topically, transdermally, transmucosally, or through another administration approach.

[0075] The one or more therapeutic agents may be delivered to the subject enterally. For example, the one or more therapeutic agents may be administered to the subject orally, nasally, rectally, sublingually, sub-labially, buccally, topically, or through an enema. In such cases, the one or more therapeutic agents may be formulated into a tablet, capsule, drop or other formulation. The formulation may be configured to be delivered enterally.

[0076] The one or more therapeutic agents may be delivered to the subject par enterally. For example, the one or more therapeutic agents may be administered via injection into a location of the subject. The location may comprise the central nervous system, and the one or more therapeutic agents may be delivered epidurally, intracerebrally, intracerebroventricularly, etc. The location may comprise the skin, and the one or more therapeutic agents may be delivered epicutaneously. For instance, the one or more therapeutic agents may be formulated in a transdermal patch, which can deliver the one or more therapeutic agents to the skin of a subject. The one or more therapeutic agents may be delivered sublingually and/or bucally, extra-amniotically, nasally, intra-arterially, intra-articularly, intravavernously, intracardiacally, intradermally, intralesionally, intramuscularly, intraocularly, intraosseously, intraperitoneally, intrathecally, intrauterinely, intravaginally, intravenously, intravesically, intravitreally, subcutaneously, trans-dermally, perivascularly, transmucosally, or through another route of administration. In some cases, the one or more therapeutic agents may be delivered topically.

[0077] The one or more therapeutic agents may be formulated into an aerosol, pill, tablet, capsule (e.g., asymmetric membrane capsule), pastille, elixir, emulsion, powder, solution, suspension, tincture, liquid, gel, dry powder, vapor, droplet, ointment, patch, or a combination thereof. For instance, the one or more therapeutic agents may be formulated in a gel or polymer and delivered via a thin film.

[0078] In some instances, the one or more therapeutic agents may be delivered to the subject using a targeted delivery approach (e.g., for targeted delivery to the tumor site) or using a delivery approach to increase uptake of a cell of the one or more therapeutic agents. The delivery approach may comprise magnetic drug delivery (e.g., magnetic nanoparticle-based drug delivery), an acoustic targeted drug delivery approach, a self-microemulsifying drug delivery system, or other delivery approach. In some cases, the one or more therapeutic agents may be formulated for targeted delivery or for increased uptake of a cell. For example, the one or more therapeutic agents may be formulated with another agent, which may improve the solubility, hydrophobicity, hydrophilicity, absorbability, half-life, bioavailability, release profile, or other property of the one or more therapeutic agents. For example, the one or more therapeutic agents may be formulated with a polymer which may control the release profile of the one or more therapeutic agents. The one or more therapeutic agents may be formulated as a coating or with a coating (e.g., bovine submaxillary mucin coatings, polymer coatings, etc.) to alter a property of the one or more therapeutic agents (e.g., bioavailability, pharmacokinetics, etc.).

[0079] In some instances, the one or more therapeutic agents may be formulated using retrometabolic drug design. In such cases, the one or more therapeutic agents may be assessed for metabolic effects in a cell, and a new formulation comprising a derivative (e.g., chemically synthesized alternative or engineered variant) may be designed to change a property of the one or more therapeutic agents (e.g., to increase efficacy, minimize undesirable side effects, alter bioavailability, etc.).

Examples

Example 1- Identification of AURKA and ARID 1 A as a synthetic lethal pair [0080] Synthetic lethal candidates in cancer cells can be mined from literature and public data, and further refinement of candidates can be performed using, for example, considerations such as multi-omics analysis, evaluation of tumor type (e.g. primary tumor), cell lines, target tractability, biomarker prevalence, etc. In one example of such synthetic lethal candidates screening, ARID 1 A and AURKA may be identified as a synthetic lethal pair due to, for example, higher frequency of ARID 1 A in cancer cells compared to non-cancer cells and higher expression levels of AURKA in cancer cells compared to non-cancer cells. Investigated cancer types may include, but are not limited to: acute myeloid leukemia (DAML), adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BUCA), brain lower grade glioma (UGG), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), cholangiocarcinoma (CHOU), chromic myelogenous leukemia (UCML), adenocarcinoma (CO AD), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), mesothelioma (MESO), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), sarcoma (SARC), skin cutaneous melanoma (SKCM), testicular germ cell tumors (TGCT), thymoma (THYM), thyroid carcinoma (THCA), uterine carcinosarcoma (UCS), uterine corpus endometrial carcinoma (UCEC), and uveal melanoma (UVM).

[0081] For instance, FIG. 5 shows a plot of frequency (Y-axis) of ARID1 A inactivation or deficiency in different cancer types (X-Axis). In certain types of cancers (e.g., UCEC), the frequency of mutation of ARID1 A can be as high as about 40%. In certain other cancer types (e.g., STAD, BLCA), the frequency of mutation of ARID 1 A may be greater than 20%. In various cancer types, the deficiency of ARID 1 A leading to inactivation may include, in non-limiting examples: hypermethylation, deep deletion, or mutation in the ARID 1 A gene.

[0082] FIG. 6 shows a plot of expression of AURKA (X-Axis) in different cancer types (Y- Axis). In all cancer cell types presented, increased expression of AURKA is observed. FIG. 7 shows a plot of the expression level of AURKA in cancer versus normal (non-cancer) cells (Y-Axis, displayed as a -loglO(Wilcox p-value)) in various cancer types (X-Axis). In certain types of cancer, e.g., BRCA, the expression level of AURKA is elevated compared to non-cancer cells. FIG. 8 shows a scatterplot of expression level (Y-Axis) of AURKA in either cancer (tumor) cells (n=1093) compared to normal cells (n=l 12). As can be noted from the plot, AURKA expression in breast cancer cells may be significantly higher than in normal cells. Altogether, FIGS. 6-8 demonstrate that AURKA may be more highly expressed in various cancer types.

[0083] Further screening of ARID 1 A and AURKA as a possible synthetic lethal pair may be performed. For instance, the expression level of AURKA across a population of cells that have inactive or deficient ARID 1 A may be compared to the expression level of AURKA across a population of cells that have a normal or wild-type genotype or phenotype of ARID 1 A. FIG. 9 shows a scatterplot of expression level (Y-Axis) of AURKA in two populations of cells, either having the inactive or deficient ARID1 A (e.g., mutated ARID1 A) (n=271 cells) or having the wild- type ARID 1 A (n=9990 cells). The population of cells having the ARID 1 A deficiency has a higher AURKA expression level than the population of cells having the wild-type ARID 1 A. “TCGA” refers to the Cancer Genome Atlas.

[0084] FIG. 10 shows a plot of expression level of mutation occurrences of ARID 1 A compared to the expression level of AURKA (Y-Axis, displayed as a -logl0(Wilcox p-value)) in various cancer types (X-Axis). FIGS. 9-10 indicate that in various cancer types, AURKA may be highly expressed and ARID 1 A may be mutated. These data may suggest that ARID 1 A and AURKA may be a potential candidate of a synthetic lethal pair, and that knockdown of both genes may result in cell death, or that knockdown of AURKA in ARID1 A-deficient cells may result in cell death.

Example 2- AURKA and ARID 1 A as a synthetic lethal pair

[0085] AURKA-ARIDIA synthetic lethality may be tested experimentally. In one specific approach, the AURKA and ARID 1 A genes may be knocked down or knocked out of a cell’s genome using a combinatorial genetics CRISPR approach (e.g., combinatorial genetics en masse (CombiGEM)). In such an example, a DNA construct may be generated. The DNA construct may comprise an AURKA gRNA to direct an endonuclease (e.g., a Cas protein) to the AURKA gene, as well as an ARID 1 A gRNA to direct an endonuclease (e.g., a Cas protein) to the ARID 1 A gene. The AURKA gRNA and ARID 1 A gRNA may comprise a sequence homologous or complementary to a sequence on the endogenous AURKA gene and ARID 1 A gene, respectively. In some instances, the DNA construct may also comprise replacement genes to replace AURKA and ARID 1 A in the genome (e.g., dysfunctional sequences, random DNA sequences).

[0086] Control DNA constructs may also be generated. For example, to determine if the gene pair is synthetic lethal, it may be important to monitor the effect of disrupting AURKA and ARID 1 A individually as well as the combination of the gene pair. Moreover, it may be important to monitor the effect of a negative control, in which a DNA construct comprising an ineffective gRNA e.g.., non-specific gRNA as a “non-cutting” control for one or both genes may be constructed. Another example of a negative control construct may comprise a vehicle control. In some examples, the negative control may comprise a gRNA to disrupt a gene such as adeno-associated virus integration site (AAVS1). In some cases, a positive control may also be used. The positive control may comprise, for instance, a DNA construct comprising a gRNA for a polymerase (e.g., an RNA polymerase, e.g., POUR2D), which can demonstrate that knockout (and the delivery mechanisms of doing so) of a gene that is essential for cell viability or proliferation results in lethality. In another example of a positive control, knockout of two genes known to be a synthetic lethal pair (e.g., methylthioadenosine phosphorylase (MTAP) and protein arginine methyltransferase 5 (PRMT5)) may be performed, e.g., using DNA constructs comprising gRNA directed to each of the known synthetic lethal genes.

[0087] The DNA constructs may then be introduced to cancer cells (e.g., liver cancer cells), which may comprise cells from a primary source (e.g., isolated from a tumor or cancer) or a cell line. An endonuclease, e.g., Cas9, may also be introduced to the cancer cells. The Cas9 may then replace, edit, or delete the AURKA and ARID 1 A genes in the treated cells, and in some cases, replace the AURKA and ARID 1 A genes in the genomes with the replacement genes in the DNA constructs. Proliferation or viability of the cells may then be monitored over time to determine the effectiveness of the treatment. The viability of the cells may be normalized or compared to a negative control or control population of cells that are not treated. [0088] FIGS. 11A-11B show example data of a CRISPR-based approach to knock out AURKA and ARID 1 A. FIG. 11A illustrates bar plots of cell viability fraction (which can be measured as absorbance using a viability assay, for example, a PrestoBlue assay) as a function of the DNA construct introduced. Abs represents absorbance, which can be indicative of cell count. The DNA constructs can be used for knockout and can comprise: (i) a dual-negative control (NTC-NTC) sequence, (ii) a polymerase (POLR2D) sequence as a positive control for knockout of an essential gene, which can optionally be paired with a negative control sequence (NCC), (iii) MTAP knockout, which can optionally be paired with a negative control sequence, (iv) a PRMT5 knockout, which can optionally be paired with a negative control sequence, (v) a MTAP-PRMT5 dual knockout control, (vi) an ARID 1 A sequence for knockout, which can optionally be paired with a negative control sequence, (vii) an AURKA sequence for knockout, which can optionally be paired with a negative control gRNA and (viii) ARID 1 A and AURKA sequences for knockout. The positive control sequence can be a DNA construct comprising a dysfunctional RNA polymerase gene (e.g., POUR2D gene) to replace the endogenous POUR2D gene, or the DNA construct may be configured to knock down or knock out a polymerase gene. The positive control sequence may be used, for example, to determine that the DNA constructs function as expected, e.g., that knock out of a gene essential for DNA replication, and thus cell proliferation, results in decreased cell viability. [0089] The viability of the treated cells can be normalized to a negative control (e.g., non-treated cells, or cells treated with DNA constructs comprising scrambled gRNA or comprising normal copies of AURKA and ARID 1 A). As can be seen in FIG. 11 A, the negative control groups of cells (NTC-NTC) have viability that is highest amongst the tested groups and to which the rest of the groups are normalized. The positive control (POUR2D-NCC), where the cells are treated with a DNA construct to knockout a polymerase, results in dramatically decreased normalized viability, as expected. The control (MTAP-PRMT5), where the cells are treated with a DNA construct to knockout MTAP and PRMT5, also results in decreased viability compared to the negative control groups. The cells that are treated with a single gene knockout, either ARID 1 A or AURKA, also show reduced viability compared to the negative control groups. Knock out of ARID 1 A and AURKA results in much lower viability than the single-knockout of ARID 1 A and the negative controls. Error bars represent standard deviation, n=3.

[0090] FIG. 11B illustrates another example of bar plots of cell viability (which can be measured using a viability assay, for example, a PrestoBlue assay) as a function of the DNA construct introduced. FIG. 11B shows two additional experimental replicates. The viability can be measured as a fraction of viable cells compared to a negative control. The DNA constructs can be used for knockout and can comprise: (i) a dual-negative control (NTC-NTC) sequence, (ii) a polymerase (POLR2D) sequence as a positive control for knockout of an essential gene, which can optionally be paired with a negative control sequence (NCC), (iii) MTAP sequence for knockout, which can optionally be paired with a negative control sequence (NCC), (iv) PRMT5 sequence for knockout, which can optionally be paired with a negative control sequence (NCC), (v) MTAP and PRMT5 sequences, which can serve as a positive synthetic lethal control, (vi) ARID 1 A sequence for knockout, which can optionally be paired with a negative control sequence, (vii) AURKA sequence for knockout, which can optionally be paired with a negative control gRNA and (viii) an ARIDl and AURKA sequences for dual knockout.

[0091] The viability of the treated cells can be normalized to a negative control (e.g., non-treated cells, or cells treated with DNA constructs comprising scrambled gRNA or comprising normal copies of AURKA and ARIDl A). Similar to FIG. 11A, the negative control groups of cells (NTC- NTC) in FIG. 11B have viability that is highest amongst the tested groups. The positive control (POUR2D-NCC), where the cells are treated with a DNA construct to knockout a polymerase, results in dramatically decreased normalized viability, as expected. The positive control (MTAP-PRMT5), where the cells are treated with a DNA construct to knockout MTAP and PRMT5, also results in decreased viability compared to the negative control groups, as well as single gene knockouts of MTAP or PRMT5 alone. The cells that are treated with a single gene knockout, either ARIDl A or AURKA, also show reduced viability compared to the negative control groups. Knock out of ARIDl A and AURKA results in significantly lower viability than the single- knockout of ARIDl A (p<0.0005). Error bars represent standard deviation, n=2.

[0092] FIGS. 12A-12B show example data of a CRISPR-based approach to knock out AURKA and ARIDl A. FIG. 12A illustrates a line plot of cell growth (which can be measured as cell confluency) as a function of the time elapsed for a variety of DNA constructs introduced. Cell growth and proliferation is measured based on the confluency of the cells in, for example, a well, a dish, a tube, etc. The DNA constructs that can be used for knockout and can comprise: (i) a dual negative control (NCCsgl-NCCsg3) sequence, (ii) a negative control (AAVSl-NCCsgl) sequence (iii) a polymerase (POUR2D) sequence as a positive control for knockout of an essential gene, which can optionally be paired with a negative control sequence (NCC), (iv) an ARIDl A sequence for knockout, which can optionally be paired with a negative control sequence, (v) an AURKA sequence for knockout, which can be optionally paired with a negative control gRNA, (vi) an ARID 1 A and AURKA sequences for knockout, and (vii) a MYC sequence for knockout, which can be optionally paired with a negative control gRNA and which can be another target gene for knockout, alternatively or in addition to AURKA.

[0093] The growth of the treated cells over time can be monitored. As can be seen in FIG. 12A, the negative control groups of cells (NCCsgl-NCCsg3 and AAVSl-NCCsgl) have confluency that is highest amongst the tested groups. The positive control (POUR2D-NCC), where the cells are treated with a DNA construct to knockout a polymerase, results in dramatically decreased normalized viability, as expected. The MYC knockout also results in decreased viability compared to the negative control groups. The cells that are treated with an ARID 1 A single gene knockout have slightly reduced viability (confluency) compared to the negative control, and the cells treated with an AURKA single gene knockout have substantially lower viability compared to the negative control groups. Knock out of ARID 1 A and AURKA results in much lower viability than the single knockout of ARID 1 A and the negative controls and similar viability to the positive control.

FIG. 12B illustrates another line plot of cell growth (which can be measured as cell confluency) as a function of the time elapsed for a variety of DNA constructs introduced. FIG. 12B shows an experimental replicate of FIG. 12A. Similar to the results shown in FIG. 12A, the negative control groups of cells (NCCsgl-NCCsg3 and AAVSl-NCCsgl) in FIG. 12B have confluency that is highest amongst the tested groups. The positive control (POUR2D-NCC), where the cells are treated with a DNA construct to knockout a polymerase, results in dramatically decreased normalized viability, as expected. The MYC knockout also results in decreased viability compared to the negative control groups. The cells that are treated with an ARID 1 A single gene knockout have slightly reduced viability (confluency) compared to the negative control, and the cells treated with an AURKA single gene knockout have also slightly lower viability compared to the negative control groups. Knock out of both ARID 1 A and AURKA results in much lower viability than the single knockout of ARID 1 A and AURKA and the negative controls.

[0094] Altogether, these results support that ARID 1 A and AURKA may be synthetic lethal. In such cases, treatment of ARIDlA-deficient cells with a therapeutically effective amount of one or more therapeutic agents that cause decreased activity level or expression of AURKA may be a viable treatment option. [0095] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.