Cancer treatment using siRNA to modulate PRDM2 / RIZ protein expression

Abstract

It is an inhibitory RNA molecule that specifically inhibits mammalian RIZ2 expression and has therapeutic effects on cell proliferative diseases such as cancer.

Description

Background Art

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 024,624, filed May 14, 2020, which is hereby incorporated by reference in its entirety.

[0002] Cancer diseases remain a major health concern worldwide as the second leading cause of death. According to the World Health Organization (WHO), nearly 10 million people are estimated to die from cancer worldwide in 2020, including 2.26 million new cases of breast cancer and then 2.21 million cases of lung cancer. Small interfering RNA (siRNA) is a novel and emerging therapeutic agent that has shown promising results in several diseases. Lymphoma is a cancer that originates from lymphocytes. T-cell lymphoma (TCL) is a lymphoma that originates from T cells, which account for approximately 7% of all non-Hodgkin lymphomas in the United States. Common subtypes of TCL include the following. Peripheral T-cell lymphoma not otherwise specified (PTCLNOS), anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), and cutaneous T-cell lymphoma (CTCL). Each type of TCL has its own unique medical conditions and symptoms. Small interfering RNA (siRNA) is a novel and emerging therapeutic agent that has shown promising results in several diseases. Considering the complexity and severity of cancer, there is a continuing need for the development of improved and precisely targeted therapies.

Summary of the Invention

[0003] Disclosed herein is a method of inhibiting cell proliferation, the method comprising contacting a population of cells with a composition comprising a retinoblastoma protein-interacting zinc finger protein 2 (RIZ2) inhibitor. In one embodiment, the RIZ2 inhibitor decreases the expression of RIZ2 in the cells. In one embodiment, the RIZ2 inhibitor decreases the expression of RIZ2 mRNA in the cells. In one embodiment, the RIZ2 inhibitor decreases the expression of RIZ2 protein in the cells.

[0004] In some embodiments, RIZ2 mRNA is reduced by at least 10% in cells compared to the expression of housekeeping genes. In some embodiments, RIZ2 mRNA is reduced by at least 10% compared to a control cell population. In some embodiments, the control cell population is a population of cells that have not been in contact with a composition containing the RIZ2 inhibitor.

[0005] In some embodiments, RIZ2 mRNA expression is reduced by at least 50% compared to the control cell population.

[0006] In some embodiments, RIZ2 mRNA expression is reduced by at least 80% compared to the control cell population.

[0007] In one embodiment, the RIZ2 inhibitor further increases RIZ1 expression in a cell population. In some embodiments, the composition alters the ratio of RIZ1 mRNA expression levels to RIZ2 mRNA expression levels. In some embodiments, the composition alters the ratio of RIZ1 protein expression levels to RIZ2 protein expression levels. In some embodiments, the RIZ2 inhibitor alters the ratio of RIZ1 to RIZ2 mRNA expression levels to at least 1.1 times.

[0008] In some embodiments, the RIZ2 inhibitor alters the ratio of RIZ1 to RIZ2 mRNA expression levels by at least 1.5 times. In some embodiments, the RIZ2 inhibitor alters the ratio of RIZ1 to RIZ2 mRNA expression levels by at least 2 times.

[0009] In one embodiment, the RIZ2 inhibitor further reduces the hypermethylation of RIZ1.

[0010] In some embodiments of the methods described herein, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are cancer cells.

[0011] In some embodiments of the methods described herein, the cells are derived from tissues such as breast, colon, endometrium, esophagus, stomach, glioma, kidney, liver, lung, lymphoma, melanoma, meningioma, myeloma, nasopharynx, neuroblastoma, ovary, pancreas, parathyroid gland, pituitary gland, prostate, thyroid gland, or uterine tissue. In some embodiments, the cancer cells are derived from hematological malignancies.

[0012] In some embodiments, the cancer cells originate from solid tumors.

[0013] In some embodiments, contact involves contacting a population of cells with a composition containing a RIZ2 inhibitor. In some embodiments, contact results in a reduction of at least 10% of the cell population. In some embodiments, contact results in a reduction of about 90% of the cell population.

[0014] In some embodiments, inhibiting cell proliferation includes reducing the cell mass by at least 10%. In some embodiments, inhibiting cell proliferation includes reducing the cell mass by about 50%.

[0015] In one embodiment of the method disclosed herein, contacting cells further includes incorporating a RIZ2 inhibitor into the cells.

[0016] In one embodiment, the RIZ2 inhibitor comprises a non-natural compound. In one embodiment, the RIZ2 inhibitor is a synthetic drug. In one embodiment, the RIZ2 inhibitor comprises a peptide. In one embodiment, the RIZ2 inhibitor comprises a complexed polypeptide.

[0017] In one embodiment, the RIZ2 inhibitor comprises one or more polynucleotides.

[0018] In some embodiments, the RIZ2 inhibitor comprises a synthetic polynucleotide. In some embodiments, one or more polynucleotides comprise one or more modified nucleotides. In some embodiments, the composition comprising the RIZ2 inhibitor comprises one or more polynucleotides bound to a delivery system.

[0019] In one embodiment, the delivery system comprises one or more of the following: lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, or combinations thereof.

[0020] In some embodiments, the delivery system includes lipids. In some embodiments, the delivery system includes PEGylated lipids. In some embodiments, the delivery system includes a cell targeting portion. In some embodiments, the cell targeting portion is cell type specific. In some embodiments, the cell targeting portion is a ligand that selectively binds to a receptor on a target cell. In some embodiments, the cell targeting portion is an antibody that binds to a cell surface molecule on a target cell. In some embodiments, the cell targeting portion is a single-chain antibody or antibody fragment. In some embodiments, the cell targeting portion is a peptide. In some embodiments, the cell targeting portion is a cell-permeable peptide. In some embodiments, the cell targeting portion is a cyclic peptide. In some embodiments, the cell targeting portion is an aptamer. In some embodiments, the delivery system includes liposomes. In some embodiments, the delivery system includes nanoparticles. In some embodiments, the RIZ2 inhibitor includes an RNA molecule. In some embodiments, the RIZ2 inhibitor includes an inhibitory RNA molecule.

[0021] In one embodiment, the inhibitory RNA molecule disclosed herein hybridizes to at least 10 adjacent nucleic acid bases on the PRDM2 gene or PRDM2 gene product. In some embodiments, the inhibitory RNA is about 10 to 21 nucleotides in length. In some embodiments, the inhibitory RNA molecule is double-stranded. In some embodiments, the RNA molecule contains one or more modified nucleotides.

[0022] In some embodiments of the methods disclosed herein, the RIZ2 inhibitor comprises an inhibitory double-stranded RNA comprising the sequence of SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments of the methods disclosed herein, the RIZ2 inhibitor comprises an inhibitory double-stranded RNA comprising the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 10.

[0023] In some embodiments of the methods disclosed herein, the RIZ2 inhibitor comprises an inhibitory double-stranded RNA comprising the sequence of SEQ ID NO: 6 or a sequence having at least 90% identity to SEQ ID NO: 6. In some embodiments of the methods disclosed herein, the RIZ2 inhibitor comprises an inhibitory double-stranded RNA comprising the sequence of SEQ ID NO: 6 or a sequence of SEQ ID NO: 11.

[0024] This specification discloses a method for treating a cell proliferation disorder or impairment in a subject, comprising administering a composition comprising a RIZ2 inhibitor to the subject.

[0025] This specification discloses a method for selecting a patient as a subject requiring administration of a composition containing a RIZ2 inhibitor, comprising (i) detecting an increase in the RIZ1 methylation level in the subject's biological sample compared to a control sample or control value, or (ii) detecting a decrease in the RIZ1 mRNA level compared to a control sample or control value, wherein the subject is determined to require administration of a composition containing a RIZ2 inhibitor if the biological sample of the subject shows at least a 1.1-fold increase in methylated RIZ1 or at least a 1 / 2 decrease in the RIZ1 mRNA level compared to a control sample or control value, and the control sample is a sample from a clinically healthy individual, and the control value is the average value of RIZ1 methylation levels from samples of two or more clinically healthy individuals.

[0026] In some embodiments of the methods disclosed herein, the proliferative disorder or impairment is cancer.

[0027] In some embodiments, the cancer is cancer of the breast, colon, endometrium, esophagus, stomach, glioma, kidney, liver, lung, lymphoma, melanoma, meningioma, myeloma, nasopharynx, neuroblastoma, ovary, pancreas, parathyroid, pituitary, prostate, thyroid, or uterine tissue.

[0028] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer.

[0029] In some embodiments of the methods disclosed herein, administering comprises administering an effective amount of an RIZ2 inhibitor.

[0030] In some embodiments, the effective amount of the RIZ2 inhibitor is an amount that reduces at least one parameter of a disease associated with a cell proliferative disorder.

[0031] In some embodiments, the cell proliferative disease is cancer and the effective amount of RIZ2 reduces the number of cancer cells. In some embodiments, the cell proliferative disease is a solid tumor and the effective amount of RIZ2 reduces the tumor mass.

[0032] In some embodiments, the composition comprising the RIZ2 inhibitor comprises one or more polynucleotides conjugated to a delivery system. In some embodiments, the one or more polynucleotides comprise inhibitory RNA molecules. In some embodiments, the inhibitory RNA molecule is a double-stranded RNA molecule comprising at least 90% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments, the inhibitory RNA molecule is a double-stranded RNA molecule comprising at least 90% sequence identity to the sequence of SEQ ID NO: 6.

[0033] In some embodiments, the inhibitory RNA molecule is a double-stranded RNA molecule comprising at least 90% sequence identity to any one of the sequences listed in Table 2.

[0034] In some embodiments, the composition further comprises a delivery system. In some embodiments, the delivery system comprises a lipid and / or a cell-targeting moiety. In some embodiments, the lipid is a PEGylated lipid. In some embodiments, the cell-targeting moiety is a cell-permeable peptide, a cyclic peptide, an antibody or a fragment thereof, or an aptamer.

[0035] Furthermore, this specification discloses pharmaceutical compositions comprising (a) a RIZ2 inhibitor and (b) a pharmaceutically acceptable excipient. In some embodiments of the pharmaceutical compositions disclosed herein, the RIZ2 inhibitor comprises one or more polynucleotide molecules and a delivery system. In some embodiments, the RIZ2 inhibitor comprises an inhibitory RNA molecule. In some embodiments, the inhibitory RNA molecule is hybridized to at least 10 adjacent nucleic acid bases on the PRDM2 gene or PRDM2 gene product.

[0036] In some embodiments, the inhibitory RNA molecule is about 10 to 21 nucleotides long. In some embodiments, the inhibitory RNA molecule is double-stranded. In some embodiments, the inhibitory double-stranded RNA contains the sequence of SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the inhibitory double-stranded RNA contains the sequence of SEQ ID NO: 13 or a sequence having at least 90% identity to SEQ ID NO: 13. In some embodiments, the inhibitory double-stranded RNA contains the sequence of SEQ ID NO: 13 or a sequence having at least 90% identity to SEQ ID NO: 14. In some embodiments, the delivery system includes a cell-targeting portion. In some embodiments, the cell-targeting portion is a ligand and an antibody or antibody fragment, a single-chain antibody, a peptide or aptamer. In some embodiments, it is a cell-permeable peptide.

[0037] In some embodiments, the delivery system comprises one or more of the following: lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, or combinations thereof. In some embodiments, the RIZ2 inhibitor comprises an siRNA that (i) contains a 10-21 nucleotide double-stranded RNA homologous to the human PRDM2 / RIZ2 gene, (ii) is covalently bound to a PEG molecule, and (iii) is bound to a cell target moiety, the cell target moiety being an aptamer. In some embodiments, the delivery system comprises liposomes. In some embodiments, the delivery system comprises nanoparticles. In some embodiments, the nanoparticles comprise lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, and combinations thereof. In some embodiments, the nanoparticles comprise a calcium phosphosilicate complex. In some embodiments, the pharmaceutical composition further comprises a calcium phosphosilicate complex conjugated with the RIZ2 inhibitor. In some embodiments, the pharmaceutical composition further comprises one or more chemotherapeutic agents.

[0038] Further features of any of the aforementioned multifunctional molecules, nucleic acids, vectors, host cells, or methods include one or more of the embodiments listed below.

[0039] Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein, or can confirm them by conventional experimentation alone. Such equivalents are intended to be encompassed by the embodiments listed below.

[0040] Further features of any of the aforementioned multifunctional molecules, nucleic acids, vectors, host cells, or methods include one or more of the embodiments listed below. [Brief explanation of the drawing]

[0041] [Figure 1]

[0041] Figure 1 shows an example of one embodiment of the present invention, comprising a drug delivery system containing an siRNA payload complementary to RIZ mRNA. In this example, the drug delivery system is PEGylated and carries a target peptide that specifically directs the payload to cancer cells or cancer stem cells. To illustrate that the drug delivery system may contain one or more additional payloads, nanoparticles are shown containing a combination of small or large molecules in addition to the anti-RIZ siRNA. Such additional payloads may be currently approved chemotherapeutic agents, repurposed drugs, or any drug that effectively helps to kill cancer cells or control the growth or metastasis of cancer cells. [Figure 2]

[0042] Figure 2 shows the survival rate of human lung cancer cells (cell line A549) after exposure to siRNA directed at PRDM2. [Figure 3]

[0043] Figure 3 shows the RIZ1 mRNA and RIZ2 mRNA levels in A549 lung cancer cells after exposure to ARIZ-011. [Figure 4]

[0044] Figure 4 shows the survival rates of human colon cancer cells (cell line HCT116) and normal colon cells (cell line CCD112) after exposure to ARIZ-011. [Figure 5]

[0045] Figure 5 shows the RIZ1 mRNA levels and RIZ2 mRNA levels in HCT116 cancer cells after exposure to ARIZ-011. [Figure 6]

[0046] Figure 6 shows the survival rates of human lung cancer cells (cell line A549) and normal epithelial cells (cell line CCD112) after exposure to ARIZ-047 (with or without cisplatin). [Figure 7]

[0047] Figure 7 shows the RIZ1 mRNA levels and RIZ2 mRNA levels in A549 lung cancer cells after exposure to ARIZ-047. [Figure 8]

[0048] Figure 8 shows the survival rate of multiple myeloma cells KMS11 after cell treatment with ARIZ-011. [Figure 9]

[0049] Figure 9 shows a c(RGDfk)-PEG-MAL-siRNA construct that self-assembles to form a nanoparticle drug delivery system targeting the integrin αvβ3 receptor. [Figure 10]

[0050] Figure 10 shows illustrative data illustrating tumor regression in an A549 lung cancer xenograft model. Nanoparticle-delivered siRNA (ARIZ-047) showed the most promising effect in controlling tumor growth.

[0042]

[0051] The numbered accompanying drawings in this specification are provided for illustrative purposes only and are not intended to limit the scope in any way. [Modes for carrying out the invention]

[0043]

[0052] This disclosure is based on the important finding that the balance between RIZ1 and RIZ2 levels in mammalian cells is a determinant of a stable, normal cellular life cycle, and that imbalance leads to changes in the cell cycle, which can result in hyperproliferative disorders such as hyperplasia or cancer. This disclosure is based on a further groundbreaking development: RIZ2 can be specifically targeted to address this imbalance and thereby prevent or improve hyperproliferative disorders such as cancer.

[0044]

[0053] Current chemotherapy treatments have limited effectiveness. While commonly used anticancer drugs can provide temporary remission of tumors and potentially prolong the patient's life, they are generally not effective in treating cancer because they often do not completely eliminate it, leading to recurrence. Small molecule chemotherapy is currently the most widely used treatment for cancer, but its effects are largely nonspecific.

[0045]

[0054] Chemotherapy drugs have serious nonspecific toxic effects, causing incidental damage not only to cancer cells that are intended to be controlled and destroyed, but also to normal, healthy cells and tissues. As a result, patients suffer severe side effects due to toxicity. Immunotherapy was expected to have lower toxicity and improve survival rates, but despite this expectation, the prospects for cancer treatment and good long-term patient outcomes have only improved incrementally. Targeted therapies and immunotherapies are increasingly used as supplements or alternatives to conventional chemotherapy, but such therapies generally lack long-term efficacy because cancer commonly adapts, rapidly develops resistance, and evades the effects of targeted therapy. For the reasons mentioned above, it is rare for chemotherapy, immunotherapy, and targeted therapy to be effective in curing cancer. Cancer may be curable by addressing the root cause, for example, by overcoming the underlying genetic abnormalities that cause cells to become cancerous in the first place and give rise to this devastating disease. This approach may involve regulating the expression of proteins that act as key regulators, suppressing tumorigenesis in normal cells but becoming driving factors for cancer cell formation when abnormally expressed.

[0046]

[0055] Of particular interest is cancer therapy that focuses on altering the expression of PRDM class tumor suppressor genes. PRDM is a family of genes and proteins that control cell growth, proliferation, survival, and mobility. Studies have shown that alterations in PRDM expression and activity are typically among the initial changes in normal cells that lead to cancer cell formation. Abnormal expression of PRDM genes is strongly involved in a wide variety of cancer types. PRDM is associated as a causative gene and causative protein in solid tumors such as breast, colon, stomach, liver, lung, melanoma, prostate, and other cancers, as well as in hematological malignancies such as leukemia, lymphoma, and myeloma.

[0047] definition

[0056] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art of the field to which this invention pertains. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, unless otherwise specified, the following terms have the meanings set forth below. Where used herein, terms are intended to describe specific cases only and are not intended to limit them. Where used herein, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise specified by the context. Furthermore, to the extent that the terms “including,” “include,” “having,” “has,” “with,” or variations thereof are used in either the detailed description of the invention and / or the claims, such terms are intended to be as comprehensive as the term “comprising.”Naturally, terms such as “comprise,” “comprised,” and “comprising” may have meanings attributable to them in U.S. patent law, for example, these terms can mean “include,” “included,” and “including,” and terms such as “consisting essentially of” and “consist essentially of” also have meanings attributable to them in U.S. patent law, for example, these terms may include elements not explicitly listed, but exclude elements found in the prior art or elements that affect the fundamental or novel features of the present invention. Nothing in this specification is intended as a warranty.

[0048]

[0057] "Drug" means any small molecule compound, antibody, nucleic acid molecule, polypeptide, or fragment thereof.

[0049]

[0058] The terms “polynucleotide” and “nucleic acid” are used herein without distinction and refer to polymers of nucleotides of any length, including DNA and RNA, such as mRNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA polymerase or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid may be mRNA transcribed in vitro. In some embodiments, the polynucleotide administered using the method of the present invention is mRNA.

[0050]

[0059] In the context of two or more nucleic acids or polypeptides, the term “identical” or “matching” rate refers to two or more sequences or subsequences that are identical or have a specific proportion of identical nucleotide or amino acid residues when compared and aligned (with gaps introduced as necessary) to the maximum extent possible without considering any conserved amino acid substitutions as part of sequence identity. The matching rate can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid sequences or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variations. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity when compared and aligned to the maximum extent possible, as measured using sequence comparison algorithms or by visual inspection.

[0051]

[0060] As used herein, cell proliferative disorders or hyperproliferative disorders are diseases or disorders associated with increased cell proliferation, most often uncontrolled cell proliferation, resulting from the cessation of function of one or more cell cycle-related genes. Exemplary hyperproliferative disorders are neoplasia and cancer. The cell cycle is the periodic interval between two consecutive G0 / G1 phases (or two consecutive G1 / M phases) in the life cycle of a cell, which normally remains constant in cell type under optimal environmental conditions. Cell cycle abnormalities can be disorders that disrupt the normal periodic interval between two consecutive G0 / G1 phases (or two consecutive G1 / M phases) in the life cycle of a cell, which may generally be caused by the disruption of cell cycle regulators. An example cell cycle regulator is cyclin D. In some embodiments, RIZ1 and / or RIZ2 may be considered cell cycle regulators. In some embodiments, RIZ1 and / or RIZ2 may be considered major regulators of the cell cycle in that they ubiquitously control the life cycle, or in that they control milestones in the cell's life cycle regardless of cell type, location, or environmental conditions.

[0061] Hyperplasia can be defined as a state of rapid cell proliferation, for example, as a result of disruption of cell cycle regulators, such as checkpoint inhibitors. Neoplasia is a state in which cells have undergone neoplastic transformation, such as malignant transformation into cancer.

[0052]

[0062] RNA interference and siRNA. RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by small interfering RNAs (siRNAs) (Zamore et al, 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Patent Publication, International Publication No. 99 / 61631) is usually called post-transcriptional gene silencing or RNA silencing, and in fungi it is also called quelling. The presence of long dsRNA in cells stimulates the activity of a ribonuclease III enzyme called Dicer (Bass, 2000, Cell, 101, 235; Zamore et al, 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in processing dsRNA into shorter fragments known as small interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Small interfering RNAs derived from dicers activity are typically about 21–23 nucleotides long and contain a double-stranded body of about 19 base pairs (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicers are also involved in the removal of small transient RNAs (stRNAs), which are 21 and 22 nucleotides long, from precursor RNAs of conserved structures involved in translational regulation (Hutvagner et al., 2001, Science, 293, 834).RNAi reactions are also characterized by an endonuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA double-stranded body. The cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA double-stranded body (Elbashir et al., 2001, Genes Dev., 15, 188).

[0053]

[0063] The term "inhibition" is often used interchangeably with "reduction" or "reduction in expression levels," but it refers to a decrease in the level of a particular component being inhibited, which may be partial (e.g., 20%, 50%, 75%, 80%) or complete (about 100%). For example, a RIZ2 inhibitor may inhibit the RIZ2 protein in cells by about 50% or about 100%.

[0054]

[0064] When used in the context of disease, injury, or disability, the terms “treatment,” “to treat,” “to alleviate,” etc., are used herein to generally mean the attainment of a desired pharmacological and / or physiological effect, and may also be used to mean improving, alleviating, and / or reducing the severity of one or more symptoms of the condition being treated. Such effect may be preventive in that it completely or partially delays the onset or recurrence of the disease, condition, or its symptoms, and / or therapeutic in that it results in a partial or complete cure of the disease or symptoms and / or adverse effects caused by such disease or symptoms. As used herein, “treatment” encompasses all treatments of diseases or symptoms in mammals, particularly humans, and includes: (a) preventing the onset of the disease or symptoms in a subject that may be predisposed to the disease or symptoms but has not yet been diagnosed with such disease or symptoms; (b) inhibiting the disease or symptoms (e.g., preventing its onset); or (c) alleviating the disease or symptoms (e.g., causing regression of the disease or symptoms, resulting in improvement of one or more symptoms).

[0055] PRDM genes and ecology

[0065] This disclosure relates to methods and compositions for regulating the expression and function of one or more PRDM genes, such as the PRDM2 gene. Generally, PR / SET ([Su(var)3-9, Enhancer of Zeste, and Trithorax]) domain 2 (PRDM2) defines a large group of histone methyltransferases (HMTs), and belongs to the PRDM (positive regulatory domain) gene family, a subfamily of Kruppel-like zinc finger gene products, which currently includes 19 members in humans. PR domains have protein-binding interfaces, and some of them can accommodate the universal methyl donor S-adenosylmethionine (SAM) and thus perform function as lysine methyltransferases (KMTs). The PRDM (PRDI-BF1 and RIZ homologous domain-containing) protein family members belong to the histone / protein methyltransferase (PRDM) superfamily, but these members are characterized by a conserved N-terminal PR domain with methyltransferase activity and a zinc finger array at the C-terminus. Members of this class are characterized by the presence of a PR domain and a different number of Zn fingers. Experimental evidence shows that PRDM proteins play a crucial role in gene expression regulation, altering chromatin structure directly, via methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins function as transcription factors, controlling the expression of a vast number of other genes involved in developmental processes including growth, differentiation, proliferation, mobility, and survival. Therefore, PRDM proteins are major transcriptional regulators that drive and maintain cellular state transitions in response to developmental signals. While some PRDM proteins normally function as tumor suppressors, dysregulation of their expression, or abnormal expression of specific isoforms, can lead to changes that drive the development and progression of cancer. Many cancer types are strongly associated with dysfunction of specific PRDM proteins.In these tumors, the expression of normal PRDM protein may be abnormally low. PRDM protein has a dual function: mediating effects induced by different cellular signals, such as steroid hormones, and regulating the expression of growth factors. Therefore, PRDM protein plays a central role in the transmission of signals that control cell proliferation and differentiation, and consequently, neoplastic transformation (Zazzo et al., Biology (Basel). 2013 Mar;2(1):107-141). The PR domain shares high homology with catalytic SET (Su(var)3-9 (Suppressor of variegation 3-9), Enhancer of zeste, and Trithorax) domains that define the histone methyltransferase group (Xiao B. et al., Curr. Opin. Struct. Biol. 2003;13:699-705). The human genome contains 17 genes encoding PR / SET proteins, all of which, except PRDM11, have varying numbers of Zn finger domains (Fumasoni I. et al., BMC Evol. Biol. 2007;7:187). PRDM proteins play a central role in signaling that controls cell proliferation and differentiation, and consequently, neoplastic transformation (Fog CK et al., BioEssays. 2011;34:50-60). A common characteristic of PRDM family genes is their expression in different molecular forms, either through alternative splicing or the action of different promoters. Furthermore, some genes in this family are expressed in two selective forms: one lacking the PR domain (PR-minus), but otherwise identical to the other PR-containing product (PR-plus) (PRDM1, PRDM2, PRDM3, PRDM16) [(Gyory et al., J.Immunol, 2003, 170:3125-3133), (Liu L., et al., J.Biol.Chem. 1997, 272:2984-2991)].Other genes encode proteins that differ in whether or not they have a Zn-finger domain (PRDM6, PRDM9).

[0056]

[0066] PRDM1 and PRDM2 were initially identified as Blimp-1 (B lymphocyte-induced maturation protein-1) and RIZ (retinoblastoma-interacting zinc finger protein), respectively, but have two promoters encoding PR-plus and PR-minus isoforms. The PRDM1 promoter is located upstream of exon 1 and exon 4, respectively. These transcription start sites in the following two promoters guide the following: PRDI-BF1 (positive regulatory domain I binding factor 1)α (PR-plus)e and PRDI-BF1β (PR-minus), differing only in the presence of a PR domain. Similar to PRDM1, PRDM2 expresses two proteins, PRDM2a / RIZ1 (PR-plus) and PRDM2b / RIZ2 (PR-minus), through differential transcription initiated by the two promoters. One promoter of PRDM2 is located upstream of the open reading frame in the region containing exon 1a, while the second promoter is located within the range of intron 5 and exon 6. PRDM2 was initially identified as a retinoblastoma-interacting zinc finger protein (RIZ) and a GATA-3 binding protein through functional screening of human cDNA libraries in independent studies (Sorrentino, A., et al., 2018, Volume 1861, Issue 7, Pages 657-671). Subsequently, a PRDM2 variant was isolated from a cDNA expression library of human monocytic leukemia cell lines, which was named the MTE-binding protein zinc finger type (MTB-ZF).

[0057] [Table 1-1]

[0058] [Table 1-2]

[0059]

[0068] To date, enzyme activity has only been experimentally demonstrated for a few family members: PRDM9 (towards H3K4me3, H3K9me1 / 3, H3K18me1, H3K36me3, and H4K20me1 / 2), PRDM2, PRDM3, and PRDM16 (H3K9me1), and PRDM8 (H3K9me3) (Xie, M. et al., J. Biol. Chem., 1997, 272:42, 2636-26366). Furthermore, the activity of PRDM6 has been reported, although the nature of this activity requires further elucidation.

[0060]

[0069] PRDM2, or retinoblastoma-binding protein (RIZ), encodes a red blood cell binding protein and the PRDI-BF1 / BLIMP1 transcriptional repressor, which promotes B lymphocyte maturation. RIZ can have two isoforms: PRDM2a (RIZ1) and PRDM2b (RIZ2). Both proteins are widely expressed in mammalian cells. RIZ1 is identical to RIZ2, except for the presence of an additional 201 residues at the amino terminus, which in RIZ1 contains a PR domain. These additional residues in RIZ1 are known to confer specific functions to the RIZ1 protein. An internal promoter generates RIZ2, which lacks the RIZ1 PR domain. This PR domain represents the major functional motif within the RIZ1 amino-terminal region. The PR domain is a derivative of the SET domain and can function as a protein-binding interface in chromatin-mediated gene expression regulation.

[0061]

[0070] The following is an example of a sequence of the N-terminal region containing a PR domain within RIZ1, which is absent in RIZ2, with the presumed PR domain shown in bold:

[0062] [Table A]

[0063]

[0072] RIZ2 is characterized by the absence of the segment indicated as sequence number 19, which is present in RIZ1.

[0064]

[0073] An example RIZ2 protein sequence is provided below. MRDSAEGPKEDEEKPSASALEQPATLQEVASQEVPPELATPAPAWEPQ PEPDERLEAAACEVNDLGEEEEEEEEEDEEEEEDDDDDELEDEGEEEAS MPNENSVKEPEIRCDEKPEDLLEEPKTTSEETLEDCSEVTPAMQIPRTKE EANGDVFETFMFPCQHCERKFTTKQGLERHMHIHISTVNHAFKCKYCG KAFGTQINRRRRHERRHEAGLKRKPSQTLQPSEDLADGKASGENVASKD DSSPPSLGPDCLIMNSEKASQDTINSSVVEENGEVKELHPCKYCKKVFG THTNMRRHQRRVHERHLIPKGVRRKGGLEEPQPPAEQAQATQNVYVP STEPEEEGEADDVYIMDISSNISENLNYYIDGKIQTNNNTSNCCDVIEMES ASADLYGINCLLTPVTVEITQNIKTTQVPVTEDLPKEPLGSTNSEAKKRR TASPPALPKIKAETDSDPMVPSCSLSLPLSISTTEAVSFHKEKSVYLSSKL KQLLQTQDKLTPAGISATEIAKLGPVCVSAPASMLPVTSSRFKRRTSSPP SSPQHSPALRDFGKPSDGKAAWTDAGLTSKKSKLESHSDSPAWSLSGRD ERETVSPPCFDEYKMSKEWTASSAFSSVCNQQPLDLSSGVKQKAEGTGK TPVQWESVLDLSVHKKHCSDSEGKEFKESHSVQPTCSAVKKRKPTTCML QKVLLNEYNGIDLPVENPADGTRSPSPCKSLEAQPDPDLGPGSGFPAPTV ESTPDVCPSSPALQTPSLSSGQLPPLLIPTDPSSPPPCPPVLTVATPPPPLLPT VPLPAPSSSASPHPCPSPLSNATAQSPLPILSPTVSPSPSPIPPVEPLMSAASP GPPTLSSSSSSSSSSSSFSSSSSSSSPSPPPLSAISSVVSSGDNLEASLPMISFK QEELENEGLKPREEPQSAAEQDVVVQETFNKNFVCNVCESPFLSIKDLTK HLSIHAEEWPFKCEFCVQLFKDKTDLSEHRFLLHGVGNIFVCSVCKKEFA FLCNLQQHQRDLHPDKVCTHHEFESGTLRPQNFTDPSKAHVEHMQSLPE DPLETSKEEEELNDSSEELYTTIKIMASGIKTKDPDVRLGLNQHYPSFKPPP FQYHHRNPMGIGVTATNFTTHNIPQTFTTAIRCTKCGKGVDNMPELHKHI LACASASDKKRYTPKKNPVPLKQTVQPKNGVVVLDNSGKNAFRRMGQP KRLNFSVELSKMSSNKLKLNALKKKNQLVQKAILQKNKSAKQKADLKN ACESSSHICPYCNREFTYIGSLNKHAAFSCPKKPLSPPKKKVSHSSKKGGH SSPASSDKNSNSNHRRRTADAEIKMQSMQTPLGKTRARSSGPTQVPLPSSS FRSKQNVKFAASVKSKKPSSSSLRNSSPIRMAKITHVEGKKPKAVAKNHS AQLSSKTSRSLHVRVQKSKAVLQSKSTLASKKRTDRFNIKSRERSGPVVT RSLQLAAAADLSENKREDGSAKQELKDFRNFL (Sequence ID 20).

[0065]

[0074] The sequence of sequence number 20 aligns perfectly with RIZ1 starting from amino acid residue 202 for RIZ1. As shown below, the query sequence is RIZ1 and the subject sequence is RIZ2. For example, PRDM2 / RIZ1 is the sequence of NM_012231. For example, PRDM2 / RIZ2 is the sequence of NM_001007257. Sequence NM_012231 is an exemplary human RIZ1 sequence. Sequence NM_001007257 is an exemplary human RIZ2 sequence.

[0066] [Table B-1]

[0067] [Table B-2]

[0068]

[0075] Both RIZ1 and RIZ2 bind to Sp-1-like DNA elements with high GC content and repress the transcription of the early promoter of Simianvirus 40, but RIZ1 is a stronger repressor than RIZ2, suggesting that the PR domain of RIZ1 regulates transcription (Huang, S., J. Biol. Chem., Vol. 273, No. 26, Issue of June 26, pp. 15933-15939).

[0069]

[0076] While the RIZ1 protein is expressed in normal tissues, decreased or absent RIZ1 expression levels and / or increased RIZ2 expression levels are frequently detected in many human malignant tissues and cancer cell lines. As has been proposed for other PRDM proteins, this "yin and yang" imbalance in the amounts of the two protein products may be important for neoplastic transformation. Furthermore, exclusive negative selection of RIZ1 over RIZ2 (specifically genetically or epigenetically mediated decrease in expression) appears to be a common feature across various human cancers. This finding suggests that RIZ1 may possess tumor suppressor activity, and that RIZ2 is required for tumorigenesis by promoting cell proliferation through its cell division activity. The putative endogenous pro-proliferative oncogenicity of RIZ2 is associated with the first cluster (residues 359-507) of Zn finger domains present in both RIZ1 and RIZ2. Indeed, stably transfected MCF7 cells expressing this cluster of Zn fingers showed increased proliferation rates compared to control cells under both estrogen-deficient and estrogen-stimulated conditions, as well as higher expression levels of cyclin D1 and A and reduced responsiveness to anti-estrogen growth inhibitory effects. Furthermore, via proteomic mass spectrometry-based methods, forced expression of the PRDM2 gene Zn finger domain in MCF-7 cells correlated with the differentiation expression of proteins associated with various types of cancer or involved in cell proliferation and differentiation, such as keratin K8, the glycolytic enzyme α-enolase, the acid protease cathepsin D, and nucleoside diphosphate kinase A. Changes in the expression of these proteins may contribute to the observed elevated proliferation rates. The oncogenic validity of RIZ2 is likely inhibited by the presence of the PR domain in the RIZ1 protein. The significant functional differences in chromatin regulation between RIZ1 and RIZ2 are attributed to the PR domain that confers specific functions to RIZ1, but this may be due to their opposing roles in tumorigenesis.

[0070]

[0077] The imbalance in the amounts of RIZ1 and RIZ2, where the PR-positive isoform is typically lost or downregulated, and the PR-negative isoform is always present at higher levels in cancer cells, can be a significant cause of malignancy. Interestingly, the RIZ1 isoform also implies a key target of estradiol action downstream of its interaction with hormone receptors. Furthermore, the imbalance between these two products may also be the molecular basis for other human diseases. In one embodiment of the present invention shown in Figure 1, the drug delivery vehicle comprises a protective layer (e.g., liposome PEGylation) to extend the drug lifetime in the bloodstream and shield the drug delivery system from destruction by the immune system. In one embodiment, the drug delivery system is specifically directed to cancer cells via ligands that target receptors or other parts on the surface of tumor cells or cancer stem cells. The ligands may be proteins, peptides, or other classes of molecules that have the ability to bind to target cancer cells with high specificity and affinity. This targeted drug delivery system needs to concentrate the payload at the target site, as well as protect against damage to healthy cells. At the same time, this needs to increase efficacy (with lower drug doses), improve safety, and consequently lead to higher therapeutic indicators.

[0071]

[0078] In other embodiments, the drug delivery vehicle is In addition to inhibitory siRNA, it can carry a payload to enhance the anti-cancer efficacy of drug formulations. Such additional payloads are not limited to, but could include large or small molecule chemotherapeutic agents.

[0072] Regulation of PRDM2 gene expression

[0079] PRDM2 / RIZ1 can be controlled via methylation. In one embodiment, hypermethylation of RIZ2 can be targeted for cancer therapy. For example, one study observed that the frequency of PRDM2 / RIZ1 methylation was approximately 73% in tumors compared to 20% in distant lung tissue (Tan S. et al., Oncotargets and Therapy 2018:11,2991-3002).

[0073]

[0080] The two RIZ isoforms regulate cellular function in a "yin and yang" manner (Di Zazzo, Biology 2016, 5, 54; doi: 10.3390), thereby producing dually favored, opposite responses. Specifically, the RIZ1 protein acts as a tumor suppressor, inhibiting cancer cells in the G2 / M phase of the cell cycle and promoting apoptosis, while the RIZ2 protein acts as a proto-oncogene, promoting cell division. In other words, an imbalance in the levels of RIZ1 and RIZ2 may be a significant cause of cancer progression. This view is supported by laboratory findings that silencing or deregulation of RIZ1 expression, associated with increased RIZ2 expression, has been observed in various human cancers, including hepatocellular carcinoma, leukemia, malignant lymphoma, breast cancer, colorectal cancer, thyroid cancer, and others.

[0074]

[0081] In one embodiment, the present disclosure provides a method for controlling PRDM2 gene expression. In one embodiment, the method can control RIZ2 expression. In some embodiments, the method can be used to reduce RIZ2 expression. In one embodiment, RIZ2 expression is reduced in cells using the method described herein. In some embodiments, the method described herein is used to reduce RIZ2 expression in cells within a mammalian system. In some embodiments, the mammalian system is a human system.

[0075]

[0082] In one embodiment, this specification provides a method for reducing the level of RIZ2 in cells that express RIZ2 abnormally high compared to normal cells, where normal cells are healthy cells or cells taken from a healthy individual, and a healthy individual may be an individual without clinical signs of disease or symptoms. In some embodiments, cells with abnormally high RIZ2 expression are cells with cell cycle dysregulation. In some embodiments, cells exhibiting abnormally high RIZ2 expression are cells exhibiting hyperplasia. In some embodiments, cells with abnormally high RIZ2 expression are cancer cells.

[0076]

[0083] In some embodiments, this specification provides a method for specifically reducing RIZ2 expression in cancer cells or hyperplastic cells, which exhibit abnormally high RIZ2 expression. In some embodiments, cells with abnormally high RIZ2 expression also exhibit decreased RIZ1 expression. In some embodiments, the method provided herein can reduce RIZ2 expression and simultaneously increase RIZ1 expression. In some embodiments, the method provided herein restores the RIZ1-RIZ2 balance in cells. In some embodiments, the method provided herein enables increased RIZ1 expression and decreased RIZ2 expression. In some embodiments, the method provided herein enables increased RIZ1 expression by decreasing RIZ2 expression. In some embodiments, the method provided herein includes contacting cells with a RIZ2 inhibitor.

[0077]

[0084] This specification provides a method for reducing RIZ2 expression and simultaneously increasing RIZ1 expression in cells expressing a higher RIZ2 level than normal cells, wherein the cells expressing a higher RIZ2 level than normal cells also exhibit a lower RIZ1 level than normal cells, the method comprising contacting the cells with a RIZ2 inhibitor described herein. In some embodiments, normal cells are cells exhibiting a normal cell division cycle. In some embodiments, this specification provides a method for restoring the RIZ1-RIZ2 balance in cells. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.1 times. For example, in some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.2 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.3 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.4 times. In some embodiments, the ratio of RIZ1 to RIZ2 mRNA expression level changes to at least 1.5 times. In some embodiments, the ratio of RIZ1 to RIZ2 mRNA expression level changes to at least 1.6 times. In some embodiments, the ratio of RIZ1 to RIZ2 mRNA expression level changes to at least 1.7 times. In some embodiments, the ratio of RIZ1 to RIZ2 mRNA expression level changes to at least 1.8 times. In some embodiments, the ratio of RIZ1 to RIZ2 mRNA expression level changes to at least 1.9 times. In one embodiment, the ratio of RIZ1 to RIZ2 mRNA expression level changes to at least 2 times.

[0078]

[0085] In some embodiments, the method provided herein involves contacting cells with a composition comprising a retinoblastoma protein-interacting zinc finger protein 2 (RIZ2) inhibitor. In some embodiments, the RIZ2 inhibitor is a non-natural drug, such as a synthetic drug. In some embodiments, the RIZ2 inhibitor is a small molecule. In some embodiments, the small molecule is designed to be specific to inhibit RIZ2 and not to inhibit RIZ1. In some embodiments, the RIZ2 inhibitor comprises a peptide. In some embodiments, the RIZ2 inhibitor is a peptide that specifically inhibits RIZ2 in cells and comprises at least about 4 amino acids, at least about 4 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, or at least about 20 amino acids, or more than 20 amino acids. In some embodiments, the RIZ2 inhibitor comprises a complexed polypeptide. In some embodiments, the complexed polypeptide comprises at least one bioactive polypeptide (RIZ2 inhibitor) that can reduce RIZ2 levels and a portion that, when applied in vivo, provides stability, provides a pathway across the cell membrane, or allows the bioactive peptide to be directed to a specific cell, tissue, or organ. In some embodiments, the RIZ2 inhibitor comprises one or more polynucleotides. In some embodiments, the RIZ2 inhibitor comprises synthetic polynucleotides. In some embodiments, the RIZ2 inhibitor comprises synthetic polynucleotides such as siRNAs.

[0079]

[0086] In some embodiments, the method comprises contacting cells with a RIZ2 inhibitor present in an amount sufficient to decrease RIZ2 levels and increase RIZ1 levels in the cells, such that the levels of RIZ2 and RIZ1 are comparable to those of normal cells, or the ratio of RIZ1 to RIZ2 is similar to that of normal cells, i.e., cells with a normal cell growth rate, such as a normal cell cycle.

[0080]

[0087] In some embodiments, the method described herein is a method for inhibiting cell proliferation, comprising contacting a population of cells with a composition comprising a RIZ2 inhibitor.

[0081]

[0088] In some embodiments, when the method described herein is applied to a population of cells, it reduces the cell proliferation rate in the cell population from an overgrowth state to a proliferation rate similar to that of normal, healthy cells.

[0082]

[0089] In some embodiments, the methods described herein, when applied to a population of cells, result in a reduction of the number of cells in the population by at least 10%. In some embodiments, contacting cells with a RIZ2 inhibitor results in a reduction of the number of cells in a population by at least about 15%, or at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.

[0083]

[0090] In some embodiments, contacting cells with a RIZ2 inhibitor results in a reduction of approximately 90% in the number of cells in a population. In some embodiments, inhibiting cell proliferation includes reducing the cell mass by at least 10%. In some embodiments, contacting cells with a RIZ2 inhibitor results in a reduction of at least approximately 15%, or at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, or at least approximately 90%. %.In some embodiments, contacting cells with a RIZ2 inhibitor results in a reduction of the cell mass of a population of cells by at least about 15%, or at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.

[0084]

[0091] In some embodiments, inhibiting cell proliferation includes reducing the cell mass by about 50%. In some embodiments, inhibiting cell proliferation includes reducing the cell mass by about 60%. In some embodiments, inhibiting cell proliferation includes reducing the cell mass by about 65%. In some embodiments, inhibiting cell proliferation includes reducing the cell mass by about 70%. In some embodiments, inhibiting cell proliferation includes reducing the cell mass by about 75%.

[0085]

[0092] In some embodiments, the methods described herein include a method for treating hyperproliferative disorder in a subject, for example, a human subject, by administering a therapeutically effective dose of a RIZ2 inhibitor to the subject, wherein the hyperproliferative disorder in the subject is reduced or improved when the inhibitor is administered at a certain dose and appropriate time intervals. In some embodiments, the hyperproliferative disorder is cancer.

[0086] PRDM2 siRNA design

[0093] This specification provides inhibitory RNA molecules suitable for inhibiting RIZ2 expression. In some embodiments, specific RNA molecules are suitable for therapeutic applications. Examples include synthetic small nucleic acid molecules such as small interfering nucleic acids (siNAs), small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), and small hairpin RNAs (shRNAs) that can regulate intracellular gene expression by RNA inference (RNAi). The siNA molecules of the present invention can be chemically modified. By using chemically modified siNAs, various properties of native siRNA molecules can be enhanced, including increased resistance to nuclease degradation in vivo and / or improved cellular uptake. The chemically modified siNA molecules of the present invention provide useful reagents and methods for a variety of therapeutic, cosmetic, medicinal, prophylactic, diagnostic, agricultural, target validation, genome discovery, genetic engineering, and pharmacogenomic applications.

[0087]

[0094] In some embodiments, the siRNA binds to at least 5, 6, 7, 8, 9, and 10 consecutive nucleotides of the PRDM2 gene, which can bind to at least 5, 6, 7, 8, 9, and 10 consecutive nucleotides.

[0088]

[0095] In some embodiments, the siRNA binds to at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides of the RIZ1 or RIZ2 gene, which can then bind to at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides.

[0089]

[0096] In some embodiments, the siRNA binds to at least 5, 6, 7, 8, 9, 10 consecutive nucleotides that can bind to at least 5, 6, 7, 8, 9, 10 consecutive nucleotides of the RIZ2 gene.

[0090]

[0097] In some embodiments, the siRNA binds to at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides of the RIZ2 gene, rather than to the RIZ1 gene.

[0091]

[0098] In some embodiments, the siRNA specifically binds to at least the 5, 6, 7, 8, 9, and 10 consecutive nucleotides of the RIZ2 gene, primarily affecting RIZ2 gene expression without adversely impacting RIZ1 expression levels.

[0092]

[0099] In one embodiment, the disclosure provides one or more siRNA sequences that can reduce (e.g., silence) the expression of the RIZ2 gene without adversely affecting, and in particular without reducing, the expression of the RIZ1 gene. More specifically, the specification provides siRNA sequences that reduce or inhibit the expression of the RIZ2 gene and induce the expression of the RIZ1 gene. In some embodiments, the siRNA sequences provided herein can reduce or inhibit human RIZ2 expression without inhibiting human RIZ1 expression. In one embodiment, the disclosure is based on the surprising and unexpected finding that one or more siRNAs targeting a common region of the RIZ2 and RIZ1 genes primarily inhibit RIZ2 but leave RIZ1 intact, even though the siRNAs share homology to both the gene and the gene product.

[0093]

[0100] In some embodiments, the siRNA is designed to target a region between 1 and 500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 10 and 500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 100 and 500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 200 and 500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 500 and 1000 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 200 and 2000 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 500 and 2500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 1000 and 2500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 1600 and 2200 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 1800 and 2200 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 1900 and 1950 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 1920 and 1940 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 2000 and 3500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 1000 and 5000 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 1500 and 3000 nucleotides from the 5' end of the RIZ2 mRNA.In some embodiments, the siRNA is designed to target a region between 2500 and 3500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 3500 and 5000 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 4000 and 5000 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 4250 and 4500 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 4300 and 4400 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 4500 and 5000 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 4550 and 4700 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 4560 and 4600 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 4500 and 6000 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 5000 and 6140 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 6050 and 6155 nucleotides from the 5' end of the RIZ2 mRNA. In some embodiments, the siRNA is designed to target a region between 6090 and 6120 nucleotides from the 5' end of the RIZ2 mRNA.

[0094]

[0101] An exemplary list of sequences, including the positive and negative strands of siRNA, as demonstrated in the exemplary tests of the present invention disclosed herein, is provided below.

[0095] [Table 2]

[0096]

[0103] This specification provides for a number of double-stranded polynucleotide compositions comprising the sense strands and antisense strands shown in Table 2, where the left column of Table 2 contains the sense strand sequences of each double-stranded polynucleotide pair, and the right column within the same row contains the antisense strand sequences. In one embodiment, the length of the siRNA is 21 nucleotides. In one embodiment, the siRNA includes a single nucleotide overhang of 1, 2, or 3 nucleotides at the 5' or 3' end.

[0097]

[0104] Accordingly, in one embodiment, a RIZ2 inhibitor is provided herein that comprises a double-stranded polynucleotide including the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 10.

[0098]

[0105] In one embodiment, a RIZ2 inhibitor comprising a double-stranded polynucleotide including the sense strand of SEQ ID NO: 6 and the antisense strand of SEQ ID NO: 11 is provided herein.

[0099]

[0106] In one embodiment, the RIZ2 inhibitor is provided herein, comprising a double-stranded polynucleotide including the sense strand of SEQ ID NO: 2 and the antisense strand of SEQ ID NO: 12.

[0100]

[0107] In one embodiment, a RIZ2 inhibitor comprising a double-stranded polynucleotide including the sense strand of SEQ ID NO: 3 and the antisense strand of SEQ ID NO: 13 is provided herein.

[0101]

[0108] In one embodiment, a RIZ2 inhibitor comprising a double-stranded polynucleotide including the sense strand of SEQ ID NO: 4 and the antisense strand of SEQ ID NO: 14 is provided herein.

[0102]

[0109] In one embodiment, a RIZ2 inhibitor is provided herein that comprises a double-stranded polynucleotide including the sense strand of SEQ ID NO: 5 and the antisense strand of SEQ ID NO: 15.

[0103]

[0110] In one embodiment, a RIZ2 inhibitor is provided herein that comprises a double-stranded polynucleotide including the sense strand of SEQ ID NO: 7 and the antisense strand of SEQ ID NO: 16.

[0104]

[0111] In one embodiment, a RIZ2 inhibitor is provided herein that comprises a double-stranded polynucleotide including the sense strand of SEQ ID NO: 8 and the antisense strand of SEQ ID NO: 17.

[0105]

[0112] In one embodiment, a RIZ2 inhibitor is provided herein that comprises a double-stranded polynucleotide including the sense strand of SEQ ID NO: 9 and the antisense strand of SEQ ID NO: 18.

[0106]

[0113] In one embodiment, the present invention provides a double-stranded polynucleotide composition for inhibiting RIZ2 mRNA expression. In one embodiment, the double-stranded polynucleotide may arise without any modifications. In one embodiment, one or more nucleotides in the sense molecule or antisense molecule are modified. For example, the sense and antisense strands of ARIZ-0047 have the same nucleotide sequence as ARIZ-0011 but include modified nucleotides, for example, the sense strand includes a disulfide modification at the 5' end. In one embodiment, the oligomer includes a modified nucleotide, for example, 2'-methoxyuridine, which interrupts a sequence of unmodified nucleotides, such as the 9th nucleotide residue from the 5' end of SEQ ID NO: 6. In one embodiment, the sense or antisense strand may include multiple modifications, for example, the antisense strand of ARIZ-0047 (SEQ ID NO: 11) includes two 5'-P'-methoxyuridine residues in tandem, and the terminal 3'-dinucleotide at the 3' end includes a phosphorothioate bond.

[0107]

[0114] In this specification, one or more modifications to either the sense or antisense strand of any of the double-stranded polynucleotide compositions presented herein in Table 1 are considered. In some embodiments, one or both of the strands are designed to contain modifications. In some embodiments, one strand may contain a interrupted or gapped motif, and the other strand may contain a gapped motif, a hemimer motif, a blockmer motif, a fully modified motif, a positionally modified motif, or alternating motifs. An "interrupted" or "gapped" motif contains a modified nucleoside that interrupts a sequence of nucleosides such that a sequence of nucleotides is divided into two, preferably three, regions, for example, two outer regions as adjacent inner regions on both sides. These regions are interrupted and separated from each other by modifications of at least a nucleoside-containing (different) sugar group. In some embodiments, nucleosides having different sugar groups include oligomeric compounds pD-ribonucleosides, or 2'-modified nucleosides, or 4'-thio-modified nucleosides, or 4'-thio-2'-modified nucleosides, or bicyclic sugar-modified nucleosides.

[0108]

[0115] In some embodiments, the internal region or gap generally contains pD-ribonucleosides, but may be a sequence of sugar-modified nucleosides. In some embodiments, the nucleosides located in the gap of the gapped oligomer compound have different sugar groups than their wings. In some embodiments, the gapped oligomer compound is "symmetrical". In some embodiments, the gapped oligomer compound is "asymmetrical". A gapmer in which each wing has the same uniform sugar modification may be a symmetric gapped oligomer compound. A gapmer in which each wing has different uniform modifications is referred to as an asymmetric gapped oligomer compound. In some embodiments, such gapped oligomer compounds may have wings containing, for example, 4'-thio-modified nucleosides (symmetric gapmer) and a gap containing pD-ribonucleosides or modified nucleosides other than 4'-thio-modified nucleosides. In some embodiments, a gapped asymmetric oligomeric compound may comprise one wing containing a 2'-OCH3 modified nucleoside and the other wing containing a 4'-thio modified nucleoside, with an internal region (gap) containing a p D-ribonucleoside or a sugar-modified nucleoside other than a 4'-thio or 2'-OCH3 modified nucleoside.

[0109]

[0116] In some embodiments, each strand of the composition of the present invention may be modified to play a specific role, for example, in the siRNA pathway. The antisense strand may be modified at its 5' end to enhance its role in one region of the RISC, while the 3' end of 5 may be modified differently to enhance its role in a different region of the RISC.

[0110]

[0117] In one embodiment, the siRNA includes a T overhang. In some embodiments, the T overhang includes a single uridine (indicated without distinction from thymidine (T) in the sequences presented herein) or two uridine nucleotide residues in tandem (e.g., dTdT).

[0111]

[0118] In some embodiments, one or more residues are linked by phosphorothioate bonds.

[0112]

[0119] In one embodiment, the use of any one of the RIZ2 siRNAs described herein for the treatment of, for example, a hyperproliferative disorder such as cancer. In one embodiment, the use of any one of the RIZ2 siRNAs described herein for preparing a pharmacopoeia suitable for the treatment of, for example, a hyperproliferative disorder such as cancer.

[0113]

[0120] ARIZ siRNA-011 to ARIZ siRNA-014 are complementary to sequences common to both the RIZ1 mRNA transcript and the RIZ2 mRNA transcript. ARIZ-015 is complementary to the last 19 bases of the 3' end of the RIZ2 mRNA and contains 10 nucleotides specific to the RIZ2 mRNA, corresponding to the four terminal amino acids specific to RIZ2, as well as 9 nucleotides common to both the RIZ1 mRNA and the RIZ2 mRNA. ARIZ-047 has the same sequence as ARIZ-011, except for the base modifications shown.

[0114]

[0121] ARIZ siRNA-062 to ARIZ siRNA-064 are complementary to the sequences specific to RIZ2 mRNA.

[0115]

[0122] In one embodiment, the present specification provides an siRNA that targets only RIZ2 and leaves RIZ1 mRNA intact. In another embodiment, the present specification provides an siRNA that targets only RIZ1 and does not affect RIZ2 mRNA. In yet another embodiment, the present specification provides an siRNA that targets RIZ2 mRNA, leaves RIZ1 mRNA intact, and exerts an inhibitory effect on RIZ2 mRNA. Despite the fact that the siRNA disclosed herein can target either RIZ1 mRNA or RIZ2 mRNA, the applicant's findings provide the surprising result that the siRNA specifically inhibits RIZ2 mRNA. Even more surprising is the finding that the siRNA results in an increase in RIZ1 mRNA levels, as presented herein. Even more surprising, and this finding is disclosed herein for the first time, is that the siRNA of the present invention can preferentially induce cell death in cancer cells while leaving non-cancer cells intact. This is highly unexpected given that the siRNA can target regions common to both RIZ2 mRNA and RIZ1 mRNA, and therefore has the potential to inhibit both mRNAs.

[0116]

[0123] In one embodiment, the siRNA provided herein targets RIZ2 mRNA, which is present at higher concentrations in a given cell, such as a diseased cell or a cell with hyperproliferative disorder, such as a cancer cell, compared to the simultaneously low concentrations of RIZ1 mRNA present in diseased cells, such as cancer cells. While not intended to be bound by any theory, if the siRNA of the present invention targets the more targetable RIZ2 mRNA, which is present at higher concentrations than RIZ1 mRNA in diseased cells, the siRNA may inhibit RIZ2 expression, potentially leading to a simultaneous increase in RIZ1 mRNA. The increase in RIZ1 mRNA may help reset cell cycle homeostasis, and cell death may be achieved in abnormally proliferating cells, such as cancer cells.

[0117]

[0124] In some embodiments, a RIZ2 inhibitor containing any one of the siRNAs in Table 2 reduces RIZ2 mRNA expression in treated cells by at least 5% compared to untreated cells. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 6%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 7%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 8%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 9%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 10%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 11%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 12%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 13%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 14%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 15%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 16%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 17%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 18%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 19%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 20%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 21%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 22%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 23%.In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 24%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 25%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 26%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 27%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 28%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 29%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 30%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 31%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 32%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 33%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 34%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 35%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 36%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 37%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 38%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 39%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 40%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 41%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 42%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 43%.In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 44%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 45%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 46%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 47%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 48%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 49%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 50%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 51%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 52%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 53%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 54%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 55%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 56%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 57%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 58%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 59%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 60%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 61%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 62%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 63%.In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 64%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 65%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 66%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 67%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 68%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 69%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 70%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 71%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 72%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 73%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 74%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 75%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 76%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 77%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 78%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 79%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 80%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 81%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 82%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 83%.In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 84%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 85%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 86%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 87%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 88%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 89%. In some embodiments, RIZ2 siRNA reduces RIZ2 mRNA expression by at least 90%.

[0118]

[0125] In some embodiments, RIZ2 siRNA reduces the expression of RIZ2 mRNA in cells compared to the expression of housekeeping genes. Exemplary housekeeping genes are GAPDH or beta-actin genes. In some embodiments, RIZ2 siRNA reduces the expression of RIZ2 mRNA compared to the expression of RIZ2 mRNA in control cells or a control cell population. Exemplary housekeeping genes are GAPDH or beta-actin genes.

[0119]

[0126] In some embodiments, this specification provides a method for restoring the RIZ1-RIZ2 balance in cells. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.1 times. For example, in some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.2 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.3 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.4 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.5 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.6 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.7 times. In some embodiments, the ratio of RIZ1 to the RIZ2 mRNA expression level is changed to at least 1.8 times. In some embodiments, the ratio of RIZ1 to RIZ2 mRNA expression levels is changed to at least 1.9 times. In one embodiment, the ratio of RIZ1 to RIZ2 mRNA expression levels is changed to at least 2 times.

[0120]

[0127] In some embodiments, the RIZ2 inhibitor is an siRNA that reduces the proliferation of diseased cells (e.g., cancer cells) by at least 10%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 15%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 20%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 25%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 30%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 35%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 40%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 45%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 50%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 55%. In some embodiments, the RIZ2 inhibitor siRNA reduces cancer cell proliferation by about 60%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 65%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 70%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 75%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 80%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 81%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 82%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 83%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 84%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 85%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 86%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 87%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 88%.In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 89%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 90%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 91%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 92%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 93%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 94%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 95%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 96%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 97%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 98%.

[0121]

[0128] In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 35%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 40%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 45%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 50%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 55%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 60%. In some embodiments, RIZ2 inhibitor siRNA reduces cancer cell proliferation by approximately 65%. In some embodiments, RIZ2 inhibitor siRNA kills approximately 70% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 75% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 80% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 81% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 82% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 83% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 84% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 85% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 86% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 87% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 88% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 89% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 90% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 91% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 92% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 93% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 94% of cancer cells. In other embodiments, RIZ2 inhibitor siRNA kills approximately 95% of cancer cells.In some embodiments, RIZ2 inhibitor siRNA kills approximately 96% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 97% of cancer cells. In some embodiments, RIZ2 inhibitor siRNA kills approximately 98% of cancer cells.

[0122]

[0129] In one embodiment, RIZ2 inhibitor siRNA can reduce tumor cell mass by at least about 10%. In one embodiment, RIZ2 inhibitor siRNA can reduce cell mass (e.g., tumor cell mass) by about 50% compared to untreated cells or a control cell population. In some embodiments, the reduction in cell mass can be greater than 50%, for example, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0123]

[0130] In one embodiment, the RIZ2 inhibitor siRNA has no significant effect on non-cancer cells. In another embodiment, the RIZ2 inhibitor siRNA kills cancer cells by approximately 2 times and 2.2 times.

[0124]

[0131] In some embodiments, one or more siRNAs are formulated into pharmaceutical compositions for use in the treatment of hyperproliferative disorders or neoplasia, such as breast cancer, colon cancer, endometrial cancer, esophageal cancer, glioma, kidney cancer, leukemia, lymphoma, lung cancer, liver cancer, parathyroid cancer, pituitary cancer, meningioma, myeloma, neuroblastoma, prostate cancer, or thyroid cancer.

[0125] siRNA delivery

[0132] In one embodiment, the siRNA of the present invention is encapsulated in liposomes.

[0133] In another embodiment, the siRNA of the present invention is an unencapsulated double-stranded molecule.

[0126]

[0134] In one embodiment, the siRNA of the present invention complexes with a cell targeting moiety. In one embodiment, the siRNA of the present invention binds to a cell targeting moiety. In one embodiment, the siRNA binds to a cell targeting moiety that can target cell surface elements. In an exemplary embodiment, the cell targeting moiety targets cancer cells or hyperproliferative cells.

[0127]

[0135] In one embodiment, the cell-targeting portion is a ligand and an antibody or antibody fragment, a single-chain antibody, a peptide or aptamer. In one embodiment, the cell-targeting portion is a cell-permeable peptide. In some embodiments, the delivery system includes one or more of the following: lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, or combinations thereof.

[0128]

[0136] In some embodiments, the delivery system includes cyclodextrin.

[0129]

[0137] In some embodiments, the delivery system includes chitosan.

[0130]

[0138] In some embodiments, the delivery system includes a carbohydrate polymer.

[0131]

[0139] In some embodiments, the delivery system includes an elastin-like polymer (ELP). In one embodiment, the targeted delivery system includes a passive targeted nanocarrier. The “passive” targeted nanocarrier utilizes an vascular permeability and retention enhancement (EPR) effect.

[0132]

[0140] In some embodiments, specific molecules or ligands on the surface of cancer cells are targeted, for example, with antibodies or fragments thereof. For example, single-chain anti-prostate stem cell antigen (PSCA) antibodies (scAb) as specific "address tags" for prostate cancer targeted imaging and therapy. PSCA) (Ling Y, Wei K, Luo Y, Gao X, Zhong S. Dual docetaxel / superparamagnetic iron oxide loaded nanoparticles for both targeting magnetic resonance imaging and cancer therapy. Biomaterials. 2011;32:7139-50). For example, Hadjipanayis et al. used an anti-epidermal growth factor receptor (EGFR) deletion mutant antibody to create iron oxide nanoparticles for targeted imaging and therapeutic treatment of glioblastoma (Hadjipanayis CG, Machaidze R, Kaluzova M, Wang L, Schuette AJ, Chen H. et al. EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma. Cancer Res. 2010;70:6303-12). For example, Chen and Shuai et al. developed a CD3 single-chain antibody (scAb) for gene delivery to T cells. CD3 )A functionalized nonviral polymeric vector was used (Chen G, Chen W, Wu Z, Yuan R, Li H, Gao J. et al. MRI-visible polymeric vector bearing CD3 single chain antibody for gene delivery to T cells for immunosuppression. Biomaterials. 2009;30:1962-70). Any of these techniques can be adapted to the siRNA delivery mechanism intended herein.

[0133]

[0141] In one embodiment, the siRNA is bound to one or more lipid components, such as cationic lipids. In one embodiment, the siRNA is bound to one or more lipid components, such as at least cationic and anionic lipids. In one embodiment, the siRNA is modified with polyethylene glycol (PEG) molecules. In one embodiment, the siRNA is bound in a complex containing one or more lipids and PEG. In one embodiment, the siRNA is bound in a complex containing one or more lipids and PEG, wherein the PEG is of a specified length. In one embodiment, the PEG contains a chain having about 20 to about 120 carbon atoms. In one embodiment, the PEG contains a chain having about 40 to 120 carbon atoms. In one embodiment, the PEG contains a chain having about 60 to 120 carbon atoms. In one embodiment, the PEG contains a chain having about 80 to 120 carbon atoms. In one embodiment, the PEG contains a chain having about 100 to 120 carbon atoms. In one embodiment, the PEG contains a chain having about 20 to 40 carbon atoms. In one embodiment, the PEG contains a chain having about 20 to 60 carbon atoms. In one embodiment, PEG comprises a chain having about 20 to 80 carbon atoms. In another embodiment, PEG comprises a chain having about 20 to 100 carbon atoms. In some embodiments, PEG is about 2000 kDa.

[0134]

[0142] In one embodiment, the siRNA of the present invention may be delivered via nanoparticles. In one embodiment, the nanoparticles may be lipid nanoparticles. In one embodiment, the nanoparticles may include calcium phosphate nanoparticles. In some embodiments, the calcium phosphate nanoparticles include multiple siRNAs.

[0135]

[0143] In one embodiment, the nanoparticles release siRNA in vivo in a non-toxic, stable, and degradable manner. In one embodiment, the nanoparticles contain a calcium phosphosilicate complex. The calcium phosphosilicate complex is stable and non-toxic.

[0136]

[0144] In some embodiments, as illustrated in Figure 1, the nanoparticles are referred to as "nanojackets."

[0137]

[0145] In one embodiment, the nanoparticles (e.g., lipid nanoparticles, calcium phosphosilicate, etc.) include particles with a diameter of 80 to 250 nm.

[0138]

[0146] In one embodiment, the siRNA of the present invention can bind to a cell target moiety. In one embodiment, the siRNA may be designed so that a 5'- or 3' overhang can be chemically crosslinked to the target moiety. In one embodiment, the siRNA is bound to the target moiety via a linker. In some embodiments, the linker is a non-reactive short linker, such as a short peptide linker. In one embodiment, the linker may be a bioactive peptide linker. In some embodiments, the linker is a short PEG molecule containing 10 to 12 carbon atoms in the chain. In some embodiments, the PEG molecule is crosslinked to the 5' or 3' end of a single strand of siRNA. In one embodiment, the crosslinker is a maleimide-functionalized bi-conjugational linker. In one embodiment, the crosslinker is a short polymer. In one embodiment, the polymer is a functional polymer, a complex polymer, or a copolymer, such as PLGA-PEG, PLA-PEG, PCA-PEG, lipid PEG, or polylysine PEG.

[0139]

[0147] In some embodiments, the siRNA delivery system is a SNALP (Solid Nucleic Acid Lipid Nanoparticle) technology that utilizes cationic lipids or charge-converting lipids having polyethylene glycol (PEG) surface groups.

[0140]

[0148] In some embodiments, the siRNA delivery system is a cyclodextrin-based delivery system.

[0141]

[0149] In some embodiments, siRNA is complexed with aptamers, either directly or via a linker. Aptamers are synthetically manageable ligands for both diagnostic and therapeutic purposes and are often referred to as nucleic acid ligands, or "oligobodies" or "chemoantibodies." Aptamers that bind to cell surface receptors are readily taken up into cells. Aptamers are characterized by their ability to fold into complex tertiary structures and bind to their targets with high affinity (equilibrium dissociation constant, ranging from low nM to high pM) and specificity. Isolation of aptamers specific to targets of interest involves multiple iterations of a process called SELEX (systematic evolution of ligands by exponential enrichment) (Tuerk and Gold, 1990, Proc. Natl. Acad. Sci. USA. 89, 6988-6992). In the SELEX process, an aptamer library is incubated with a protein target. Then, the aptamers bound to the protein are specifically recovered. These sequences are amplified by PCR or RT-PCR. Then, single-stranded RNA or DNA sequences representing the recovered sequences are generated from these PCR products and used in subsequent selection rounds. Exemplary aptamers known to those skilled in the art include tenascin-C aptamers (TTA1), PMSA aptamers, CTLA-4 aptamers, and HIV-bivalent aptamers, depending on the type of cancer, target, etc. (Thiel et al., Oligonucleotides. 2009 Sep 1;19(3):209-222).

[0142] Pharmaceutical composition

[0150] This specification provides pharmaceutical compositions comprising RIZ2 inhibitors, which also include pharmaceutically acceptable components. In some embodiments, the RIZ2 inhibitor comprises one or more polynucleotide molecules and a delivery system. In some embodiments, the RIZ2 inhibitor comprises an inhibitory RNA molecule, such as a double-stranded siRNA molecule. In some embodiments, the inhibitory RNA molecule is hybridized to at least 10 adjacent nucleic acid bases on the PRDM2 gene or PRDM2 gene product. In some embodiments, the inhibitory RNA molecule is about 10 to about 21 nucleotides in length. In some embodiments, the RIZ2 inhibitor comprises one or more siRNAs.

[0143]

[0151] This specification provides siRNA formulated as a pharmaceutical composition for administration by subcutaneous injection.

[0144]

[0152] This specification provides siRNA formulated as a pharmaceutical composition for administration by intravenous injection.

[0145]

[0153] This specification provides siRNA formulated as a pharmaceutical composition for systemic administration.

[0146]

[0154] This specification provides siRNA formulated as a pharmaceutical composition for topical administration.

[0147]

[0155] In one embodiment, a pharmaceutically acceptable excipient may be pathogen-free water. In one embodiment, a pharmaceutically acceptable excipient may be a suitable buffer at a neutral pH, such as phosphate-buffered saline. In some embodiments, a pharmaceutically acceptable excipient may be a weakly acidic aqueous solution. In some embodiments, a pharmaceutically acceptable excipient may include glycols, glycerin, DMSO, soluble components, sugars, salts, etc. In some embodiments, the formulation may include fillers such as sucrose, trehalose, mannitol, glycine, lactose, and / or raffinose, which impart the desired consistency to the formulation and / or provide stability to the formulation components. In some embodiments, excipients commonly used for topical administration, as known to those skilled in the art, may be used.

[0148]

[0156] This specification provides pharmaceutical compositions comprising inhibitory double-stranded RNA containing the sequence of SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, this specification provides pharmaceutical compositions comprising inhibitory double-stranded RNA containing the sequence of SEQ ID NO: 10 or a sequence having at least 90% identity with SEQ ID NO: 10. This specification provides pharmaceutical compositions comprising inhibitory double-stranded RNA containing the sequence of SEQ ID NO: 1 as a sense strand and the sequence of SEQ ID NO: 10 as an antisense strand.

[0149]

[0157] This specification provides a pharmaceutical composition comprising inhibitory double-stranded RNA comprising the sequence of SEQ ID NO: 6 or a sequence having at least 90% identity with SEQ ID NO: 6. In some embodiments, this specification provides a pharmaceutical composition comprising inhibitory double-stranded RNA comprising the sequence of SEQ ID NO: 11 or a sequence having at least 90% identity with SEQ ID NO: 11. This specification provides a pharmaceutical composition comprising inhibitory double-stranded RNA comprising the sequence of SEQ ID NO: 6 as a sense strand and the sequence of SEQ ID NO: 11 as an antisense strand.

[0150]

[0158] This specification provides pharmaceutical compositions comprising inhibitory double-stranded RNA. In one embodiment, this specification provides a RIZ2 inhibitor comprising a double-stranded polynucleotide comprising the sense strand of SEQ ID NO: 2 or a sequence having at least 90% identity with SEQ ID NO: 2, and the antisense strand of SEQ ID NO: 12 or a sequence having at least 90% identity with SEQ ID NO: 12.

[0151]

[0159] In one embodiment, this specification provides a pharmaceutical composition comprising a RIZ2 inhibitor containing a double-stranded polynucleotide comprising the sense strand of SEQ ID NO: 3 or a sequence having at least 90% identity with SEQ ID NO: 3, and the antisense strand of SEQ ID NO: 13 or a sequence having at least 90% identity with SEQ ID NO: 13.

[0152]

[0160] In one embodiment, this specification provides a pharmaceutical composition comprising a RIZ2 inhibitor containing a double-stranded polynucleotide comprising the sense strand of SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, and the antisense strand of SEQ ID NO: 14 or a sequence having at least 90% identity with SEQ ID NO: 14.

[0153]

[0161] In one embodiment, this specification provides a pharmaceutical composition comprising a RIZ2 inhibitor containing a double-stranded polynucleotide comprising the sense strand of SEQ ID NO: 5 or a sequence having at least 90% identity with SEQ ID NO: 5, and the antisense strand of SEQ ID NO: 15 or a sequence having at least 90% identity with SEQ ID NO: 15.

[0154]

[0162] In one embodiment, this specification provides a pharmaceutical composition comprising a RIZ2 inhibitor containing a double-stranded polynucleotide comprising the sense strand of SEQ ID NO: 7 or a sequence having at least 90% identity with SEQ ID NO: 7, and the antisense strand of SEQ ID NO: 16 or a sequence having at least 90% identity with SEQ ID NO: 16.

[0155]

[0163] In one embodiment, this specification provides a pharmaceutical composition comprising a RIZ2 inhibitor containing a double-stranded polynucleotide comprising the sense strand of SEQ ID NO: 8 or a sequence having at least 90% identity with SEQ ID NO: 8, and the antisense strand of SEQ ID NO: 17 or a sequence having at least 90% identity with SEQ ID NO: 17.

[0156]

[0164] In one embodiment, this specification provides a pharmaceutical composition comprising a RIZ2 inhibitor containing a double-stranded polynucleotide comprising the sense strand of SEQ ID NO: 9 or a sequence having at least 90% identity with SEQ ID NO: 9, and the antisense strand of SEQ ID NO: 18 or a sequence having at least 90% identity with SEQ ID NO: 18.

[0157]

[0165] In some embodiments, the pharmaceutical composition comprises one or more siRNAs as described above, and an siRNA delivery system such as liposomes or nanoparticles disclosed herein. In some embodiments, the pharmaceutical composition having a delivery system comprises a cell-targeting moiety, the cell-targeting moiety being a ligand and an antibody or antibody fragment, a single-chain antibody, a peptide or aptamer. In some embodiments, the cell-targeting moiety is a cell-permeable peptide. In some embodiments, the delivery system comprises one or more of lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, or combinations thereof.

[0158]

[0166] This specification provides a pharmaceutical composition comprising a RIZ2 inhibitor containing siRNA, wherein (i) it contains a double-stranded RNA of 10 to 21 nucleotides homologous to the human PRDM2 / RIZ2 gene, (ii) it is covalently bound to a PEG molecule, and (iii) it is bound to a cell target region, the cell target region being an aptamer.

[0159]

[0167] In one embodiment, the pharmaceutical composition comprises one or more chemotherapeutic agents. Exemplary chemotherapeutic agents include, but are not limited to, obinutuzumab, bendamustine, chlorambucil, cyclophosphamide, ibrutinib, methotrexate, cytarabine, dexamethasone, cisplatin, bortezomib, fludarabine, idelalisib, acalabrutinib, lenalidomide, venetoclax, cyclophosphamide, ifosfamide, etoposide, pentostatin, melphalan, carfilzomib, ixazomib, panobinostat, daratumumab, elotuzumab, thalidomide, lenalidomide, or pomalidomide, or combinations thereof.

[0160]

[0168] In one embodiment, the therapeutic composition may include additional agents, such as checkpoint inhibitors, such as PD1 inhibitors, PDL1 inhibitors, or CTLA4 inhibitors, or combinations thereof.

[0161] Treatment and diagnostic methods

[0169] The therapeutic compositions described above, comprising one or more inhibitory RNA molecules targeting RIZ2, may be used to treat cell proliferation disorders in a subject. In some embodiments, the subject is human. In one embodiment, the cell proliferation disorder is cancer. In one embodiment, the cell proliferation disorder is associated with, or a consequence of, cell cycle deregulation, loss of cell cycle checkpoint inhibition, and / or RIZ1 / RIZ2 imbalance. It is well known that the histone H3K9 methyltransferase activity of RIZ1 plays a crucial role in the negative regulation of cell proliferation, and that RIZ1 expression is reduced in various cancers. RIZ2 was known to be increased in cancer cells, but its biological activity was not clear. It was thought to be primarily a non-functional counterpart of RIZ1, and it was hypothesized that RIZ2 acts as a negative regulator of RIZ1 function, but mediates the proliferative effects of estrogen through the regulation of survival and differentiation gene expression. J Cell Physiol 2012 Mar;227(3):964-75. Furthermore, RIZ2 is a transcript cleaved at a shorter N-terminus compared to RIZ1, and is a translation product; the former is unrelated to RIZ1 and therefore had fewer opportunities for manipulation.

[0162]

[0170] This disclosure provides a method for targeting RIZ2 and a composition for inhibiting RIZ2 independently of RIZ1.

[0163]

[0171] This specification provides therapeutic methods and compositions for specifically targeting and manipulating RIZ2. In one embodiment, the therapeutic composition comprises a pharmaceutical composition comprising a RIZ2-specific siRNA for the treatment of a cell proliferation disorder. In one embodiment, the cell proliferation disorder is cancer.

[0164]

[0172] This specification provides a method for treating cancer, which comprises administering a pharmaceutical composition described in any of the above sections to a subject in need. As a result of research in this disclosure, it is now known that RIZ2 offers a very promising therapeutic target, and that not only can RIZ2 be selectively deexpressed in cancer cells, but inhibition of RIZ2 can lead to increased expression of RIZ1. Furthermore, RIZ / RIZ2 imbalance, for example, deexpression of RIZ1 and increase of RIZ2, is a regulation at a significantly upstream node in the cell cycle, which is common to many cancers, and therefore this therapy is applicable to a wide range of cancers. For example, in one embodiment, the methods and compositions described herein are therapeutically applicable to solid tumors. In one embodiment, the methods and compositions described herein are therapeutically applicable to liquid cancer. In one embodiment, the cancer is a cancer of the breast, colon, endometrium, esophagus, stomach, glioma, kidney, liver, lung, lymphoma, melanoma, meningioma, myeloma, nasopharyngeal, neuroblastoma, ovarian, pancreatic, parathyroid, pituitary, prostate, thyroid, or uterine tissue.

[0165]

[0173] In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is lung cancer. It is intended that any cancer may be treated using the methods and compositions described herein. Examples of cancers include, but are not limited to, cancer, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers are listed below, and also include: squamous cell carcinoma (e.g., squamous cell carcinoma of epithelium), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer or cancer of the stomach including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, penile cancer, and head and neck cancer. The term "cancer" includes primary malignant cells or tumors (for example, those in which the cells have not metastasized to any other part of the body of the subject besides the original malignant tumor or tumor site) and secondary malignant cells or tumors (for example, those resulting from metastasis, migration of malignant cells or tumor cells to a secondary site different from the original tumor site).

[0166]

[0174] In other embodiments, RIZ2 inhibitors are used to treat hyperproliferative disorders, such as hyperplasia, cancer, or hyperproliferative connective tissue disorders (e.g., hyperproliferative fibrosis), as described above and herein. In one embodiment, the hyperproliferative fibrosis is polysystemic or organ-specific. Exemplary hyperproliferative fibrosis include, but are not limited to, polysystemic (e.g., systemic sclerosis, multifocal fibrous sclerosis, scleroderma graft-versus-host disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, scleroderma) and organ-specific disorders (e.g., fibrosis of the eye, lung, liver, heart, kidney, pancreas, skin, and other organs). In other embodiments, the disorder is selected from cirrhosis or tuberculosis. In other embodiments, the disorder is leprosy.

[0167]

[0175] In one embodiment, the RIZ2 inhibitor is specifically designed to target and treat lung cancer. In some embodiments, the RIZ2 inhibitor is formulated for aerosol delivery to the lungs. In some embodiments, the RIZ2 inhibitor is formulated for systemic delivery via intravenous injection.

[0168]

[0176] In one embodiment, a RIZ2 inhibitor containing ARIZ-047 siRNA is designed to treat lung cancer.

[0169]

[0177] In one embodiment, ARIZ-047 does not exhibit toxic effects on living cells or experimental animals.

[0170]

[0178] In some embodiments, a therapeutic composition containing a RIZ2 inhibitor may be administered concurrently with a chemotherapeutic agent. The chemotherapeutic agent may be cyclophosphamide, doxorubicin, vincristine, prednisone, or rituximab, or a combination thereof. Other chemotherapeutic agents include obinutuzumab, bendamustine, chlorambucil, cyclophosphamide, ibrutinib, methotrexate, cytarabine, dexamethasone, cisplatin, bortezomib, fludarabine, idelalisib, acalabrutinib, lenalidomide, venetoclax, cyclophosphamide, ifosfamide, etoposide, pentostatin, melphalan, carfilzomib, ixazomib, panobinostat, daratumumab, elotuzumab, thalidomide, lenalidomide, or pomalidomide, or a combination thereof. "Concurrent administration" refers to the administration of two or more therapeutic drugs or pharmaceutical compositions during the course of treatment. Such concurrent administration can be simultaneous or sequential. Sequential administration of the later-administered therapeutic drug or pharmaceutical composition may occur at any point during the course of treatment with a RIZ2 inhibitor.

[0171]

[0179] In one embodiment, the RIZ2 inhibitor siRNA may be administered concurrently with cisplatin.

[0172]

[0180] In one embodiment, using the siRNA described herein (e.g., ARIZ-047) in conjunction with the aforementioned drugs may allow for lower concentrations or doses of the drug to achieve therapeutic effects, thereby offering the advantage of reducing drug-related toxic effects in cancer patients.

[0173]

[0181] The administration of the pharmaceutical compositions envisioned herein may be carried out using prior art, including, but not limited to, infusion, infusion, or parenteral administration. In some embodiments, parenteral administration includes infusion or injection into the intravascular, intravenous, intramuscular, intraarterial, subarachnoid space, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, and intrasternal regions.

[0174]

[0182] In one embodiment, a pharmaceutical composition containing a RIZ2 inhibitor may be administered once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every eight days, once every nine days, once every ten days, once every eleven days, once every twelve days, once every thirteen days, or once every fourteen days. In one embodiment, a pharmaceutical composition containing a RIZ2 inhibitor may be administered once every fifteen days. In one embodiment, a pharmaceutical composition containing a RIZ2 inhibitor may be administered once every twenty days. In one embodiment, a pharmaceutical composition containing a RIZ2 inhibitor may be administered once every one month. In one embodiment, a pharmaceutical composition containing a RIZ2 inhibitor may be administered once every two months, once every three months, once every four months, once every five months, or once every six months.

[0175]

[0183] In one embodiment, the Specified provides a method for selecting a patient to be administered a composition containing a RIZ2 inhibitor, comprising (i) detecting an increase in the RIZ1 methylation level in a biological sample of the subject compared to a control sample or control value, or (ii) detecting a decrease in the RIZ1 mRNA level compared to a control sample or control value, wherein the subject is determined to be in need of administration of a composition containing a RIZ2 inhibitor if the biological sample of the subject shows at least a 1.1-fold increase in methylated RIZ1 or at least a 1 / 2 decrease in the RIZ1 mRNA level compared to a control sample or control value, and the control sample is a sample from a clinically healthy individual, and the control value is the average value of RIZ1 methylation levels from samples of two or more clinically healthy individuals.

[0176]

[0184] In some embodiments, RIZ2 inhibitors reduce hypermethylation of the RIZ1 gene.

[0177] kit

[0185] The present invention also provides kits comprising RIZ2 inhibitors. Some aspects of this disclosure provide kits for the treatment of cancer or hyperproliferative disorders. In one embodiment, the kit is a neoplasia treatment kit.

[0178]

[0186] In one embodiment, the kit may include a therapeutic composition comprising the RIZ2 inhibitor described herein in a appropriately labeled bottle indicating the concentration and instructions for use, which is stored under temperature and environmental conditions that ensure the safety and efficacy of the therapeutic active ingredient.

[0179]

[0187] In one embodiment, the kit may include a set of detectors for detecting RIZ1 mRNA and RIZ2 mRNA in a biological sample. The set of detectors may include a set of primers, e.g., a set of forward and reverse primers for detecting RIZ1 levels, and a set of forward and reverse primers for detecting RIZ2 levels. In some embodiments, RIZ1 levels are measured in sputum samples to serve as a biomarker of novation. In one embodiment, the kit includes one or more components for detecting RIZ1 and RIZ2 levels (e.g., mRNA, protein, etc.) in a biological sample from a human. In some embodiments, the kit may include a diagnostic unit for detecting RIZ1 methylation. In one embodiment, RIZ1 methylation can be detected using methylation-based sequencing. In one embodiment, RIZ1 methylation can be detected using one or more methylation-based primers. The kit may include one or more vials containing primers, reagents, enzymes, buffers, etc. Each vial is labeled with its components and concentrations. The kit includes at least one instruction manual regarding the use of the kit.

[0180]

[0188] The neoplasia treatment kit includes instructions for using modified immune cells in the treatment of neoplasia.

[0181]

[0189] The detection kit includes instructions regarding its components, safety parameters, and usage guidelines.

[0182]

[0190] In some embodiments, a single kit may include components for both a treatment kit and a detection kit.

[0183]

[0191] The implementation of the present invention employs prior art in molecular biology (including recombination techniques), microbiology, cell biology, biochemistry, and immunology, which is well within the understanding of those skilled in the art, unless otherwise suggested. Such techniques are fully described in the literature, for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and can be considered in themselves when constructing and carrying out the present invention. Techniques that are particularly useful with respect to specific embodiments will be described in the following sections.

[0184]

[0192] Embodiment 1. A method for inhibiting cell proliferation, comprising contacting a population of cells with a composition comprising a retinoblastoma protein-interacting zinc finger protein 2 (RIZ2) inhibitor. 2. The method according to Embodiment 1, wherein a RIZ2 inhibitor reduces the expression of RIZ2 in cells. 3. The method according to Embodiment 1 or 2, wherein the RIZ2 inhibitor reduces the expression of RIZ2 mRNA in cells. 4. The method according to Embodiment 3, wherein RIZ2 mRNA is reduced by at least 10% in cells compared to the expression of housekeeping genes. 5. The method according to Embodiment 3 or 4, wherein RIZ2 mRNA is reduced by at least 10% compared to the control cell population. 6. The method according to Embodiment 5, wherein the control cell population is a cell population that has not been in contact with the composition containing the RIZ2 inhibitor. 7. The method according to Embodiment 5, wherein RIZ2 mRNA expression is reduced by at least 50% compared to the control cell population. 8. The method according to Embodiment 5, wherein RIZ2 mRNA expression is reduced by at least 80% compared to the control cell population. 9. The method according to any one of Embodiments 1 to 8, wherein the RIZ2 inhibitor further increases the expression of RIZ1 in a cell population. 10. The method according to Embodiment 9, wherein the composition alters the ratio of the RIZ1 mRNA expression level to the RIZ2 mRNA expression level. 11. The method according to Embodiment 9, wherein the composition alters the ratio of the RIZ1 protein expression level to the RIZ2 protein expression level. 12. The method according to Embodiment 9 or 10, wherein the RIZ2 inhibitor alters the ratio of RIZ1 to the RIZ2 mRNA expression level to at least 1.1 times. 13. The method according to Embodiment 9 or 10, wherein the RIZ2 inhibitor alters the ratio of RIZ1 to the RIZ2 mRNA expression level to at least 1.5 times. 14. The method according to Embodiment 9 or 10, wherein the RIZ2 inhibitor alters the ratio of RIZ1 to the RIZ2 mRNA expression level by at least twofold. 15. The method according to any one of Embodiments 1 to 8, wherein the RIZ2 inhibitor further reduces the hypermethylation of RIZ1. 16. The method according to Embodiment 1, wherein the cells are mammalian cells. 17. The method according to Embodiment 1, wherein the cells are human cells. 18. The method according to Embodiment 1, wherein the cells are cancer cells. 19. The method according to Embodiment 1, wherein the cells are derived from tissue which is breast, colon, endometrium, esophagus, stomach, glioma, kidney, liver, lung, lymphoma, melanoma, meningioma, myeloma, nasopharynx, neuroblastoma, ovary, pancreas, parathyroid gland, pituitary gland, prostate, thyroid gland, or uterine tissue. 20. The method according to Embodiment 18, wherein the cancer cells are derived from blood cancer. 21. The method according to Embodiment 8, wherein the cancer cells are derived from a solid tumor. 22. The method according to Embodiment 1, wherein contact is made by bringing a population of cells into contact with a composition comprising a RIZ2 inhibitor. 23. The method according to Embodiment 22, wherein contact results in a reduction of at least 10% in the number of cells in a population of cells. 24. The method according to Embodiment 22, wherein contact results in a reduction of approximately 90% in the number of cells in a population of cells. 25. The method according to Embodiment 1 or 22, wherein inhibiting cell proliferation includes reducing the cell mass by at least 10%. 26. The method according to Embodiment 25, wherein inhibiting cell proliferation reduces the cell mass by approximately 50%. 27. The method according to Embodiment 1, further comprising bringing cells into contact with each other to introduce a RIZ2 inhibitor into the cells. 28. The method according to Embodiment 1, wherein the RIZ2 inhibitor comprises a non-natural compound. 29. The method according to Embodiment 28, wherein the RIZ2 inhibitor is a synthetic drug. 30. The method according to Embodiment 29, wherein the RIZ2 inhibitor comprises a peptide. 31. The method according to Embodiment 29, wherein the RIZ2 inhibitor comprises a complexed polypeptide. 32. The method according to Embodiment 29, wherein the RIZ2 inhibitor comprises one or more polynucleotides. 33. The method according to Embodiment 32, wherein the RIZ2 inhibitor comprises a synthetic polynucleotide. 34. The method according to Embodiment 33, wherein one or more polynucleotides include one or more modified nucleotides. 35. The method according to Embodiment 1, wherein the composition comprising a RIZ2 inhibitor comprises one or more polynucleotides conjugated to a delivery system. 36. The method according to Embodiment 35, wherein the delivery system comprises one or more of the following: lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, or combinations thereof. 37. The delivery system is the method according to embodiment 35, comprising lipids. 38. The delivery system is the method according to Embodiment 35, comprising a PEGylated lipid. 39. The delivery system is the method according to embodiment 35, comprising a cell targeting portion. 40. The method according to Embodiment 39, wherein the cell targeting portion is cell type specific. 41. The method according to Embodiment 39, wherein the cell targeting portion is a ligand that selectively binds to a receptor on a target cell. 42. The method according to Embodiment 39, wherein the cell targeting portion is an antibody that binds to a cell surface molecule on a target cell. 43. The method according to Embodiment 42, wherein the cell targeting portion is a single-chain antibody or antibody fragment. 44. The method according to Embodiment 39, wherein the cell targeting portion is a peptide. 45. The method according to Embodiment 44, wherein the cell-targeting portion is a cell-permeable peptide. 46. ​​The method according to embodiment 44 or 45, wherein the cell targeting portion is a cyclic peptide. 47. The method according to Embodiment 39, wherein the cell targeting portion is an aptamer. 48. The delivery system is the method according to embodiment 35, comprising liposomes. 49. The delivery system is the method according to embodiment 35, comprising nanoparticles. 50. The method according to any one of Embodiments 1 to 49, wherein the RIZ2 inhibitor comprises an RNA molecule. 51. The method according to Embodiment 50, wherein the RIZ2 inhibitor comprises an inhibitory RNA molecule. 52. The method according to Embodiment 51, wherein the inhibitory RNA molecule hybridizes to at least 10 adjacent nucleic acid bases on the PRDM2 gene or the PRDM2 gene product. 53. The method according to Embodiment 50, wherein the inhibitory RNA is approximately 10 to 21 nucleotides in length. 54. The method according to Embodiment 50, wherein the inhibitory RNA molecule is double-stranded. 55. The method according to Embodiment 50, wherein the RNA molecule comprises one or more modified nucleotides. 56. The method according to any one of Embodiments 1 to 55, wherein the RIZ2 inhibitor comprises an inhibitory double-stranded RNA having a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 10. 57. The method according to any one of Embodiments 1 to 55, wherein the RIZ2 inhibitor comprises an inhibitory double-stranded RNA having a sense strand having the sequence of SEQ ID NO: 6 and an antisense strand having the sequence of SEQ ID NO: 11. 58. The method according to any one of Embodiments 1 to 55, wherein the RIZ2 inhibitor comprises an inhibitory double-stranded RNA molecule including a sense strand and an antisense strand selected from Table 2. 59. A method for treating a cell proliferation disorder or impairment in a subject, comprising administering a composition comprising a RIZ2 inhibitor to the subject. 60. A method for selecting patients who require administration of a composition containing a RIZ2 inhibitor, the method being: (i) Detect an increase in the RIZ1 methylation level in the target biological sample compared to a control sample or control value, or (ii) This includes detecting a decrease in RIZ1 mRNA levels compared to a control sample or control value, Compared to a control sample or control value, a 1.1-fold increase in methylated RIZ1 or a 1 / 2 decrease in RIZ1 mRNA levels in the subject's biological sample indicates that the subject requires administration of a composition containing a RIZ2 inhibitor, and The control sample was a sample from a clinically healthy individual. The control value is the mean RIZ1 methylation level from samples of two or more clinically healthy individuals, according to the method. 61. The method according to Embodiment 59, wherein a cell proliferation disorder or disorder is cancer. 62. The method according to Embodiment 61, wherein the cancer is cancer of the breast, colon, endometrium, esophagus, stomach, glioma, kidney, liver, lung, lymphoma, melanoma, meningioma, myeloma, nasopharyngeal, neuroblastoma, ovarian, pancreatic, parathyroid, pituitary, prostate, thyroid, or uterine tissue. 63. The method according to embodiment 61, wherein the cancer is a solid tumor. 64. The method according to embodiment 62, wherein the cancer is lung cancer. 65. The method according to Embodiment 59, wherein administration includes administering an effective amount of a RIZ2 inhibitor. 66. The method according to embodiment 65, wherein an effective dose of the RIZ2 inhibitor is an amount that reduces a parameter of at least one disease associated with cell proliferation disorder. 67. The method according to Embodiment 66, wherein a cell proliferation disorder is cancer, and an effective amount of RIZ2 reduces the number of cancer cells. 68. The method according to embodiment 66, wherein the cell proliferation disorder is a solid tumor, and an effective amount of RIZ2 reduces the tumor volume. 69. The method according to Embodiment 59 or 60, wherein the composition comprising a RIZ2 inhibitor comprises one or more polynucleotides conjugated to a delivery system. 70. The method according to Embodiment 69, wherein one or more polynucleotides include an inhibitory RNA molecule. 71. The method according to Embodiment 70, wherein the inhibitory RNA molecule is a double-stranded RNA comprising a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 10. 72. The method according to Embodiment 70, wherein the inhibitory RNA molecule is a double-stranded RNA comprising a sense strand having the sequence of SEQ ID NO: 6 and an antisense strand having the sequence of SEQ ID NO: 11. 73. The method according to Embodiment 1, further comprising a delivery system. 74. The delivery system comprises a lipid and / or cell-targeting moiety, as described in any one of Embodiments 73. 75. The method according to Embodiment 74, wherein the lipids include PEGylated lipids. 76. The method according to Embodiment 74, wherein the cell-targeting portion is a cell-permeable peptide, a cyclic peptide, an antibody or a fragment thereof, or an aptamer. 77. A pharmaceutical composition comprising (a) a RIZ2 inhibitor and (b) a pharmaceutically acceptable excipient. 78. The RIZ2 inhibitor is a pharmaceutical composition according to the above embodiment, comprising one or more polynucleotide molecules and a delivery system. 79. The RIZ2 inhibitor is a pharmaceutical composition according to Embodiment 78, comprising an inhibitory RNA molecule. 80. The pharmaceutical composition according to Embodiment 77, wherein the inhibitory RNA molecule hybridizes to at least 10 adjacent nucleic acid bases on the PRDM2 gene or PRDM2 gene product. 81. The pharmaceutical composition according to Embodiment 77, wherein the inhibitory RNA molecule is approximately 10 to 21 nucleotides in length. 82. The pharmaceutical composition according to Embodiment 77, wherein the inhibitory RNA molecule is double-stranded. 83. The pharmaceutical composition according to Embodiment 77, wherein the inhibitory double-stranded RNA comprises a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 10. 84. The pharmaceutical composition according to Embodiment 77, wherein the inhibitory double-stranded RNA comprises a sense strand and an antisense strand selected from Table 2. 85. The pharmaceutical composition according to Embodiment 77, wherein the inhibitory double-stranded RNA comprises a sense strand having the sequence of SEQ ID NO: 6 and an antisense strand having the sequence of SEQ ID NO: 11. 86. The delivery system is the pharmaceutical composition according to Embodiment 78, comprising a cell-targeting portion. 87. The pharmaceutical composition according to Embodiment 86, wherein the cell targeting portion is a ligand and an antibody or antibody fragment, a single-chain antibody, a peptide or aptamer. 88. The pharmaceutical composition according to Embodiment 86, which is a cell-permeable peptide. 89. The pharmaceutical composition according to Embodiment 86, wherein the delivery system comprises one or more of the following: lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, or combinations thereof. 90. RIZ2 inhibitors are (i) Containing siRNA containing a 10-21 nucleotide double-stranded RNA homologous to the human PRDM2 / RIZ2 gene, (ii) Covalently bonded to the PEG molecule, (iii) It binds to the cell target region, The pharmaceutical composition according to Embodiment 78, comprising the siRNA wherein the cell targeting portion is an aptamer. 91. The delivery system comprises liposomes, the pharmaceutical composition according to Embodiment 78. 92. The delivery system is the pharmaceutical composition according to Embodiment 78, comprising nanoparticles. 93. The pharmaceutical composition according to Embodiment 92, wherein the nanoparticles include lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, and combinations thereof. 94. The pharmaceutical composition according to Embodiment 92, wherein the nanoparticles comprise a calcium phosphosilicate complex. 95. The pharmaceutical composition according to Embodiment 90, further comprising a calcium phosphosilicate conjugate bound to a RIZ2 inhibitor. 96. The pharmaceutical composition according to Embodiment 77, further comprising one or more chemotherapeutic agents.

[0185]

[0193] Reference All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent specifically and individually indicated that it is invoked by reference. [Examples]

[0186] Example 1: Effect of RIZ2 siRNA

[0194] The following examples are provided to those skilled in the art to provide a complete disclosure and description of how to prepare and use the assays, screenings, and therapeutic methods of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention.

[0187]

[0195] The following describes an exemplary demonstration of the siRNA design described herein regarding its effective cytotoxicity against human lung cancer cells using the A549 cell line.

[0188]

[0196] Methods: A549 cells were seeded in 96-well plates (1,000 cells / well) and, after 24 hours, transfected with 100 nM siRNA using 0.2 μl of DharmaFECT 1 (Dharmacon, Inc.) according to the manufacturer's instructions. The medium was changed after 2 days, and after 4 days, the percentage cell viability was determined compared to untreated cells using an MTS cell viability assay. RIZ1 mRNA levels and RIZ2 mRNA levels were determined by qPCR in control and siRNA-transfected cells. RNA was extracted from cells 48 hours after transfection, and mRNA expression levels were detected using PRDM isoform A-specific and PRDM isoform C-specific PCR primers. RIZ mRNA expression levels were determined by comparison with GAPDH mRNA expression levels. Scrambled siRNA was used as a negative control, and Tox (transfection control siRNA, commercially available) was used as a positive control.

[0189]

[0197] Results and Discussion: Figure 2 shows the cell viability of A549 lung cancer cells after exposure to various siRNAs, including ARIZ-011, ARIZ-012, ARIZ-013, ARIZ-014, and ARIZ-015, using the method described in Example 1 above. The viability of A549 lung cancer cells decreased with the application of ARIZ-011, ARIZ-012, and ARIZ-015. However, ARIZ-011 showed very promising results.

[0190]

[0198] The relative levels of RIZ1 mRNA and RIZ2 mRNA in A549 cells after treatment with siRNA ARIZ-011 were examined, and as shown in Figure 3, ARIZ-011 showed not only a nearly 75% decrease in RIZ2 mRNA but also a simultaneous increase in RIZ1 mRNA. This was a surprising and unexpected result, associated not only with the decrease in RIZ2 mRNA in the cancer cell line but also with the restoration of the RIZ1 / RIZ2 ratio imbalance.

[0191]

[0199] In summary, the results demonstrated that PRDM2 siRNA was effective in killing A549 lung cancer cells. siRNA ARIZ-011 was the most effective of the siRNAs examined, reducing relative cell viability by up to 30% after a single exposure to the siRNA.

[0192]

[0200] siRNA ARIZ-015, a RIZ2-specific siRNA containing 10 nucleotides unique to the 3' end of RIZ2 mRNA, was less effective in killing A549 cells (cell viability 70%) than ARIZ-011 (cell viability 30%), which is complementary to both RIZ1 mRNA and RIZ2 mRNA.

[0193]

[0201] The results of the RIZ mRNA assay shown in Figure 3 were surprisingly unexpected, as the apparent knockdown of the oncoprotein RIZ2 by siRNA ARIZ-011 occurred simultaneously with an increase in the expression of the tumor suppressor protein RIZ1.

[0194] Example 2: Effect of simultaneous administration of RIZ2 siRNA and chemotherapy drugs

[0202] This is an exemplary demonstration that the co-administration of PRDM siRNA with the chemotherapy drug (5-fluorouracil) enhances the killing of human colon cancer cells with minimal impact on healthy cells.

[0195]

[0203] Methods: Cells (cell line HCT116) were seeded in 96-well plates (1,000 cells / well), and after 24 hours, siRNA ARIZ-011 was transfected at 100 nM using 0.2 μl of DharmaFECT 1 (Dharmacon, Inc.) according to the manufacturer's instructions. Where applicable, 5-fluorouracil (25 μM) was added 1 day after transfection. The medium (containing 5-fluorouracil where applicable) was changed after 2 days, and at 4 days, the percentage cell viability was determined compared to untreated cells.

[0196]

[0204] Results and Discussion: As shown in Figure 4, simultaneous administration of ARIZ-011 siRNA and 5FU resulted in a 97% decrease in the cell viability of HCT116 colon cancer cells, whereas a single dose of either siRNA or 5FU resulted in a decrease of approximately 72-74%. Similar to the findings reported in Example 1, RIZ2 siRNA selectively reduced the expression level of RIZ2 in HCT116 cells, while a simultaneous increase in RIZ1 mRNA was observed (Figure 5).

[0197]

[0205] While the siRNA ARIZ-011 alone was effective in killing HCT116 colon cancer cells (74% killed at a 100 nM dose), it was comparable to the chemotherapeutic drug 5-fluorouracil (5-FU) at a 25 μM concentration (72%). These results demonstrate that siRNA can further enhance the effectiveness of chemotherapeutic drugs. RIZ2 siRNA increases the mRNA of the tumor suppressor RIZ1, which is known to kill cancer cells by causing cell cycle arrest.

[0198]

[0206] Example 3: siRNA modification

[0207] Exemplary modifications of nucleic acid bases in designed siRNAs are demonstrated and tested as follows. For siRNA ARIZ-011, the modified siRNA is designed as ARIZ-047 (Table 1), sequence 6, as shown. Essentially, a 5'-disulfide moiety is incorporated, and one uridine in the sense strand of the siRNA backbone is replaced with a 2'-methoxyuridine. Modifications to the non-sense strand are also shown in the right-hand column of sequence 6.

[0199]

[0208] As shown in Figure 6, siRNA ARIZ-047 alone (without cisplatin) was highly effective in killing A549 lung cancer cells (92% cell death) with only limited harm to healthy cells (normal colon fibroblasts CCD-112). These results demonstrate that modified siRNA (ARIZ-047) is highly effective in killing cancer cells at a dose of 20 nM, which is one-fifth the dose of unmodified ARIZ-011, while having minimal impact on normal cells. Furthermore, ARIZ-047, when used in combination with cisplatin, was significantly more effective than ARIZ-047 alone (98% cell death), but caused substantially greater harm to healthy cells than ARIZ-047 alone (50% death).

[0200]

[0209] Similar to previous data for lung and colon cancer, the RIZ mRNA assay results for lung cancer cells, shown in Figure 7, show an increase in RIZ1 mRNA and a decrease in RIZ2 mRNA. Here again, these results, which demonstrate an effect on RIZ1 levels, further suggest that increased RIZ2 expression leads to a decrease in RIZ1 expression.

[0201]

[0210] Example 4: Effect of siRNA on multiple myeloma cells

[0211] In this example, the efficacy of siRNA ARIZ-011 was further tested in the multiple myeloma cell line KMS-11. The KMS-11 cell line was obtained from a commercially available supplier and originally derived from cells of four multiple myeloma patients. These cells possessed plasma cell characteristics. Using a protocol similar to that described in the previous section, ARIZ-011 was observed to play a significant role in KMS-11 cell death in culture medium, comparable to that of Tox-positive controls, at approximately 80% (Figure 8). These results indicate that ARIZ-011 is effective across multiple cancer cell lines and could serve as a means for developing cancer therapies that target dysregulation and hyperproliferation at cell cycle stages.

[0202]

[0212] Example 5: Targeted Drug Delivery Design

[0213] An exemplary design for a targeted drug delivery system for cancer treatment using siRNA disclosed herein is shown herein. The carrier is a tumor-targeting, self-assembling peptide nanoparticle consisting of a cyclic peptide target ligand conjugated with 8-amino-3,6-dioxaoctanoic acid-β-maleimidopropionic acid conjugated with anti-RIZ siRNA, which can specifically deliver the siRNA payload to cancer cells expressing the target receptor in animal models.

[0203]

[0214] The aforementioned siRNA ARIZ-047 is complexed with the cyclic peptide RGDfk to form (RGDfk) / siRNA (Figure 9). This siRNA binds to a PEG molecule via a short linker and has RGDfk at the other end.

[0204]

[0215] Example 6. In vivo testing of siRNA efficacy

[0216] A lung adenocarcinoma tumor model with A549 cells was prepared using female thymus-deficient nude mice. Mice carrying A549 lung cancer tumor xenografts were intravenously injected with c(RGDfk) / siRNA once daily. Tumor size was monitored in relation to the effect of c(RGDfk) / siRNA on the mice. After 14 days, the mice were sacrificed, and the tumors were excised and evaluated. Blood and other tissues were collected to examine mRNA levels for RIZ1, RIZ2, and other genes of interest by RT-PCR.

[0205]

[0217] The use of targeted drugs, such as siRNA molecules conjugated with RGDfk, as described here eliminates the need for liposomes or other nanoparticle-based delivery systems. This reduces or avoids the toxicity associated with the use of liposomes or other types of nanoparticle delivery systems, which can result from their accumulation in the liver and other organs.

[0206]

[0218] Example 7: In vivo delivery using liposomes

[0219] In this exemplary study, a targeted drug delivery system for cancer therapy, containing calcium phosphate nanoparticles that present a target ligand, is used to specifically deliver a PRDM2 siRNA payload to cancer cells in an animal model.

[0207]

[0220] Nanoparticles capable of supporting ARIZ-047 and further presenting a target peptide c (RGDfk) that targets human A549 lung cancer cells are prepared according to the method described by Parette et al. (U.S. Patent No. 10,226,424). Briefly, a 21-base pair or 25-base pair siRNA is first complexed with a PEG moiety. In phosphoamide chemistry, this involves complexing an siRNA with a 5'PO4 terminal group with an amine-terminated methoxyPEG molecule, while in thioether chemistry, this involves complexing an siRNA with a 5'-sulfhydryl terminal group with a maleimide-terminated methoxyPEG molecule. To achieve phosphoamide complexation, 0.12 M N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 47.6 μM single-stranded siRNA (inactive chain containing a 5'PO4 group), and 0.238 M amine-PEG are dissolved in 0.1 M imidazole at pH 6 and incubated at 50°C for 18 hours. This reaction typically yields 15–30% complexation. Alternatively, adding 0.1 M MES buffer, pH 4, to the phosphoamide reaction mixture yields a complexation efficiency of 75–90%. In thioether chemistry, 200 μM single-stranded siRNA (inactive strand containing a 5'PO4 group) is combined with 0.476 M amine-PEG in the presence of 0.02 M DTT in 0.1 M Tris HCl buffer, and the reaction mixture is incubated at 25°C for 18 hours. This reaction yields approximately 50% complexation. These reactions were carried out using 2 kDa and 5 kDa PEG molecules. After complexation, the inactive (sense) strand is annealed to the uncomplexed active (antisense) RNA strand in the reaction mixture by heating to 70°C and then slowly cooling. For highly efficient (75-90%) phosphoamide reactions, uncomplexed, annealed siRNA containing either a 5'PO4 group in both the active (antisense) and inactive (sense) chains, or one 5'PO4 group in the active (antisense) chain, is added to the reaction mixture as described below to facilitate desalting and purification.The presence of 5'PO4 groups in the inert (sense) chain results in particles with a more negative charge, while the absence of these groups causes the particle's charge to shift towards neutrality.

[0208]

[0221] The particles are washed by centrifugation filtration with 5% glucose containing calcium and phosphate to remove unincorporated siRNA and siRNA-PEG complexes. Alternatively, unincorporated siRNA, Ca, Cl, Na, and PO4 can be isolated from the suspension by ultracentrifugation at 132,000 g, resulting in particle collection of the bottom 10% of the sample volume. Separating the bottom 10% of the sample volume containing these particles reduces the unincorporated residual components by up to 90%. siRNA capture within the particle range is sequence-dependent and ranges from 10 to 25% of the siRNA added in the synthesis. The synthesis described herein yields calcium phosphate nanoparticles containing siRNA with a unimodal particle size distribution of 160 nm or less, as shown below.

[0209]

[0222] This nanoparticle preparation was administered intravenously to mice having A549 lung cancer tumor xenografts as described in the previous examples. The mice were then administered siRNA at a dose of 1 mg / kg of ARIZ-047 encapsulated in the nanoparticles described in this section. In some embodiments, these nanoparticles are referred to as "nanojackets." The mice were then observed for tumor size and any toxic effects of the treatment to determine therapeutic efficacy. Most surprisingly, ARIZ-047 showed effective control of tumor growth over the observed 25 days (Figure 10). Tumor growth was suppressed by approximately 50% compared to the non-siRNA reference control. Claims at the time of international application [Section 1] A method for inhibiting cell proliferation, comprising contacting a population of cells with a composition comprising a retinoblastoma protein-interacting zinc finger protein 2 (RIZ2) inhibitor. [Section 2] The method according to claim 1, wherein the RIZ2 inhibitor reduces the expression of RIZ2 mRNA in the cells. [Section 3] The method according to claim 1 or 2, wherein the RIZ2 inhibitor reduces the expression of the RIZ2 protein in the cells. [Section 4] The method according to claim 2, wherein the RIZ2 mRNA is reduced by at least 10% compared to the expression of housekeeping genes in the cells. [Section 5] The method according to claim 2 or 4, wherein the RIZ2 mRNA is reduced by at least 10% compared to a control cell population, and the control cell population is a cell population that has not been in contact with the composition containing the RIZ2 inhibitor. [Section 6] The method according to claim 5, wherein the expression of RIZ2 mRNA is reduced by at least 50% compared to the control cell population. [Section 7] The method according to claim 5, wherein the expression of RIZ2 mRNA is reduced by at least 80% compared to the control cell population. [Section 8] The method according to claim 1, wherein the RIZ2 inhibitor alters the ratio of RIZ1 to the RIZ2 mRNA expression level to at least 1.1 times. [Section 9] The method according to claim 1, wherein the RIZ2 inhibitor alters the ratio of RIZ1 to the RIZ2 mRNA expression level to at least 1.5 times. [Section 10] The method according to claim 1, wherein the RIZ2 inhibitor alters the ratio of RIZ1 to the RIZ2 mRNA expression level by at least twofold. [Section 11] The method according to the claim, wherein the RIZ2 inhibitor further reduces hypermethylation of RIZ1. [Section 12] The method according to claim 1, wherein the cells are mammalian cells. [Section 13] The method according to claim 1, wherein the cells are human cells. [Section 14] The method according to claim 1, wherein the cells are cancer cells. [Section 15] The method according to claim 1, wherein the composition comprising a RIZ2 inhibitor comprises one or more polynucleotides bound to a delivery system. [Section 16] The method according to claim 15, wherein the RIZ2 inhibitor is a double-stranded RNA molecule comprising a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 10. [Section 17] The method according to claim 15, wherein the RIZ2 inhibitor is a double-stranded RNA molecule comprising a sense strand having the sequence of SEQ ID NO: 6 and an antisense strand having the sequence of SEQ ID NO: 11. [Section 18] The method according to claim 15, wherein the delivery system comprises one or more of the following: lipids, cyclodextrins, chitosan, carbohydrate polymers, elastin-like polymers (ELPs), calcium phosphate polymers, or combinations thereof. [Section 19] The method according to claim 1, wherein the cells are derived from tissue which is breast, colon, endometrium, esophagus, stomach, glioma, kidney, liver, lung, lymphoma, melanoma, meningioma, myeloma, nasopharynx, neuroblastoma, ovary, pancreas, parathyroid gland, pituitary gland, prostate, thyroid gland, or uterine tissue. [Section 20] A method for treating a cell proliferation disorder or impairment in a subject, comprising administering a composition comprising a RIZ2 inhibitor to the subject. [Section 21] A method for selecting patients who require administration of a composition containing a RIZ2 inhibitor, the method being: (i) Detect an increase in the RIZ1 methylation level in the target biological sample compared to a control sample or control value, or (ii) This includes detecting a decrease in RIZ1 mRNA levels compared to a control sample or control value, Compared to the control sample or the control value, a 1.1-fold increase in methylated RIZ1 or a 1 / 2 decrease in RIZ1 mRNA levels in the subject's biological sample indicates that the subject needs to be administered the composition containing the RIZ2 inhibitor. The aforementioned control sample is a sample from a clinically healthy individual. The control value is the mean RIZ1 methylation level from samples of two or more clinically healthy individuals, according to the method. [Section 22] The method according to claim 20, wherein the cell proliferation disorder or disorder is cancer. [Section 23] The method according to claim 1, 19, or 20, further comprising a delivery system. [Section 24] A pharmaceutical composition comprising (a) a RIZ2 inhibitor and (b) a pharmaceutically acceptable excipient. [Section 25] The pharmaceutical composition according to claim 24, wherein the RIZ2 inhibitor is a double-stranded RNA molecule comprising a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 10. [Section 26] The pharmaceutical composition according to claim 24, wherein the RIZ2 inhibitor is a double-stranded RNA molecule comprising a sense strand having the sequence of SEQ ID NO: 6 and an antisense strand having the sequence of SEQ ID NO: 11. [Section 27] The aforementioned RIZ2 inhibitor is (i) Containing siRNA containing a 10-21 nucleotide double-stranded RNA homologous to the human PRDM2 / RIZ2 gene, (ii) Covalently bonded to the PEG molecule, (iii) It binds to the cell target region, The pharmaceutical composition according to claim 24, comprising the siRNA wherein the cell target portion is an aptamer. [Section 28] The pharmaceutical composition according to claim 24, further comprising nanoparticles containing a calcium phosphosilicate complex incorporating the RIZ2 inhibitor. [Section 29] The pharmaceutical composition according to claim 24, further comprising one or more chemotherapy agents.

Claims

1. A pharmaceutical composition for use as a pharmacopoeia in the treatment of a cell proliferation disorder or cancer, comprising (a) a RIZ2 inhibitor and (b) a pharmaceutically acceptable excipient, wherein the RIZ2 inhibitor comprises a double-stranded inhibitory RNA molecule and a delivery system; The inhibitory RNA molecule is 10 to 21 nucleotides long, hybridizes to at least 10 adjacent nucleic acid bases on the PRDM2 gene or PRDM2 gene product, and contains one or more modified nucleotides. A pharmaceutical composition in which the one or more modified nucleotides include 2'-methoxyuridine, 5'-P-methoxyuridine, 2'-fluorodeoxyuridine, a nucleotide containing a 5'-disulfide moiety, and a nucleotide having a phosphorothioate bond.

2. The pharmaceutical composition according to claim 1, wherein the RIZ2 inhibitor comprises a sense strand having the sequence of SEQ ID NO: 1, or a modified sequence of SEQ ID NO: 1 containing one or more modified nucleotides, and an antisense strand having the sequence of SEQ ID NO: 10, or a modified sequence of SEQ ID NO: 10 containing one or more modified nucleotides.

3. The RIZ2 inhibitor is [Math 1] A sense strand having the sequence of (SEQ ID NO: 6) (where 4 is 2'-methoxyuridine), and [Math 2] Antisense chain having the sequence (wherein 4 is 2'-methoxyuridine; 6 is 2'-fluorodeoxyuridine; the bolded and underlined part is a phosphorothioate) (SEQ ID NO: 11) A pharmaceutical composition according to claim 1 or 2, comprising:

4. The RIZ2 inhibitor is (i) siRNA containing 10-21 nucleotide double-stranded RNA homologous to the human PRDM2 / RIZ2 gene; (ii) A PEG molecule bonded to (iii); (iii) Cell targeting moiety A pharmaceutical composition according to claim 1 or 2, comprising:

5. The pharmaceutical composition according to claim 1 or 2, further comprising nanoparticles incorporating the RIZ2 inhibitor.

6. The pharmaceutical composition according to claim 1 or 2, further comprising one or more chemotherapeutic agents.

7. The pharmaceutical composition according to claim 1 or 2, wherein the siRNA includes a protruding portion of 1, 2, or 3 nucleotides at its 5' or 3' end.

8. The pharmaceutical composition according to claim 7, wherein the siRNA includes a T overhang, and the T overhang comprises a single thymidine (T) or two thymidine nucleotide residues in tandem (e.g., dTdT).

9. The pharmaceutical composition according to claim 5, wherein the nanoparticles are calcium phosphate nanoparticles.

10. The aforementioned nanoparticles (i) siRNA comprising a sense strand having the sequence of SEQ ID NO: 6 and an antisense strand having the sequence of SEQ ID NO: 11; (ii) PEG molecule, and (iii) The pharmaceutical composition according to claim 5, comprising a cell targeting portion.

11. The pharmaceutical composition according to claim 10, wherein the cell targeting portion is selected from a ligand, an antibody, a cell-permeable peptide, a cyclic peptide, or an aptamer.

12. The pharmaceutical composition according to claim 10, wherein, when the siRNA is tested in vitro, it reduces the expression of RIZ2 mRNA in cells but does not reduce the expression of RIZ1 mRNA.

13. The pharmaceutical composition according to claim 10, wherein when the RIZ2 inhibitor is introduced into cells, it reduces the expression of RIZ2 mRNA in the cells and changes the ratio of RIZ1 to the RIZ2 mRNA expression level to at least 1.1 times.

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