EP4684016A2 - Methods and materials for treating cancer - Google Patents

Abstract

Disclosed are compositions comprising microRNAs. Methods of use, including treatment with immunotherapy, are also disclosed.

Description

METHODS AND MATERIALS FOR TREATING CANCER

CLAIM OF PRIORITY

This application claims priority to U.S. Patent Application Serial Nos. 63/562,170, filed March 6, 2024, and 63/453,282, filed on March 20, 2023. The entire contents of each are hereby incorporated by reference.

ST TEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under CAI 77322 and DA039562 awarded by National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

This document relates to compositions and methods of treating subjects with cancer using microRNAs and immunotherapy.

BACKGROUND

A microRNA (abbreviated miRNA) is a small non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression. The ability of miRNAs to regulate gene expression illustrates their potential as a therapeutic.

There are over 200 different known cancers that afflict human beings. Cancer causes millions of deaths a year worldwide and rates are also rising as more people live to an older age and urbanization causes more stress. It is anticipated that one in eight people currently alive will eventually die of cancer. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness. Malignant tumors are the second leading cause of death in the United States, after heart disease.

There remains a need to develop successful therapeutics for the treatment of cancer.

SUMMARY

Provided herein are methods of treating a subject having cancer, the method including: (a) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor; and (b) administering to the subject a therapeutically effective amount of a composition that modulates expression of one or more microRNAs (miRNAs). Also provided here are methods of treating a subject having cancer, the method including: (a) determining that a cancer cell in the subject expresses aberrant expression of one or more miRNAs compared to a control sample; (b) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor; and (c) administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs.

One aspect of this document features methods of treating a subject having cancer, the method including: (a) determining that a cancer cell in the subject expresses aberrant expression of one or more miRNAs compared to a control sample; and (b) administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs. Also provided herein are methods of treating a subject having cancer, the method including administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs, where the subject expresses aberrant expression of one or more miRNAs compared to a control sample.

In another aspect, this document features methods for decreasing an immune response in a subject having cancer including administering to the subject: (a) a therapeutically effective amount of an immune checkpoint inhibitor; and (b) a therapeutically effective amount of a composition that modulates expression of one or more miRNAs. Also provided herein are methods for increasing an immune response in a subject having cancer including administering to the subject: (a) a therapeutically effective amount of an immune checkpoint inhibitor; and (b) a therapeutically effective amount of a composition that modulates expression of one or more miRNAs. For example, the immune response can include tumor immune cell infiltration, Granzyme B expression, phagosome formation, complement cascade gene expression, proinflammatory gene expression, Cxcr4 signaling, or innate and immune adaptive immune regulation. In some cases, the tumor immune cell infiltration can include CD45⁺ cell infiltration, CD69⁺ cells, T cell infiltration including CD8⁺ T cells, CD4⁺ T cells. Ml macrophages, or gamma delta T cells (y5 T cells). In some cases, administering the composition can increase expression of the one or more miRNAs. In other cases, administering the composition can decrease expression of the one or more miRNAs. In some embodiments, the one or more miRNAs is miR-16, miR-29a, miR-150, miR-125b- 5p, miR-15b, miR-let7f, miR-26b, miR-21, miR-466g, miR-383, miR-181a, miR-30a, or any combination thereof. In some embodiments, the one or more miRNAs is miR-106a, miR-25, miR-1983, miR-93, miR-30c, miR-30d, miR-18a, miR-425, miR-17, miR-92a, miR-872, or any combination thereof. In some cases, the one or more miRNAs is miR-25, miR-17, miR-92a, or any combination thereof.

In some cases, the one or more miRNAs include miR-16, miR-29a, miR-150. miR-15b, miR-let7f, miR-26b, miR-21, miR-466g, miR-383, miR-181a, miR-30a, miR- 106a, miR-25, miR 1983, miR-93, miR-30c, miR-30d, miR-18a, miR-425, miR- 17, miR- 92a, miR-125b, miR-20b, miR-99b, miR-92a, miR-17, miR-19a, miR-20a, miR-19b, or miR-872. In some embodiments, expression of miR-25 and miR-106a are decreased therapeutically. In some embodiments, expression of miR-29 and miR-150 are increased therapeutically. In some embodiments, expression of miR-25, miR-17, miR-92a, miR-21, and miR-Let7f are decreased therapeutically. In some cases, the one or more microRNAs can include at least 2 to at least 20 miRNAs.

In some embodiments, the aberrant expression is an increase in expression of the one or more miRNAs compared to a control sample. For example, the aberrant expression can be a decrease in expression of the one or more miRNAs compared to a control sample. The control sample can be from a subject that does not have cancer. In some cases, the control sample can be from a subject that has cancer but has not been administered the immune checkpoint inhibitor.

Also provided herein are methods of restoring expression of one or more miRNA within a mammal, where the method includes administering a composition to a mammal identified as having a cancer, where the expression of one or more miRNAs is restored relative to a non-cancerous tissue. In some embodiments, the composition is delivered as a recombinant plasmid, an overexpression plasmid construct, a viral vector, non-viral vector, a RISC, crRNA, or an antisense oligomer.

In some embodiments, the composition includes a recombinant plasmid, vectors, miRNA-duplexes, primary-miRNA (pri-miRNA). or precursor-miRNA (pre-miRNA), and/or a delivery reagent such as a lipophilic reagent; a lipofectin, Lipofectamine, cellfectin, a polycation, or a liposome. The composition can further include a sequence that encodes a miRNA. In some embodiments, the vector is a viral vector, or a non-viral vector. The viral vector can be an AAV. a lentivirus, an adenovirus, an adeno-associated virus, retrovirus, or a herpes simplex virus. The non-viral vector can be a liposome, exosome, an extracellular vesicle, a polymer, a nanoparticle, a peptide, or a dendrimer. In some cases, the composition includes an inhibitory nucleic acid molecule that can decrease expression of the one or more miRNAs. For example, the inhibitory nucleic acid molecule can be a shRNA, a recombinant plasmid, a siRNA, a micro RNA, an antisense oligomer, a crRNA, or an RNA-induced silencing complex (RISC). In some cases, the one or more miRNAs can include modifications, where the modification can be a nonnatural intemucleotide linkage, a modified backbone, a substituted sugar moiety, a sugar/backbone modification, an unnatural base pair (UBP), a locked nucleic acid (LNA), a phosphorothioate modification, a cholesterol conjugation, a lipid group conjugation, an antagomir, a 5’ and/or a 3’ end modification, or a chemical modification including a 2’- OMethyl modification, uridylation, A^-methyladenosine (m⁶A), 5-methylcytosine (m⁵C), A¹ -methyladenosine (m¹ A), JV⁷-methy 1 guanosine (m⁷G), A^-acetylcytosine (ac⁴C), pseudouridine ( ), or adenosine-to-inosine (A-to-I).

In one aspect, this document features in vitro methods of identifying a miRNA that modulates an immune response, the method including: (a) treating cells with an immune checkpoint inhibitor; (b) obtaining a biopsy sample from a subject, where the biopsy sample is a solid tissue, a blood sample, a serum sample, tumor interstitial fluid (TIF), or a plasma sample; (c) processing the biopsy sample for miRNA detection method; and (d) detecting a change in miRNA expression relative to a control cell. For example, the detection method can include qRT-PCR, RNA immunoprecipitation, RNA sequencing, RNA-fluorescence in situ hybridization (FISH), single cell genetic and epigenetic analysis, or combinations thereof.

Also provided herein are methods of diagnosing whether a subject has, or is at risk of developing cancer, the method including: (a) measuring the level of at least one miRNA in a test sample from a subject, where the at least one miRNA is miR-25, miR- 17, or miR 92a, and (b) where an alteration in the level of the miRNA in the test sample, relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk of developing cancer. In another aspect, this document provides methods of diagnosing whether a subject has, or is at risk of developing cancer, the method including: (a) measuring the level of at least one miRNA in a test sample from a subject, where the at least one miRNA is one or more of miR- 106a. and (b) where an alteration in the level of the miRNA in the test sample, relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk of developing, cancer. In some embodiments, the administering of the immune checkpoint inhibitor and/or the composition is via oral delivery, rectal delivery, intranasal delivery, injection; infusion; intravascular administration such as intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature; peri- and/or intra-tissue injection such as peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection; subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest; inhalation, or any combination thereof. The subject can be a mammal, optionally where the mammal is a human. In some embodiments, the cancer is a cancer cell present in a tissue or an organ. The cancer cell is a neoplastic cell. For example, the cancer can be a solid tumor. In some cases, solid tumor can be a breast cancer, a lung cancer, a prostate cancer, a glioma, a melanoma, an ovarian cancer, a colorectal cancer, a glioblastoma, a liver cancer, a pancreatic cancer, a kidney cancer, a bladder cancer, a thyroid cancer, a sarcoma, a stomach cancer, a head and neck cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a thymoma, a soft tissue sarcoma, a bone cancer, a testicular cancer, a penile cancer, a gallbladder cancer, a uterine sarcoma, an adrenal gland cancer, an ampullary cancer, a hepatic angiosarcoma, a nasal, or a paranasal sinus cancer. In some embodiments, the cancer is a blood cancer. For example, the blood cancer can be a leukemia, a lymphoma, a myeloma, a myelodysplastic syndrome, or a myeloproliferative neoplasm.

In some embodiments, the administering of the immune checkpoint inhibitor and the composition is performed at the same time. In some embodiments, the subject has previously been treated with immune checkpoint inhibitor. For example, the immune checkpoint inhibitor can be an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG- 3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGF-beta receptor. The inhibitor of PD-1 can be nivolumab, pembrolizumab, or pidilizumab. The inhibitor of PD- L1 can be atezolizumab. avelumab, or durvalumab. The inhibitor of CTLA-4 can be ipilimumab or tremelimumab. The inhibitor of LAG-3 can be relatlimab. The inhibitor of CEACAM can be CM24. The inhibitor of VISTA can be CA-170, VISTA-IN-3, or VISA-IN-2M. The inhibitor of BTLA can be INBRX-106, PF-04518600. cudarolimab, or HFB200603. The inhibitor of TIGIT can be Vibostolimab, Etigilimab, Domvanalimab. Ociperlimab, or Tiragolumab. The inhibitor of TIM-3 can be sym023, cobolimab, sabatolimab, INCAGN2390, BMS-986258, SHR-1702, RO7121661, or LY3321367. The inhibitor of TGF-0 receptor can be GW-788388, LY2109761, galunisertib, SB-431542, or repsox.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. Anti-PD-1 therapy model and experimental strategy. (FIGS. 1A and IB) Mouse B16 melanoma cells were subcutaneously injected into C57BL/6 wildtype mice. Mice were divided into three treatment arms: 1) non-treatment control (NTC) B16 cells, 2) GV AX monotherapy, or 3) GV AX and anti-PD-1 (GP) combination therapy. After tumor inoculation, mice were treated with GV AX monotherapy on day 1 and 4, followed by treatment with anti-PD-1 on day 6 and day 9. (FIGS. 1C-1E) Tumor growth was monitored for 12 days and at the end point, tumor tissues were dissected, and RNA was extracted from each group.

FIGS. 2A-2C. Identification of miRNAs altered during the course of immunotherapy. NanoString miR profiling methods were used to quantify miRNA expression in the tumor tissues. (FIG. 2A) RNA extracted from the tumors were subjected to small RNA-sequencing and differential expression analysis was performed. (FIGS. 2B and 2C) Volcano plot was generated to identify miRNAs altered during the course of the treatments.

FIGS. 3A-3B. Bioinformatics analysis of miRNAs altered during the course of immunotherapy. (FIGS. 3A and 3B) Heatmap was generated to show clustering of upregulated and downregulated expression of miRNAs when mice were treated with either GV AX monotherapy or combination therapy (GP).

FIGS. 4A-4B. Analysis of miRNA expression in tumors dissected from GV AX and combination therapy (GP) groups. Tumors extracted from mice treated with GV AX monotherapy and combination therapy (GP) ere analyzed using qRT-PCR. FIG. 4A shows upregulated miRNAs in tumors treated with the combination therapy (GP). FIG. 4B shows downregulated miRNAs in tumors treated with the combination therapy.

FIGS. 5A-5B. Analysis of miRNA expression in tumors dissected from NTC and combination therapy (GP) groups. Tumors extracted from mice treated with NTC and combination therapy (GP) were analyzed using qRT-PCR. FIG. 5A show s upregulated miRNAs in tumors treated with the combination therapy (GP). FIG. 5B shows downregulated miRNAs in tumors treated with the combination therapy.

FIGS. 6A-6J. CRISPR Knockout of miR-29. (FIG. 6A) Design of sgRNAs targeting miR-29 family including miR-29a. (FIG. 6B) RT-PCR confirmed successful knockout (KO) of miR-29a expression in cell line. (FIG. 6C) Nuclease digestion confirmed successful target cleavage by the sgRNAs. (FIG. 6D) MTS assay was used to test the role of miR-29a in cell proliferation in vitro. (FIG. 6E) Mice were injected with either NTC B16 cells or miR-29a KO B16 cells. To test the effects of miR-29 KO on immunotherapy treatment, each group was treated with/without combination therapy (GP). and tumor growth was monitored for 15 days. (FIG. 6F) Kaplan-Meier survival curve was plotted to analyze the effect of miR-29 KO when combined with the combination therapy (GP). (FIG. 6G) Individual animal data under indicated treatments. (FIG. 6H) miR-29a overexpressing (OE) Bl 6 cell line was created, and the overexpression was confirmed using qRT-PCR. (FIG. 61) The effects of miR-29a overexpression on cell proliferation was assessed in B 16 cells. (FIG. 6J) Mice were injected with either NTC Bl 6 cells or miR-29a OE B16 cells. To test the effects of miR- 29a OE on treatment, each group was treated with/without anti-PD-1 therapy, and tumor growth was monitored for 15 days.

FIGS. 7A-7K. CRISPR Knockout of miR-150. For B16 and melanoma models, treatment is a combination of GV AX and anti-PD-1 antibody (GP). For colon and breast cancer models, the treatment is anti-PD-1 therapy or combination of anti-PD-1 and anti- CTLA4 antibodies when indicated. (FIG. 7A) Design of sgRNAs targeting miR-150. (FIG. 7B) RT-PCR confirmed successful knockout (KO) of miR-150 expression. (FIG. 7C) Nuclease digestion confirmed successful target cleavage by the sgRNAs. (FIG. 7D) MTS assay was used to test the role of miR-150 in cell proliferation in vitro. (FIG. 7E) Mice were injected with either NTC B16 cells or miR-150 KO B16 cells. To test the effects of miR-150 KO on immunotherapy treatment, each group was treated with/without immunotherapy, and tumor growth was monitored for 15 days. (FIG. 7F) Kaplan-Meier survival curve was plotted to analyze the effect of miR-150 KO when combined with treatment (FIG. 7G) Individual animal data under indicated treatments. (FIG. 7H) miR-150 overexpressing (OE) B16 cell line was created, and the overexpression was confirmed using qRT-PCR. (FIG. 71) The effects of miR-150 overexpression on cell proliferation was assessed in B16 cells. (FIG. 7 J) Mice were injected with either NTC B16 cells or miR-150 OE B16 cells to test the effects of miR- 150 OE on treatment. Each group was treated with/without anti-PD-1, and tumor growth was monitored for 15 days. (FIG. 7K) Cell proliferation was evaluated for miR-29a OE and miR-150 OE B16 cells. FIGS. 8A-8G. CRISPR Knockout of miR-25. (FIG. 8A) Design of sgRNAs targeting miR-25. (FIG. SB) RT-PCR confirmed successful knockout (KO) of miR-25 expression in cell line. (FIG. 8C) Nuclease digestion confirmed successful target cleavage by the sgRNAs. (FIG. 8D) MTS assay was used to test the role of miR-25 in cell proliferation in vitro. (FIG. 8E) Mice were injected with either NTC B16 cells or miR-25 KO B16 cells. To test the effects of miR-25 KO on immunotherapy treatment, each group was treated with/without immunotherapy, and tumor grow th was monitored for 15 days. (FIG. 8F) Kaplan-Meier survival curve was plotted to analyze the effect of miR-25 KO when combined with immunotherapy treatments. (FIG. 8G) Individual animal data under indicated treatments.

FIGS. 9A-9G. CRISPR Knockout of miR-106a. (FIG. 9A) Design of sgRNAs targeting miR-106a. (FIG. 9B) RT-PCR confirmed successful knockout (KO) of miR- 106a expression in cell line. (FIG. 9C) Nuclease digestion confirmed successful target cleavage by the sgRNAs. (FIG. 9D) MTS assay was used to test the role of miR-106a in cell proliferation in vitro. (FIG. 9E) Mice were injected with either NTC B16 cells or miR-106a KO B16 cells. To test the effects of miR-106a KO on immunotherapy treatment, each group was treated with/without immunotherapy, and tumor growth was monitored for 15 days. (FIG. 9F) Kaplan-Meier survival curve was plotted to analyze the effect of miR-106a KO when combined with immunotherapy treatments. (FIG. 9G) Individual animal data under indicated treatments.

FIGS. 10A-10G. CRISPR Knockout of miR-125b. (FIG. 10A) Design of sgRNAs targeting miR-125b. (FIG. 10B) RT-PCR confirmed successful knockout (KO) of miR-125b expression in cell line. (FIG. 10C) Nuclease digestion confirmed successful target cleavage by the sgRNAs. (FIG. 10D) MTS assay was used to test the role of miR- 125b in cell proliferation in vitro. (FIG. 10E) Mice were injected with either NTC Bl 6 cells or miR-125b KO Bl 6 cells. To test the effects of miR-125b KO on immunotherapy treatment, each group was treated with/without immunotherapy, and tumor growth was monitored for 15 days. (FIG. 10F) Kaplan-Meier survival curve was plotted to analyze the effect of miR-125b KO when combined with immunotherapy treatments. (FIG. 10G) Individual animal data under indicated treatments.

FIGS. 11A-11G. CRISPR Knockout of miR-16-l/miR-16-2. (FIG. 11A) Design of sgRNAs targeting miR-16-l/miR-16-2. (FIG. 11B) RT-PCR confirmed successful knockout (KO) of miR-16-l/miR-16-2expression in cell line. (FIG. 11C) Nuclease digestion confirmed successful target cleavage by the sgRNAs. (FIG. 11D) MTS assay was used to test the role of miR-16-l/miR-16-2 in cell proliferation in vitro. (FIG. HE) Mice were injected with either NTC B16 cells or miR-16-l/miR-16-2 KO B16 cells. To test the effects of miR-16-l/miR-16-2 KO on immunotherapy treatment, each group was treated with/without immunotherapy, and tumor growth was monitored for 15 days. (FIG. HF) Kaplan-Meier survival curve was plotted to analyze the effect of miR-16-l/miR-16- 2 KO when combined with immunotherapy treatments. (FIG. HG) Individual animal data under indicated treatments.

FIGS. 12A-12D. CRISPR Knockout of miR-21. (FIG. 12A) RT-PCR confirmed successful knockout (KO) of miR-21 expression in cell line. (FIG. 12B) MTS assay was used to test the role of miR-21 in cell proliferation in vitro. (FIG. 12C) Mice were injected with either NTC B16 cells or miR-21 KO B16 cells. To test the effects of miR-21 KO on immunotherapy treatment, each group was treated with/without the combination therapy (GP), and tumor growth was monitored for 15 days. (FIG. 12D) Mice were injected with either NTC B16 cells or Let-7f KO B16 cells. To test the effects of Let-7f KO on immunotherapy treatment, each group was treated with/without immunotherapy, and tumor growth was monitored for 15 days.

FIGS. 13A-13M. miR-25 deficiency enhances tumor responses to anti-PD-1 treatment. (FIGS. 13A) Experimental design to investigate the role of miRNAs in anti- PD-1 therapy. B16 mouse melanoma cells were injected subcutaneously into C57BL/6 wild-type mice (5 x 10⁵/mouse). Control group received non-treatment control (NTC) Bl 6 cells. All mice were subcutaneously injected with GV AX (irradiated B16-GM-CSF cells) on day 1 and 4 to elicit an anti-B16 immune response. Anti-PD-1 antibody (200 pg/mouse) was intraperitoneally injected on day 6, 9, and 12 (or as indicated for individual experiments). Similar experiments were performed for MC38 and 4T1 cells. The cells were inoculated in BALB/c mice and were treated with anti-PD-1 antibody on indicated days. GP: GV AX + aPDl combination treatment. (FIG. 13B) In vivo growth of NTC, GV AX treated, and combination of GV AX and anti-PD-1 (GP) treated mice. Data are show n as mean ± SEM of indicated total number of mice per group. (FIG. 13C) Volcano plot of small RNA-seq data from combination therapy treated Bl 6 tumors and the NTC B16 tumors from FIG. 13A. (FIG. 13D) RT-qPCR was used to analyze the miRNA expression in the combination therapy treated B16F10 tumors and the NTC Bl 6 tumors from FIG. 13A. Each dot represents one mouse. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p <0.001 by Student’s t-tests. (FIGS. 13E) RT-qPCR was used to measure miRNA-25 knockout efficacy in indicated cell lines. Data are shown as mean ± SEM of n = 3. ***p <0.001, ****p <0.0001 by Student’s t-tests. (FIGS. 13F) Left: In vivo tumor growth of NTC and miR-25 KO B16F10 with or without combination therapy. Data are shown as mean ± SEM of indicated total number of mice per group. ****p <0.0001 by two-way ANOVA. Right: Kaplan-Meier survival curves of mice treated with different treatment groups. **** p<0.0001 by Long-rank (Mantel-Cox) test. GP: GV AX + aPDl. (FIG. 13G) Left: In vivo tumor growth of NTC and miR-25 KO MC38 with or without anti-PD-1 therapy. Data are shown as mean ± SEM of indicated total number of mice per group. ***p <0.001 by two-way ANOVA. Right: Kaplan-Meier survival curves of mice treated with different treatment groups. **** p<0.0001 by Long-rank (Mantel- Cox) test. (FIG. 13H) Left: In vivo tumor growth of NTC and miR-25 KO 4T1 with or without anti-PD-1 therapy. Data are shown as mean ± SEM of indicated total number of mice per group. ***p <0.001 by two-way ANOVA. Right: Kaplan-Meier survival curves of mice treated with different treatment groups. **** p<0.0001 by Long-rank (Mantel- Cox) test. (FIG. 131) In vivo tumor growth of NTC and miR-25/miR-17/miR-106a/miR- 92a KO B16F10 with combination therapy. (FIGS. 13J-13M) MC38 colorectal cancer model was established. GP: GV AX + aPDl. Data are mean ± SEM of n=5 mice per group. **p <0.01, ****p <0.0001 by two-way ANOVA.

FIGS. 14A-14I. Deficiency of miR-25 or miR-17/92 family enhances tumor responses to anti-PD-1 immunotherapy. (FIG. 14A) Tumor growth in individual C57BL/6J mice for the experiments shown in FIG. 13A. (FIG. 14B) Design of sgRNAs for miR-25 deletion. (FIG. 14C) T7EN1 assay was performed to detect DNA cleavage and to confirm successful knockout of miR-25. (FIG. 14D) In vitro proliferation assay of non-targeting control (NTC) sgRNAs and miR-25 targeting sgRNAs treated B16F10 (left). MC38 (middle), and 4T1 (right). Data are shown as mean ± SEM of n = 6. Data were analyzed by two-way ANOVA. (FIG. 14E) Tumor growth in individual C57BL/6J mice for the experiments shown in FIG. 13F. GP: GV AX + aPDl. (FIG. 14F) Experimental outline of anti-PD-1 treatment for MC38 and 4T1. MC38 tumor cells w ere injected into C57BL/6J mice on day 0 and followed up with anti-PD-1 every three days from day 9 until death. 4T1 tumor cells were injected into Balb/c mice on day 0 and followed up with anti-PD-1 every three days from day 9 until death. (FIG. 14G) Tumor growth in individual C57BL/6J mice for the experiments shown in FIG. 13G. (FIG. 14H) Tumor growth in individual Balb/c mice for the experiments shown in FIG. 13H. (FIG. 141) Tumor growth in individual C57BL/6J mice for the experiments shown in FIG. 131. GP: GV AX + aPDl.

FIGS. 15A-15K. Deficiency of miR-25 alters tumor immune infiltration and activates innate immune sensing. (FIG. 15A) Flow cytometry quantification of total infiltrating immune cells isolated from B16 NTC GP and miR-25 KO GP tumors. Data are shown as mean ± SEM. Each dot represents one mouse. ****p < 0.0001 by Student’s t-tests. GP: GVAX + aPDl. (FIG. 15B) Flow cytometry' quantification of CD8+ T cells isolated from B 16 NTC GP, miR-25 KO GP tumors. Data are shown as mean ± SEM. Each dot represents one mouse. **p < 0.01 by Student’s t-tests. GP: GV AX + aPDl. (FIG. 15C) Flow cytometry quantification of CD4+ T cells isolated from Bl NTC GP, miR-25 KO GP tumors. Data are shown as mean ± SEM. Each dot represents one mouse. ****p < 0.0001 by Student’s t-tests. GP: GV AX + aPDl. (FIG. 15D) Flow cytometry quantification of tumor-resident macrophages isolated from B 16 NTC GP, miR-25 KO GP tumors. Data are shown as mean ± SEM. Each dot represents one mouse. ****p < 0.0001, **p < 0.01 by Student’s t-tests. GP: GV AX + aPDl. (FIG. 15E) Flow cytometry quantification of splenocytes isolated from B16 NTC GP, miR-25 KO GP tumor-bearing mice. Data are shown as mean ± SEM. Each dot represents one mouse. *p < 0.05 by Student’s t-tests. GP: GV AX + aPDl. (FIG. 15F) Top upregulated pathways analyzed by Ingenuity Pathway Analysis (IP A) based on differentially expressed genes identified by RNA-seq from B16 NTC GP, miR-25 KO GP tumors. GP: GV AX + aPDl. (FIG. 15G) Representative differentially expressed phagosome formation genes from the tumor RNA-seq experiments in FIG. 15F. GP: GV AX + aPDl. (FIG. 15H) Representative images of CD45, C3 and F4/80 IHC staining from indicated group of tumors. Arrows indicate interactions betw een positive stained cells and tumor cells. Representative of n=3 independent experiments. GP: GVAX + aPDl. (FIG. 151) Percentage of AEC positive area was calculated by ImageJ from 5 x fields for indicated antibodies in FIG. 15H. Data are shown as mean ± SEM of n=3. *p < 0.05, **p <0.01 by Student’s t-tests. GP: GV AX + aPDl. (FIG. 15 J) Experimental outline for macrophage depletion experiments. NTC GP and miR-25 KO GP B16 tumor-bearing mice were intravenously injected with 200 pl liposome clodronate on day 8 and 11 to deplete macrophages. Tumor challenge: B16 cells. GP: GV AX + aPDl. (FIG. 15K) Left: In vivo growth of NTC and miR-25 KO B16 tumors in mice treated as described in FIG. 15J. Data are shown as mean ± SEM of indicated total number of mice per group. ****p <0.0001 by two-way ANOVA. Right: Tumor weight from FIG. 15J on day 13. Each dot represents one mouse. *p < 0.05 by Student’s t-tests. GP: GV AX + aPDl. LC: liposome clodronate.

FIGS. 16A-16L. Immune infiltration analysis of miR-25-KO tumors. (FIG. 16A) Representative dot plots showing the gating strategy for T cells and B cells. (FIG. 16B) Representative dot plots showing the gating strategy for MDSCs. (FIG. 16C) Representative dot plots showing the gating strategy⁷ for DC and macrophages. (FIGS. 16D-16H) FACS quantification of immune cells isolated from B16 NTC GP and miR-25- KO GP tumors. Tumor-infiltrating cells were analyzed using the gating in shown in FIGS. 16A-16C. Data are presented as the mean ± SEM. Each dot represents one mouse. *p < 0.05, **p < 0.01, ****p <0.0001 by Student’s t-tests. GP: GVAX + aPDl. (FIG. 161) Top upregulated pathways analyzed by Ingenuity' Pathway Analysis (IP A) based on differentially expressed genes in tumor RNA-seq isolated from B 16 NTC GP and miR- 106a KO GP tumors with anti-PD-1 therapy from FIG. 131. (FIG. 16J) Tumor growth in individual C57BL/6J mice for the experiments shown in FIG. 15K. GP: GV AX + aPDl . LC: liposome clodronate. (FIG. 16K) Flow cytometry' quantification of spleen macrophages on day 13 from FIG. 15K. Data are shown as mean ± SEM of n=4. Each dot represents one mouse. *p<0.05 by Student’s t-tests. GP: GV AX + aPDl. LC: liposome clodronate. (FIG. 16L) Flow cytometry quantification of tumor macrophages on day 13 from FIG. 15K. Data are show n as mean ± SEM of n=4. Each dot represents one mouse. **p < 0.01, ***p <0.001 by Student’s t-tests. GP: GV AX + aPDl. LC: liposome clodronate.

FIGS. 17A-17H. Single cell RNA-seq reveals innate immune activation in miR- 25 deficient tumor microenvironment (TME). (FIG. 17A) UMAP plots of scRNA-seq data showing the tumor composition of miR-25 KO MC38 and the control (NTC) tumors under the same anti-PD-1 treatment. (FIG. 17B) Changes in cell clusters between NTC treated anti-PD-1 and miR-25 KO treated anti-PD-1. *p<0.05, **p < 0.01 by Student’s t- tests. (FIG. 17C) Changes in the proportion of different cell clusters between NTC treated anti-PD-1 and miR-25 KO treated anti-PD-1. (FIG. 17D) Volcano plot showing top upregulated genes in Ml macrophages in the anti-PDl treated miR-25 KO tumors versus the NTC control. (FIG. 17E) Volcano plot showing top upregulated genes in CAF in anti-PD-1 treated miR-25 KO tumors versus the NTC control. (FIG. 17F) Top enriched path ays analyzed by Ingenuity Pathway Analysis (IP A) based on the differentially expressed genes in Ml macrophages isolated from anti-PD-1 treated miR- 25 KO tumors and NTC tumors. (FIG. 17G) Upper: Violin plots of the up-regulated macrophage signature genes in macrophages. Bottom: Violin plots of the dow n -regulated macrophage signature genes in macrophages. Each dot represents a single cell, and the shapes represent the expression distribution. The remaining cells are depicted on the x axis. (FIG. 17H) Expression of T-cell receptor (TCR) and B-cell receptor (BCR) upstream regulated genes in T cells and B cells. The color intensity indicates the average expression level in a cluster and the circle size reflects the percentage of expressing cells within each cluster.

FIGS. 18A-18E. Single cell RNA-seq analysis of miR-25-KO tumors. (FIG. 18A) Cell clusters were identified by specific marker expression. (FIG. 18B) Upregulated pathways comparing Ml macrophage cluster cells to M2 cluster. (FIG. 18C) UMAP plots showing expression of Ifltl and IfltS in all cell clusters from FIG. 17A. (FIG. 18D) Complement gene C3, Clsl, Cls2, Clra, Clrb and Clrl in identified cell clusters. (FIG. 18E) UMAP plots showing expression of Cxcr4 in all cell clusters from FIG. 17A.

FIGS. 19A-19K. Augmentation of immunotherapy by miR-25 deficiency depends on IFN-y release. miRNA induced silencing complex, and Sdc3 expression. (FIG. 19A) NTC GP and miR-25 KO GP B16 tumor-bearing mice were intraperitoneally injected with 1.25 mg/kg of antibodies against IFN-y on day 8 and 11 to deplete the cytokine. Tumor challenge: Bl 6 cells. Data are shown as mean ± SEM of indicated total number of mice per group. *p < 0.05, ****p <0.0001 by two-way ANOVA. GP: GV AX + aPDl. (FIG. 19B) Flow cytometry quantification of splenocytes isolated from the mice treated from (FIG. 19A) on day 13. Data are shown as mean ± SEM. Each dot represents one mouse. **p < 0.01, ****p <0.0001 by Student’s t-tests. GP: GV AX + aPDl. (FIG. 19C) In vivo tumor growth of NTC GP, miR-25 single KO GP, Ago2 single KO GP, and miR- 25 plus Ago2 double KO GP B16 tumors. Data are shown as mean ± SEM of indicated total number of mice per group. ****p <0.0001 by two-way ANOVA. GP: GV AX + aPDl. (FIG. 19D) Venn diagrams describing the strategies to identify potential miR-25 targets. Gene expression analyses, by RNA-seq data from three indicated experiments, were compared with miR-25 targets predicted by TargetScan. GP: GV AX + aPDl. (FIG. 19E) Firefly luciferase activities measured in the B 16 cell transfected with the reporter plasmid containing equal dose of either wild-type (WT) or mutant (MT) miR-25 binding sites of Sdc3 3’ UTR, together with equal dose of miR-25 or scrambled negative control RNAs. Data are shown as mean ± SD of n=5. ***p<0.001 by Student’s t-tests. (FIG. 19F) RNA immunoprecipitation (RIP) assays w ere performed in B16 cells at indicated time points of IFN-y treatment. Anti-Ago2 or control IgG immunoprecipitates were subjected to RT-qPCR analysis of Sdc3 mRNA (top) or immunoblotting of Ago2 (bottom). Data are shown as mean ± SEM of n = 3. **p <0.01, ***p<0.001 by Student’s t-tests. (FIG. 19G) Left: RT-qPCR of Sdc3 mRNA normalized to Gapdh in B16 cells at indicated time points of IFN-y treatment. Data are shown as mean ± SEM of n = 3. **p <0.01, ***p <0.001, ****p <0.0001 by Student’s t-tests. Right: Representative immunoblotting of Sdc3. Statl and Beta-actin (Act-b) in B16 cells at indicated time points of IFN-y treatment. (FIG. 19H) Left: RT-qPCR of Sdc3 mRNA normalized to Gapdh in MC38 cells at indicated time points of IFN-y treatment. Data are shown as mean ± SEM of n = 3. **p <0.01, ****p <0.0001 by Student's t-tests. Right: Representative immunoblotting of Sdc3. Statl and Beta-actin (Act-b) in MC38 cells at indicated time points of IFN-y treatment. (FIG. 191) Left: RT-qPCR of Sdc3 mRNA normalized to Gapdh in 4T1 cells at indicated time points of IFN-y treatment. Data are shown as mean ± SEM of n = 3. **p <0.01, ****p <0.0001 by Student's t-tests. Right: Representative immunoblotting of Sdc3. Statl and Beta-actin (Act-b) in 4T1 cells at indicated time points of IFN-y treatment. (FIG. 19J) Representative immunoblotting of Sdc3 and Gapdh in B16 cells transduced with NTC sgRNAs and Sdc3 -targetting sgRNAs. (FIG. 19K) In vivo tumor grow th of NTC GP, miR-25 single KO GP, Sdc3 single KO GP, and miR-25 plus Sdc3 double KO GP B16 tumors. Data are shown as mean ± SEM of indicated total number of mice per group. ****p <0.0001 by two-way ANOVA. GP: GVAX + aPDl.

FIGS. 20A-20K. Exploration and validation of miR-25 target. (FIG. 20A) Tumor growth in individual C57BL/6I mice for the experiments shown in FIG. 19B. GP: GV AX + aPDl. (FIG. 20B) Representative immunoblotting of Ago2 and Gapdh of B16 cells with indicated gene knockout. (FIG. 20C) Representative immunoblotting of Ago2 and Gapdh in Bl 6 cells from NTC, miR-25 single knockout, Ago2 single knockout and miR- 25 plus Ago2 double knockout. (FIG. 20D) Tumor grow th in individual C57BL/6J mice for the experiments show n in FIG. 19D. (FIG. 20E) Predicted pairing of the target region in Sdc3 3’UTR and miRNA seed region (highlighted) including miR-25-3p, miR-92a-3p, miR-363-3p, miR-367-3p, miR-92b-3p, and miR-32-5p. Prediction and pairing was generated by using TargetScan. (FIG. 20F) qRT-PCR of miR-25 in in vitro B16 cells at indicated time points of IFN-y treatment. Data are shown as mean ± SEM of n = 3. ****p <0.0001 by Student’s t-tests. (FIG. 20G) qRT-PCR of miR-25 in in vitro MC38 cells at indicated time points of IFN-y treatment. Data are shown as mean ± SEM of n = 3. ****p <0.0001 by Student’s t-tests. (FIG. 20H) qRT-PCR of miR-25 in in vitro 4T1 cells at indicated time points of IFN-y treatment. Data are shown as mean ± SEM of n = 3. **p <0.01 ****p <0.0001 by Student’s t-tests. (FIG. 201) In vitro proliferation assay ofNTC sgRNAs and Sdc3 targeting sgRNAs transduced B16cells. Data are shown as mean ± SEM of n = 6. *p <0.05, ****p <0.0001 by two-way ANOVA. (FIG. 20J) Tumor growth in individual C57BL/6J mice for the experiments shown in FIG. 19K. GP: GV AX + aPDl. (FIG. 20K) Representative images of Sdc3 IHC staining in indicated group of tumors and time points representative of n=3 independent experiments. GP: GV AX + aPDl.

FIGS. 21A-21G. miR-25 and its target SDC3 expression influences innate immune responses and immune infiltration in tumors from melanoma patients. (FIG. 21A) miR-25 expression in melanoma specimens based on TCGA SKCM datasets. (FIG. 2 IB) Kaplan-Meier survival analysis of SKCM patients stratified into low or high miR- 25 expression. (FIG. 21C) Top upregulated pathways analyzed by Ingenuity Path ay Analysis (IP A) based on differential expressed genes (DEGs) in miR-25 low expressing SKCM samples comparing to miR-25 high expressing samples. (FIG. 21D) Representative immune gene sets enriched in GSEA of DEGs comparing lower miR-25 to miR-25 high samples. (FIG. 21E) CIBERSORT estimation of different immune cells presented in miR-25 low SKCM patient samples compared to miR-25 high. Data are shown as median of n=88. Each dot represents one patient. **p < 0.01, ***p <0.001, ****p <0.0001 by Student’s t-tests. (FIG. 21F) Correlation analysis of miR-17/92a members and the phagosome formation representative genes. MIR17HG was used as a positive correlation control. (FIG. 21G) Heatmap of miR-25 and innate immune gene expression in responders and non-responders treated with anti-PD-1. Each row represents one gene, and each column represents one patient. Patients are divided by responders and non-responders by RECIST. CR: complete response; PR: partial response; PD: progressive disease; SD: stable disease. Data analyzed from GSE91061.

FIG. 22 Proposed model for selective pressure from intact immune system. Cancer cells in tumors are composed of a mixture of high/low immunogenic cells due to heterogeneity. However, functional immune system selectively eliminates the cancer cells incapable of repressing their immunogenicity related molecules such as Sdc3. On the other hand, the cancer cells with reduced immunogenicity or with the ability to reduce the molecules when sensing ‘danger signal’ such as IFN-y can survive and proliferate. This selective process may play critical roles in tumor cells escaping the immunosurveillance and, thus contributing to initial resistance when treated with immunotherapies.

DETAILED DESCRIPTION

Immune checkpoint blockade (ICB) therapy (e.g., anti-CTLA-4, anti-PD-1, anti- PD-L1, or anti-TIM3) has revolutionized cancer treatment, but the clinical benefits are limited. Most patients do not respond to ICB, and many develop resistance to the treatment. Thus, combating resistance (e.g., primary and acquired resistance) to ICB therapy remains an unmet clinical need. MicroRNAs (miRNAs) are a class of small noncoding RNAs (~22 nucleotides in length) that bind to mRNA untranslated regions, and facilitate suppression of protein translation or enhance mRNA decay. MicroRNAs play critical roles in regulating protein expression and many pathophysiological processes.

The methods and materials provided herein identified specific miRNAs that are altered during the course of various immunotherapy regimens. As provided herein, combination therapy with immune checkpoint inhibitors (e.g., anti-PD-1 and GVAX) and compositions that modulate expression of one or more miRNAs (e.g.. miR-25, miR 106a, miR-let7f, miR17, miR-21, miR-92a, miR 29, and miR- 150) enhances the efficacy of immune checkpoint inhibitors. For example, deletion of specific miRNAs (e.g., miR-25, miR 106a, miR17, miR-21, miR-92a, and miR-let7f) can sensitize tumors to immune checkpoint inhibitors. In addition, effects of miR-25 in enhancing the efficacy of immunotherapy depends on tumor resident macrophages. IFN-y release in the tumor microenvironment (TME), miRISC (miRN A induced silencing complex) protein Argonaut2, and miR-25-mediated repression of a membrane proteoglycan protein Syndecan3 (Sdc3). In some cases, overexpression of specific miRNAs (e g., miR 29 and miR- 150) can sensitize tumors to immune checkpoint inhibitors.

In summary, the present disclosure includes methods and materials for treating cancer by a combination therapy using immune checkpoint inhibitors and compositions that modulate expression of one or more miRNAs. Thus, miRNAs identified herein can serve as novel therapeutic targets for enhancing the efficacy of immunotherapy in treating cancers. MicroRNAs

MicroRNAs (miRNAs or miRs) are small non-coding RNA transcripts ~ 19-22 nucleotides that regulate gene expression at posttranscriptional levels. miRNAs are transcribed from genome into primary microRNAs (pri-miRNAs) and processed into precursor microRNAs (pre-miRNAs) that mature into microRNAs. Mature miRNAs are loaded onto the Argonaute (Ago) proteins to form a miRNA-induced silencing complex (miRISC). By binding to the mRNA untranslated regions. miRISC suppresses protein translation or enhances mRNA decay. The mRNA targeting specificity of miRNAs is controlled by many factors, including base pairing between the miRNA 5' seed sequence and mRNA 3'-UTR sequence, cooperativity⁷ between multiple miRNA-binding sites, and the position of miRNA-binding sites in the targeted mRNA. Therefore, individual miRNAs are capable of repressing the translation of hundreds of target mRNAs. As a result, miRNAs are known to play pivotal roles in post-transcriptional regulation of numerous biological processes.

In some embodiments, the miRNA is a one or more miRNAs including miR-16, miR-29a, miR-150, miR-15b, miR-let7f. miR-26b, miR-21, miR-466g, miR-383. miR-181a, miR-30a, miR-106a, miR-25, miR 1983, miR-93, miR-30c, miR-30d, miR- 18a, miR-425, miR- 17, miR-92a, miR-125b, miR-20b, miR-99b, miR-92a, miR-17, miR- 19a, miR-20a, miR-19b, miR-872, or any combinations thereof.

In some instances, treatment includes one microRNA of interest. In some instances, treatment includes multiple microRNAs (e.g., 2, 3, 4. 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more).

In some instances, the disclosure includes a sequence that encodes a miRNA or a sequence that encodes a miRNA hairpin. A hairpin can be a DNA or RNA sequence that, when processed by a cell, generates the miRNA or miRNA hairpin. The encoding sequence may be, for example: a portion of the genome of a DNA virus, a portion of the genome of an RNA vims, a pri-miRNA sequence or a pre-miRNA sequence. Depending on the nature of the sequence that encodes the miRNA or miRNA hairpin, the encoding sequence may include a sequence that is (i) identical to the miRNA or miRNA hairpin sequence, (ii) complementary to the miRNA or miRNA hairpin sequence, or (iii) reverse complementary to the miRNA or miRNA hairpin sequence. A sequence that encodes pre- miRNA or pri-miRNA may include a cassette. In some instances, the one or more microRNAs are incorporated into a viral vector to mediate transfer to a cell. Additional expression constructs encoding other therapeutic agents as described herein may also be transferred via viral transduction using infectious viral particles, for example, by transformation with an adeno-associated virus (AAV) of the present disclosure. Alternatively, a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus may be used. Similarly, nonviral methods which include, but are not limited to, direct delivery of DNA such as by perfusion, naked DNA transfection, liposome mediated transfection, encapsulation, and receptor-mediated endocytosis may be employed. These techniques are well known to those of skill in the art, and the particulars thereof do not lie at the crux of the present disclosure and thus need not be exhaustively detailed herein. For example, a viral vector is used for the transduction of cancer cells to deliver a therapeutically significant polynucleotide to the cell. The virus may gain access to the interior of the cell by a specific means such as receptor-mediated endocytosis, or by non-specific means such as pinocytosis.

A vector used herein can be engineered to deliver genes of interest (e.g., the one or more microRNAs) by deleting the internal nonrepeating portion of the vector genome (e.g., the rep and cap genes) and inserting a heterologous gene between the ITRs. The heterologous gene is typically functionally or operatively linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in the patient's target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included.

In some instances, an AAV vector can include an adeno-associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, the rep and/or cap genes, but retain functional flanking 1TR sequences. In some embodiments, the AAV vector is derived from an adeno-associated virus serotype AAV1. Despite the high degree of homology, the different serotypes have tropisms for different tissues. The receptor for AAV1 is unknown; however, AAV1 is known to transduce skeletal and smooth muscle more efficiently than AAV2. Without being bound by theory, since most of the studies have been done with pseudotyped vectors in which the vector DNA flanked with AAV2 ITR is packaged into capsids of alternate serotypes, it is clear that the biological differences are related to the capsid rather than to the genomes.

Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, for example, by the insertion, deletion, or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging.

Cancer

Provided herein are methods for treating cancer in a subject having cancer. In some instances, the methods include (a) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor, and (b) administering to the subject a therapeutically effective amount of a composition that modulates expression of one or more microRNAs (miRNAs). Also provided herein are methods of treating a subject having cancer, the method including (ajdetermining that a cancer cell in the subject expresses aberrant expression of one or more miRNAs compared to a control sample, (b) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor, and (c) administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs.

Also provided herein are methods of diagnosing whether a subject has, or is at risk for developing, cancer. In some instances, the methods include (a) measuring the level of at least one miRNA in a test sample from the subject, where the at least one miRNA is one or more of miR-25, miR-17, or miR 92a, and (b) where an alteration in the level of the miRNA in the test sample, relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk for developing, cancer. Also provided herein are methods of diagnosing whether a subject has, or is at risk for developing, cancer, the method including, (a) measuring the level of at least one miRNA in a test sample from the subject, wherein the at least one miRNA is one or more of miR- 106a, and (b) wherein an alteration in the level of the miRNA in the test sample, relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk for developing, cancer. In some embodiments, the cancer is a solid tumor. For example, the solid tumor can be a breast cancer, a lung cancer, a prostate cancer, a colorectal cancer, a glioma, a melanoma, an ovarian cancer, a liver cancer, a pancreatic cancer, a kidney cancer, a bladder cancer, a thyroid cancer, a sarcoma, a stomach cancer, a head and neck cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a thymoma, a soft tissue sarcoma, a bone cancer, a testicular cancer, a penile cancer, a gallbladder cancer, a uterine sarcoma, an adrenal gland cancer, an ampullary cancer, a hepatic angiosarcoma, a nasal, or a paranasal sinus cancer. In some embodiments, the cancer is a blood cancer. For example, the blood cancer can be a leukemia, a lymphoma, a myeloma, a myelodysplastic syndrome, or a myeloproliferative neoplasm. In some embodiments, the cancer is a cancer cell (e.g., neoplastic cell). For example, the cancer cell can be present in a tissue or an organ.

Methods of Treatment

Provided herein are methods for treating cancer in a subject having cancer, the method including, (a) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor, and (b) administering to the subject a therapeutically effective amount of a composition that modulates expression of one or more microRNAs (miRNAs). Also provided herein are methods of treating a subject having cancer, the method including (a)delermining that a cancer cell in the subject expresses aberrant expression of one or more miRNAs compared to a control sample, (b) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor, and (c) administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs. In some cases, the aberrant expression is an increase in expression of the one or more miRNAs compared to a control sample. For example, the one or more miRNAs can be miR-16, miR-29a, miR-150, miR-125b-5p, miR-15b, miR-let7f, miR-26b, miR-21, miR-466g, miR-383, miR-181a, miR-30a, or any combination thereof. In some cases, the aberrant expression is a decrease in expression of the one or more miRNAs compared to a control sample. For example, the one or more miRNAs can be miR-106a, miR-25, miR 1983, miR-93, miR-30c, miR-30d, miR-18a, miR-425, miR-17, miR-92a, miR-125b, miR-20b, miR-99b, miR-92a, miR-17, miR-19a, miR-20a, miR-19b, miR-872, or any combination thereof. For example, the control sample can be from a subj ect that does not have a cancer. In other cases, the control sample can be from a subject that has cancer but has not been administered the immune checkpoint inhibitor.

The methods described herein can be used to treat cancer in a mammal, optionally wherein the mammal is a human. Examples of mammals that can have cancer and can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, and rats. In some cases, a human having cancer can be treated by administering (a) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor, and (b) administering to the subject a therapeutically effective amount of a composition that modulates expression of one or more microRNAs (miRNAs). In some embodiments, the subjects have an increased risk of developing cancer (e.g., breast cancer, or lung cancer). For example, the subject may have a family history and/or personal history of cancer.

Methods for treating a mammal having cancer (e.g., breast cancer) can include identifying the mammal as having cancer. Examples of methods for identifying the mammal as having cancer include, without limitation, physical examination, laboratory tests (e.g., blood, urine, and/or circulating tumor cells (CTCs)), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, genetic tests, or other methods of identifying cancer as known in the art.

In some embodiments, the one or more miRNAs include miR-16, miR-29a, miR-150, miR-15b, miR-let7f, miR-26b, miR-21, miR-466g, miR-383, miR- 181a, miR-30a, miR-106a, miR-25, miR 1983, miR-93, miR-30c, miR-30d, miR-18a, miR-425. miR- 17. miR-92a, miR- 125b. miR-20b, miR-99b, miR- 92a, miR- 17, miR- 19a, miR-20a, miR-19b, or miR-872. In some embodiments, expression of miR-25, miR-17, miR- 92a, miR-21, miR-Let7f, and miR- 106a can be decreased therapeutically. In some embodiments, expression of miR-29 and miR-150 can be increased therapeutically.

Also provided herein are methods for decreasing an immune response in a subject having cancer including administering to the subject, (a) a therapeutically effective amount of an immune checkpoint inhibitor, and (b) a therapeutically effective amount of a composition that modulates expression of one or more miRNAs. Also provided herein are methods for increasing an immune response in a subject having cancer including administering to the subject, (a) a therapeutically effective amount of an immune checkpoint inhibitor, and (b) a therapeutically effective amount of a composition that modulates expression of one or more miRNAs. For example, inducing an immunogenic response can include enhancing T cell response, T cell number and/or T cell infiltration into the tumor and/or around the tumor margin, thereby reducing the risk that the subject will develop cancer. In some embodiments, the immune response comprises tumor immune cell infiltration, Granzyme B expression, phagosome formation, complement cascade gene expression, proinfl ammatory gene expression, Cxcr4 signaling, or innate and adaptive immune regulation. For example, phagosome formation can include elevated expression of complement genes: Clra, Clqb, C Iqc, C3arl, C3, immunoglobulin genes: Ilgam. Ilgb2. and toll like receptor genes: 77r7, Tlr8, Tlr9. In some cases, the tumor immune cell infiltration comprises CD45⁺ cell infiltration, CD69⁺ cells, T cell infiltration including CD8⁺ T cells, CD4⁺ T cells, Ml macrophages, and gamma delta T cells (y5 T cells). For example, an innate and adaptive immune regulation include expansion and activation of T cell and B cell, and Treg and MDSC suppression. In some cases, CD4⁺ T cells expressed more TCR upstream genes including Ccr7, Cxcr4. Socs3 and Tcf7. In other cases, B cells in the expressed more BCR upstream regulators like Atp6v0c, Gls, Sial 3. and Tubb4b.

Methods for treating a subject (e.g., a mammal) having cancer using an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNA (e.g., miR-25, miR-17. and miR-92a) provided herein can be effective to reduce the number of cancer cells in the mammal. In some cases, the methods and materials provided herein can be used as described herein to reduce the number of cancer cells in the mammal by, for example, 10, 20, 30, 40. 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to reduce the volume of one or more solid tumors in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, treating a mammal having cancer using immune checkpoint inhibitor and a composition that modulates expression of one or more miRNA provided herein can be effective to eliminate the cancer cells in the mammal. For example, an immune checkpoint inhibitor can include inhibitors of PD- 1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4, or TGF-beta receptor. The PD-1 inhibitors can include nivolumab, pembrolizumab, or pidilizumab, or any combinations thereof. The PD-L1 inhibitors can include atezolizumab, avelumab, or durvalumab. The CTLA-4 inhibitors can include ipilimumab and/or tremelimumab. The LAG-3 inhibitors can include relatlimab. the inhibitor of CEACAM is CM24. the inhibitor of VISTA is CA-170, VISTA-IN-3, or VISA-IN-2 M. The BTLA inhibitors can include INBRX-106, PF-04518600, cudarolim HFB200603. The TIGIT inhibitors can include Vibostolimab, Etigilimab, Domvanalimab, Ociperlimab, or Tiragolumab. The TIM-3 inhibitors can include sym023, cobolimab, sabatolimab, INCAGN2390, BMS-986258, SHR-1702, RO7121661, or LY3321367. The TGF -beta receptor inhibitors can include GW-788388, LY2109761, galunisertib, SB- 431542, or repsox.

In cases where a composition that modulates expression of one or more miRNA provided herein and a composition that modulates expression of one or more miRNAs are provided separately, the administration of an immune checkpoint inhibitor provided herein can be in any order relative to the administration of a composition that modulates expression of one or more miRNA provided herein. For example, an immune checkpoint inhibitor provided herein can be administered to a mammal prior to, concurrent with, or following administration of a composition that modulates expression of one or more miRNAs to the mammal.

In some cases, the methods and materials provided herein can be used to improve survival of a mammal (e.g., a human) having cancer. For example, a mammal in need thereof (e.g., a mammal having cancer such as a human having cancer) can be administered using an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNA (e.g., miR-25, miR-17, and miR-92a) to improve survival of the mammal. For example, the methods and materials described herein can be used to improve the survival of a mammal having cancer by, for example, 10, 20. 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the methods and materials described herein can be used to improve the survival of a mammal having cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

Also provided here are method of identifying a miRNA that modulates an immune response, the method including, (a) treating cells with an immune checkpoint inhibitor,

(b) obtaining a biopsy sample from the subject, wherein the biopsy sample is a solid tissue, a blood sample, a serum sample, tumor interstitial fluid (TIF), or a plasma sample

(c) processing the biopsy sample for miRNA detection method, and (d) detecting a change in miRNA expression relative to a control cell. In some embodiments, the detection method comprises qRT-PCR, RNA immunoprecipitation, RNA sequencing, RNA-fluorescence in situ hybridization (FISH), single cell genetic and epigenetic analysis, or combinations thereof. For example, tumor tissues dissected from the subject can be mechanically or enzymatically digested into cells (e.g., single cells). The single cells can then be processed following methods well known in the art to prepare for RNA sequencing. In some cases, tumor tissues can be digested into cells and extract the RNA using kits well known in the art to perform qRT-PCR and/or RNA immunoprecipitation. In some cases, tumor interstitial fluid (TIF) can be used as a bipsy sample.

Also provided herein are methods of diagnosing whether a subject has, or is at risk for developing, cancer, the method including, (a) measuring the level of at least one miRNA in a test sample from the subject, wherein the at least one miRNA is one or more of miR-25, miR-106a, miR-17, or miR 92a, and (b) wherein an alteration in the level of the miRNA in the test sample, relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk for developing, cancer.

Also disclosed are compositions comprising one or more of the microRNAs disclosed herein for use in the treatment of cancer. Further provided are use of any one of the microRNAs disclosed herein for the manufacture of a medicament, where the medicament is intended for treatment of cancer (e g., any cancer described herein).

Pharmaceutical composition

The methods and materials provided herein includes composition comprising a recombinant plasmid, vectors, miRNA-duplexes, primary -miRNA (pri-miRNA). or precursor-miRNA (pre-miRNA), and/or a deliver}’ reagent such as a lipophilic reagent; a lipofectin, Lipofectamine, cellfectin, a poly cation, or a liposome. In some embodiments, the composition further comprises a sequence that encodes a miRNA. In some embodiments, the vector is a viral vector, or a non- viral vector. In some embodiments, the viral vector is an AAV, a lentivirus, an adenovirus, an adeno-associated virus, retrovirus, or herpes simplex virus. In some embodiments, the non-viral vector is a liposome, exosome, an extracellular vesicle, a polymer, a nanoparticle, a peptide, or a dendrimer. For example, a nanoparticle can be a lipid nanoparticle.

In some embodiments, the composition comprises an inhibitory nucleic acid molecule that can decrease expression of the one or more miRNAs. In some embodiments, the composition further comprises a sequence that encodes a miRNA. In some embodiments, the inhibitory nucleic acid molecule is a shRNA, a recombinant plasmid, a siRNA, a micro RNA, an antisense oligomer, a crRNA, or a RNA-induced silencing complex (RISC). In some embodiments, the one or more miRNAs is selected from miR-16, miR-29a, miR-150, miR-125b-5p, miR-15b, miR-let7f, miR-26b, miR-21, miR-466g, miR-383, miR-181a, miR-30a, miR-106a, miR-25, miR 1983. miR-93, miR- 30c, miR-30d, miR-18a, miR-425. miR-17, miR-92a, miR-125b, miR-20b. miR-99b. miR-92a, miR-17, miR-19a, miR-20a, miR-19b, miR-872, or any combination thereof. In some embodiments, the one or more miRNAs is selected from miR-25, miR-92a, or any combination thereof.

In some embodiments, the one or more miRNAs comprise modifications, where the modification is a non-natural intemucleotide linkage, a modified backbone, a substituted sugar moiety, a sugar/backbone modification, an unnatural base pair (UBP), a locked nucleic acid (LNA), a phosphorothioate modification, a cholesterol conjugation, a lipid group conjugation, an antagomir. a 5’ and/or a 3’ end modification, or a chemical modification including a 2‘-OMethyl modification, uridylation. A⁶ -methyladenosine (m⁶A), 5-methylcytosine (m⁵C), A'-methyladenosine (m¹ A), A⁷-methylguanosine (m⁷G), A⁴-acetylcytosine (ac⁴C), pseudouridine ( ), or adenosine-to-inosine (A-to-I). For example, the 5‘ and/or 3‘ ends modification can be capping with NH2, dye molecules, cholesterols, or lipid groups. .

In some instances, the modification is a posttranscriptional RNA modification of a microRNA. Posttranscriptional modifications are disclosed in Wang et al., EMBO I (2022) 42: ell 1673, which is incorporated by reference in its entirety. In some instances, the modification is an adenosine N6-methylation (m6A). In some instances, the modification is an N6,2'-O-dimethylation (m6Am).

Posttranscriptional RNA modification is emerging as an important epigenetic regulatory circuit in multiple pathophysiological contexts. N6-methyladenosine (m6A) is the most abundant mRNA modification in mammals and is generally located at the 5'- and 3 '-untranslated regions (UTRs) and stop codons (Domimssini et al. 2012; Meyer et al, 2012; Schwartz et al, 2014). The abundance of m6A in cellular RNA metabolism is regulated by the activity of the methyltransferases METTL3/14 (Liu et al, 2014) (“writers'’), the YTH family of m6A-binding proteins (Wang et al, 2014, 2015; Xiao et al, 2016: Li et al, 2017) (“readers”), and demethylases such as ALKBH5 (Zheng et al. 2013) (“erasers”). Recent studies have identified roles for m6A in various diseases (Hess et al, 2013; Satterlee et al, 2014) including cancer (Barbieri et al, 2017; Vu et al, 2017; Su et al, 2018; Han et al, 2019; Paris el al, 2019; Yang el al, 2019), and in the response of cancer to immunotherapy. For example, knockdown of YTHDF1 and the demethylase FTO were shown to enhance the response of melanoma tumors to anti-PD-1 therapy (Han et al, 2019; Yang et al. 2019). In addition, we recently showed that deletion of ALKBH5 sensitized melanoma tumors to immunotherapy and prolonged mouse survival through a mechanism involving modulation of lactate levels in the TME, which, in turn, regulated accumulation of immunosuppressive T regulatory⁷ lymphocytes (Tregs) and myeloid-derived suppressor cell (MDSCs) in the TME (Li et al. 2020). We also found that inhibition of m6A modification by depletion of Mettl3 and Mettll4 enhanced the response of pMMR-MSI-L CRC and melanoma ta anti-PD-1 treatment (Wang et al, 2020). Consequently, inhibitors targeting enzy mes regulating m6A pathways are being developed as potential new⁷ therapeutics to treat multiple cancers (Li et al, 2020; Huff et al, 2021, 2022; Yankova et al, 2021).

Another abundant RNA modification located near the mRNA cap structure is the dimethylated N6,2'-O-dimethyladenosine (m6Am) (Wei et al, 1975; Keith et al, 1978). m6Am is located at the first transcribed nucleotide in ~ 30% of cellular mRNAs and thus can have a major influence on the transcriptome (Wei et al, 1975). Recent studies identified the methylase phosphorylated CTD-interacting factor 1 (PCIF1) as the enzyme that catalyzes m6A methylation of 2'-O-methylated adenosine at the 5'-end of mRNAs (Akichika et al, 2019; Boulias et al, 2019; Sendinc et al, 2019; Sun et al, 2019). However, the pathophysiological roles of m6Am and PCIF1 are unclear. Two recent studies revealed the function of PCIF1 in modulating HIV and VSV pathogenesis and immune responses (Tartell et al, 2021; Zhang et al, 2021). During HIV infection, viral protein Vpr degrades PCIF1 and reprograms human T cells m6Am methylome. In VSV, PCIF1 modifies viral mRNA cap structure and attenuates the antiviral effects of interferon-beta (Tartell et al, 2021). In the present study, we set out to determine the role of m6Am modification in CRC and its response to anti-PD-1 immunotherapy. We found that PC1F1 is highly expressed in human CRC tumors and correlates with poor patient prognosis and that PCIF1 -mediated m6Am modification of the classical oncogene FOS promotes CRC malignant behaviors and suppresses the response to anti-PD-1 immunotherapy in mouse tumor models. Thus, our results identify a novel role for m6Am in tumor biology and also raise the possi bi 1 i ty that m6Am-regulating pathways could serve as therapeutic targets for cancer both directly and by sensitizing refractory tumors to ICB therapy. Methods of formulating suitable pharmaceutical compositions are know n in the art, see, e.g., Remington: The Science and Practice of Pharmacy , 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions 20 used for parenteral, intradermal, intramuscular, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany. NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fl ui di ty can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-dry ing, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In some embodiments, administering a therapeutically effective amount of an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs (e.g., miR-25, miR-17, and miR-92a) provided herein, to a subject identified as having a cancer includes intravenous, intradermal, subcutaneous, intratumoral, intramuscular, or subcutaneous administration. Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

In some embodiments, the administering of the immune checkpoint inhibitor and/or the composition that modulates expression of one or more miRNAs (e.g., miR-25, miR-17, and miR-92a) provided herein are via oral delivery, rectal delivery', intranasal delivery', injection; infusion; intravascular administration such as intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature; peri- and/or intra-tissue injection such as peri- tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection; subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest; inhalation, or any combination thereof. An immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs (e.g., miR-25, miR-17, and miR-92a) provided herein, can be administered to a mammal (e.g., a human) having cancer in any appropriate amount (e.g., any appropriate dose). In some cases, an effective dose of an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs can be a flat dose. In some cases, an effective dose of an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs can be based on the body of a mammal (e.g., a human) to be treated as described herein. An effective amount of an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs can be any amount that can treat a mammal having cancer without producing significant toxicity to the mammal. In some cases, an effective amount of an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs can be from about 10 mg/kg to about 1000 mg/kg (e.g., per day). For example, an effective amount of an immune checkpoint inhibitor can be from about 10 mg/kg to about 1000 mg/kg. In some cases, an effective amount of a composition that modulates expression of one or more miRNAs can be from about 10 mg/kg to about 1000 mg/kg. The effective amount of an immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the cancer in the mammal being treated may require an increase or decrease in the actual effective amount administered.

An immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs (e.g., miR-25, miR-17, and miR-92a) provided herein, can be administered to a mammal (e.g.. a human) having cancer at any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having cancer without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a day to about once a month, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.

An immune checkpoint inhibitor and a composition that modulates expression of one or more miRNAs (e.g., miR-25. miR-17, and miR-92a) provided herein, can be administered to a mammal (e.g., a human) having cancer for any appropriate duration. An effective duration can be any duration that can treat a mammal having cancer without producing significant toxicity' to the mammal. For example, the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration.

Dosage, toxicity and therapeutic efficacy of the therapeutic compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit high therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to minimize and reduce side effects. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compositions used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models. Such information can be used to more accurately determine useful doses in humans.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

Example 1: Methods and Materials

Cell lines

All studies were conducted in accordance with approved IRB protocols by the University⁷ of California, San Diego. All animal work was approved by the Institutional Review Board at the University of California, San Diego and was performed in accordance with Institutional Animal Care and Use Committee guidelines. The mouse B16F10melanoma cell line, B16-GM-CSF, MC38 and CT26 cell lines were used. All cells were cultured in high-glucose DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Gibco) and 50 U/ml penicillin-streptomycin (Gibco) in a humidified 5% CO2 atmosphere. Mycoplasma was tested monthly.

Mouse Model and Treatments

Animal studies and procedures were approved by the UCSD Institutional Animal Care and Use Committee. Female C57BL/6J and BALB/c wild-type mice were obtained from The Jackson Laboratory and housed in the UCSD specific-pathogen free facility. B6.129S2-TcratmlMom/J (Tcra-/-) mice, which are CD4⁺ and CD8⁺ T cell deficient, were obtained from The Jackson Laboratory and bred on-site. For the standard protocol, B6 mice (aged 9-12 weeks at use) were injected subcutaneously (s.c.) with 5 x io⁵ B16 cells (NTC control, miR-25-KO. Ago2-KO. or Sdc3-KO.) into the left flank on day 0, and then injected with 10⁶ irradiated (100 Gy) B16-GM-CSF cells (GV AX) into the opposite flank on day 1 and 4 to elicit an anti-tumor immune response. Mice were then injected intraperitoneally (i.p.) with 10 mg/kg (-200 pg/mouse) of rat monoclonal anti-mouse PD- 1 Ab (Bio X Cell, clone 29F.1A12) on the days 6. 9 and 12. For anti-PD-1 Ab treatment alone, mice were implanted with B16 cells and treated with antibody on day 6, 9 and 12. For the Treg depletion experiments, mice were injected as described above and were additionally injected i.p. with rat anti-mouse CD25 Ab (Bio X Cell, clone 7D4) on day 1 IFor CT26 model, 2 x io⁶ CT26 cells (NTC control or KO generated as described below) were implanted into the flank on day 0, mice were then injected intraperitoneally (i.p.) with 10 mg/kg (-200 pg/mouse) of rat monoclonal anti-mouse PD-1 Ab (Bio X Cell, clone 29F. 1 A12) on the days 11,14,17,20 and 23. For the macrophage depletion experiments, mice were injected as described above and additionally injected intravenously (i.v.) with liposome clodronate (Liposoma BV, Netherlands) on day 8 and 11. For the IFN-Ab depletion experiments, mice were injected as described above and additionally injected i.p. with rat anti-mouse IFNy Ab (Bio X Cell, clone XMG1.2) on day 8 and 11. For MC38 model, 0.5 x 10⁶ MC38 cells (NTC control or miR-25-KO generated as described above) were implanted into C57BL/6J mice flank on day 0. mice were then injected i.p. with 10 mg/kg (200 pg/mouse) of anti-mouse PD-1 Ab (Bio X Cell, clone 29F. 1A12) from day 9 until death. For 4T1 model, 2 x 10⁶ 4T1 cells (NTC control or miR-25-KO generated as described above) were implanted into BABL/C mice flank on day 0. and then injected i.p. with 10 mg/kg (200 pg/mouse) of anti-mouse PD-1 Ab (Bio X Cell, clone 29F.1A12) from day 9 until death. Tumors were measured every 3 days beginning on day 7. Measurements of the longest dimension (length, L) and the longest perpendicular dimension (width, W) were taken to calculate tumor volume: (L x W²)/2. Mice were euthanized by CO2 inhalation and cervical dislocation when tumors reached 2.0 cm in length, and the day of sacrifice was taken as the date of death for the purpose of the survival experiments.

CRISPR/Cas9-Mediated Generation of Knockout Cell Lines

NTC and KO cell lines were generated using at least three sgRNA sequences per gene. sgRNAs were cloned into the lentiCRISPR V2 vector by Golden Gate assembly. Lentiviruses were generated by transfecting HEK293T cells with the sgRNA-expressing vectors, packaging plasmid (psPAX2), and envelope plasmid (pMD2.G) in Opti-MEM medium (ThermoFisher). After 4 to 6 hours post transfection, the medium was replaced by DMEM withl0% FBS. After 48 hours of transfection, the supernatants containing the lentivirus were collected and spin-transduced to mouse tumor cells. Transduced cells were selected by puromycin or blasticidin (InvivoGen), and finally KO efficiency was determined by qPCR, immunoblotting or T7EN1 assay.

T7EN1 assay

The genomic DNA was extracted by Quick-DNA Kit (Zymo Research). DNA fragments spanning target sites of CRISPR/CAS9 were PCR amplified by Q5 Hot Start High-Fidelity 2x Master Mix (NEB). Total 200 ng of PCR products was denatured and reannealed in 1 x NEBuffer 2 (NEB) in a thermocycler with following steps: 95 °C 5 minutes, 95 - 85 °C -2 °C/s, 85 - 25 °C -0. 1 °C/s, hold at 4 °C. 10 U T7EN1 enzymes (M0302L, NEB) was added to the hybridized PCR products. The reaction mix was incubated at 37 °C for 15 minutes. Reactions were stopped by adding 2 pl 0.5 M EDTA. The products were finally separated by 2% polyacrylamide gel electrophoresis (PAGE), stained with SYBR Safe DNA Gel Stain (Thermo) and the images were captured by ChemiDoc™ XRS (Bio-Rad).

Flow Cytometry of Tumor-Infiltrating Immune Cells

Tumors were excised from mice using sterile techniques, weighed, mechanically diced, and then incubated with complete RPMI medium plus collagenase P (2 mg/ml, Sigma- Aldrich) and DNase I (50 pg/ml, Sigma- Aldrich) for 10-20 minutes with gentle shaking every 5 minutes. Single-cell suspensions were filtered through a 70-pm filter and resuspended in FACS staining buffer. Red blood cells were lysed by addition of lysis reagent. Cells were incubated with TruStain fcX anti-mouse CD 16/32 Abs (BioLegend) and then with either Zombie Aqua Live/Dead fixable dye (BioLegend; for cells to be labeled for surface and intracellular proteins) or Calcein violet 450 AM Live/Dead (eBiosciences; for cells to be labeled only for surface markers). The cells were labeled with the appropriate combinations of antibodies against cell surface markers. For intracellular protein staining, cells were fixed, permeabilized. and stained with the appropriate Abs. Finally, the cells were resuspended in FACS staining buffer and analyzed on a BD FACSCanto (UCSD Flow Cytometry' core). BD CompBeads were used to optimize fluorescence settings (552845. BD Biosciences). Fluorescence-minus-one, unstained, and single-stained cells were also used to set gates. The following anti-mouse antibodies were used for flow cytometry: CD45 (clone 30-F 11), CD8 (clone 53-6.7), CD4 (clone RM4-5), CD3s (clone 145-2C11), NKl.l(clone PK136), FoxP3 (clone MF- 14), granzyme B (clone 25-8898-82), B220 (clone RA3-6B2), CD69 (clone H1.2F3), CDl lb (clone MI/70), Ly6G (clone 1A8). Ly6C (clone HK1.4), MHC-II (clone M5/114.15.2), F4/80 (BM8), CD206(clone C068C2). and CD24-(clone MI/69). All Abs were from BioLegend except anti-granzyme B (eBioscience). qRT-PCR and RNA-Seq

Total RNA was extracted from cultured cells using Quick-RNA Miniprep Plus Kit (Zymo Research) according to the manufacturer’s instructions. Freshly dissected mouse tumors were weighed and immediately homogenized in TRIzol (Thermo Fisher Scientific). The lysates were centrifuged, and RNA was isolated from the supernatants using Direct-zol RNA Miniprep Plus kit (Zymo Research). All RNAs were treated with DNase I. cDNAs were synthesized using an iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR was used SsoAdvanced Universal SYBR Green PCR SuperMix (1725270. BioRad). Gene expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels and are expressed as the relative fold-change in expression compared with the control condition. miRNAs were normalized to U6 snRNA and presented as the relative fold-change to the controls. For RNA-Seq, total RNA was isolated from NTC or KO tumors (two biological replicates). Sequencing was performed by HiSeq 4000 at the IGM Genomics Center, UCSD. Fastqc was used to perform quality control on sequencing data, and Cutadapt was used to remove adapters and trim reads. The preprocessed reads were then aligned to the Mus musculus genome (ml 9 GENCODE data) using STAR. The raw gene count for each sample was obtained by Htseq2 (strand - reverse) and was normalized using the built-in method (median of ratios) in DEseq2. Differential gene expression was analyzed by DEseq2 using a cut-off p value of 0.05.

IFNy Stimulation of Melanoma Cells In Vitro

B16 cells were plated at a density of 50000/well in 12-well plates in complete DMEM medium with DPBS (vehicle control) or IFNy (100 ng/ml, BioLegend) for 48 hours. The cells were then collected, RNA was extracted, and gene expression levels were determined by qRT-PCR.

Tumor Dissociation and Single Cell RNA-Seq

Tumors treated with anti-PD-1 were harvested on day 18. Tumors were mechanically cut and enzymatically digested with mouse tumor dissociation kit (Miltenyi Biotec) as manual instructed. The tissue dissociation was performed as follows: 1 x MACS program h_tumor_03 using gentleMACS Octo Dissociator (Miltenyi Biotec). After dissociation, cells were passed through a 70 pm filter and washed with DMEM containing 10% FBS (Gibco). The samples were then incubated with Ammonium Chloride Solution (STEMCELL technologies) to deplete red blood cells (RBCs). Dead cells were removed by Dead Cell Removal Kit (Miltenyi Biotec) to provide more than 90% viability’ of single cells. 20,000 single cells per sample were loaded on the 1 Ox Chromium system and encapsulated with Single cell 3’ HT Library & Gel Bead Kit (10* Genomics). Single-cell gene expression were generated according to the manufacturer’s instructions. cDNAs and libraries were submitted to TapeStation for quality control. Completed libraries were sequenced on NovaSeq (Illumina) platforms at a targeted median read depth of 30.000 reads per cell.

Sequencing Data Processing and Analysis

FastQC was used to perform quality control on sequencing data, and Cutadapt was used to remove adapters and trim reads. The preprocessed reads were then aligned to the Mus musculus genome (mmlO) using STAR. The raw gene count for each sample was obtained by Htseq2 and was normalized using the built-in method in DEseq2. Differential gene expression was analyzed by DEseq2 using a cut-off p value of 0.05. For single cell RNA-seq, the sequencing data were analyzed using the Cell Ranger Single-Cell Software Suite (10X Genomics). Ingenuity Pathway Analysis (IPA) uses a network generation algorithm to segment the map between molecules. For canonical pathway analysis, the - log (p-value) > 1.3 was taken as threshold and the z-score > 2 was defined as significant activation, while z-score < -2 indicated significant inhibition.

Cell Proliferation Assay

Cells were incubated in 96-well plates with 1000 cells at 0 hour timepoint. 20 pL CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was added to each well at each timepoint and incubated for 1 hour. The absorbance was measured at the wavelength 490 nm every 24 hours. The absorbances were normalized to average of 24-hour post seeding and presented as the relative fold-change compared to the 24-hour values.

Western Blot Analysis

Cells or fresh isolated mouse tumors were lysed in lysis buffer (60 mM Tris HC1, 2% SDS, 10% glycerol, complete EDTA-free protease inhibitor, 500 U/ml benzonase nuclease) by pipetting or homogenization. Samples were clarified by centrifugation and protein concentrations were determined with a BCA protein assay kit (Pierce). Aliquots of 50-150 pg of protein were resolved by 10% Tris-Glycine or 4-12% Bis-Tris Plus PAGE and the proteins were transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with antibodies. After washing, the membranes were incubated for 1 hour at room temperature with secondary antibody. Finally, the blots were developed using ECL and imaged.

Immunohis tochemis try

The staining analysis followed the previous description. Briefly, slides of paraffin-embedded from fresh mouse tissue were deparaffinized in xylene and rehydrated in graded ethanol (5 min in 100%, 5 min in 95%, and 5 min in 75%) and then washed by PBS containing 0.3% Triton X-100 (Sigma-Aldrich) (PBST) for three times. Sections w ere treated with antigen retrieval with Tris/EDTA buffer pH 9.0, rinsed three times with PBST, incubated with 3% H2O2 in PBS at 37°C for 10 minutes. After blocking with 10% normal goat serum (50062Z, Abeam) for 1 hour, tissue slides were incubated at 4°C overnight with primary antibodies overnight at 4 °C, followed by biotinylated secondary antibody for 1 hour at room temperature, and then incubated with peroxidase conjugated avidin biotin complex for 1 hour at room temperature. The sections were incubated with AEC chromogen substrate developing agent and imaged using a Keyence microscope.

RNA Immunoprecipitation (RIP)

RNA immunoprecipitation assays were performed as previously published with some modifications. Briefly, cells were lysed in 2 ml of polysome lysis buffer containing 100 mM KC1, 5 mM MgC12, 10 mM HEPES, pH 7.0, 0.5% Nonidet P-40, 1 mM dithiothreitol, lOO U/ml RNasin RNase inhibitor, 2 mM vanadyl ribonucleoside complexes, and protease inhibitor cocktail and centrifuged. The supernatants were collected. Lysates were precleared with protein A magnetic beads, and 10% of the lysate was removed and reserved as the lysis input sample. The remainder of the precleared lysates were incubated with anti-Ago2 antibody (ab 186733. Abeam) at 4°C overnight and then mixed ith 100 pl protein A magnetic beads per sample and incubated at 4°C for 4 h. The beads were washed three times with polysome lysis buffer and then three times with polysome lysis buffer containing 1 M urea. About 10% of the sample was removed and reserved to determine IP efficiency. The remainder of the sample was incubated with 100 pl of polysome lysis buffer containing 0. 1% SDS and 30 pg proteinase K at 50°C for 30 min to elute RNA. The eluates were mixed with 1 volume (100 pl) of phenol- chloroform-isoamyl alcohol and centrifuged for 1 min to separate the phases, and the upper aqueous phase was recovered. The elution step was repeated twice, and the aqueous phases were combined. RNA was purified using an RNA Clean & Concentrator kit (ZYMO). The purified input sample and IP-eluted RNA were reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad) and analyzed by qPCR.

Luciferase assay

An 80-bp fragment of the Sdc3 3’ UTR containing the conserved miR-25 binding sites was inserted into a luciferase reporter plasmid (pMIR-REPORT™ miRNA Expression Reporter Vector System. Invitrogen). To test binding specificity, sequences that interact with the miR-25 seed sequence were mutated from GTGCAAT to CACGTTA, and the synthetic Sdc3 3’ UTR mutant fragment was inserted into the same reporter plasmid. For the luciferase reporter assays, B16 cells were cultured in 96-well plates and each well was co-transfected with 0.05 pg reporter plasmid, 0.05 pg - galactosidase expression plasmid (Invitrogen), and equal amounts (10 pmol) of miR-25 mimic or the scrambled negative control RNAs using Lipofectamine 3000 (Invitrogen). The -galactosidase was used as a control. The cells were assayed using a luciferase assay kit 24-hour post transfection (Promega).

Gene Expression and Estimated Immune Infiltration on Human Tumors

Data was downloaded for 352 metastatic SKCM patients’ miR-25 expression data using TCGAbiolinks. Patient samples were sorted by log2 miR-25 expression and 88 lowest and 88 highest miR-25 patient barcode were extracted with clinical information. TCGAanalyze_survival was used to visualize the survival difference. Corresponding RNA-seq data including STAR counts was downloaded by GDCquery and processed by TCGAanalyze_Preprocessing and TCGAanalyze_Normalization. Differential expression was done by TCGAanalyze DEA. DEGs were defined as fold change (FC) > 1 and p- value < 0.05. For 1PA canonical pathway analysis, the -log (p-value) > 1.3 was taken as threshold and the z-score > 2 was defined as significant activation, while z-score < -2 indicated significant inhibition. Gene set enrichment analysis (GSEA) w as also used to perform analysis of the datasets. The up-regulated pathways were defined by a normalized enrichment score (NES) > 0 and the downregulated pathways were defined by an NES < 0. Pathways with an FDR-P value < 0.05 were chosen as significantly enriched pathways. For estimated immune infiltration in the samples, the leukocyte signature matrix LM22 (547 genes) which discriminates 22 types of tumor-infiltrating immune cells was used for CIBERSORT analysis.

Normalized gene expression data from above were processed with the CIBERSORT web tool (http://cibersort.stanford.edu/) setting no quantile normalization and 1.000 permutations as parameters.

Statistical Analysis

Data are presented as the mean ± standard error (SEM) unless otherwise indicated. Group means were compared by Student’s t-test or Two-Way ANOVA. P < 0.05 was considered statistically significant.

Example 2: Anti-PD-1 therapy model and experimental strategy

To identify miRNA (miRs) expressed at different stages during cancer immunotherapy treatment that can regulate the effectiveness and escape mechanisms of the treatment, mice were injected with melanoma cancer cell line (B16F10) (FIG. 1A). Mice were either left untreated or vaccinated with GV AX on day 1 and day 4, followed by treatment with anti-PD-1 on day 6 and day 9. It was shown that combination treatment of GV AX and anti-PD-1 (GP) showed a significant decrease in tumor growth compared to GV AX alone and non-treatment control (NTC) group (FIGS. IB- IE).

Example 3: Identification of miRNAs with altered expression during immunotherapy treatment

To identify miRNAs with altered expression during the course of immunotherapy treatment, tumor RNA extracted from NTC group, GV AX (irradiated B16 cells secreting granulocyte-macrophage colony stimulating factor (GM-CSF)) monotherapy group and GP combination therapy group were subjected to small RNA sequencing (FIG. 2A). Differential expression analysis showed that compared to the NTC. the combination treatment (GP) increased the expression of miR-36a, miR-1839-3p, miR-706, miR-3099, and miR-2183. Compared to the GVX monotherapy, the combination treatment (GP) decreased the expression of miR-1903 (FIGS. 2B and 2C). Bioinformatic analysis revealed distinct clustering of up regulated and downregulated miRNAs by GV AX monotherapy and combination therapy (GP) (FIGS. 3A and 3B). Quantitative real time PCR (qRT-PCR) comparing the expression of key upregulated and downregulated miRNAs in combination treatment (GP) group and GV AX monotherapy group showed that most of the downregulated miRNAs belong to the miR- 17/92 miRNA family (FIGS. 4A and 4B). The changes in the expression of these miRNAs was also confirmed when NTC group was compared with the combination treatment (GP) group (FIGS. 5A and 5B). Thus, upregulated miRNAs (miR- 16, miR-29a, miR- 150, and miR- 125b) and downregulated miRNAs (miR- 106a and miR-25) were tested for downstream experiments.

Example 4: CRISPR Knockout and overexpression of miRNAs and regulation of cancer Immunotherapy

To test the function of the miR-29. miR-150, miR-25. miR-106a, miR-21, Let-7f. miR- 125b, and miR- 16-1 /miR 16-2 in regulating responses to immunotherapy, single guide RNAs (sgRNAs) targeting these miRNAs and their respective families were designed (FIGS. 6A-6C, FIGS. 7A-7C, FIGS. 8A-8C, FIGS. 9A-9C, FIGS. 10A-10C, FIGS. 11A-11C, and FIGS. 12A-C). Knocking out miR-29, miR-150. and miR-16-1 significantly increased in vitro cell proliferation (FIG. 6D, FIG. 7D and FIG. 11D). However, knocking out miR-25, miR-106a, miR-21 and miR-125b did not affect in vitro cell proliferation (FIG. 8D, FIG. 9D, FIG. 10D, and FIG. 12B). When treated w ith the combination therapy (GP) in vivo, miR-29a and miR-150 knockout tumors showed accelerated growth and larger tumor volumes compared to the NTC tumors (FIGS. 6E and 6G, FIGS. 7E and 7G) but miR-125b and miR-16 knockout tumors did not show any significant differences compared to the NTC tumors (FIG. 10E, FIG. 10G, FIG. HE and FIG. 11G) However, knocking out untreated miR125b slowed the tumor growth resulting in smaller tumor volumes compared to the untreated NTC tumors (FIG. 10E). On the other hand, w hen treated with immunotherapy in vivo, knocking out miR-25, miR106a, Let-7f, and miR-21 significantly delayed tumor growth and reduced tumor volumes compared to the NTC group (FIG. 8E, FIG. 8G, FIG. 9E, FIG. 9G, and FIGS. 12C and 12D). Mice bearing miR-29a and miR-150 knockout tumors when treated with immunotherapy showed lower survival rate than the NTC tumors (FIG. 6F, FIG. 7F). On the contrary, mice bearing miR-25, miR- 125b knockout tumors when treated with the combination therapy (GP) showed better survival than the NTC tumors (FIG. 8F, FIG.10F). Treating miR-106, miR16 knockout tumors with the combination therapy (GP) did not significantly improve their survival (FIG. 9F, FIG. 11F). The study also showed that overexpression of miR-29a and miR-150 significantly reduced in vitro cell proliferation (FIGS. 6H-I, FIGS. 7H-I and FIG. 7K) and enhanced effects of anti-PD-1 treatment (FIG. 6J and FIG. 7J)

Example 5: miR-25 deficiency enhances tumor responses to anti-PD-1 immunotherapy

To determine the role of miRNAs in modulating the response to anti-PD-1 therapy, poorly immunogenic murine melanoma Bl 6F 10 cells were subcutaneously injected into immunocompetent C57BL/6J mice on day 0 and vaccinated with GV AX cells (irradiated B16 cells secreting granulocyte-macrophage colony stimulating factor (GM-CSF)) on the opposite flank on day 1 and 4. The mice were then treated with anti- PD-1 monoclonal antibodies on day 6, 9 and 12 (FIG. 13A). Compared to non-treatment control (NTC), tumor grow th in combination therapy treated group (GV AX + aPD-1) was significantly reduced but the same was not observed for the GV AX monotherapy and anti-PD-1 monotherapy (FIG. 13B and FIG. 14A). To investigate the change in miRNA expression during immunotherapy, tumor RNAs were isolated from each group on day 13 and small RNA sequencing was performed. Differential expression analysis showed that miR-25 and miR-17-92a family members were downregulated (FIG. 13C). Quantitative real time PCR (qRT-PCR) further validated the decrease in the expression of these miRNAs in tumors that responded to therapy (FIG. 13D). This showed that miR-25 and miR-17-92a family members may play important roles in responding to anti-PD-1 therapy. To test the function of these miRNAs, sgRNAs targeting miR-25 genomic DNA loci were designed and incorporated with CRISPR-Cas9 knockout plasmid (FIG. 14B) using benchling (www.benchling.com/crispr). CRISPR/Cas9 can edit genome DNA sequences and result insertions or deletions (indels), which will be recognized by T7EN1 assay. To confirm the successful knockout of miR-25, T7EN1 assay was used. The assay detected DNA cleavage at the genomic loci of miR-25 (FIG. 14E). Also, knocking out miR-25 in B16 (melanoma), MC38 (colorectal cancer), and 4T1 (triple-negative breast cancer) cell lines was able to decrease the level of miR-25 up to 90% (FIG. 13E). Compared to the NTC, knocking out miR-25 in B16, MC38, and 4T1 cell lines did not affect in vitro cell proliferation (FIG. 14D). In vivo studies showed that miR-25 depletion did not affect B16 tumor growth in mice. How ever, when these mice were treated with a combination therapy of GV AX and anti-PD-1 (GP) as described in FIG. 13 A, growth of miR-25 KO tumors was significantly reduced (FIG. 13F and FIG. 14E). To test the effects of miR-25 KO on MC38 (FIGS. 13J-13M) and 4T1 cells when treated with anti- PD-1, MC38 and 4T1 inoculated mice were treated with anti-PD-1 from day 9 and every three days until death (FIG. 14F). Similar to B16. in both MC38 and 4T1 tumors, miR-25 depletion did not affect the in vivo tumor growth, but sensitized the tumors to anti-PD-1 immunotherapy (FIG. 13F). When treated with anti-PD-1, mice bearing miR-25- deficient tumors survived much longer than the NTC (FIGS. 13F-13H and FIGS. 14G- 14H). Similar to miR-25 KO models, deletion of miR-92a, miR-106a, and miR-17 also significantly reduced tumors size when treated with the combination therapy (GP) (FIG. 131 and FIG. 141). Altogether, these results indicate that depletion of miR-25 and the miR-17-92a family members can enhance the tumors' response to anti-PD-1.

Example 6: miR-25 deficiency modifies tumor immune infiltration and activates innate immune sensing

To examine the changes in miR-25-deficient tumor microenvironment (TME), NTC and miR-25 KO cells inoculated mice were treated with GP. On day 13, tumors were isolated and analyzed by flow cytometry. Compared to the NTC tumors, miR-25 KO tumors showed a significant increase (around 3~4 fold) in total tumor infiltrating CD45⁺ cells, which includes all hematopoietic cells (FIG. 15A). Although CD8⁺ T cell numbers per gram tumor increased (FIG. 16D). the ratio of CD8⁺ T cell in CD45⁺ didn’t show significant change (FIG. 15B, left). Similarly, CD4⁺ T cell numbers per gram tumor also increased (FIG. 16E) but the ratio of CD45⁺ didn’t show significant change (FIG. 15C, left). The results also showed presence of active CD8⁺ T cells with higher Granzyme B expression (FIG. 15B, right), and fewer suppressive CD4⁺ regulation T cells (Treg cells) (FIG. 15C, right), indicating that miR-25 KO had constructed an antitumor microenvironment. Dendritic cells (DC), myeloid derived suppressive cells (MDSC), and tumor resident macrophages (Macs) were also analyzed (FIG. 15D and FIGS. 16F-16H). Notably, macrophage numbers per gram tumor did not change but the ratio of CD45⁺ was significantly reduced (FIG. 15D, left and middle). Specifically, CD206, also known as mannose receptor C type 1 (Mrcl), a marker to identify the M2 macrophage, were reduced to 1/3 (FIG. 15D right). To investigate whether miR-25 KO affects circulating leukocytes, splenocytes were analyzed. miR-25 KO tumors did not affect the spleen macrophage population but a slight increase in active B cells with higher CD69 expression was observed (FIG. 15E).

RNA sequencing of miR-25 -deficient tumors revealed a remarkable up-regulation of pathways related to pathogen recognition such as phagosome formation, role of hypercytokinemia/hyperchemokinemia in the pathogenesis of influenza, role of pattern recognition receptors in recognition of bacteria and viruses, pyroptosis signaling pathway, TREM1 Signaling and Fey receptor-mediated phagocytosis in macrophages and monocytes (FIG. 15F). Phagosome formation representative genes including innate sensing complement genes Clra, Clqb, Clqc. C3arl, C3, immunoglobulin genes Itgam, Itgb2, and toll like receptor genes Tlr7, Tlr8, Tlr9 were elevated in miR-25 KO GP tumors (FIG. 15G). In addition, miR-106a-KO tumor RNA-seq also revealed similar enriched pathways (FIG. 161), indicating other miR- 17/92 family members may function similar to miR-25 in influencing the TME.

To seek further specific changes in the TME, Immunohistochemistry (IHC) staining was performed for leukocyte marker CD45, complement C3, and macrophage marker F4/80 (FIG. 15H). To amplify target signal, 3-Amino-9-Ethylcarbazole (AEC) was used, which produces a red product, and hematoxylin was used to stain the nuclei of the cells. Under 5x magnification, it was evident that miR-25 KO GP tumors were immersed with more CD45 leukocytes and more C3 complement but proportion of macrophages remained unchanged (FIG. 151). The IHC results were consistent with the flow cytometry and the RNA-seq results. More detailed interactions between cells are shown with 20x lens (FIG. 15H). In the NTC GP group, CD45 leukocytes and the tumor cells formed a clear boundary without much interaction (FIG. 15H). However, in the miR-25 KO GP group, CD45⁺ leukocytes were not only increased by numbers, but also dived into tumor cells as indicated by the arrows (FIG. 15H). The cellular interaction difference became more pronounced in macrophages despite the similar positive area of F4/80. Interestingly, C3 positive cells did not interact with the tumor cells as the CD45 and F4/80 positive cells did, which suggests that the complement activation may occur in a different cell subtype (FIGS. 15H-15I). These results together show that the NTC GP TME is immune silenced whereas the miR-25 -KO GP TME is more pro-inflammatory.

To demonstrate the functional role of macrophages, liposome clodronate was used to deplete macrophages (FIG. 15 J). Liposome clodronate was intravenously injected twice to completely abolish the miR-25-KO effect, leading to larger tumor volumes and heavier tumor weights (FIG. 15K). The macrophage depletion by confirmed by measuring the spleen and tumor infiltrating macrophages. Both depletion groups showed fewer macrophages in splenocytes and tumors (FIGS. 16K-16L). Taken together, these findings suggest that miR-25 KO GP tumors can enhance anti-tumor immunity by activating whole leukocyte infiltration and complement cascade via macrophages.

Example 7: Single cell RNA-seq reveals innate immune activation in miR-25- defificent tumor microenvironment

To investigate the cellular and genetic details of TME composition, miR-25 KO GP tumors were isolated and subjected to single cell RNA sequencing (scRNA-seq). MC38 is a more immunogenic solid tumor than other tumor models and the in vitro and in vivo effects of miR-25 KO on B16 tumors were also observed in MC38 tumors (FIG 13G). Thus, scRNA-seq was also performed on the MC38 tumors. After quality control and filtering, we recovered 51,971 transcriptomes of live single cells. Unsupervised clustering gave rise to 19 cell clusters. The cell clusters exhibiting high similarities were annotated as the same cell subsets, and the remaining were manually annotated by their signature genes (FIG. 17A and FIG. 18A ). A significant increase in the leukocyte (Ptprc⁺) infiltration was observed in the TME of miR-25 KO tumor treated with anti-PD- 1 (9066 leukocyte per gram of tumor) compared to the NTC tumor treated with anti-PD-1 (5124 leukocyte per gram of tumor (FIG. 15A). Notably, the number of macrophages decreased more than half (FIG. 19B). Ml macrophages are proinflammatory, phagocytic, and can initiate an immune response; while M2 macrophages are associated with wound healing and tissue repair, and often favor immune suppression and eventually result tumor progression. The study revealed a specific subset of macrophages with a metabolic signature that was different from other macrophages (FIG. 18B). This subset of macrophages was annotated as ‘Ml ’ macrophage and the rest of macrophages as “M2”. When the Ml and M2 macrophages were analyzed separately, there was a decrease in the macrophage infiltration from an average of 11% to 6% in the miR-25 KO tumor. This decrease was attributed to the M2 macrophages. Consistent with the B16 model, the population of other tumor infiltrating leukocytes was upregulated but the increase was not statistically significant except for the gamma delta T cells (y8 T cells) (FIG. 17B).. This suggests that miR-25 KO tumor treated with anti-PDl immunotherapy may elicit the innate immunity. Taking the average ratio of each cell types, the population of macrophages and tumor cells were shown to decrease while T cell population expanded from 1/3 to about 1/2 of the TME (FIG. 17C).

Since macrophages are the key regulators in TME and immunity' (FIGS. 15 J- 15K), both Ml and M2 subsets were investigated. Differential expression analysis showed a significant elevation in proinflammatory genes including FkbpS, Nlpr3, Jakl, II7r, and Crebbp in miR-25-KO tumor associated Ml (FIGS. 17D and 17G). Ml macrophage elevating and reducing signaling pathways are shown in FIG. 17F. Among them. Nrf2-mediated oxidative stress response, HIF-la signaling, pyroptosis signaling pathway. FTL3 signaling in hematopoietic progenitor cells and iNOS signaling are closely related to Ml macrophage polarization. Specifically, the same pyroptosis signaling pathway was also observed in the B 16 RNA-seq result (FIG. 15F). The expression of Nlrp3 significantly increased in the Ml macrophages of miR-25 KO tumors compared to the control (FIG. 17D). In addition, according to the Bl 6 RNA-seq data, NlrplO, Caspl, Casp4, (ibp2.3.4.5. Gzma and Tlrl.2, 7,8,9, 13 were all enriched in the miR-25-KO GP B16 tumors (FIG. 15F). Interestingly, the expression of Ifitl , Ifit2, Ifit3 and another type I interferon induced gene, Sljh4 decreased in both Ml and M2 subsets in the miR25-KO tumors compared to the control (FIG. 17F and FIG. 17G). In addition, interferon signaling gene suppression was analyzed in the TME. UMAP expression analysis of genes show ed that the interferon genes were specifically repressed in macrophages, while Ifitl and Ifit3 expressions were enhanced in y5 T cells and CD8 T cells (FIG. 18C).

Next, the complement genes were analyzed which were enriched in the B16 model shown in FIG. 15G. Although the macrophages expressed some of the complement genes, most of the complement genes seemed to arise from cancer-associated fibroblasts (CAF) (FIG. 18D). Expression of the complement classic activation genes including Clsl, Clra, and C4b were significantly elevated in miR-25-KO tumor associated fibroblast (FIG. 17E). The C3 gene, the central component of complement cascade, was also highly upregulated in the miR-25-KO tumor associated fibroblast (FIG. 17E).

To test whether the miR-25 -deficient tumors can elicit an adaptive immunity in the TME, percent expression of T-cell receptor (TCR) and B-cell receptor (BCR) upstream genes were analyzed in different cell subsets. It was shown that CD4⁺ T cells expressed more TCR upstream genes such as Ccr7, Cxcr4, Socs3 and Tcf7. B cells in the miR-25-deficient tumors expressed more BCR upstream regulators like Atp6v0c, Gls, Stcit3, and Tubb4b (FIG. 17H). Noteworthy, Cxcr4 signaling was enhanced in CD4⁺ T cell, B cell and NK cells (FIG. 18E). All the above data suggests that when treated with anti-PD-1, macrophages can recognize miR-25 KO tumors by inflammasome formation and activates the adaptive immunity in the TME.

Example 8: miR-25 represses the expression of Syndecan3

Inflammasome activation is initiated by pattern-recognition receptors (PRRs) responding PAMPs or DAMPs. To test if miR-25 regulates PAMPs or DAMPs upon the release of IFN-y caused by the anti-PD-1 treatment, IFN-y depletion experiment was started on day 8 (one day before the tumor growth curve starts to separate). IFN-y depletion using anti-IFN-y fully abolished miR-25-KO effect in the B 16 tumors (FIG. 19A and FIG. 20A). Notably, although the NTC GP group treated with anti-IFN-y significantly decreased the population of spleen MHCII⁺ macrophages compared to the NTC GP group without the anti-IFN-y treatment, miR-25 KO GP tumor-bearing mice treated with anti-IFN-y presented a steady augment of MHCII⁺ spleen macrophages, suggesting the tumor resident macrophages may have already been activated on day 8, and IFN-y is indispensable for them to stay in the TME (FIG. 19B).

To test if miR-25 regulates its target genes through minimal miRNA-induced silencing complex (miRISC) by inducing target mRNA degradation and translational repression. Ago2 expression was tested in miR-25 KO B16 cells and other miR-17/92 family member KO B16 cells. The expression of Ago2 did not change in the miR-25 KO compared to the NTC. Ago2 depletion w as confirmed by immunoblotting (FIG. 20B). A miR-25 and Ago2 double knockout model w as generated in the Bl 6 cell line to test the tumor growth in vivo with the GP treatment (FIG. 20C). Similar to miR-25 KO GP group, Ago2 KO also induced tumor repression. The double knockout of Ago2 and miR- 25 did not show a difference compared to the single Ago2 KO (FIG. 19C and FIG. 20D). These results demonstrate that miR-25 regulation of cancer immunotherapy depends on the miRISC and is affected by the bona fide functions of miR-25. To identify potential miR-25 targets, four datasets were overlapped: Bl 6 miR-25 KO GP tumor RNA-seq, which includes TME genes; in vitro RNA-seq data of miR-25-KO B16 cells; in vitro RNA-seq data of miR-25-KO B16 cells treated with IFN-y (48 hours); and predicted miR-25 potential targets based on Targetscan (www. targetscan.org) (FIG. 19D). The only downregulated gene common to all datasets was Syndecan3 (Sdc3).

Since miRNAs regulate gene expression by binding to the 3’UTR sequences of target genes, seed sequences of miR-25 and other family members including miR-92a perfectly base paired 3’UTR of Sdc3 (FIG. 20E). To test the function of this miR-25 seed region in targeting Sdcs3, a luciferase reporter plasmid was designed. A sequence containing the predicted miR-25 binding site was inserted downstream of the firefly luciferase reporter plasmid. Subsequently, Bl 6 cells were transfected with the plasmid, with either miR-25 mimics or scrambled negative control RNA. The luciferase activity w as decreased by half when transfected with miR-25 mimics. However, when the binding sequence in Sdc3 was mutated, the luciferase activity was unchanged between the control and miR-25 overexpression (FIG. 19E).

IFN-y depletion using anti-IFN-y fully abolished the miR-25-KO effect in B16 tumors (FIG. 19A and FIG. 20A). To determine the changes in Ago2 bound Sdc3 mRNA in the presence and absence of IFN-y, Ago2 immunoprecipitation coupled with qPCR (RIP-qPCR) w as used. The percentage of Ago2 bound Sdc3 transcripts was higher in miR-25 KO B16 cells compared to the NTC group without the IFN-y treatment (labeled as 0 hour) (FIG. 19F). However, there was no difference in Ago2 bound Sdc3 mRNA expression between the NTC and the miR-25 KO cells after 72-hour of IFN-y treatment. To analyze the kinetics of IFN-y effects on Ago2-Sdc3 interactions, RT-qPCR and immunoblotting of Sdc3 at different time points. Sdc3 mRNA w as repressed in the NTC Bl 6 cells, whereas the miR-25 KO Bl 6 cells consistently showed upregulation of Sdc3 mRNA levels. Specifically, at the 72-hour timepoint, Sdc3 mRNA expression reached the highest level in miR-25 KO cells (FIG. 19G left). Correspondingly, immunoblotting showed a decrease in the expression of SDC3 protein in NTC group treated with IFN-y, but a gradual increase in the expression of SDC3 was observed in miR-25 KO cells (FIG. 19G, right). These results show that in NTC cells, miR-25 repressed sdc3 expression by miRISC degradation in the presence of IFN-y. These findings were confirmed in MC38 and 4T1 cells, using RIP-qRT-PCR and immunoblotting. In MC38 and 4T1 cells, both mRNA and protein expression of SDC3 were downregulated by IFN-y stimulation, whereas in the miR-25 KO cells. SDC3 expression was steadily expressed. (FIG. 19H and FIG. 191). The kinetics of Sdc3 mRNA and protein expression in NTC and miR-25 KO cells were different in all three cell lines, presumably due to cell-intrinsic differences. For example, in 4T1 cells, Sdc3 transcript level was higher at 0 hour and was stimulated by IFN-y at 24-hour time point. However, Sdc3 mRNA expression consistently decreased after the 24-hour time point in NTC. The mRNA suppression of Sdc3 was released by miR-25 KO, and the Sdc3 protein expression in miR-25-KO 4T1 cells was markedly higher than the controls at each timepoint (FIG. 191). With varying degree in multiple cells, miR-25 expression was persistent in Bl 6, and was enhanced by IFN-y treatment in MC38 and 4T1 (FIGS. 20F-20H).

To test the function of Sdc3, sgRNAs targeting the genomic loci of Sdc3 was designed and confirmed the knockout of Sdc3 in B16 cells (FIG. 1J). Sdc3 KO enhanced tumor cell proliferation in vitro (FIG. 201). A miR-25 and Sdc3 double KO model was also generated. Double KO abolished the miR-25 GP effects (FIG. 19K and FIG. 20J). In B16 NTC GP tumors, the expression of Sdc3 was reduced after 72-hour of anti-PD-1 treatment, whereas the miR-25-KO GP showed the opposite trend with lower expression of Sdc3 on day 6 but a significant enhancement was observed on day 9 (FIG. 20K), which was consistent with the in vitro immunoblotting results ((FIG. 19G). Taken together, these results indicate that miR-25 in tumor cells reduce the Sdc3 expression during the anti-PD-1 treatment.

Example 9: miR-25 and miR-17-92 family members suppress innate immunity in melanoma patients

To investigate the role of miR-25 in melanoma, small RNA and mRNA expression data in TCGA was used. Small RNA expression data from 352 skin cutaneous metastatic melanoma (SKCM, TM) patient samples were divided by miR-25 expression into: lowest miR-25 expression and highest miR-25 expression (FIG. 21A). Lower miR- 25 expressing patients showed prolonged survival (FIG. 21B). Next, the corresponding tumor RNA sequencing data for the samples were analyzed. Ingenuity pathway analysis (IP A) indicated phagosome formation as the top upregulated pathway in the TME (FIG. 21C), which was also shown as a top enriched pathways in the B16 mouse data (FIG. 15F). Gene Set Enrichment Analysis (GSEA) showed that the top two upregulated signaling pathways were immune response related IL6-JAK-STAT3 and complement pathways (FIG. 21D). Furthermore, consistent with the B16 flow cytometry results (FIGS. 2A and 2B) and the single cell RNA-seq data (FIGS. 3A and 3B), CIBERSORT analysis indicated that tumors with lower miR-25 expression were estimated to be infiltrated with fewer MO macrophages, more gamma delta T cells, more CD8 T cells, and significantly higher memory activated CD4 T cells (FIG. 21E). The study also assessed the correlation between miR-25, other miR- 17/92 family members and the innate immune response genes including complement system. All the selected innate immune response genes were negatively correlated with the miR-17/92 family members by Huber regression, while the positive control MIR17HG showed a strong and robust positive correlation (FIG. 21F). Finally, we assessed miR-25 and the innate immune gene expression in anti-PD-1 treated patients. To evaluate the expression of miR-25 and the innate immune genes in anti-PD-1 treated patients, the patients were divided by responders (complete response or partial response to anti-PD-1 treatment) and nonresponders (progressive diseases or stable diseases). When sorted by miR-25 expression levels, the responders showed a negative correlation with miR-25 expression but a positive correlation with the innate immune genes such as C3, CIS. C/R. and C1QA. However, these genes were overall silenced in non-responders (FIG. 21G). These results suggest important roles of miR-25 in cancer immunity.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having cancer, the method comprising:

(a) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor; and

(b) administering to the subject a therapeutically effective amount of a composition that modulates expression of one or more microRNAs (miRNAs).

2. A method of treating a subject having cancer, the method comprising:

(a) determining that a cancer cell in the subject expresses aberrant expression of one or more miRNAs compared to a control sample;

(b) administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor; and

(c) administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs.

3. The method of either claim 1 or 2, wherein the one or more miRNAs include miR- 16, miR-29a. miR-150. miR-15b, miR-let7f. miR-26b, miR-21. miR-466g, miR- 383, miR-181a, miR-30a, miR-106a, miR-25, miR 1983, miR-93, miR-30c, miR- 30d, miR-18a, miR-425, miR- 17, miR-92a, miR-125b, miR-20b, miR-99b, miR- 92a, miR-17, miR-19a, miR-20a, miR-19b, or miR-872.

4. The method of either claim 1 or 3, wherein expression of miR-25 and miR-106a can be decreased therapeutically.

5. The method of either claim 1 or 3, wherein expression of miR-29 and miR-150 can be increased therapeutically.

6. The method of either claim 1 or 3, wherein expression of miR-25, miR-17, miR- 92a, miR-21, and miR-Let7f can be decreased therapeutically.

7. A method of treating a subject having cancer, the method comprising:

(a) determining that a cancer cell in the subject expresses aberrant expression of one or more miRNAs compared to a control sample; and (b) administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs.

8. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition that modulates expression of the one or more miRNAs, wherein the subject expresses aberrant expression of one or more miRNAs compared to a control sample.

9. The method of either claim 7 or 8, wherein the one or more miRNAs include miR- 16, miR-29a, miR-150, miR-15b, miR-let7f, miR-26b, miR-21, miR-466g, miR-383, miR-181a, miR-30a, miR-106a, miR-25. miR 1983, miR-93, miR-30c, miR-30d, miR-18a, miR-425, miR- 17, miR-92a, miR-125b, miR-20b, miR-99b, miR-92a, miR-17, miR-19a, miR-20a, miR-19b, or miR-872.

10. The method of either claim 7 or 8, wherein expression of miR-25 and miR-106a can be decreased therapeutically.

11. The method of either claim 7 or 8, wherein expression of miR-29 and miR-150 can be increased therapeutically.

12. The method of either claim 7 or 8, wherein expression of miR-25, miR-17, miR- 92a, miR-21 and miR-Let7f can be decreased therapeutically.

13. The method of either claim 7 or 8, wherein the aberrant expression is an increase in expression of the one or more miRNAs compared to a control sample.

14. The method of either claim 7 or 8, wherein the aberrant expression is a decrease in expression of the one or more miRNAs compared to a control sample.

15. The method of any one of claims 7-14, wherein the control sample is from a subject that does not have cancer.

16. The method of any one of claims 7-14, wherein the control sample is from a subject that has cancer but has not been administered the immune checkpoint inhibitor.

17. A method for decreasing an immune response in a subj ect having cancer comprising administering to the subject:

(a) a therapeutically effective amount of an immune checkpoint inhibitor; and

(b) a therapeutically effective amount of a composition that modulates expression of one or more miRNAs.

18. A method for increasing an immune response in a subj ect having cancer comprising administering to the subject:

(a) a therapeutically effective amount of an immune checkpoint inhibitor; and

(b) a therapeutically effective amount of a composition that modulates expression of one or more miRNAs.

19. The method of either claim 17 or 18. wherein the immune response comprises tumor immune cell infiltration, Granzyme B expression, phagosome formation, complement cascade gene expression, proinflammatory gene expression, Cxcr4 signaling, or innate and immune adaptive immune regulation.

20. The method of claim 19, wherein the tumor immune cell infiltration comprises CD45⁺ cell infiltration, CD69⁺ cells, T cell infiltration including CD8⁺ T cells, CD4⁺ T cells, Ml macrophages, or gamma delta T cells (y8 T cells).

21. The method of any one of claims 1-20. wherein administering the composition increases expression of the one or more miRNAs.

22. The method of claim 21, wherein the one or more miRNAs is miR-16, miR-29a, miR-150. miR-125b-5p, miR-15b, miR-let7f, miR-26b, miR-21, miR-466g, miR- 383, miR-181a, miR-30a, or any combination thereof.

23. The method of any one of claims 1-20. wherein administering the composition decreases expression of the one or more miRNAs.

24. The method of claim 23, wherein the one or more miRNAs is miR-106a, miR-25, miR-1983, miR-93, miR-30c, miR-30d, miR-18a, miR-425. miR-17, miR-92a, miR-872, or any combination thereof.

25. The method of claim 23, wherein the one or more miRNAs is miR-25, miR-17, miR-92a. or any combination thereof.

26. The method of any one of claims 1-25, wherein the composition comprises a recombinant plasmid, vectors, miRNA-duplexes, primary-miRNA (pri-miRNA), or precursor-miRNA (pre-miRNA), and/or a delivery reagent such as a lipophilic reagent; a lipofectin, Lipofectamine, cellfectin, a poly cation, or a liposome.

27. The method of any one of claims 1-26, wherein, the composition further comprises a sequence that encodes a miRNA.

28. The method of claim 26, wherein the vector is a viral vector, or a non-viral vector.

29. The method of claim 26, wherein the viral vector is an AAV, a lentivirus, an adenovirus, an adeno-associated virus, retrovirus, or a herpes simplex virus.

30. The method of claim 26, wherein the non-viral vector is a liposome, exosome, an extracellular vesicle, a polymer, a nanoparticle, a peptide, or a dendrimer.

31. The method of any one of claims 1-25. wherein the composition comprises an inhibitory nucleic acid molecule that can decrease expression of the one or more miRNAs.

32. The method of claim 31, wherein the inhibitory nucleic acid molecule is a shRNA, a recombinant plasmid, a siRNA, a micro RNA, an antisense oligomer, a crRNA, or a RNA-induced silencing complex (RISC).

33. The method of any one of claims 1-32, wherein the one or more miRNAs comprise modifications, wherein the modification is a non-natural intemucleotide linkage, a modified backbone, a substituted sugar moiety, a sugar/backbone modification, an unnatural base pair (UBP), a locked nucleic acid (LNA). a phosphorothioate modification, a cholesterol conjugation, a lipid group conjugation, an antagomir, a 5' and/or a 3’ end modification, or a chemical modification including a 2‘-OMethyl modification, uridylation, N⁶- methyladenosine (m⁶A), 5-methylcytosine (m⁵C), A¹ -methyladenosine (m¹ A), N⁷- methylguanosine (m⁷G), A⁴-acetylcytosine (ac⁴C), pseudouridine ('P), or adenosine-to-inosine (A-to-I).

34. The method of any one of claims 1-33. wherein the administering of the immune checkpoint inhibitor and/or the composition is via oral delivery, rectal delivery, intranasal delivery, injection; infusion; intravascular administration such as intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature; peri- and/or intra-tissue injection such as peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection; subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest; inhalation, or any combination thereof.

35. The method of any one of claims 1-34, wherein the subject is a mammal, optionally wherein the mammal is a human.

36. The method of any one of claims 1-35. wherein the cancer is a cancer cell present in a tissue or an organ.

37. The method of claim 36, wherein the cancer cell is a neoplastic cell.

38. The method of any one of claims 1-37. wherein the cancer is a solid tumor.

39. The method of claim 38, wherein the solid tumor is a breast cancer, a lung cancer, a prostate cancer, a glioma, a melanoma, an ovarian cancer, a colorectal cancer, a glioblastoma, a liver cancer, a pancreatic cancer, a kidney cancer, a bladder cancer, a thyroid cancer, a sarcoma, a stomach cancer, a head and neck cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a thymoma, a soft tissue sarcoma, a bone cancer, a testicular cancer, a penile cancer, a gallbladder cancer, a uterine sarcoma, an adrenal gland cancer, an ampullary cancer, a hepatic angiosarcoma, a nasal, or a paranasal sinus cancer.

40. The method of any one of claims 1-37, wherein the cancer is a blood cancer.

41. The method of claim 40, wherein the blood cancer is a leukemia, a lymphoma, a myeloma, a myelodysplastic syndrome, or a myeloproliferative neoplasm.

42. The method of any one of claims 1-41, wherein the one or more microRNAs comprises at least two to at least 20 miRNAs.

43. An in vitro method of identifying a miRNA that modulates an immune response, the method comprising:

(a) treating cells with an immune checkpoint inhibitor;

(b) obtaining a biopsy sample from a subject, wherein the biopsy sample is a solid tissue, a blood sample, a serum sample, tumor interstitial fluid (TIF), or a plasma sample;

(c) processing the biopsy sample for miRNA detection method; and

(d) detecting a change in miRNA expression relative to a control cell.

44. The method of claim 43, wherein the detection method comprises qRT-PCR, RNA immunoprecipitation, RNA sequencing, RNA-fluorescence in situ hybridization (FISH), single cell genetic and epigenetic analysis, or combinations thereof.

45. A method of diagnosing whether a subject has, or is at risk of developing cancer, the method comprising: (a) measuring the level of at least one miRNAin a test sample from a subject wherein the at least one miRNA is miR-25, miR-17, or miR 92a, and

(b) wherein an alteration in the level of the miRNA in the test sample, relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk of developing cancer.

46. A method of diagnosing whether a subject has, or is at risk of developing cancer, the method comprising:

(a) measuring the level of at least one miRNA in a test sample from a subject, wherein the at least one miRNA is one or more of miR- 106a, and

(b) wherein an alteration in the level of the miRNA in the test sample, relative to the level of a corresponding miRNA in a control sample, is indicative of the subject either having, or being at risk of developing, cancer.

47. The method of any one of claims 1-46, wherein the administering of the immune checkpoint inhibitor and the composition is performed at the same time.

48. The method of any one of claims 1-46. wherein the subject has previously been treated with immune checkpoint inhibitor.

49. The method of any one of claims 1-48. wherein the immune checkpoint inhibitor is an inhibitor of PD-1. PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM. VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4, or TGF-beta receptor.

50. The method of claim 49, wherein the inhibitor of PD-1 is nivolumab, pembrolizumab, or pidilizumab.

51. The method of claim 49, wherein the inhibitor of PD-L1 is atezolizumab, avelumab, or durvalumab.

52. The method of claim 49, wherein the inhibitor of CTLA-4 is ipilimumab or tremelimumab.

53. The method of claim 49, wherein the inhibitor of LAG-3 is relatlimab.

54. The method of claim 49, wherein the inhibitor of CEACAM is CM24.

55. The method of claim 49, wherein the inhibitor of VISTA is CA-170, VISTA-IN-3, or VISA-IN-2M.

56. The method of claim 49, wherein the inhibitor of BTLA is INBRX-106, PF- 04518600, cudarolimab, or HFB200603.

57. The method of claim 49, wherein the inhibitor of TIGIT is Vibostolimab, Etigilimab, Domvanalimab, Ociperlimab, or Tiragolumab.

58. The method of claim 49, wherein the inhibitor of TIM-3 is sym023, cobolimab, sabatolimab, INCAGN2390, BMS-986258, SHR-1702, RO7121661, or LY3321367.

59. The method of claim 49, wherein the inhibitor of TGF-0 receptor is GW-788388. LY2109761, galunisertib, SB-431542, or repsox.

60. A method of restoring expression of one or more miRNA within a mammal, wherein the method comprises administering a composition of claim 26 to a mammal identified as having a cancer, wherein the expression of one or more miRNAs is restored relative to a non-cancerous tissue.

61. The method of claim 60, wherein the one or more miRNAs is miR-29a or miR150.

62. The method of either claim 60 or 61, wherein the composition is delivered as a recombinant plasmid, an overexpression plasmid construct, a viral vector, non- viral vector, a RISC, crRNA. or an antisense oligomer.

63. The method of claim 62, wherein the viral vector is an AAV, a lentivirus, an adenovirus, an adeno-associated virus, retrovirus, or a herpes simplex virus.

64. The method of claim 62, wherein the non-viral vector is a liposome, exosome, an extracellular vesicle, a polymer, a nanoparticle, a peptide, or a dendrimer.