Inhibiting cancer chemoresistance and metastasis by targeting doublecortin-like kinase 1 (DCLK1)

Abstract

A method of treating a Doublecortin-like kinase 1 (DCLKl)-expressing cancer and inhibiting tumor metatastasis in a subject in need of such treatment by (1) providing a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1, (2) providing a therapeutically effective amount of a platinum-based anticancer drug, and (3) administering to the subject a combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug. The SAMiRNA nanoparticle and the platinum-based anticancer drug may be administered together or separately.

Description

ELECTRONICALLY TRANSMITTED: JUNE 21, 2024 INHIBITING CANCER CHEMORESISTANCE AND METASTASIS BY TARGETING DOUBLECORTIN-LIKE KINASE 1 (DCLK1) STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] Not Applicable. CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63 / 509,946, filed June 23, 2023, the content of which is hereby expressly incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The instant application contains, as a separate part of the present disclosure, a Sequence Listing which has been submitted via EFS-Web in computer readable form as an XML file. The Sequence Listing, created June 19, 2024, is named “5835.227wo Sequence Listing.xml,” and is 21,275 bytes in size. The entire contents of the Sequence Listing are hereby incorporated herein by reference. BACKGROUND

[0004] Epithelial ovarian cancer (OvCa) is the fifth leading cause of cancer-related deaths among women (1). Among the different histological subtypes, high-grade serous ovarian carcinoma (HGSOC) is the most lethal and the most prevalent accounting for more than 75% of the cases (1). First-line treatment includes cytoreductive surgery in combination with platinum- and taxane-based chemotherapy followed by targeted maintenance therapy in specific populations. However, 60-80% of patients relapse within 12 to 18 months of initial response primarily due to the emergence of resistance to chemotherapeutics (2, 3). Platinum- resistant OvCa patients have a dismal median survival of 9 to 12 months (4). Thus, it is important to understand the molecular mechanisms associated with chemoresistance to improve therapeutic outcomes in these patients.

[0005] Doublecortin-like kinase 1 (DCLK1) is a serine / threonine kinase known to regulate microtubule polymerization, neurogenesis, and neuronal migration. It exists as two mainisoforms i.e., long (DCLK1-L, ∼80 to 82 kDa) and short form (DCLK1-S, ∼45–50 kDa); where the long form has a microtubule-binding domain and a kinase domain, while the short form only contains the kinase domain (5, 6). DCLK1 overexpression and dysregulation is associated with malignant progression and metastasis in kidney, lung, bladder, and esophageal cancers (7, 8, 9, 10, 11, 12). DCLK1 is also recognized as a putative tumor stem cell marker in colorectal and pancreatic carcinogenesis (13, 14). Several studies have demonstrated the role of DCLK1 in modulating tumor cell pluripotency, drug resistance, epithelial-to-mesenchymal transition (EMT) (15), and tumor immunity through the recruitment of immunosuppressive myeloid-derived suppressor cells (MDSCs) (16). However, the role of DCLK1 in pathophysiology, chemotherapeutic response, and recurrence in HGSOC disease has been largely unknown. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Aspects of several embodiments of the present disclosure are illustrated in the appended drawings. The appended drawings only illustrate several embodiments and are not intended to be limiting of the scope of the inventive concepts disclosed herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0007] FIG.1A shows a representative western blot for total DCLK1 expression (DCLK1- L and DCLK1-S) in whole cell lysates (WCL) derived from ovarian cancer (OvCa) cells compared to fallopian tube epithelial cells (FTE187) and human ovarian surface epithelial cells (HOSE) when grown under adherent / 2D conditions; GAPDH – loading control, n≥3.

[0008] FIG.1B shows a representative western blot for total DCLK1 expression (DCLK1- L and DCLK1-S) in whole cell lysates (WCL) derived from ovarian cancer (OvCa) cells compared to fallopian tube epithelial cells (FTE187) and human ovarian surface epithelial cells (HOSE) when grown under suspended / 3D conditions; GAPDH – loading control, n≥3.

[0009] FIG.1C shows a representative western blot of DCLK1 in WCLs from OvCa cells and FTE187 cells comparing expression under 2D and 3D conditions, n≥3.

[0010] FIG. 1CA shows that DCLK1 is over-expressed in cisplatin-resistant spheroids. mRNA expression of DCLK1 in OVCAR-8 and OVCAR-8 CPR spheroids as quantified by RNA sequencing (Microarray data available at the Gene Expression Omnibus database, GSE45553). Statistical analysis was performed using a two-tailed unpaired t-test. ****p<0.0001.

[0011] FIG. 1D shows a representative western blot for total DCLK1 expression in WCL derived from cisplatin-sensitive (parental, P) and cisplatin-resistant (CPR) OvCa cell lines, β- actin – loading control, n≥3.

[0012] FIG.1E shows representative images of IHC staining for DCLK1 in a tumor tissue microarray (TMA) containing major histological subtypes of epithelial ovarian cancers. Scale bar - 50 μm, 100 μm (inset).

[0013] FIG.1F shows the quantification of the results of FIG.1E.

[0014] FIG. 1G shows a Kaplan-Meier survival plot showing higher DCLK1 expression associated with shorter progression-free survival in serous OvCa patients. Plot was created in KM Plotter (http: / / kmplot.com). Statistical analysis was performed using a log-rank test where p<0.05 was considered significant. HR, hazard ratio.

[0015] FIG. 1H shows a Kaplan-Meier survival plot showing higher DCLK1 expression associated with overall survival in serous OvCa patients. Plot was were created in KM Plotter (http: / / kmplot.com). Statistical analysis was performed using a log-rank test where p<0.05 was considered significant. HR, hazard ratio.

[0016] FIG. 2A shows cisplatin dose-response curves for cisplatin-sensitive (OVCAR-8) and cisplatin–resistant (OVCAR-8 CPR) spheroids. Results were obtained from ≥3 independent experiments and are represented as the Mean ±SEM.

[0017] FIG.2B shows representative images of OVCAR-8 and OVCAR-8 CPR spheroids treated with or without cisplatin (IC75). Scale bar: 500 μm.

[0018] FIG. 2C shows quantification of cisplatin IC50values in OVCAR-8 v. OVCAR-8 CPR spheroids. Results were obtained from ≥3 independent experiments and are represented as the Mean ±SEM. Statistical analysis was performed using two-tailed unpaired t-tests, *p<0.05; **p<0.01.

[0019] FIG.2CA shows the sensitivity of cisplatin-resistant spheroids to DCLK1 inhibitor. Representative western blot of whole cell lysates (WCL) from OVCAR-4 cells stably transduced with DCLK1 WT and DCLK1 kinase domain mutant (DCLK1 D511N and DCLK1 D533N) over-expression plasmids; β-actin - loading control. Results were obtained from ≥3 independent experiments.

[0020] FIG. 2CB shows dose-response curves and interpolated IC50 values for DCLK1- IN-1 (DCLK1 inhibitor-1) in OVACR-8 and OVCAR-8 CPR spheroids. Results were obtained from ≥3 independent experiments and represent mean ±SEM.

[0021] FIG. 2CC shows representative images of OVCAR-8 and OVCAR-8 CPR spheroids treated with different concentrations of DCLK1-IN-1 (IC95). Results were obtained from ≥3 independent experiments and represent mean ±SEM. Scale bar: 500 μm.

[0022] FIG.2D shows cisplatin dose-response curves in OVCAR-4 and DCLK1 WT over- expressing (OE) spheroids. Results were obtained from ≥3 independent experiments and are represented as the Mean ±SEM.

[0023] FIG.2E shows representative images of OVCAR-4 and DCLK1 WT OE spheroids treated with or without cisplatin (IC95). Scale bar: 500 μm.

[0024] FIG. 2F shows quantification of cisplatin IC50 values in OVCAR-4 and DCLK1 WT OE spheroids. The results demonstrate that high DCLK1 expression is associated with cisplatin resistance (CPR) in OvCa spheroids. Results were obtained from ≥3 independent experiments and are represented as the Mean ±SEM. Statistical analysis was performed using two-tailed unpaired t-tests, *p<0.05; **p<0.01.

[0025] FIG.2G shows DCLK1-IN-1 dose-response curves in OVCAR-4 and DCLK1 WT OE spheroids. Results were obtained from ≥3 independent experiments and are represented as the Mean ±SEM.

[0026] FIG. 2H shows representative images of OVCAR-4 WT and DCLK1 WT OE spheroids treated with or without DCLK1-IN-1 (IC95). Scale bar: 500 μm.

[0027] FIG. 2I shows quantification of DCLK1-IN-1 IC50values for OVCAR-4 and DCLK1 WT OE spheroids. Results were obtained from ≥3 independent experiments and are represented as the Mean ±SEM. Statistical analysis was performed using two-tailed unpaired t-tests, *p<0.05; **p<0.01.

[0028] FIG. 2J shows effects of two anti-DCLK1 siRNA treatments (SAMiRNA #9, SAMiRNA #11) on DCLK1 activity. (A) Both siRNA#9 and siRNA#11 caused knock down of DCLK1 in vitro. (B) BxPC3 cells were treated with SAMiRNA nanoparticles containing siRNA#9 and siRNA#11. SAMiRNA#11 nanoparticles had significant DCLK1 protein inhibition (isoform 82 kDa = 44% inhibition at 0.1 μM; 74% inhibition at 1.0 μM) (isoform 47kDa = 12% inhibition at 0.1 μM; 27% inhibition at 1.0 μM) (indicated with *) in the BxPC3 cells when compared with Vehicle-treated cells.

[0029] FIG.2K shows results of tumor xenograft experiments with CBT-611S, a DLCK1- specific SAMiRNA. (A) A schematic representation of the tumor xenograft experimental design. Mice were injected with BxPCe tumor cells and tumors were allowed to grow. On day 16, SAMiRNA treatment was initiated. Tumor volumes were measured and plotted. (B) At day 25, average tumor volumes of the two treatments began to diverge and continued to divergeuntil the end of the experiment at 46 days after injection. (C) After 46 days of tumor growth, CBT-611S treatment resulted in significant inhibition (p<0.05) of tumor volumes.

[0030] FIG. 3A shows cisplatin dose-response curves in OVCAR-8 CPR DCLK1 KO (sgDCLK1 #1 and sgDCLK1 #2) spheroids. Results obtained from ≥3 independent experiments. Results represent the mean ±SEM.

[0031] FIG.3B shows representative images of OVCAR-8 CPR sgCtrl, sgDCLK1#1, and sgDCLK1#2 spheroids treated with or without cisplatin (IC95). Results obtained from ≥3 independent experiments. Scale bar: 500 μm.

[0032] FIG.3C shows quantification of cisplatin IC50 values in OVCAR-8 and OVCAR-8 CPR spheroids. Results obtained from ≥3 independent experiments. Results represent the mean ±SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test. *P<0.05; **p<0.01; ***p<0.001.

[0033] FIGS.3CA shows DCLK1-IN-1 sensitivity in OV8 CPR knockout spheroids using a dose-response curve for DCLK1-IN-1 (DCLK1 inhibitor) in OVACR-8 CPR DCLK1 KO spheroids. Results were obtained from ≥3 independent experiments and represent mean ±SEM.

[0034] FIG.3CB shows representative images of OVCAR-8 CPR sgCtrl, sgDCLK1#1, and sgDCLK1#2 spheroids treated with different concentrations of DCLK1-IN-1 (IC95). Results were obtained from ≥3 independent experiments and represent mean ±SEM. Scale bar: 500 μm.

[0035] FIG. 3CC shows quantification of DCLK1-IN-1 IC50 values in OVCAR-8 CPR DCLK1 KO spheroids. Results were obtained from ≥3 independent experiments and represent mean ±SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *p<0.05.

[0036] FIG. 3D shows cisplatin dose-response curves in OVCAR-4 spheroids transduced with DCLK1 WT and DCLK1 kinase domain mutant (D511N and D533N) OE plasmids. Results obtained from ≥3 independent experiments. Results represent the mean ±SEM.

[0037] FIG.3E shows representative images of OVCAR-4, DCLK1 WT, DCLK1 D511N, and DCLK1 D533N OE spheroids treated with or without cisplatin (IC75). Results obtained from ≥3 independent experiments. Scale bar: 500 μm.

[0038] FIG. 3F shows quantification of cisplatin IC50 values in OVCAR-4, DCLK1 WT, DCLK1 D511N, and DCLK1 D533N OE spheroids. Results obtained from ≥3 independent experiments. Results represent the mean ±SEM. Statistical analysis was performed using one- way ANOVA followed by Sidak’s multiple comparison test. *P<0.05; **p<0.01; ***p<0.001.

[0039] FIG. 3G shows representative images of OVCAR-8 CPR spheroids treated with cisplatin and DCLK1-IN-1 alone and in combination. Results obtained from ≥3 independent experiments. Scale bar: 500 μm.

[0040] FIG. 3H shows isobologram and combination index values for cisplatin and DCLK1-IN-1 in OVCAR-8 CPR spheroids calculated using CompuSyn software. Results obtained from ≥3 independent experiments.; ns: not significant. KO, Knockout; OE, Over- expression

[0041] FIG.4A shows quantification of the OVCAR-8 CPR DCLK1 KO spheroid growth assay performed using the Incucyte Live-Cell Imaging system. Gowth is significantly inhibited in sgDCLK1 #1, and sgDCLK1 #2. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test, **p<0.01; ***p<0.001; ****p<0.0001.

[0042] FIG. 4B shows representative images of spheroids derived from OVCAR-8 CPR sgCtrl, sgDCLK1 #1, and sgDCLK1 #2 at 0 h and 144 h time points. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Scale bar: 300 μm.

[0043] FIG. 4C shows representative 12-day clonogenic assay images and quantification in OVCAR-4, DCLK1 WT, DCLK1 D511N, and DCLK1 D533N OE cells. DCLK1 promotes OvCa cell proliferation. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test, **p<0.01; ***p<0.001; ****p<0.0001.

[0044] FIG.4D shows representative transwell migration assay images and quantification in OVCAR-8 CPR sgCtrl, sgDCLK1 #1, and sgDCLK1 #2 for 16 h. DCLK1 promotes OvCa cell migration. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Scale bar: 500 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test, **p<0.01; ***p<0.001; ****p<0.0001.

[0045] FIG. 4DA shows cell proliferation under adherent / 2D conditions monitored in OVCAR-8 CPR DCLK1 KO cells monitored for 120 h. Results show that DCLK1 promotes 2D cell proliferation. Results obtained from ≥3 independent experiments and represent mean ±SEM. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05; **p<0.01; ****p<0.0001.

[0046] FIG. 4DB shows cell proliferation under adherent / 2D conditions monitored in OVCAR-4 DCLK1 WT and kinase-domain mutant OE cells, monitored for 120 h. Results show that DCLK1 promotes 2D cell proliferation. Results obtained from ≥3 independentexperiments and represent mean ±SEM. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05; **p<0.01; ****p<0.0001.

[0047] FIG. 4DC shows representative images of spheroid invasion assay in ES2 GFP- tagged spheroids treated with DCLK1-IN-1 (4 µM), monitored using IncuCyte (magnification 4x). Results show that DCLK1 promotes spheroid invasion. Results obtained from ≥3 independent experiments and represent mean ±SEM. Scale bar: 800 μm.

[0048] FIG.4DD shows quantification of spheroid invasion assay performed in FIG.4DC. Results obtained from ≥3 independent experiments and represent mean ±SEM. Statistical analysis was performed using two-way ANOVA followed by Sidak's multiple comparisons test. *p<0.05; **p<0.01; ****p<0.0001.

[0049] FIG. 4E shows representative transwell migration assay images and quantification in OVCAR-4 DCLK1 WT, mutant OE, and control cells for 24 h. DCLK1 promotes OvCa cell migration. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Scale bar: 500 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test, **p<0.01; ***p<0.001; ****p<0.0001.

[0050] FIG. 4F shows representative 24 h-transwell invasion assay images and quantification in OVCAR-8 CPR DCLK1 KO and control cells. DCLK1 promotes OvCa cell invasion. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Scale bar: 500 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test, **p<0.01; ***p<0.001; ****p<0.0001.

[0051] FIG. 4G shows representative 24 h-transwell invasion assay images and quantification in OVCAR-4, DCLK1 WT, DCLK1 D511N, and DCLK1 D533N OE cells. DCLK1 promotes OvCa cell invasion. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Scale bar: 500 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test, **p<0.01; ***p<0.001; ****p<0.0001.

[0052] FIG.5A shows results of a Luciferase assay performed in OVCAR-8 CPR DCLK1 KO cells to assess TGFβ signaling pathway activation. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *p<0.05; **p<0.01; ***p<0.001.

[0053] FIG. 5AA shows representative western blot evaluation of pSMAD2 and SMAD2 expression in whole cell lysates (WCL) from DCLK1 knockout OVCAR-8 CPR spheroids; GAPDH - loading control. DCLK1 knockout exhibits reduced canonical TGFβ signalingactivation. Results obtained from ≥3 independent experiments and represent mean ±SEM. Statistical analysis was performed using one-way ANOVA followed by two-tailed unpaired t- test. *p<0.05; ***p<0.001; ****p<0.0001.

[0054] FIG. 5AB shows quantification of the results of FIG. 5AA. Results obtained from ≥3 independent experiments and represent mean ±SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05; ***p<0.001; ****p<0.0001.

[0055] FIG. 5AC shows results of qRT-PCR for EMT markers performed using cDNA obtained from OVCAR-4 and OVCAR-8 CPR spheroids. Cisplatin-resistant spheroids have high expression of mesenchymal markers. Results obtained from ≥3 independent experiments and represent mean ±SEM.

[0056] FIG. 5B shows qRT-PCR results for epithelial-to-mesenchymal transition (EMT) markers performed using cDNA obtained from OVACR-8 CPR sgCtrl, sgDCLK1#1, and sgDCLK1 #2 spheroids. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *p<0.05; **p<0.01; ***p<0.001.

[0057] FIG.5C shows qRT-PCR results for EMT markers performed using cDNA obtained from OVCAR-4 and DCLK1 WT OE spheroids. FIGS. 5B-C show DCLK1 modulates EMT in cisplatin-resistant OvCa spheroids. Results were obtained from ≥3 independent experiments and represent the mean ±SEM. Statistical analyses were performed using two-tailed unpaired t-test, *p<0.05; **p<0.01; ***p<0.001.

[0058] FIG. 6A shows schematic of the orthotopic cisplatin-resistant spheroid xenograft mouse model test process. Spheroids derived from OVCAR-8 CPR luc cells were injected intraperitoneally in nude mice and subjected to various treatments.

[0059] FIG. 6B is a graphic showing how tumor-bearing mice were randomized into 7 groups to receive cisplatin, DCLK1-IN-1, cisplatin + DCLK1-IN-1, scrambled SAMiRNA, siDCLK1 SAMiRNA, cisplatin + siDCLK1 SAMiRNA, or vehicle control.

[0060] FIG. 6C shows that combining DCLK1-IN-1 with cisplatin was effective in reducing tumor weights in the peritoneal cavity of mice. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; *p<0.05; **p<0.01.

[0061] FIG. 6D shows that combining DCLK1-IN-1 with cisplatin was effective in reducing visible metastases in the peritoneal cavity of mice. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; *p<0.05; **p<0.01.

[0062] FIG. 6E shows representative BLI of the tumor-bearing mice after receiving vehicle, cisplatin, DCLK1-IN-1, or a cisplatin / DCLK1-IN-1 combination.

[0063] FIG. 6F shows quantified results of the treatments of FIG. 6E. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; *p<0.05; **p<0.01.

[0064] FIG.7A shows that inhibiting DCLK1 in combination with cisplatin reduces tumor weight in an orthotopic platinum-resistant model of HGSOC. 6–8 week old female athymic nude mice received intraperitoneal injections of spheroids derived from OVCAR-8 CPR luc cells (1 million cells / mouse). Treatment with cisplatin, siNC, siDCLK1, and a siNC / siDCLK1 combination was initiated one week post-injection. Mice were euthanized when moribund and total tumor weights (mg) were measured in different drug treated groups at the end of study. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. **P<0.01, ns: non significant.

[0065] FIG. 7B shows that inhibiting DCLK1 in combination with cisplatin reduces metastasis in orthotopic platinum-resistant model of HGSOC. 6–8 week old female athymic nude mice received intraperitoneal injections of spheroids derived from OVCAR-8 CPR luc cells (1 million cells / mouse). Treatment with cisplatin, siNC, siDCLK1, and a siNC / siDCLK1combination was initiated one week post-injection. Mice were euthanized when moribund and the total number of visible metastased nodules in peritoneal cavities of mice receiving different drug treatments were counted by 2 independent researchers. Statistical analysis was performed using one-way ANOVA followed by two-tailed unpaired t-test. **P<0.01, ns: non significant.

[0066] FIG.7C shows the body weights of mice that received intraperitoneal injections of different drug treatments during the study duration. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post-hoc test. **P<0.01, ns: non significant.

[0067] FIG. 8A shows representative BLI of the tumor-bearing mice receiving scrambled SAMiRNA, cisplatin, siDCLK1 SAMiRNA, and a cisplatin / siDCLK1 SAMiRNA combination.

[0068] FIG.8B shows quantified results of the treatments of FIG.8A.

[0069] FIG. 8C shows representative H&E stains and IHC for DCLK1 in tumors of mice receiving DCLK1-IN-1, cisplatin, and a cisplatin / DCLK1-IN-1 combination.

[0070] FIG.8D shows quantified results of the treatments of FIG.8C.

[0071] FIG.8E shows representative H&E stains and IHC for DCLK1 in tumors of mice receiving siDCLK1 SAMiRNA, cisplatin, and a cisplatin / siDCLK1 SAMiRNA combination.

[0072] FIG.8F shows quantified results of the treatments of FIG.8E.

[0073] FIG.8G shows that treatment with the DCLK1 inhibitor DCLK1-IN-1 and cisplatin alone and / or in combination in vivo has no significant effect on body weight (g) of female athymic nude mice. Statistical analysis was performed using RM two-way ANOVA.

[0074] FIG.8H shows that treatment with the DCLK1 inhibitor siDCLK1 SAMiRNA and cisplatin alone and / or in combination in vivo has no significant effect on body weight (g) of female athymic nude mice. Statistical analysis was performed using RM two-way ANOVA.

[0075] FIG. 8I shows representative H&E staining of the liver, spleen, and kidney tissue isolated from tumor-bearing mice at the end of the study of FIGS. 8G and 8H that received different treatments. Scale bar: 50 μm.

[0076] FIG.9 is a schematic showing the role of DCLK1 in mediating cisplatin resistance in OvCa. The graphical summary indicates that DCLK1 high expression modulates chemoresistance and EMT in tumor cells, which promotes various pro-metastatic phenotypes. These phenotypes can be reversed by inactivating DCLK1 (genetic / pharmacological) in vitro and result in curbing tumor metastasis in vivo. ABBREVIATIONS

[0077] Abbreviations used herein may include: BCA: Bicinchoninic acid, BLI: Bioluminescent imaging, BRCA1: Breast cancer gene 1, CI: Combination index, CPR: Cisplatin-resistance, CRISPR / Cas9: Clustered regularly interspaced palindromic repeats / CRISPR-associated protein 9, CV: crystal violet, DPBS: Dulbecco’s phosphate-buffered saline, DCLK1: Doublecortin-like kinase 1, DCLK1-IN-1: DCLK inhibitor-1, CAS No.2222635-15-4 DCLK1-L: DCLK1 long variant, DCLK1-S: DCLK1 short variant, ECL: Enhanced chemiluminescence, EGFR: Epidermal growth factor receptor, E / M: Epithelial and mesenchymal,EMP: Epithelial-mesenchymal plasticity, EMT: Epithelial-to-mesenchymal transition, FBS: fetal bovine serum, FFPE: Formalin-fixed, paraffin-embedded, FTE cell: fallopian tube epithelial cell, H&E: Hematoxylin and Eosin, HGSOC: High-grade serous ovarian carcinoma, HOSE: Human normal ovarian surface epithelial cells, IC50: Inhibitory concentration resulting in 50% of maximal effect, IHC: Immunohistochemistry, KO: Knock out, MDSC: Myeloid-derived suppressor cell, mRNA: messenger Ribonucleic acid, ns: not significant, OE: Over-expression, OvCa: Ovarian cancer, RIPA: Radioimmunoprecipitation assay, RNA: Ribonucleic acid, PBS: Phosphate-buffered saline, poly-HEMA: poly (2-hydroxyethyl methacrylate), PVDF: Polyvinylidene difluoride, qRT-PCR: Quantitative reverse transcription-polymerase chain reaction, SAMiRNA: Self-assembled-micelle inhibitory RNA, SFM: serum-free medium, sgCtrl: Single guide control sgDCLK1: Single guide DCLK1 RNA, sgRNA: Single guide RNA, siDCLK1: Small interfering DCLK1 KO RNA, siRNA: Small interfering RNA, SMAD: Suppressor of Mothers against Decapentaplegic, TGFβ: Transforming growth factor beta, TMA: Tissue Microarray, WCL: whole-cell lysate, and WT: Wild-type.DETAILED DESCRIPTION

[0078] In the present disclosure it is shown that DCLK1 is clinically relevant, as high DCLK1 expression correlates significantly with worsened progression-free and overall survival in HGSOC patients. Using various model systems, the role of DCLK1 in chemoresistance and the promotion of a pro-metastatic phenotype of OvCa spheroids is demonstrated in vitro. Further, the present results show that combining cisplatin with DCLK1 inhibitors (e.g., small molecule DCLK1 specific kinase inhibitor [DCLK1-IN-1] and novel DCLK1-targeting self-assembled-micelle inhibitory RNA [SAMiRNA] nanoparticles) reduced OvCa tumor growth and metastasis in vivo. Collectively, these findings identify DCLK1 as a driver of chemoresistance and tumorigenesis in HGSOC and demonstrate that inhibiting DCLK1 provides a novel therapeutic strategy for mitigating cisplatin sensitivity, disease recurrence, and metastasis. In the methods of the present disclosure, cisplatin may be substituted with one or more other platinum-based anti-cancer drugs such as, but not limited to, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, oxoplatin. In the methods of the present disclosure, the cancer which is treated may be selected from the group including cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast.

[0079] Before further describing various embodiments of the peptides, peptide compounds, compositions, and methods of the present disclosure in more detail by way of exemplary description, examples, and results, it is to be understood that the peptides, peptide compounds, compositions, and methods of the present disclosure are not limited in application to the details of specific embodiments and examples as set forth in the following description. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. As such, the language used herein is intended to be given the broadest possible scope and meaning, and the embodiments and examples are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the present disclosure. However, it will be apparent to a person having ordinary skill in the art that the present disclosure may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessarycomplication of the description. It is intended that all alternatives, substitutions, modifications, and equivalents apparent to those having ordinary skill in the art are included within the scope of the present disclosure. Thus, while the peptides, peptide compounds, compositions, and methods of the present disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the peptides, peptide compounds, compositions, and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the inventive concepts.

[0080] All patents, published patent applications, and non-patent publications mentioned in the specification or referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0081] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those having ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0082] As utilized in accordance with the methods and compositions of the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0083] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer inclusive therein. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0084] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the contextclearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150- 200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, includes ranges of 1-20, 10- 50, 50-100, 100-500, and 500-1,000, for example. Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10 includes 9, 8, 7, etc. all the way down to the number one (1). An amino acid sequence having a length in a range of 12 to 50 amino acids, for example, refers to a peptide or oligopeptide oligonucleotide having at least 12 amino acids and less than 51 amino acids, and includes any range bounded by two different integers in said range of 12, 13, 14, 15, 16, 17, 18, 19, ,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 amino acids, including for example 18 to 25, 20 to 24, or 20-22.

[0085] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0086] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0087] Throughout this application, the terms “about” or “approximately” are used to indicate that a value includes the inherent variation of error for the composition, the method used to administer the composition, or the variation that exists among the study subjects. Asused herein the qualifiers “about” or “approximately” are intended to include not only the exact value, amount, degree, orientation, or other qualified characteristic or value, but are intended to include some slight variations due to measuring error, manufacturing tolerances, stress exerted on various parts or components, observer error, wear and tear, and combinations thereof, for example. The term “about” or “approximately,” where used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass, for example, variations of ± 25%, or ± 20%, or ± 15%, ± 10%, or ± 5%, or ± 1%, or ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. As used herein, the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, the term “substantially” means that the subsequently described event or circumstance occurs at least 90% of the time, or at least 95% of the time, or at least 98% of the time.

[0088] As used herein any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment and may be included in other embodiments. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment and are not necessarily limited to a single or particular embodiment.

[0089] Where used herein the term “active agent” refers to a presently disclosed or described oligonucleotide, or a compound, complex, coacervate, and / or conjugate comprising the oligonucleotide. By “biologically active” is meant the ability of an active agent to modify the molecular, biochemical, or physiological system of a cell, organ, or organism, without reference to how the active agent has its physiological effects. In one non-limiting embodiment, biologically active refers to the ability of an active agent to target and interfere with the normal function of the DCLK1 gene, DCLK1 mRNA, or DCLK1 protein.

[0090] The term “pharmaceutically acceptable” refers to compounds and compositions which are suitable for administration to humans and / or animals without undue adverse side effects such as toxicity, irritation and / or allergic response commensurate with a reasonable benefit / risk ratio. The compounds of the present disclosure may be combined with one or more pharmaceutically-acceptable excipients, including carriers, vehicles, and diluents which may improve solubility, deliverability, dispersion, stability, and / or conformational integrity of the compounds or conjugates thereof.

[0091] As used herein, “pure,” or “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other object species in the composition thereof), and particularly a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, more particularly more than about 85%, more than about 90%, more than about 95%, or more than about 99%. The term “pure” or “substantially pure” also refers to preparations where the object species is at least 60% (w / w) pure, or at least 70% (w / w) pure, or at least 75% (w / w) pure, or at least 80% (w / w) pure, or at least 85% (w / w) pure, or at least 90% (w / w) pure, or at least 92% (w / w) pure, or at least 95% (w / w) pure, or at least 96% (w / w) pure, or at least 97% (w / w) pure, or at least 98% (w / w) pure, or at least 99% (w / w) pure, or 100% (w / w) pure.

[0092] Where used herein, the pronoun “we” is intended to refer to all persons involved in a particular aspect of the investigation disclosed herein and as such may include non-inventor laboratory assistants and non-inventor collaborators working under the supervision of the inventor(s).

[0093] Non-limiting examples of animals within the scope and meaning of this term include dogs, cats, rats, mice, guinea pigs, chinchillas, horses, goats, cattle, sheep, zoo animals, Old and New World monkeys, non-human primates, and humans.

[0094] “Treatment” refers to therapeutic treatments. “Prevention” refers to prophylactic or preventative treatment measures or reducing the onset of a condition or disease. The term “treating” refers to administering the composition to a subject for therapeutic purposes and / or for prevention. Non-limiting examples of modes of administration include oral, topical, retrobulbar, subconjunctival, transdermal, parenteral, subcutaneous, intranasal, intramuscular, intraperitoneal, intravitreal, and intravenous routes, including both local and systemic applications. The term “topical” is used herein to define a mode of administration through an epithelial surface, such as but not limited to, the skin, eye, or internal epithelial surfaces. In addition, the compositions of the present disclosure may be designed to provide delayed, controlled, extended, and / or sustained release using formulation techniques which are well known in the art.

[0095] The terms “therapeutic composition” and “pharmaceutical composition” refer to a composition containing a peptide as described herein that may be administered to a subject by any method known in the art or otherwise contemplated herein, wherein administration of the composition brings about a therapeutic effect as described elsewhere herein.

[0096] The term “effective amount” refers to an amount of a peptide or peptide compound which is sufficient to exhibit a detectable therapeutic, amelioration, or treatment effect in a subject without excessive adverse side effects (such as substantial toxicity, irritation and allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner of the present disclosure. The effective amount for a subject will depend upon the subject’s type, size and health, the nature and severity of the condition to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0097] The term “ameliorate” means a detectable or measurable improvement in a subject’s condition or a symptom thereof. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the condition, or an improvement in a symptom or an underlying cause or a consequence of the condition, or a reversal of the condition. A successful treatment outcome can lead to a “therapeutic effect,” or “benefit” of ameliorating, decreasing, reducing, inhibiting, suppressing, limiting, controlling, or preventing the occurrence, frequency, severity, progression, or duration of a condition, or consequences of the condition in a subject.

[0098] A decrease or reduction in worsening, such as stabilizing the condition, is also a successful treatment outcome. A therapeutic benefit therefore need not be complete ablation or reversal of the condition, or any one, most or all adverse symptoms, complications, consequences or underlying causes associated with the condition. Thus, a satisfactory endpoint may be achieved when there is an incremental improvement such as a partial decrease, reduction, inhibition, suppression, limit, control or prevention in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal of the condition (e.g., stabilizing), over a short or long duration of time (e.g., seconds, minutes, hours).

[0099] As used herein, the phrase "biologically active" refers to a substance that has activity in a biological system (e.g., in a cell (e.g., isolated, in culture, in a tissue, in an organism), in a cell culture, in a tissue, in an organism, etc.). For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. It will be appreciated by those skilled in the art that often only a portion or fragment of a biologically active substance is required (e.g., is necessary andsufficient) for the activity to be present; in such circumstances, that portion or fragment is considered to be a "biologically active" portion or fragment.

[0100] As used herein, the term "small molecule" means a low molecular weight organic compound that may serve as an enzyme substrate or regulator of biological processes. In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, provided nanoparticles further include one or more small molecules. In some embodiments, the small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, one or more small molecules are encapsulated within the nanoparticle. In some embodiments, small molecules are non-polymeric. In some embodiments, in accordance with the present disclosure, small molecules are not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc. In some embodiments, a small molecule is a therapeutic. In some embodiments, a small molecule is an adjuvant. In some embodiments, a small molecule is a drug.

[0101] In some embodiments, provided agents and / or compositions comprising such agents may be provided in particles. Particles as used in this context means nanoparticles or microparticles (or in some instances larger particles) which can consist in whole or in part of provided agent(s) and / or other therapeutic agent(s) as described herein. Such particles may contain the agent(s) and / or compositions in a core surrounded by a coating, including, but not limited to, an enteric coating. The agent(s) and / or compositions also may be dispersed throughout the particles. The agent(s) and / or compositions also maybe adsorbed into the particles. The particles maybe of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the agent(s) and / or compositions, any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles maybe microcapsules which comprise one or more provided agents in a solution or in a semi-solid state. The particles may be of virtually any shape.

[0102] According to various embodiments, both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering provided agent(s) and / or compositions. Such polymers maybe natural or synthetic polymers. In many embodiments, a polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels which may comprise, for example, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethylmethacrylates), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(laurylmethacrylate), poly(phenylmethacrylate), poly(methylacrylate), poly(isopropylacrylate), poly(isobutylacrylate), and poly(octadecylacrylate). In some embodiments, provided agents and / or compositions comprising such agents maybe contained in controlled release systems.

[0103] The term "controlled release" in this context is intended to refer to any drug- containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations.

[0104] The term "sustained release" (also referred to as "extended release") is used in this context in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that in certain particular (but non- limiting) embodiments, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in this context its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release." In some embodiments, use of a long-term sustained release implant maybe particularly suitable for treatment of chronic conditions with one or more provided agents. "Long-term" release, as used in this context, means that an implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and in certain non-limiting embodiments, 30-60 days. Long- term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described elsewhere herein.

[0105] The term “mutant” or “variant” is intended to refer to a protein, peptide, nucleic acid or organism which has at least one amino acid or nucleotide which is different from the wild type version of the protein, peptide, nucleic acid, or organism and includes, but is not limited to, point substitutions, multiple contiguous or non-contiguous substitutions, chimeras, or fusion proteins, and the nucleic acids which encode them.

[0106] The term "homologous" or “% identity” as used herein means a nucleic acid (or fragment thereof) or a protein (or a fragment thereof) having a degree of homology to the corresponding natural reference nucleic acid or protein that may be in excess of 70%, or in excess of 80%, or in excess of 85%, or in excess of 90%, or in excess of 91%, or in excess of 92%, or in excess of 93%, or in excess of 94%, or in excess of 95%, or in excess of 96%, or in excess of 97%, or in excess of 98%, or in excess of 99%. For example, in regard to peptides or polypeptides, the percentage of homology or identity as described herein is typically calculated as the percentage of amino acid residues found in the smaller of the two sequences which align with identical amino acid residues in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to assist in that alignment (as set forth by Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972)). In one embodiment, the percentage homology as described above is calculated as the percentage of the components found in the smaller of the two sequences that may also be found in the larger of the two sequences (with the introduction of gaps), with a component being defined as a sequence of four, contiguous amino acids. Also included as substantially homologous is any protein product which may be isolated by virtue of cross-reactivity with antibodies to the native protein product. Sequence identity or homology can be determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A non-limiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul (Proc. Natl. Acad. Sci. USA (1990) 87:2264-2268), modified as in Karlin & Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877).

[0107] In one embodiment “% identity” represents the number of amino acids or nucleotides which are identical at corresponding positions in two sequences of a protein having the same activity or encoding similar proteins. For example, two amino acid sequences each having 100 residues will have 95% identity when 95 of the amino acids at corresponding positions are the same.

[0108] Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller (CABIOS (1988) 4:11-17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman (Proc. Natl. Acad. Sci. USA (1988) 85:2444-2448).

[0109] Another algorithm is the WU-BLAST (Washington University BLAST) version 2.0 software (WU-BLAST version 2.0 executable programs for several UNIX platforms). This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, Methods in Enzymology (1996) 266:460-480; Altschul et al., J Molec Biol. (1990) 215:403-410; Gish & States, Nature Genetics (1993) 3:266-272; Karlin & Altschul, Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877; all of which are incorporated by reference herein).

[0110] In addition to those otherwise mentioned herein, mention is made also of the programs BLAST, gapped BLAST, BLASTN, BLASTP, and PSI-BLAST, provided by the National Center for Biotechnology Information. These programs are widely used in the art for this purpose and can align homologous regions of two amino acid sequences. In all search programs in the suite, the gapped alignment routines are integral to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.

[0111] Specific amino acids may be referred to herein by the following designations: alanine: ala or A; arginine: arg or R; asparagine: asn or N; aspartic acid: asp or D; cysteine: cys or C; glutamic acid: glu or E; glutamine: gln or Q; glycine: gly or G; histidine: his or H; isoleucine: ile or I; leucine: leu or L; lysine: lys or K; methionine: met or M; phenylalanine: phe or F; proline: pro or P; serine: ser or S; threonine: thr or T; tryptophan: trp or W; tyrosine: tyr or Y; and valine: val or V.

[0112] The terms “oligonucleotide,” "polynucleotide," or “nucleic acid,” as used herein, include any nucleotide sequence which encodes a variant, chimeric, or mutant peptide including polynucleotides in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The DNA may be double-stranded or single- stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. The polynucleotide sequence encoding a mutant peptide or encoding a therapeutically-effective fragment of a mutant peptide can be substantially the same as the coding sequence of the endogenous coding sequence as long as it encodes a biologically active mutant peptide. Further, the mutant peptide, or therapeutically-effective fragment of a mutant peptide may be expressed using polynucleotide sequence(s) which differ in codon usage due to the degeneracies of the genetic code or allelic variations.

[0113] As noted above, the peptides of the present disclosure, and the nucleic acids which encode them, include peptide and nucleic acid variants which comprise additional conservative substitutions. For example, the variant peptides include, but are not limited to, variants that are not exactly the same as the sequences disclosed herein, but which have, in addition to the substitutions explicitly described for various sequences listed herein, conservative substitutions of amino acid residues which do substantially not impair the agonistic or antagonistic activity or properties of the variants described herein. Examples of such conservative amino acid substitutions include, but are not limited to, ala to gly, ser, or thr; arg to gln, his, or lys; asn to asp, gln, his, lys, ser, or thr; asp to asn or glu; cys to ser; gln to arg, asn, glu, his, lys, or met; glu to asp, gln, or lys; gly to pro or ala; his to arg, asn, gln, or tyr; ile to leu, met, or val; leu to ile, met, phe, or val; lys to arg, asn, gln, or glu; met to gln, ile, leu, or val; phe to leu, met, trp, or tyr; ser to ala, asn, met, or thr; thr to ala, asn, ser, or met; trp to phe or tyr; tyr to his, phe or trp; and val to ile, leu, or met.

[0114] The present constructs or antigen-binding portions thereof can be formulated into compositions for delivery to a mammalian subject. The composition can be administered alone and / or mixed with a pharmaceutically acceptable vehicle or excipient. Suitable vehicles are, for example (but not by way of limitation), water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, the vehicle can contain minor amounts of auxiliary substances such as (but not limited to) wetting or emulsifying agents, pH buffering agents, or adjuvants. The compositions of the present disclosure can also include ancillary substances,such as (but not limited to) pharmacological agents, cytokines, or other biological response modifiers.

[0115] Furthermore, the compositions can be formulated into compositions in either neutral or salt forms. Pharmaceutically acceptable salts include (but are not limited to) the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, and procaine.

[0116] Compositions can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight, and condition of the subject, the particular composition used, and the route of administration. In one non-limiting embodiment, a single dose of the composition according to the disclosure is administered. In other non-limiting embodiments, multiple doses are administered. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, degree of immunoprotection desired, or whether the composition is used for prophylactic or curative purposes. For example, in certain non-limiting embodiments, the composition is administered once per month, twice per month, three times per month, every other week, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily, twice a day, or three times a day. The duration of treatment (i.e., the period of time over which the composition is administered) can vary, depending on any of a variety of factors, e.g., subject response. For example, the composition can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.

[0117] The dosage of an administered active agent for humans will vary depending upon factors such as (but not limited to) the patient's age, weight, height, sex, general medical condition, and previous medical history. In certain non-limiting embodiments, the recipient is provided with a dosage of the active agent that is in the range of from about 1 mg to about 1000 mg as a single infusion or single or multiple injections, although a lower or higher dosage also may be administered. In certain non-limiting embodiments, the dosage may be in the range offrom about 25 mg to about 100 mg of the active agent per square meter (m2) of body surface area for a typical adult, although a lower or higher dosage also may be administered. Non-limiting examples of dosages of the active agent that may be administered to a human subject further include 1 to 500 mg, 1 to 70 mg, or 1 to 20 mg, although higher or lower doses may be used. Dosages may be repeated as needed, for example (but not by way of limitation), once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. It may also be given less frequently, such as (but not limited to) every other week for several months, or more frequently, such as twice weekly or by continuous infusion.

[0118] In at least one embodiment, the present disclosure is directed to a dosing regimen involving administration of the multispecific construct such as disclosed elsewhere herein. The dosing regimen may comprise multiple dosing cycles (e.g., wherein the first dosing cycle is a step-up, fractionated dosing cycle). The doses may range from about 0.02 mg to about 2.0 mg (e.g., from about 0.02 to about 1.8 mg, from about 0.02 to about 1.6 mg, from about 0.02 to about 1.4 mg, from about 0.02 to about 1.2 mg, from about 0.05 to about 1.8 mg, from about 0.1 to about 1.8 mg, from about 0.4 to about 1.8 mg, from about 0.6 to about 1.8 mg, from about 0.8 to about 1.8 mg, from about 0.5 to about 1.5 mg, from about 0.8 to about 1.2 mg; e.g., about 1 mg), from about 0.05 mg to about 4.0 mg (e.g., from about 0.05 to about 3.5 mg, from about 0.05 to about 3.0 mg, from about 0.05 to about 2.5 mg, from about 0.05 to about 2.2 mg, from about 0.1 to about 3.5 mg, from about 0.5 to about 3.5 mg, from about 1.0 to about 3.5 mg, from about 1.5 to about 3.5 mg, from about 1.8 to about 3.5 mg, from about 1.0 to about 3.0 mg, from about 1.5 to about 2.5 mg; e.g., about 2 mg).

[0119] In some embodiments, the dose may range from 50 mg to 200 mg (e.g., from 50 mg to 175 mg, from 50 mg to 150 mg, from 50 mg to 125 mg, from 50 mg to 100 mg, from 50 mg to 75 mg, from 50 mg to 70 mg, from 52 mg to 100 mg, from 52 mg to 75 mg, from 50 mg to 180 mg, from 55 mg to 150 mg, from 55 mg to 100 mg, from 55 mg to 70 mg, from 55 mg to 65 mg, from 58 mg to 62 mg; e.g., about 60 mg). In some embodiments, the dose may be about 60 mg. In some embodiments, the dose is about 1 mg. In some embodiments, the dose is about 2 mg.

[0120] In some embodiments, the dose is from 20 mg to 200 mg (e.g., from 20 mg to 175 mg, from 20 mg to 150 mg, from 20 mg to 100 mg, from 20 mg to 75 mg, from 30 mg to 175 mg, from 40 mg to 175 mg, from 45 mg to 175 mg, from 50 mg to 175 mg, from 30 mg to 150 mg, from 40 mg to 100 mg, from 45 mg to 75 mg, from 50 mg to 70 mg, from 55 mg to 65 mg, from 58 mg to 62 mg; about 20 mg, about 30 mg, about 45 mg, or e.g., about 60 mg). In some embodiments, the dose is from about 12 mg to about 48 mg (e.g., from about 12 mg to about42 mg, from about 12 mg to about 36 mg, from about 12 mg to about 30 mg, from about 18 mg to about 48 mg, from about 18 mg to about 42 mg, from about 24 mg to about 42 mg, from about 27 mg to about 42 mg, from about 24 mg to about 36 mg, from about 27 mg to about 33 mg, from about 28 mg to about 32 mg; e.g., about 24 mg, about 27 mg, about 30 mg, about 33 mg, or about 36 mg).

[0121] In some embodiments, the dosing regimen comprises administration of a loading dose, such as from 20 mg to 200 mg (e.g., from 20 mg to 175 mg, from 20 mg to 150 mg, from 20 mg to 100 mg, from 20 mg to 75 mg, from 30 mg to 175 mg, from 40 mg to 175 mg, from 45 mg to 175 mg, from 50 mg to 175 mg, from 30 mg to 150 mg, from 40 mg to 100 mg, from 45 mg to 75 mg, from 50 mg to 70 mg, from 55 mg to 65 mg, from 58 mg to 62 mg; e.g., about 60 mg). In some embodiments, the dose is from about 12 mg to about 48 mg (e.g., from about 12 mg to about 42 mg, from about 12 mg to about 36 mg, from about 12 mg to about 30 mg, from about 18 mg to about 48 mg, from about 18 mg to about 42 mg, from about 24 mg to about 42 mg, from about 27 mg to about 42 mg, from about 24 mg to about 36 mg, from about 27 mg to about 33 mg, from about 28 mg to about 32 mg; e.g., about 24 mg, about 27 mg, about 30 mg, about 33 mg, or about 36 mg).

[0122] In some non-limiting embodiments, the active agent is provided in a concentration of about 1 nM, about 5 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 500 nM, about 550 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 ^M, about 2 ^M, about 3 ^M, about 4 ^M, about 5 ^M, about 6 ^M, about 7 ^M, about 8 ^M, about 9 ^M, about 10 ^M, about 15 ^M, about 20 ^M, about 25 ^M, about 30 ^M, about 35 ^M, about 40 ^M, about 45 ^M, about 50 ^M, about 60 ^M, about 70 ^M, about 75 ^M, about 80 ^M, about 90 ^M, about 100 ^M, about 125 ^M, about 150 ^M, about 175 ^M, about 200 ^M, about 250 ^M, about 300 ^M, about 350 ^M, about 400 ^M, about 500 ^M, about 600 ^M, about 700 ^M, about 750 ^M, about 800 ^M, about 900 ^M, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM, about 300 mM, about400 mM, about 500mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, about 10 M, about 15 M, about 20 M, about 25 M, about 30 M, about 35 M, about 40 M, about 45 M, about 50 M, about 75 M, about 100 M, or any range in between any two of the aforementioned concentrations, including said two concentrations as endpoints of the range, or any number in between any two of the aforementioned concentrations.

[0123] When administered orally, the present compositions may be protected from digestion. This can be accomplished either by complexing the construct or antigen-binding portion thereof with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the construct or antigen-binding portion thereof in an appropriately resistant carrier such as (but not limited to) a liposome, e.g., such as shown in U.S. Patent No.5,391,377.

[0124] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. For topical transdermal administration, the agents are formulated into ointments, creams, salves, powders, and gels. Transdermal delivery systems can also include (for example but not by way of limitation) patches. The present compositions can also be administered in sustained delivery or sustained release mechanisms. For example, biodegradeable microspheres or capsules or other biodegradeable polymer configurations capable of sustained delivery of a peptide can be included herein.

[0125] For inhalation, the present compositions can be delivered using any system known in the art, including (but not limited to) dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. For example (but not by way of limitation), the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another aspect, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include (for example but not by way of limitation) air jet nebulizers.

[0126] In one aspect, the pharmaceutical formulations comprising compositions or nucleic acids, antibodies or fragments thereof are incorporated in lipid monolayers or bilayers, such as (but not limited to) liposomes, such as shown in U.S. Patent Nos. 6,110,490; 6,096,716;5,283,185; and 5,279,833. In other aspects, non-limiting embodiments of the disclosure include formulations in which the polypeptides or nucleic acids have been attached to the surface of the monolayer or bilayer of the liposomes. Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art, such as (but not limited to) those disclosed in U.S. Patent Nos.4,235,871; 4,501,728; and 4,837,028.

[0127] In one aspect, the compositions are prepared with carriers that will protect the construct or fragment thereof against rapid elimination from the body, such as (but not limited to) a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as (but not limited to) ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[0128] The constructs and fragments thereof in general may be formulated to obtain compositions that include one or more pharmaceutically suitable excipients, surfactants, polyols, buffers, salts, amino acids, or additional ingredients, or some combination of these. This can be accomplished by known methods to prepare pharmaceutically useful dosages, whereby the active compound is combined in a mixture with one or more pharmaceutically suitable excipients. Sterile phosphate-buffered saline is one non-limiting example of a pharmaceutically suitable excipient.

[0129] Non-limiting examples of routes of administration of the compositions described herein include parenteral injection, e.g., by subcutaneous, intramuscular, or transdermal delivery. Other forms of parenteral administration include (but are not limited to) intravenous, intraarterial, intralymphatic, intrathecal, intraocular, intracerebral, or intracavitary injection. In parenteral administration, the compositions will be formulated in a unit dosage injectable form such as (but not limited to) a solution, suspension, or emulsion, in association with a pharmaceutically acceptable excipient. Such excipients are inherently nontoxic and nontherapeutic. Non-limiting examples of such excipients include saline, Ringer's solution, dextrose solution, and Hanks' solution. Nonaqueous excipients such as (but not limited to) fixed oils and ethyl oleate may also be used. An alternative non-limiting excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as (but not limited to) substances that enhance isotonicity and chemical stability, including buffers and preservatives. The constructs can be delivered or administered alone or as pharmaceutical compositions by any means known in the art, such as (but not limited to) systemically, regionally, or locally; by intra-arterial, intrathecal (IT), intravenous (IV), parenteral, intra-pleural cavity, topical, oral, orlocal administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa).

[0130] Administration can be (for example but not by way of limitation) parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Administration can also be localized directly into a tumor. Administration into the systemic circulation by intravenous or subcutaneous administration is typical. Intravenous administration can be, for example (but not by way of limitation), by infusion over a period such as (but not limited to) 30-90 min or by a single bolus injection.

[0131] Formulated compositions comprising the constructs can be used (for example but not by way of limitation) for subcutaneous, intramuscular, or transdermal administration. Compositions can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. Compositions can also take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0132] The compositions may be administered in solution. The formulation thereof may be in a solution having a suitable pharmaceutically acceptable buffer, such as (but not limited to) phosphate, Tris (hydroxymethyl) aminomethane-HCl, or citrate, and the like. Buffer concentrations should be in the range of 1 to 100 mM. The formulated solution may also contain a salt, such as (but not limited to) sodium chloride or potassium chloride in a concentration of 50 to 150 mM. An effective amount of a stabilizing agent such as (but not limited to) mannitol, trehalose, sorbitol, glycerol, albumin, a globulin, a detergent, a gelatin, a protamine, or a salt of protamine may also be included.

[0133] As used herein, the term "RNA interference" or "RNAi" refers to the silencing or decreasing of gene expression by siRNAs. It is the process of sequence-specific, post- transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene. The gene may be endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector that is not integrated into the genome. The expression of the gene is either completely or partially inhibited. RNAi inhibits the gene by compromising the function of a target RNA, completely or partially. Both plants and animals mediate RNAi by the RNA-induced silencing complex (RISC); a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (e.g., approximately 22 nucleotides) derived from the double-stranded RNA trigger, although the protein components of this activity are unknown. However, the 22-nucleotide RNA sequencesare homologous to the target gene that is being suppressed. Thus, the 22-nucleotide sequences appear to serve as guide sequences to instruct a multicomponent nuclease, RISC, to destroy the specific mRNAs. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21 to 23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease, and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart.

[0134] As used herein, the term "siRNA" refers to a short interfering RNA. In some embodiments, siRNAs comprise a duplex, or double-stranded region, of about 18-25 nucleotides long; often siRNAs contain from about two to four unpaired nucleotides at the 3' end of each strand. At least one strand of the duplex or double-stranded region of a siRNA is substantially homologous to or substantially complementary to a target RNA molecule. The strand complementary to a target RNA molecule is the "antisense strand"; the strand homologous to the target RNA molecule is the "sense strand", and is also complementary to the siRNA antisense strand. siRNAs may also contain additional sequences; non-limiting examples of such sequences include linking sequences, or loops, as well as stem and other folded structures. siRNAs appear to function as key intermediaries in triggering RNA interference in invertebrates and in vertebrates, and in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants.

[0135] In at least certain embodiments, the active agents (e.g., oligonucleotides) may have strand lengths comprising, for example, approximately 12 to 50, or 18 to 40, or 20 to 30 nucleotides, including a targeting sequence (i.e., a seed sequence) that is complementary to a target sequence of a nucleic acid which comprises a portion of an AR coregulator, such as an AR coregulator as listed elsewhere herein, a pre-mRNA transcribed from an AR coregulator, and / or (2) a mature mRNA processed from said pre-mRNA. For example, when an oligonucleotide binds to the target sequence of a preprocessed mRNA, it effectively inhibits splicing at the normal splice acceptor site and thus produces a splice variant mRNA, leading to truncated or otherwise aberrant versions of the encoded protein upon translation, or when the oligonucleotide binds to the target region of a mature mRNA, it effectively inhibits proper translation of the mRNA into an encoded protein.

[0136] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally-occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an "A," a "G," a uracil "U" or a "C"). The term nucleobase also includes non-natural bases as described below. The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid." The term "oligonucleotide" generally refers to a molecule of between about 3 and about 100 nucleobases in length. The term "polynucleotide" generally refers to at least one molecule of greater than about 100 nucleobases in length. These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double-stranded molecule that comprises a complementary strand or "complement" of a particular sequence comprising a molecule. As used herein, a single- stranded nucleic acid may be denoted by the prefix "ss," and a double-stranded nucleic acid by the prefix "ds. The terms "polynucleotide sequence" or “nucleic acid,” as used herein, include any polynucleotide sequence which encodes a peptide or fusion protein (or polypeptide) including polynucleotides in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The RNA or DNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

[0137] Where a strand is designated herein as RNA, and thus comprises uracil (U) nucleobases, the present disclosure is also directed to an equivalent DNA sequence where the U nucleobase is replaced with a thymine (T) nucleobase. For example, where an RNA active agent described herein comprises the seed sequence GUCUGA, the equivalent DNA active agent comprises the seed sequence GTCTGA.

[0138] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred (but not by way of limitation). Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[0139] Therefore, in the context of the present disclosure, the term "oligonucleotide" refers to an oligomer or polymer of RNA or DNA or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring nucleobases, sugars and synthetic heterocycles and covalent internucleoside (backbone) linkages which function similarly. Such modified or substituted non-natural oligonucleotides , as compared to native (natural) forms may have desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0140] Where used herein, the term “oligonucleotide,” is also intended to include linked nucleobase sequences containing modified backbones comprising non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Further, for the purposes of this specification, the term “nucleoside” is intended to refer to a nucleobase linked to a ribose or deoxyribose sugar (a natural nucleoside), and to a nucleobase linked to a non-ribose or non-deoxyribose heterocycle, e.g., a morpholine structure (a non-natural, or modified, nucleoside or other structures described elsewhere herein). Thus, a series of such modified, non-natural, nucleosides linked together via an internucleoside backbone can also be considered to be an oligonucleotide (a non-natural, or modified, oligonucleotide). Further, the term “sugar” where used herein in the context of a nucleoside, is intended to include “non-sugar” heterocyclic compounds, such as morpholines, as the portion of the internucleoside backbone which is linked to the nucleobase.

[0141] Oligonucleotides useful in the compounds and methods disclosed herein also include those comprising entirely or partially of naturally occurring nucleobases. Naturally occurring nucleobases as defined herein, include adenine, guanine, thymine, cytosine, and uracil. Although 5-methylcytosine (5-me-C) is technically a naturally occurring nucleobase, for the purposes of the present disclosure it will be included in the list of non-natural (a.k.a., modified) nucleobases.

[0142] As noted above, oligonucleotides of the present disclosure may further include those comprised entirely or partially of modified nucleobases and their corresponding nucleosides. These modified nucleobases include, but are not limited to, 5-uracil (pseudouridine), dihydrouracil, inosine, ribothymine, 5-me-C, 7-methylguanine, hypoxanthine, xanthine, 5- hydroxymethyl cytosine, 2-aminoadenine, 2-methyladenine, 6-methyladenine, 2- propyladenine, N6-adenine, N6-isopentenyladenine, 2-methylthio-N6-isopentenyladenine, 2-methylguanine, 6-methylguanine, 2-propylguanine, 1-methylguanine, 7-methylguanine, 2,2- dimethylguanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, dihydrouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5- carboxymethylaminomethyl-2-thiouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 5-methyl- 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 5-carboxymethylaminomethyluracil, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, alkynyl derivatives of pyrimidine bases including 5-propynyl uracil, and 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 4-thiouracil, 8-halo-adenines, 8-amino adenine, 8-thiol adenine, 8- thioalkyl adenine, 8-hydroxyl adenine, 5-trifluoromethyl uracil, 3-methylcytosine, 5- methylcytosine, 5-trifluoromethyl cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2- amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 8-halo-guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanine, 8-hydroxyl guanine, 7-deazaadenine, 3- deazaguanine, 3-deazaadenine, beta-D-galactosylqueosine, beta-D-mannosylqueosine, 1- methylinosine, 2,6-diaminopurine, queosine, tricyclic pyrimidines, phenoxazine cytidine(1H- pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), and phenothiazine cytidine (1H-pyrimido[5,4- b][1,4]benzothiazin-2(3H)-one.

[0143] The present disclosure also encompasses oligonucleotides which comprise targeting sequences (base sequences) that are complementary to particular nucleic acid target sequences taught herein. A nucleic acid is a "complement" or is "complementary" to another nucleic acid when it is capable of base-pairing with the other nucleic acid according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. Polynucleotides (nucleic acids) are described as "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides.

[0144] More particularly, "complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific bindingoccurs between the oligonucleotide and the DNA or RNA target, and as such, as is understood in the art, the targeting sequence of an antisense oligonucleotide of the present disclosure need not be 100% complementary to that of its target sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence of the DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. An oligonucleotide and a target sequence are thus complementary to each other when a sufficient number of nucleobases of the oligonucleotide can hydrogen bond with the corresponding nucleobases of the target sequence, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as an AR coregulator).

[0145] For example, an oligonucleotide in which 18 of 20 nucleobases of the oligonucleotide are complementary to a target sequence, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligonucleotide which is 18 nucleobases in length having three noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid, or are distributed in non- contiguous positions, would have 83% overall complementarity with the target sequence.

[0146] In other embodiments, the seed sequence of the antisense oligonucleotide provided herein is fully complementary (i.e.100% complementary) to a target sequence of a nucleic acid, e.g., of an AR coregulator. As used herein, "fully complementary" means each nucleobase of the referenced portion of an oligonucleotide (e.g., the seed sequence) is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid.

[0147] The term "target sequence" where used herein refers to a contiguous series of nucleobases in a specific nucleotide sequence (target region), for example of an mRNA. The term “target sequence” refers to a sequence that is a subsequence (portion or segment) of the target region, or to the entire sequence of the target region. A target sequence may include the 5’ terminal nucleobase of a nucleic acid sequence plus adjacent internal nucleobases of the sequence, or the 3’ terminal nucleobase plus adjacent internal nucleobases of the sequence, or only internal nucleobases within the sequence, or the target sequence may be 100% identical to the target region. In certain embodiments, a nucleic acid compound of the present disclosurecomprises an oligonucleotide having a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of a target sequence of a nucleic acid target region to which it is targeted.

[0148] The terms "complementary" and "antisense" can be used interchangeably. Complementary also refers to polynucleotide sequences that are substantially complementary (antisense) over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches.

[0149] In certain embodiments, oligonucleotides of the present disclosure are synthesized using one or more modified nucleotides. As used herein, the terms "modified" and "modification" when used in the context of the constituents of a nucleotide monomer, i.e., sugar, nucleobase and internucleoside linkage (backbone), refer to non-natural changes to the chemical structure of these naturally occurring constituents or the substitutions of these constituents with non-naturally occurring ones, i.e., mimetics. For example, the "unmodified" or "naturally occurring" sugar ribose (of RNA) can be modified by replacing the hydrogen at the 2'-position of ribose with a methyl group. Similarly, the naturally occurring internucleoside linkage of nucleic acids is a 3' to 5' phosphodiester linkage that can be modified, in one embodiment, by replacing one of the non-bridging oxygen atoms of the phosphate linker with a sulfur atom to create a phosphorothioate linkage. Modified oligonucleotides are structurally distinguishable, but functionally interchangeable with naturally occurring or synthetic unmodified oligonucleotides and usually have enhanced properties such as increased resistance to degradation by exonucleases and endonucleases, or increased binding affinity.

[0150] As noted above, in certain embodiments, modifications to the oligonucleotides of the present disclosure encompass substitutions or changes in internucleoside linkages, sugar moieties, or nucleobases. Where used herein in reference to an oligonucleotide, the term “non- natural” or "unnatural" refers to an oligonucleotide which comprises at least one modification in an internucleoside linkage, a sugar, and / or a nucleobase thereof, wherein such modified internucleoside linkage, modified sugar, and / or modified nucleobase is not found naturally in DNA or RNA (unless specifically defined otherwise herein)

[0151] Non-naturally occurring internucleoside linkages of the oligonucleotides of the present disclosure include those that contain a phosphorus atom and also those that do not contain a phosphorus atom. Numerous phosphorus-containing modified oligonucleotidebackbones are known in the art and may be used in the oligonucleotides of the present disclosure. Examples of phosphorus-containing internucleoside linkages of non-natural (modified) oligonucleotide backbones which may occur in the presently disclosed oligonucleotides include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoramidite, phosphorodiamidate, morpholino, phosphotriester, aminoalkylphosphotriester, phosphonate, chiral phosphorothioates, methyl and other alkyl phosphonates including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage, and oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e., a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof) linkages. Examples of U.S. patents that teach the preparation of such phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050.

[0152] As noted above, in some embodiments, the internucleoside linkages are without phosphorus atoms and may instead comprise short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. In further embodiments, the non-naturally occurring internucleoside linkages are uncharged and in others, the linkages are achiral. In some embodiments, the non-naturally occurring internucleoside linkages are uncharged and achiral, such as peptide nucleic acids (PNAs).

[0153] It is understood that the sequence set forth in each sequence or SEQ ID NO contained herein is independent of any modification to sugar moieties, internucleoside linkages, or nucleobases of the sequence, unless otherwise specified. As such, antisense oligonucleotides of the present disclosure may be defined by a complementary correspondence to a sequence or SEQ ID NO disclosed herein, or segment thereof, and may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Other embodiments of oligonucleotide backbones include siloxane backbones; sulfide, sulfoxide andsulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2component parts. Examples of U.S. patents that teach the preparation of such non-phosphorus containing oligonucleotides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439.

[0154] In certain oligonucleotide mimetics of the present disclosure, both the sugar moiety and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with non- natural groups. One such oligomeric compound is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Examples of U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262.

[0155] The oligonucleotides described herein stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. The oligonucleotides can include a non-natural nucleoside linkage such as a phosphorothioate linkage as the first, second, and / or third internucleotide linkage at the 5' or 3' end of the oligonucleotide sequence. In certain embodiments, the oligonucleotides can include a 2'- modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O- methoxyethyl (2'- O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'- O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O--N-methylacetamido (2'-O-NMA) nucleotide. In a particular embodiment, the oligonucleotides include at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-O-methyl modification.

[0156] As noted elsewhere herein, the oligonucleotide can be further modified so as to be conjugated to an organic moiety such as a biogenic molecule that is selected to improve stability, distribution and / or cellular uptake of the oligonucleotide, e.g., cholesterol, forming the nucleic acid compound of the present disclosure. Such an organic moiety can be attached,e.g., to the 3' or 5' end of the oligonucleotide, and / or at the 2' position of the sugar moiety of a nucleotide of the oligonucleotide, such as the 2' ribose position.

[0157] The nucleic acid compound can further be in isolated form or can be part of a pharmaceutical composition, such as a pharmaceutical composition formulated for parental administration. The pharmaceutical compositions can contain one or more nucleic acid compounds, and in some embodiments will contain two or more inhibitory nucleic acid compounds, each one directed to a different target gene.

[0158] The oligonucleotides can be delivered in any of a variety of forms, including in liposomes as described above, and via expression vectors. The oligonucleotide can be endogenously expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors for example. Viral vectors suitable for producing the presently disclosed oligonucleotides capable of reducing expression or activity of an AR coregulator can be constructed based on, but not limited to, adeno-associated virus, retrovirus, lentivirus, adenovirus, or alphavirus. The recombinant vectors which contain a nucleic acid for expressing the oligonucleotides disclosed herein can be delivered as described above and can persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of the oligonucleotides. Such vectors can be repeatedly administered as necessary. Once expressed, in one embodiment, the oligonucleotides may interact with the target RNA and inhibit mRNA activity for example. The delivery vehicles (vectors) for the oligonucleotides optionally comprise an expression construct which includes an enhancer sequence, a promoter sequence, and other sequences necessary for expression of the products of the oligonucleotide sequence desired to be produced. In one embodiment, the promoter is cell-specific. The term "cell-specific" means that the particular promoter selected for the recombinant vector can direct expression of the selected transgene only in a particular cell type. As one example, the promoter is specific for expression in prostate cells. A number of viruses can be used in connection with the methods described herein, including papovaviruses, e.g., SV40, adenovirus, vaccinia virus, adeno-associated virus, herpesviruses including HSV and EBV, and retroviruses of avian, murine, and human origin. In certain embodiments, lentiviral vectors can be used in connection with the methods described herein. In certain embodiments, the lentiviral vector can be a doxycycline-inducible lentiviral vector engineered to express one or more shRNAs or siRNAs.

[0159] Specific vectors which may be used include, but are not limited to, adeno-associated virus vectors (e.g., as disclosed in U.S. Pat. Nos. 5,139,941, 5,436,146, and 5,622,856), an attenuated or gutless adenoviral vectors, (e.g., as disclosed in U.S. Pat. No. 5,935,935),lentiviral vectors (such as are disclosed in U.S. Pat. Nos.5,665,577; 5,994,136; and 6,013,516), plasmids or synthetic (non-viral) vectors (such as disclosed in U.S. Pat. Nos. 4,394,448 and 5,676,954), and / or nanoparticles (such as disclosed, for example, in U.S. Patents 6,217,912; 7,514,098; and 8,323,618), retroviral vectors (such as are disclosed in U.S. Pat. Nos.5,672,510; 5,707,865; and 5,817,491), herpes virus vectors (such as are disclosed in U.S. Pat. No. 5,288,641), and sindbis virus vectors and papilloma virus vectors (such as are disclosed in EP 820 773). The vectors may be either monocistronic, bicistronic, or multicistronic. A recombinant vector (e.g., lenti-, parvo-, AAV) sequence can be packaged as a “particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle can also be referred to as a “rAAV.” Such particles include proteins that encapsulate or package the vector genome. Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins.

[0160] Thus, the oligonucleotides of the present disclosure may be used as a form of gene therapy. The term “gene therapy” as used herein means genetic modification of cells by the introduction of exogenous DNA or RNA into these cells, such as via an expression vector containing the oligonucleotide, for the purpose of expressing or replicating one or more peptides, polypeptides, proteins, oligonucleotides, or polynucleotides in vivo for the treatment or prevention of disease or deficiencies in humans or animals. Examples of gene therapy are disclosed for example in U.S. Pat. No. 5,399,346. Any suitable route of administration of the oligonucleotide-containing vector may be employed. For example, parenteral (subcutaneous, subretinal, suprachoroidal, intramuscular, intravenous, transdermal) and like forms of administration may be employed. Dosage formulations include injections, implants, or other known and effective gene therapy delivery methods.

[0161] Delivery of the oligonucleotide-expressing vectors can be systemic, such as by intravenous or intra-muscular administration, direct administration to a tumor site, such as a prostate tumor, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell. The therapeutic and / or pharmaceutical compositions, in non-limiting embodiments, contain viral particles per dose in a range of, for example, from about 104to about 1011particles, from about 105to about 1010particles, or from about 106to about 109particles. In the context of AAV vectors, vector genomes are provided in in a range of, for example, from about 104to about 1014vector genomes, from about 105to about 1013vector genomes, from about 106to about 1013vector genomes, from about 107to about 1013vectorgenomes, from about 108to about 1013vector genomes, or from about 109to about 1013vector genomes. Such doses / quantities of AAV vector are useful in the methods set forth herein.

[0162] Because nucleases that cleave the phosphodiester linkages are expressed in almost every cell, unmodified nucleic acid molecules such as the inhibitory oligonucleotides of the present disclosure may be modified to resist degradation, as described above for example. Other biogenic molecules may be conjugated to the oligonucleotides to improve their ability to resist degradation, target certain cells, or to cross barriers like cell membranes or the blood brain barrier. Examples of biogenic molecules that can be conjugated to the oligonucelotides include lipids such as, but not limited to, stearic acid, palmitic acid, docosanoic acid, docosahexanoic acid, docosahexaenoic acid, cholesterol, tocopherol, and other C12-C22 saturated or unsaturated fatty acids; peptides such as but not limited to, cell-penetrating peptides (CPPs) such as penetratin, HIV-1 Tat peptides, pVEC-Cadherin 615-634, polyarginines (6-12), and transportan, linear and cyclic RGD-containing peptides, and SPACE peptide; receptor-specific ligands; aptamers (synthetic oligoribonucleotides); antibodies or antibody fragments; CpG-containing oligonucleotides; polyamines, such as spermine and spermidine; polymers such as dendrimers and polyethylene glycols (e.g., PEG 0.6 kDa -5,000 kDa); and saccharides such as N-acetylgalactosamine (GalNAc) and cyclodextrins. The biogenic molecule may be conjugated to the oligonucleotide by any suitable means, such as via linker or a cleavable bond such as but not limited to disulfide, thioether, pH sensitive (e.g., hydrazone or carboxymethylmaleic anhydride), or ethylene glycol.

[0163] The oligonucleotides or nucleic acid compounds of the present disclosure may be delivered in the form of nanoparticles and microparticles which encapsulate the nucleic acid compounds within liposomes of cationic lipids or within PEG, for example. These delivery systems can enhance intracellular delivery either by protecting the nucleic acid compound from nuclease degradation and / or by promoting absorptive endocytosis. Further, the addition of dioleylphosphatidylethanolamine to liposome delivery systems results in the destabilization of endosomal membranes and promotion of release of the oligonucleotide after endocytosis. The nucleic acid compounds can be administered to cells by a variety of other methods known to those of skill in the art, including, but not limited to, ionophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors. In one example, the nucleic acid compounds can be delivered via the nanoparticle system shown in U.S. Patent Application Publication 2019 / 0255088. The liposomes may comprise amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent Nos.4,235,871; 4,501,728; 4,837,028; and 4,737,323.

[0164] In certain embodiments, the nanoparticles which contain the nucleic acid compounds of the present disclosure may comprise a pharmaceutically acceptable carrier such as, but not limited to, poly(ethylene-co-vinyl acetate), PVA, partially hydrolyzed poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyl acetate-co-vinyl alcohol), a cross- linked poly(ethylene-co-vinyl acetate), a cross-linked partially hydrolyzed poly(ethylene-co- vinyl acetate), a cross-linked poly(ethylene-co-vinyl acetate-co-vinyl alcohol), poly-D,L-lactic acid, poly-L-lactic acid, polyglycolic acid, PGA, copolymers of lactic acid and glycolic acid, polycaprolactone, polyvalerolactone, poly (anhydrides), copolymers of polycaprolactone with polyethylene glycol, copolymers of polylactic acid with polyethylene glycol, polyethylene glycol; fibrin, Gelfoam™ (which is a water-insoluble, off-white, nonelastic, porous, pliable gel foam prepared from purified gelatin and water for injection), and combinations and blends thereof. Copolymers can comprise from about 1% to about 99% by weight of a first monomer unit such as ethylene oxide and from 99% to about 1% by weight of a second monomer unit such as propylene oxide. Blends of a first polymer such as gelatin and a second polymer such as poly-L-lactic acid or polyglycolic acid can comprise from about 1% to about 99% by weight of the first polymer and from about 99% to about 1% of the second polymer.

[0165] The oligonucleotides or nucleic acid compounds can be delivered directly by systemic administration such as using oral formulations or stereotactic injection into prostate or prostate tumor, typically in saline with chemical modifications to enable uptake, or other methods described elsewhere herein. In certain embodiments, such as when the oligonucleotide of the nucleic acid compound has a phosphorothioate backbone, the oligonucleotide binds to serum proteins, slowing excretion by the kidney. The aromatic nucleobases also interact with other hydrophobic molecules in serum and on cell surfaces. In certain embodiments, siRNA delivery systems involve complexing the RNA with cationic and neutral lipids, although encouraging results have also been obtained using peptide transduction domains and cationic polymers. Including PEGylated lipids in the formulation prolongs the circulating half-life of the particles.

[0166] As noted, one type of optimization of single-stranded DNA or RNA oligonucleotides is the use of chemical modifications to increase the nuclease resistance such as the introduction of phosphorothioate ("PS") linkages in place of the phosphodiester bond.This modification improves protection from digestion by nucleases. PS linkages also improved binding to serum proteins in vivo, increasing half-life and permitting greater delivery of active compound to tissues. Chemical modifications to subunits of the nucleotides can also improve potency and selectivity by increasing binding affinity of oligonucleotides for their complementary sequences. Examples of such modifications to the nucleoside sugars include 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), and 2'-O-methoxyethyl (2'-MOE) RNA, and others as discussed elsewhere herein. Even more affinity can be gained using oligonucleotides modified with locked nucleic acid (LNA), which contains a methylene bridge between the 2' and 4' position of the ribose. This bridge "locks" the ribose ring in a conformation that is ideal for binding, leading to high affinity for complementary sequences. Related bridged nucleic acid (BNA) compounds have been developed and share these favorable properties. Their high affinity has permitted the development of far shorter oligonucleotides than previously thought possible which nonetheless retain high potency. The chemistry for introducing 2'-O-Me, 2'- MOE, 2'-F, or LNA into oligonucleotides is compatible with DNA or RNA synthesis, allowing chimeras with DNA or RNA bases to be easily obtained. This compatibility allows the properties of chemically modified oligonucleotides to be fine-tuned for specific applications, which is a major advantage for development that makes LNAs and other BNAs convenient tools for many applications.

[0167] Therapeutic administration of the active agents described herein include any method by which a nucleic acid (e.g., DNA or RNA), as known to one of ordinary skill in the art. For treatment of aggressive prostate cancer, delivery may be via, for example, oral administration and / or injection into the prostate gland or tumor or both.

[0168] In certain embodiments, the active agents can be delivered to an organelle, a cell, a tissue, a tumor or an organism via one or more injections (i.e., a needle injection), such as, for example, orally, subcutaneously, intradermally, intramuscularly, intravenously, or intraperitoneally.

[0169] A described inhibitory nucleic acid or other active agent can be incorporated into pharmaceutical compositions suitable for administration. For example, pharmaceutical compositions can comprise one or more the active agents and a pharmaceutically acceptable carrier.

[0170] Then active agent may be provided in a sustained release composition. The use of immediate or sustained release compositions depends on the nature of the condition being treated. If the condition consists of an acute or over-acute disorder, treatment with an immediate release form can be conducted over a prolonged release composition. Alternatively,for certain preventative or long-term treatments, a sustained release composition may be appropriate.

[0171] The active agent can be administered in a single dose or in multiple doses. Where the administration of the active agent is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the active agent can be directly into the tissue at or near the site of aberrant or unwanted target gene expression. Multiple injections of the active agent can be made into the tissue, for example, into the prostate gland, into the prostate tumor, or near the tumor.

[0172] In addition to treating pre-existing aggressive or non-aggressive prostate cancers, active agents of the disclosure can be administered prophylactically in order to prevent or slow the conversion of a non-aggressive prostate cancer to an aggressive form. The active agent can be employed in combination therapies, meaning that the present compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutic agents or medical procedures. The combination of therapies (therapeutic agents or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed can achieve a desired effect for the same disorder (for example, a compound described herein can be administered concurrently with another therapeutic agent used to treat the same disorder), or they can achieve different effects (e.g., control of any adverse effects).

[0173] Turning now to specific embodiments of the present disclosure, cancer remains a major health burden worldwide, and new therapeutic approaches are needed to improve the treatment of this disease. RNA interference (RNAi) has emerged as a promising therapeutic strategy for the treatment of cancer and other diseases, as it provides a mechanism for specifically silencing disease-causing genes. However, the effective delivery of small interfering RNA (siRNA) remains a major challenge in realizing the full therapeutic potential of RNAi. The present disclosure is therefore directed to, in at least certain embodiments, novel self-assembled micelles of highly specific siRNA silencing agents and their use for the delivery of siRNA for targeting the tumor stemness and EMT protein Doublecortin-like kinase 1 (DCLK1), which is upregulated in many solid tumor cancers. Dysregulation by DCLK1 drives tumorigenesis, and metastasis in solid tumors. Furthermore, elevated expression of DCLK1 in patients with solid tumors is associated with poor overall survival.

[0174] In various embodiments, the advantages of the presently disclosed technology include increased cellular uptake without the requirement of transfection reagents, improvedstability and circulation, reduction of adverse stimulation of the immune response, reduced off- target toxicity, specific silencing of a cancer stemness and EMT promoting genes, and improved efficacy against solid tumor cancers. These advantages represent a substantial improvement of nanoparticle and liposome-based RNA silencing based delivery platforms. This technology can be used in the cancer related context depending on the contribution of DCLK1-related signaling on tumor related outcomes including metastasis, inhibition, or prevention. The route of administration can be, but is not limited to, intravenous, intraperitoneal, or intertumoral, or by any other effective means such as oral, subcutaneous, or transdermal administration. Additionally, given the critical role of tumor stemness in promoting the highly proliferative features of tumorigenesis, these agents can be used in combination with traditional and or other therapeutic modalities.

[0175] SAMiRNA is a self-assembling, micelle-forming double-conjugated RNAi with the oligonucleotide in the middle flanked by a hydrophobic and hydrophilic end that is stable in the circulation due to reduced metabolic clearance. The hydrophobic interactions drive the molecular assembly into the micellar structure, allowing for targeting moieties to be added to the hydrophilic end.

[0176] The linker chemistry used in the present disclosure allows for effective endosomal escape and the SAMiRNA hides the unmodified double-stranded RNA from triggering innate immune stimulation, thus reducing the side effects associated with excessive stimulation of the immune system. The siRNA design platform comprises a unique siRNA synthesis algorithm that allows for creation of highly specific siRNAs that can knockdown specific RNA moieties of virtually any gene product. Large scale synthesis and high-quality detection protocols, RT- qPCR enables quantitative detection of RNA silencing efficacy.

[0177] The SAMiRNA technology designed herein for DCLK1 knockdown is particularly important due to multifunctional activities of DCLK1, which are increasingly becoming recognized as being isoform dependent, human and animal versions of DCLK1 are generated by alternative promoter usage and alternative splicing events. These characteristics of DCLK1 often require RNA knockdown at distinct domains within the protein. For example, human DCLK1 has been determined to have 2 long isoforms and 2 short isoforms generated by alpha and beta promoters respectively. Although the isoforms share kinase domain, there are differences in the N-terminal, microtubule binding domain and C-terminal, non-kinase domain. SAMiRNA thus has the potential to dissect the functional differences that exists within distinct DCLK1 domains.

[0178] The SAMiRNA synthesis platform does not merely encapsulate the siRNA sequences but self-assembles the siRNA sequence into the micelle. Thus, SAMiRNA identifies novel compatible target siRNA sequences. creating a highly specific DCLK1 targeted SAMiRNA. The SAMiRNA synthesis process used herein is further described in U.S. Provisional Patent Application Ser. No. 63 / 509,946, to which the present application claims priority.

[0179] Based on this approach we generated novel proprietary DCLK1 siRNA / SAMiRNA formulations. We used a high throughput screening protocol based on an algorithmic screening method (Bioneer Inc). We identified 17 specific siRNA sequences that targeted all four human DCLK1 isoforms. The 17 candidates were subject to physicochemical characterization (Zeta potential and HPLC analysis). Based on mRNA analysis 2 candidates were chosen for further evaluation due to greater than 36 percent inhibition of DCLK1 mRNA expression in BXPC3 cells compared to scrambled control. Dose curve experiments using candidate SAMiRNA molecules targeting DCLK1 revealed DCLK1 knock down in vitro, with corresponding reduction in DCLK1 protein expression in vivo (FIG.2J). METHODS

[0180] DCLK1 expression was evaluated in HGSOC cell lines, and cisplatin-sensitive and cisplatin-resistant (CPR) isogenic cell lines using western blot and in ovarian tumor tissue microarray by immunohistochemistry. We used a pharmacologic inhibitor (DCLK1-IN-1; CAS No. 2222635-15-4) and genetic manipulation (CRISPR-Cas9 knockout and over-expression) to perform 3D drug sensitivity assays, migration, invasion, and proliferation assays. Targeted gene expression profiling was performed in DCLK1 knockout CPR spheroids. An orthotopic intraperitoneal CPR spheroid xenograft model was used to assess the efficacy of a novel combination of DCLK1 inhibition with cisplatin. Reagents and Cell Culture

[0181] Human OvCa cell lines OVCAR-8, IGROV-1, OVCAR-8 CPR, and IGROV-1 CPR (cisplatin-resistant) were a generous gift from Dr. Alexander S Brodsky (Brown University, Providence, RI) (17). Immortalized normal fallopian tube epithelial cells (FTE187 and FTE188) and TykNu were a kind gift from Dr. Danny Dhanasekaran (University of Oklahoma Health Sciences Center [OUHSC], Oklahoma City, OK). Human normal ovarian surface epithelial cells (HOSE), OVCAR-3, OVCAR-4, MeSOV, SNU-119, ES2, and Kuramochi were a kind gift from Dr. Doris M. Benbrook (OUHSC). The cell lines were profiled via short tandem repeat profiling to confirm their identity. Cell lines were cultured in RPMI (OVCAR-8, OVCAR-8 CPR, IGROV-1, SNU-119, MeSOV, OVCAR-3, OVCAR-4,and Kuramochi), McCoy’s 5A (ES2), and MEM (TykNu) media supplemented with 10% fetal bovine serum (FBS). The cell lines were tested periodically for mycoplasma using Mycoplasma PCR Detection Kit (abm®, G238), and if found positive, mycoplasma-free stocks were instead used. Experiments were performed on cells within 15 passages post-thaw. DCLK1 inhibitor, DCLK1-IN-1 (Tocris, 7285), and cisplatin (Sigma-Aldrich, P4394) were reconstituted according to the manufacturer’s protocol. A proprietary SAMiRNA nanoparticle that targets DCLK1 (siDCLK1) and a scramble control SAMiRNA (siScrambled) were provided by COARE Holding, Inc. DCLK1 overexpression, CRISPR-Cas9 engineering, and generation of stable cell lines

[0182] OVCAR-4 stable cell lines over-expressing wild-type DCLK1 (DCLK1 WT; long variant) and kinase domain mutants (DCLK1 D511N and DCLK1 D533N) were generated using lentiviral transduction, and selection using blasticidinS (3 µg / ml). The plenti_DCLK1 (RRID: Addgene_163625), plenti_DCLK1 D511N (RRID: Addgene_163626), and plenti_DCLK1 D533N (RRID: Addgene_163627) plasmids were a gift from Dr. Kenneth Westover (18). CRISPR / Cas9 technology was used to knock out DCLK1 in OVCAR-8 CPR cells. Briefly, sgRNA targeting sequences were designed using cloud-based software (www.benchling.com). The following DCLK1-targeting sense (DNA) and corresponding antisense (RNA) oligos, sgDCLK#1: 5′-CACCGAGTAGAGAGCTGACTACCAG-3′ (SEQ ID NO:1) and 5′-CUGGUAGUCAGCUCUCUACUCGGUG-3′ (SEQ ID NO:2); and sgDCLK1#2: 5′-CACCGAGTAGAGAGCTGACTACCA-3′ (SEQ ID NO:3) and 5′- UGGUAGUCAGCUCUCUACUCGGUG-3′ (SEQ ID NO:4), were annealed and ligated into BsmB1 cut lentiCRISPRv2 vector (RRID: Addgene_52961) following previously published methodology (19). OVCAR-8 CPR cells were transduced and selected with 2 µg / ml puromycin. Luciferase-tagged OVCAR-8 CPR stable cell lines (OVCAR-8 CPR luc) were generated using lentiviral transduction of Lenti-Labeler™ plasmid (pLL-CMV-Luciferase- T2A-Puro, System Biosciences, LL150PA-1), and puromycin selection. Spheroid Drug Sensitivity and Combination Index

[0183] OvCa cells lines (OVCAR-8, OVCAR-8 CPR, OVCAR-8 CPR DCLK1 KO, OVCAR-4, OVCAR-4 DCLK1 WT, OVCAR-4 DCLK1 D511N, and OVCAR-4 DCLK1 D533N) were seeded (1,000-2,000 cells / well) in 96-well clear round bottom ultra-low attachment microplates (Corning®, 7007) and incubated at 37°C in 5% CO2for 48 – 72 hours, to allow for spheroid formation. Subsequently, the spheroids were treated with varying concentrations of DCLK1-IN-1 and cisplatin for 48 and 96 hours, respectively to determine the IC50 of individual drugs. In combination treatment, OVCAR-8 CPR spheroids were exposed todifferent concentrations of the inhibitors simultaneously. Images of spheroids treated with different concentrations of the inhibitors were taken at the endpoint using a bright field microscope (Nikon Microscope Eclipse TE2000-U). Spheroid viability was assessed using CellTiter-Glo®3D Cell Viability Assay (Promega, G9683) according to the manufacturer’s protocol. Briefly, 100 µl CellTiter-Glo®3D reagent was added to each well and placed on a shaker for 40 minutes. The contents of each well of the plate were then transferred separately in single wells of MicroliteTM1+ plates (ThermoFisher Scientific, 7571) and the luminescence was read with Synergy H1 Microplate Reader (BioTek). Spheroid survival was calculated as the percentage of the control (vehicle-treated) group. To assess the combined effect of DCLK1- IN-1 and cisplatin on platinum-resistant OvCa cells, a Combination index (CI) was used. The CI value was calculated using CompuSyn software (CompuSyn, Inc.) which is based on the Chou-Talalay Method (20), and the combined effect was classified as follows: CI < 1 for a synergistic effect; CI > 1 for an antagonistic effect, and CI = 1 for an additive effect. Immunoblot

[0184] Western blotting was performed on whole-cell lysates (WCLs) generated from adherent / monolayer and suspended / spheroid cell cultures. For suspended cells, 250,000- 400,000 cells were plated in poly-HEMA (Sigma-Aldrich, P3932; 12 mg / mL in 95% ethanol)- coated plates for 24 to 48 hours. Cell clusters were centrifuged to obtain a pellet. WCLs were prepared using RIPA buffer (PierceTM,89901) and Protease and Phosphatase Inhibitor (PierceTM, A32961). Protein concentration was determined using the BCA Protein Assay kit (PierceTM, 23228). Equal amounts of lysates (15–30 μg) were electrophoresed and transferred to PVDF membranes. Ponceau S Stain (Sigma-Aldrich, P7170) was used to stain total protein on the membrane to obtain a nonspecific band. Membranes were blocked using EveryBlot Blocking Buffer (Bio-Rad, 12010020) for 10 minutes post-transfer, and incubated overnight at 4°C with primary antibody. After secondary antibody incubation, membranes were analyzed using Clarity MaxTMWestern ECL Substrate (Bio-Rad, 1705062). Antibody sources and dilutions are listed in Table 1.Table 1. Antibodies and antibody dilutions used for Western blots and IHCProliferation, Migration, and Invasion Assay

[0185] Proliferation assays were performed using IncuCyte®S3 Live-Cell Analysis Instrument (Sartorius) and colony formation assays. Total cell numbers were counted using the Countess II automated cell counter (Life Technologies). For the proliferation assays under 2D conditions, 2,500 OVCAR-8 CPR and 2,000 OVCAR-4 cells were seeded in 96-well plates (TPP™, 92096) and placed in IncuCyte. For the spheroid growth assay, 1,000 OVCAR-8 CPR cells were plated in 96-well clear round bottom ultra-low attachment microplates (Corning®, 7007) and then placed in IncuCyte. After incubation for the indicated times live-cell images were obtained using a 10x objective lens (4 images / well for adherent cells and 1 image / well for single spheroids) within the instrument, and percentage confluence (adherent cells) and brightfield area (spheroid) was analyzed using IncuCyte 2020A software. For colony formation assays, 500 cells / well (OVCAR-4) were plated in 6-well plates and cultured for 12 days with media replacement every 3 to 4 days. On day 12, the colonies were fixed using 70% ethanol and stained with 0.4% crystal violet (CV). Images were taken using GelCountTMColony Counter (Optronix) and quantifications were performed using GelCount Software (version 1.1.8.0). The migration assays were performed using Transwell 8-µm cell culture inserts (BD Falcon, 353097). Briefly, 10,000 cells / well (OVCAR-8 CPR) and 40,000 cells / well (OVCAR- 4) were plated in serum-free medium (SFM) on a transwell filter and allowed to migrate to a medium containing 10% FBS. After 16-24 hours, cells from above the membrane were wiped with cotton swabs, and cells at the bottom were fixed in 10% formalin and stained with 0.05% CV. Cell migration was analyzed by counting cells using a bright field microscope (NikonMicroscope Eclipse TE2000-U) and ImageJ. For adherent cells, invasion assays were performed using 8-µm Transwell cell culture inserts (BD Falcon, 353097), after coating the filters with diluted Matrigel (1 mg / ml; Corning®, 354234) in SFM. Cells (100,000-200,000 cells / well) were plated on the Matrigel and allowed to invade to medium containing 20% FBS for 24 hours. Cell invasion was analyzed as in the cell migration assays. For assessing spheroid invasion, ES2 cells (1,500 cells / well) were seeded in a 96-well clear round bottom ultra-low attachment microplates (Corning®, 7007) and incubated for 24 hours to allow spheroid formation. Culturex Spheroid Invasion Extracellular Matrix (R&D Systems, 3500-096-03) was added on top and allowed to polymerize for 1 hour at 37°C, followed by the addition of medium containing 20% FBS. DCLK1-IN-1 was added to the spheroids. The spheroids were monitored for 4 days using IncuCyte. Live-cell images were obtained using a 4x objective lens within the instrument, and invading cell area was quantified using IncuCyte 2020A software. Quantitative real-time polymerase chain reaction

[0186] Total RNA was extracted from the cell pellets using TRIzol reagent (Invitrogen, 15596026) and purified using Monarch RNA Cleanup Kit (New England BioLabs, T2050L). RNA concentration was quantitated using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). qRT-PCR assays were performed using EvaGreen 2x qPCR MasterMix (Bullseye). Analysis was performed using Bio-Rad CFX96. Primer sequences qRT-PCR assays are listed in Table 2. Table 2. Primer sequences used in qRT-PCR assays. Genes are human. Gene Forward Reverse E-cadherinTGGCGTCTGTAGGAAAGCAGCAGAATCAGATargeted Gene Expression Profiling

[0187] Total RNA was extracted from OVCAR-8 CPR sgCtrl and DCLK1 KO spheroids using the method described above. Targeted mRNA expression profiling was performed using nCounter®Tumor Signaling 360™ Panel (NanoString Technologies) composed of 780 genes (including internal reference genes) involved in cellular energetics, sustained proliferation, evasion of growth suppression, genomic instability, resistance to cell death, EMT, and metastasis. Normalization of housekeeping genes for the quantification of gene expression levels, normalization of the positive control for background correction, and data analysis were performed using the nSolverTManalysis software (NanoString Technologies). Transcripts with an RNA count < 20 for all samples were excluded because they were considered unexpressed. The mean and standard deviation (SD) were calculated for each group. Only genes with significant (p < 0.05) fold changes (< –2 or > +2) were considered differentially expressed. TGFβ / SMAD Luciferase Reporter Assay

[0188] The TGFβ / SMAD signaling activity was measured using the SBE4-luc reporter construct, a gift from Dr. Bert Vogelstein (RRID: Addgene_16495) (21). Briefly, control or DCLK1 knockout OVCAR-8 CPR cells were seeded in a 6-well plate and were co-transfected with 1 µg SBE4-luc reporter and 20 ng renilla luciferase vector (pRL-TK, Promega) using X- tremeGENE 360 transfection reagent (Roche, 08724121001). At 24 h post-transfection, cell lysates were assayed according to the manufacturer’s protocol using Dual-Luciferase®Reporter Assay System (Promega, E1910). The luminescence was read with Synergy H1 Microplate Reader (BioTek). Orthotopic cisplatin-resistant spheroid mouse model

[0189] The animal experiment was performed according to protocols approved by the Institutional Animal Care and Use Committee at OUHSC. For this study, 6-to-8-week-old female athymic nude mice (Envigo) were used. One million OVCAR-8 CPR luc cells were plated on a 100 mm poly-HEMA-coated plate to form spheroids for 48 hours. The spheroids were centrifuged to obtain a loose pellet that was re-suspended in 150 µl of sterile PBS. This spheroid suspension was injected intraperitoneally in each mouse using a 21G needle and mice were randomized into 7 groups for different treatments. One week post-spheroid injection, treatment was initiated for the different groups: 1. Vehicle control, 2. Cisplatin (5 mg / kg), 3. DCLK1-IN-1 (25 mg / kg), 4. Scrambled SAMiRNA (30 mg / kg), 5. siDCLK1 SAMiRNA (30 mg / kg), 6. Cisplatin combined with DCLK1-IN-1, and 7. Cisplatin combined with siDCLK1 SAMiRNA. All treatments were administered intraperitoneally every other day. Mouse weightmeasurements and bioluminescent imaging (BLI) were performed weekly. Imaging was performed with the IVIS Spectrum In Vivo Imaging System (PerkinElmer). Mice were administered D-luciferin potassium salt (100 mg / kg; Goldbio, LUCK) in sterile DPBS (Gibco, 14190-136) intraperitoneally, anesthetized using isoflurane, and then imaged. Living Image software (version 2.5.5) was used to collect and analyze images. Regions of interest covering the entire peritoneal cavity were selected, including tumors, and total photon counts were determined. In some cases, mice that received combination therapy appeared to be moribund with extreme weight loss; however, no statistical trends were observed. Mice were euthanized 30 days after injection (n = 6 mice per group). The tumor colonies were counted, collected, and weighed. Excised tumor tissues and organs (e.g., liver, kidney, and spleen) were stained with H&E. Immunohistochemistry (IHC) staining for DCLK1 was performed on the tumor tissue isolated from mice. Quantification was done using HALO®software (Indica Labs, v3.2.1851). Antibody sources and dilutions are listed in Table 1 above. Tissue Microarray (TMA)

[0190] The commercially available TMA (CHTN OvCa2 Ovarian Carcinoma Survey; four 0.6 mm cores / case) with de-identified formalin-fixed, paraffin-embedded (FFPE) patient tissue samples representing major histological types of epithelial OvCa and associated clinicopathological data (age, histologic subtype, and stage) was used to assess DCLK1 expression. Carcinoma tumor types included serous papillary, clear cell, endometrioid adenocarcinoma, and mucinous adenocarcinoma (12 cases each). DCLK1 staining quantification analysis was performed using HALO®software (Indica Labs, v3.2.1851), and the H-score was calculated. Synthesis of SAMiRNA nanoparticles

[0191] Non-limiting examples of the methods of synthesizing self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles are shown in the U.S. Provisional Patent Application Ser. No.63 / 509,946. Statistics

[0192] GraphPad Prism version 9.4.1 for Windows (GraphPad Software) was used for all statistical analyses. A two-tailed unpaired Student t-test was used to compare pairs of conditions. One-way ANOVA nonparametric followed by Tukey’s / Dunnett’s / Sidak’s post hoc test was used to compare more than two conditions. A two-way ANOVA followed by post hoc tests was used to analyze data from time-course experiments. A P value of <0.05 was denoted as statistical significance.RESULTS

[0193] High DCLK1 expression significantly correlates with poor overall and progression- free survival in HGSOC patients. DCLK1 was differentially expressed in 3D and 2D OvCa cultures, with high expression in CPR spheroids relative to their sensitive controls. DCLK1 inhibition effectively re-sensitized CPR OvCa spheroids to cisplatin, reducing cell proliferation, migration, and invasion. In addition, DCLK1 kinase activity is critical for mediating CPR. A combination of cisplatin and DCLK1-IN-1 showed a synergistic cytotoxic effect against OvCa spheroids. Targeted gene expression profiling revealed that DCLK1 inhibition in CPR OvCa spheroids significantly affects pathways related to TGFβ signaling, EMT, Myc, immortality, and stemness. A significant reduction of tumor burden with combined DCLK1 inhibition (DCLK1-IN-1 / novel siDCLK1 self-assembled micelle) and cisplatin in vivo was observed. High DCLK1 expression is associated with poor prognosis in OvCa patients.

[0194] A panel of human OvCa cell lines, FTE cells (FTE188 and FTE187), and HOSE cells grown under adherent (2D) and suspended (3D) conditions were screened for DCLK1 expression to elucidate the role of DCLK1 in OvCa. We observed that OvCa cells differentially express both the long and short variants of DCLK1 under 2D and 3D conditions (FIGS.1A-B) but at a higher or similar level than that observed in FTE187 cells or HOSE cells. This differential expression was independent of their classification i.e., p53 or BRCA1 mutation status, or high-grade serous, non-serous, or clear cell carcinoma (22). Interestingly, OVCAR- 3 and OVCAR-8 cell lines, established from ovarian tumors of patients refractory to cisplatin and high-dose carboplatin (23), showed an upregulation of the DCLK1-L and DCLK1-S form when grown under suspended conditions, respectively (FIG. 1C). Further, we evaluated DCLK1 mRNA expression using RNA seq data from previously established isogenic pairs of cisplatin-resistant (CPR) OVCAR-8 spheroids (OVCAR-8 and OVCAR-8 CPR) (17). The platinum-resistant spheroids showed a 20% increase in DCLK1 expression relative to that in their sensitive controls (FIG.1CA). These findings were confirmed at the protein level, where IGROV-1 CPR and OVCAR-8 CPR spheroids showed a 1.2- and 2.5-fold upregulation in total DCLK1 relative to IGROV-1 and OVCAR-8 spheroids, respectively (FIG. 1D). Collectively, this data indicates that DCLK1 has a role in mediating CPR in the context of OvCa cell spheroids. Analysis of DCLK1 expression in a human ovarian carcinoma TMA including major histological subtypes revealed elevated DCLK1 levels in serous papillary carcinoma and endometrial adenocarcinoma compared to normal fallopian tubes (FIGS. 1E-F). A meta- analysis (24, 25) further showed that increased DCLK1 expression was significantly correlatedwith worsened progression-free survival and overall survival in patients with serous ovarian cancer (FIGS. 1G-H). Together this data demonstrates the pathological and clinical significance of DCLK1 in the context of ovarian cancer. Increased DCLK1 levels correlate with cisplatin resistance.

[0195] Ovarian tumor cell aggregates or spheroids are involved in peritoneal dissemination, pose a significant impediment to treatment efficacy, and contribute to disease recurrence (26). Thus, in vitro, cultured spheroid models are an invaluable tool to improve our understanding of OvCa biology and test novel anticancer therapeutics and their combinations. We developed OVCAR-8 and OVCAR-8 CPR spheroids and tested cisplatin sensitivity by quantifying cellular viability. Cisplatin-resistant spheroids showed a shift in the dose-response curve to the right relative to sensitive control (FIG.2A). Morphologically, the OVCAR-8, and OVCAR-8 CPR spheroids were similar at the start of treatment (FIG. 2B). In the presence of cisplatin, the OVACR-8 spheroids decreased in size, but cisplatin-resistant spheroids continued to grow and lost their well-defined edges (FIG.2B). OVACR-8 CPR cells showed a significant ~3-fold increase in cisplatin IC50 compared to OVACR-8 cells when grown as spheroids (FIG. 2C, 15.3µM vs. 5.1µM). This data collectively demonstrated that the OVCAR-8 CPR cells were indeed resistant to cisplatin when cultured in suspended conditions.

[0196] To determine the causal role of DCLK1 in mediating cisplatin resistance, we then examined if constitutive ectopic over-expression of DCLK1 in deficient cell lines would confer resistance. We first stably overexpressed wild-type DCLK1 (DCLK1 WT) and kinase-domain mutants (D511N and D533N) in OVCAR-4 cells (FIG.2CA). Over-expression of DCLK1 WT (OVCAR-4 WT OE) resulted in a curve-shift towards the right relative to control cells when grown under suspended conditions (FIG. 2D). Interestingly, OVCAR-4 cells produced multicellular aggregates instead of compact spheroids as observed with OVCAR-8 cells, indicative of HGSOC heterogeneity (FIGS.2B, 2F). Visual inspection of the cisplatin-treated spheroids revealed that the control spheroids appeared dense relative to the DCLK1 WT OE spheroids (FIG. 2E). Further, OVCAR-4 DCLK1 WT OE cells showed a significant increase in cisplatin IC50 compared to OVCAR-4 cells when grown as spheroids (FIG.2F, 16.4µM vs. 6.4µM). This data indicates that high DCLK1 expression alone is sufficient to confer CPR in OvCa spheroids.

[0197] Subsequently, we tested the sensitivity of a highly selective small-molecule inhibitor of DCLK1 that targets the kinase domain (DCLK1-IN-1) in our model systems (27). We observed that over-expression of DCLK1 WT in OVCAR-4 spheroids made them more sensitive to DCLK1-IN-1 as illustrated by the left curve shift, spheroid size reductions, andlower IC50 values relative to control spheroids (OVCAR-4) (FIGS.2G-I, 9.9µM vs.15.7µM). Similar observations were also noted in OVCAR-8 and OVCAR-8 CPR spheroids (FIGS.2CB- CC) indicating that DCLK1 is indeed a druggable target in ovarian cancer.

[0198] FIG. 2J shows effects of two anti-DCLK1 siRNA treatments (SAMiRNA #9, SAMiRNA #11) on DCLK1 activity. Both siRNA#9, and siRNA#11 (5´- GGCGACUUGCCUGAGCGCG-3´ - SEQ ID NO:21), caused knock down of DCLK1 in vitro. BxPC3 cells were treated with SAMiRNA nanoparticles containing siRNA#9 and siRNA#11. SAMiRNA#11 nanoparticles had significant DCLK1 protein inhibition (isoform 82 kDa = 44% inhibition at 0.1 µM; 74% inhibition at 1.0 µM) (isoform 47kDa = 12% inhibition at 0.1 µM; 27% inhibition at 1.0 µM) in the BxPC3 cells when compared with Vehicle-treated cells.

[0199] The present disclosure also includes anti-DCLK1 siRNA treatments that comprise SEQ ID NO:21 and that further include up to 20 optional nucleotides at each end thereof (5’- X1-20 GGCGACUUGCCUGAGCGCGX1-20-3´ - SEQ ID NO:22). Each of the optional nucleotides of SEQ ID NO:22 (i.e., X1-20at the 5’ and 3’ ends thereof) can be any of the natural or modified, non-natural bases disclosed herein (including, but not limited to, adenine, guanine, thymine, cytosine, uracil, 5-uracil (pseudouridine), dihydrouracil, inosine, ribothymine, 5- methylcytosine (5-me-C), 7-methylguanine, hypoxanthine, xanthine, 5-hydroxymethyl cytosine, 2-aminoadenine, 2-methyladenine, 6-methyladenine, 2-propyladenine, N6-adenine, N6-isopentenyladenine, 2-methylthio-N6-isopentenyladenine, 2-methylguanine, 6- methylguanine, 2-propylguanine, 1-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 2- thiouracil, 2-thiothymine, 2-thiocytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, dihydrouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-carboxymethylaminomethyl-2- thiouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- methoxycarboxymethyluracil, 5-methoxyuracil, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, 5-carboxymethylaminomethyluracil, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, alkynyl derivatives of pyrimidine bases including 5- propynyl uracil, and 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 4- thiouracil, 8-halo-adenines, 8-amino adenine, 8-thiol adenine, 8-thioalkyl adenine, 8-hydroxyl adenine, 5-trifluoromethyl uracil, 3-methylcytosine, 5-methylcytosine, 5-trifluoromethyl cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 8-halo-guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanine, 8-hydroxyl guanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, beta-D- galactosylqueosine, beta-D-mannosylqueosine, 1-methylinosine, 2,6-diaminopurine, queosine,tricyclic pyrimidines, phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), and phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one, and the like).

[0200] In addition, one or more nucleotides of SEQ ID NO:21 (and / or the corresponding sequence in SEQ ID NO:22) can be substituted with a corresponding non-natural nucleotide; for example, but not by way of limitation, a cytosine (C) could be replaced with a non-natural derivative of C (such as, but not limited to, 5-me-C, 5-hydroxymethyl cytosine, 2-thiocytosine, 4-acetylcytosine, 5-propynyl cytosine, 6-azo cytosine, 5-methylcytosine, 5-trifluoromethyl cytosine, phenoxazine cytidine, phenothiazine cytidine, and the like).

[0201] FIG.2K shows results of tumor xenograft experiments with CBT-611S, a DLCK1- specific SAMiRNA. (A) A schematic representation of the tumor xenograft experimental design. Mice were injected with BxPCe tumor cells and tumors were allowed to grow. On day 16, SAMiRNA treatment was initiated. Tumor volumes were measured and plotted. (B) At day 25, average tumor volumes of the two treatments began to diverge and continued to diverge until the end of the experiment at 46 days after injection. (C) After 46 days of tumor growth, CBT-611S treatment resulted in significant inhibition (p<0.05) of tumor volumes. DCLK1 inhibition re-sensitizes OvCa spheroids to cisplatin.

[0202] To determine the necessity of DCLK1 in mediating CPR, we stably knocked out (KO) DCLK1 using CRISPR-Cas9 technology in OVCAR-8 CPR cells expressing high endogenous DCLK1. OVCAR-8 CPR DCLK1 KO (sgDCLK1 #1 and sgDCLK1#2) spheroids were re-sensitized to cisplatin relative to OVCAR-8 CPR control (sgCtrl) spheroids as demonstrated by a left shift in the dose-response curves (FIG.3A). Cisplatin treated OVCAR- 8 sgDCLK1 #1 and #2 spheroids exhibited reduced spheroid size and / or disruption when compared to control spheroids (FIG. 3B). We observed statistically significantly lower cisplatin IC50 values in sgDCLK1 #1 and #2 spheroids when compared to that observed in sgCtrl spheroids (FIG. 3C, 6.2µM, 3.9 µM vs. 14.7µM). The IC50values in OVCAR-8 sgDCLK1 #1 and #2 spheroids were comparable to those observed in sensitive OVCAR-8 spheroids. In addition, DCLK1 KO altered the sensitivity of OVCAR-8 CPR spheroids to the DCLK1 inhibitor (DCLK1-IN-1) as observed by curve shift to the right in dose-response curves and significant ~2-fold change in the IC50value, indicating that the inhibitor is specific to DCLK1 in our model system (FIGS. 3CA-CC). Collectively, this data shows that DCLK1 mediates CPR in OvCa.

[0203] Further, spheroids derived from OVCAR-4 cells over-expressing DCLK1 kinase- dead mutants (D511N and D533N) were more sensitive to cisplatin relative to control spheroids (OVCAR-4) as demonstrated by a left dose-response curve-shift, spheroid morphologicalchanges, and reduced cisplatin IC50 values (8.7µM, 5.2µM vs.16.3µM) (FIGS.3D-F). These data also indicate that the over-expression of the kinase-dead mutants (D511N and D533N), unlike DCLK1 WT, rescued the sensitivity to cisplatin; indicating that the kinase domain is important for conferring drug resistance. Having demonstrated that ectopic expression of DCLK1 was sufficient to confer cisplatin resistance, we tested the necessity of the endogenous protein for resistance, by measuring the effect of co-treatment of cisplatin with DCLK1 inhibitor (DCLK1-IN-1) in OVCAR-8 CPR spheroids. We observed robust sensitization of spheroids to cisplatin when used in combination with DCLK1-IN-1 in the CPR spheroids. In fact, we observed a strongly synergistic effect as indicated by the combination index values of <1 observed at different dose levels (e.g., ED50, ED75, etc.) (FIGS.3G, H). DCLK1 increases OvCa cell proliferation, migration, and invasion in vitro.

[0204] Using the spheroid area (as a surrogate for proliferation), we observed a significant decrease in the area of OVCAR-8 CPR sgDCLK1 #1 and #2 spheroids compared to sgCtrl spheroids (FIGS. 4A-B). As OVCAR-4 cells do not form compact spheroids like OVACR-8 CPR cells (e.g., FIGS.2E, 2H), we used the colony formation assay to evaluate the effects of DCLK1 over-expression and kinase domain inactivation on cell proliferation. We observed a significant increase in total colony count in OVCAR-4 DCLK1 WT OE cells compared to OVCAR-4 cells, and this increase was reversed to control levels in OVCAR-4 cells over- expressing DCLK1 kinase-dead mutants (D511N and D533N) (FIG.4C). However, we did not observe significant differences in colony diameter, area, average density, or average volume between the different groups. We also evaluated the effect of DCLK1 KO (OVCAR-8 CPR) and over-expression (OVCAR-4) on cell proliferation under adherent (2D) conditions, using area confluence as a parameter to quantify proliferation. Our data showed significantly reduced proliferation in OVCAR-8 CPR sgDCLK1 #1 and #2 cells relative to sgCtrl cells (FIG.4DA). Further, cell proliferation was significantly increased in OVCAR-4 DCLK1 WT cells compared to OVCAR-4 cells. Between the two-kinase domain mutants, cell proliferation of OVCAR-4 DCLK1 D511N was similar to control cells, but OVCAR-4 DCLK1 D533N cells did show a significant increase in cell proliferation compared to OVCAR-4 cells. However, it was less relative to OVCAR-4 DCLK1 WT cells (FIG.4DB). Together this data indicates that DCLK1 expression is sufficient to increase proliferation in OvCa cells in vitro both under 3D and 2D conditions.

[0205] We used a transwell chamber assay to study the effect of DCLK1 on OvCa cell movement (cell migration) to distal sites for establishing metastasis (cell invasion) in vitro. DCLK1 KO in OVCAR-8 CPR cells significantly reduced migration and invasion comparedto sgCtrl cells (FIGS.4D, 4F). In contrast, OVCAR-4 DCLK1 WT cells exhibited a significant increase in cell migration and invasion relative to control cells. Further, over-expression of the kinase-dead mutants (D511N and D533N) reversed the migratory and invasive phenotypes in vitro (FIGS.4E, 4G). In addition, DCLK1-IN-1 efficiency in inhibiting spheroid invasion was tested using ES2 cells that have endogenous DCLK1 expression. We observed a significant reduction in the invasive area around the ES2 spheroids when treated with DCLK1-IN-1 relative to the vehicle control group (FIGS. 4DC-4DD) indicating that the phenotype was specific to DCLK1. Thus, together these data demonstrate that DCLK1 expression is both required and sufficient to increase OvCa pro-metastatic phenotypes of migration and invasion. DCLK1 promotes epithelial-mesenchymal transition (EMT) in cisplatin-resistant spheroids.

[0206] To gain an understanding of the mechanism(s) by which DCLK1 might mediate chemoresistance and various pro-metastatic phenotypes, we next examined alterations to molecular signaling pathways using the Nanostring nCounter®Tumor Signaling 360™ Panel. The mRNA expression of 780 genes mapped to more than 40 tumor-signaling pathways was determined in OVCAR-8 CPR DCLK1 KO (sgDCLK1 #1) and sgCtrl spheroids. A heatmap of this normalized data for all the genes (equal variance) was generated using unsupervised clustering indicative of data reproducibility among the replicates. Genetic ablation of DCLK1 resulted in attenuation of the genes involved in TGFβ, HIPPO, MET, and MYC signaling pathways and inhibition of glutamine metabolism, EMT, and pathways related to immortality and stemness. We also observed an increase in antigen presentation and EGFR signaling pathways. TGFβ signaling pathways have been shown to trigger EMT thereby contributing to tumor progression, metastasis, and emergence of therapy resistance (28). Further, to validate these data, we performed a TGFβ reporter assay and western blotting, in which we observed a reduced activation of canonical TGFβ signaling with DCLK1 knockout in OVCAR-8 CPR cells (FIG. 5A, FIGS. 5AA-AB). To understand the role of DCLK1 in modulating EMT, we performed qRT-PCR analyses evaluating levels of different EMT-related genes and transcription factors (e.g., E-cadherin, Vimentin, Slug, Snail, Zeb-1, Zeb-2, etc.) in OvCa spheroids. At the basal level, we observed high Vimentin, Zeb-1, Zeb-2, Twist1, Snail, and low E-cadherin levels in OVCAR-8 CPR spheroids relative to OVCAR-4 spheroids indicating that the CPR spheroids were indeed more mesenchymal (FIG. 5AC). Further, DCLK1 KO in OVCAR-8 CPR spheroids resulted in a significant decrease in Vimentin, Snail, and Zeb-2. We also observed a decrease in E-cadherin and Zeb-1 expression; however, it was not statistically significant (FIG. 5B). Over-expression of DCLK1 WT in OVCAR-4 spheroids caused asignificant increase in Vimentin (~12-fold), and Snail (~5-fold) expression with the most dramatic upregulation in Slug (~125-fold), Twist1 (~40-fold), Zeb-2 (~60-fold), and Zeb-1 (~20-fold) (FIG.5C). A 2-fold increase in E-cadherin was also observed in OVCAR-4 DCLK1 WT spheroids relative to control (FIG.5C). Collectively, this data indicates that high DCLK1 expression in OvCa spheroids causes a shift from an epithelial to a more mesenchymal phenotype. In addition, genetic ablation of DCLK1 in chemoresistant OvCa spheroids resulted in a shift from a more mesenchymal to an epithelial phenotype. Combined inhibition of DCLK1 with cisplatin reduces intraperitoneal metastasis of cisplatin-resistant HGSOC spheroids in vivo.

[0207] Since we observed a synergistic effect following simultaneous combination treatment with cisplatin and DCLK1-IN-1 in vitro (FIG.3H), we sought to determine whether this novel combination strategy was efficacious in reducing tumor burden and / or metastasis of chemoresistant OvCa cells in vivo. To this end, to mimic advanced OvCa, we injected OVCAR- 8 CPR luc-tagged spheroids intraperitoneally in mice. At 7 days post-implantation, the tumor- bearing mice received different treatments (FIGS.6A-B).

[0208] Compared to the control groups (vehicle and scrambled SAMiRNA), the single treatment groups, i.e., cisplatin, DCLK1-IN-1, and siDCLK1 SAMiRNA (also referred to elsewhere herein as siRNA #11, SAMiRNA #11, and SAMiRNA DCLK1 #11) showed no significant difference in tumor weight and / or the number of visible metastases, while the combination treatment (DCLK1-IN-1 with cisplatin) exhibited a significant reduction in tumor weight and metastases (FIGS. 6C-D, 7A-B). In the siDCLK1 SAMiRNA and cisplatin combination treatment group, we observed a significant decrease in the number of metastases, and a modest decrease in the tumor weight that was not statistically significant compared to the control group (FIGS.7A-B). Bioluminescent imaging (BLI) did show that the combination of DCLK1 inhibition (using either DCLK1-IN-1 or siDCLK1 SAMiRNA) with cisplatin was effective in reducing tumor burden; however, we observed high variability in the BLI signal among the groups which could be due to the smaller number of mice that were imaged per group (n=3) (FIGS. 6E-F, 8A-B). Nevertheless, quantification of tumor weights and visible metastases at the end of the present work established the in vivo efficacy of the novel combination in CPR OvCa. Further, IHC analyses of the tumor tissue taken from mice receiving different treatments revealed that cisplatin treatment increased DCLK1 expression in the tumors in mice. Inhibiting DCLK1 in combination with cisplatin was effective in reducing DCLK1 expression (FIGS.8C-F).

[0209] The single treatment groups did not significantly influence the body weights of the mice. However, we did observe a decrease in the mouse body weights in the combination treatment groups relative to the control groups, albeit with no statistical difference (FIGS.8G- H). No overt ascites formation was present in either group. No significant abnormalities were observed in the liver, kidneys, and spleen tissue of the mice that received different treatments as assessed by a pathologist by H&E staining (FIG. 8I). In conclusion, these results demonstrate for the first time that inhibiting DCLK1 (e.g., using DCLK1-IN-1 or a siDCLK1 SAMiRNA) in combination with cisplatin was effective in reducing tumor growth and peritoneal metastatic implantation (a hallmark of HGSOC) in a platinum-resistant setting in vivo. DISCUSSION

[0210] HGSOC has the highest mortality rate among gynecological cancers that is ascribed to an advanced-stage at diagnosis and platinum-resistant relapses. Treatment of recurrent HGSOC is very challenging due to (1) a high degree of intra-tumor and inter-tumor heterogeneity between the primary tumors and metastatic sites, (2) the emergence of multi- drug resistance, (3) the modest efficacy of targeted therapies and / or immune checkpoint inhibitors, and (4) a limited availability of alternative treatment options due to treatment-related toxicities (29, 30). Thus, it is critical to identify unique drug targets to overcome chemoresistance and inhibit disease progression in OvCa patients.

[0211] DCLK1 is a microtubule-associate protein kinase, best known for its role as a tuft cell and cancer stem cell marker in gastrointestinal cancers (31). Multiple studies have shown its role in the development and progression of colon, pancreatic, renal, non-small cell lung, and esophageal squamous cell cancers (10, 32, 33, 34, 35). Herein, we report that DCLK1 is critical for mediating chemoresistance and pro-metastatic phenotypes in HGSOC. We established that OvCa cell lines express variable levels of DCLK1 when they are cultured under adherent and suspended conditions. Studies in colon cancer cells have shown the use of an alternate- promoter (β) within intron 5 of the DCLK1 gene resulting in the expression of the short variant (DCLK1-S), while the 5’ (α)-promoter regulates the expression of the long variant (DCLK1- L) (36). The present results show differential expression of both DCLK1-L and DCLK1-S variants in high-grade serous and clear-cell ovarian carcinoma cell lines. This could be due to differences in the transcriptional regulation of the α / β promoters in the hDCLK1-gene in OvCa cells. Using OvCa spheroids as our model systems, we showed that DCLK1 is both necessary and sufficient to mediate CPR in OvCa cell lines. In addition, we observed that inactivation of the kinase domain (using kinase dead versions of DCLK i.e., DCLK1 D511N and D533N)could re-sensitize the resistant OvCa spheroids to cisplatin, illustrating the importance of the kinase domain of DCLK1 in regulating this phenotype. Using pharmacologic inhibitors and genetic manipulation of DCLK1, we demonstrated that the increased pro-metastatic phenotype of OvCa cells including cell proliferation, migration, and invasion, were specific to DCLK1. These findings were consistent with studies using siRNA-mediated DCLK1 knockdown in esophageal squamous cell carcinoma (37).

[0212] We used a highly specific small molecule inhibitor of DCLK1, DCLK1-IN-1 (27), and a proprietary SAMiRNA nanoparticle that targets DCLK1 (siDCLK1) developed by COARE Holding, Inc to validate DCLK1 as a therapeutic target in chemoresistant HGSOC. In the present work, the DCLK1 inhibitor DCLK1-IN-1 was not only effective in reducing spheroid invasion but also showed a synergistic cytotoxic effect with cisplatin in platinum- resistant OvCa spheroid model in vitro. These findings were also validated in vivo, where DCLK1 inhibition (DCLK1-IN-1 / siDCLK1 SAMiRNA) combined with cisplatin effectively reduced peritoneal dissemination of OvCa. Tumor growth was reduced but the most striking differences were in the number of metastases compared to vehicle control. This indicates that DCLK1 is important for the survival and / or proliferation of the tumor cells and promotes their seeding at distant metastatic sites in the peritoneal cavity. Remarkably, DCLK1-IN-1 or siDCLK1 SAMiRNA as single agents at the indicated dose levels were not effective, but therapeutic benefits could be achieved at higher doses. The body weight changes observed in the groups receiving combination treatment were mainly attributed to the use of cisplatin (known to cause bone marrow suppression and nephrotoxicity), as DCLK1-IN-1 is well tolerated in mice at doses up to 100 mg / kg (27, 38).

[0213] Several studies have described different molecular mechanisms involved in drug resistance including altered drug efflux / influx because of dysregulation of multidrug- resistance transporters, presence of cancer stem cells, and / or altered expression of anti- apoptotic proteins (39). Panneerselvam J et al, have shown the involvement of ABCD-4 drug transporter in DCLK1-mediated CPR in non-small cell lung carcinoma (34). Studies by an independent group have shown that OVCAR-8 CPR spheroids do not have significantly different intracellular cisplatin concentrations compared to sensitive parental controls indicating a mechanism of resistance that is independent of drug transporter expression in OvCa (17). In recent years, epithelial-mesenchymal plasticity (EMP) and the tumor microenvironment have emerged as key players to impact the development of drug resistance in cancer. In the present work, targeted gene expression profiling has shown that DCLK1 is closely associated with the regulation of TGFβ, MET, and EMT pathways. We observedincreased expression of genes related to antigen presentation and EGFR signaling with DCLK1 KO, which could be potentially exploited as a novel combination strategy with immunotherapy and / or EGFR inhibitors in future studies. EMT is a highly dynamic and reversible process and the presence of intermediate cellular states where the tumor cells co-express both epithelial and mesenchymal markers (E / M hybrid phenotypes) can drive therapy resistance (40). In the present model systems, although we observed massive upregulation in mesenchymal markers, E-cadherin levels were also significantly increased in DCLK1 high-expression spheroids indicating an E / M hybrid state. This transitory E / M hybrid stage of tumor cells also has been shown to display cancer stem cell features, as so it is yet to be seen if DCLK1 is a putative marker for stemness in OvCa (41, 42). Further, DCLK1 inactivation reduced Zeb-2, which in turn led to reduced Vimentin levels. Studies are underway to identify the mechanism by which DCLK1 regulates Zeb-2 in modulating EMT and identify novel substrates that could cause chemoresistance and promote OvCa metastasis. Importantly, we showed that high DCLK1 expression in HGSOC patients was significantly associated with poor overall and progression- free survival, suggesting a pathological significance of DCLK1 in OvCa.

[0214] Results of the present disclosure show that DCLK1 promotes chemoresistance and metastasis of high-grade serous OvCa (HGSOC) and is a druggable target for OvCa. Targeting DCLK1 in combination with existing chemotherapy will be a novel therapeutic approach to prevent OvCa recurrence in a clinical setting. We observed a dramatic reduction in the presence of metastasis compared to either agent alone, demonstrating superior effectiveness of SAMiRNA and cisplatin used in combination. To summarize, we have shown that DCLK1 promotes pro-metastatic phenotypes and resistance to chemotherapy by modulating EMT. We have shown the preclinical efficacy of a novel combination strategy of combining cisplatin with a DCLK1 inhibitor to reduce peritoneal metastasis in cisplatin-resistant HGSOC (FIG.9). Thus, the present work demonstrates that DCLK1 is a drug target that is useful for modulating tumor progression and chemoresistance in HGSOC.

[0215] The present disclosure in at least one embodiment is directed to a method of treating a Doublecortin-like kinase 1 (DCLK1)-expressing cancer in a subject in need of such treatment, including the steps of (a) providing a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1, (b) providing a therapeutically effective amount of a platinum-based anticancer drug, and (c) administering to the subject a combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug, wherein the SAMiRNA nanoparticle andthe platinum-based anticancer drug are administered together or separately. The nucleotide sequence of the RNA oligonucleotide may be selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO: 4. The anti-DCLK1 RNA oligonucleotide may be selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense deoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA). The platinum- based anticancer drug may be selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin. The DCLK1-expressing cancer may be selected from the group consisting of cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast. In another embodiment, the present disclosure is directed to an oligonucleotide composition, comprising: a self-assembled micellar nanoparticle comprising an RNA oligonucleotide having the nucleotide sequence SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, or SEQ ID NO: 4, wherein the RNA oligonucleotide has inhibitory activity against the expression or biological activity of DCLK1. In another embodiment, the present disclosure is directed to a method of inhibiting tumor metastasis of a Doublecortin-like kinase 1 (DCLK1)-expressing cancer in a subject in need of such treatment, including (a) providing a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1, (b) providing a therapeutically effective amount of a platinum-based anticancer drug, and (c) administering to the subject a combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug, wherein the SAMiRNA nanoparticle and the platinum-based anticancer drug are administered together or separately. The nucleotide sequence of the RNA oligonucleotide may be selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO: 4. The anti-DCLK1 RNA oligonucleotide may be selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense deoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA). The platinum-based anticancer drug may be selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin. The DCLK1-expressing cancer may be selected from the group consisting of cancers of the ovary, pancreas, kidney, smallintestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast.

[0216] It will be understood from the foregoing description that various modifications and changes may be made in the various embodiments of the present disclosure without departing from their true spirit. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. Thus, while embodiments of the present disclosure have been described herein so that aspects thereof may be more fully understood and appreciated, it is not intended that the present disclosure be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the inventive concepts as defined herein. Thus, the examples described above, which include particular embodiments, will serve to illustrate the practice of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as of the principles and conceptual aspects of the inventive concepts. Changes may be made in the formulations and compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the present disclosure. NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0217] Illustrative embodiment 1. A method of treating a Doublecortin-like kinase 1 (DCLK1)-expressing cancer in a subject in need of such treatment, comprising: providing a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1; providing a therapeutically effective amount of a platinum-based anticancer drug; and administering to the subject a combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug, wherein the SAMiRNA nanoparticle and the platinum-based anticancer drug are administered together or separately.

[0218] Illustrative embodiment 2. The method of Illustrative embodiment 1, wherein the nucleotide sequence of the RNA oligonucleotide is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO: 4.

[0219] Illustrative embodiment 3. The method of Illustrative embodiment 1 or 2, wherein the anti-DCLK1 RNA oligonucleotide is selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisensedeoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA).

[0220] Illustrative embodiment 4. The method of any of Illustrative embodiments 1-3, wherein the platinum-based anticancer drug is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin.

[0221] Illustrative embodiment 5. The method of any of Illustrative embodiments 1-4, wherein the DCLK1-expressing cancer is selected from the group consisting of cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast.

[0222] Illustrative embodiment 6. An oligonucleotide composition, comprising: a self- assembled micellar nanoparticle comprising an RNA oligonucleotide having the nucleotide sequence SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, or SEQ ID NO: 4, wherein the RNA oligonucleotide has inhibitory activity against the expression or biological activity of DCLK1.

[0223] Illustrative embodiment 7. A method of inhibiting tumor metastasis of a Doublecortin-like kinase 1 (DCLK1)-expressing cancer in a subject in need of such treatment, comprising: providing a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1; providing a therapeutically effective amount of a platinum-based anticancer drug; and administering to the subject a combination treatment comprising the SAMiRNA nanoparticle and the platinum- based anticancer drug, wherein the SAMiRNA nanoparticle and the platinum-based anticancer drug are administered together or separately.

[0224] Illustrative embodiment 8. The method of Illustrative embodiment 7, wherein the nucleotide sequence of the RNA oligonucleotide is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO: 4.

[0225] Illustrative embodiment 9. The method of Illustrative embodiment 7 or 8, wherein the anti-DCLK1 RNA oligonucleotide is selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense deoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA).

[0226] Illustrative embodiment 10. The method of any of Illustrative embodiments 7-9, wherein the platinum-based anticancer drug is selected from the group consisting of cisplatin,carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin.

[0227] Illustrative embodiment 11. The method of any of Illustrative embodiments 7-10, wherein the DCLK1-expressing cancer is selected from the group consisting of cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast.

[0228] Illustrative embodiment 12. A combination treatment for use in a method of treating a Doublecortin-like kinase 1 (DCLK1)-expressing cancer in a subject in need of such treatment, the combination treatment comprising a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1, and a therapeutically effective amount of a platinum-based anticancer drug; and the method comprising the step of administering to the subject the combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug, wherein the SAMiRNA nanoparticle and the platinum-based anticancer drug are administered together or separately.

[0229] Illustrative embodiment 13. The combination treatment of Illustrative embodiment 12, wherein the nucleotide sequence of the RNA oligonucleotide is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO: 4.

[0230] Illustrative embodiment 14. The combination treatment of Illustrative embodiment 12 or 13, wherein the anti-DCLK1 RNA oligonucleotide is selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense deoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA).

[0231] Illustrative embodiment 15. The combination treatment of any of Illustrative embodiments 12-14, wherein the platinum-based anticancer drug is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin.

[0232] Illustrative embodiment 16. The combination treatment of any of Illustrative embodiments 12-15, wherein the DCLK1-expressing cancer is selected from the group consisting of cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast.

[0233] Illustrative embodiment 17. A combination treatment for use in a method of inhibiting tumor metastasis of a Doublecortin-like kinase 1 (DCLK1)-expressing cancer in a subject in need of such treatment, the combination treatment comprising a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1 and a therapeutically effective amount of a platinum-based anticancer drug; and wherein the method comprises the step of administering to the subject the combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug, wherein the SAMiRNA nanoparticle and the platinum-based anticancer drug are administered together or separately.

[0234] Illustrative embodiment 18. The combination treatment of Illustrative embodiment 17, wherein the nucleotide sequence of the RNA oligonucleotide is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO: 4.

[0235] Illustrative embodiment 19. The combination treatment of Illustrative embodiment 17 or 18, wherein the anti-DCLK1 RNA oligonucleotide is selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense deoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA).

[0236] Illustrative embodiment 20. The combination treatment of any of Illustrative embodiments 17-19, wherein the platinum-based anticancer drug is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin.

[0237] Illustrative embodiment 21. The combination treatment of any of Illustrative embodiments 17-20, wherein the DCLK1-expressing cancer is selected from the group consisting of cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast. REFERENCES

[0238] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. In addition, the following is not intended to be an Information Disclosure Statement; rather, an Information Disclosure Statement in accordance with the provisions of 37 CFR § 1.97 will be submitted separately.1. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72(1):7-33. 2. Colombo N, Lorusso D, Scollo P. Impact of Recurrence of Ovarian Cancer on Quality of Life and Outlook for the Future. Int J Gynecol Cancer.2017;27(6):1134-40. 3. van Zyl B, Tang D, Bowden NA. Biomarkers of platinum resistance in ovarian cancer: what can we use to improve treatment. Endocr Relat Cancer.2018;25(5):R303-R18. 4. Davis A, Tinker AV, Friedlander M. "Platinum resistant" ovarian cancer: what is it, whom to treat and how to measure benefit? Gynecol Oncol.2014;133(3):624-31. 5. Chandrakesan P, Panneerselvam J, May R, Weygant N, Qu D, Berry WR, et al. DCLK1-Isoform2 Alternative Splice Variant Promotes Pancreatic Tumor Immunosuppressive M2-Macrophage Polarization. Mol Cancer Ther.2020;19(7):1539-49. 6. Patel O, Roy MJ, Kropp A, Hardy JM, Dai W, Lucet IS. Structural basis for small molecule targeting of Doublecortin Like Kinase 1 with DCLK1-IN-1. Commun Biol. 2021;4(1):1105. 7. Chandrakesan P, Weygant N, May R, Qu D, Chinthalapally HR, Sureban SM, et al. DCLK1 facilitates intestinal tumor growth via enhancing pluripotency and epithelial- mesenchymal transition. Oncotarget.2014;5(19):9269-80. 8. Vega KJ, May R, Sureban SM, Lightfoot SA, Qu D, Reed A, et al. Identification of the putative intestinal stem cell marker doublecortin and CaM kinase-like-1 in Barrett's esophagus and esophageal adenocarcinoma. J Gastroenterol Hepatol.2012;27(4):773-80. 9. Kalantari E, Ghods R, Saeednejad Zanjani L, Rahimi M, Eini L, Razmi M, et al. Cytoplasmic expression of DCLK1-S, a novel DCLK1 isoform, is associated with tumor aggressiveness and worse disease-specific survival in colorectal cancer. Cancer Biomark. 2022;33(3):277-89. 10. Ding L, Yang Y, Ge Y, Lu Q, Yan Z, Chen X, et al. Inhibition of DCLK1 with DCLK1- IN-1 Suppresses Renal Cell Carcinoma Invasion and Stemness and Promotes Cytotoxic T-Cell- Mediated Anti-Tumor Immunity. Cancers (Basel).2021;13(22). 11. Shafiei S, Kalantari E, Saeednejad Zanjani L, Abolhasani M, Asadi Lari MH, Madjd Z. Increased expression of DCLK1, a novel putative CSC maker, is associated with tumor aggressiveness and worse disease-specific survival in patients with bladder carcinomas. Exp Mol Pathol.2019;108:164-72. 12. Yan R, Fan X, Xiao Z, Liu H, Huang X, Liu J, et al. Inhibition of DCLK1 sensitizes resistant lung adenocarcinomas to EGFR-TKI through suppression of Wnt / beta-Catenin activity and cancer stemness. Cancer Lett.2022;531:83-97.13. Nakanishi Y, Seno H, Fukuoka A, Ueo T, Yamaga Y, Maruno T, et al. Dclk1 distinguishes between tumor and normal stem cells in the intestine. Nat Genet.2013;45(1):98- 103. 14. Bailey JM, Alsina J, Rasheed ZA, McAllister FM, Fu YY, Plentz R, et al. DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer. Gastroenterology.2014;146(1):245-56. 15. Liu ZQ, He WF, Wu YJ, Zhao SL, Wang L, Ouyang YY, et al. LncRNA SNHG1 promotes EMT process in gastric cancer cells through regulation of the miR- 15b / DCLK1 / Notch1 axis. BMC Gastroenterol.2020;20(1):156. 16. Yan R, Li J, Xiao Z, Fan X, Liu H, Xu Y, et al. DCLK1 Suppresses Tumor-Specific Cytotoxic T Lymphocyte Function Through Recruitment of MDSCs via the CXCL1-CXCR2 Axis. Cell Mol Gastroenterol Hepatol.2022;15(2):463-85. 17. Chowanadisai W, Messerli SM, Miller DH, Medina JE, Hamilton JW, Messerli MA, et al. Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors. PLoS One.2016;11(3):e0151089. 18. Liu Y, Ferguson FM, Li L, Kuljanin M, Mills CE, Subramanian K, et al. Chemical Biology Toolkit for DCLK1 Reveals Connection to RNA Processing. Cell Chem Biol. 2020;27(10):1229-40 e4. 19. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods.2014;11(8):783-4. 20. Chou TC. Drug combination studies and their synergy quantification using the Chou- Talalay method. Cancer Res.2010;70(2):440-6. 21. Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein B, et al. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell.1998;1(4):611-7. 22. Domcke S, Sinha R, Levine DA, Sander C, Schultz N. Evaluating cell lines as tumour models by comparison of genomic profiles. Nat Commun.2013;4:2126. 23. Johnson SW, Laub PB, Beesley JS, Ozols RF, Hamilton TC. Increased platinum-DNA damage tolerance is associated with cisplatin resistance and cross-resistance to various chemotherapeutic agents in unrelated human ovarian cancer cell lines. Cancer Res. 1997;57(5):850-6. 24. Gyorffy B. Discovery and ranking of the most robust prognostic biomarkers in serous ovarian cancer. Geroscience.2023.25. Lanczky A, Gyorffy B. Web-Based Survival Analysis Tool Tailored for Medical Research (KMplot): Development and Implementation. J Med Internet Res. 2021;23(7):e27633. 26. Shield K, Ackland ML, Ahmed N, Rice GE. Multicellular spheroids in ovarian cancer metastases: Biology and pathology. Gynecol Oncol.2009;113(1):143-8. 27. Ferguson FM, Nabet B, Raghavan S, Liu Y, Leggett AL, Kuljanin M, et al. Discovery of a selective inhibitor of doublecortin like kinase 1. Nat Chem Biol.2020;16(6):635-43. 28. Dudas J, Ladanyi A, Ingruber J, Steinbichler TB, Riechelmann H. Epithelial to Mesenchymal Transition: A Mechanism that Fuels Cancer Radio / Chemoresistance. Cells. 2020;9(2). 29. Luyckx M, Squifflet JL, Bruger AM, Baurain JF. Recurrent High Grade Serous Ovarian Cancer Management. In: Lele S, editor. Ovarian Cancer. Brisbane (AU)2022. 30. Alkema NG, Wisman GB, van der Zee AG, van Vugt MA, de Jong S. Studying platinum sensitivity and resistance in high-grade serous ovarian cancer: Different models for different questions. Drug Resist Updat.2016;24:55-69. 31. Westphalen CB, Asfaha S, Hayakawa Y, Takemoto Y, Lukin DJ, Nuber AH, et al. Long-lived intestinal tuft cells serve as colon cancer-initiating cells. J Clin Invest. 2014;124(3):1283-95. 32. Ge Y, Fan X, Huang X, Weygant N, Xiao Z, Yan R, et al. DCLK1-Short Splice Variant Promotes Esophageal Squamous Cell Carcinoma Progression via the MAPK / ERK / MMP2 Pathway. Mol Cancer Res.2021;19(12):1980-91. 33. Chandrakesan P, Yao J, Qu D, May R, Weygant N, Ge Y, et al. Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells. Mol Cancer.2017;16(1):30. 34. Panneerselvam J, Mohandoss P, Patel R, Gillan H, Li M, Kumar K, et al. DCLK1 Regulates Tumor Stemness and Cisplatin Resistance in Non-small Cell Lung Cancer via ABCD-Member-4. Mol Ther Oncolytics.2020;18:24-36. 35. Westphalen CB, Takemoto Y, Tanaka T, Macchini M, Jiang Z, Renz BW, et al. Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis. Cell Stem Cell.2016;18(4):441-55. 36. O'Connell MR, Sarkar S, Luthra GK, Okugawa Y, Toiyama Y, Gajjar AH, et al. Epigenetic changes and alternate promoter usage by human colon cancers for expressing DCLK1-isoforms: Clinical Implications. Sci Rep.2015;5:14983.37. Zhang L, Zhou S, Guo E, Chen X, Yang J, Li X. DCLK1 inhibition attenuates tumorigenesis and improves chemosensitivity in esophageal squamous cell carcinoma by inhibiting beta-catenin / c-Myc signaling. Pflugers Arch.2020;472(8):1041-9. 38. Perse M. Cisplatin Mouse Models: Treatment, Toxicity and Translatability. Biomedicines.2021;9(10). 39. Assaraf YG, Brozovic A, Goncalves AC, Jurkovicova D, Line A, Machuqueiro M, et al. The multi-factorial nature of clinical multidrug resistance in cancer. Drug Resist Updat. 2019;46:100645. 40. De Las Rivas J, Brozovic A, Izraely S, Casas-Pais A, Witz IP, Figueroa A. Cancer drug resistance induced by EMT: novel therapeutic strategies. Arch Toxicol.2021;95(7):2279-97. 41. Kim S, Han Y, Kim SI, Kim HS, Kim SJ, Song YS. Tumor evolution and chemoresistance in ovarian cancer. NPJ Precis Oncol.2018;2:20. 42. Chhetri D, Vengadassalapathy S, Venkadassalapathy S, Balachandran V, Umapathy VR, Veeraraghavan VP, et al. Pleiotropic effects of DCLK1 in cancer and cancer stem cells. Front Mol Biosci.2022;9:965730.

Claims

What is claimed is:

1. A method of treating a Doublecortin-like kinase 1 (DCLK1)-expressing cancer in a subject in need of such treatment, comprising: providing a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1; providing a therapeutically effective amount of a platinum-based anticancer drug; and administering to the subject a combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug, wherein the SAMiRNA nanoparticle and the platinum-based anticancer drug are administered together or separately.

2. The method of claim 1, wherein the nucleotide sequence of the RNA oligonucleotide is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO:

4.

3. The method of claim 1, wherein the anti-DCLK1 RNA oligonucleotide is selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense deoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA).

4. The method of claim 1, wherein the platinum-based anticancer drug is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin.

5. The method of claim 1, wherein the DCLK1-expressing cancer is selected from the group consisting of cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast.

6. An oligonucleotide composition, comprising: a self-assembled micellar nanoparticle comprising an RNA oligonucleotide having the nucleotide sequence SEQ ID NO: 21, SEQ IDNO:22, SEQ ID NO: 2, or SEQ ID NO: 4, wherein the RNA oligonucleotide has inhibitory activity against the expression or biological activity of DCLK1.

7. A method of inhibiting tumor metastasis of a Doublecortin-like kinase 1 (DCLK1)- expressing cancer in a subject in need of such treatment, comprising: providing a self-assembled micelle interfering ribonucleic acid (SAMiRNA) nanoparticle comprising a therapeutically effective amount of an RNA oligonucleotide having inhibitory activity against the expression or biological activity of DCLK1; providing a therapeutically effective amount of a platinum-based anticancer drug; and administering to the subject a combination treatment comprising the SAMiRNA nanoparticle and the platinum-based anticancer drug, wherein the SAMiRNA nanoparticle and the platinum-based anticancer drug are administered together or separately.

8. The method of claim 7, wherein the nucleotide sequence of the RNA oligonucleotide is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 2, and SEQ ID NO:

4.

9. The method of claim 7, wherein the anti-DCLK1 RNA oligonucleotide is selected from the group consisting of short interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), antisense RNA, antisense deoxyribonucleic acid (antisense DNA), chimeric antisense DNA / RNA, and microRNA (miRNA).

10. The method of claim 7, wherein the platinum-based anticancer drug is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, lobaplatin, miriplatin, tetraplatin, iproplatin, satraplatin, ormaplatin, and oxoplatin.

11. The method of claim 7, wherein the DCLK1-expressing cancer is selected from the group consisting of cancers of the ovary, pancreas, kidney, small intestine, colon, rectum, stomach, esophagus, cervix, uterus, endometrium, bladder, testes, head, brain, neck, lung, bone, blood, liver, and breast.

Images