RNA therapeutics for consumptive hypothyroidism induced osteoporosis and estrogen receptor positive breast cancer metastases

Abstract

Described herein are compositions, pharmaceutical compositions, kits, and methods of use relating to anti-Dio3os antisense RNA. In embodiments, antisense Dio3os compositions, kits, and methods are described to improve symptoms of osteoporosis, for example, osteoporosis induced by a disorder characterized by a thyroid hormone imbalance (i.e., hyperthyroidism or hypothyroidism). In embodiments, antisense Dio3os compositions, kits, and methods are described to improve sensitivity of cancer cells to anti-cancer therapeutics, for example, sensitivity of ER+ breast cancer or other breast cancer cells, thyroid cancer cells, prostate cancer cells, hepatocellular cancer cells, pancreatic cancer cells, and ovarian cancer cells that are sensitized or otherwise non-responsive to cancer therapeutics (for example, aromatase inhibitors or HDAC inhibitors). Combination therapies are also contemplated utilizing antisense RNA according to the present disclosure and other drugs, for example, thyroid and anti-cancer medications.

Description

[0001] TH Docket #: 222120-2090; U24-076

[0002] RNA THERAPEUTICS FOR CONSUMPTIVE HYPOTHYROIDISM INDUCED OSTEOPOROSIS AND ESTROGEN RECEPTOR POSITIVE BREAST CANCER METASTASES

[0003] CROSS-REFERENCE TO RELATED APPLICATION^]

[0004] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Serial No. 63 / 688,603 filed on August 29, 2024, the entire contents of which are incorporated herein by reference as if set forth in its entirety.

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on August 29, 2025, is named “222120_2090_sequence_listing.xml” and is 70,568bytes in size.

[0007] BACKGROUND

[0008] It is well recognized that an imbalance of thyroid hormones (TH) production causes hypothyroidism or hyperthyroidism, respectively, and can lead to abnormalities in bone growth and maturation during development and in bone density / fragility, and repair in adulthood. Although treatment can slow bone loss and improve bone density, osteoporosis has no cure because of the complex genetic and epigenetic mechanisms involved in this disorder that regulate the intracellular activity of thyroid hormones. Endocrine resistance remains a major clinical challenge in the treatment of estrogen receptor-positive (ER+) breast cancer, particularly among postmenopausal patients receiving aromatase inhibitors (Als). Up to 40% of ER+ tumors develop resistance to Als, resulting in relapse and 60-75% bone-dominant metastasis. Recent studies have identified specific non-coding RNAs as mechanistic drivers of drug therapy resistance in breast cancer, with emerging evidence suggesting they may also contribute to the increased risk of bone metastasis through dysregulation of bone epigenetic changes and controlling tumor gene expression.

[0009] Accordingly, there is a need to address the aforementioned deficiencies and inadequacies and, a need for novel osteogenic and anti-cancer therapeutics.

[0010] SUMMARY

[0011] Described herein are compositions, pharmaceutical compositions, kits, and methods relating to anti-Dio3os antisense RNA (ASO) and anti-Dio3os siRNA.

[0012] Described herein are compositions. In certain aspects, compositions described herein can comprise one or more anti-Dio3os antisense RNA, one or more anti-Dio3os siRNA, or any combination of any thereof. In certain aspects, compositions described herein can consist TH Docket #: 222120-2090; U24-076 essentially of or consist of one or more anti-Dio3os antisense RNA, one or more anti-Dio3os siRNA, or any combination of any thereof. In embodiments, each of the one or more anti- Dio3os antisense RNA, one or more anti-Dio3os siRNA, or any combination of any thereof contain at least 2 or more modified nucleotides. In certain aspects, the one or more anti- Dio3os antisense RNA comprise a nucleotide sequence that is at least 90% identical to any one or more of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In certain aspects, the one or more anti-Dio3os antisense RNA comprise a nucleotide sequence that is at least 95% identical to any one or more of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.

[0013] In certain aspects, the one or more anti-Dio3os antisense RNA comprise a nucleotide sequence of at least one or more of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In certain aspects, the one or more anti-Dio3os antisense RNA comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 34, SEQ ID NO: 35, or both. In certain aspects, the one or more anti-Dio3os antisense RNA comprise a nucleotide sequence of SEQ ID NO: 34, SEQ ID NO: 35, or both. In certain aspects, the one or more anti-Dio3os antisense RNA consist essentially of a nucleotide sequence of SEQ ID NO: 34, SEQ ID NO: 35, or both.

[0014] In certain aspects, the one or more anti-Dio3os siRNA RNA comprise one or more nucleotide sequences that are at least 90% identical to any one of more of SEQ ID NOs: 45- 50. In certain aspects, the one or more anti-Dio3os siRNA RNA comprise one or more nucleotide sequences that are at least 90% identical to any one of more of SEQ ID NOs: 45- 50. In certain aspects, the one or more anti-Dio3os siRNA RNA consist essentially of one or more or at least two nucleotide sequences that are at least 90% identical to any one or more of SEQ ID NOs: 45-50.

[0015] In certain aspects, one or more anti-Dio3os antisense RNA, siRNA, or both is present in a therapeutically effective amount. In certain aspects, the therapeutically effective amount is an amount effective to improve symptoms of osteoporosis in a subject in need thereof. In certain aspects, the subject in need thereof has or is suspected of having primary osteoporosis, secondary osteoporosis, or osteoporosis as a symptom of a disorder characterized by a thyroid hormone imbalance. In certain aspects, the therapeutically effective amount is an amount effective to increase sensitivity of one or more cancer cells to anti-cancer therapeutics. In certain aspects, the one or more cancer cells comprise ER+ breast cancer or other breast cancer cells, thyroid cancer cells, prostate cancer cells, hepatocellular cancer cells, pancreatic cancer cells, and ovarian cancer cells. In certain aspects, the anti-cancer therapeutics comprise an aromatase inhibitor, a histone deacetylase inhibitor, or a lactate dehydrogenase A (LDHA) inhibitor.

[0016] Described herein are pharmaceutical compositions. In certain aspects, described herein are pharmaceutical compositions, comprising as described herein and a pharmaceutically acceptable carrier. TH Docket #: 222120-2090; U24-076

[0017] Described herein are kits. In certain aspects, described herein is a kit for increasing osteogenesis or sensitivity of cancer cells, comprising a composition or a pharmaceutical composition described herein and instructions for use. In certain aspects, one or more components of the composition is lyophilized.

[0018] Described herein are methods of reducing the severity of one or more symptoms of osteoporosis in a subject, comprising administering a composition or a pharmaceutical composition described herein to a subject in need thereof. In certain aspects, the subject in need thereof has or is suspected of having a disorder causing osteoporosis, for example, menopause or a cancer. In certain aspects, the disorder causing osteoporosis is a disorder characterized by a thyroid hormone imbalance.

[0019] Described herein are methods of increasing sensitivity of cancer cells to anti-cancer therapeutics. In certain aspects, such methods comprise administering a composition or a pharmaceutical composition described herein. In certain aspects, the subject in need thereof has or is suspected of having ER+ breast cancer or other breast cancer, thyroid cancer, prostate cancer, hepatocellular cancer, pancreatic cancer, or ovarian cancer.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Many aspects of the disclosed devices and methods can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the relevant principles. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0022] FIGs. 1A-1G: Dio3-Dio3os locus and IncRNA Dio3os function are critical to osteogenesis: (FIG. 1A) Central Hypothesis: IncRNA Dio3os recruits NuRD complex to activate Dio3 (cis) and to repress OB-specific genes (trans) by promoting NuRD deacetylase activity. (FIG. 1B) Levels of Dio3os and Dio3 showed higher expression during early MC3T3- E1 differentiation. (FIG. 1C) Higher promoter accessibility (ATAC-seq) and active H3 modifications (ChlP-seq) of the Dio3-Dio3os promoter support higher transcriptional activities during osteoblast growth. (FIG. 1D) Dio3os, Dio3, Runx2, and Ocn mRNA upon overexpression of Dio3os. (FIG. 1E) Knockouts of Dio3os exons using CRISPR-Cas9. AEx1. Exon 1 (90 nt); 2) AEx1-4: Exons 1-4, including the intronic regions (2.7 kb). Lower panel: mRNA levels for Dio3os, Dio3 (left), and Ocn (right). (FIG. 1F) Insertion of polyadenylation signal at the end of the second (-621 nt), third (-1546 nt), and fourth exons (-2784 nt) of Dio3os gene using CRISPR-Cas9. Lower panel: mRNA levels for Dio3os, Dio3 (left), and Ocn (right). (FIG. 1H) Dio3os transcription repression by dCas9-KRAB. Lower panel: Dio3os, Dio3 (left), and Ocn (right) mRNA levels. TH Docket #: 222120-2090; U24-076

[0023] FIGs. 2A-2F: Phenotypic characterization of OB-specific CRISPRi mice to study adolescent bone synthesis (1 month). (FIG. 2A, female & FIG. 2B, male) Longitudinal microCT scans from CRISPRi + targeting sgRNA (T-sgRNA) (right) and non-targeting sgRNA (NT-sgRNA) (left) mice. Mice were sacrificed at 1 -month of age after receiving 10 mg / kg / day of 4-OH-TAM (l / P) and sgRNA lentivirus (109virus particles / kg / day, l / V) for four consecutive days. Femoral trabecular and cortical microCT scans of one-month-old female (FIG. 2C) and male (FIG. 2D) from CRISPRi + T-sgRNA (right) and NT-sgRNA (left) mice. MicroCT quantitation of femoral trabecular bone of female (FIG. 2E) and male (FIG. 2F) from CRISPRi + T-sgRNA (black bar) and A / T-sgRNA (grey bar) mice. Representative graphs indicate bone volume over tissue volume, trabecular number, thickness, and space. Quantitation data here comply with CRISPRi, n=7 (T-sgRNA) and n=5 (NT-sgRNA) female and n=7 CRISPRi (T- sgRNA) and n=6 (NT-sgRNA) male mice. * p < 0.05, “ p < 0.005, *** p < 0.0005. 4-OH-Tam: 4-hydroxytamoxifen. NT-sgRNA: non-targeting sgRNA, and T-sgRNA: targeting sgRNA.

[0024] FIGs. 3A-3F: Phenotypic characterization of OB-specific CR / SPRa mice to study adolescent bone synthesis (1 month). (FIG. 3A, female & FIG. 3B, male) Longitudinal microCT scans from CR / SPRa + targeting sgRNA (T-sgRNA) (right) and non-targeting sgRNA (NT-sgRNA) (left) mice. Mice were sacrificed at 1 -month of age after receiving 10 mg / kg / day of 4-OH-TAM (l / P) and sgRNA lentivirus (109virus particles / kg / day, l / V) for four consecutive days. Femoral trabecular and cortical microCT scans of one-month-old female (FIG. 3C) and male (FIG. 3D) from CR / SPRa + T-sgRNA (right) and NT-sgRNA (left) mice. MicroCT quantitation of femoral trabecular bone of female (FIG. 3E) and male (FIG. 3F) from CR / SPRa + T-sgRNA (black bar) and A / T-sgRNA (grey bar) mice. Representative graphs indicate bone volume over tissue volume, trabecular number, thickness, and space. Quantitation data here comply with CRISPRa, n=7 (T-sgRNA) and n=5 (NT-sgRNA) female and n=7 CR / SPRa (T- sgRNA) and n=6 (NT-sgRNA) male mice. * p < 0.05, ** p < 0.005, *** p < 0.0005. 4-OH-Tam: 4-hydroxytamoxifen. NT-sgRNA: non-targeting sgRNA, and T-sgRNA: targeting sgRNA.

[0025] FIGs. 4A-4C: ATAC sequencing verified globally enhanced chromatin accessibility in bone-specific genes in Dio3os knockout cells (FIG. 4A) Upper panel showing peaks annotated to the nearest transcription start site (TSS) of genes or regulatory elements in the promoter when compared Dio3os knockout, Dio3os knockout+T4 over control MC3T3-E1 cells. Lower panel: Heatmaps with transposase hypersensitive sites (THSs) of Dio3os regulated promoters in control, Dio3os knockout, and Dio3os knockout+T4 MC3T3-E1 cells. The regions in the heatmaps are ranked from highest ATAC-seq signal (top) to lowest (bottom) and aligned to mm10 mouse genome. (FIG. 4B) Barplot showing the accessibility distribution of reproducible peaks of all seven regions. Bottom scale showing percentage of open chromatin within 1-3 kb promoter and other regions of a gene. (FIG. 4C) Alp staining in TH Docket #: 222120-2090; U24-076 control, Dio3os knockout, and Dio3os knockout+T4 MC3T3-E1 cells at day 14 of osteoblast differentiation.

[0026] FIGs. 5A-5C: RNA and ATA-seq together uncovered a group of IncRNA Dio3os regulated thyroid hormone-responsive and osteoblast-specific genes. (FIG. 5A) RNA- seq analysis uncovered 285 increased and 367 decreased out of total 652 genes significantly altered in, Dio3os knockout when compared with control MC3T3-E1 cells. FIG. 5B is a heatmap of gene expression with Iog2 fold change values in control, Dio3os knockout MC3T3- E1 cells. Thyroid hormone cis-regulatory elements were revealed using Ensembl and Transfac bioinformatics. The color scale is based on the relative fold change in expression. FIG. 5C illustrates genomic tracks of ATAC-seq data were derived from standard protocol. X- axis, genomic coordinates; Y-axis, normalized ATAC-seq read counts. Gene names are shown at the top and transcription direction bottom. The RNA-seq and ATAC-seq data were aligned to the mm10 mouse genome.

[0027] FIGs. 6A-6C: RNA immunoprecipitation with Dio3os followed by mass spectrometry analysis identified NuRD complex as Dio3os interactome. (FIG. 6A) Volcano plot showing quantitative protein profiles that are interacting with IncRNA Dio3os (-Iog10(adjusted P) > 20, log2(FC) > 0.5) between in vitro transcribed, biotin-labeled control luciferase and Dio3os RNAs. NuRD complex members are presenting in red. (FIG. 6B) Peptide count from protein profiling showing enrichment of RBBP7, MBD3, CHD3, MTA1 , MTA2, CHD4, HDAC1 , P66A, and P66B NuRD members and co-repressor SIN3B. (FIG. 6C) Reverse pulldown of Dio3os with MBD3, MTA1 and HDAC1 antibodies using UV-crosslinked MC3T3-E1 sonicated whole cell lysate. Y-Axis represents fold enrichment over non-specific IgG pulldown.

[0028] FIG. 7: Dio3os recruits NuRD complex to the Dio3 and Ocn promoters. ChIP assays with indicated antibodies against MTA1 , MTA2, MBD3, HDAC1 , and H3K27ac to determine the binding on Dio3os and Ocn proximal promoters in control (-sgRNA) and Dio3os.

[0029] FIG. 8: Levels of Dio3os showed higher expression in head and neck squamous carcinoma (FaDu), ER+ breast cancer (MCF-7), and prostate cancer (PC3) cells. Normal squamous, breast epithelial, and LNCaP (less metastatic) cells were used as controls.

[0030] FIGs. 9A-9C: (FIG. 9A) Upper panel: Dio3os exon deletion was performed using CRISPR cas9 + exon-specific sgRNAs. WT, exon 1 deleted (A1 ) , Exons 1-4 deleted (A1-A4), and exons 2-4 deleted (A2-A4). Lower panel: MC3T3-E1 cells were transfected with WT and exon deleted Dio3os promoter-Luc plasmids, + Hdacl , and Hdac1+ Hdacl inhibitor, and luciferase activity was measured 48 hrs. post-transfection. (FIG. 9B) Dio3os RNA-IP-Mass spec Volcano plot shows quantitative protein profiles interacting with IncRNA Dio3os. HDAC1 is among the highest interacting proteins presented in red. (FIG. 9C) RNA expression analysis of Dio3os 48 hrs. post using two Dio3os antisense NAT 1 and NAT 2) treatment. TH Docket #: 222120-2090; U24-076

[0031] FIGs. 10A-10F are micrographs of proliferation of MCF-7 (estrogen-sensitive breast cancer) cells with and without antisense Dio3os RNA (FIGs. 10A, 10D), with and without antisense Dio3os RNA and Estradiol (FIGs. 10B, 10E), and with and without antisense Dio3os RNA, Estradiol, and the aromatase inhibitor (Al) letrozole (FIGs. 10C, 10F).

[0032] FIG. 11 shows Dio3os promotes phosphorylation of the RB protein in highly invasive ER+ breast cancer cell MCF7.

[0033] FIG. 12 is a graph depicting highly invasive estrogen-deprived ER+ breast cancer cell MCF7-5C cells increase Dio3os and aerobic glycolysis marker Ldha.

[0034] FIG. 13 is a graph showing antisense RNAs ASO159, 566, 306, and siRNAs 1 ,2, and 3 successfully reduce Dio3os expression.

[0035] FIG. 14 is a graph showing a combination of ASO159 or siRNA3 with the HDAC inhibitor SAHA further reduced Dio3os expression.

[0036] FIG. 15 is a graph showing co-treatment with an LDH inhibitor or the aromatase inhibitor (Al) anastrozole enhances Dio3os suppression. Notably, despite the cells being resistant to Al , ASO 159 in combination with anastrozole appears to reverse this resistance, highlighting a promising therapeutic strategy for treating Al-resistant breast cancer.

[0037] FIG. 16: DepMap CRISPR / siRNA screening in three highly invasive metastatic ER+ breast cancer cell lines (MCF7, T47D, and KPL1), compared to estrogen-deprived Al-resistant T47D cells’ RNA sequencing, identifies 25 tumor suppressors with low Z-scores, suggesting potential Dio3os-regulated pathways.

[0038] DETAILED DESCRIPTION

[0039] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0041] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and TH Docket #: 222120-2090; U24-076 features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0042] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of genetics, biochemistry, molecular biology, cellular biology, tissue culture, therapeutic administrations and the like.

[0043] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0044] Definitions

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.

[0046] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0047] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject-matter.

[0048] The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context, for example, ±5%, ±4%, ±3%, ±2%, etc.

[0049] Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered TH Docket #: 222120-2090; U24-076 to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0050] As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions can reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0051] Those skilled in the art will appreciate that the term “composition”, as used herein, can be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition can be of any suitable form - e.g. , gel, liquid, solid, etc.

[0052] A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential to a particular aspect or embodiment, but other elements or steps can be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and can also include additional elements or steps that do not materially affect the basic and novel TH Docket #: 222120-2090; U24-076 characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of' one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of (or "consists of) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step can be substituted for that element or step.

[0053] In this disclosure, "consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0054] As used herein, “Improved,” “increased” or “reduced,” or grammatically comparable comparative terms, indicate values that are relative to a baseline value or reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained or expected in the absence of treatment or with a comparable reference agent or control. Alternatively, or additionally, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g. , prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance.

[0055] As used herein, “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, TH Docket #: 222120-2090; U24-076 polypeptide, protein, antibody, or fragments thereof, do not require “isolation” to distinguish it from its naturally occurring counterpart.

[0056] As used herein, the term “encode” refers to principle that DNA can be transcribed into RNA, which can then be translated into amino acid sequences that can form proteins

[0057] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0058] As used herein, the term “specific binding” or “preferential binding” can refer to non- covalent physical association of a first and a second moiety wherein the association between the first and second moieties is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 times as strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs. Binding of two or more entities may be considered specific if the equilibrium dissociation constant, Kd, is 10~3M or less, 10~4M or less, 10~5M or less, 10~6M or less, 10~7M or less, 10~8M or less, 10~9M or less, 10~10M or less, 10-11M or less, or 10“12M or less under the conditions employed, e.g., under physiological conditions such as those inside a cell or consistent with cell survival. In some embodiments, specific binding can be accomplished by a plurality of weaker interactions (e.g., a plurality of individual interactions, wherein each individual interaction is characterized by a Kd of greater than 10-3M). In some embodiments, specific binding, which can be referred to as “molecular recognition,” is a saturable binding interaction between two entities that is dependent on complementary orientation of functional groups on each entity. Examples of specific binding interactions include primer-polynucleotide interaction, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metalchelate interactions, hybridization between complementary nucleic acids, etc.

[0059] As used herein, the term “recombinant” or “engineered” can generally refer to a non- naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc. Recombinant or engineered can also refer to the polypeptide encoded by the recombinant nucleic acid. Non-naturally TH Docket #: 222120-2090; U24-076 occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

[0060] As used herein, “variant” can refer to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains essential and / or characteristic properties (structural and / or functional) of the reference polynucleotide or polypeptide. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. The differences can be limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in nucleic or amino acid sequence by one or more modifications at the sequence level or post-transcriptional or post-translational modifications (e.g., substitutions, additions, deletions, methylation, glycosylations, etc.). A substituted nucleic acid may or may not be an unmodified nucleic acid of adenine, thiamine, guanine, cytosine, uracil, including any chemically, enzymatically or metabolically modified forms of these or other nucleotides. A substituted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. “Variant” includes functional and structural variants.

[0061] As used herein, "organism", "host", and "subject" refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single isolated eukaryotic cell or cultured cell or cell line, or as complex as a mammal, including a human being, and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans).

[0062] As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0063] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet TH Docket #: 222120-2090; U24-076 website, or as recorded presentation. Instructions can comprise one or multiple documents and are meant to include future updates.

[0064] Reference throughout this specification to “one embodiment”, “an embodiment”, “another embodiment”, “some embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” “in another embodiment”, or “in some embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but they may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other, features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0065] A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample or condition. For example, a test sample can include cells exposed to a test condition or a test agent, while the control is not exposed to the test condition or agent (e.g., negative control). The control can also be a positive control, e g., a known primary cell or a cell exposed to known conditions or agents, for the sake of comparison to the test condition. A control can also represent an average value gathered from a plurality of samples, e.g., to obtain an average value. For therapeutic applications, a sample obtained from a patient suspected of having a given disorder or deficiency can be compared to samples from a known normal (non-deficient) individual. A control can also represent an average value gathered from a population of similar individuals, e.g., patient having a given deficiency or healthy individuals with a similar medical background, same age, weight, etc. A control value can also be obtained from the same individual, e.g., from an earlier-obtained sample, prior to the disorder or deficiency, or prior to treatment. One of skill will recognize that controls can be designed for assessment of any number of parameters.

[0066] The term “biological sample” encompasses a variety of sample types obtained from an organism or a cell line. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term includes a clinical sample, and includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. TH Docket #: 222120-2090; U24-076

[0067] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0068] The term “normal” as used in the context of “normal cell,” is meant to refer to a cell of an untransformed phenotype or exhibiting a morphology of a non-transformed cell of the tissue type being examined. A “cancer cell” refers to a cell of a cancer that can be identified by abnormalities in, for example, cell growth or proliferation (e.g., uncontrolled growth or proliferation), regulation of the cell cycle, cell mobility, cell-cell interaction, or metastasis, etc.

[0069] The term “clinical well-being” as used herein, refers to a state or degree of clinical or physiological wellness or health of a patient. A clinician can evaluate a patient’s clinical wellbeing by physical examination or performing one or more tests or assays.

[0070] “Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity of a described target protein (or encoding polynucleotide), e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term “modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression or bind to, partially or totally block stimulation or protease inhibitor activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a described target protein or bind to, stimulate, increase, open, activate, facilitate, enhance activation or protease inhibitor activity, sensitize or up regulate the activity of described target protein (or encoding polynucleotide), e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists and agonists (e.g., small chemical molecules, antibodies and the like that function as either agonists or antagonists). Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to cells expressing the described target protein and then determining the functional effects on the described target protein activity, as described above. Samples or assays comprising described target protein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition of a described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of the described target protein is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or more higher.

[0071] The terms “administering,” “delivering,” and “introducing,” can be used interchangeably to indicate the introduction of a therapeutic composition or agent (e.g., cells, exosomes, and / or natriuretic peptides) into the body of a subject. The therapeutic composition TH Docket #: 222120-2090; U24-076 or agent can be administered through any appropriate means that results in the delivery of at least a portion of the composition or agent to a desired location in the subject such that the composition or agent retains its therapeutic capability. Useful methods of delivering the therapeutic include, but are not limited to, intravenous delivery, subcutaneous delivery, intradermal delivery, intracoronary delivery, intracardiac delivery, oral delivery, or any combination thereof.

[0072] The term “administered continuously” refers to the continuous delivery of a therapeutic agent, e.g., compound, molecule, peptide, biologic, chemical, etc. over a 24-hour period or longer.

[0073] The term “therapeutically effective amount” refers to an amount of therapeutic agent effective to treat ( / .e., reduce the severity of or eliminate) at least one symptom of a disease or disorder in a subject. In other words, such an amount is sufficient to bring about a beneficial or desired clinical effect. The “therapeutically effective amount” of the agent for administration may vary based upon the desired activity, the diseased state of the subject being treated, the dosage form, method of administration, subject factors such as the subject's sex, genotype, weight and age, the underlying causes of the condition or disease to be treated, the route of administration and bioavailability, the persistence of the administered agent in the body, evidence of natriuresis and / or diuresis, the type of formulation, and the potency of the agent.

[0074] As used herein, the terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

[0075] The terms “therapy,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms, e.g. , of a neurodegenerative disorder or neuronal injury. As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, improved cognitive function or coordination, increase in survival time or rate, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.

[0076] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotide described TH Docket #: 222120-2090; U24-076 herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are nonlimiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0077] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0078] As used herein, “cDNA” refers to a DNA sequence that is complementary to an RNA transcript in a cell. It is a man-made molecule. Typically, cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates.

[0079] As used herein with reference to the relationship between DNA, cDNA, cRNA, RNA, protein / peptides, and the like “corresponding to” or “encoding” (used interchangeably herein) refers to the underlying biological relationship between these different molecules. As such, one of skill in the art would understand that operatively “corresponding to” can direct them to determine the possible underlying and / or resulting sequences of other molecules given the TH Docket #: 222120-2090; U24-076 sequence of any other molecule which has a similar biological relationship with these molecules. For example, from a DNA sequence an RNA sequence can be determined and from an RNA sequence a cDNA sequence can be determined.

[0080] As used herein, “gene” can refer to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. The term gene can refer to translated and / or untranslated regions of a genome. “Gene” can refer to the specific sequence of DNA that is transcribed into an RNA transcript that can be translated into a polypeptide or be a catalytic RNA molecule, including but not limited to, tRNA, siRNA, piRNA, miRNA, long- non-coding RNA and shRNA.

[0081] As used herein, the term “exogenous DNA” or “exogenous nucleic acid sequence” or “exogenous polynucleotide” refers to a nucleic acid sequence that was introduced into a cell, organism, or organelle via transfection. Exogenous nucleic acids originate from an external source, for instance, the exogenous nucleic acid may be from another cell or organism and / or it may be synthetic and / or recombinant. While an exogenous nucleic acid sometimes originates from a different organism or species, it may also originate from the same species (e.g., an extra copy or recombinant form of a nucleic acid that is introduced into a cell or organism in addition to or as a replacement for the naturally occurring nucleic acid). Typically, the introduced exogenous sequence is a recombinant sequence.

[0082] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et a / . (2001) Gene Therapy 8:1 -4 and TH Docket #: 222120-2090; U24-076

[0083] Prochiantz (2007) Nat. Methods 4:119-20. The skilled artisan would understand that nucleic acids according to the present disclosure can be created with any known method in the art, including, utilizing an expression vector for expression of the nucleic acid followed by subsequent purification.

[0084] The word "expression" or "expressed" as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0085] Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression" the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. Expression of a transfected gene can further be accomplished by transposon- mediated insertion into to the host genome. During transposon-mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that allow insertion into the host genome as well as subsequent excision.

[0086] The term "plasmid" refers to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0087] The term "episomal" refers to the extra-chromosomal state of a plasmid in a cell. Episomal plasmids are nucleic acid molecules that are not part of the chromosomal DNA and replicate independently thereof.

[0088] The term “exogenous” refers to a molecule or substance (e.g. , nucleic acid or protein) that originates from outside a given cell or organism. Conversely, the term “endogenous” refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0089] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. In some embodiments, vectors of use in the invention are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". Thus, an "expression vector" is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell. In some embodiments the gene of interest is operably TH Docket #: 222120-2090; U24-076 linked to another sequence in the vector, e.g., a promoter. Vectors include non-viral vectors such as plasmids and viral vectors.

[0090] A "viral vector" is a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of nucleic acids or a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In particular, viral vectors can also be utilized and packaged within a functional virus, where the virus then infects a cell and delivers the viral vector to the cell through viral transduction.

[0091] The term “operably linked” refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence, such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operably linked nucleic acid sequences need not be physically adjacent to each other. The term “operably linked” also refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence.

[0092] The terms "regulatory sequence" and "promoter" are used interchangeably herein, and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operatively linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell- type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally occurring form of a protein. In some instances, the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required for initiating transcription of a specific gene.

[0093] "Expression cassette" refers to a polynucleotide comprising a promoter or other regulatory sequence operably linked to a sequence encoding a protein or IncRNA described herein.

[0094] The term “siRNA” refers to a ribonucleic acid that forms a double stranded RNA, which double stranded RNA can reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. In the context of this invention, the term “siRNA” thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, TH Docket #: 222120-2090; U24-076 an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. The sequence of the siRNA can correspond to the full-length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0095] The term “shRNA” refers generally to an siRNA that is introduced into a cell as part of a larger DNA construct. Typically, such constructs allow stable expression of the siRNA in cells after introduction, e.g., by integration of the construct into the host genome.

[0096] An "antisense" oligonucleotide or polynucleotide is a nucleotide sequence that is substantially complementary to a target polynucleotide or a portion thereof and can specifically hybridize to the target polynucleotide.

[0097] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids. The term “protein” as used herein refers to either a polypeptide or a dimer (i.e., two) or multimer ( / .e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions. The terms “portion” and “fragment” are used interchangeably herein to refer to parts of a polypeptide, nucleic acid, or other molecular construct.

[0098] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. TH Docket #: 222120-2090; U24-076

[0099] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0100] The term “a recombination of amino acid sequences,” in the context of a peptide, refers to a change or variation in the amino acid sequence of a reference peptide, such that the biological properties of the reference peptide are maintained after the amino acid sequence change. For example, the recombination of amino acid sequence may be a conservative amino acid substitution or an amino acid sequence modification (addition, deletion or substitution) to produce a chimeric peptide.

[0101] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of the naturally occurring amino acids, unnatural amino acids and chemically modified amino acids. Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Zhang et al. “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4): 581-87 (2013); Xie et al. “Adding amino acids to the genetic repertoire, "Curr. Opin. Chem. Biol. 9(6): 548-54 (2005); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0102] In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows, for example: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V). “Protein” and “Polypeptide” can refer to a molecule composed of one or more chains of amino acids in a specific order. The term protein is used interchangeable with “polypeptide.” The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins can be involved in the structure, function, and regulation of various functions.

[0103] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. A side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post- translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0104] The term “identity” or “substantial identity,” as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% TH Docket #: 222120-2090; U24-076 sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0105] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0106] A “comparison window,” as used herein, includes reference to a segment of any one of the numbers of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0107] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul etal. (1977)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be TH Docket #: 222120-2090; U24-076 increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1 , N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0108] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01 , more preferably less than about 105, and most preferably less than about 1020. Discussion

[0109] Described herein are compositions, pharmaceutical compositions, and methods relating to anti-sense RNA to the long non-coding RNA (IncRNA) Dio3os (also referred to herein as “anti-Dio3os RNA” or “anti-Dio3os RNAs”). In particular, compositions and pharmaceutical compositions relating to novel anti-Dio3os RNAs are described herein. Methods of use, including methods of treatment, which comprise administering anti-Dio3os RNA[s] to subjects in need thereof are also described herein.

[0110] In some aspects, the combined use of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) offers complementary mechanisms for effective target inhibition. ASOs are particularly effective against nuclear-enriched or regulatory long non-coding RNAs (IncRNAs) by inducing RNase H-mediated degradation or blocking RNA-protein interactions, while siRNAs cleave cytoplasmic transcripts via the RISC pathway. This dual approach can ensure broad coverage across subcellular compartments, enhances on-target effect reproducibility, and allows for optimized delivery tailored to specific tissues. Ultimately, it boosts confidence in target validation, offers flexibility for clinical translation, aligns with regulatory standards, and mitigates development risks for RNA-based therapies. TH Docket #: 222120-2090; U24-076

[0111] In some aspects, using a dual approach with ASO and siRNA provides good coverage across different parts of the cell. It enhances the consistency of targeted effects and enables tailored delivery to specific tissues. These aspects can increases confidence in validating targets, offering flexibility for clinical use, meeting regulatory requirements, and reducing risks in developing RNA-based therapies. In certain aspects, the combination may be ASO+ASO, ASO+siRNA, or ASO+ASO+siRNA. The skilled artisan would also recognize, that, according to the present disclosure, one or more ASO may be utilized in compositions, kits, and methods described herein; one or more siRNA may be utilized in compositions, kits, and methods herein; or any combination of any one or more ASO and any one or more siRNA.

[0112] I. INTRODUCTION

[0113] It is well recognized that an imbalance of thyroid hormones (TH) production causes hypothyroidism or hyperthyroidism, respectively, and can lead to abnormalities in bone growth and maturation during development and in bone density / fragility and repair in adulthood. Although treatment can slow bone loss and improve bone density, osteoporosis has no cure because of the complex genetic and epigenetic mechanisms involved in this disorder that regulate the intracellular activity of thyroid hormones.

[0114] Aspects of the present disclosure are based, in part, on preliminary studies indicating that the Dio3 imprinted locus and Dio3os IncRNA are critical regulators for osteogenesis. LncRNAs are a unique class of long noncoding RNA transcripts that influence the chromatin state of protein-coding genes and universally control biologic processes, including osteoblastogenesis. However, the mechanism of IncRNA function in this process is still unclear.

[0115] The present Inventors discovered a IncRNA named Dio3os in the proximity of iodothyronine deiodinase 3 (Dio3), coded by the opposite strand of the Dio3 locus, which was decreased during osteoblast differentiation. The bi-directional promoter activates the transcription of Dio3os. ChIP and promoter activity assays showed that HIF1 a and HDAC1 directly bind and activate, while RUNX2 and Brg1 repress both Dio3 and Dio3os transcription. These findings directly suggest that the local control of the Dio3 chromatin state by Dio3os is linked to T4 inactivation, which inhibits osteoblast differentiation.

[0116] Furthermore, Dio3os function is independent of BMP2 but mediated through nuclear hormone or vitamin D receptors in regulating osteoblast differentiation. Dio3os bound proteomics analysis demonstrated a significant association with the HDAC1-led nucleosomal remodeling deacetylase (NuRD) complex. It was also found that Dio3os directly controls the chromatin accessibility and modifications of a group of TH-controlled bone-forming genes. CRISPR-CAS9 mediated Dio3-Dio3os repression or knockout significantly decreased local Dio3 and increased non-local OB-specific gene expression. In mice, CRISPR-mediated OB- TH Docket #: 222120-2090; U24-076 specific loss (CRISPRi) and gain (CRISPRa) of Dio3-Dio3os locus transcription greatly affected the synthesis, mineral density, cortical and trabecular structure of long bone. These findings have firmly established the pivotal role of Dio3os in regulating TH signaling during bone synthesis and maintenance. It achieves this by activating Dio3 locally and repressing the transcription of distant OB-specific genes, a key discovery of our research.

[0117] An imbalance of thyroid hormones (THs), specifically the active T3 hormone production and functionality, causes hyperthyroidism or hypothyroidism, both of which may lead to anomalies in adolescent bone formation, compromise bone remodeling, and increase bone fragility in adulthood1 4. Hyperthyroidism is a leading cause of secondary osteoporosis, and osteoporosis-related fractures affect 50% of women and 25% of men aged >50 years58. Again, hypothyroidism, with an incidence of 1 in 800, affects more than 8 million people in the USA with multiple skeletal abnormalities1 9 10. Although treatment options to slow bone loss and improve bone density are available, there is no cure for osteoporosis11 13due to the complex genetic and epigenetic mechanisms involved in the disorder14. This is likely because intracellular tissue-specific TH activity regulation in osteoblasts has yet to be fully understood.

[0118] TH activity in bone cells is further regulated by DIO315, a member of a family of deiodinases that fine-tune the intracellular TH functions in a tissue-specific and spatiotemporal manner16 18. While in osteoblast, DIO2 converts T4 to active T3, DIO3 converts T3 and T4 to the irreversible inactive forms T2 and rT3. Thus, it prevents or limits access of T3 to osteoblast cells. DIO3 is expressed in all skeletal lineages1920and may be critical for bone health as indicated by perinatal thyrotoxicosis, adult hypothyroidism, and impaired growth in mice following knockout of Dio321 22. Additionally, consumptive hypothyroidism, a recently discovered rare early childhood disorder23, appears to be due to aberrant Dio3 expression that impairs endochondral ossification1.

[0119] Recently identified IncRNAs, including Dio3os, functionally contribute to osteogenesis24, osteogenic signaling25 26, lineage commitment27, differentiation2831, and osteosarcoma3233. Genome-wide association studies (GWAS) revealed that genetic variants in 30 loci were robustly associated with thyroid hormone function3439. One of the novel loci implicated in thyroid hormone metabolism is DIO3-DIO3OS40. Preliminary library screening of human IncRNAs indicated that the Dio3 opposite strand (Dio3os) IncRNA is involved in osteogenesis. Genomic studies confirmed that Dio3 and IncRNA Dio3os (the sense and antisense transcripts) expressed from the Dio3 gene imprinted locus in mice and humans22 41. Recent mechanistic studies strongly suggest that the genomic loci that produce Dio3os regulates local Dio3 expression and contributes to TH turnover and bone formation42.

[0120] There is a myriad of clinical relevance of hypothyroidism and Dio3-Dio3os function associated skeletal anomalies. Delayed bone maturation and stuppled epiphyses are significant abnormalities due to hypothyroidism43 44 48. Congenital and juvenile acquired TH Docket #: 222120-2090; U24-076 hypothyroidism resulted in delayed skeletal development1. Similarly, severe hypothyroidism ceases ossification, causes skeletal dysplasia, and dislocates the hip49. On bone turnover, hypothyroidism reduces osteoclast activity and bone resorption5052. Dio3- Dio3os dysregulation causes consumptive hypothyroidism, a severe condition that affects 0.1- 0.7 percent and can lead to osteoporosis due to the autoimmune disease, Hashimoto's thyroiditis53. Genetic variants in the Dio3os gene had a higher anemia risk54. Therefore, osteoporosis due to anemia may be orchestrated by Dio3os55. Osseous metastases of thyroid cancer, a significant clinical problem, are poorly studied, and 70% of patients with osseous metastases historically died within 4 years5660. Recently, Chen et al.61, uncovered that Dio3os activation regulates obesity. Research has shown that people with obesity have a lower bone density and an increased risk of fractures, osteoporosis, osteo, and rheumatoid arthritis62. Meta-analyses of genes revealed a potential role for the DIO3 gene in osteoarthritis susceptibility63. While peripheral thyroid replacement therapy is currently being used to induce “catch up” growth in such juveniles, the results never achieve full growth, and the congenital hypothyroid child is left with a height deficit.

[0121] An extensive GWAS identified Dio3-Dio3os as a novel genomic locus implicated in thyroid function and dysfunction40, suggesting that Dio3os may regulate bone development and maintenance. Several reports indicated that Dio3os regulation facilitates osteolysis and is linked to multiple cancers (e.g., thyroid56, prostate, and breast64, hepatocellular65, pancreatic66, and ovarian cancers67). Plasma IncRNA expression profiling identified upregulated Dio3os in patients with Crohn's disease (CD), which is also associated with bone loss68. While IncRNA-dependent mechanisms play crucial roles in controlling gene expression in every biological process6970

[0122] II. ANTI-DIO3OS ANTISENSE RNAS

[0123] IncRNAs have been arbitrarily defined as non-coding transcripts of more than 200 nucleotides (200 nt). Characterized examples have indicated that RNAs participate in virtually all levels of genome organization, cell structure and gene expression, through RNA-RNA, RNA-DNA and RNA-protein interactions, often involving repeat elements, including small interspersed nuclear elements in 3' untranslated regions. These interactions are involved in the regulation of chromatin architecture and transcription, splicing (especially by antisense IncRNAs), protein translation and localization, and other forms of RNA processing, editing, localization and stability.

[0124] Many IncRNAs are involved in the regulation of cell differentiation and development in animals and plants. They also have roles in physiological processes such as (in mammals) the p53-mediated response to DNA damage, V(D)J recombination and class switch recombination in immune cells, cytokine expression, endotoxic shock, inflammation and TH Docket #: 222120-2090; U24-076 neuropathic pain, cholesterol biosynthesis and homeostasis, growth hormone and prolactin production, glucose metabolism, cellular signal transduction and transport pathways, synapse function and learning, and have roles in the response to various biotic and abiotic stresses in plants. There is also an emerging association of IncRNAs with cell membrane and with ribozymes. Additional information on IncRNAs can be found, for example, in Mattick, J.S., Amaral, P.P., Carninci, P. et al. Long non-coding RNAs: definitions, functions, challenges and recommendations. Nat Rev Mol Cell Biol 24, 430-447 (2023).

[0125] The IncRNA Dio3os (human variant having NCBI Gene ID: 64150, mouse variant having NCBI Gene ID: 353504, the entire NCBI contents for both of which are incorporated by reference as if fully set forth herein) functionally contributes to osteogenesis, osteogenic signaling, lineage commitment, differentiation, and osteosarcoma. Genome-wide association studies (GWAS) have recently revealed that genetic variants in 30 loci were robustly associated with thyroid hormone function. One of the novel loci implicated in thyroid hormone metabolism is DIO3-DIO3OS. Preliminary library screening of human IncRNAs indicated that the Dio3 opposite strand (Dio3os) IncRNA is involved in osteogenesis. Genomic studies confirmed that Dio3 and IncRNA Dio3os (the sense and antisense transcripts) expressed from the Dio3 gene imprinted locus in mice and humans. Recent mechanistic studies strongly suggest that the genomic loci that produce Dio3os regulates local Dio3 expression and contributes to TH turnover and bone formation.

[0126] Described herein are compositions comprising one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof and siRNAs. Such antisense RNA and siRNA as described herein are capable of binding to, knocking down, digesting, or otherwise inhibiting the action of the IncRNA Dio3os. In embodiments, described herein are compositions comprising nucleic acids that encode for one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof and siRNA[s], In embodiments, described herein are compositions comprising one or more anti- Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof, siRNAs, or both ( / .e., one or more ASOs described herein, one or more siRNAs as described herein, or one or more ASOs plus one or more siRNAs, individually or in any combination of any thereof (or nucleic acids encoding such) and one or more secondary therapeutics, for example, a therapeutic for hyperthyroidism, a therapeutic for hypothyroidism, an anti-cancer therapeutic, or any combination thereof. In some aspects the secondary therapeutic is one or more of an aromatase inhibitor, a histone deacetylase inhibitor, or a lactate dehydrogenase A (LDHA) inhibitor. In embodiments, sequences of anti-Dio3os antisense RNA[s] are provided in Tables 2-5 below.

[0127] In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ TH Docket #: 222120-2090; U24-076

[0128] ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or

[0129] 34-43.

[0130] In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti- Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 1- 10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.

[0131] In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , TH Docket #: 222120-2090; U24-076

[0132] 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or

[0133] 34-43.

[0134] In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35.

[0135] In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35 In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about TH Docket #: 222120-2090; U24-076

[0136] 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of S SEQ ID NOs: 12, 13, 34, or 35.

[0137] In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35.

[0138] In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti- Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 1- 10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.

[0139] In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] TH Docket #: 222120-2090; U24-076 that have about 75% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.

[0140] In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti- Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 1- 10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.

[0141] In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or TH Docket #: 222120-2090; U24-076 more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35.

[0142] In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs:

[0143] 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 12,

[0144] 13, 34, or 35.

[0145] In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure TH Docket #: 222120-2090; U24-076 can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 34 or 35. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 12, 13, 34, or 35. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os antisense RNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of S SEQ ID NOs: 12, 13, 34, or 35.

[0146] The skilled artisan would understand that different combinations of anti-Dio3os antisense RNA can be utilized according to aspects of the present disclosure. For example, and without intending to be limiting, compositions comprising / consisting essentially of / consisting of both nucleotides having or encoding nucleotides having about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NOs: 12, 13, 34, or 35 (or nucleic acids comprising such) are contemplated according to the present disclosure.

[0147] In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os siRNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os siRNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os siRNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os siRNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more anti-Dio3os siRNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 45-50.

[0148] In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os siRNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os siRNA[s] that have about 80% sequence identity TH Docket #: 222120-2090; U24-076 to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os siRNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os siRNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more anti-Dio3os siRNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 45-

[0149] 50.

[0150] In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os siRNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os siRNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os siRNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os siRNA[s] that have about 90% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more anti-Dio3os siRNA[s] that have about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 45-50.

[0151] In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti- Dio3os siRNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 90% sequence identity to that of S SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can comprise one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 90% sequence identity, about TH Docket #: 222120-2090; U24-076

[0152] 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 45-50.

[0153] In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 90% sequence identity to that of S SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist essentially of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 45-50.

[0154] In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 75% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti- Dio3os siRNA[s] that have about 80% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 85% sequence identity to that of SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 90% sequence identity to that of S SEQ ID NOs: 45-50. In embodiments, compositions according to the present disclosure can consist of one or more nucleotides encoding one or more anti-Dio3os siRNA[s] that have about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about TH Docket #: 222120-2090; U24-076

[0155] 97% sequence identity, about 98% sequence identity, about 99%, or about 100% sequence identity to that of SEQ ID NOs: 45-50. siRNA of the present disclosure can comprise a sense and an antisense strand pair as components of a composition. Antisense and sense strands examples are provided in Example 13 and the sequence listing (for example, SEQ ID NO: 45 and SEQ ID NO:46; SEQ ID NO: 45 and SEQ ID NO:48; SEQ ID NO: 45 and SEQ ID NO:50; SEQ ID NO: 47 and SEQ ID NO:46; SEQ ID NO: 47 and SEQ ID NO:48; SEQ ID NO: 47 and SEQ ID NO:50; SEQ ID NO: 49 and SEQ ID NO:46; SEQ ID NO: 49 and SEQ ID NO:48; SEQ ID NO: 49 and SEQ ID NO:50).

[0156] Furthermore, the skilled artisan would recognize that any one or more nucleotides according to the present disclosure can be chemically modified to impart stability on the ASO or siRNA. Such modifications include, without intending to be limiting: 2'-0-methoxyethyl (2'MOE), Phosphodiester and Phosphorothioate (PS); 2'-O-methyl modified ribose (2 -OMe); 2 -O-methoxyethyl modified ribose (2'-MOE); 2’fluoro (2 -F); Locked nucleic acid (LNA): (Constrained ethyl (cEt); Tricyclo-DNA (tcDNA); Phosphorodiamidate morpholino oligos (PMO); Peptide nucleic acid (PNA); 5-methyl-cytosine (m5C); and -acetylgalactosamine (GalNAc). Additional modifications are discussed, for example, in Adachi H, Hengesbach M, Yu YT, Morais P. From Antisense RNA to RNA Modification: Therapeutic Potential of RNA- Based Technologies. Biomedicines. 2021 May 14;9(5):550. doi: 10.3390 / biomedicines9050550. PMID: 34068948; PMCID: PMC8156014, the entirety of which is incorporated by reference as if fully set forth herein.

[0157] In embodiments, on the 5’ end, any one or more of the first nucleotide, second nucleotide, third nucleotide, forth nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide, eight nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelve nucleotide, thirteenth nucleotide, fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide, or twentieth nucleotide can be a modified nucleotide. In embodiments, the first through the fifth nucleotides from the 5’ end, 3’ end, or both can all each contain modified nucleotides according to any sequence disclosed herein. In embodiments, the modification can be a 2’MOE modification. In certain aspects, any one or more of SEQ ID NOs 1 to 50 can contain first through the fifth nucleotides from the 5’ end, 3’ end, or both modified nucleotides. In certain aspects, any one or more of SEQ ID NOs 1 to 50 contain first through the fifth nucleotides from the 5’ end, 3’ end, or both modified nucleotides, wherein each nucleotide at those initial 5’ five nucleotides or 3’ five terminal nucleotides is modified.

[0158] In embodiments, the secondary therapeutic can be a medication or treatment for a disorder characterized by a thyroid hormone imbalance ( / .e., hyperthyroidism or hypothyroidism). Such thyroid medications or treatments include methimazole (also called TH Docket #: 222120-2090; U24-076 thiamazole), carbimazole, propylthiouracil, beta blockers (such as metoprolol or propranolol), radioactive iodine, or surgery ( / .e., thyroid gland removal). In other embodiments, such medications include levothyroxine.

[0159] In embodiments, a secondary therapeutic can be an aromatase inhibitor. In embodiments, the aromatase inhibitor can be letrozole or anastrozole.

[0160] In certain aspects, Aromatase inhibitors include: Anastrozole (Arimidex, AstraZeneca) - nonsteroidal, reversible; Letrozole (Femara, Novartis) - nonsteroidal, reversible; and Exemestane (Aromasin, Pfizer) - steroidal, irreversible.

[0161] When patients relapse or progress on or after Als, in certain aspsects, combination therapy may be used to overcome resistance: CDK4 / 6 inhibitors (Palbociclib - Ibrance, Pfizer; Ribociclib - Kisqali, Novartis; Abemaciclib - Verzenio, Eli Lilly) Combined with Als or fulvestrant, improves PFS and OS.

[0162] In embodiments, a secondary therapeutic can be a histone deacetylase inhibitor ( / .e., an HDAC inhibitor). In embodiments, the HDAC inhibitor can be an HDACI, HDACII, HDACIII, or HDACIV inhibitor. In embodiments, the HDAC inhibitor is an HDACI inhibitor. In embodiments, the HDACI inhibitor comprises Vorinostat (also referred to herein as “SAHA”). In embodiments, pan HDAC inhibitors can be utilized as a secondary therapeutic, such as Vorinostat (Zolinza, also referred to as “SAHA”), Romidepsin (Istodax), Belinostat (Beleodaq), Panobinostat (Farydak), and Chidamide (Epidaza). Furthermore, selective HDAC inhibitors with activity against HDAC1 and other class I HDACs, may be combined with anti-Dio3os antisense RNA described herein (for example, NAT1 / NAT2, i.e. SEQ ID NOs: 34 and 35).

[0163] Additional therapeutics or secondary therapeutics that can used, in particular for subjects with ER+breast cancer, include: HDAC inhibitors (esp. entinostat), which can reverse endocrine resistance by restoring ER expression and modifying epigenetic silencing. HDAC inhibitors studied in breast cancer include entinostat (leading candidate, in phase III trials with exemestane), and FDA-approved hematology HDAC inhibitors such as vorinostat, panobinostat, belinostat, and romidepsin.

[0164] In additional embodiments, Lactate Dehydrogenase A (LDHA) inhibitors can be a secondary therapeutic and combined with anti-Dio3os antisense RNAs, for example, 1- (phenylseleno)-4-(trifluoromethyl) benzene (PSTMB), NHI-Glc-2, or oxamic acid sodium. Other examples include FX11 , galloflavin, oxamate, NCI-006 / GNE-140, and derivatives. Entinostat and other epigenetic drugs sometimes indirectly reduce LDHA expression, and combination regimens with immunotherapy or metabolic drugs are an active research frontier mTOR inhibitor (Everolimus - Afinitor, Novartis) and PI3Ka inhibitors may also be utilized according to the present disclosure (Alpelisib - Piqray, Novartis, for example). Combined with fulvestrant for PIK3CA-mutated tumors after Al progression (SOLAR-1 trial). Selective estrogen receptor degrader (SERD). Fulvestrant (Faslodex, AstraZeneca) is TH Docket #: 222120-2090; U24-076 standard after Al resistance. Next-generation oral SERDs (elacestrant, camizestrant, giredestrant, etc.) (elacestrant was FDA-approved in 2023); other endocrine agents; and Tamoxifen (SERM) may still be used in certain settings after Al failure.

[0165] Additional therapeutics or secondary therapeutics that can used in any combination with any composition as described herein include: levothyroxine (Synthroid, Levoxyl, etc.; for hypothyroidism); bisphosphonates (Fosamax, Reclast), denosumab (Prolia), or anabolic agents (Forteo, Evenity), depending on patient profile and fracture risk (bone loss from hypothyroidism).

[0166] III. KITS AND PACKAGING

[0167] The compositions ( / .e., those comprising, consisting essentially of, or consisting of one or more anti-Dio3os antisense RNA[s], anti-Dio3os siRNAs, or both described herein) can be utilized in the preparation of a kit. In some embodiments, kits are provided for carrying out any of the methods described herein. The kits of this disclosure may comprise a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.

[0168] In some instances, one of the containers may comprise a composition as described in this disclosure that is, or can be, detectably labeled. The kit may also have containers containing buffer(s) and / or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic or fluorescent label. In some embodiments, the kit comprises separate containers containing compositions described herein and a detectable label.

[0169] A composition as described in this disclosure for use in improving osteoporosis and / or cancer symptoms in subjects may be delivered in a pharmaceutical package or kit to doctors and patients. Such packaging is intended to improve patient convenience and compliance with the treatment plan. Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit or pharmaceutical package further comprises instructions for use (e.g., for administering according to a method as described herein).

[0170] In some embodiments, a pharmaceutical package or kit comprises unit dose forms of a composition or components of compositions described herein. In some embodiments, the pharmaceutical package or kit further comprises unit dose forms of one or more of an additional therapeutic, for example, another medicament used for treatment of a disorder in a patient.

[0171] In one embodiment, the kit or pharmaceutical package comprises a composition as described herein in a defined, therapeutically effective dose in a single unit dosage form or as TH Docket #: 222120-2090; U24-076 separate unit doses. The dose and form of the unit dose (e.g., tablet, capsule, immediate release, delayed release, etc.) can be any doses or forms as described herein.

[0172] In one embodiment, the kit or pharmaceutical package includes doses suitable for multiple days of administration, such as one week, one month, or three months.

[0173] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, kits containing single or multi-chambered pre-filled syringes are included. In certain embodiments, kits containing one or more containers of a formulation described in this disclosure are included.

[0174] IV. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0175] Compositions comprising one or more anti-Dio3os antisense RNA[s], anti-Dio3os siRNAs, or both of the present disclosure and a pharmaceutically acceptable carrier are also provided. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions can be used in a subject with primary or secondary osteoporosis (in particular due to a thyroid hormone imbalance such as hypothyroidism) or a cancer subject (such as one with ER+ breast cancer or other breast cancer, thyroid cancer, prostate cancer, hepatocellular cancer, pancreatic cancer, and ovarian cancer) would benefit from any of the one or more anti-Dio3os antisense RNA[s], anti-Dio3os siRNAs, or both described herein. Compositions comprising co-therapies are also contemplated, in which a single pharmaceutical composition may be made comprising one or more anti-Dio3os antisense RNA[s], anti-Dio3os siRNAs, or both and one or more additional active agents that are intended as a co-therapy (e.g., thyroid disorder therapeutics and other anti-cancer therapeutics which may be utilized with the one or more anti-Dio3os antisense RNA[s], anti- Dio3os siRNAs, or both ).

[0176] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for I.V. administration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); TH Docket #: 222120-2090; U24-076 fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof.

[0177] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buffered saline. In certain embodiments, a composition comprising one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising or consisting of one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof, one or TH Docket #: 222120-2090; U24-076 more anti-Dio3os siRNA, or any combination of any thereof disclosed herein can be formulated as a lyophilizate using appropriate excipients. In some instances, appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof. Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some instances, the cryo-preservative may be sucrose or trehalose. In some instances, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.

[0178] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

[0179] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH may be 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6,

[0180] 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6,

[0181] 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, or 8.5. In some instances, the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3. In some instances, the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5- 6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5. In some instances, the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0- 4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5. In an embodiment, the pH is 7.2.

[0182] In certain embodiments when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof is formulated as a sterile, isotonic solution and properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, which can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used and can have the effect of promoting TH Docket #: 222120-2090; U24-076 sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0183] In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, one or more anti-Dio3os antisense RNA[s], one or more anti- Dio3os siRNA, or any combination of any thereof that are administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized, and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof (or EV comprising such). In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0184] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving one or more anti-Dio3os antisense RNA[s], one or more anti- Dio3os siRNA, or any combination of any thereof in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, International Application Publication No. WO / 1993 / 015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U.S. Patent No. 3,773,919; U.S. Patent No. 5, 594,091 ; U.S. Patent No. 8,383,153; U.S. Patent No. 4,767,628; International Application Publication No. WO1998043615, Calo, E. et al. (2015) Eur. Polymer J 65:252-267 and European Patent No. EP 058,481), including, for example, chemically synthesized polymers, starch based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1993) Biopolymers 22:547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al. (1981) J Biomed Mater Res. 15: 167-277; and Langer (1982) Chem Tech 12:98-105), ethylene vinyl acetate (Hsu and Langer (1985) J Biomed Materials Res 19(4):445-460) or poly-D(-)-3-hydroxybutyric acid (European Patent No. EP0133988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent No. EP 036,676; and U.S. Patent Nos. 4,619,794 and 4,615,885). TH Docket #: 222120-2090; U24-076

[0185] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0186] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0187] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having dried nucleic acid and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0188] In certain embodiments, the effective amount of a pharmaceutical composition comprising one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which the one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0189] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof in the formulation used. In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain TH Docket #: 222120-2090; U24-076 embodiments, appropriate dosages can be ascertained through use of appropriate doseresponse data.

[0190] In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of a combination therapy may be administered by different routes.

[0191] In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

[0192] In certain embodiments one or more anti-Dio3os antisense RNA[s], one or more anti- Dio3os siRNA, or any combination of any thereof can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the nucleic acids. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by a subject’s immune system or by other detrimental factors from the surrounding tissues.

[0193] V. METHODS OF USE AND TREATMENT

[0194] As described herein, the present disclosure provides a method of treating a subject with osteoporosis (e.g., secondary osteoporosis, i.e., osteoporosis as a symptom of a disorder characterized by a thyroid hormone imbalance, i.e., specifically hypothyroidism) and / or a cancer (e.g., ER+ breast cancer, thyroid cancer, prostate cancer, hepatocellular cancer, pancreatic cancer, and ovarian cancer))( / .e., a subject “in need thereof’), comprising administering to the subject a therapeutically effective amount of compositions (i.e., those comprising, consisting essentially of, or consisting of miRNA and / or miRNA mimics described TH Docket #: 222120-2090; U24-076 herein) according to the present disclosure. In some embodiments, the subject has or is determined to have osteoporosis as a result of a thyroid hormone imbalance or a cancer.

[0195] The compositions described herein are useful in, inter alia, methods for treating primary or secondary osteoporosis or cancer in a subject. As used herein, the term subject means a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats and sheep. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have osteoporosis, or a disorder characterized by osteoporosis being present as a symptom. In embodiments, the disorder characterized by osteoporosis as a symptom is one of a thyroid hormone imbalance, such as hyperthyroidism or hypothyroidism. In some embodiments, the subject is diagnosed with osteoporosis as a symptom is one of a thyroid hormone imbalance, such as hyperthyroidism or hypothyroidism. In some embodiments, the subject is a human that is suspected of having osteoporosis as a symptom is one of a thyroid hormone imbalance, such as hyperthyroidism or hypothyroidism. In embodiments, the subject may have or be suspected of having a treatment-resistant cancer and osteoporosis.

[0196] In some embodiments, the subject is one having a treatment-resistant cancer, for example ER+ breast cancer, or thyroid, prostate, and breast, hepatocellular, pancreatic, and ovarian cancers.

[0197] As used herein, administer or administration refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0198] The compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), intranasal spraying, intracranial injection, or intrathecal injection (IT). The injection can be in a bolus or a continuous infusion. Techniques for preparing injectate or infusate delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Remington's Pharmaceutical Sciences, ' Sth edition, 1990, Mack Publishing). Those of skill in the art can readily determine the various parameters and conditions for producing antibody injectates or infusates without resort to undue experimentation. In some TH Docket #: 222120-2090; U24-076 embodiments, compositions as described herein can be therapeutically delivered to a subject by way of intravenous administration.

[0199] Administration can be achieved by, e.g., topical administration, local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501 ,856; 5,164,188; 4,863,457; and 3,710,795. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

[0200] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and the like.

[0201] Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or a symptom thereof, in particular, ameliorating deficits in bone density or anti-cancer therapeutic efficacy resulting from the disorder. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., decreased or depressed osteogenesis or bone volume, density, or length compared to the same level of bone volume, density, or length in a subject without the disorder or lessening in the severity or progression.

[0202] Thus, treating or treatment includes ameliorating at least one parameter or symptom, in particular bone volume or bone growth. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for improving osteogenesis in a subject by administering a composition as described in this disclosure is considered to be a treatment or therapeutic, for example, if there is a 5-10% improvement in bone volume or bone growth according to the TH Docket #: 222120-2090; U24-076 measured or observed parameter in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more (or any percent improvement in between 10% and 100%) as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0203] Symptoms of osteoporosis include back pain, loss of height, stooped posture, broken bones, joint pant, weak grip strength, and bones that fracture or break more readily than normal.

[0204] As used herein, the term “therapeutically effective amount” or effective amount refers to an amount of active components of a therapeutic composition (such as a composition comprising anti-Dio3os antisense RNA[sj) thereof that, when administered to a subject, is effective to treat a disease or disorder (or ameliorate a symptom thereof, for example, lack of a symptom of osteoporosis), e.g., a thyroid disorder characterized by bone loss. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects. A suitable dose capable of improving osteogenesis or sensitivity of cancer cells in a subject, can depend on a variety of factors including the particular construct used and whether it is used concomitantly with other therapeutic agents. Generally, a therapeutically effective amount may vary with the subject’s age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g. , doctor or nurse). A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. The dosage of the therapeutically effective amount may be adjusted by the individual physician or veterinarian in the event of any complication. In some instances, a therapeutically effective amount may vary from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, most preferably from about 0.2 mg / kg to about 2 mg / kg, in one or more dose administrations daily, for one or several days. In embodiments, for systemic delivery, antisense RNAs might be given subcutaneously or intravenously. The subcutaneous dosage is generally 250-300 mg once weekly or 20-30 mg / kg intravenously once a week.

[0205] A pharmaceutical composition can include a therapeutically effective amount of one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof described herein. Such effective amounts can be readily determined by one of ordinary skill in the art as described above. Considerations include the effect of the TH Docket #: 222120-2090; U24-076 administered anti-Dio3os antisense RNA[s], or the combinatorial effect of the one or more anti- Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof with one or more additional active agents (thyroid disorder therapeutics or anti-cancer therapeutics, for example), if more than one agent is used in or with the pharmaceutical composition. In certain aspects, the doses can be about 1 , about 0.5, about 0.1 , about 0.05, or about 0.01 mg / kg, or any intervening dose between about 0.01 mg / kg and 1 mg / kg.

[0206] Suitable human doses of any of the anti-Dio3os antisense RNA[s] described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531 ; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

[0207] Toxicity and therapeutic efficacy of such anti-Dio3os antisense RNA[s] can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD5o (the dose lethal to 50% of the population) and the ED5o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. An anti-Dio3os antisense RNA[s] that exhibits a high therapeutic index is preferred. While constructs that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

[0208] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof lies generally within a range of circulating concentrations of the one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For the one or more anti- Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC50 (i.e., the concentration of the construct - e.g., antibody - which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration is desired, cell culture or animal TH Docket #: 222120-2090; U24-076 models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

[0209] In some embodiments, a composition or component of a composition described herein ( / .e., one or more anti-Dio3os antisense RNA[s], one or more anti-Dio3os siRNA, or any combination of any thereof) can be administered to a subject as a monotherapy. Alternatively, the composition or component of a composition described herein can be administered in conjunction with secondary therapeutics for the thyroid disorder or cancer. For example, the composition can be administered to a subject at the same time, prior to, or after, a second therapy. In some embodiments, the composition or component of a composition described herein, and the one or more additional active agents are administered at the same time. Optionally, the composition or component of a composition described herein is administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one or more additional active agents are administered first in time and the anti-Dio3os antisense RNA[s] composition or component of a composition described herein is administered second in time. Optionally, the composition or component of a composition described herein, and the one or more additional agents are administered simultaneously in the same or different routes.

[0210] In embodiments, the secondary therapeutic can be a medication or treatment for a disorder characterized by a thyroid hormone imbalance ( / .e., hyperthyroidism or hypothyroidism). Such thyroid medications or treatments include methimazole (also called thiamazole), carbimazole, propylthiouracil, beta blockers (such as metoprolol or propranolol), radioactive iodine, or surgery ( / .e., thyroid gland removal). In other embodiments, such medications include levothyroxine.

[0211] In embodiments, a secondary therapeutic can be an aromatase inhibitor. In embodiments, the aromatase inhibitor can be letrozole.

[0212] In embodiments, a secondary therapeutic can be a histone deacetylase inhibitor (i.e., an HDAC inhibitor). In embodiments, the HDAC inhibitor can be an HDACI, HDACII, HDACIII, or HDACIV inhibitor. In embodiments, the HDAC inhibitor is an HDACI inhibitor. In embodiments, the HDACI inhibitor comprises Vorinostat. In embodiments, pan HDAC inhibitors can be utilized as a secondary therapeutic, such as Vorinostat (Zolinza), Romidepsin (Istodax), Belinostat (Beleodaq), Panobinostat (Farydak), and Chidamide (Epidaza). Furthermore, selective HDAC inhibitors with activity against HDAC1 and other class I HDACs, may be combined with anti-Dio3os antisense RNA described herein (for example, NAT1 / NAT2, i.e. SEQ ID NOs: 34 and 35).

[0213] In additional embodiments, Lactate Dehydrogenase A (LDHA) inhibitors can be a secondary therapeutic and combined with anti-Dio3os antisense RNAs, for example, 1- (phenylseleno)-4-(trifluoromethyl) benzene (PSTMB), NHI-Glc-2, or oxamic acid sodium. TH Docket #: 222120-2090; U24-076

[0214] A composition as described herein can replace or augment a previously or currently administered therapy, such as previously prescribed bone enhancement therapeutic or anticancer therapeutic.

[0215] Monitoring a subject (e.g., a human patient) for an improvement in osteoporosis or cancer therapy, as defined herein, means evaluating the subject for a change in symptoms as reported by clinical imaging (e.g., MRI for breast cancer mass imaging and Diagnostic mammogram), blood tests, bone density / volume tests (e.g., dual-energy x-ray absorptiometry (DXA) scan), or self-reporting by the patient. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality.

[0216] In some instances, the one or more anti-Dio3os antisense RNA[s], one or more anti- Dio3os siRNA, or any combination of any thereof can be administered via virus-like particles. Virus-like particles (VLPs) comprise viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0217] In some instances, the one or more anti-Dio3os antisense RNA[s], one or more anti- Dio3os siRNA, or any combination of any thereof can be administered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0218] In another aspect, provided is a method of improving osteoporosis or cancer in a subject, the method comprising administering to the patient a composition comprising a vector that comprises a nucleic acid sequence encoding antisense RNA as described in this disclosure.

[0219] There are a number of compositions and methods which can be used to deliver the nucleic acid molecules to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in art and readily adaptable for use with the compositions and methods described herein.

[0220] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without undesired degradation and include a promoter yielding TH Docket #: 222120-2090; U24-076 expression of the nucleic acid molecule and / or adapter polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61 :1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virology 57:267- 74 (1986); Davidson et al., J. Virology 61 :1226-39 (1987); Zhang et al., BioTechniques 15:868- 72 (1993). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. In some instances, the nucleic acid molecules according to the present disclosure can be delivered via extracellular vesicles or virus-like particles.

[0221] Non-viral based delivery methods can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding the compositions comprising antisense RNA, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wl), Clonetech (Pal Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[0222] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., p-actin promoter or EF1 a promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fused to the p- actin promoter). Of course, promoters from the host cell or related species are also useful herein. TH Docket #: 222120-2090; U24-076

[0223] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0224] The promoter and / or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and / or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and / or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the beta-actin promoter, the EF1 a promoter, and the retroviral long terminal repeat (LTR).

[0225] The vectors also can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. TH Docket #: 222120-2090; U24-076

[0226] In certain embodiments, the effective amount of a pharmaceutical composition comprising compositions of the present disclosure to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which a composition is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0227] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the active components in the formulation used. Such pharmacokinetic parameters are well known in the art, l.e., the rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:61 1-617; Groning (1996) Pharmazie 51 :337-341 ; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; the latest Remington's, supra). In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0228] In some cases, the dosage (of the active components] or compositions as described herein) ranges from about 0.0001 to 100 mg / kg, and more usually 0.01 to 20 mg / kg, of the patient’s body weight. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or within the range of 0.1-20 mg / kg. In certain examples, the compositions thereof can be administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg once every other day at least four times. An exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. In some cases, the treatment comprises administering a composition according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period. In some cases, the treatment discontinues for a period of time before the same or a different dosing regimen resumes. For example, a patient may be on a dosing regimen for two weeks, off for a week, on for another two weeks, and so on. Dosage regimens for TH Docket #: 222120-2090; U24-076 compositions of this disclosure include 0. 1 mg / kg body weight, 0.3 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, or 10 mg / kg via intravenous administration, with the compositions being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks.

[0229] In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of a combination therapy may be administered by different routes.

[0230] In certain aspects, compositions according to the present disclosure can be administered as a co-therapy with other therapeutic agents. Other examples of therapeutic agents include otherthyroid medications and cancer medications (aromatase inhibitors and / or HDAC inhibitors, for example).

[0231] While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

[0232] Other features, objects, and advantages of the present invention are apparent in the description that follows. It should be understood, however, that the description, while exemplifying certain embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

[0233] VI. EXAMPLES

[0234] Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

[0235] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the TH Docket #: 222120-2090; U24-076 compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is in atmosphere. Standard temperature and pressure are defined as 25 °C and 1 atmosphere.

[0236] EXAMPLE 1 Dio3-Dio3os Locus is Critical in TH Metabolism and Osteogenesis

[0237] Several experiments were performed to test see the role that Dio3os IncRNA plays in activating neighboring Dio3 and inhibits non-neighboring OB-specific genes (FIG. 1A). First, murine Dio3os was uncovered from a human IncRNA library, transcribed as opposite strand RNA from Dio3-Dio3os locus, and its expression decreased along with Dio3 during osteoblast differentiation (FIG. 1 B). AT AC and ChlP-sequencing assays demonstrated higher transcriptional activity during OB growth due to higher chromatin accessibility and elevated H3K4me3 and H3K27ac at the promoter and H3K36me3 modifications at the gene body (FIG. 1C). The moderate H3K4me1 modifications in the coding regions of Dlo3os indicated the possibility of a functional distal enhancer (FIG. 1C). Dio3os overexpression showed increased Dio3os and Dio3 but decreased Runx2 and Ocn expression (FIG. 1 D). Next, different CR / SPR editing tools were used to decipher the contributions of the Dio3-Dio3os locus in osteogenesis. First, CRISPR-Cas9 targeted deletion of all four exons (AE1-4), including introns (without disturbing D / o3 transcription and the functionality) significantly decreased Dio3os and Dio3 but increased OB-specific Ocn gene compared to exon 1 deletion only (AE1) (FIG. 1 E). Second, CR / SPR-mediated polyA addition of Dio3os exons 2, 3, and 4 (without affecting Dio3 transcription) noticeably halted Dlo3os and Dio3 RNA transcriptions; still induced Ocn transcription (Twenty modified antisense RNAs were designed (XL file attached). Ten are human and ten are mouse. We tested two murine antisense RNAs (see Example 10), including MMENSMUSE00001474554-27 (NAT 1) and MMENSMUSE00001474554-145 (NAT2)(FIG. 1F). Finally, recruitment of dCas9-KRAB on Exons 2 and 3 of Dlo3os (without impacting the typical Dio3 transcription start site); we achieved a 60-80% reduction in Dlo3os and Dlo3 expression while increasing up to 3.5- fold Ocn expression (FIG. 1 G).

[0238] EXAMPLE 2: Osteoblast -Specific Loss (CRISPRi) or Gain (CRISPRa) of Dio3-Dio3os

[0239] Locus Transcription Will Affect the Synthesis, Mineral Density, Cortical and Trabecular Structure, Cellular Detail, Quality, and Quantity of Long Bone

[0240] The inventors postulated that OB-specific repression of Dio3-Dio3os may significantly increase the synthesis and quality of bone at 1 month (adolescent). To test this hypothesis, TH Docket #: 222120-2090; U24-076 conditional CRISPR inhibition (CRISPRi) mice were utilized, predicting that OB- specific Co1a1-CreERT2 recombinase in the presence of 4-OH Tamoxifen would remove the loxP-flanked STOP cassette (LSL) to allow dCas9-KRAB fusion protein expression, and with sgRNA would silence the Dio3-Dio3os locus through dCas9-KRAB binding. Kim JE et al. 200473successfully evidenced that Col1a1 -ERT2 Cre, expressed explicitly in osteoblasts and odontoblasts of both embryos and postnatal mice, and bone specificity of Cre expression increased 60-160-fold compared to other tissues. Additionally, the inducible Cre recombinase activity was not detected in chondrocytes.

[0241] First, homozygous (CRISPRi) (LSL-dCas9-KRAB+lCol1a1-CreERT2+) mice were genotyped using primers derived from dCas9-KRAB and transgene Col1a1-Cre recombinase and used to study uCT and histomorphometric analysis. Preliminary longitudinal uCT scans showed higher bone formation in both 1 -month-old males and females due to CRISPRi of Dio3-Dio3os locus compared to non-targeting sgRNA (FIG. 2A, 2B). Dio3- Dio3os CRISPRi increased trabecular and cortical bones (FIGs. 2C, 2D) in both sexes. Further quantitation of pCT scans indicated a significant increase in bone volume to tissue volume (BV / TV), trabecular number (Th. N), and thickness (Tb. Th) but a decrease in trabecular space (Th. Sp) in females (FIG. 2E) and males (FIG. 2F) CRISPRi mice. Histomorphometric analysis (Table 1) of 1-month-old female and male femurs indicated a significant increase in bone volume / tissue volume (BV / TV), bone perimeter trabecular thickness, trabecular number, number of osteoblasts over the bone surface but a decrease in trabecular space and a slight reduction in osteoclast numbers in male mice when the Dio3os was repressed using CRISPRi in mature osteoblast cells.

[0242] Further testing was done to examine OB-specific activation of Dio3-Dio3os and its significant impact on decreasing the synthesis and quality of bone at 1 month (adolescent). CRISPR activation (CRISPRa; LSL-dCas9-SPH) mice were utilized to generate the present data. The homozygous (CRISPRa) (LSL-dCas9-SPH+ / +; Col1a1-CreERT2*) mice were genotyped using primers derived from dCas9-SPH and transgene Col1a1-Cre recombinase and used to study uCT and histomorphometric analysis. Preliminary longitudinal uCT scans showed diminished bone formation in both 1-month-old males and females due to CRISPRa of Dio3-Dio3os locus (FIGs. 3A, 3B). Dio3-Dio3os CRISPRa remarkably increased trabecular and cortical bones (FIGs. 3C, 3D) in both sexes. Further quantitation of pCT scans indicated a significant reduction in the ratio of bone volume to tissue volume (BV / TV), trabecular number (Tb. N), and thickness (Tb. Th) but an increase in trabecular space (Th. Sp) in female (FIG. 3E) and male (FIG. 3F) CRISPRa mice. Histomorphometric analysis (Table 1 below) of 1-month-old female and male femurs indicated a substantial reduction in bone volume / tissue volume (BV / TV), bone perimeter trabecular thickness, trabecular number, number of osteoblasts over the bone surface but an increase in trabecular space and TH Docket #: 222120-2090; U24-076 osteoclast numbers in male mice when the Dio3os was activated using CRISPRa in mature osteoblast cells.

[0243] Female Male Female Male

[0244] Table 1 : Femur Bone Histomorphometric Analysis of CRISPRi and CRISPRa

[0245] Female and Male Mice. BV / TV: Bone Volume, Tissue Volume; N. Ob: Number of Osteoclast; BS: Bone Surface.

[0246] EXAMPLE 3: CRISPR-CAS9 Mediated Deletion of Dio3os Results in Robust Chromatin Openness in Osteoblast-Specific Promoters

[0247] Based on initial findings (FIGs. 4A-4C and FIGs. 5A-5C), the interaction of Dio3os, rT3, and its interacting NuRD complex was examined and thought to strictly inhibit the chromatin accessibility of TH responsive osteoblast-specific genes. To understand this trans chromatin regulatory mechanism of Dio3-Dio3os, MC3T3-E1 cells were transduced with Lentiviral Cas9 and D / o3os-specific sgRNA to delete Dio3os. PlotHeatmap analysis of ATAC-seq findings revealed a significant increase of accessible regions to the nearest transcription start site (TSS) of osteoblast-specific gene promoters due to Dio3os CRISPR knockout (FIG. 4A). We observed no additional changes when Dio3os CRISPR cells were treated with T4 (FIG. 4A, right). Further analysis revealed that Dio3os knockout increased the distribution of TF binding within >=1 kb of TSS compared to 2-3 kb upstream (FIG. 4B). We observed non-significant changes of TF binding when Dio3os CRISPR cells were treated with T4 (FIG. 4B, bottom). ALP staining of Dio3os KO cells showed a robust increase in ALP staining when compared with WT and T4 treated cells (FIG. 4C). TH Docket #: 222120-2090; U24-076

[0248] EXAMPLE 4: CRISPR-CAS9 Mediated Knockout of Dio3os Results in Robust Upregulation of OB-specific TH Responsive Genes with Highly Accessible Chromatin

[0249] To test the hypothesis that Dio3os and rT3 promote chromatin compaction for TH regulated gene expression, RNA-seq in MC3T3-E1 cells was performed, and cells were transduced with lentiviral Cas9 and D / o3os-specific sgRNA to delete Dio3os. Next, genomewide gene expression changes in WT and Dio3os knockout cells were examined and found that knockout of the Dio3os significantly activated osteoblast transcription programs for enhanced gene expression. RNA-sequencing findings revealed 652 genes differentially expressed. Among them, 285 genes were significantly upregulated and 367 genes down- regulated (FIG. 5A). Heatmap profile uncovered 22 genes, including Col1a1, Ocn, Itgb7, Alpl, Adamtsl, and Adamts4, responsible for matrix maturation and ossification were increased >2- fold due to CRISPR knockout of Dio3os (FIG. 5B). Dio3os knockout also rose Runx2 and Sp7, two essential transcription factors involved in osteogenesis (FIG. 5B). Bioinformatics analysis further confirmed that all highly expressed genes have thyroid response elements (TRE) on their proximal promoter (FIG. 5B, right). To verify whether higher gene expression directly correlates with chromatin openness, ATAC-seq results were examined in WT and Dio3os knockout cells. It was discovered that the chromatin accessibility of 22 TH responsive factors were increased, including Ocn, Col1a1, Runx2, and Sp7, and have higher expression due to Dio3os CRISPR knockout (FIGs. 5B, 5C). These findings indicate that the dio3-Dio3os-NuRD axis mediated chromatin repression of TH responsive activators is essential for TH balance during osteogenesis.

[0250] EXAMPLE 5: Dio3os RA / A-NuRD Complex Interaction is Crucial for TH Signaling in Osteoblast

[0251] Results indicated that Dio3os and NuRD associate to form a non-coding RNA-protein chromatin regulatory complex. This complex with rT3 supervises osteoblast-specific gene promoters responsive to TH and controls the osteoblast transcription program supporting physiologic bone synthesis and maintenance. To identify the IncRNA Dio3os interacting proteins, Dio3os RNA immunoprecipitation (IP) mass spectrometry (MS) was performed in WT and Dio3os CRISPR knockout MC3T3-E1 cells. Volcano plot peptide analysis of Dio3os IP-MS identified NuRD complex members, including HDAC1 , MBD3, CHD3, P66a / p, RBBP7, MTA2, and transcriptional corepressor SIN3B interacted with Dio3os (FIG. 6A). Furthermore, peptide count enrichment from MS analysis indicated that CHD3 (33-fold) and CHD4 (29-fold) have high-affinity interaction compared to other members of the NuRD complex (FIG. 6B). During NuRD mediated chromatin remodeling, MBD3 directly binds to open DNA (CGCGCG) and interacts with CHD3 / 4 and HDAC1 to catalyze promoter TH Docket #: 222120-2090; U24-076 deacetylation. The binding NuRD complex to Dio3os was further confirmed by reverse pulldown using anti MBD3, CHD3, and HDAC1 antibodies. Reverse pulldown with MC3T3-E1 UV crosslinked whole cell lysate demonstrated significant enrichment (14-16%) of Dio3os interaction with MBD3, CHD3, and HDAC1 (FIG. 6C). These results indicate that Dio3os interacts with the NuRD complex and provides a scaffold for strictly controlling TH cellular function.

[0252] EXAMPLE 6: Dio3os is Essential for NuRD Complex Recruitment

[0253] Our findings indicated that Dio3os recruits MTA1 & 2, MBD3, HDAC1 , the NuRD factors, and H3K27ac to Dio3-Dio3os and Ocn promoters. To understand the role of Dio3os in recruiting the NuRD complex to Dio3-Dio3os and OB-specific Ocn promoters, ChIP assays were perfomed in WT and Dio3os knockout MC3T3-E1 cells. ChIP assays with CR / SPR CAS9 deleted Dio3os showed decreased recruitment of NuRD members, namely MTA1 & 2, MBD3, and HDAC1 in both Dio3-Dio3os and Ocn promoters (FIG. 7). We also found that Dio3os deletion decreased H3K27 acetylation in the Dio3-Dio3os promoter but increased in the Ocn promoter (FIG. 7).

[0254] EXAMPLE 7: Dio3os Expression Increases in Multiple Cancer Cells

[0255] Initial findings described herein, which investigated the expression of Dio3os in several cancer cells, including the ER+ breast cancer cell line MCF-7, the head and neck squamous cell carcinoma cell line FaDu, and the metastatic prostate cancer cell line PC3, have the potential to significantly impact the field. The increased expression of Dio3os in highly metastatic and invasive cancer lines (FIG. 8) Underscores the crucial role of IncRNA Dio3os in regulating the growth, invasion, and metastasis of ER-positive breast cancer, head and neck squamous cell carcinoma, and prostate cancer.

[0256] EXAMPLE 8; Diseases that can be Targeted Besides Hypothyroidism / lnduced Bone Loss

[0257] It has been reported that IncRNA Dio3os is associated with several types of cancers, including thyroid (Wang, M. et al. J. Cell Commun. Signal. 2021), prostate (Gururajan M et al. Clin Cancer Res. 2014), breast (Chen, X. et al. Nat Commun. 2022), hepatocellular (Wang Z et al. Cancer Manag Res. 2020), pancreatic (Kang Cui et al. Cancer Cell Int. 2019), and ovarian cancers (Mitra R et al. Nat Commun. 2017).

[0258] Genetic variants in the Dio3os gene had a higher anemia risk (Toxqui, L. and Vaquero, M.P., 2015. Nutrients). Recently, Chen et al. (Chen, YT et al. Nat Commun 2021), uncovered that Dio3os activation regulates obesity. TH Docket #: 222120-2090; U24-076

[0259] EXAMPLE 9: Clinically Approved HDAC Inhibitor(s) Along with the Antisense RNAs for Superior Therapeutic Effects

[0260] The present inventors have discovered that Dio3os Exons 2 and 3 are crucial for binding and function of HDAC1 . The present inventors have observed that HDAC1 increases the expression of Dio3os, but the HDAC1 inhibitor Vorinostat (SAHA) reduces it (see FIG. 9A).

[0261] Additionally, the mass spectrometry analysis of Dio3os IP revealed a significant threefold increase in HDAC1 binding (FIG. 9B). Dio3os antisense NAT 1 and NAT2 decrease Dio3os expression by 60% (FIG. 9C). It seems that HDAC1 inhibitors suppress the transcription of Dio3os, while antisense NAT 1 and NAT2 degrade Dio3os post-transcriptionally. Combining clinically approved HDAC1 inhibitors with the antisense RNAs can lead to enhanced therapeutic effects.

[0262] EXAMPLE 10: Antisense RNA sequence for Dio3os

[0263] Twenty modified antisense RNAs were designed. Ten are human and ten are mouse. Two murine antisense RNAs were tested, including MMENSMUSE00001474554-27 (NAT 1 ;

[0264] SEQ ID NO: 12) and MMENSMUSE00001474554-145 (NAT2; SEQ ID NO: 13). Tables 2, 3, 4, and 5 below show examples of antisense RNAs according to the present disclosure. 2MOE modifications were provided where noted.

[0265] TH Docket#: 222120-2090; U24-076

[0266] Table 3 (Above); Antisense Mouse RNA According to the Present Disclosure - 2'MOE Gapmer Designs (♦ = modified nucleotide *- Unique nucleotide)

[0267] Table 4 (Above); Antisense Human RNA According to the Present Disclosure ~ Affinity Plus Gamer Designs (+ ~ modified nucleotide Unique nucleotide)

[0268]

[0269] Table 5 (Above): Antisense Human RNA, According to the Present Disciosure ~ 2'MOE Gapmer Designs

[0270] TH Docket #: 222120-2090; U24-076

[0271] EXAMPLE 11 Materials and Methods for Examples 1-10

[0272] 1. Mice

[0273] MC3T3-E1 Subclone 4 cells (CRL-2593) were obtained from ATCC (https: / / www.atcc.Org / products / all / CRL-2593.aspx#generalinformation) were cultured in alpha-MEM media supplemented with 10% FBS and 1 % Penicillin / Streptomycin (Sigma- Aldrich) (100U / mL, 100ug / mL respectively). Primary calvarial cells were isolated from homozygous CRISPRi and CRISPRa and Dio3os CRISPR mice and were maintained in a- MEM medium containing 10% fetal bovine serum plus 1 % Penicillin / Streptomycin (100U / mL, 100ug / mL respectively) cocktail. Upon reaching 90-95% confluency, cells were differentiated using ascorbic acid (100 ug / ml) and B-glycerol phosphate (5 mM).

[0274] MC3T3-E1 cells were treated with 20 ng of TGF or 10-8 M Vitamin D, 300 ng / ml BMP2, and 100-1000ng / ml of T4, T3, T2, and rT3 thyroid hormones for24 h priorto harvesting. Medium was replaced every 2 days for the duration of all experiments. All cells were maintained at 37 °C in a humidified 5% CO2 environment.

[0275] 2. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

[0276] Total RNA was isolated using TRIzol™ Reagent (Catalog number: 15596026, ThermoFisher Scientific), column purified, and cDNA was reverse transcribed using the Invitrogen First Strand cDNA Synthesis system with Superscript III reverse transcriptase using oligo(dT) or gene-specific primers. The qPCR reactions were performed using SYBR chemistry with either Applied Biosystems or NEB Syber Green qPCR master mixes. Results were quantified using the delta-delta CT method and shown as Log-2-fold change over control. The amplification experiments were performed in triplicate with gene-specific primers and probes.

[0277] 3. Chromatin immunoprecipitation assays (ChlPs)

[0278] ChIP experiments were performed as described (Hassan et al. Nature and PNAS). Chromatin immunoprecipitation assays (ChlPs) were performed with 1 % formaldehyde crosslinked soluble chromatins. Crosslinked osteoblast cells were quenched by adding glycine to a final concentration of 250 mM for 10 min and washed twice with 1XPBS. Cell pellets were resuspended and lysed in lysis buffer (150 mM NaCI, 50 mM Tris-HCI pH 8.0, 1 % NP40, 25 pM MG-132, and 1X Complete® protease inhibitor cocktail (Roche, Indianapolis, IN). After 10 min on ice, cells were sonicated in a Bioruptor (Diagenode Inc.) to obtain DNA fragments of 200~500 bp. Chromatins were pre-cleared by centrifugation at 15K rpm for 20 min. Soluble chromatins were immunoprecipitated for 16 h with 10 pg of each non-specific IgG control (Santa Cruz Biotechnology) and specific antibodies, followed by 1 h with 60 pl of Protein A / G conjugated Agarose beads. Protein A / G beads-bound protein-DNA complexes were washed with the following buffers: low salt (20 mM Tris-CI, pH 8.1 , 150 mM NaCI, 1 % Triton X-100, 2 mM EDTA, 1X complete protease inhibitor), high salt (20 mM Tris-CI, pH 8.1 , 500 mM NaCI, TH Docket #: 222120-2090; U24-076

[0279] 1% Triton X-100, 2 mM EDTA), LiCI salt (10 mM Tris-CI, pH 8.1 , 250 mM LiCI, 1% deoxycholate, 1% NP-40, 1mM EDTA) and twice in TE (10 mM Tris-CI, pH 8.1, 1 mM EDTA). Protein-DNA complexes were eluted in 1% SDS and 100 mM NaHCO3. Crosslinks were reversed by incubation overnight in elution buffer and 300 mM sodium acetate, pH 5.2. DNA was then extracted, purified, precipitated, and resuspended in TE for qPCR using primers indicated in Table 2. For comparative analysis, a quantitative measure reflects the amount of non-specific genomic DNA region precipitated with a specific antibody (i.e.,) relative to the non-specific antibody (i.e., normal IgG). Pull-down samples were normalized to a non-specific genomic region amplicon control. Results are shown as percent chromatin pull-down with samples normalized to input.

[0280] 4. MicroCT Analysis

[0281] Femurs were dissected from mice and fixed in 70% ethanol for microCT (pCT). Cortical and trabecular bone properties were measured using a high resolution Scanco 40 pCT. Samples were placed in a 12 mm diameter sample holder and scanned at 12 pm resolution, 55kVp, 145 pA with 200ms integration time. For cortical bone, samples were scanned midshaft with 25 slices at a 737.3 mgHA / ccm threshold. Trabecular bone scans started at the growth plate with 209 slices per sample, with the region of interest starting where the chondrocytes region ends. For analysis 100 slices were used at 451.2 mgHA / ccm threshold. Data was analyzed using the pCT Evaluation Program (v.6.5-2, Scanco Medical). Nomenclature, symbols, and units used are according to the Nomenclature Committee of the American Society for Bone and Mineral Research <24>.

[0282] 5. Histology and Histomorphometry

[0283] Samples were dissected and fixed in 70% ethanol for plastic packaging. Histomorphometry analysis of bone parameters, including osteoblast and osteoclast numbers, were analyzed using Trichrome staining. Plastic embedded sections were used to observe osteoclast activity using Tartrate-resistant acid phosphatase (TRAP). Histomorphometric changes were performed and analyzed by histomorphometry and molecular analysis core of the UAB bone center. All histomorphometric data was analyzed using BioQuant Osteo software, with two data sets collected by blinded readers.

[0284] 6. RNA and AT AC sequencing

[0285] ATAC and RNA-seq were used to study the correlation of chromatin accessibility for osteoblast-specific gene expression. Primary calvarial OBs derived from Dio3-Dio3os CRISPR MC3T3-E1 cells were transduced with adeno / lentiviral sgRNAs and Cre recombinase, and cells were stimulated with osteogenic differentiation media for 10 days. On days 3 and 10, cells were processed for transposome preparation, oligonucleotide ligations, transposition, reverse transcription, library preparation, and sequencing were performed according to procedures Dr. Buenrostro and his group 88 described recently. Libraries were TH Docket #: 222120-2090; U24-076 sequenced on the Next-seq platform (Illumina) using a 150-cycle High-Output Kit or a 200- cycle S1 kit. ATAC-seq and RNA-seq were performed according to procedures recently described 89. AT AC peaks, peak-gene cis-association, and DORC identification located ± 50 kb or ± 500 kb around annotated TSSs were examined. We calculated the -Iog10(p-value) (KS test) of the Spearman correlation between mean normalized DORC gene expression and DORC score, and the TF motifs enriched in the DORC. We manually selected TFs that regulate DORCs.

[0286] 7. Dio3os-RNAIP Mass Spectrometry

[0287] To identify the composition of the D / o3os-rT3 interacting NuRD complex proteins due to Dio3os loss during differentiation, we isolated nuclear extract from control and Dio3os CR / SPR MC3T3-E1 cells on day 3 and immunoprecipitated with MBD3, CHD3, and HDAC1 for MS analysis. The cells were transduced with Adeno / Lentiviral Cre, +- targeting, and nontargeting sgRNAs. MS and peptide count analysis were performed in the DAB Mass Spectrometry facility. Details of mass spectrometry analysis were described in several publications 97,98. After pulldown with non-specific IgG, anti-MBD3, anti-CHD4, and anti- HDAC1 antibodies, we performed MS, compared results, and quantified proteomics.

[0288] Outlier data points were removed by a ROUT (Q = 1 %) outlier test. The significance was determined for all comparisons by a two-way ANOVA with Tukey's post hoc test and Benjamini-Hochberg FDR correction (q - 5%). We used the SEQUEST algorithm supported by Proteome Discoverer (version 1 .4) to analyze differential peptide / protein spectral counts with high-accuracy MS2 (Orbitrap) data sets. Furthermore, the Significance Analysis of the INTeractome (SAINT), the database of protein sequence and functional information, UniProt, the database of antibodies-based protein profiling in cells, Protein Atlas, and the database of protein subcellular localization to link the D / o3os-interactome to regulatory function were analyzed.

[0289] 8. Statistical Power and Analysis

[0290] Assuming a Type I error rate of 0.05, we detected 80% power to see the variation in groups, which equated to Cohen’s effect size (f) of 0.32 for the Analysis of Variance test. Continuous variables were reported as means with 95% confidence intervals, SDs, and ranges. For experiments with two or more groups and two factors (e.g., genotype and sgRNA), we employed two-way ANOVA tests. We used pairwise group comparisons to a False Discovery Rate of 0.05. We used Graph Pad Prism version 9.2.0 and Origin 10.5.10ssoftware for statistical analysis. Relationships between micro-CT parameters and in vitro osteoclastogenesis were determined by Pearson correlation. In RNA and AT AC sequencing, Analysis of Variance methods was used for all independent observation comparisons TH Docket #: 222120-2090; U24-076 between control and D / o3os CRISPR groups. Regarding statistical power, we utilized a False Discovery Rate approach to maintain a moderate control over Type I error rates, but not as strict as a Bonferroni correction, thus providing more statistical power. We implemented EdgeR Bioconductor package 92 using false discovery rate (FDR)-adjusted p-values93. Differentially expressed genes (FDR < 0.10) between control and experimental groups were identified. We utilized a two-tailed paired t-test Pathway and other annotation analysis 94,95 to interpret differentially expressed genes (DEGs), particularly the GSEA70 method.

[0291] EXAMPLE 12: Dio3os Antisense RNAs Promising New Strategy for Reversing Therapeutic Al Resistance in ER-positive Human Breast Cancer

[0292] 1. Results

[0293] The MCF-7 breast cancer cells are estrogen receptor-positive. Treatment with 10 nM of 17-beta estradiol increased MCF-7 proliferation, as shown in FIGs. 10A and 10B. Essentially, the hormone activates estrogen receptors, promoting tumor growth and the development of hormone-dependent cancers. 17-beta-estradiol (20 nM) and the aromatase inhibitor (Al) letrozole (20 nM) were combined in ER+ MCF-7 cells. The findings showed that the Al partially counteracted the proliferative effects of the exogenously added estradiol and showed phenotype-like MCF-7 cells (FIG. 10A), resulting in reduced proliferation compared to treatment with estradiol alone, as depicted in FIG. 10C.

[0294] Understanding the interaction between estradiol and aromatase inhibitors is not just crucial in breast cancer therapy, but it also has the potential to revolutionize the field by limiting the estrogen-driven growth of cancer cells. Recent research suggests that IncRNA Dio3os is upregulated in Al-resistant breast cancer and clinically associated with Al resistance. When Dio3os was overexpressed in ER+ MCF-7 cells, it was found that the cells became loosely packed and exhibited a motile mesenchymal state, possibly indicating resistance to Al, as shown in FIG. 10D.

[0295] Furthermore, treating these overexpressed cells with 17-beta estradiol and Al revealed that Al helped 70-80% of MCF-7 cells overcome the resistance, as depicted in FIG. 10E. However, treating these MCF-7 cells with Dio3 antisense RNAs (NAT 1 and NAT 2) rendered almost all the cells sensitive to the aromatase inhibitor letrozole, as illustrated in FIG. 10F. These findings underscore the importance of targeting Dio3os with antisense RNAs, as it will provide new strategies for reversing therapeutic Al resistance in ER-positive breast cancers.

[0296] Currently, in postmenopausal patients, the third-generation Al drugs (letrozole, anastrozole, and exemestane) have become the first-line of endocrine therapy for ER-positive breast tumors with therapeutic benefits superior to tamoxifen, once a classical endocrine drug for breast cancer. However, resistance to aromatase inhibition accounts for the majority of failures in adjuvant endocrine therapy. Our findings indicate that targeting Dio3os with TH Docket #: 222120-2090; U24-076 antisense RNAs can render almost all the cells sensitive to the aromatase inhibitor letrozole, providing a promising new strategy for reversing therapeutic Al resistance in ER-positive breast cancers

[0297] 2. Materials and Methods

[0298] Human breast cancer cell lines MCF-7 were obtained from the American Type Culture Collection (ATCC), along with MCF-7:WS8 and estrogen-deprived MCF-7:5C, which were a gift from Dr. Clodia Osipo of Loyola University Chicago, USA. These cell lines were cultured in RPMI 1640 medium (Gibco™) supplemented with 10% fetal bovine serum, both with and without estrogen, in accordance with standard protocols. All the cell lines were authenticated by short tandem repeat profiling before use and tested negative for mycoplasma contamination. We transfected 1 ug of Dio3os overexpression clone, 20uM anti-Dio3os antisense RNA oligonucleotides per 6 wells using X-tremeGENE™ 360 T ransfection Reagent (#8724105001 , Roche) at 70 % confluency for 48 hrs following the manufacturer’s recommendations. Cells were treated with 20 nM each of 17-beta estradiol and Letrozole (Millipore Sigma, # E8875 and #L6545).

[0299] EXAMPLE 13 Targeting Dio3os overcomes Al resistance in ER+ breast cancer

[0300] Overexpression of Dio3os in MCF7 cells increases phosphorylated RB (pRB), while Dio3os antisense ASO159 treatment reduces both Dio3os expression and pRB levels (FIG.

[0301] 11). These data suggest that aberrant RB phosphorylation may act downstream of Dio3os dysregulation, promoting cell cycle re-entry and contributing to Al-resistant breast cancer phenotypes with high metastatic potential. Messenger RNA analysis using RT-qPCR analysis showed elevated Dio3os and metabolic marker Ldha in estrogen-deprived MCF7-5C cells, that promote Al resistance (FIG. 12). It is thought that LDHA-driven metabolic reprogramming contributes to aromatase inhibitor resistance by promoting aerobic glycolysis and NAD+- dependent activation of CDK4 / 6, leading to RB phosphorylation, E2F activation, and estrogen- independence cell cycle progression. Six (6) antisense oligonucleotides (ASOs) and siRNAs targeting Dio3os were designed, all of which reduced its expression. Among them, ASO159 showed the strongest efficacy (FIG. 13). Antisense oligonucleotide (ASO)-mediated knockdown of Dio3os significantly reduces its expression, disrupting its scaffold function in recruiting histone deacetylases (e g., HDAC1 / NuRD complex) to tumor gene promoters. This loss of deacetylation permits increased HIF1 a-dependent transcriptional activation of tumorpromoting genes. When combined with HDAC (SAHA) and LDH inhibitors, ASO159 further reduced Dio3os levels (FIG. 14). Co-treatment with an LDH inhibitor orthe aromatase inhibitor (Al) anastrozole enhances Dio3os suppression. Notably, despite the cells being resistant to Al, ASO 159 in combination with anastrozole appears to reverse this resistance, highlighting a promising therapeutic strategy for treating Al-resistant breast cancer (FIG. 15). TH Docket #: 222120-2090; U24-076

[0302] CRISPR / siRNA screening from the DepMap portal (https: / / depmap.org / portal / cell_line / ACH- 000019?tab=overview) in metastatic ER+ lines (MCF7, T47D, KPL1), compared to estrogen- deprived Al-resistant T47D cells’ RNA sequencing, identifies 25 tumor suppressors with low Z-scores, suggesting potential Dio3os-regulated pathways. Very soon, we will identify and define Dio3os-regulated oncogenic and tumor-suppressor pathways for combined and highly effective antisense therapy (FIG. 16).

[0303] Additional embodiments:

[0304] ASO 566.2: 5’- +C*+C*+G*A*G*A*A*G*A*A*G*T*T*+T*+C*+G-3’

[0305] ASO 306.2: 5’- +G*+T*+G*C*T*G*G*T*T*T*G*G*G*+T*+T*+A-3’ ASO 159.2: 5’- +A*+A*+G*A*G*T*G*T*G*G*A*T*C*+A*+G*+G-3’ + = modified nucleotide *= Unique nucleotide

[0306] Embodiments of siRNA: dsiRNAI (sense strand): 5’- rCrUrUrUrCrArGrArCrUrUrArGrGrUrGrGrUrArArArGrUGA-3’ dsiRNAI (anti-sense strand): 5’- rUrCrArCrUrUrUrArCrCrArCrCrUrArArGrUrCrUrGrArArArGrArG-3’ dsiRNA2 (sense strand): 5’- rGrGrGrUrArGrArGrCrArArGrArGrArUrArGrUrUrCrUrCTT-3’ dsiRNA2 (anti-sense strand): 5’- rArArG r ArG rArArC rU r ArU rC rU rC rU rU rG rC rU rC rU rArC rC rC rAr A-3’ dsiRNA3 (sense strand): 5’- rGrArCrUrUrArGrGrUrGrGrUrArArArGrUrGrArGrArArGAG-3’ dsiRNA3 (anti-sense strand): 5’- rC rll rC rll rll rC rU rC rArC rU rll rll rArC rC rArC rC rU rArArG rU rC rU rG-3’ TH Docket #: 222120-2090; U24-076

[0307] Table 6 (Above): Antisense Human RNA According to the Present Disclosure - Affinity Plus Gamer Designs (+ = modified nucleotide *= Unique nucleotide)

[0308] References Related to Examples 1-11 of the Present Disclosure:

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[0310] 2. Tuchendler D, Bolanowski M. The influence of thyroid dysfunction on bone metabolism. Thyroid Res. 2014 Dec 20;7(1):12. doi: 10.1186 / s13044-014-0012-0. eCollection 2014. PMID: 25648501

[0311] 3. Baqi L, Payer J, Killinger Z, et al. Thyrotropin versus thyroid hormone in regulating bone density and turnover in premenopausal women. Endocr Regul. 2010; 44:57-63.

[0312] 4. Paul TL, Kerrigan J, Kelly AM, Braverman LE, Baran DT. Long-term L-thyroxine therapy is associated with decreased hip bone density in premenopausal women. JAMA. 1988; 259:3137-3141.

[0313] 5. Delitala AP, Scuteri A, Doria C. Thyroid Hormone Diseases and Osteoporosis. J Clin Med. 2020 Apr 6;9(4): 1034. doi: 10.3390 / jcm9041034. PMID: 32268542

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[0316] 8. Mizobuchi M, Towler D, Slatopolsky E. Vascular calcification: the killer of patients with chronic kidney disease. J Am Soc Nephrol. 2009 Jul;20(7): 1453-64. doi:

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[0382] It should be emphasized that the above-described embodiments are merely examples of possible implementations. Many variations and modifications may be made to the above- TH Docket #: 222120-2090; U24-076 described embodiments without departing from the principles of the present disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

TH Docket #: 222120-2090; U24-076CLAIMSAt least the following is claimed:1) A composition, comprising: one or more anti-Dio3os antisense RNA, one or more anti-Dio3os siRNA, or any combination of any thereof.2) The composition of claim 1 , wherein each of the one or more anti-Dio3os antisense RNA, one or more anti-Dio3os siRNA, or any combination of any thereof contain at least 2 or more modified nucleotides.3) The composition of claims 1 or 2, wherein the one or more anti-Dio3os antisense RNA comprises a nucleotide sequence that is at least 90% identical to any one or more of of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.4) The composition of any one of claims 1 to 3, wherein the one or more anti-Dio3os antisense RNA comprises a nucleotide sequence that is at least 95% identical to any one or more of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.5) The composition of any one of claims 1 to 4, wherein the one or more anti-Dio3os antisense RNA comprises a nucleotide sequence of at least one or more of SEQ ID NOs: 1-10, 12-21 , 23-32, or 34-43.6) The composition of any one of claims 1 to 5, wherein the one or more anti-Dio3os antisense RNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 35, or any combination of any thereof.7) The composition of any one of claims 1 to 6, wherein the one or more anti-Dio3os antisense RNA comprises a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 35, or any combination of any thereof.8) The composition of any one of claims 1 to 7, wherein the one or more anti-Dio3os antisense RNA consist essentially of a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 35, or any combination of any thereof.TH Docket #: 222120-2090; U24-0769) The composition of any one of claims 1 to 5, wherein the one or more anti-Dio3os siRNA RNA comprises one or more nucleotide sequences that are at least 90% identical to any one of more of SEQ ID NOs: 45-50.10) The composition of any one of claims 1 to 6, wherein the one or more anti-Dio3os siRNA RNA comprises one or more nucleotide sequences that are at least 90% identical to any one of more of SEQ ID NOs: 45-50.1 1) The composition of any one of claims 1 to 7, wherein the one or more anti-Dio3os siRNA RNA consist essentially of one or more or at least two nucleotide sequences that are at least 90% identical to any one or more of SEQ ID NOs: 45-5012) The composition of any one of claims 1 to 1 1 , wherein the one or more anti-Dio3os antisense RNA, anti-Dio3os siRNA, or both are present in a therapeutically effective amount.13) The composition of claim 12, wherein the therapeutically effective amount is an amount effective to improve symptoms of osteoporosis in a subject in need thereof.14) The composition of claim 13, wherein the subject in need thereof has or is suspected of having primary osteoporosis, secondary osteoporosis, or osteoporosis as a symptom of a disorder characterized by a thyroid hormone imbalance.15) The composition of any one of claims 1 to 14, wherein the therapeutically effective amount is an amount effective to increase sensitivity of one or more cancer cells to anti-cancer therapeutics.16) The composition of claim 15, wherein the one or more cancer cells comprise ER+ breast cancer or other breast cancer cells, thyroid cancer cells, prostate cancer cells, hepatocellular cancer cells, pancreatic cancer cells, and ovarian cancer cells.17) The composition of any one of claims 1 to 16, wherein the anti-cancer therapeutics comprise an aromatase inhibitor, a histone deacetylase inhibitor, or a lactate dehydrogenase A (LDHA) inhibitor.18) A pharmaceutical composition, comprising a composition according to any one of claims 1 to 17; andTH Docket #: 222120-2090; U24-076 a pharmaceutically acceptable carrier.19) A kit for increasing osteogenesis or sensitivity of cancer cells, comprising: a composition of any one of claims 1 to 17 or a pharmaceutical composition of claim 15; and instructions for use.20) The kit of claim 19, wherein the composition is lyophilized.21) A method of reducing the severity of one or more symptoms of osteoporosis in a subject, comprising: administering a composition of any one of claims 1 to 17 or a pharmaceutical composition of claim 18 to a subject in need thereof.22) The method of claim 21 , wherein the subject in need thereof has or is suspected of having a disorder causing osteoporosis.23) The method of claim 21 or 22, wherein the disorder causing osteoporosis is a disorder characterized by a thyroid hormone imbalance.24) A method of increasing sensitivity of cancer cells to anti-cancer therapeutics, comprising: administering a composition of any one of claims 1 to 17 or a pharmaceutical composition of claim 18 to a subject in need thereof.25) The method of claim 24, wherein the subject in need thereof has or is suspected of having ER+ breast cancer or other breast cancer, thyroid cancer, prostate cancer, hepatocellular cancer, pancreatic cancer, or ovarian cancer.

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