Compositions for demethylation of bard1 and methods of use thereof for treating cancer

Abstract

Provided are compositions and methods for modulating methylation and / or expression of one or more target nucleic acids in a cell. Also provided are methods of making the disclosed compositions and methods of treating a disease or condition associated with increased methylation of a target nucleic acid by administering a disclosed composition.

Description

COMPOSITIONS FOR DEMETHYLATION OF BARD1 ANDMETHODS OF USE THEREOF FOR TREATING CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application Serial No. 63 / 569,069, filed March 22, 2024, the content of which is incorporated herein by this reference as if fully set forth herein.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under grants P30 CAO 16672, CAI 77909, and R35 CA209904 awarded by The National Institutes of Health, grants W81XWH-21-1-0361 and W81XWH-22-1-0138 awarded by the Department of Defense, and grant R01CA258433 awarded by the National Cancer Institute. The government has certain rights in the invention.BACKGROUND

[0003] Epigenetic changes (e.g., methylation of gene promoter or gene body) are well known to play important roles in tumor development, growth, and resistance to cancer therapy. Aberrant DNA methylation has also been implicated in other conditions such as neurodegenerative disorders, cardiovascular, autoimmune, and many other diseases. This process is a critical means by which cells containing the same DNA template can differentiate into a spectacular array of different cell types. Promoter methylation, typically occurring within CpG islands, results in powerful repression of transcription, primarily by recruiting repressor proteins or chromatin modifiers that enhance the binding of DNA to histones. In addition, gene body DNA methylation also can regulate gene expression.SUMMARY OF THE DISCLOSURE

[0004] The terms “invention,” “the invention,” “this invention,” “the present invention,” “this disclosure,” “the present disclosure,” and the like, as used in this document, are intended to refer broadly to all of the subject matter of this patent application and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Covered embodiments of the disclosure are defined by the claims, not this summary. This summary isa high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0005] In one aspect, constructs are provided, comprising a nucleic acid that encodes a guide ribonucleic acid (gRNA) that hybridizes to a breast cancer 1 (BRCAl)-associated RING domain 1 (BARD1) nucleic acid; and a nucleic acid encoding a fusion polypeptide comprising the RNA-guided dCas9 operably linked to a demethylation domain. In some embodiments, the nucleic acid encoding the gRNA comprises a nucleic acid having at least 90% sequence identity with any one of SEQ ID NOs: 1-4. In certain embodiments, the nucleic acid encoding the gRNA comprises any one of SEQ ID NOs: 1-4. In some embodiments, the dCas9 comprises an amino acid sequence encoded by a nucleic acid having at least 90% identity with SEQ ID NO: 6. In certain embodiments, the dCas9 is encoded by a nucleic acid that comprises SEQ ID NO: 6. In some embodiments, the demethylation domain comprises a ten-eleven translocation methylcytosine dioxygenase 1 (TET1) catalytic domain. In some embodiments, the TET1 catalytic domain comprises an amino acid sequence encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO: 7. In certain embodiments, the TET1 catalytic domain comprises an amino acid sequence encoded by a nucleic acid that comprises SEQ ID NO: 7. In some embodiments, the construct further comprises a first promoter operably linked to the nucleic acid encoding the gRNA and / or a second promoter operably linked to the nucleic acid encoding the fusion polypeptide.

[0006] Vectors are provided that comprise any one of the disclosed constructs. In some embodiments, the vector is a plasmid, phage, or virus. In some embodiments, the vector comprises SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0007] Compositions are provided that comprise any one of the disclosed constructs or any one of the disclosed vectors and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a neutral lipid. In certain embodiments, the neutral lipid is l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).

[0008] In another aspect, compositions are provided that comprise a complex for modulating methylation of a breast cancer 1 (BRCAl)-associated RING domain 1 (BARD1) nucleic acid in a cell, comprising: a guide RNA (gRNA) that hybridizes to a BARD1 nucleic acid in thegenome of the cell and interacts with an RNA-guided nuclease dead Cas9 (dCas9); and a fusion polypeptide comprising the RNA-guided dCas9 operatively linked to a demethylation domain, wherein the gRNA and the fusion polypeptide are linked, wherein the RNA-guided dCas9 interacts with the gRNA and specifically binds to the BARD1 nucleic acid, and wherein the demethylation domain operatively linked to the dCas9 modulates demethylation of the BARD1 nucleic acid. In some embodiments, the compositions comprise two or more distinct complexes, wherein each distinct complex comprises a distinct BARD1 gRNA and the fusion polypeptide. In some embodiments, the gRNA and the fusion polypeptide are covalently linked by chemical crosslinking or UV cross-linking. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions comprise a neutral lipid. In certain embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC).

[0009] Cells are provided that comprise any one of the disclosed compositions. In some embodiments, methylation of BARD 1 in the cell is decreased by introduction of the disclosed composition into the cell.

[0010] In another aspect, methods of treating a subject in need thereof are provided. The methods comprise administering to a cell of the subject a therapeutically effective amount of any one of the disclosed compositions. In some embodiments, the subject has a disease or condition having hypermethylation of a BARD1 gene. In some embodiments, the cell is ex vivo. In some embodiments, the subject has cancer. The cancer may be, for example, selected from a group consisting of lymphoma, myeloma, breast cancer, colon cancer, colorectal cancer, lung cancer, skin cancer, pancreatic cancer, renal (kidney) cancer, testicular cancer, bladder cancer, cervical cancer, ovarian cancer, uterus cancer, prostate cancer, head and neck, laryngeal cancer, nasopharyngeal cancer, gastric cancer, adrenal cancer, follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), leukemia, chronic lymphocytic leukemia (CLL), and marginal zone lymphoma. In some embodiments, the cancer is ovarian cancer. In some embodiments, the subject has a primary cancer. In some embodiments, the subject has metastatic cancer. In some embodiments, the subject has relapsed or recurrent cancer. In some embodiments, the subject has cancer that has not responded to or has become resistant to an anti-cancer therapy. In some embodiments, the subject has become resistant to an anti -vascular endothelial growth factor (VEGF) therapy. In certain embodiments, the anti -VEGF therapy is an anti -VEGF antibody. For example, the anti -VEGF antibody is bevacizumab.

[0011] In some embodiments, the methods further comprise administering to the subject at least a second anti-cancer therapy. In some embodiments, the second anti-cancer therapy is chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti-angiogenic therapy, or cytokine therapy. In some embodiments, the disclosed composition and the second anti-cancer therapy are administered simultaneously. In other embodiments, the disclosed composition and the second anti-cancer therapy are administered sequentially. In some embodiments of the treatment methods, the methylation of BARD 1 in the subject is decreased after administration of the composition.

[0012] In another aspect, methods of making a complex for modulating methylation of a breast cancer 1 (BRCAl)-associated RING domain 1 (BARD1) nucleic acid in a cell are provided. The methods comprise linking a BARD1 guide RNA (gRNA) with a fusion polypeptide; wherein the BARD1 gRNA hybridizes to a BARD1 nucleic acid and interacts with an RNA-guided nuclease dead Cas9 (dCas9); wherein the fusion polypeptide comprises the RNA-guided dCas9 operatively linked to a demethylation domain; wherein the RNA- guided dCas9 interacts with the gRNA and specifically binds to the BARD1 nucleic acid; and wherein the demethylation domain demethylates the BARD1 nucleic acid. In some embodiments, the gRNA and the fusion protein are covalently linked by chemical crosslinking or UV crosslinking.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0014] FIGS. 1A-1F show that the addition of 5-azacytidine (Aza) upon development of resistance to anti-vascular endothelial growth factor antibody (AV A) therapy effectively restores sensitivity to AVA. FIG. 1 A is a graph showing tumor weight in each treatment group for mice injected with HT1080 cells (Data represented as mean ± SD; n = 9 mice for control IgG; n = 10 mice for Aza treatment; n = 10 mice for B20 antibody treatment, and n = 9 mice for combination group), ns, not significant. FIG. IB is a graph showing the tumor volume in each treatment group for mice injected with HT1080 cells (Data represented as mean ± SD; n = 9 mice for control IgG; n = 10 mice for Aza treatment; n = 10 mice for B20 antibodytreatment and n = 9 mice for combination group). FIG. 1C is a graph showing the tumor weight in each treatment group in the patient-derived xenograft (PDX) model (data represented as mean ± SD; n = 7 mice for control IgG; n = 8 mice for Aza treatment; n = 6 mice for B20 antibody treatment and n = 8 mice for combination group). FIG. ID is a graph showing the number of nodules in each treatment group in the patient-derived xenograft (PDX) model (Data represented as mean ± SD; n = 7 mice for control IgG; n = 8 mice for Aza treatment; n = 6 mice for B20 antibody treatment and n = 8 mice for combination group). FIG. IE is a graph showing the tumor weight in each treatment group in the SKOV3ipl-luc ovarian cancer mouse model (Data represented as mean ± SD; n = 10 mice for control IgG; n = 10 mice for Aza treatment; n = 10 mice for B20 antibody treatment and n = 10 mice for combination group). FIG. 1H is a graph showing the number of nodules in each treatment group in the SKOV3ipl-luc ovarian cancer mouse model (data represented as mean ± SD; n = 10 mice for control IgG; n = 10 mice for Aza treatment; n = 10 mice for B20 antibody treatment and n = 10 mice for combination group). Data presented as mean values ± SD, determined by two-tailed Student’s t-test for FIGS. 1A, 1C, ID, IE, and IF and two-way ANOVA Tukey' s multiple comparisons for FIG. IB.

[0015] FIGS. 2A-2E show that treatment with azacytidine (Aza) leads to genomic changes. FIG. 2A is an overlay of the tumor-specific methylated probes and mRNA expression data in the B20 and Aza+B20 combination treatment groups in the SKOV3ipl-luc mouse model. Topexpression value in fold change. Bottom- b values (1- Methylated and 0-Unmethylated). FIG. 2B shows the relative mRNA expression of BARD 1 in each treatment group in the SKOV3ipl- luc mouse model. FIG. 2C shows the relative mRNA expression of BARD1 in normal fallopian tube epithelium (FTE) cells, HIO180 cells, and ovarian cancer cell lines. FIG. 2D shows the protein expression of BARD1 in each treatment group in the SKOV3ipl-luc mouse model. FIG. 2E shows the protein expression of BARD 1 in normal FTE cells, non-transformed ovarian surface epithelial cells (HIO180), and ovarian cancer cell lines.

[0016] FIGS. 3A-3G show BARD1 plays a role in tumor angiogenesis in ovarian cancer cells. FIG. 3A shows tube formation assay results in RF24 endothelial cells treated with BARD1 siRNA or control siRNA. FIG. 3B shows tube formation in RF24 cells conditioned in medium from SKOV3ipl cells treated with BARD1 siRNA or control siRNA. FIG. 3C shows hemoglobin levels in mice treated with conditioned medium collected from BARD1 siRNA or control siRNA-treated SKOV3ipl cells. FIG. 3D provides Gene Ontology analysis of gene and pathway enrichment in BARD 1 -silenced SKOV3ipl-luc cells. FIG. 3E shows angiogenesis array results in SKOV3ipl-luc cells treated with BARD1 siRNA (lower panel) or controlsiRNA (top panel). For FIGS. 3F-3G, a TetOn system was used in which mice were fed doxycycline (Dox) chow to knock down BARD1 and then treated with bevacizumab (anti- vascular endothelial growth factor antibody) alone or in combination with azacytidine (Aza). FIG. 3F shows tumor weight in each treatment group, with or without BARD1 knockdown, in the in vivo TetOn experiment (Data represented as mean ± SD; n = 10 mice for untreated; n = 10 mice for untreated (Dox food); n = 8 mice for Bev antibody treatment; n = 10 mice for Bev antibody treatment (Dox food); n = 9 mice for combination group and n = 9 mice for combination group (Dox food)). FIG. 3G shows relative mRNA expression of BARD1 in each treatment group, with or without BARD1 knockdown, in the in vivo TetOn experiment. Data are presented as means ± SD, determined by two-tailed Student’s t-test for FIGS. 3 A, 3B, 3C, 3F, and 3G.

[0017] FIGS. 4A-4O show BARD1 epigenetic modulation occurs under hypoxic conditions. FIGS. 4A-4B show relative mRNA expression levels of HIFa and BARD1 in SKOV3ipl cells grown under normoxic (UT) and hypoxic conditions (1% O2): (FIG. 4 A) HIFa and (FIG. 4B) BARD1. Data are presented as means ± SD (n = 3 samples per group). FIG. 4C shows relative DNMT3A mRNA expression levels in SKOV3ipl cells grown under normoxic (UT) and hypoxic conditions (1% 02). FIG. 4D shows relative DNMT3A mRNA expression levels in SKOV3ipl-luc cells treated with HIFa versus control siRNA. FIG. 4E shows the protein expression levels of HIFla in normoxic (UT) and hypoxic (C0CI2) conditions FIG. 4F shows the protein expression of BARD 1 under normoxic (UT) and hypoxic (C0CI2) conditions. FIG. 4G is a graph showing the level of BARD1 mRNA in SKOV3ipl-luc cells treated with HIFa versus control siRNA. FIG. 4H shows chromatin immunoprecipitation (ChIP) results showing HIFla levels under hypoxic conditions (right bar in each pair) and normoxic conditions (left bar in each pair). Data are presented as means ± SD (n = 3 samples per group). *P < 0.05. FIG. 41 shows HIFla binding to the promoter region of BARD 1 at site 4 under hypoxic (right bar in each pair) and normoxic (left bar in each pair) conditions. Data are presented as means ± SD (n = 3 samples per group). *P < 0.05. FIG. 4J shows relative mRNA expression levels of TET1 under hypoxic and normoxic conditions. FIGS. 4K-4L show an anti -turn or effect of targeted demethylated BARD1 in SKOV3ipl mouse model. FIG. 4K shows the tumor weight in mice treated with DOPC-dCas9-TETl-sgBARDl-3 (sgBARDl-3) or sgcontrol (no sgRNA). Data are presented as means ± SD (n = 12 mice per group). The body weight of the mice was similar, with the control mice having slightly higher body weight (data not shown) FIG. 4L shows relative mRNA expression of BARD 1 in mice treated with sgBARDl-3 or sgcontrol by qRT-PCR (Student t-test was used). Data are presented as means ± SEM (n = 4 mice per group).FIG. 4M shows tumor weight in mice treated with sgBARDl-3, alone or in combination with bevacizumab (Bev). Data are presented as means ± SEM (n = 13 mice for untreated, n = 12 mice for DOPC-TETl-sgcontrol, n = 12 mice for DOPC-TETl-sgBARDl-3, n = 12 mice for DOPC-TETl-sgcontrol + Bev antibody treatment and n = 12 mice for DOPC-TET1- sgBARDl-3 + Bev antibody treatment group). FIG. 4N shows the number of tumor nodules in mice treated with sgBARDl-3, alone or in combination with bevacizumab (Bev). Data are presented as means ± SEM (n = 13 mice for untreated, n = 12 mice for DOPC-TETl-sgcontrol, n = 12 mice for DOPC-TETl-sgBARDl-3, n = 12 mice for DOPC-TETl-sgcontrol + Bev antibody treatment and n = 12 mice for DOPC-TETl-sgBARDl-3 + Bev antibody treatment group). FIG. 40 the tumor weight in mice treated with sgBARDl-1 or control. Data are presented as means ± SEM (n = 8 mice per group). Data are presented as means ± SD or SEM, determined by two-tailed Student’s t-test for FIGS. 4A-4O.

[0018] FIG. 5 shows proposed mechanisms of the epigenetic regulation of BARD 1 with anti- vascular endothelial growth factor antibody (AV A) therapy. Prolonged AVA treatment frequently triggers adaptive resistance, resulting in tumor growth. With progressive tumor hypoxia, DNMT3A expression is increased through HIFla. DNMT3A, in turn, methylates BARD1, leading to reduced BARD1 expression in cancer cells. This decrease in BARD1 expression is associated with increased secretion of proangiogenic factors, ultimately promoting angiogenesis and tumor progression. Targeted demethylation of BARD1 leads to increased gene expression and reduced angiogenesis.

[0019] FIGS. 6A-6D show addition of 5-azacytidine (Aza) upon development of resistance to anti-vascular endothelial growth factor antibody (AVA) therapy effectively restores sensitivity to AVA. FIG. 6A shows mouse body weight in each treatment group in mice injected with HT1080 cells. Data represented as mean ± SD; n = 9 mice for control IgG; n = 10 mice for Aza treatment; n = 10 mice for B20 antibody treatment and n = 9 mice for combination group. FIG. 6B shows mouse body weight in each treatment group in the PDX (MDA-OVCA- 1) model. Data represented as mean ± SD; n = 7 mice for control IgG; n = 8 mice for Aza treatment; n = 6 mice for B20 antibody treatment and n = 8 mice for combination group. FIG. 6C shows ascites volume in each treatment group in mice injected with MDA-OVCA-1 cells. Data represented as mean ± SD; n = 7 mice for control IgG; n = 8 mice for Aza treatment; n = 6 mice for B20 antibody treatment and n = 8 mice for combination group. FIG. 6D shows the mouse body weight in each treatment group in the SKOV3ipl-luc mouse model. Data represented as mean ± SD; n = 10 mice for control IgG; n = 10 mice for Aza treatment; n = 10 mice for B20 antibody treatment and n = 10 mice for combination group.

[0020] FIGS. 7A-7E show BARD1 knockdown in SKOV3ipl and overexpression in OVCAR8ipl cells. FIG. 7A provides the densitometric analysis of BARD 1 protein expression. FIGS. 7B-7C show the relative mRNA expression (FIG. 7B) and protein expression (FIG. 7C) of BARD1 in SKOV3ipl cells treated with BARD1 siRNA. FIGS. 7D-7E show the relative mRNA expression (FIG. 7D) and protein expression (FIG. 7E) of BARD1 in OVCAR8ipl cells transfected with BARD 1 -expressing pReceiver-Lvl22 lentiviral particles.

[0021] FIGS. 8A-8H show BARD1 plays a role in tumor angiogenesis in ovarian cancer cells. FIG. 8 A shows quantification of angiogenesis factors upregulated in mice with BARD1- silenced SKOV3ipl-luc cells. FIG. 8B provides Gene Ontology analysis of gene and pathway enrichment in OVCAR8 cells with BARD1 overexpression. FIG. 8C shows relative mRNA expression of BARD 1 in SKOV3ipl cells after treatment with doxycycline for 24 hours. Data are presented as the means ± SD (n = 3 samples per group). FIG. 8D shows mouse body weight in each treatment group, with or without BARD1 knockdown, in the in vivo TetOn experiment. Data represented as mean ± SD; n = 10 mice for untreated; n = 10 mice for untreated (Dox food); n = 8 mice for Bev antibody treatment; n = 10 mice for Bev antibody treatment (Dox food); n = 9 mice for combination group and n = 9 mice for combination group (Dox food). FIG. 8E shows relative mRNA expression levels of BARD1 after treatment with BARD1 siRNA or control siRNA in SKOV3ipl-luc cells. Data are presented as the means ± SEM (n = 3 samples per group). FIG. 8F shows relative mRNA expression levels of BRCA1 after treatment with BARD1 siRNA or control siRNA in SKOV3ipl-luc cells. Data are presented as the means ± SEM (n = 3 samples per group). FIG. 8G shows relative mRNA expression levels of ESMI after knockdown of BARD1 or BRCA1 in SKOV3ipl-luc cells. Data are presented as the means ± SEM (n = 3 samples per group). FIG. 8H shows relative mRNA expression levels of FN1 after knockdown of BARD1 or BRCA1 in SKOV3ipl-luc cells. Data are presented as the means ± SEM (n = 3 samples per group). FIG. 81 provides a Spearman correlation of BARD1 with ESMI expression (data from The Cancer Genome Atlas, TCGA). FIG. 8J shows tube formation in RF24 cells conditioned in medium with BRCA1 or BARD1 knockdown. Data are presented as the means ± SD (n = 6 samples per group).

[0022] FIGS. 9A-9M show BARD1 epigenetic modulation occurs under hypoxic conditions. FIGS. 9A-9B show relative mRNA expression levels of (FIG. 9A) HIFla and (FIG. 9B) BARD1 in SKOV3ipl cells treated for 24 h with cobalt chloride (CoCh; HIFla stabilizer). FIG. 9C shows relative HIFla mRNA expression levels in SKOV3ipl-luc cells transfected with HIFla siRNA or control siRNA. FIG. 9D provides a correlation between DNMT3A and HIFla expression levels (data from The Cancer Genome Atlas). FIG. 9E shows HIFla bindingto the promoter region of BARD 1 at site 1 under hypoxic and normoxic conditions. Data are presented as means ± SD (n = 3 samples per group). *P < 0.05. FIG. 9F shows relative TET1 mRNA expression in SKOV3ipl-luc cells transfected with HIFla siRNA or control siRNA. FIG. 9G shows relative BARD1 mRNA expression levels after dCas9-TETl-sgcontrol or - sgBARDl transfection. FIG. 9H shows mouse body weight in mice treated with DOPC-dCas9- TETl-sgcontrol or -sgBARDl -3. FIG 91 shows mouse body weight in mice treated with sgBARDl-3, alone or combined with bevacizumab (Bev). Data are presented as means ± SEM (n = 13 mice for untreated, n = 12 mice for DOPC-TETl-sgcontrol treatment, n = 12 mice for DOPC-TET1 -sgBARDl -3 treatment, n = 12 mice for DOPC-TETl-sgcontrol + Bev antibody treatment and n = 12 mice for DOPC-TET1 -sgBARDl -3 + Bev antibody treatment group). FIG. 9J shows relative BARD1 mRNA expression levels in mice treated with sg-BARDl-3, alone or in combination with Bev. Data are presented as means ± SEM (n = 3 mice for untreated, n = 3 mice for DOPC-dCas9-TETl-sgcontrol, n = 3 mice for DOPC-dCas9-TETl- sgBARDl-3, n = 3 mice for DOPC-dCas9-TETl-sgcontrol + Bev antibody treatment and n = 3 mice for DOPC-dCas9-TETl -sgBARDl -3 + Bev antibody treatment group). FIG. 9K shows mouse body weight in mice treated with DOPC-dCas9-TETl-sgcontrol or -sgBARDl-1. Data are presented as means ± SEM (n = 8 mice per group). FIGS. 9L-9M show an anti-tumor effect for targeted methylated BARD1 combined with bevacizumab in SKOV3ipl in vivo model according to aspects of this disclosure, as shown by tumor weight (FIG. 9L) and number of tumor nodules (FIG. 9M) (Student t-test was used).

[0023] FIGS. 10A-10C show BARD1 expression was decreased in hypoxic conditions according to aspects of this disclosure. Relative expression levels of HIFla and BARD1 under normoxic and hypoxic conditions. HIFla (FIG. 10 A), BARD1 mRNA (FIG. 10B), and HIFla and BARD1 (FIG. 10C) protein expression levels in 1% O2 by qRT-PCR (FIGs. 10A-10B) and Western blotting (FIG. 10C). (Student t-test was used).

[0024] FIGS. 11A-11B show CHMP4A and CRIP1A expression in SKOV3ipl in vivo samples treated with Aza and AVA (B20). Quantitative PCR (qPCR) analysis of (FIG. 11 A) CHMP4A and (FIG. 11B) CRIP1A expression in SKOV3ipl tumors from mice treated with Aza, AVA (B20), or the combination (Aza+AVA) compared to the control group. Expression levels were normalized to housekeeping gene (18S), and statistical analysis was performed using Student’s t-test. No significant differences in CHMP4A or CRIP 1 A expression were observed between treatment groups. Error bars represent standard deviation (SD). n=3 mice / group

[0025] FIGS. 12A-12B show validation of siRNA transfection efficiency for CHMP4A and CRIP1 knockdown. qPCR analysis of CHMP4A and CRIP1 mRNA levels in SKOV3ipl cells transfected with three different siRNAs for each gene, along with control siRNA and untreated (UT) controls. siRNA 1 was selected for further analyses based on its highest knockdown efficiency 48 hours post-transfection. Gene expression was normalized to housekeeping gene (18S), confirming effective knockdown 48 hours post-transfection.

[0026] FIG. 13 shows an angiogenesis array of CHMP4A- and CRIP 1 -knockdown SKOV3ipl cells. Conditioned media were collected from SKOV3ipl cells transfected with CHMP4A siRNA, CRIP1 siRNA, control siRNA, or left untreated (UT). Angiogenesis array analysis was performed to evaluate the secretion of angiogenic factors. No significant differences in angiogenesis-related protein levels were detected between groups.

[0027] FIGS. 14A-14B show BARD1 expression in HeyA8 and HeyA8-MDR tumors following chemotherapy treatment. qPCR analysis of BARD 1 mRNA levels in HeyA8 tumors (FIG. 14 A) treated with cisplatin and HeyA8-MDR tumors (FIG. 14B) treated with paclitaxel. Gene expression was normalized to housekeeping gene (18S), and comparisons were made between treated and control groups. Statistical analysis was performed using Student’s t-test. No significant changes in BARD1 expression were observed in response to chemotherapy in either model. Error bars represent standard deviation (SD).

[0028] FIGS. 15A-15C show the effect of BARD1 overexpression on tumor burden in OVCAR8ipl xenografts. (FIG. 15A) qRT-PCR showing BARD1 mRNA expression. (FIG. 15B) Tumor weight and mouse body weight (FIG. 15C) in mice implanted with OVCAR8ipl cells expressing BARD1 or WT cells (n = 10 mice / group). Data are presented as mean ± SD. Statistical analysis was performed using a student' s t-test.

[0029] FIGS. 16A-16C show the impact of BARD1 modulation on tumor burden in a BRCAl-mutant model. (FIG. 16A) Tumor weight (FIG. 17B) Mouse weight (FIG. 17C) Ascites volume of COV362ipl xenografts following treatment with control IgG and the combination (Aza+B20). Statistical analysis was performed using Student’s t-test. Error bars represent standard deviation (SD). n = 11 mice per group.

[0030] FIG. 17 shows BARD1 expression in COV362ipl tumors following Aza and AVA (B20) treatment. qPCR analysis of BARD 1 mRNA levels in tumors from mice treated with control and the combination (Aza+AVA). expression was normalized to housekeeping gene (18S), and error bars represent standard deviation (SD). Statistical analysis was performed using Student’s t-test. n = 3 mice per group.DETAILED DESCRIPTION

[0031] Despite widespread use of anti-vascular endothelial growth factor (VEGF) antibody (AV A) for cancer therapy, most patients develop progressive disease after initially responding to AVA. Therefore, novel strategies are needed to understand and overcome AVA resistance. Here, an unexpected role of BRCA1 -associated RING domain 1 (BARD1) was identified in overcoming adaptive resistance to AVA. The effects of epigenetic modulation on cellular response to AVA were investigated; in particular, the molecular effects of modulation of both global and targeted DNA methylation. Using in vitro assays, BARD1 was identified as being specifically involved in angiogenesis. Sequential treatment with azacytidine overcame AVA therapy resistance in cancer models in vivo. The data further demonstrated that specifically targeting BARD1 with either small-interfering RNA or CpG-targeted demethylation reduced tumor growth when combined with AVA therapy (compared with AVA alone) in mouse models of ovarian cancer. These results identify a previously unrecognized role for BARD1 in tumor angiogenesis and show that targeted restoration of BARD 1 can enhance the efficacy of AVA therapy in cancer models.A. TERMINOLOGY

[0032] A number of terms and concepts are discussed below. They are intended to facilitate the understanding of various embodiments of the present disclosure in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present disclosure, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present disclosure, the description provided throughout the present document and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present disclosure and interpreted in the context of the present document and / or the accompanying figures.

[0033] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitionsherein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0034] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0035] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of those certain elements.” As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0036] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, for example, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0037] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0038] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotidedescribed herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are nonlimiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after the assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and singlestranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0039] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated.

[0040] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L- amino acids, entirely D-amino acids, or a mixture of L and D amino acids. The term “protein” as used herein refers to either a polypeptide or a dimer (i.e., two) or multimer (z.e., three or more) of single chain polypeptides. The single-chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions. The terms “portion” and “fragment” are used interchangeably herein to refer to parts of a polypeptide, nucleic acid, or other molecular construct.

[0041] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of the naturally occurring amino acids, unnatural amino acids and chemically modified amino acids. Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Zhang et al. “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4): 581- 87 (2013); Xie et al. “Adding amino acids to the genetic repertoire,” Curr. Opin. Chem. Biol. 9(6): 548-54 (2005); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0042] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a signaling moiety, such as a fluorop hore or a radiolabel. A side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post- translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0043] The term “identity” or “substantial identity,” as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% sequence identity to a reference sequence (e.g., any one of SEQ ID NOS: 1-12). Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0044] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test andreference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0045] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0046] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Set. USA 89: 10915 (1989)).

[0047] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'L Acad. Set. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10'5, and most preferably less than about IO'20.

[0048] Other terms used in the fields of recombinant nucleic acid technology, microbiology, immunology, antibody engineering, and molecular and cell biology as used herein will be generally understood by one of ordinary skill in the applicable arts.B. COMPOSITIONS

[0049] Compositions are provided for targeted demethylation of a gene (e.g., target nucleic acid) associated with a disease or condition (e.g., cancer). In some embodiments, increased methylation of the gene in a subject indicates the presence of the disease or condition, poor prognosis of the subject, and / or likelihood that the subject will not respond to a therapy for the disease or condition. Target nucleic acids include any nucleic acid sequence, including genes, for which it is desirable to modulate methylation or expression using a construct or complex described herein. DNA, for example, double-stranded DNA, can include the target nucleic acid, and a complex can bind to a site within the target nucleic acid, adjacent to, or in proximity to the target nucleic acid. DNA includes genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. Target nucleic acids include endogenous (or naturally occurring) nucleic acids and exogenous (or foreign) nucleic acids. In certain embodiments, the target nucleic acid is endogenous genomic DNA. In some embodiments, the construct comprises dCas9-TETl- sgBARDl.

[0050] In some embodiments, the constructs include a nucleic acid encoding a guide ribonucleic acid (gRNA) that hybridizes to a target nucleic acid; and a nucleic acid encoding a fusion polypeptide comprising the RNA-guided dCas9 operably linked to a demethylation domain. In some embodiments, the target nucleic acid is a BRC Al -associated RING domainprotein 1 (BARD1) nucleic acid. In certain embodiments, the BARD1 nucleic acid encodes a human BARD1 polypeptide. The human BARD1 protein is 777 amino acids and contains a RING finger domain, four ankyrin repeats, and a tandem BRCA1 C terminus (BRCT) domain. BARD1 displays a dual role in cancer development and progression as it acts as a tumor suppressor and an oncogene. BARD1 gene has been identified as an important tumor suppressor gene in certain cancers, such as breast, ovarian, and uterine cancers. BARD1 also has a number of isoforms that differ from the full-length BARD1 (resulting from nonsense and frameshift mutations, or deletions) and that have been associated with susceptibility to various cancers including neuroblastoma, lung, breast, and cervical cancers. In some embodiments, the BARD1 polypeptide is encoded by a nucleic acid having at least 60% sequence identity with SEQ ID NO: 12. That is, the BARD polypeptide is encoded by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12, or any value within these percentages. In some embodiments, the BARD1 polypeptide is encoded by a nucleic acid comprising SEQ ID NO: 12 (GenBank Accession No. Accession No. NM_000465.4).

[0051] Site-specific gRNAs (also referred to single guide RNAs, sgRNAs, or gRNAs) guide the complexes described herein to specific target nucleic acids and may be designed using methods known in the art. A gRNA can be designed to guide a complex to a promoter region of a gene, a region in proximity to the promoter region of the gene, or within the gene. Once the complex binds to its target, the complex (e.g., the demethylation domain of the fusion protein in the complex) may modulate methylation or expression of the gene by interacting with the promoter region, region in proximity to the promoter region of the gene, or within the gene. Similarly, other regulatory regions can be targeted, including, but not limited to, core promoters, proximal promoters, enhancer regions, silencers, and insulators. Because each gRNA hybridizes to a unique sequence of a target nucleic acid, each complex in the compositions described herein is a distinct complex that binds to a unique target nucleic acid. A “promoter” refers to one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0052] As used herein, a gRNA is a single guide RNA sequence that interacts with dCas9 of the fusion protein and specifically binds to or hybridizes to a target nucleic acid within the genome of the cell, such that the gRNA and the fusion protein co-localize to the target nucleicacid in the genome of the cell. Each gRNA includes a first nucleotide sequence that hybridizes to a target nucleic acid. The first nucleotide sequence includes a crRNA sequence that hybridizes to the target nucleic acid and provides sequence specificity, and a second RNA (tracrRNA) sequence that hybridizes to the crRNA. Each gRNA also includes a second nucleotide sequence that interacts with or binds to dCas9.

[0053] In certain embodiments, each gRNA in the complexes is complementary to a unique pre-defined target nucleic acid sequence. In some embodiments, the length of the gRNA is between about 10 to about 200 nucleotides. Therefore, the length of the gRNA can be about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or any length in between these lengths. It is understood that the gRNA does not have to be complementary to the entire target nucleic acid sequence as long as the gRNA can hybridize to the target nucleic acid and the complex can bind to the target nucleic acid in a site-specific manner. One of skill in the art would know how to vary the length of complementarity in order to increase binding specificity and / or decrease offsite binding of the gRNA and / or the complex. As used herein, the term “complementary” or “complementarity” refers to base pairing between nucleotides or nucleic acids, for example, and not to be limiting, base pairing between a gRNA and a target nucleic acid. Complementary nucleotides are, generally, A and T (or A and U), and G and C.

[0054] In some embodiments, the target nucleic acid is a BARD1 target nucleic acid. In some embodiments, the BARD1 target nucleic acid is a promoter region of the gene, a region in proximity to the promoter region of the gene, or within the gene. In some embodiments, the nucleic acid encoding the gRNA comprises a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or any value therein, to any one of SEQ ID NOS: 1-4 or its complement. In some embodiments, the gRNA comprises a nucleic acid having at least 90% sequence identity with any one of SEQ ID NOs: 1-4 or its complement. In certain embodiments, the gRNA comprises any one of SEQ ID NOs: 1-4 or its complement.

[0055] The construct also includes a nucleic acid encoding dCas9. As used herein, the term “Cas9” means a Cas9 protein or a fragment thereof present in any bacterial species that encodes a Type II CRISPR / Cas9 system. See, for example, Makarova el al.. Nature Reviews, Microbiology, 9: 467-477 (2011), including supplemental information, hereby incorporated by reference in its entirety. For example, the Cas9 protein or a fragment thereof can be from Streptococcus pyogenes. Full-length Cas9 is an endonuclease comprising a recognition domain and two nuclease domains (HNH and RuvC, respectively). In the amino acid sequence, HNH is linearly continuous, whereas RuvC is separated into three regions, one left of the recognition domain, and the other two right of the recognition domain flanking the HNH domain. Cas9from Streptococcus pyogenes is targeted to a genomic site in a cell by interacting with a guide RNA that hybridizes to a 20-nucleotide DNA sequence that immediately precedes an NGG motif recognized by Cas9.

[0056] In the disclosed compositions and methods, Cas9 is a nuclease-deficient Cas9 (nuclease-dead Cas9, dCas9) that has been modified to inactivate Cas9 nuclease activity. Modifications include, but are not limited to, altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. For example, and not to be limiting, D10A and H840A mutations can be made in Cas9 from Streptococcus pyogenes to inactivate Cas9 nuclease activity. Other modifications include removing all or a portion of the nuclease domain of Cas9, such that the sequences exhibiting nuclease activity are absent from Cas9. Accordingly, a dCas9 may include polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The dCas9 retains the ability to bind to DNA. Accordingly, dCas9 includes the polypeptide sequence or sequences required for DNA binding but includes modified nuclease sequences or lacks the nuclease sequences responsible for nuclease activity.

[0057] In some embodiments, the dCas9 protein is a full-length Cas9 sequence from S. pyogenes lacking the polypeptide sequence of the RuvC nuclease domain and / or the HNH nuclease domain and retaining the DNA binding function. In some embodiments, the dCas9 protein sequences have at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity, or any value within these percentages to Cas9 polypeptide sequences lacking the RuvC nuclease domain and / or the HNH nuclease domain and retains DNA binding function. In some embodiments, the dCas9 comprises an amino acid sequence encoded by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity, or any value within these percentages to SEQ ID NO: 6. In some embodiments, the dCas9 comprises an amino acid sequence encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO: 6. In certain embodiments, the dCas9 is encoded by a nucleic acid that comprises SEQ ID NO: 6.

[0058] In the disclosed compositions and methods, the demethylation domain may comprise, for example, a ten-eleven translocation methylcytosine dioxygenase (TET) or a catalytic domain thereof. In some embodiments, the demethylation domain is a TET1, TET2, or TET3, or a catalytic domain thereof. In some embodiments, the demethylation domain comprises a TET1 catalytic domain. The TET1 catalytic domain is encoded by a nucleic acid having at 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity, or any value within these percentages to SEQ ID NO: 7 and retains its catalytic activity. In some embodiments, the TET1 catalytic domain comprises an amino acid sequence encoded by a nucleic acid having at least90% sequence identity with SEQ ID NO: 7 and retains its catalytic activity. In certain embodiments, the TET1 catalytic domain comprises an amino acid sequence encoded by a nucleic acid that comprises SEQ ID NO: 7.

[0059] In some embodiments of the constructs, a first promoter is operably linked to the nucleic acid encoding the gRNA and / or a second promoter is operably linked to the nucleic acid encoding the fusion polypeptide. Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., P-actin promoter or EFla promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fused to the P-actin promoter). Promoters from the host cell or related species are also useful herein. The promoter can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, a steroid, an antibiotic, or a metal. A physically regulated promoter can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter region can act as a constitutive promoter to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter can be active in all eukaryotic cells, independent of cell type. Promoters of this type include, for example, the CMV promoter, the ubiquitin C promoter, the SV40 promoter, the beta-actin promoter, the EFla promoter, and the retroviral long terminal repeat (LTR).

[0060] In some embodiments, the construct comprises dCas9-TETl-sgBARDl. In certain embodiments, the construct comprises a nucleic acid that has at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity, or any value within these percentages to any one of SEQ ID NOS: 8-11. In some embodiments, the construct comprises a nucleic acid that has at least 90% sequence identity with any one of SEQ ID NOS: 8-11. In certain embodiments, the construct comprises any one of SEQ ID NOS: 8-11.

[0061] Vectors comprising any one of the disclosed constructs are also provided. In some embodiments, the vector is a plasmid, phage, or virus. Plasmid vectors can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformedmammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase.

[0062] Viral vectors include, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus, sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also included are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., 1997, Retroviruses, Cold Spring Harbor Laboratory Press, which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., 1987, J. Virology 61 : 1213-20; Massie et al., 1986, Mol. Cell. Biol. 6:2872-83; Haj-Ahmad et al., 1986, J. Virology 57:267-74; Davidson et al., 1987, J. Virology 61 : 1226-39; Zhang et al., 1993, BioTechniques 15:868-72). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. Constructs also can be delivered via virus-like particles.

[0063] In some embodiments, compositions are provided that include one or more of the disclosed constructs or vectors and a pharmaceutically acceptable carrier (excipient). A pharmaceutically acceptable carrier (excipient) is a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. The carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein.

[0064] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 2P1Edition, Philip P. Gerbino, ed., Lippincott Williams & Wilkins (2006). In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous and / or intravenous administration. In certain embodiments, the formulation comprises an appropriate amount of a pharmaceuticallyacceptable salt to render the formulation isotonic. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the construct, vector, or complex.

[0065] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be sterile water for injection, physiological saline solution, buffered solutions like Ringer’s solution, dextrose solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-bufferedsaline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH-controlling buffer such as phosphate-buffered saline or acetate-buffered saline.

[0066] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery (e.g., through injection by intravenous, intraperitoneal, intracerebral (intra- parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intra-ocular, intraarterial, intraportal, or intralesional routes). Preparations for parenteral administration can be in the form of a pyrogen-free, parenterally acceptable aqueous solution (i.e., water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media) comprising a disclosed construct or vector in a pharmaceutically acceptable vehicle. Preparations for parenteral administration can also include non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection.

[0067] In another aspect, a composition comprising a complex for modulating methylation of a gene is provided. In some embodiments, the complex comprises a guide RNA that hybridizes to a target nucleic acid in the genome of the cell; and a fusion polypeptide comprising an RNA-guided dCas9 operatively linked to a demethylation domain. The gRNA and the fusion polypeptide may be covalently linked, for example, by chemical crosslinking or UV cross-linking. The RNA-guided dCas9 interacts with the gRNA and specifically binds to the target nucleic acid, and the demethylation domain operatively linked to the dCas9 modulates demethylation of the target nucleic acid. In some embodiments, the target nucleic acid is a BARD1 nucleic acid. In some embodiments, the gRNA is any one of the gRNAs disclosed herein, and the dCas9-demethylation domain fusion protein is any one of the fusion proteins disclosed herein. In some embodiments, the composition comprises two or more distinct complexes, wherein each distinct complex comprises a distinct BARD1 gRNA.

[0068] Any of the constructs or compositions described herein may be included in a nanoparticle for delivery. In some embodiments, the compositions comprise a neutral lipid. In certain embodiments, the neutral lipid is l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC). Liposomes have been used for drug delivery, and cationic liposomes are described in PCT publications W002 / 100435A1, W003 / 015757A1, and WO04029213A2; U.S. Pat. Nos. 5,962,016, 5,030,453, and 6,680,068; and U.S. Patent Application 2004 / 0208921. Aprocess of making liposomes is also described in W004 / 002453 Al . Furthermore, neutral lipids have been incorporated into cationic liposomes (e.g., Farhood etal., 1995). Cationic liposomes have been used to deliver siRNA to various cell types (Sioud and Sorensen, 2003; U.S. Patent Application 2004 / 0204377; Duxbury et al., 2004; Donze and Picard, 2002). Neutral liposomes were used to deliver therapeutic antisense oligonucleotides in U.S. Patent Application 2003 / 0012812 and siRNA in U.S. Pat. No. 8,067,390 and WO 2006 / 113679. Lipid-based nanoparticles (NPs) are in use in about 30% of all FDA-approved drugs, which is due to their unique qualities such as biocompatibility, high bioavailability, high safety, non-toxicity, high efficiency, and high tissue distribution.

[0069] The neutral phospholipid of the presently disclosed compositions has an essentially neutral charge, either because it comprises a neutral phospholipid or it comprises a net neutral charge. A phospholipid is a lipid molecule that has a hydrophilic head containing a phosphate group and two hydrophobic tails derived from fatty acids, joined by an alcohol residue (e.g., a glycerol molecule). In some embodiments, a neutral phospholipid includes, but is not limited to, a di oleoyl-sn-glycero-3 -phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidyl-choline (DMPC), distearoylphosphatidylcholine (DSPC), 1- myristoyl-2-palmitoyl phosphatidyl-choline (MPPC), l-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl- 2-palmitoyl phosphatidylcholine (SPPC), dimyristyl phosphatidylcholine (DMPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DAPC), l,2-diarachidoyl-sn-glycero-3- phosphocholine (DBPC), l,2-dieicosenoyl-sn-glycero-3 -phospho-choline (DEPC), palmitoyloeoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphophatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloeoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and a combination thereof. In certain embodiments, the phospholipid component can include a singletype of neutral phospholipid. In other embodiments, a phospholipid component can include 2, 3, 4, 5, 6, or more kinds or types of neutral phospholipids.

[0070] In some embodiments, a lipid component can have an essentially neutral charge because it comprises a positively charged lipid and a negatively charged lipid. The lipid component may further comprise a neutrally charged lipid(s) or phospholipid(s). The positively charged lipid may be a positively charged phospholipid. The negatively charged lipid may be a negatively charged phospholipid. The negatively charged phospholipid may be a phosphatidylserine, such as dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidyl serine (DPPS), or brain phosphatidylserine (BPS). The negatively charged phospholipid may be a phosphatidylglycerol, such as dilauryloylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), or dioleoylphosphatidylglycerol (DOPG). In certain embodiments, the composition further comprises cholesterol or polyethyleneglycol (PEG). In certain embodiments, a phospholipid is a naturally-occurring phospholipid. In other embodiments, a phospholipid is a synthetic phospholipid.

[0071] Also provided is a cell comprising any one of the disclosed constructs, vectors, or compositions. In some embodiments, the methylation of BARD1 in the cell is decreased by introduction of the construct, vector, or composition in the cell.C. METHODS OF USE

[0072] Methods are provided for the targeted demethylation of a gene associated with a disease or condition by administering to the cell an effective amount of a composition disclosed herein. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo.

[0073] Also provided are methods for the treatment of a subject that has a disease or condition having hypermethylation of a gene by administering to a cell an effective amount of a composition that demethylates the gene as disclosed herein. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. The subject to be treated by any of the methods described herein may have one of various of different cancers, including, but not limited to, lymphoma, myeloma, breast cancer, colon cancer, colorectal cancer, lung cancer, skin cancer, pancreatic cancer, renal (kidney) cancer, testicular cancer, bladder cancer, cervical cancer, ovarian cancer, uterus cancer, prostate cancer, head and neck, laryngeal cancer, nasopharyngeal cancer, gastric cancer, adrenal cancer, follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), leukemia, chronic lymphocytic leukemia (CLL), or marginal zone lymphoma. Incertain embodiments, the cancer is ovarian cancer. In some embodiments, the subject may have a primary cancer. In other embodiments, the subject may have metastatic cancer. In some embodiments, the subject has relapsed or recurrent cancer. In some embodiments, the subject has cancer that has not responded to other treatments or has become resistant to other anticancer therapies or treatments. In some embodiments, the subject has become resistant to anti- vascular endothelial growth factor (VEGF) therapy. In some embodiments, the anti-VEGF therapy is an anti-VEGF antibody. In certain embodiments, the anti-VEGF antibody is bevacizumab.

[0074] As used throughout, subject can be a vertebrate, more specifically a mammal (e.g., a human, monkey, horse, cat, dog, cow, pig, sheep, camel, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, “patient” or “subject” may be used interchangeably and includes human and veterinary subjects. The compositions described herein are useful for treating cancer in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have cancer or other non-cancerous malignancy associated with hypermethylation or increase methylation. In some embodiments, the subject is diagnosed with a cancer.

[0075] “ Treat,” “treatment,” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treating” or “treatment” may refer to any indicia of success in the treatment or amelioration of cancer. Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or any one or more symptoms thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. “Treating” or “treatment” includes the administration of an agent to impede growth of a cancer, to do one or more of the following: cause a cancer to shrink by weight or volume (i.e., shrink from a first weight or volume to a second weight or volume, wherein the second weight or volume is less than the first), delay or prevent metastasis, extend the expected survival time of the subject, or extend the expected time to progression of the tumor, or the like. Thus, in the disclosedmethods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a cancer in a subject by administering a pharmaceutical composition as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0076] In some embodiments, the gene that is hypermethylated is BARD1, and the composition comprises an agent that demethylates BARD1. In certain embodiments, the BARD1 gene is hypermethylated at a CpG island selected from the group consisting of cg26049092, cg08563601, cg24607575, or cg24715680, or a combination thereof. In some embodiments, the composition comprises a construct for the targeted demethylation of the BARD1 gene. In some embodiments, the composition comprises a dCas9-TETl-sgBARDl.

[0077] The compositions that demethylate a gene (e.g., BARD1) may be used as a monotherapy or as a combination therapy with another agent for the treatment of the disease or disorder (e.g., an anti-cancer therapy). The therapies may be used to treat the subject in either order. For example, in some embodiments, a subject may be treated with both an anti-vascular endothelial growth factor (VEGF) therapy, such as an anti-VEGF antibody (AV A) (e.g., bevacizumab), and a composition for the targeted demethylation of BARD1. In some embodiments of this combination therapy, the subject may be treated first with an anti-VEGF therapy. Following the treatment of the subject with the anti-VEGF therapy, a composition for the targeted demethylation of BARD 1 may be administered to cells from the subject ex vivo, and the cells could then be returned to the subject. In other embodiments of combination therapy, a composition for the targeted demethylation of BARD 1 may be administered to cells from the subject ex vivo and returned to the subject, prior to the treatment of the subject with an anti-VEGF therapy.

[0078] In some embodiments the combination therapy may include, for example, chemotherapy, radiotherapy, surgical therapy, immunotherapy, or radioimmunotherapy. Exemplary anti-cancer agents (also referred to below as therapeutic agents) include chemotherapeutic agents, radiotherapeutic agents, and immunotherapeutic agents, as well ascombinations thereof. The terms “contacted” and “exposed,” when applied to a cell in this context, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.

[0079] As referred to herein, a chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (such as TARCEVA®, Genentech / OSI Pharm.), bortezomib (such as VELCADE®, Millenium Pharm.), fulvestrant (such as FASLODEX®, AstraZeneca), sutent (such as SU11248, Pfizer), letrozole (such as FEMARA®, Novartis), imatinib mesylate (such as GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (such as Eloxatin®, Sanofi), 5 -fluorouracil (5-FU), leucovorin, rapamycin (also known as sirolimus) (such as RAPAMUNE®, Wyeth), lapatinib (such as TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (such as SCH 66336), sorafenib (such as BAY43-9006, Bayer Labs.), capecitabine (such as XELODA®, Roche), docetaxel (such as TAXOTERE®), and gefitinib (such as IRESSA®, Astrazeneca), AG1478, AG1571 (such as SU 5271; Sugen Inc.), alkylating agents such as thiotepa and cyclosphosphamide (such as CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethio-phosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin yi1and calicheamicin Oi1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (such as ADRIAMYCIN®, including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Trametes Versicolor polysaccharide-K (Krestin, PSK) (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine (cytosine arabinoside, “Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (such as TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ (a Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL)), and doxetaxel (such as TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (such as GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine (such as NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0080] Chemotherapeutic agents, as used herein, also refers to (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY 117018, onapristone, and toremifene (such as FARESTON®); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (such as MEGASE®), exemestane (such as AROMASIN®), formestanie, fadrozole, vorozole (such as RIVISOR®), letrozole (such as FEMARA®), and anastrozole (such as ARIMIDEX®); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) VEGF receptor and angiogenesis inhibitors (including ribozymes such as ANGIOZYME®) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN-7® vaccine (plasmid / lipid complex containing the DNA sequences encoding HLA-B7 and 132 microglobulin), LEUVECTIN® vaccine (plasmid DNA expression vector encoding interleukin-2 (IL-2) complexed with a lipid delivery vehicle (DMRIE / DOPE)), and VAXID® vaccine (patient-specific naked DNA vaccine); IL-2 or aldesleukin (such as PROLEUKIN®); topoisomerase 1 inhibitors (such as TOPOTECAN®); gonadotropinreleasing hormone antagonists (such as ABARELIX®); (x) anti-angiogenic agents such as bevacizumab (such as AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0081] In some embodiments, the treatment methods provided herein may further comprise administering an immunosuppressive agent such as an immune checkpoint inhibitor as part of the method. These treatments work by “taking the brakes off’ the immune system (are immunosuppressive), allowing it to mount a stronger and more effective attack against cancer. Several different types of checkpoint inhibitors, targeting different checkpoints or “brakes” on immune cells, are currently in use. Immune checkpoint proteins that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), TROP2 (also known as CD276), B and T lymphocyte attenuator (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), HLA- DRB 1, ICOS (also known as CD278), HLA-DQA1, HLA-E, indoleamine 2,3 -dioxygenase 1 (IDO1), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG-3, also known as CD223), Mer tyrosine kinase (MerTK), NKG7, 0X40 (also known as CD134), programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1, also known as CD274), PDCD1LG2,PSMB 10, ST A Tl, T cell immunoreceptor with 1g and ITIM domains (TI GIT), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain 1g suppressor of T cell activation (VISTA, also known as C10orf54). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4. Exemplary immunosuppressive agents are PD-1 inhibitors (such as nivolumab and pembrolizumab), PD-L1 inhibitors (such as atezolizumab, durvalumab, and avelumab), and CTLA-4 inhibitors (such as ipilimumab). In one example, the second form of cancer therapy comprises a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA4 inhibitor. In some instances, combinations of such inhibitors can be administered. In some instances, the PD-L1 inhibitor, the PD-1 inhibitor, and / or the CTLA4 inhibitor may be an inhibitory antibody that binds specifically to PD-L1, PD-1, or CTLA4, respectively.

[0082] In some instances, the treatment methods provided herein may further comprise administering radiation therapy to the subject. Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are types of radiation used for cancer treatment. The radiation may be delivered by a machine outside the body (external-beam radiation therapy), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy). Systemic radiation therapy uses radioactive substances, such as radioactive iodine, which travel in the blood to kill cancer cells.EXAMPLES

[0083] The following examples are offered to illustrate, but not to limit, the present disclosure.EXAMPLE 1. OVERCOMING RESISTANCE TO ANTLVEGF THERAPY VIA EPIGENETIC REGULATION OF BARD1A. INTRODUCTION

[0084] Anti-vascular endothelial growth factor (VEGF) antibody (AV A) therapy has emerged as a promising treatment for numerous cancers, including ovarian cancer. Although surgery and chemotherapy are effective treatments, many cancers become resistant to chemotherapy after an initial response. Moreover, resistance to AVA therapy is common and represents a significant clinical obstacle that limits its effectiveness. Thus, a deeper understanding of the molecular mechanisms underlying AVA resistance is critical to effectively overcome this problem and improve patient outcomes.

[0085] Bevacizumab is a humanized monoclonal antibody that targets VEGF and is approved by the US Food and Drug Administration, the European Medicines Agency, and many other regulatory agencies worldwide for use in patients with ovarian and other cancers. Despite its broad activity in both preclinical and clinical settings, a high percentage of patients eventually develop disease resistance to this treatment. Development of AVA resistance may be associated with diverse mechanisms, such as upregulation of pro-angiogenic factors in the tumor microenvironment or acquisition of mutations and epigenetic changes leading to progressive tumor growth and / or increased invasiveness.

[0086] Epigenetic changes (e.g., methylation of gene promoter or gene body) are well known to play important roles in tumor development, growth, and resistance to chemotherapy. Beneficial effects of hypomethylating agents in reversing platinum drug resistance in patients with ovarian, colon and other cancers have been reported. DNA methylation inhibitors such as 5-azacytidine (Aza) and 5-aza-2'-deoxycytidine have been extensively used in patients with hematologic disorders and other neoplasms since their approval by the FDA more than a decade ago. However, global DNA demethylating agents result in intolerable toxicities that are likely due to the broad effects of such drugs across the genome. Therefore, targeted epigenetic modulation such as demethylation of specific genes in cancer treatment may prove useful. Integrating site-specific methylation / demethylation with AVA holds promise for overcoming resistance and enhancing outcomes. Although epigenetic changes are known to be important in many aspects of tumor biology, their relevance in the context of tumor microenvironment (TME)-targeted drugs (e.g., AVA) remains unclear. Thus, the investigators studied whether such epigenetic changes could occur in response to the dynamic alteration of AVA-induced TME.

[0087] As discussed in the following examples, methylation and transcriptional changes in orthotopic ovarian cancer models with emergence of AVA resistance were examined. To understand the epigenetic alteration during AVA-induced resistance, a series of in vitro (methylation array, RNA Sequencing) and in vivo (orthotopic ovarian cancer models) were performed. The combination of 5' -Azacytidine (5' -Aza) with B20 (murine anti-VEGF antibody) or bevacizumab (human anti-VEGF antibody) treatment effect was subsequently compared to control mice receiving no therapy, and those receiving 5' -Aza or B20 monotherapy with gross necropsy, assessment of tumor weight, and disease burden.

[0088] A targeted demethylation strategy for BARD1 was developed to enhance the effectiveness of AVA. A novel dCas9-CRISPR-based system was used for targeted BARD1 CpG demethylation (derived by fusing the deactivated Cas9 [dCas9] with the TET1 catalyticdomain) and was delivered in vivo using a nanoliposomal carrier. To directly demethylate BARD1, liposomal dCas9-TETl-sgBARDl in a single expression plasmid vector was used. Four pairs of sgRNAs were designed to target BARD1, and these sgRNAs were cloned into each dCas9-TETl vector, under the CMV promoter to drive expression. These plasmids were incorporated into the neutral lipid l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).

[0089] A unique molecular mechanism underlying AVA resistance driven by methylation of BARD 1 (BRCA1 -associated RING domain 1) was identified. A targeted demethylation strategy for BARD1 was developed and used to enhance the effectiveness of AVA. A novel dCas9-CRISPR-based system was developed for targeted BARD1 CpG demethylation (derived by fusing the deactivated Cas9 [dCas9] with the TET1 catalytic domain) and was delivered in vivo using a nanoliposomal carrier. The data show that this targeted approach could elicit robust antitumor effects when combined with AVA therapy in multiple cancer models. Collectively, the findings provide a novel approach for CRISPR-based BARD 1 -targeted DNA demethylation using a liposomal delivery system and combinatorial treatment strategy to overcome AVA resistance.B. MATERIALS AND METHODS

[0090] Cell maintenance. Human ovarian cancer cells, SKOV3ipl-luc, SKOV3-TR, OVCAR5, A2780ip2, HeyA8, OVCAR3 and OVCAR8ipl, were cultured in RPMI-1640. OVCAR5 and MDA-OVCA-1 (PDX from chemotherapy -resistant patient tumor) cells were cultured in Dulbecco modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA). Human fibrosarcoma cells, HT1080, were cultured in Eagle minimum essential medium with 5% FBS. Cell culture was performed at 37 °C in a 5% CO2 incubator with 95% humidity. Cell lines were routinely tested for mycoplasma contamination. All cell lines were authenticated by the Cytogenetics & Cell Authentication Core at The University of Texas MD Anderson Cancer Center. Cells were used within ~20 passages after thawing for in vitro experiments and ~10 passages after thawing for in vivo experiments.

[0091] Protein extraction and Western blot analysis Total protein lysates were prepared using ice-cold RIPA lysis buffer (25 mM Tris»HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Pittsburgh, PA). Protein was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 30 ug of protein was used for Western blot analysis using 4-20% and 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation gel . The membranes were then blocked and incubated in primary antibody dilutedin 5% milk in Tris-buffered saline-Tween-20 overnight at 4 °C. Membranes were probed with a horseradish peroxidase-conjugated secondary antibody depending on their primary antibodies and developed with ECL substrate (Thermo Fisher Scientific). Quantification of band intensity was done using Imaged software.

[0092] RNA isolation, cDNA synthesis, and real-time PCR Tumor tissues or cells of different types / conditions were harvested in Trizol (Life Technologies). Total RNA was extracted using the Direct-zol RNA mini prep kit (Zymo Research) according to the manufacturer’ s instructions. A total of I ug of R A was used for preparing cDNA with random hexamer oligonucleotide primers using a Verso cDNA synthesis kit (Thermo Fisher Scientific) according to the supplier’s recommended protocol. Gene-specific primers were used for realtime PCR in a 7500 Real-Time PCR System (Applied BioSystems, Carlsbad, CA) using 2X SYBR green master mix (Applied BioSystems). The expression of 18S rRNA in each sample was used to normalize the expression of the gene(s) of interest. Fold change in expression was calculated using the comparative 2AAt lmethod.

[0093] BARD1 gene silencing by siRNA. siRNA targeted to BARD 1 , BRCA1 , and HIF 1 a were purchased from Sigma-Aldrich (Woodlands, TX). In vitro, transient transfection was performed as described previously. Briefly, siRNA (2 pg) was incubated with 6 pL of Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) for 20 minutes at room temperature and then added to cells cultured in 6-well plate at 60% confluence. siRNA with a nonspecific function that shared no sequence homology with any known mRNA in a BLAST search was used to control the target siRNA (Mission siRNA Universal Negative Control#l, Sigma). At selected time intervals, cells were harvested to measure mRNA levels of BARD1, BRCA1, ESMI, and FN1 using quantitative reverse-transcriptase PCR.

[0094] BARD1 overexpression. A BARD 1 -expressing OVCAR8 cell line was generated by transduction with a BARD1 plasmid (EX-Q0613-Lvl22, GeneCopoeia, Inc). Lentivirus particles containing BARD1 open reading frames were produced using the Lenti-Pac HIV Expression Packaging Kit protocol (HPK-LvTR-20; GeneCopoeia) and transduced into OVCAR8 cells (1 x 106) that were previously plated in a complete DMEM 10-cm culture dish and incubated overnight at 37 °C with 5% CO2. Virus particles and polybrene were diluted in Opti-MEM and added to the cells the following day, and the cells were incubated overnight at 37 °C with 5% CO2. The next day, the media was replaced with complete media, and after 48 hours, the infected cells were analyzed for transient expression of GFP-BARD1 using a fluorescent microscope. Antibiotic (Puromycin) selection was started for 6 days, and GFP+cells were then sorted by fluorescence-activated cell sorting and used for further experiments.

[0095] DNA methylation arrays. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples and were hybridized to the Illumina Infmium 450K Human DNA Methylation Beadchip array to obtain DNA methylation profiles according to the manufacturer’s instructions.

[0096] Microarray analysis. Total RNA was extracted from tumor samples of control IgG, Aza, B20, and combination groups using RNeasy mini kit (Qiagen). A NanoDrop spectrophotometer (Thermo Fisher Scientific) was used for the assessment of both RNA quality and RNA quantity. Total RNA (700 ng) was labeled and hybridized to Bead Chips according to the manufacturer’s protocols (Illumina, San Diego, CA). We scanned Bead Chips with an Illumina BeadArray Reader, and all array normalization, filtering, and statistical analysis were performed using BRB- Array Tools (National Cancer Institute).

[0097] RNA sequencing. Total RNA was extracted using Direct-zol RNA Miniprep Plus kit (ZYMO Research, Irvine, CA, USA) according to the manufacturer’ s protocol. RNA quality was determined by RNA integrity number using a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and the samples were shipped to Novogene (Sacramento, CA, USA) for RNA-Seq analysis on the Illumina NovaSeq 6000 platform. Gene expression level was calculated using the number of mapped reads. Fragments per kilobase of transcript sequence per millions of base pairs sequenced (FPKM) was used to estimate gene expression levels; this method takes into consideration the effects of both sequencing depth and gene length on counting of fragments. Downstream analysis was performed using a combination of programs, including hisat2, DEseq2, and ClusterProfiler software.

[0098] Promoter analysis and ChIP assay. ChIP assays were performed at the MD Anderson Cancer Center Epigenomics Profiling Core. SKOV3ipl-luc cells were cultured in hypoxic (1% oxygen) and normoxic (20% oxygen) conditions for 16 hours. Next, the cells were crosslinked with formaldehyde and subjected to ChIP with antibodies specific to HIFla, DNMT3A, AML-1, DNMT1A or normal IgG as control. ChIP assays were performed as described previously with modifications. DNMTla was used as negative and AML-1 was used as positive control. The DNA region of interest was detected by SYBR real-time quantitative PCR, and enrichment relative to input was calculated.

[0099] Proteome profiler human angiogenesis array. Supernatants from cells were collected and applied to the array membranes following the manufacturer’s instructions (R&D Systems, MN, U SA). The measurement of the signal was performed according to the instructions of the manufacturer and the pixel density of each array spot was analyzed using Imaged software program (National Institutes of Health).

[0100] In vivo studies. All mouse studies were approved by the Institutional Animal Care and Use Committee at MD Anderson. All animal experiments were performed with 4- to 6- week-old female athymic nude (NCr-nude) and NOD / SCID mice obtained from Taconic Biosciences (Rensselaer, NY). Five mice per cage were housed under pathogen-free conditions at a constant temperature and humidity. All mice were fed a regular diet (unless otherwise specified) and water ad libitum according to American Association for Laboratory Animal Science guidelines and the US Public Health Service Policy on Human Care and Use of Laboratory Animals. Mice were euthanized via cervical dislocation if found moribund by the investigators, who have approval from the IACUC committee. Female athymic nude mice (6- 8 weeks old) were intraperitoneally injected with 1 x 106SKOV3ipl-luc and SKOV3-TetOn- shBARDl. HT1080 cells were subcutaneously injected. MDA-OVCA-1 (PDX, IRB no: PAIS- 0441) chunks were implanted into the peritoneal cavity. Mice were randomly assigned to experimental groups after cell injection. For the SKOV3ipl model, adaptive resistance to AVA was established by injecting luciferase-labeled cells, as described previously. After 3 weeks, each mouse received initial treatments with a control IgG antibody or B20, a murine monoclonal VEGF-A antibody (5 mg / kg, injected intraperitoneally twice weekly), and tumor growth was monitored by IVIS imaging (Xenogen, Alameda, CA). After 3 weeks, the mice were divided into B20-sensitive and B20-resistant groups according to their responses to the drug.

[0101] To analyze the effect of knockdown of BARD 1 in an in vivo model, we injected 1x106SKOV3ipl-shBARDl-TetON cells intraperitoneally in 0.2 mL of Hank’s balanced saline solution. Mice were divided into 6 groups: standard diet groups are group 1 (UT), group 3 (shBARDl+Bev), group 5 (shBARDl + Bev + Aza); doxycycline-diet (200 mg / kg) groups are group 2 (UT), group 4 (shBARDl +Bev), group 6 (shBARDl + Bev + Aza).

[0102] Tumor growth was monitored, and mice were euthanized 6-10 weeks after inoculation. Samples were processed and stored for histopathologic analyses. Once mice in any group became moribund, all mice were euthanized by cervical dislocation. Individuals who performed the necropsies were blinded to the treatment group assignments. Tumor weights, nodule numbers, and the presence of ascites were recorded at the time of gross necropsy. All tumor tissues were dissected and samples were snap-frozen for later DNA, RNA, or protein analyses, fixed in formalin for paraffin embedding, or snap-frozen in optimal cutting temperature compound (Miles, Inc, Elkhart, IN), for frozen slide preparation.

[0103] Bevacizumab (Avastin) was obtained from MD Anderson Cancer Center pharmacy. The AVA B20 (Genentech) is a cross-species reactive, function-blocking monoclonal antibodytargeting both human and murine VEGF-A. Azacytidine (Vidaza) was obtained from MD Anderson Cancer Center pharmacy. Bevacizumab, B20 (5 mg / kg, twice per week), and Aza were administered intraperitoneally (5 mg / kg, three times per week).

[0104] Targeted demethylation liposomal delivery system. DOPC and dCas9-TETl- sgRNA-BARDl were mixed in the presence of excess tertiary butanol at a ratio of 1 : 10 (w / w) DNA / DOPC. Tween-20 was added to the mixture in a ratio of 1 : 19 Tween-20 :DNA / DOPC. The mixture was vortexed, frozen in an acetone / dry ice bath, and lyophilized. Before in vivo administration, this preparation was hydrated with phosphate-buffered saline without Ca+and Mg+2at a concentration of 50 or 25 pg / mL to achieve the desired dose of 100 pL per injection.

[0105] Statistical analyses. All in vitro experiments were done at least in technical triplicates with differences by group assessed by the Student / -test or analysis of variance (for comparison of all groups). For RNA sequencing, first, the raw read count was normalized (mainly to correct the sequencing depth); next, the statistical model was used to calculate the hypothesis test’s probability (P value). Finally, multiple hypothesis test corrections were used to obtain false discovery rate values. For in vivo experiments, tumor weight was analyzed by one-way analysis of variance, and groups were compared by analysis of variance. GraphPad Prism software (version 8.0.0) was used for all analyses. All data are presented as mean ± SD or mean ± SEM and two-sided unless otherwise indicated. The P values were two-tailed, and values less than 0.05 were considered significant.EXAMPLE 2. AZA RESTORES SENSITIVITY TO AVA

[0106] Given the lack of data on the role of demethylating drugs such as Aza on sensitivity to AVA therapy, an in vivo experiment was performed using the orthotopic SKOV3ipl-luc ovarian cancer mouse model. Tumor-bearing mice were treated with the anti-VEGF antibody B20 (targets both human and mouse VEGF) as monotherapy until they developed resistance to the drug and tumors were obtained at various time points that demonstrated either sensitivity or resistance. Treatment of the AVA-resistant mice continued with either B20 alone or combination of B20 and Aza (treated in twice weekly) (data not shown). To measure genomewide methylation levels from AVA-resistant tumors, tumor DNA samples were isolated from this model and used with the Illumina Infinium Human Methylation 450k BeadChip array that provides quantitative methylation measurement at the single-CpG-site level. It was found that multiple methylation events occurred with emerging resistance to AVA, which were neutralized by Aza alone as well as in combination treatment (data not shown).

[0107] To assess the efficacy of Aza along with AVA therapy, this combination was tested in a series of models. First, the HT1080 fibrosarcoma model was tested, which is well-known to be highly angiogenic. As shown in Figures 1A and IB, tumor weight and volume were significantly decreased in the combination group compared with the control and monotherapy groups, and no significant changes were observed in mouse body weight (FIG. 6A). The effect of Aza and B20 combination therapy was also tested in a platinum-resistant patient-derived xenograft (PDX) model, in which the PDX tumor was implanted into the peritoneal cavity of NOD / SCID mice. The mice were then treated with Aza or B20 monotherapies or in combination. Tumor weight (Figure 1C) and number of nodules (FIG. ID) were significantly decreased in the combination group compared with the control and monotherapy groups; the mouse body weight was lower in the treatment groups (FIG. 6B), reflecting lower ascites production (FIG. 6C). Similarly, in the orthotopic SKOV3ipl-luc ovarian cancer mouse model, the combination treatment of Aza and B20 led to significantly reduced tumor burden and number of nodules compared with monotherapies or control with no significant change in mouse body weight (FIGS. IE, IF, 6D).EXAMPLE 3. HIGH-THROUGHPUT ANALYSES REVEAL SIGNIFICANT GENOMIC CHANGES AFTER AZA THERAPY

[0108] Given the robust efficacy observed with Aza at the emergence of AVA resistance, both genetic and epigenetic differences were investigated among treatment groups in the SKOV3ipl-luc model described above by comparing gene expression (mRNA array) combined with a methylation array (HumanMethylation450 platform). The focus was on methylated loci with 0-value above 0.6 in AVA-resistant tissues, and 43,830 tumor-specific methylated loci (43,830 / 485,513, 9%) were selected to be compared between the B20 and combination groups. These results were overlaid with mRNA expression data. Genes altered by more than 1.5-fold were considered part of a candidate gene signature of AVA resistance. To identify genes that exhibited reduced methylation levels alongside elevated expression in the combination treatment group relative to the B20 monotherapy group, the methylation array data set was integrated with the gene expression data. Subsequently, a visual representation was generated highlighting the top genes based on their fold change (FIG. 2A). Among them, mRNA (FIG. 2B) and protein (FIG. 2D) levels of BARD1 were found to be increased in response to Aza therapy in adaptive AVA-resistant tumors from mice; moreover, this increase was significantly higher in the combination group (FIG. 7A). Thus BARD1 methylation and reduced expression was identified as a lead candidate with AVA resistance. Pathway analysisindicated that BARD1 has a role in DNA repair, homology-directed repair, and cell cycle (data not shown).

[0109] Before performing additional in vitro experiments to assess the biological effects of BARD1 knockdown or overexpression in ovarian cancer, the expression level was determined in normal fallopian tube epithelium (FTE), non-transformed ovarian surface epithelial cells (HIO180), and a panel of ovarian cancer cell lines (SKOV3ipl, SKOV3-TR, A2780ipl, HeyA8, OVCAR3, OVCAR5, and OVCAR8ipl). Under normoxic conditions (20% Ch at 37 °C), BARD1 mRNA (FIG. 2C) and protein (FIG.2E) expression levels were highest in the SKOV3ipl cells and lowest in OVCAR8ipl cells; therefore, the SKOV3ipl cell line was chosen for further knockdown experiments and OVCAR8ipl for overexpression experiments. Treatment of SKOV3ipl cells with siRNA targeting BARD1 resulted in >90% knockdown of BARD1 protein levels within 72 hours compared with a nontargeting (control) siRNA (FIGS. 7B-7C). BARD1 was also expressed in OVCAR8ipl cells using pReceiver-Lvl22 plasmid, which resulted in >7.5-fold higher BARD1 mRNA expression compared to parental cells (FIG. 7D) and 5-fold higher in protein level (FIG. 7E). Thus, BARD 1-2 siRNA and a stable OVCAR8-BARD1 -Clone-2 were selected for further experiments.EXAMPLE 4. BARD1 PLAYS A ROLE IN TUMOR ANGIOGENESIS

[0110] To determine whether the effects of BARD1 could be direct (i.e., endothelial cells) or indirect (mediated via cancer cells) on angiogenesis, BARD1 was silenced in RF24 endothelial cells using siRNA and assessed the ability of these cells to form tubes. No significant difference was observed in tube formation in cells treated with BARD1 siRNA compared with control siRNA (FIG. 3A). To assess for potential indirect effects, BARD1 was silenced in SKOV3ipl cells for 48 hours and the conditioned medium (CM) was collected. RF24 cells were cultured in the CM for 8 hours and then subjected to tube formation assay. The BARD1 siRNA medium-treated RF24 cells exhibited a significant increase in tube formation compared with control siRNA medium-treated cells (FIG. 3B), indicating that the effect of BARD 1 silencing is indirect on angiogenesis.[OHl] To understand the effect of BARD1 silencing on tumor angiogenesis pathways, RNA sequencing of BARD 1 -silenced SKOV3ipl cells was performed. Results showed that 952 genes were differentially expressed: 268 were downregulated and 684 were upregulated (DESeq2 padj<=0.05 |log2FoldChange|>=1.0) in the BARD1 siRNA-treated group compared with the control siRNA group. Among these genes, several pro-angiogenic genes, including FN1, ESMI, CXCL8, ETS1, PTX3, CCL 2, and others, were found to be upregulated. Moreover,enrichment analysis with Gene Ontology showed an increase in the angiogenesis pathway after BARD1 knockdown (FIG. 3D).

[0112] To further validate the role of BARD1 in tumor growth, the conditioned medium after BARD1 knockdown in SKOV3ipl cells was collected for an in vivo Matrigel plug assay. Silencing BARD1 resulted in significant increase in angiogenesis, as reflected by the highest hemoglobin level compared with other groups (FIG. 3C). To further confirm the effect of BARDI on angiogenesis, conditioned medium from BARD1 silenced SKOV3ip1 cells was tested with an array for angiogenesis-related proteins. Several angiogenic factors, such as IL- 8, uPA, PTX3, thrombospondin- 1 and others were more abundant when BARDI was silenced compared with the control (FIGS. 3E, 8A) Next, expression of BARDI was ectopically increased in OVCAR8ipl cells and then the cells were subjected to RNA sequencing. There was increased expression of several pro-angiogenic genes after BARDI silencing compared to reduction of these genes in BARDI -overexpressing cells (FIG. 8B).

[0113] To determine whether BARDI silencing affects tumor growth and progression, a TetOn system was developed for the SKOV3ipl model. Treatment of SKOV3ipl-shBARDl- TetOn cells with doxycycline for 24 hours resulted in >80% reduction in BARDI expression in vitro (FIG. 8C). The same TetOn model was used in vivo and it was found that knockdown of BARDI (animals received dox-containing food) led to increased tumor burden in the control group (BARDF) compared with BARD1+group. The same trend was observed for group 2 (BARDF compared with BARD1+group treated with bevacizumab [AV A] alone) and group 3 (BARDF compared with BARD1+treated with bevacizumab and Aza; FIG. 3F). Tumor weight and BARDI expression were inversely correlated (FIG. 3G) and no significant change was observed in mouse body weight (FIG. 8D).

[0114] BARD I is an obligatory partner of breast cancer type 1 susceptibility' protein (BRCAl), and there is evidence that BARDI is also a tumor suppressor. To test the hypothesis that BARDI has an independent role in angiogenesis, BARDI and BRCAl knockdown groups were compared in vitro and it was found that the expression of BARDI and BRCAl was positively correlated (FIGS. 8E-8F). BARDI knockdown resulted in significant changes in mRNA expression levels of pro-angiogenic genes ESMI and FN1 whereas no significant differences were observed after BRCAl knockdown (FIGS. 8G-8H). The mRNA expression of BARDI and ESMI was compared in high-grade serous ovarian cancer samples from the publicly available TCGA database, and it was determined that they are positively correlated with each other (R=0.36, p value= 2.5e-14) (FIG. 81).

[0115] To determine if the effect of BARD1 has a role independent of BRCA1 on angiogenesis, BARD I and BRCA1 were silenced in SKOV3ipl cells using siRNA for 48 hours and the conditioned medium was collected. Then, RF24 cells were cultured in the conditioned medium for 8 hours following subjected to tube formation assay. As shown in FIG. 8 J, RF24 cells treated with BARD1 silenced-conditioned medium showed a signficant increase in tube formation compared with control siRNA medium-treated cells; whereas no changes were observed in cells treated with BRC Al -silenced conditioned medium, indicating that. BARD I has a BRC Al -independent role on angiogenesis.EXAMPLE 5. BARD1 EPIGENETIC MODULATION OCCURS UNDER HYPOXIC CONDITIONS

[0116] Because AVA therapy leads to progressive hypoxia in the tumor, the mechanisms by which DNMT3A and BARD1 expression could be altered in hypoxic conditions were investigated. BARD1 mRNA and protein expression levels in SKOV3ipl cells under hypoxic conditions (1% O2) were determined, and it was determined that BARD1 expression levels were significantly decreased compared to normoxic conditions (FIGS. 4A-4B, 4E-4F). Extending this finding further, SKOV3ipl cells were incubated with a HIF la-stabilizing compound (C0CI2 for 24 hours under normoxic conditions), and it was determined that BARD1 mRNA expression levels were significantly lower after 24 hours of C0CI2 incubation (FIGS. 9A-9B).

[0117] To determine whether HIF la blockade would abrogate the effects of hypoxia on BARD1, a significant increase in BARD1 mRNA expression was observed compared with untreated and control siRNA-treated cells when silenced HIF la using siRNA in SKOV3ipl cells under hypoxic conditions and (FIGS. 4G, 9C). Next, to determine whether DNMT3A is altered in hypoxic conditions, DNMT3A expression levels were checked and found to be higher under hypoxic conditions (FIGS. 4C). Furthermore, HIFla siRNA-treated cells showed decreased DNMT3 A mRNA expression (FIG. 4D). DNMT3 A levels were positively correlated with HIFla expression in human ovarian cancer samples (FIG. 9D, TCGA). Next, promoter analysis and chromatin immunoprecipitation (ChIP) assay were performed. The binding sites for HIFla were checked within a 2-kb region spanning the DNMT3A promoter using TF SEARCH, and AML1 was chosen as positive control for the binding and DNMTla as negative control. Upon promoter analysis, HIFl a was predicted to bind to the DNMT3A promoter region. ChIP assays revealed that the binding of HIFla to the promoter region wassignificantly higher under hypoxic conditions than under normoxic conditions, suggesting that the increase in DNMT3 A under hypoxic conditions is HIF la-dependent (FIG. 4H).

[0118] To investigate whether hypoxic conditions affect BARD1 expression, SKOV3ipl cells were exposed to hypoxia (1% Ch or treatment with CoCh) for 48 hours. To understand whether HIFla is a transcription factor of BARD 1, SKOV3ipl cells were first exposed to hypoxia for 48 hours and a ChIP assay was performed. HIFla binding to the promoter region of BARD1 at sites 1 and 4 was significantly enriched under hypoxic conditions compared with normoxic conditions (FIGS. 41, 9E). It was also checked whether DNA demethylases are altered by hypoxia. The mRNA expression level of TET1 was reduced under low oxygen conditions (FIG. 4J). Moreover, HIFla siRNA-treated cells had increased TET1 mRNA expression (FIG. 9F).EXAMPLE 6. LIPOSOMAL DEACTIVATED CAS9-CRISPR SYSTEM DEMETHYLATES BARD1

[0119] Multiple CpG island sites were identified in the BARD1 gene (cg26049092, cg08563601, cg24607575, cg24715680) from adaptive AVA-resistant tumors in the methylation array, and that methylation is associated with reduced BARD1 expression. The molecular mechanism of this methylation regulation is still unknown. Therefore, the effects of manually inducing demethylation were tested. To accomplish this, a nanoengineered demethylation system targeting BARD1 was developed. To directly demethylate BARD1, a liposomal dCas9-TETl-sgBARDl in a single expression plasmid vector was used. Four pairs of sgRNAs were designed to target BARD1, one against the gene body and three against the transcription start site (TSS), and these sgRNAs were cloned into each dCas9-TETl vector, under the CMV promoter to drive expression. These sgRNAs were tested in vitro by transfecting SKOV3ipl cells and selection of sgRNA-1 and -3 for further studies (FIG. 9G). The dCas9-TETl-sgBARDl plasmids were scaled up and produced for in vivo experiments. These plasmids were incorporated into the neutral lipid l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC).

[0120] To determine whether targeted demethylated BARD1 would have antitumor effects in vivo, an orthotopic SKOV3ipl-luc ovarian cancer mouse model was used. Mice were treated with DOPC-dCas9-TETl-sgcontrol- (control) and DOPC-dCas9-TETl-sgBARDl-3 (sgBARDl-3) given IV for 6 weeks. Tumor weight in the sgBARDl-3 group was reduced compared with the control group (FIG. 4K) with no significant change in mouse body weight(FIG. 9H). BARD1 expression was significantly increased in sgBARDl-3 treated group compared with control (FIG. 4L).

[0121] Next, to determine whether demethylated BARD1 would combine well with AVA therapy in the SKOV3ipl model, sgBARDl-3 was combined with bevacizumab (Bev), and the results showed that sgBARDl-3 alone led to a significant decrease in tumor burden compared with untreated or control groups. Moreover, tumor burden was significantly reduced in the sgBARDl-3 and bevacizumab combination group compared with the sgcontrol group and bevacizumab alone group (FIGS. 4M-4N). No significant change was observed in mouse body weight in any group (FIG. 91). BARD1 expression was significantly increased in sgBARDl -3- treated and in combination tumor samples compared with controls (FIG. 9 J). To check for any potential off-target effects, the efficacy of another sgBARDl was tested. SKOV3ipl tumorbearing mice were treated with DOPC-dCas9-TETl -sgcontrol- (control) and DOPC-dCas9- TETl-sgBARDl-1 (sgBARDl-1) for 5 weeks. Tumor burden in the sgBARDl-1 group was significantly reduced compared with the control group (FIG. 40). No significant change was observed in mouse body weight in any group (FIG. 9K).EXAMPLE 7. BARD1 TARGETED APPROACH RESULTS IN ROBUST ANTITUMOR EFFECTS

[0122] The data from preceding examples identified epigenetic modulation as a fundamental mechanism of adaptive resistance to AVA. In particular, BARD1 leads to profound upregulation of alternative pro-angiogenic pathways (FIG. 5). A highly specific liposomal dCas9-TETl -sgBARDl approach was developed and tested for targeted BARD1 CpG demethylation. This resulted in BARD 1 -specific demethylation in vivo (without impacting other genes), which had a robust antitumor effect in combination with AVA. This combined therapy has the potential to expand the utility and efficacy of bevacizumab in treating ovarian cancer, particularly in AVA-resistant disease.

[0123] Anti-VEGF drugs have been approved for the treatment of various cancer types. Although improvements in objective response and progression-free survival have been observed with anti-angiogenic therapy, its impact on overall survival for patients is limited owing to various factors, including the development of resistance. The mechanisms of this adaptive resistance are likely diverse and not fully understood. Accumulating evidence indicates that alterations in the tumor microenvironment are important for facilitating this adaptive response. Stromal cell recruitment might also underlie resistance to AVA. We and others have focused on interactions between various cell types such as endothelial cells,platelets, pericytes, macrophages and cancer-associated fibroblasts in the context of the response to AVA.

[0124] The present findings indicate that alterations in DNA methylation patterns contribute to the development of resistance to AVA. Epigenetic therapies hold promise in cancer treatment, yet their broad effects can result in undesirable toxicities such as hematologic abnormalities, gastrointestinal disturbances, or the potential for secondary malignancies. To overcome these challenges, a highly specific liposomal dCas9-TETl-sgRNA delivery system was developed and tested, which minimized off-target effects and nonspecific toxicities. This strategy enhances treatment specificity and reduces toxicity linked to broad epigenetic drugs. By reversing abnormal DNA methylation, site-specific demethylation could restore key gene expression in AVA response, enhancing tumor cell sensitivity to AVA. This approach could counteract AVA resistance mechanisms, ultimately enhancing overall treatment efficacy. Combining epigenetic and AVA therapies offers synergy, sensitizing tumor cells to AVA, augmenting response, and overcoming AVA resistance.

[0125] This study is the first systematic approach to combine demethylation agents with bevacizumab to overcome AVA resistance. Moreover, a targeted demethylation approach was successful in preclinical models of ovarian cancer. These findings identified a plausible predictive marker for response to AVA therapies and demonstrated the potential use of demethylating drugs for reversing resistance to AVA. Collectively, the findings represent a significant advance in gene-specific epigenetic editing, with broad implications and high translational potential for drugs to improve the therapeutic outcomes of patients with cancer.EXAMPLE 8. ANTITUMOR EFFECT IS SPECIFIC TO BARD1

[0126] Further experiments were performed to determine if the robust antitumor effects of the targeted approach were specific to BARD 1 ’ s role in AVA resistance. CHMP4A and CRIP 1 showed no significant changes across treatment groups, suggesting a limited role in adaptive resistance mechanisms. These findings further support BARDl’s relevance in Aza-induced adaptive resistance, distinguishing it from other candidates (FIGS. 11 A-l IB).

[0127] To further investigate the functional relevance of BARD1, CHMP4A, and CRIP1 in ovarian cancer, a comparative analysis was conducted focusing on their potential roles in angiogenesis. BARD1 was prioritized due to its well-established involvement in DNA repair, tumor suppression, and its interaction with VEGF signaling pathways, which are critical for tumor angiogenesis. In contrast, the functional impact of CHMP4A and CRIP1 in this context remained unclear. To assess their potential roles, SKOV3ipl cells were transfected withCHMP4A or CRIP1 siRNA, alongside control siRNA and untreated (UT) groups. qPCR analysis confirmed efficient knockdown of CHMP4A and CRIP1 48 hours post-transfection (FIGS. 12A-12B). Supernatants collected after 48 hours were subjected to an angiogenesis array, which revealed no significant changes in angiogenic factor secretion across groups. These findings suggest that CHMP4A and CRIP1 do not significantly influence angiogenesis, further supporting the selection of BARD 1 as a key candidate for further investigation, given its functional relevance to tumor progression and response to anti-vascular therapies (AV A).

[0128] To determine if there were differences in the secretion of angiogenic factors, conditioned media were collected from SKOV3ipl cells transfected with CHMP4A siRNA, CRIP1 siRNA, control siRNA, or left untreated (UT). No significant differences in angiogenesis-related protein levels were detected between groups (FIG. 13).

[0129] To understand the molecular mechanisms underlying adaptive resistance to anti- VEGF (AV A) therapy, BARD1 expression and methylation changes were examined in this context. Since anti-VEGF therapy primarily targets the tumor microenvironment, experiments were conducted to determine whether BARD1 regulation is involved in this adaptive process. However, to assess whether BARD1 expression changes were specific to AVA resistance or part of a broader resistance mechanism, BARD1 mRNA levels following cisplatin (HeyA8) and paclitaxel (HeyA8-MDR) treatment in vivo also were studied. qPCR analysis revealed no significant changes in BARD1 expression between chemotherapy -treated and control groups, suggesting that BARD1 upregulation is specific to AVA resistance rather than a generalized response to cytotoxic agents (FIGS. 14A-14B).

[0130] To generate a stable BARD 1 -overexpressing cell line, OVCAR8ipl cells were transduced with lentiviral particles (CLP-Q0613 -Lv 122-200, GeneCopoeia), encoding full- length BARD1 under a CMV promoter with puromycin resistance. After selection, BARD1 overexpression was confirmed at mRNA level. Next, an in vivo study was performed using OVCAR8ipl cells to evaluate the impact of BARD 1 overexpression on tumor growth. The results indicate that tumors in the BARD 1 -expressing group exhibited a significant reduction in tumor burden compared to wild-type (WT) (p value= 0.0103; n = 10 mice / group). No significant differences in mouse body weight were observed. These findings support the tumorsuppressive role of BARD1 in this model (FIGS. 15A-15C).

[0131] To determine whether BARD1 modulation influences in vivo tumor growth and therapeutic response independently of BRCA1, an in vivo study was performed using the BRCAl-mutant COV362ipl model, which harbors a frameshift mutation (BRCA1 c.2288delT, p.Val763fs) leading to loss of function. Mice were treated with azacytidine (Aza), B20 (anti-VEGF therapy), or the combination (Aza+B20), and tumor burden was assessed. Tumor weight was significantly reduced in the combination group compared to controls (p < 0.0001), indicating a potential synergistic effect of Aza and B20 in this BRC Al -deficient model. Additionally, a significant decrease in mouse weight was observed in the combination group; however, this reduction was primarily associated with a decrease in ascites volume rather than systemic toxicity (FIGS. 16A-16C). These findings suggest that BARD1 modulation may impact tumor progression and response to anti-VEGF therapy in BRC Al -deficient tumors, further supporting its role in tumor biology.

[0132] To assess the impact of Aza and AVA (B20) treatment on BARD1 expression in the BRCAl-mutant COV362ipl model, BARD1 mRNA expression on tumors from mice treated with control or the combination (Aza+AVA) was determined. BARD1 expression was significantly increased in the combination group compared to control (FIG. 17).

[0133] To assess the specificity of BARD1 demethylation in vivo, an in-silico analysis was performed to evaluate potential off-target effects of the gRNAs used in the study. The exact locations of each gRNA were validated and assessed for possible unintended binding sites to ensure specificity. Using BLAST, it was confirmed that each gRNA overlapped with the CpG sites of interest, aligning with the intended target regions. Additionally, Cas-OFFinder was employed to predict potential off-target binding sites, identifying only a limited number of sites within a two-base mismatch tolerance, with most located in non-coding regions or introns (data not shown). Specifically, for gRNA-3, which was primarily used in the study, off-target predictions were identified at Chr6 (intergenic), ChrlO (R3HCC1L, last intron), Chrl9 (LSM14A, intron), and Chr3 (intergenic). Notably, all Chr2 off-target sites overlapped with the intended BARD1 target region, further supporting the specificity of the approach. These findings confirm that the observed BARD1 demethylation was target-specific without significantly altering the methylation status of other genes, supporting the conclusion that the system achieved BARD 1 -specific epigenetic modulation in vivo (data not shown).

[0134] Disclosed herein are materials, compositions, and methods that can be used for, can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can bemade to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

[0135] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The following description provides further non-limiting examples of the disclosed compositions and methods.REFERENCES1 Mei, C. et al. Anti-angiogenic therapy in ovarian cancer: Current understandings and prospects of precision medicine. Front Pharmacol 14, 1147717 (2023). https: / / doi.org: 10.3389 / fphar.2023.11477172 Siegel, R. L., Miller, K. D., Wagle, N. S. & Jemal, A. Cancer statistics, 2023. CA Cancer J Clin 73, 17-48 (2023). https: / / doi.org: 10.3322 / caac.217633 Lengyel, E. Ovarian cancer development and metastasis. Am J Pathol 177, 1053-1064 (2010). https: / / doi.org: 10.2353 / ajpath.2010.1001054 Perren, T. J. et al. A phase 3 trial of bevacizumab in ovarian cancer. N Engl J Med 365, 2484-2496 (2011). https: / / doi.org: 10.1056 / NEJMoal 1037995 Casanovas, O., Hicklin, D. J., Bergers, G. & Hanahan, D. Drug resistance by evasion of anti angiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 8, 299-309 (2005). https: / / doi.org: 10.1016 / j.ccr.2005.09.0056 Monk, B. J., Minion, L. E. & Coleman, R. L. Anti -angiogenic agents in ovarian cancer: past, present, and future. Ann Oncol 27 Suppl 1, i33-i39 (2016). https: / / doi.org: 10.1093 / annonc / mdw0937 Bergers, G. & Hanahan, D. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 8, 592-603 (2008). https: / / doi.org: 10.1038 / nrc24428 Eichten, A. et al. Resistance to Anti-VEGF Therapy Mediated by Autocrine IL6 / STAT3 Signaling and Overcome by IL6 Blockade. Cancer Res 76, 2327-2339 (2016). https: / / doi.org: 10.1158 / 0008-5472. CAN-15-14439 Baker, E. K. & El-Osta, A. The rise of DNA methylation and the importance of chromatin on multi drug resistance in cancer. Exp Cell Res 290, 177-194 (2003). https: / / doi.org: 10.1016 / s0014-4827(03)00342-210 Chen, C. C. et al. Changes in DNA methylation are associated with the development of drug resistance in cervical cancer cells. Cancer Cell Int 15, 98 (2015). https: / / doi.org: 10.1186 / sl2935-015-0248-311 Zeller, C. et al. Candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer identified by methylome and expression profiling. Oncogene 31, 4567-4576 (2012). https: / / doi.org: 10.1038 / onc.2011.611Fu, S. et al. Phase lb-2a study to reverse platinum resistance through use of a hypomethylating agent, azacitidine, in patients with platinum-resistant or platinum- refractory epithelial ovarian cancer. Cancer 117, 1661-1669 (2011). https: / / doi.org: 10.1002 / cncr.25701Glasspool, R. M. et al. A randomised, phase II trial of the DNA-hypomethylating agent 5- aza-2'-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer. Br J Cancer 110, 1923- 1929 (2014). https: / / doi.org: 10.1038 / bjc.2014.116Plumb, J. A., Strathdee, G., Sludden, J., Kaye, S. B. & Brown, R. Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLHl gene promoter. Cancer Res 60, 6039-6044 (2000).Pohlmann, P. et al. Phase II trial of cisplatin plus decitabine, a new DNA hypomethylating agent, in patients with advanced squamous cell carcinoma of the cervix. Am J Clin Oncol 25, 496-501 (2002). https: / / doi.org: 10.1097 / 00000421-200210000-00015Worm, J., Kirkin, A. F., Dzhandzhugazyan, K. N. & Guldberg, P. Methylation-dependent silencing of the reduced folate carrier gene in inherently methotrexate-resistant human breast cancer cells. J Biol Chem 276, 39990-40000 (2001). https: / / doi.org: 10.1074 / jbc.M103181200Misra, R. M., Bajaj, M. S. & Kale, V. P. Vasculogenic mimicry of HT1080 tumour cells in vivo: critical role of HIF-1 alpha-neuropilin- 1 axis. PLoS One 7, e50153 (2012). https: / / doi. org: 10.1371 / journal. pone.0050153Gorodetska, I., Kozeretska, I. & Dubrovska, A. BRCA Genes: The Role in Genome Stability, Cancer Sternness and Therapy Resistance. J Cancer 10, 2109-2127 (2019). https: / / doi.org: 10.7150 / jca.30410Mersch, J. et al. Cancers associated with BRCA1 and BRCA2 mutations other than breast and ovarian. Cancer 121, 269-275 (2015). https: / / doi.org: 10.1002 / cncr.29041Tarsounas, M. & Sung, P. The antitumorigenic roles of BRCA1-BARD1 in DNA repair and replication. Nat Rev Mol Cell Biol 21, 284-299 (2020). https: / / doi.org: 10.1038 / s41580- 020-0218-zTang, Z. et al. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45, W98-W102 (2017). https: / / doi.org: 10.1093 / nar / gkx247Gao, X. N. et al. AML1ZETO cooperates with HIFlalpha to promote leukemogenesis through DNMT3a transactivation. Leukemia 29, 1730-1740 (2015). https: / / doi.org: 10.1038 / leu.2015.56Wagner, M. J. et al. Preclinical Mammalian Safety Studies of EPHARNA (DOPC Nanoliposomal EphA2-Targeted siRNA). Mol Cancer Ther 16, 1114-1123 (2017). https: / / doi.org: 10.1158 / 1535-7163.MCT-16-0541Glassman, D. et al. Exploiting metabolic vulnerabilities after anti-VEGF antibody therapy in ovarian cancer. iScience 26, 106020 (2023). https: / / doi.org: 10.1016 / j.isci.2023.106020 Ma, S. et al. The role of tumor microenvironment in resistance to anti -angiogenic therapy. FlOOORes 7, 326 (2018). https: / / doi.org: 10.12688 / flOOOresearch.11771.1Vasudev, N. S. & Reynolds, A. R. Anti -angiogenic therapy for cancer: current progress, unresolved questions and future directions. Angiogenesis 17, 471-494 (2014). https: / / doi.org: 10.1007 / sl0456-014-9420-yHartmann, S., Bhola, N. E. & Grandis, J. R. HGF / Met Signaling in Head and Neck Cancer: Impact on the Tumor Microenvironment. Clin Cancer Res 22, 4005-4013 (2016). https: / / doi. org: 10.1158 / 1078-0432. CCR-16-0951Dalton, H. J. et al. Macrophages Facilitate Resistance to Anti-VEGF Therapy by Altered VEGFR Expression. Clin Cancer Res 23, 7034-7046 (2017). https: / / doi.org: 10.1158 / 1078- 0432.CCR- 17-0647Huijbers, E. J. etal. Role of the tumor stroma in resistance to anti-angiogenic therapy. Drug Resist Updat 25, 26-37 (2016). https: / / doi.org: 10.1016 / j.drup.2016.02.002LaFargue, C. J. et al. Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L. Nat Commun 14, 2407 (2023). https: / / doi.org: 10.1038 / s41467-023-36910-5Kaminskas, E., Farrell, A. T., Wang, Y. C., Sridhara, R. & Pazdur, R. FDA drug approval summary: azacitidine (5-azacytidine, Vidaza) for injectable suspension. Oncologist 10, 176-182 (2005). https: / / doi.org: 10.1634 / theoncologist.10-3-176Schwartsmann, G. et al. A phase I trial of cisplatin plus decitabine, a new DNA- hypomethylating agent, in patients with advanced solid tumors and a follow-up early phase II evaluation in patients with inoperable non-small cell lung cancer. Invest New Drugs 18, 83-91 (2000). https: / / doi.org: 10.1023 / a: 1006388031954Sweder, K. Recent advances in understanding / assessing toxicity to the epigenome. FlOOORes 6, 96 (2017). https: / / doi.org: 10.12688 / fl000research.9649.1Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001). https: / / doi.org: 10.1006 / meth.2001.1262Landen, C. N., Jr. et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 65, 6910-6918 (2005). https: / / doi.org: 10.1158 / 0008-5472.CAN-05-0530Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5, 621-628 (2008). https: / / doi.org: 10.1038 / nmeth.1226Jain, A. K. et al. LncPRESSl Is a p53-Regulated LncRNA that Safeguards Pluripotency by Disrupting SIRT6-Mediated De-acetylation of Histone H3K56. Mol Cell 64, 967-981 (2016). https: / / doi.org: 10.1016 / j.molcel.2016.10.039Van, H. T. et al. Methyl-lysine readers PHF20 and PHF20L1 define two distinct gene expression-regulating NSL complexes. J Biol Chem 298, 101588 (2022). https: / / doi.org: 10.1016 / j.jbc.2022.101588SEQUENCES

Claims

CLAIMS1. A construct comprising: a) a nucleic acid encoding a guide ribonucleic acid (gRNA) that hybridizes to a breast cancer 1 (BRCAl)-associated RING domain 1 (BARD1) nucleic acid; and b) a nucleic acid encoding a fusion polypeptide comprising the RNA-guided dCas9 operably linked to a demethylation domain.

2. The construct of claim 1, wherein the nucleic acid encoding the gRNA comprises a nucleic acid having at least 90% sequence identity with any one of SEQ ID NOs: 1-4.

3. The construct of claim 1, wherein the nucleic acid encoding the gRNA comprises any one of SEQ ID NOs: 1-4.

4. The construct of any one of claims 1-3, wherein the dCas9 comprises an amino acid sequence encoded by a nucleic acid having at least 90% identity with SEQ ID NO: 6.

5. The construct of any one of claims 1-3, wherein the dCas9 is encoded by a nucleic acid that comprises SEQ ID NO: 6.

6. The construct of any one of claims 1-5, wherein the demethylation domain comprises a ten-eleven translocation methylcytosine dioxygenase 1 (TET1) catalytic domain.

7. The construct of claim 6, wherein the TET1 catalytic domain comprises an amino acid sequence encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:7.

8. The construct of claim 6, wherein the TET1 catalytic domain comprises an amino acid sequence encoded by a nucleic acid that comprises SEQ ID NO: 7.

9. The construct of any one of claims 1-8, further comprising a first promoter operably linked to the nucleic acid encoding the gRNA and / or a second promoter operably linked to the nucleic acid encoding the fusion polypeptide.

10. A vector comprising the construct of any one of claims 1-9.

11. The vector of claim 10, wherein the vector is a plasmid, phage, or virus.

12. The vector of claim 10 or 11, wherein the vector comprises SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

13. A composition comprising the construct of any one of claims 1-9 or the vector of any one of claims 10-12, and a pharmaceutically acceptable carrier.

14. The composition of claim 13, wherein the composition further comprises a neutral lipid.

15. The composition of claim 14, wherein the neutral lipid is l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC).

16. A composition comprising a complex for modulating methylation of a breast cancer 1 (BRCAl)-associated RING domain 1 (BARD1) nucleic acid in a cell, comprising: a) a guide RNA (gRNA) that hybridizes to a BARD1 nucleic acid in the genome of the cell and interacts with an RNA-guided nuclease dead Cas9 (dCas9); and b) a fusion polypeptide comprising the RNA-guided dCas9 operatively linked to a demethylation domain, wherein the gRNA and the fusion polypeptide are linked, wherein the RNA-guided dCas9 interacts with the gRNA and specifically binds to the BARD1 nucleic acid, and wherein the demethylation domain operatively linked to the dCas9 modulates demethylation of the BARD1 nucleic acid.

17. The composition of claim 16, comprising two or more distinct complexes, wherein each distinct complex comprises a distinct BARD1 gRNA and the fusion polypeptide.

18. The composition of claim 16 or 17, wherein the gRNA and the fusion polypeptide are covalently linked by chemical crosslinking or UV cross-linking.

19. The composition of any one of claims 16-18, further comprising a pharmaceutically acceptable carrier.

20. The composition of any one of claims 16-19, comprising a neutral lipid.

21. The composition of claim 20, wherein the neutral lipid is l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC).

22. A cell comprising the composition of any one of claims 13-21.

23. The cell of claim 22, wherein methylation of BARD1 in the cell is decreased by introduction of the composition in the cell.

24. A method of treating a subj ect in need thereof, comprising administering to a cell of the subject a therapeutically effective amount of the composition of any one of claims 13-21.

25. The method of claim 24, wherein the subject has a disease or condition having hypermethylation of a BARDl gene.

26. The method of claim 24 or 25, wherein the cell is ex vivo.

27. The method of any one of claims 24-26, wherein the subject has cancer.

28. The method of claim 27, wherein the cancer is selected from a group consisting of lymphoma, myeloma, breast cancer, colon cancer, colorectal cancer, lung cancer, skin cancer, pancreatic cancer, renal (kidney) cancer, testicular cancer, bladder cancer, cervical cancer, ovarian cancer, uterus cancer, prostate cancer, head and neck, laryngeal cancer, nasopharyngeal cancer, gastric cancer, adrenal cancer, follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), leukemia, chronic lymphocytic leukemia (CLL), and marginal zone lymphoma.

29. The method of claim 27, wherein the cancer is ovarian cancer.

30. The method of any one of claims 27-29, wherein the subject has a primary cancer.

31. The method of any one of claims 27-29, wherein the subject has metastatic cancer.

32. The method of any one of claims 27-29, wherein the subject has relapsed or recurrent cancer.

33. The method of any one of claims 27-29, wherein the subject has cancer that has not responded to or has become resistant to an anti-cancer therapy.

34. The method of claim 33, wherein the subject has become resistant to anti-vascular endothelial growth factor (VEGF) therapy.

35. The method of claim 34, wherein the anti -VEGF therapy is an anti-VEGF antibody.

36. The method of claim 35, where the anti-VEGF antibody is bevacizumab.

37. The method of any one of claims 24-36, further comprising administering to the subject at least a second anti-cancer therapy.

38. The method of claim 37, wherein the second anti-cancer therapy is chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy, radioimmunotherapy, phototherapy, gene therapy, surgery, hormonal therapy, epigenetic modulation, anti-angiogenic therapy, or cytokine therapy.

39. The method of claim 37 or 38, wherein the composition of any one of claims 13-21 and the second anti-cancer therapy are administered simultaneously.

40. The method of claim 37 or 38, wherein the composition of any one of claims 13-21 and the second anti-cancer therapy are administered sequentially.

41. The method of any one of claims 24-40, wherein the methylation of BARD 1 in the subject is decreased after administration of the composition.

42. A method of making a complex for modulating methylation of a breast cancer 1 (BRCAl)-associated RING domain 1 (BARD1) nucleic acid in a cell, comprising linking a BARD1 guide RNA(gRNA) with a fusion polypeptide; wherein the BARD1 gRNA hybridizes to a BARD1 nucleic acid and interacts with an RNA-guided nuclease dead Cas9 (dCas9); wherein the fusion polypeptide comprises the RNA-guided dCas9 operatively linked to a demethylation domain; wherein the RNA-guided dCas9 interacts with the gRNA and specifically binds to the BARD1 nucleic acid; and wherein the demethylation domain demethylates the BARD1 nucleic acid.

43. The method of claim 42, wherein the gRNA and the fusion protein are covalently linked by chemical crosslinking or UV crosslinking.

Images