Modulation of SPP1 gene and OPN protein expression, methods and applications thereof

Abstract

An antisense oligonucleotide (ASO) that is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene, the mRNA having a start codon in an open reading frame encoding the SPP1 or OPN, and the target sequence including the start codon in the open reading frame of the mRNA. Binding of the ASO to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN. Alternatively, an ASO wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene. Also double-stranded DNA or RNA molecules capable of binding to a target sequence in a mRNA or pre-mRNA of a SPP1 or OPN gene. Additionally, pharmaceutical compositions and methods of treatments using the ASOs or double-stranded DNA or RNA molecules.

Description

MODULATION OF SPP1 GENE AND OPN PROTEIN EXPRESSION, METHODS AND APPLICATIONS THEREOF

[0001] This application claims priority from U.S. Provisional Application No.63 / 738,687, filed December 24, 2024. The entirety of all the aforementioned application is incorporated herein by reference.SEQUENCE LISTING

[0002] A Sequence Listing in XML format entitled "Paine 0001. xml", which was created December 17, 2025, and is 12,041 bytes, is incorporated herein by reference in its entirety.BACKGROUND

[0003] Abnormal gene and / or protein expression can cause various disorders.Researchers have explored the delivery of exogenous nucleotides into cells to restore the function of missing genes or to regulate the expression of target proteins or genes. Recently, oligonucleotide-based therapies have gained attention for their potential to prevent the progression of many disorders by targeting nucleotides and modulating the expression of abnormally expressed proteins.

[0004] The present application addresses the foregoing limitations and provides novel strategies for the design and use of antisense oligonucleotides (ASOs), as well as doublestranded ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecules to selectively increase or decrease translation of proteins from their endogenous genes.SUMMARY

[0005] An aspect of the application is an ASO comprising 8-50 nucleotides, wherein the ASO is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN.

[0006] An aspect of the application is an ASO comprising 8-50 nucleotides, wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the ASO to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN.

[0007] An aspect of the application is a double-stranded ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5 ’-3’ strand, wherein the molecule is capable of binding to a target sequence in a mRNA of a SPP1 or OPN gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises thestart codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN.

[0008] An aspect of the application is a double-stranded RNA or DNA molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5 ’-3’ strand, wherein the molecule is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the molecule to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN.

[0009] An aspect of the application is a pharmaceutical composition comprising an ASO comprising 8-50 nucleotides, wherein the ASO is capable of binding to a target sequence in a mRNA of a SPP1 or OPN gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0010] An aspect of the application is a pharmaceutical composition comprising an ASO comprising 8-50 nucleotides, wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the ASO to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0011] An aspect of the application is a pharmaceutical composition comprising a RNA or DNA molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5 ’-3’ strand, wherein the molecule is capable of binding to a target sequence in a mRNA of a SPP1 or OPN gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0012] An aspect of the application is a pharmaceutical composition comprising a RNA or DNA molecule comprising - 8-50 nucleotides, wherein the molecule is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the molecule to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0013] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising an ASO comprising 8-50 nucleotides, wherein the ASO is capable of binding to a target sequence in a mRNA of a SPP1 or OPN gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0014] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising an ASO comprising 8-50 nucleotides, wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the ASO to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0015] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising a RNA or DNA molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5’-3’ strand, wherein the molecule is capable of binding to a target sequence in a mRNA of a SPP1 or OPN gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0016] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising a RNA or DNA molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5’-3’ strand, wherein the molecule is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the molecule to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0017] All features of exemplary embodiments which can be described in this disclosure and can be not mutually exclusive can be combined with one another. Elements of one embodiment can be utilized in the other embodiments without further mention. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with any accompanying Figures.BRIEF DESCRIPTION OF THE DRAWING

[0018] FIG. 1, Panel A shows SPP1 gene and, Fig. 1, Panel B, shows translated splice variants OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

[0019] FIG. 2 shows a proposed molecular mechanism for PMOs such as Hu-01, through steric blocking, which involves binding to the mRNA (around the start codon), thereby preventing ribosomal translation of proteins.

[0020] FIG. 3 shows a proposed molecular mechanism for PMOs such as Hu-03, through exon skipping by binding to the junction of an exon and an intron on pre-mRNA, preventing correct splicing. The outcome is either generating an early termination codon or generating a short protein.

[0021] FIG. 4 shows antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production. U-251 cells were treated with Hu-01, Hu-02, and Hu-03, and a scramble control (PMO-CTRL) with two concentrations (luM and lOuM) for 24 hours. lOug of total protein from cell lyses of each reaction was loaded into the indicated well, detected with anti-OPN antibody.

[0022] FIG. 5A shows antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production. U-251 cells were treated with Hu-01, Hu-03, and a scramble control (PMO-CTRL) with 3uM for 24H. 15ug of total protein (cell lysis) was loaded into the indicated well, then detected with anti-OPN antibody. FIG. 5B shows the same blot was then detected with anti-beta-actin to confirm that the same amount of samples were loaded per lane.

[0023] FIG. 6 shows antisense oligo (ASO) activities on regulating OPN (Osteopontin, or SPP1) production. U-251 cells were treated with Hu-01, Hu-03, and a scramble control (PMO-CTRL) with 3uM for 24H. 5ug cell lysis per well was loaded to a 96-well plate (duplicated each sample, n=2). OPN production was quantified by using a Human OPN detection Elisa Kit and then normalized to cell alone control. Mean of duplicate shown.

[0024] FIG. 7A shows antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production in THP-1 cells. THP-1 cells were treated with Hu-01, Hu-02, and Hu-03, and a scramble control (PMO-CTRL) at a concentration of 3uM for 48h. Cell lyses were detected with anti-OPN antibody). FIG. 7B shows the same blot was then detected with anti-beta-actin antibody. H2O represents that THP-1 cells treated with Endo-porter without ASO (delivery reagent only control).

[0025] FIG. 8 shows antisense oligo (ASO) activities on regulating OPN (Osteopontin, or SPP1) production in THP-1 cells by normalizing with the production of beta actin. THP-1 cells were treated as described in Figure 7 legend.

[0026] FIG. 9 shows dose dependent antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production in U251 cells using Hu-01 (24 hours treatment).

[0027] FIG. 10 shows time course of antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production in U251 cells. U-251 cells were treated with Hu-01 at concentrations of luM and 3uM for 24, 48, 96, and 144 hours. Cell lyses were detected with anti-OPN antibody. H2O represents Endo-porter without ASO (delivery reagent only control).

[0028] FIG. 11 shows antisense oligo (ASO) activities (western-blotting) on regulating mouse OPN (Osteopontin, or SPP1) production in mouse microglial SIM-A9 cells. SIM-A9cells were treated with Mouse-01, Mouse-02, and a scramble control (PMO-CTRL) at concentrations of lum and lOuM for 24 hours before harvesting. OPN production in cell lysis was detected with anti-mouse-OPN antibody. Cells were treated with either 5ul or 2.5ul endo-porter (Delivery reagent) as controls.

[0029] FIG. 12 shows dose dependent antisense oligo (ASO) activities (western-blotting) on regulating mouse OPN (Osteopontin, or SPP1) production in mouse microglial SIM-A9 cells. SIM-A9 cells were treated with Mouse-01, Mouse-02, and a scramble control (PMO-CTRL) at indicated concentrations for 24 hours before harvesting. OPN production in cell lysis was detected with anti-mouse-OPN antibody. Cells were treated with endo-porter (Delivery reagent) as controls.

[0030] FIG. 13 shows dose-dependent antisense oligo (ASO) activities (western-blotting) on regulating mouse OPN (Osteopontin, or SPP1) production in mouse microglial SIM-A9 cells. Cells were treated as described in Figure 12. Mouse OPN production in cell lysis was quantified by using a mouse OPN detection kit. 5ug protein from cell lysis was loaded per well in 96 well plate. Mean of duplicated samples shown.

[0031] FIG. 14 shows the cellular internalization of peptide-conjugated phosphorodiamidate morpholino oligonucleotide (PPMO) Hu-01 across three peptide constructs by U-251 glioblastoma cells. The PMO Hu-01 was covalently linked to fluorescein and one of three targeting moi eties: PPMO #1 (P7 peptide), PPMO #2 (R6B peptide), or PPMO #3 (RGD peptide). Following a 24-hour incubation with U-251 cells, the cell-associated fluorescence of the unconjugated (fluorescein-labeled) Hu-01 and the three PPMOs was measured via flow cytometry. Mean Fluorescence Intensity (MFI) was used to evaluate cellular uptake, with results for 5pM and lOpM concentrations presented for comparative analysis.

[0032] FIG. 15 shows the cellular internalization of peptide-conjugated phosphorodiamidate morpholino oligonucleotide (PPMO) Hu-01 across three peptide constructs by Hela cells. The PMO Hu-01 was covalently linked to fluorescein and one of three targeting moieties: PPMO #1 (P7 peptide), PPMO #2 (R6B peptide), or PPMO #3 (RGD peptide). Following a 24-hour incubation with Hela cells, the cell-associated fluorescence of the unconjugated (fluorescein-labeled) Hu-01 and the three PPMOs was measured via flow cytometry. Mean Fluorescence Intensity (MFI) was used to evaluate cellular uptake, with results for 5pM and lOpM concentrations presented for comparative analysis.

[0033] FIG. 16 shows the cytotoxicity of different PPMOs in U-251 cells. 5000 U-251 cells / well were seeded into a 96-well cell culture plate. After 24 hours, cells were incubated with indicated concentrations of PMO Hu-01 and three different peptide conjugated PMOs (PPMO #l-#3). After another r24 hours, cell viability was examined with a CellTiter-Glo kit (commercial kit (Promega) .

[0034] FIG. 17 shows the biological effect of three peptide-conjugated PMOs (PPMOs) on OPN production in U-251 cells. U251 cells were seeded into each well of 12-well plates with 250,000 cells / lml medium per well. After 24 hours, U-251 cells were treated with indicated concentrations of PPMO #1, #2, and #3, and three corresponding scrambled PMO controls (conjugated with P7, R6B, and RGD, respectively), along with the unconjugated PMO Hu-01. Following 24-hour incubation, cell lysates were collected and assayed for OPN production using a Human OPN detection ELISA Kit (top). Results were quantified usingof the duplicate measurements.

[0035] FIG. 18 shows the biological effect of three peptide-conjugated PMOs (PPMOs) on OPN production in U-251 cells. U251 cells were seeded into each well of 12-well plates with 250,000 cells / lml medium per well. After 24 hours, U-251 cells were treated with indicated concentrations of PPMO #1, #2, and #3, and three corresponding scrambled PMO controls (conjugated with P7, R6B, and RGD, respectively), along with the unconjugated PMO Hu-01. Following 24-hour incubation, cell culture media were collected and assayed for OPN production using a Human OPN detection ELISA Kit (top). Results were quantified using 1:20 diluted cell culture supernatants (measured in duplicate, n=2). The data shown represent the mean of the duplicate measurements.

[0036] FIG. 19 shows the biological effect of PPMO #1 (P7 peptide conjugated PMO Hu-01) and PPMO #2 (R6B peptide conjugated PMO Hu-01) on OPN production in U-251 cells.20,000 U-251 cells per well were seeded into 96 well cell culture plates. After 24 hours, cell culture medium was removed, and fresh medium containing indicated concentrations of PPMOs was added. Following 24-hour incubation, cell culture media were collected and assayed for OPN production using a Human OPN detection ELISA Kit. Results were quantified using 1:20 diluted cell culture supernatants. Each condition was quadruplicated and data represented as mean ± SEM.

[0037] FIG. 20 shows the synergistic effect of PPMO and oligo activity enhancer on reducing OPN production by U-251 cells. U-251 cells were treated with PPMO #1, a small molecule (OAE2) with oligo activity enhance properties, or both at indicated concentrations. After 24 hours, cell culture medium was collected and examined for OPN (SPP1) production by using a Human OPN detection ELISA Kit. (Triplicate wells for U251 treatment plate. For ELISA, each sample assayed in duplicate). Each condition was triplicated and data represented as mean ± SEM.

[0038] FIG. 21 shows the cytotoxicity of the combination of fixed concentration of PPMO #1 and a variety of concentrations of Oligo Activity Enhancer (OAE 2) in Hela cells (left) and U-251 cells (right). 15 000 Hela cells / well or 5000 U-251 cells / well were seeded in 96-well cell culture plates After 24 hours, cells were treated with a combination of 10 uM PPMO #1 and indicated concentrations of OAE 2. After 24 hours, cell viability was examined with a CellTiter-Glo kit (commercial kit (Promega) . Cell treatment were triplicated and data represented as mean ± SD.DETAILED DESCRIPTION

[0039] Reference will be made in detail to certain aspects and exemplary embodiments of the application, illustrating examples in the accompanying structures and figures. The aspects of the application will be described in conjunction with the exemplary embodiments, including methods, materials and examples, such description is non-limiting, and the scope of the application is intended to encompass all equivalents, alternatives, and modifications, either generally known, or incorporated here. The described aspects, features, advantages, and characteristics of the invention may be combined in any suitable manner in one or more further embodiments. One skilled in the relevant art will recognize that the invention may be practiced without one or more of the specific aspects or advantages of a particular embodiment. In other instances, additional aspects, features, and advantages may be recognized and claimed in certain embodiments that may not be present in all embodimentsof the invention. Further, one skilled in the art will recognize many techniques and materials similar or equivalent to those described here, which could be used in the practice of the aspects and embodiments of the present application. The described aspects and embodiments of the application are not limited to the methods and materials described.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value.Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to "the value," greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as "greater than or equal to 10" is also disclosed. When numbers are listed consecutively all possible ranges between those numbers listed consecutively are also disclosed, e.g., the list of 1, 2, 3, also discloses the ranges 1-2, 1-3, 2-3.Overview

[0041] The present disclosure is directed to modulation of OPN mRNA and protein expression. Human genetic modification and the alteration of gene expression have been developed to treat several disorders. In some embodiments, the delivery of exogenous deoxyribonucleic acids (DNAs) to cells has been tested for years to restore the expression of missing genes in genetic disorders. In some embodiments, gene editing has also emerged to insert, delete, or replace genes to make specific changes to DNA. In some embodiments, oligonucleotide-based therapy can be developed to halt the progression of numerous disorders by affecting ribonucleic acid (RNA) or protein expression. In some embodiments, oligonucleotides are easy to synthesize and do not require integration into the genome, making their delivery less challenging. In some embodiments, different oligonucleotide structures have been designed and classified as (i) aptamers, which modify peptide and protein structures or enhance drug delivery; (ii) double-stranded RNA interference (RNAi), which reduces gene expression; (iii) double-stranded small activating RNAs (saRNAs), which induce gene expression; and (iv) antisense oligonucleotides (ASOs). In some embodiments, ASOs are single-stranded synthetic deoxynucleotide or ribonucleotide analogues that attach to complementary RNA sequences by Watson-Crick base pairing. For example, ASOs can be between 15 and 30 nucleotides in length, that attach to complementary RNA sequences by Watson-Crick base pairing. These chemically modified oligonucleotides can target both protein-coding (messenger RNA or mRNA) and noncoding RNA molecules (microRNA) to modulate gene expression.

[0042] FIG. 1 shows SPP1 gene and translated splice variants in some embodiments. Referring to FIG 1, the translated splice variants may include, for example, OPN-a, OPN-b, OPN-c, OPN-4, or OPN-5, in some embodiments. The present disclosure relates to a methodof reducing expression levels and / or activity of SPP1 or OPN in a cell comprising administering an effective amount of the compound to the cell or an effective amount of the pharmaceutical composition to the cell. The present disclosure relates to a method of reducing expression levels and / or activity of SPP1 or OPN in a subject in need thereof comprising administering an effective amount of the compound to the subject or an effective amount of the pharmaceutical composition to the subject. In some embodiments, it is unexpectedly found that Hu-01 (SEQ. ID. NO. 1) and Hu-03 (SEQ ID. NO: 3) down-regulated, knocked down, or reduced SPP1 or OPN production. For example, in some embodiments, SEQ ID NO: 6-8, which are binding site for Hu-01, Hu-02, and Hu-03 respectively, sequences are listed in the Listing Table in the Appendix, full sequence information refer to NCBI Reference Sequence: NP_110418.1; Gene ID: 6696).

[0043] FIG. 2 and FIG. 3 illustrates a possible molecular mechanism for PMOs such as Hu-01 and Hu-03 in some embodiments. Referring to FIG. 2, and without being bound by theory, PMOs such as Hu-01 work through translational blocking. PMOs are short singlestranded DNA analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. PMOs bind to complementary sequences of target mRNA by Watson-Crick base pairing to block protein translation through steric blockade in a RNase H independent manner. They bind to mRNA and prevent the mRNA-entering ribosome so translation to protein is prevented.

[0044] Referring to FIG. 3, and without being bound by theory, PMOs such as Hu-03 work by binding to complementary sequences of pre-mRNA near exon-intron boundaries, specifically blocking splicing sites or splicing enhancers. Hu-03 binding to an exon / intron junction within a pre-mRNA thereby modulates the splicing process. This modulation induces mis-splicing of the nascent transcript, which ultimately causes a frameshift and introduces a premature termination codon (PTC) in the mature mRNA. The presence of the PTC subsequently triggers the cellular quality control mechanism known as Nonsense-Mediated mRNA Decay (NMD), leading to the rapid degradation of the faulty mRNA transcript.

[0045] In some embodiments, RNA or DNA molecules can use the same target sequence as the antisense oligonucleotides (ASOs) herein. Such embodiments can involve different types of RNAs, for which molecular mechanisms for RNA modifications other than those discussed for phosphorodiamidate morpholino oligomers (PMOs) can be, without being bound by theory, inducing the RNase Hl endonuclease activity that cleaves RNA-DNA hybrids on mRNA, RNA-Induced Silencing Complex (RISC), destabilizing pre-mRNA by inhibiting 5' cap formation / modulating 3' polyadenylation, or any combination thereof. In some embodiments, ASOs can target miRNAs and inhibit their RISC-dependent action to reduce protein translation.

[0046] In some embodiments, a relatively shorter length can reduce specificity or might cause off-target effect, while it can be more advantageous such as that more targeting sites can be identified. In some embodiments, a relatively longer length can increase specificity and can have a relatively lower target effect, e.g., due to binding kinetics, obstructions, etc. In some embodiments, a relatively longer length can be hindered by relatively limited target sites because of the secondary structures in DNA, pre-mRNA or mRNA.

[0047] In some embodiments, OPN or SPP1 is involved in multiple functions in cellular activities. SPP1 also known as Osteopontin (OPN), is a highly phosphorylated sialoprotein. In some embodiments, OPN is now considered as a regulator of diversified biological events,including developmental processes, wound healing, immunological responses, tumorigenesis, bone resorption, and calcification. In some embodiments, OPN is expressed in a variety of tissues indicates a multiplicity of functions. In some embodiments, in tumor microenvironment and some autoimmune diseases, macrophages are the major source of OPN. Thus, modulating OPN expression by the products described herein may be used in treating or preventing diseases.

[0048] For example, in some embodiments, delivering oligonucleotides such as ASOs and PMOs as therapeutics involves an approach to overcome biological barriers and ensure efficient targeting. In some embodiments, oligonucleotide delivery as therapeutics, such as ASOs and PMOs, can be achieved through various strategies to enhance their stability, cellular uptake, and target specificity. In some embodiments, it may involve chemical modifications to enhance stability and pharmacokinetics, conjugation techniques to improve cellular uptake and tissue targeting, and the use of nanocarrier systems for protection and delivery. In some embodiments, chemical modifications like phosphorothioate backbones, 2'-O-methyl sugars, and 5 -methylcytosine bases improve the oligonucleotides' drug-like properties. In some embodiments, chemical modifications, such as the introduction of phosphorothioate linkages or 2'-O-methyl groups, can improve the oligonucleotides' resistance to nuclease degradation. In some embodiments, conjugation strategies, such as GalNAc for liver targeting and cell-penetrating peptides for enhanced cellular uptake, have proven effective. In some embodiments, conjugation with cell-penetrating peptides (CPPs) like DG9 or R6G can significantly enhance cellular uptake and intracellular distribution. In some embodiments, nanocarrier systems, including lipid nanoparticles (LNPs) and polymeric nanoparticles, offer protection and improved delivery, with LNPs being particularly successful in approved therapies. In some embodiments, nanocarrier systems, including liposomes, polymeric nanoparticles, or lipid nanoparticles, can be employed to protect the oligonucleotides from degradation and facilitate their delivery to target tissues. For PMOs, which are charge-neutral, conjugation with CPPs is particularly effective in improving their cellular absorption. In some embodiments, targeted delivery can be achieved by incorporating ligands such as antibodies, aptamers, or small molecules that recognize specific cell surface receptors. In some embodiments, for a choice of delivery method, specific oligonucleotide properties, target tissue, and desired therapeutic effect, with the ultimate goal of maximizing efficacy while minimizing off-target effects and toxicity, etc., can be considered. In some aspects, the techniques described herein relate to a pharmaceutical composition including the compound and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.I. Definitions

[0049] As used herein, the articles "a," "an," and "any" refer to the grammar of one or more than one (i.e., at least one) item. For example, "an element" means one element or more than one element.

[0050] As used herein, the term “antisense oligonucleotide” or “ASO” refers to a compound comprising or consisting of an oligonucleotide or modified oligonucleotide at least a portion of which is complementary to a target nucleic acid, a target nucleotide sequence (target sequence), a target site of a nucleotide sequence (target site), or a target region of a nucleotide sequence (target region), to which it is capable of hybridizing, resulting in at least one antisense activity. In some embodiments, the ASO comprises a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% complementary to the target sequence, the target site or the target region. In some embodiments, an ASO comprises an antisense oligonucleotide conjugated to a conjugate group. In some embodiments, the conjugate group is a non-nucleotide conjugate group. An "antisense activity" refers to any detectable and / or measurable change attributable to the hybridization of an antisense oligonucleotide to its target nucleic acid, target sequence, target site or target region.

[0051] As used herein, the term “ secreted phosphoprotein” or “SPP1” or “OPN gene” refers to a gene encoding a protein involved in the attachment of osteoclasts to the mineralized bone matrix. The encoded protein is secreted and binds hydroxyapatite with high affinity. The osteoclast vitronectin receptor is found in the cell membrane and may be involved in the binding to this protein. This protein is also a cytokine that upregulates expression of interferon-gamma and interleukin-12. Several transcript variants encoding different isoforms have been found for this gene, all of which are encompassed by this application. As used herein, the term “osteopontin” or “OPN”, refers to a bone / sial oprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1), 2ar and Rickettsia resistance (Ric), and is a protein that in humans is encoded by the SPP1 gene (secreted phosphoprotein 1). The murine ortholog is SppP

[0052] As used herein, the term "mRNA" refers to an RNA molecule that encodes a protein.

[0053] As used herein, the term "pre-mRNA" refers to an RNA transcript that has not been fully processed into mRNA. A pre-RNA may include one or more introns.

[0054] As used herein, the term "nucleotide" refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates." The term "linked nucleosides" are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are present between those that are linked).

[0055] As used herein, the term "open reading frame" or "ORF" refers to the portion of the target transcript that encodes the main (or primary) protein associated with an mRNA transcript. In certain embodiments, the ORF encodes the target protein.

[0056] As used herein, the term "translation" refers to the process in which a polypeptide (e.g. a protein) is translated from an mRNA. In certain embodiments, an increase in translation refers to an increase in the number of polypeptide (e.g. a protein) molecules that are made per copy of mRNA that encodes said polypeptide.

[0057] As used herein, the terms "targeting" or "targeted to" or “target sequence” refer to the association of an antisense oligonucleotide to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense oligonucleotide targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.

[0058] As used herein, the term “disrupts translation” refers to any process or activity that impedes, reduces, prevents or negatively impacts the translation of a polypeptide from an mRNA. As used herein, the term “disrupts splicing” refers to any process or activity that impedes, reduces, prevents or negatively impacts the splicing of exons and introns of a pre-mRNA into an mRNA.

[0059] As used herein, the term “expression” refers to the expression of gene encoding a factor associated with cell activation in a cell to produce a mRNA molecule which is translated by one or more ribosomes to produce the fact or the term “expression” refers to a mRNA or a factor associated with cell activation in a cell, which is translated from a mRNA. As used herein, the term “activity” refers to one or more activities of the factor associated with cell activation, and may include, but is not limited to, one or more enzymatic activities of the factor or one or more interactions between the factor and another molecule, such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or an ion. An agent described herein may reduce the expression or activity of a factor associated with cell activation and may reduce both the expression and activity of the factor. Those of ordinary skill in the art will readily recognize that an agent that reduces the expression of a given factor will generally also reduce the activity of the factor by reducing the total number of molecules of the factor that are produced by a cell. For example, an agent described herein may increase the expression or activity of a factor associated with cell activation and may increase both the expression and activity of the factor. Those of ordinary skill in the art will readily recognize that an agent that increases the expression of a given factor will generally also increase the activity of the factor by increasing the total number of molecules of the factor that are produced by a cell.

[0060] As used herein, the term "percent identity" or “% identity” refers to the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

[0061] As used herein, the term “phosphorodiamidate morpholino oligomer” or “PMO” refers to a synthetic nucleic acid analog comprising a backbone formed by morpholine rings linked through phosphorodiamidate groups. Each morpholine unit is attached to a nucleobase via a methylene bridge, enabling sequence-specific hybridization to complementary RNA or DNA targets while conferring resistance to nucleases. As used herein, the term “peptide-conjugated phosphorodiamidate morpholino oligomer” or “PPMO” refers to a PMO covalently linked to one or more peptides, typically cell-penetrating peptides, to enhance cellular uptake and intracellular delivery. The conjugation does not alter the PMO’s hybridization properties but improves pharmacokinetic and biodistribution characteristics. As used herein, the term “2’-O-Methyl Phosphorothioate (2’0Me PS) Oligomer” refers to an oligonucleotide in which the ribose sugar of each nucleotide contains a methyl group at the 2’ -oxygen position, and the intemucleotide linkages incorporate a phosphorothioate modification (replacement of a non-bridging oxygen with sulfur). These modifications confer increased nuclease resistance and improved binding affinity to target nucleic acids. As used herein, the term “2’-O-Methoxyethyl (2’-M0E) Oligomer” refers to an oligonucleotide wherein the ribose sugar of each nucleotide is substituted at the 2’-oxygen position with a methoxyethyl group. This modification enhances duplex stability and resistance to enzymatic degradation while maintaining hybridization capability. As used herein, the term “2’-O-Methoxy ethyl Phosphorothioate” or “2’ -MOE PS Oligomer” refers to an oligonucleotide incorporating both 2’-O-methoxyethyl substitutions on the ribose sugars and phosphorothioate linkages between nucleotides. These combined modifications provide improved nuclease resistance, enhanced affinity for complementary sequences, and favorable pharmacokinetic properties. As used herein, the term “peptide nucleic acid” or “PNA” refers to a synthetic nucleic acid analog in which the sugar-phosphate backbone is replaced by a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units. Nucleobases are attached to this backbone via methylene carbonyl linkages, enabling sequence-specifichybridization with complementary DNA or RNA while imparting high chemical stability and resistance to enzymatic degradation. As used herein, the term “locked nucleic acid” or “LNA” refers to a modified nucleotide in which the ribose ring is chemically constrained by a methylene bridge connecting the 2’-oxygen and 4’-carbon atoms. This “lock” enforces a C3’-endo sugar pucker, increasing thermal stability and affinity of oligonucleotides containing LNA residues for complementary nucleic acids. As used herein, the term “LNA and 2’0Me PS Gapmer” refers to an antisense oligonucleotide comprising LNA-modified nucleotides at the termini and a central “gap” of 2’-O-methyl phosphorothioate-modified nucleotides. The design promotes high-affinity binding and facilitates RNase H-mediated cleavage of the target RNA. As used herein, the term “LNA and 2’MOE Gapmer” refers to an antisense oligonucleotide containing LNA-modified nucleotides at the ends and a central region of 2’-O-methoxyethyl-modified nucleotides. This configuration combines strong target binding with enhanced stability and RNase H activation. As used herein, the term “Gapmer of 2’0Me PS and Natural Nucleic Acids” refers to an oligonucleotide comprising a central region of unmodified nucleotides flanked by 2’-O-methyl phosphorothioate-modified nucleotides. The gap region enables RNase H activity, while the modified flanks confer nuclease resistance. As used herein, the term “Gapmer of 2’MOE PS and Natural Nucleic Acids” refers to an oligonucleotide with a central stretch of natural nucleotides flanked by 2’-O-methoxyethyl phosphorothioate-modified nucleotides, providing a balance of RNase H activation and chemical stability. As used herein, the term “unmodified or modified nucleic acids” refers to unmodified nucleic acids which are naturally occurring DNA or RNA sequences composed of standard nucleotides. Modified nucleic acids include oligonucleotides bearing chemical alterations to the sugar, base, or backbone, such as methylation, phosphorothioate substitution, or locked nucleic acid incorporation, to improve stability, affinity, or biological activity.

[0062] As used herein, the term “cell penetrating peptide” or “CPP” refers to a short peptide sequence capable of facilitating the translocation of conjugated molecules across cellular membranes. CPPs typically contain cationic or amphipathic motifs that interact with membrane components, enabling efficient intracellular delivery of nucleic acids, proteins, or other cargo.

[0063] As used herein, the term “Small Interfering RNA” or “siRNA” refers to a small interfering RNA which is a double-stranded RNA molecule typically 19-25 nucleotides in length, comprising a sense strand and an antisense strand that are partially or fully complementary. siRNAs mediate sequence-specific gene silencing through the RNA interference (RNAi) pathway by guiding the RNA-induced silencing complex (RISC) to degrade target messenger RNA. As used herein, the term “Small Segmented siRNA” or “sisiRNA” refers to a small segmented siRNA which is a modified siRNA structure in which the sense strand is divided into two or more short RNA segments that hybridize to the antisense strand, forming a discontinuous duplex. This design maintains RNAi activity while reducing off-target effects and improving stability. As used herein, the term “Short Hairpin RNA” or “shRNA” refers to a short hairpin RNA which is a single RNA transcript that folds into a stemloop structure, forming a double-stranded stem region and a loop. The stem mimics a siRNA duplex and is processed intracellularly by Dicer into functional siRNA-like molecules that mediate RNA interference. As used herein, the term “Double-Stranded RNA” or “dsRNA” refers to a double-stranded RNA which refers to an RNA molecule composed of two complementary strands forming a duplex structure. dsRNA can vary in length from short oligonucleotides to long polymers and serves as a trigger for RNA interference or other cellular pathways involving RNA recognition. As used herein, the term “MicroRNA” or “miRNA”refers to a microRNA is an endogenous, short RNA molecule typically 18-24 nucleotides in length that forms a duplex intermediate during biogenesis. Mature miRNAs guide RISC to partially complementary sequences in target mRNAs, resulting in translational repression or degradation. As used herein, the term “antagomirs” or “anti-miRs” refers to antagomirs which are chemically modified oligonucleotides designed to hybridize to and inhibit specific microRNAs. While typically single-stranded, antagomirs may form transient duplexes with their target miRNA, thereby preventing its incorporation into RISC and blocking its regulatory function. As used herein, the term “circular RNA” or “circRNA” refers to circular RNA which is a covalently closed RNA molecule forming a continuous loop without free ends. Although not inherently double-stranded, circRNAs can form intramolecular base-paired regions that resemble duplex structures and may act as sponges for microRNAs or interact with RNA-binding proteins. As used herein, the term “phosphorothioate-linked DNA” or “S-DNA” refers to a phosphorothioate-linked DNA refers to a double-stranded DNA molecule in which one or more internucleotide phosphate linkages are modified by replacing a non-bridging oxygen atom with sulfur. This modification enhances nuclease resistance while preserving the ability to form Watson-Crick base pairs in a duplex structure.

[0064] As used herein, the term "treating," "treatment," and the like relate to any treatment of a target disease or condition, including but not limited to prophylactic treatment and therapeutic treatment. "Treating" includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of a target disease or condition, such as cardiac hypertrophy or cardiac fibrosis. Those in need of treatment include those already with target disease or condition, and those in whom the target disease or condition is to be prevented. As used herein, "delaying" the development of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease in a subject.Delaying the progression of a disease may include delaying or preventing the spread of a disease occurring in a subject. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that delays the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time period and / or reduces extent of the symptoms in a given time period, as compared to the absence of such a method.Comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result. The “development” or “progression” of a disease (e.g., a fibrosis, retinopathy) refers to initial manifestations and / or ensuing progression of the disease in a subject. Development of a disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression may refer to the development or progression of symptoms of a disease (e.g., a fibrosis, retinopathy). The term “development” includes the occurrence, recurrence, and onset of a disease. As used herein “onset” or “occurrence” of a disease includes the initial onset of a disease, as well as recurrence of the disease (i.e., in a subject who has had the disease previously).

[0065] As used herein, the term "subject" refers to a mammal, e.g., humans, companion animals (e.g., dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs, horses, fowl, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, birds, and the like). As used herein, a “subject in need thereof’ refers to an individual that has a disease, a symptom of the disease, or a predisposition toward the disease. A method for treating a disease may encompass administering to a subject an agent described herein, or a composition thereof (e.g., a pharmaceutical composition) with the intention to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, a symptom of thedisease, or predisposition toward the disease in the subject. A method for treating a disease may encompass, but is not limited to, prophylaxis, wherein an agent is administered to the subject for the purpose of preventing development of the disease, for example, in a subject that is not known to have the disease but may develop or be at risk of developing the disease in the future. A composition thereof (e.g., a pharmaceutical composition), may also be used for administration to a subject in need thereof for the purpose of reducing the severity of a disease (e.g., pulmonary fibrosis) in the subject.

[0066] As used herein, a “therapeutically effective amount” or “effective amount” refers to the amount of an agent that is sufficient to elicit the desired biological response in the subject, for example, alleviating one or more symptoms of the disease (e.g., a fibrosis, retinopathy). A therapeutically effective amount may be an amount that is either administered to the subject alone or in combination with one or more other agents. Effective amounts vary, as recognized by those skilled in the art, depending on such factors as the desired biological endpoint, the pharmacokinetics of the administered agent, the particular condition or disease being treated, the severity of the condition or disease, the individual parameters of the subject, including age, physical condition, size, gender and weight, the duration of the treatment, the nature of any other concurrent therapy, the specific route of administration, and like factors that are within the knowledge and expertise of the health practitioner to determine. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual agents described herein or any combinations thereof to be used is at most the highest dose that can be safely administered to the subject according to sound medical judgment. Preferably, an effective dose is lower than the highest dose that can be safely administered to the subject. It will be understood by those of ordinary skill in the art, however, that a subject or health practitioner may select a lower dose (e.g., the minimum effective dose) in order to mitigate any potential risks of treatment, such as side effects of the treatment.

[0067] As used herein, the term “pharmaceutically acceptable carrier " refers to any substance suitable for use in administering to a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0068] As used herein, the term “pulmonary fibrosis” refers to a chronic, progressive interstitial lung disease characterized by abnormal and excessive deposition of extracellular matrix components, including collagen, within the pulmonary interstitium. This pathological remodeling leads to thickening and stiffening of lung tissue, distortion of alveolar architecture, and impaired gas exchange. Pulmonary fibrosis may occur idiopathically (idiopathic pulmonary fibrosis, IPF) or secondary to environmental exposures, autoimmune disorders, infections, or drug toxicity. Clinically, pulmonary fibrosis manifests as progressive dyspnea, reduced lung compliance, and declining pulmonary function, ultimately resulting in respiratory failure and increased mortality.II. Antisense oligonucleotides

[0069] An aspect of the application is an antisense oligonucleotide (ASO) comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, wherein the ASO is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene, wherein the mRNA comprises a start codon in an openreading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the ASO to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN. In certain embodiments of this aspect, the nucleotide sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence comprises any one of the sequences:(i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 1. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 3. In certain embodiments of this aspect, the ASO is a molecule comprising modifications selected from one or more from the group comprising: a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), a 2’-O-methyl phosphorothioate (2’OMe PS) oligomer, a 2’-O-methoxyethyl (2’-M0E) oligomer, a 2’-O-methoxyethyl phosphorothioate (2’-M0E PS) oligomer, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a LNA and 2’OMe PS gapmer, a LNA and 2’MOE gapmer, a gapmer of 2’OMe PS and natural nucleic acids, a gapmer of 2’MOE PS and natural nucleic acids, or unmodified or modified nucleic acids, a cell penetrating peptide (CPP), or a combination thereof. In certain embodiments of this aspect, the ASO is conjugated with a CPP. In certain embodiments of this aspect, the ASO is a PMO. In certain embodiments of this aspect, the ASO is a PPMO, wherein the ASO comprises a CPP conjugated with a PMO. In certain embodiments of this aspect, the PPMO is conjugated to a CPP selected from one of the group consisting of: P7, R6B, and RGD. In certain embodiments of this aspect, the CPP is P7. In certain embodiments of this aspect, the CPP is R6B. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence is SEQ ID NO 9. In certain embodiments of this aspect, the target sequence is SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence is SEQ ID NO 6. In certain embodiments of this aspect, the target sequence is SEQ ID NO 8. In certain embodiments of this aspect, the SPP1 or OPN gene is selected from one of the group consisting of SPP1, OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

[0070] An aspect of the application is an ASO comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the ASO to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN. In certainembodiments of this aspect, the nucleotide sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence comprises any one of the sequences:(i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 1. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 3. In certain embodiments of this aspect, the ASO is a molecule comprising modifications selected from one or more from the group comprising: a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), a 2’-O-methyl phosphorothioate (2’OMe PS) oligomer, a 2’-O-methoxyethyl (2’-M0E) oligomer, a 2’-O-methoxyethyl phosphorothioate (2’-M0E PS) oligomer, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a LNA and 2’OMe PS gapmer, a LNA and 2’MOE gapmer, a gapmer of 2’OMe PS and natural nucleic acids, a gapmer of 2’MOE PS and natural nucleic acids, or unmodified or modified nucleic acids, a cell penetrating peptide (CPP), or a combination thereof. In certain embodiments of this aspect, the ASO is conjugated with a CPP. In certain embodiments of this aspect, the ASO is a PMO. In certain embodiments of this aspect, the ASO is a PPMO, wherein the ASO comprises a CPP conjugated with a PMO. In certain embodiments of this aspect, the PPMO is conjugated to a CPP selected from one of the group consisting of: P7, R6B, and RGD. In certain embodiments of this aspect, the CPP is P7. In certain embodiments of this aspect, the CPP is R6B. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence is SEQ ID NO 9. In certain embodiments of this aspect, the target sequence is SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence is SEQ ID NO 6. In certain embodiments of this aspect, the target sequence is SEQ ID NO 8. In certain embodiments of this aspect, the SPP1 or OPN gene is selected from one of the group consisting of SPP1, OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

[0071] In some aspects, the techniques described herein relate to a compound including an oligonucleotide including at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 contiguous nucleotides, wherein the oligonucleotide includes a nucleotide sequence that has at least partial identity to any one of the sequences of SEQ ID NO 1-3, wherein the oligonucleotide targets mRNA or pre-mRNA encoding SPP1 or OPN. In some embodiments, the nucleotide sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences of SEQ ID NO 1-3. In some embodiments, the nucleotide sequence has at least about 90% identity to any one of the sequences of SEQ ID NO 1-3. In some embodiments, the nucleotide sequence has at least about 95% identity to any one of the sequences of SEQ ID NO 1-3. Insome embodiments, the nucleotide sequence has about 100% identity to any one of the sequences of SEQ ID NO 1-3.

[0072] In some aspects, the techniques described herein relate to a compound including an oligonucleotide including at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length, wherein the at least 8 contiguous nucleotides are at least partially complementary to any one of the sequences of SEQ ID NO 6-8, wherein the oligonucleotide targets mRNA or pre-mRNA encoding SPP1 or OPN. In some embodiments, the sequence of the oligonucleotide is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to any one of the sequences of SEQ ID NO 6-8. In some embodiments, the sequence of the oligonucleotide is at least about 90% complementary to any one of the sequences of SEQ ID NO 6-8. In some embodiments, the sequence of the oligonucleotide is at least about 95% complementary to any one of the sequences of SEQ ID NO 6-8. In some embodiments, the sequence of the oligonucleotide is about 100% complementary to any one of the sequences of SEQ ID NO 6-8.

[0073] In some embodiments, the oligonucleotide includes an antisense oligomer (ASO).In some embodiments, antisense oligonucleotides (ASO) may include, but not limited to, a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), a 2'-O-methyl phosphorothioate (2'OMe PS) oligomer, a 2'-O-methoxyethyl (2'-M0E) oligomer, a 2'-O-methoxyethyl phosphorothioate (2'-M0E PS) oligomer, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a LNA and 2'OMe PS gapmer, a LNA and 2'MOE gapmer, a gapmer of 2'OMe PS and natural nucleic acids, a gapmer of 2'MOE PS and natural nucleic acids, or unmodified or modified nucleic acids, or a combination thereof. In some embodiments of the aspects above, the compound is an antisense oligonucleotide conjugate.PMOs and PPMOs

[0074] In some embodiments, phosphorodiamidate morpholino oligomers (PMOs) are ASOs as short single-stranded DNA analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. Unlike other types of antisense oligonucleotides (ASOs) or siRNA, PMOs are relatively uncharged nucleic acid analogs, which can be relatively more or highly stable and resistant to a variety of enzymes present in biologic fluids. In some embodiments, PMOs can bind to complementary sequences of target mRNA to block protein translation through steric blockade in a RNase H independent manner. In some embodiments, PMOs can bind to complementary sequences of target pre-mRNA to prevent productive RNA splicing. In some embodiments, PMOs can be relatively neutral charge and may not relatively bind to proteins.

[0075] In some embodiments, PMOs can be designed or screened based on at least one of various types of methods. For example, in some embodiments, primary PMO candidate sequences can be confirmed by in vitro studies. For example, in some embodiments, Western-blot and ELISA data can be implemented and used for screening of PMOs.

[0076] In some embodiments, PMOs can be conjugated with one or more molecules, e.g., to form PMO conjugates. In some embodiments, PMOs can be conjugated with at least one of different types of molecules, e.g., to form PMO conjugates. In some embodiments, PMOscan be conjugated with one or more different types of molecules, e.g., to form PMO conjugates. In some embodiments, PMOs can be conjugated with different types of molecules, e.g., to form conjugated PMOs, such as Antisense PMO and Cell penetrating peptide (CPP), etc

[0077] In some embodiments, cell penetrating peptides (CPPs) can be designed or screened. For example, in some embodiments, using one primary PMO candidate, CPPs can be conjugated to generate peptide-conjugated PMOs (PPMOs). For example, in some embodiments, conjugated PPMOs were screened in vitro, e.g., by using at least one technique to test for cell uptake, cytotoxicity, and protein reduction. In some embodiments, Westernblot and ELISA data can be utilized for the testing.

[0078] For example, in some embodiments, the following PMOs as indicated in Table 1 were identified based on the disclosure of the present application.TABLE 1Sequence Length

[0079] In some embodiments, the sequence length that can be relatively effective can depend on modality.

[0080] In some embodiments, the length of ASO (single strand) can be from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or about 50 nucleotides. In some embodiments, the length of ASO (single strand) can be from 5 to about 35. In some embodiments, the length of ASO (single strand) can be from 10 to about 30.

[0081] In some embodiments, the length of PMO (morpholino, one type of ASOs) can be from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 nucleotides. In some embodiments, the length of PMO (morpholino, one type of ASOs) can be from 5 to about 35. In some embodiments, the length of PMO (morpholino, one type of ASOs) can be from 10 to about 30.

[0082] In some embodiments, the length of PMO (morpholino, one type of ASOs) can work in a shorter form such as about 14 to about 15.

[0083] In some embodiments, the length of ASO, such as PMO, can be from about 8 to about 40. In some embodiments, it can be relatively more practical if the length of ASO, such as PMO, can be from about 5 to about 40 nucleotides. In some embodiments, the length of ASO, such as PMO, can be relatively more preferably from about 12 to about 30. In some embodiments, the length of ASO, such as PMO, can be relatively more preferably from about 18 to about 28, e.g., in the case of ASO being PMO.

[0084] In some embodiments, the length of ASO such as morpholino, (one type of ASOs, but not limiting on this application) can be about 24 to about 40 nucleotides. In some embodiments, this length can relatively efficiently invade most secondary structures in mRNAs. In some embodiments, this can be a consequence of at least one of the following two factors. First, each Morpholino can be sufficiently long (25-mer) that it has a high probability of being complementary to some single-stranded segment of the target sequence -and this helps to assure efficient nucleation of pairing in spite of the extensive secondary structures characteristic of RNAs in the cell. Second, Morpholinos can have a high affinity for RNA (far higher than the affinity of S-DNA for RNA), and this high affinity allows the Morpholino to efficiently invade any secondary structures which the target sequence might be a part of. Thus, their extended length helps assure effective nucleation of pairing to the target sequence, and their high affinity helps assure that this nucleated pairing progresses to successful invasion of any proximal secondary structures which might be masking the target sequence.

[0085] In some embodiments of the aspects above, the oligonucleotide includes at least 9 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes at least 10 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes at least 12 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes at least 14 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes at least 15 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes at least 16 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes at least 18 contiguous nucleotides in length.

[0086] In some embodiments, the oligonucleotide includes up to 8 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 10 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 15 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 16 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 18 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 20 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 21 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 22 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 23 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 24 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 25 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 26 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 27 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 28 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 29 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 30 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 35 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 40 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes up to 45 contiguous nucleotidesin length. In some embodiments, the oligonucleotide includes up to 50 contiguous nucleotides in length. For the avoidance of doubt, the aforementioned lengths may refer to the total number of nucleotides in the oligonucleotide, the number of contiguous nucleotides complementary to a target sequence, or both, and may also encompass one or more portions of the oligonucleotide that are partially complementary to the target sequence.

[0087] In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 45 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 40 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 35 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 30 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 28 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 25 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 23 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 21 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 20 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 19 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 18 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 17 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 16 contiguous nucleotides in length.

[0088] In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 15 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 8 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 9 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 10 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 12 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 14 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 15 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 16 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 18 contiguous nucleotides to about 50 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 20 contiguous nucleotides to about 40 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 12 contiguous nucleotides to about 35 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 14 contiguous nucleotides to about 30 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 12 contiguous nucleotides to about 25 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 18 contiguous nucleotides to about 21 contiguousnucleotides in length. In some embodiments, the oligonucleotide includes from about 10 contiguous nucleotides to about 30 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 12 contiguous nucleotides to about 30 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 15 contiguous nucleotides to about 30 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 18 contiguous nucleotides to about 28 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 20 contiguous nucleotides to about 28 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 22 contiguous nucleotides to about 28 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 23 contiguous nucleotides to about 28 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 24 contiguous nucleotides to about 26 contiguous nucleotides in length. In some embodiments, the oligonucleotide includes from about 12 contiguous nucleotides to about 18 contiguous nucleotides in length.Sequence Stability

[0089] In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is at least about 20%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is at least about 25%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is at least about 30%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is at least about 32%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is at least about 35%.

[0090] In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is up to about 70%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is up to about 68%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is up to about 65%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 20% to about 70%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 20% to about 68%.

[0091] In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 20% to about 65%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 25% to about 70%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 25% to about 68%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 25% to about 65%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 30% to about 70%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 30% to about 68%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 30% to about 65%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 32% to about 70%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 32% to about 68%. In someembodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 32% to about 65%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 35% to about 70%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 35% to about 68%. In some embodiments, the content of guanine (G) or cytosine (C) in the at least 8 contiguous nucleotides in length is from about 35% to about 65%.

[0092] In some embodiments, a target sequence with a Guanine and Cytosine (G + C) content in the range of about 30% to about 75% can be selected. In some embodiments, a target sequence with a Guanine or Cytosine (which can be Guanine and Cytosine (G + C)) content in the range of about 32% to about 70% can be selected. In some embodiments, a target sequence with a Guanine and Cytosine (G + C) content in the range of about 33% to about 68% can be selected.

[0093] In some embodiments, a target sequence with a Guanine and Cytosine (G + C) content in the range of about 35% to 65% can be selected, e.g., to provide a good balance between efficacy and specificity. In some embodiments, to assure good solubility, the G content can be less than 4 guanines, or the G content can be less than about 36%. In some embodiments, binding affinity, stability, and Tmcan be predicted based on the length and the percentage of G and C content in the sequences.

[0094] In some embodiments of the aspects above, the at least 8 contiguous nucleotides forms a duplex with thermal stability represented by melting temperature of from about 50 °C to 150 °C. In some embodiments, the at least 8 contiguous nucleotides forms a duplex with thermal stability represented by melting temperature of from about 70 °C to 130 °C. In some embodiments, the at least 8 contiguous nucleotides forms a duplex with thermal stability represented by melting temperature of from about 80 °C to 110 °C. In some embodiments, the at least 8 contiguous nucleotides forms a duplex with thermal stability represented by melting temperature of from about 90 °C to 110 °C. In some embodiments, the at least 8 contiguous nucleotides forms a duplex with thermal stability represented by melting temperature of from about 85 °C to 115 °C. In some embodiments, the at least 8 contiguous nucleotides forms a duplex with thermal stability represented by melting temperature of from about 88 °C to 108 °C. In some embodiments, the at least 8 contiguous nucleotides forms a duplex with thermal stability represented by melting temperature of from about 90 °C to 106 °C. In some embodiments, the melting temperature can be based on 10 uM concentration of the compound comprising the at least 8 contiguous nucleotides.III. Double-stranded RNA or DNA molecules

[0095] An aspect of the application is a double-stranded ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule comprising a nucleotide sequence of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides on a 5’-3’ strand, wherein the molecule is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN. In certain embodiments of this aspect, the nucleotide sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,186%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence comprises any one of the sequences:(i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 1. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 3. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence is SEQ ID NO 9. In certain embodiments of this aspect, the target sequence is SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence is SEQ ID NO 6. In certain embodiments of this aspect, the target sequence is SEQ ID NO 8. In certain embodiments of this aspect, the SPP1 or OPN gene is selected from one of the group consisting of SPP1, OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

[0096] An aspect of the application is a double-stranded RNA or DNA molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5 ’-3’ strand, wherein the molecule is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the molecule to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN. In certain embodiments of this aspect, the nucleotide sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence comprises any one of the sequences:(i) SEQ ID NO 1, or (ii) SEQ ID NO 3. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 1. In certain embodiments of this aspect, the nucleotide sequence is SEQ ID NO 3. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 9, or (ii) SEQ ID NO 11. In certain embodiments of this aspect, the target sequence is SEQ ID NO 9. In certain embodiments of this aspect, the target sequence is SEQ ID NO 11. In certain embodiments of this aspect, the target sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence comprises any one of the sequences: (i) SEQ ID NO 6, or (ii) SEQ ID NO 8. In certain embodiments of this aspect, the target sequence isSEQ ID NO 6. In certain embodiments of this aspect, the target sequence is SEQ ID NO 8. In certain embodiments of this aspect, the SPP1 or OPN gene is selected from one of the group consisting of SPP1, OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

[0097] In some embodiments, types of RNAs or DNAs or viral vectors may include, but not limited to, small interfering RNA (siRNA), small segmented siRNA (sisiRNA), short hairpin RNA (shRNA), Double strand RNA (dsRNA), microRNA, Antagomirs (anti-miRs), Circular RNA (circRNA), Phosphorothioate-linked DNA (S-DNA), viral vector that encodes a siRNA or a shRNA that has the target sequences (such as a lentiviral vector or a recombinant adeno-associated viral vector (rAAV)), or a combination thereof.

[0098] In some embodiments, exogenous deoxyribonucleic acids (DNAs) and ribonucleic acids (RNA) directed to the aspects of the present application may have various lengths of their nucleic acid chain. For example, in some embodiments, the length can be between about 5 to about 50 nucleotides, between about 7 to about 40 nucleotides, between about 10 to 35 nucleotides, between 12 to 30 nucleotides. For example, in some embodiments, the length can be around 18 to 21 nucleotides. In some embodiments, the number of nucleotides inthe 5’-3’ single strand of DNA or RNA molecules may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

[0099] In some embodiments, the length of siRNA (double strands) can be in from about 5 to about 40. In some embodiments, the length of siRNA (double strands) can be in from about 7 to about 30. In some embodiments, the length of siRNA (double strands) can be in from about 8 to about 25. In some embodiments, the length of siRNA (double strands) can be in from about 10 to about 20.IV. Pharmaceutical Composition

[0100] An aspect of the application is a pharmaceutical composition comprising an ASO comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, wherein the ASO is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0101] An aspect of the application is a pharmaceutical composition comprising an ASO comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the ASO to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0102] An aspect of the application is a pharmaceutical composition comprising a RNA or DNA molecule comprising a nucleotide sequence of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides on a 5’-3’ strand, wherein the molecule is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0103] An aspect of the application is a pharmaceutical composition comprising a RNA or DNA molecule comprising an ASO comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, wherein the molecule is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the molecule to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers

[0104] Aspects of the application include a pharmaceutical composition comprising ASOs (or RNA or DNA molecules) as described herein, and including a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises one ormore carriers suitable for delivering the therapeutic agents to cells in a subject. Exemplary carriers for delivery include nanoparticles, lipids, liposomes, micelles, polymers, polymeric micelles, emulsions, polyelectrolyte complexes, hydrogels, microcapsules, viruses, virus-like particle (VLPs), peptides, antibodies, aptamers, small molecule chemicals, exosomes, combinations thereof, and pegylated derivatives thereof. In a particular embodiment, the pharmaceutical composition comprises a nanoparticle formulation comprising an ASO in accordance with the present application. In certain particular embodiments, the abovedescribed carriers, including nanoparticles, may be linked to selected heart tissue-specific targeting peptides or antibodies to facilitate carrier-mediated delivery of the active agents described herein to heart tissues. In certain particular embodiments, the above-described carriers, including nanoparticles, may be linked to selected lung tissue-specific targeting peptides or antibodies to facilitate carrier-mediated delivery of the active agents described herein to lung tissues.

[0105] Aspects of the application include a pharmaceutical composition comprising ASOs (or RNA or DNA molecules) as described herein, and including a pharmaceutically acceptable carrier. For example, in certain embodiments, pharmaceutical compositions include nanoparticles or liposomes covalently or non-covalently coated with a heart tissuespecific or lung tissue-specific targeting peptide or antibody. Exemplary nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, polymeric nanoparticles, nanoworms, nanoemulsions, nanogels, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanocapsules, nanospheres, nanofibers, nanohoms, nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle can produce a detectable signal, for example, through absorption and / or emission of photons (including radio frequency and visible photons) and plasmon resonance. Nanoparticles can be biodegradable or non-biodegradable. In certain embodiments, the nanoparticle is a metal nanoparticle, a metal oxide nanoparticle, or a semiconductor nanocrystal. The metal of the metal nanoparticle or the metal oxide nanoparticle can include titanium, zirconium, hafnium, vanadium, niobium, tantalum, chromium, molybdenum, tungsten, manganese, technetium, rhenium, iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel, palladium, platinum, copper, silver, gold, zinc, cadmium, scandium, yttrium, lanthanum, a lanthanide series or actinide series element (e.g., cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, and uranium), boron, aluminum, gallium, indium, thallium, silicon, germanium, tin, lead, antimony, bismuth, polonium, magnesium, calcium, strontium, and barium. In certain embodiments, the metal can be iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver, gold, cerium or samarium. The metal oxide can be an oxide of any of these materials or combination of materials. For example, the metal can be gold, or the metal oxide can be an iron oxide, a cobalt oxide, a zinc oxide, a cerium oxide, or a titanium oxide. Preparation of metal and metal oxide nanoparticles is described, for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199. In other embodiments, a polymeric nanoparticle is made from a synthetic biodegradable polymer, a natural biodegradable polymer or a combination thereof. Synthetic biodegradable polymers can include, polyesters, such as poly(lactic-co-glycolic acid)(PLGA) and polycaprolactone; polyorthoesters, polyanhydrides, polydioxanones, poly-alkyl-cyano-acrylates (PAC), polyoxalates, polyiminocarbonates, polyurethanes, polyphosphazenes, or a combination thereof. Natural biodegradable polymers can include starch, hyaluronic acid, heparin, gelatin, albumin, chitosan, dextran, or a combination thereof.

[0106] In some embodiments, the pharmaceutical composition comprises a delivery carrier, such as a nanoparticle or liposome encapsulating a pharmaceutically effective amount of the antisense oligonucleotide. In some embodiments, the pharmaceutically effective amount of an ASO (or RNA or DNA molecules) as described herein is from about 0.001 mg / mL to about 100 mg / mL (w / v) of the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically effective amount of ASO (or RNA or DNA molecules) as described herein is from about 0.1 mg / mL to about 1 mg / mL (w / v) of the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically effective amount of ASO (or RNA or DNA molecules) as described herein is from about 1 mg / mL to about 10 mg / mL (w / v) of the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically effective amount of ASO (or RNA or DNA molecules) as described herein is from about lOmg / ml to about lOOmg / ml of the pharmaceutically acceptable carrier. In some embodiments, the ASO (or RNA or DNA molecules) as described herein possesses physicochemical properties that facilitate the formulation of high-concentration compositions up to lOOmg / ml. For instance, the oligonucleotide may exhibit a reduced or neutralized net charge and a low viscosity profile at high concentrations when compared to highly charged nucleic acids. Examples of such ASO (or RNA or DNA molecules) include, but are not limited to, PMOs, as it is contemplated that their non-ionic nature facilitates high-concentration formulation. This high concentration range is particularly valuable for applications requiring low-volume administration or high delivered doses, such as localized therapies or inhalation delivery, where minimizing injection or administration volume is critical for clinical practicality and patient compliance. The concentrations and delivery carriers described herein are provided as illustrative and non-limiting examples of embodiments of the present invention.

[0107] In some embodiments, the pharmaceutical composition comprises an ASO (or RNA or DNA molecules) as described herein of the present application and a lipid moiety. Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, the lipid moiety is selected to increase distribution of a pharmaceutical agent to heart tissue or lung tissue. In certain embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to heart muscle or lung tissue.

[0108] In certain embodiments, pharmaceutical compositions provided herein include one or more ASOs (or RNA or DNA molecules) as described herein and one or more excipients. Exemplary excipients include water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and combinations thereof. In certain embodiments, the pharmaceutical compositions including one or more hydrophobic compounds, including organic solvents, such as dimethylsulfoxide.

[0109] In certain embodiments, the pharmaceutical composition provided herein comprises a co-solvent system. Co-solvent systems may include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w / v benzyl alcohol, 8% w / v of the nonpolar surfactant Polysorbate 80™ and 65% w / v polyethylene glycol 300. The proportions of such co-solventsystems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied. For example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0110] In some embodiments, the pharmaceutical composition comprises a sterile saline solution and one or more ASOs (or RNA or DNA molecules) as described herein. In certain embodiments, the pharmaceutical composition consists of a sterile saline solution and one or more ASOs (or RNA or DNA molecules) as described herein. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, the pharmaceutical composition comprises one or more ASOs (or RNA or DNA molecules) as described herein and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more ASOs and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, the pharmaceutical composition comprises one or more ASOs (or RNA or DNA molecules) as described herein and phosphate- buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more ASOs (or RNA or DNA molecules) as described herein and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.[OHl] In certain embodiments, ASOs (or RNA or DNA molecules) as described herein are admixed with pharmaceutically acceptable active and / or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions can depend on a number of criteria, including, but not limited to, route of administration, extent of disease, and / or dose to be administered. In certain embodiments for inhalation, the pharmaceutical composition is formulated to have an osmolality and viscosity suitable for aerosolization via a nebulizer.

[0112] Pharmaceutical compositions comprising ASOs (or RNA or DNA molecules) as described herein may include any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising ASOs (or RNA or DNA molecules) as described herein comprise one or more oligonucleotides, which, upon administration to an animal, such as a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of ASOs (or RNA or DNA molecules) as described herein, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active compound.Oligonucleotide Activity Enhancers

[0113] In certain aspects of the pharmaceutical compositions described herein, the pharmaceutical composition may include oligonucleotide activity enhancers (OAEs) including, but not limited to, the OAEs shown in Table 1 below.Table 1: Structures of OAEs 1-5.<<""Formulation

[0114] The pharmaceutical composition of the present application is formulated in accordance with the particular route of administration. In certain preferred embodiments, the pharmaceutical composition is formulated for administration by intravenous or intramyocardial injection. In certain embodiments, the pharmaceutical composition is formulated for administration by nebulization or inhalation. In certain preferred embodiments, delivery can be achieved via localized administration routes such as intratracheal instillation, nebulized inhalation, or intranasal delivery, which provide direct access to the respiratory tract and minimize systemic exposure.

[0115] In certain embodiments, the pharmaceutical composition is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or that serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi -dose containers. Some pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain.

[0116] In certain embodiments, the pharmaceutical composition may be formulated as inhaled formulations, including sprays, mists, or aerosols. This may be particularly preferred for treatment of a condition of the airway or lung involving fibrosis as described herein. The inhaled formulation may be for application to the upper (including the nasal cavity, pharynx and larynx) and lower respiratory tract (including trachea, bronchi and lungs). As used herein, the upper respiratory tract may include any one or more of the following regions: the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords). Typically, the lower respiratory tract includes any one or more of the following regions: the portion of the larynx below the vocal folds, trachea, bronchi and bronchioles. The lungs can be included in the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0117] For inhalation formulations, the composition or combination provided herein may be delivered via any inhalation methods known to a person skilled in the art. Such inhalation methods and devices include, but are not limited to, metered dose inhalers with propellants such as HFA or propellants that are physiologically and environmentally acceptable. Other suitable devices are breath operated inhalers, multidose dry powder inhalers and aerosol nebulizers. Aerosol formulations for use in the subject method typically include propellants, surfactants and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve. Different devices and excipients can be used depending on whether the application is to the upper (including the nasal cavity, pharynx and larynx) or lower respiratory tract (including trachea, bronchi and lungs) and can be determined by those skilled in the art. Further, processes for micronisation and nanoparticle formation for the preparation of ASOs (or RNA or DNA molecules) described herein for use in an inhaler, such as a dry powder inhaler, are also known by those skilled in the art.

[0118] Inhalant compositions may comprise liquid or powdered compositions containing the active ingredient that are suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses. Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalant solvent such as isotonic saline or bacteriostatic water. The solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's lungs. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Examples of inhalation drug delivery devices are described in Ibrahim et al. Medical Devices: Evidence and Research 2015:8 131-139, are contemplated for use in the present application.

[0119] The ASOs (or RNA or DNA molecules) as described herein may be formulated for intranasal administration, including dry powder, sprays, mists, or aerosols. This may be particularly preferred for treatment of a respiratory infection. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Alternatively, the ASOs (or RNA or DNA molecules) as described herein may be provided as a dry powder and administered to the upper respiratory tract only.Uses of pharmaceutical compositions

[0120] In some embodiments, the pharmaceutical composition is to treat a lung condition. In some embodiments, the pharmaceutical composition is to treat Idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, asthma, Chronic obstructive pulmonary disease (COPD), Pulmonary hypertension (PH or PHTN), Pulmonary arterial hypertension (PAH), lung cancer. In some embodiments, the pharmaceutical composition is to treat an ocular condition. In some embodiments, the pharmaceutical composition is to treat retinopathy of prematurity (ROP), dry age-related macular degeneration (AMD), wet AMD, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, retinal injury, or glaucoma retinopathy. In some embodiments, the pharmaceutical composition is to treat cancers, autoimmune diseases, psoriasis, fibrosis, neurodegenerative diseases liver diseases, Urinary System Diseases, heart and vascular diseases, metabolic impairments, bone conditions, or a combination thereof. In some embodiments, the pharmaceutical composition is to treat cancers, Idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, RA, atrial fibrillation or a combination thereof.

[0121] In some embodiments, the pharmaceutical composition is to treat myeloma, chronic myeloid leukemia and acute myeloid leukemia (AML), rheumatoid arthritis, inflammatory bowel disease (IBD), Systemic lupus erythematosus (SLE), psoriasis, , liver fibrosis, kidney fibrosis, muscular fibrosis, myocardial fibrosis, Alzheimer's or Parkinson's disease, Multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), Huntington's disease, acute liver failure, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), viral hepatitis, hepatocellular carcinoma, non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver diseases (MAFLD), lower urinary tract symptoms (LUTS), chronic kidney disease (CKD), atherosclerosis, hypertension, cardiovascular neurosis, atrial fibrillation, myocardial infarction, cardiomyopathy, stroke, valvular calcification, diabetes, obesity, Osteoarthritis, knee pain, osteoporosis, osteoporosis leukemia, Pulpitis, periodontitis, allergic dermatitis, asthma, Chronic obstructive pulmonary disease (COPD), Pulmonary hypertension (PH or PHTN), Pulmonary arterial hypertension (PAH), or a combination thereof.V. Methods of Treatment

[0122] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising an ASO comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, wherein the ASO is capable of binding to a target sequence in a mRNA of a SPP1 or OPN gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0123] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising an ASO comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the ASO to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0124] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising a RNA or DNA molecule comprising a nucleotide sequence of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides on a 5 ’-3’ strand, wherein the molecule is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene, wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN, wherein the target sequence comprises the start codon in the open reading frame of the mRNA, and wherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN, and a pharmaceutically acceptable carrier.

[0125] An aspect of the application is a method of treating a disease by administering a pharmaceutical composition comprising a RNA or DNA molecule comprising a nucleotide sequence of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides on a 5 ’-3’ strand, wherein the molecule is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene, wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, and wherein binding of the molecule to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN, and a pharmaceutically acceptable carrier.SPP1 or Osteopontin as a therapeutic target

[0126] In further aspects, the present disclosure provides a method as described herein for using SPP1 or Osteopontin as a therapeutic target in treating different types of diseases or conditions in a subject. For example, SPP1 or Osteopontin can be used as a therapeutic target for Fibrosis including Idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, liver fibrosis, kidney fibrosis, muscular fibrosis, myocardial fibrosis. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease (IBD), Systemic lupus erythematosus (SLE), psoriasis, etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for ocular indications, such as retinopathy of prematurity (ROP), dry age-related macular degeneration (AMD), wet AMD, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, retinal injury, or glaucoma retinopathy. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for all or most or many types of cancers including solid tumors such as lung cancer, head and neck cancers, etc, and blood cancers such as myeloma, chronic myeloid leukemia and acute myeloid leukemia (AML). In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease, Multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), Huntington’s disease, etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for liver diseases including acute liver failure, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), viral hepatitis, hepatocellular carcinoma, nonalcoholic steatohepatitis (NASH), metabolic-associated fatty liver diseases (MAFLD), etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for urinary System Diseases such as lower urinary tract symptoms (LUTS), chronic kidney disease (CKD), etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for heart and vascular diseases such as atrial fibrillation, atherosclerosis, hypertension, cardiovascular neurosis, myocardial infarction, cardiomyopathy, stroke, valvular calcification, etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for metabolic diseases such as diabetes, obesity, high blood pressures, heart conditions, etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for conditions related to a bone such as osteoarthritis, knee pain, osteoporosis, osteoporosis leukemia, etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for various different conditions, such as pulpitis, periodontitis, allergic dermatitis, asthma, etc. In some embodiments, SPP1 or Osteopontin can be used as a therapeutic target for various different conditions, such as abnormal (most are upregulated) expressions of SPP1 or OPN that have been found in patients. In some embodiments, the expression level of OPN inIPF patients is highly associated with the disease severity and survival. In some embodiments, SPP1 can be used as a biomarker and as a therapeutic target.

[0127] In further aspects, the present disclosure provides a method as described herein for using SPP1 or Osteopontin as a therapeutic target in treating different types of diseases or conditions in a subject. In some embodiments, the method is to treat an ocular condition. In some embodiments, the method is to treat retinopathy of prematurity (ROP), dry age-related macular degeneration (AMD), wet AMD, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, retinal injury, or glaucoma retinopathy. In some embodiments, the method is to treat cancers, autoimmune diseases, psoriasis, fibrosis, neurodegenerative diseases liver diseases, Urinary System Diseases, heart and vascular diseases, metabolic impairments, bone conditions, or a combination thereof. In some embodiments, the method is to treat cancers, Idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, RA, or a combination thereof. In some embodiments, the method is to treat Idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, asthma, Chronic obstructive pulmonary disease (COPD), Pulmonary hypertension (PH or PHTN), Pulmonary arterial hypertension (PAH), lung cancer. In some embodiments, the method is to treat cancers, autoimmune diseases, psoriasis, fibrosis, neurodegenerative diseases liver diseases, Urinary System Diseases, heart and vascular diseases, metabolic impairments, bone conditions, or a combination thereof. In some embodiments, the method is to treat myeloma, chronic myeloid leukemia and acute myeloid leukemia (AML), rheumatoid arthritis, inflammatory bowel disease (IBD), Systemic lupus erythematosus (SLE), psoriasis, Idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, liver fibrosis, kidney fibrosis, muscular fibrosis, myocardial fibrosis, Alzheimer's or Parkinson's disease, Multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), Huntington's disease, acute liver failure, alcoholic liver disease, non-alcoholic fatty liver disease (NAFUD), viral hepatitis, hepatocellular carcinoma, non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver diseases (MAFLD), lower urinary tract symptoms (LUTS), chronic kidney disease (CKD), atrial fibrillation, atherosclerosis, hypertension, cardiovascular neurosis, myocardial infarction, cardiomyopathy, stroke, valvular calcification, diabetes, obesity, Osteoarthritis, knee pain, osteoporosis, osteoporosis leukemia, Pulpitis, periodontitis, allergic dermatitis, asthma, Chronic obstructive pulmonary disease (COPD), Pulmonary hypertension (PH or PHTN), Pulmonary arterial hypertension (PAH), or a combination thereof.

[0128] In further aspects, the present disclosure provides a method as described herein for using SPP1 or Osteopontin as a therapeutic target in treating different types of diseases or conditions in a subject. In some embodiments, treatable fibrosis includes Idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, liver fibrosis, kidney fibrosis, muscular fibrosis, myocardial fibrosis, etc. In some embodiments, treatable lung diseases include pulmonary fibrosis, asthma, Chronic obstructive pulmonary disease (COPD), Pulmonary hypertension (PH or PHTN), Pulmonary arterial hypertension (PAH), lung cancer, etc. In some embodiments, treatable cancers include all solid tumors such as lung cancer, NSCLC, head and neck cancer, prostate cancer, breast cancer, cervical cancer, ovarian cancer, pancreatic cancer, etc. In some embodiments, treatable cancers include all blood cancers such as myeloma, chronic myeloid leukemia and acute myeloid leukemia (AML) , etc. In some embodiments, treatable diseases include autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease (IBD), Systemic lupus erythematosus (SLE), psoriasis, etc. In some embodiments, treatable diseases include ocular indications: all ocular indications include but not limited to retinopathy of prematurity (ROP), dry age-related macular degeneration (AMD), wet AMD, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, retinal injury, or glaucoma retinopathy, etc. In some embodiments, treatablediseases include neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease, Multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), Huntington’s disease, etc. In some embodiments, treatable diseases include liver diseases include acute liver failure, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), viral hepatitis, hepatocellular carcinoma, non-alcoholic steatohepatitis (NASH), metabolic-associated fatty liver diseases (MAFLD). In some embodiments, treatable diseases include Urinary System Diseases: lower urinary tract symptoms (LUTS), chronic kidney disease (CKD) , etc.

[0129] In further aspects, the present disclosure provides a method as described herein for using SPP1 or Osteopontin as a therapeutic target in treating different types of diseases or conditions in a subject. In some embodiments, treatable diseases include heart and vascular diseases: atrial fibrillation, atherosclerosis, hypertension, cardiovascular neurosis, myocardial infarction, cardiomyopathy, stroke, valvular calcification, etc. In some embodiments, treatable diseases include metabolic diseases: diabetes, obesity, etc. In some embodiments, treatable diseases includes bone-related diseases: Osteoarthritis, knee pain, osteoporosis, osteoporosis leukemia, etc. In some embodiments, treatable diseases includes other various types of diseases, such as pulpitis, periodontitis, allergic dermatitis, etc.Treatment of Pulmonary Fibrosis

[0130] In any aspect, the methods described herein may be used to prevent or treat a condition of the airway or lung involving fibrosis in a subject in need thereof. In particular, the condition of the airway or lung involving fibrosis is pulmonary fibrosis. Even more specifically, the condition of the airway or lung involving fibrosis is idiopathic pulmonary fibrosis, familial pulmonary fibrosis, pulmonary fibrosis caused by sarcoidosis, pulmonary fibrosis caused by silicosis, pulmonary fibrosis caused by asbestosis, pulmonary fibrosis caused by coal worker's pneumoconiosis, pulmonary fibrosis caused by carbon pneumoconiosis, pulmonary fibrosis caused by hypersensitivity pneumonitides, pulmonary fibrosis caused by inhalation of inorganic dust, pulmonary fibrosis caused by an infectious agent, pulmonary fibrosis caused by inhalation of noxious gases, aerosols, chemical dusts, fumes or vapors, drug-induced interstitial lung disease. The condition of the airway or lung may further involve inflammation-induced fibrosis or chronic respiratory distress associated with fibrotic tissue remodeling. In any aspect, the ASOs (or RNA or DNA molecules) are administered to the total respiratory tract, the upper respiratory tract or the lower respiratory tract.Dosage

[0131] In some embodiments, the ASO (or RNA or DNA molecules) as described herein dosage may be expressed as the amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of the ASO (or RNA or DNA molecules) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., for determining the LD50 — the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages or amounts for use in mammals (e.g., humans). The dosage or amount of an ASO (or RNA or DNA molecules) as described herein preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage or amount may vary within this range depending upon the dosageform employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. Dosage amount and interval may be adjusted individually to provide plasma or target tissue levels of the active moiety which are sufficient to maintain the desired effects.

[0132] Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g., by means of an appropriate, conventional pharmacological protocol). A physician may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to effect a beneficial therapeutic response in the patient over time, or, e.g., to reduce symptoms, or other appropriate activity, depending on the application. The dose can be determined by the efficacy of the particular formulation, and the activity, stability or serum and / or tissue half-life of the ASOs (or RNA or DNA molecules) as described herein employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose can also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular composition in a particular patient.

[0133] Optimal precision in achieving effective ASOs (or RNA or DNA molecules) as described herein concentrations within a range yielding maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the pharmaceutical composition's availability to the targeted heart tissues or lung tissues. Distribution, equilibrium, and elimination of a pharmaceutical composition may be considered when determining the optimal concentration for a treatment regimen. Generally, the pharmaceutical compositions of the present invention may be administered in a manner that maximizes efficacy and minimizes toxicity.

[0134] Moreover, the dosage administration of the compositions of the present invention may be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen and a pharmacokinetic / pharmacodynamic model may be used to determine the pharmacokinetic / pharmacodynamic profile of one or more dosage regimens. Next, one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic / pharmacodynamic response based on the particular pharmacokinetic / pharmacodynamic profile. See, e.g., US 6,747,002, which is entirely expressly incorporated herein by reference.

[0135] More specifically, the ASO(s) or RNA or DNA molecules as described herein of the present application are preferably administered intermittently, such as once every two days, twice weekly, once weekly, biweekly, monthly, or every two to six months. However, the compositions may also be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily to achieve a loading dose or for maintenance therapy. The composition is formulated for administration by injection, including, but not limited to, intravenous, subcutaneous, intramuscular, or specific organ injection (e.g., targeted pulmonary delivery). The total daily dosage of the composition may be varied over a wide range from about 0.001 mg to about 1,000 mg per patient, per administration (where an administration may be daily or intermittent). The dose may more particularly be expressed in terms of body weight, ranging from about 0.001 mg / kg to about 10 mg / kg or up to lOOmg / kg of body weight per administration.

[0136] The dosage of the ASO(s) or RNA or DNA molecules as described herein of the present application may be varied over a wide range from about 0.1 pg to about 1000 mg per adult human per day, or per week, or per month, or per quarter. For oral or targeted pulmonary administration (e.g., nebulization) administration, the compositions may be provided in dosages from about 0.1 pg to about 1000 mg of the composition or 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 milligrams of the composition for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the pharmaceutical composition is ordinarily supplied at a dosage level of from about 0.1 pg / kg to about 20 mg / kg of body weight per day, or per week, or per month, or per quarter. In one embodiment, the range is from about 0.2 pg / kg to about 10 mg / kg of body weight per day. In another embodiment, the range is from about 0.5 pg / kg to about 10 mg / kg of body weight per day, or per week, or per month, or per quarter. The pharmaceutical compositions may be administered on a regimen of about 1 to about 10 times per day or per week, or per month, or per quarter.

[0137] In the case of injections, it is usually convenient to give by an intravenous route or via a subcutaneous route in an amount of about 0.01 pg-30 mg / kg, about 0.01 pg-20 mg / kg or about 0.01-10 mg / kg per day, or per week, or per month, or per quarter to adults (at about 60 kg). In certain embodiments, injection of ASO(s) or RNA or DNA molecules is typically based on body weight such as lOmg / kg or a fixed dose for adult, such as 50mg , 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, or 600 mg in clinical, which may be for weekly, monthly, three months, or twice a year.

[0138] Doses of ASO(s) or RNA or DNA molecules as described herein of the present application can optionally include 0.0001 pg to 1,000 mg / kg / administration, or 0.001 pg to 100.0 mg / kg / administration, from 0.01 pg to 10 mg / kg / administration, from 0.1 pg to 10 mg / kg / administration, including, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and / or 100-500 mg / kg / administration or any range, value or fraction thereof, or to achieve a peak concentration (e.g., in serum or target tissues such as lung tissues) of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12.0, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 14.9, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, lOOOng / ml, or 1.1, 1.2, 1.3, 1.4, 1.5, 2, 5, 10, 20, 50, 100, 200, or 500 pg / mL, per single or multiple administration or any range, value or fraction thereof. In certain embodiments, doses of ASO(s) (or RNA or DNA molecules) as described herein can include about O.OOOlmg / kg (lOOng / kg) per single or multiple administration.

[0139] As a non-limiting example, treatment of humans can be provided as a onetime or periodic dosage of the ASO(s) or RNA or DNA molecules as described herein, ranging from about 0.0001 mg / kg to 100 mg / kg such as 0.0001, 0.001, 0.01, 0.1 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, or per week, or per month, or per quarter, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, or alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof, using single, infusion or repeated doses.Administration

[0140] Routes of administration for the therapeutic agents of the present application include oral and parenteral administration, i.e., injection, infusion, or implantation or by some other route other than the alimentary canal. Specific modes of administration include injections, such as intravenous, intramyocardial, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0141] In certain embodiments, the therapeutic agents of the present application are formulated for administration by pulmonary inhalation, including, but not limited to, aerosol, nebulization, or dry powder inhalation. In certain embodiments, the ASOs (or RNA or DNA molecules) as described herein can be delivered by pulmonary or nasal routes (e.g., via nebulized saline incorporating the ASOs). The ASOs (or RNA or DNA molecules) as described herein may be in compositions formulated for administration to the lower respiratory tract only. Limitation to the lower respiratory tract may be achieved by an amount, particularly volume and composition of form i.e. particle size, physical form whether dry powder or solution droplet, of composition that would otherwise be administered to the upper respiratory tract. Alternatively, the ASOs (or RNA or DNA molecules) as described herein may be administered via a device that ensures retention in the lower respiratory tract only.

[0142] Specifically, the ASO(s) (or RNA or DNA molecules) as described herein of the present application may be administered at least once a week over the course of several weeks. In one embodiment, the pharmaceutical compositions are administered at least once a week over several weeks to several months. In another embodiment, the pharmaceutical compositions are administered once a week over four to eight weeks. In yet another embodiment, the pharmaceutical compositions are administered once a week over four weeks.

[0143] In preferred embodiments, ASO(s) (or RNA or DNA molecules) as described herein may be administered via a staged dosing regimen comprising a loading phase and a maintenance phase. The loading phase comprises administration once per week, biweekly, or monthly for up to six or nine total doses. The maintenance phase immediately follows the loading phase and comprises administration monthly, bimonthly, or once every three, four, or six months for lifelong maintenance. This regimen may be executed via any route of administration and may be adjusted based on the individual patient’s clinical response and serum concentration.

[0144] Alternatively, the ASO(s) (or RNA or DNA molecules) as described herein of the present application may be administered at least once a week for about 2 weeks, at least once a week for about 3 weeks, at least once a week for about 4 weeks, at least once a week for about 5 weeks, at least once a week for about 6 weeks, at least once a week for about 7 weeks, at least once a week for about 8 weeks, at least once a week for about 9 weeks, at leastonce a week for about 10 weeks, at least once a week for about 11 weeks, at least once a week for about 12 weeks, at least once a week for about 13 weeks, at least once a week for about 14 weeks, at least once a week for about 15 weeks, at least once a week for about 16 weeks, at least once a week for about 17 weeks, at least once a week for about 18 weeks, at least once a week for about 19 weeks, or at least once a week for about 20 weeks, about once every 22 weeks, about once every 24 weeks, about once every 26 weeks.

[0145] Alternatively the ASO(s) (or RNA or DNA molecules) as described herein of the present application may be administered at least once a week for about 1 month, at least once a week for about 2 months, at least once a week for about 3 months, at least once a week for about 4 months, at least once a week for about 5 months, at least once a week for about 6 months, at least once a week for about 7 months, at least once a week for about 8 months, at least once a week for about 9 months, at least once a week for about 10 months, at least once a week for about 11 months, or at least once a week for about 12 months.Effect of Treatment

[0146] In some embodiments, the agent to be administered to a subject can be an agent that reduces the expression and / or activity of a factor associated with cell activation that can be disclosed herein. In some embodiments, the agent to be administered to a subject can be an agent that reduces the expression and / or activity of a factor associated with cell activation that can be upregulated. In some embodiments, the agent to be administered to a subject can be an agent that reduces the expression and / or activity of a single factor associated with cell activation. In some embodiments, the agent to be administered to a subject can be an agent that reduces the expression and / or activity of more than one factor associated with cell activation. In some embodiments, more than one agent can be administered to a subject to reduce the expression and / or activity of one or more factors associated with cell activation (i.e., 1 factor associated with cell activation, 2 factors associated with cell activation, 3 factors associated with cell activation, or more). A “decrease” in a response may be statistically significant as compared to the response produced by no ASO (or RNA or DNA molecule) as described herein or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.

[0147] In some embodiments, the treatment of the cells results in a decrease in the expression and / or activity of the factor associated with cell activation in the cells by up to about 10%, up to about 20%, up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 95%, up to about 99%, or up to about 100%, as compared to expression and / or activity of the factor associated with cell activation prior to the treatment. In some embodiments, the treatment of the cells results in reduced activation of the cells by up to about 10%, up to about 20%, up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 95%, up to about 99%, or up to about 100%, as compared activation of the lineage cells prior to the treatment. In some embodiments, the reduction of activation of the cells comprises reduction of proliferation of the cells. In some embodiments, the reduction of activation of the cells comprises the reduction of release of one or more proinflammatory cytokines or chemokines from the cells.

[0148] In some embodiments, the compound is to cause at least about 20% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments,the compound is to cause at least about 25% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 30% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 35% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 38% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 40% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 43% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 45% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN.

[0149] In some embodiments, the compound is to cause at least about 50% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 55% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 57% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 60% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 65% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 70% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 75% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 80% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN.

[0150] In some embodiments, the compound is to cause at least about 90% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 95% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 97% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause about 100% reduction in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN.Alternate method: increase in expression

[0151] The present disclosure also relates to an alternate method of increasing expression levels and / or activity of SPP1 or OPN in a cell comprising administering an effective amount of the compound of any among to the cell or an effective amount of the pharmaceutical composition of to the cell. The present disclosure relates to a method of increasing expression levels and / or activity of SPP1 or OPN in a subject in need thereof comprising administering an effective amount of the compound of to the subject or an effective amount of the pharmaceutical composition of to the subject. In some embodiments, it is unexpectedly found that Hu-02 (SEQ ID. NO: 2) up-regulated, modulated or increased SPP1 or OPN production. In some embodiments, Hu-02 binds the 5’ upstream untranslated region of SPP1 thus stabilize the mRNA and then increase OPN production.

[0152] In some embodiments, the compound is to cause at least about 20% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments,the compound is to cause at least about 25% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 30% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 35% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 38% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 40% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 43% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 45% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN.

[0153] In some embodiments, the compound is to cause at least about 50% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 55% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 57% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 60% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 65% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 70% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 75% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 80% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 90% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 95% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN.

[0154] In some embodiments, the compound is to cause at least about 100% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 110% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 120% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 150% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 200% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 250% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 300% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 350% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN. In some embodiments, the compound is to cause at least about 400% increase in translation of SPP1 or OPN from the mRNA encoding SPP1 or OPN.

[0155] The present application is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patentapplications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference.EXAMPLES

[0156] EXAMPLE 1 : Preparation for ASO and PMO measurements.

[0157] ASO and PMO measurements in the examples were prepared as described below.

[0158] All antisense oligomers (ASO) or phosphorodiamidate morpholino oligomers (PMO) were synthesized and purchased at Gene Tools (Philomath, OR USA). Cell lines (SIM-A9 and THP-1) were purchased from ATCC (Manassas, Virginia USA). U251 cells were provided by AscentGene (Gaithersburg, MD USA). Human OPN Quantification ELISA kit (DOSTOO) and mouse OPN quantification ELISA Kit (MOSTOO) were purchased from R&D System (Minneapolis, MN USA). Anti OPN antibody (human, Ab214050) was purchased from Abeam (Waltham, MA USA). Anti OPN antibody (mouse, AF808-SP) purchased from R&D System (Minneapolis, MN USA). HRP-anti-rabbit IgG (KPL, 95059-086) and HRP-anti-goat IgG (PISA51031) secondary antibodies were purchased from ThermoFisher. Anti-Actin antibody (A00702) was purchased from GenScript (Piscataway NJ USA). Endo-porter was purchased from Gene Tools (Philomath, OR USA).

[0159] Cell culture: U251 cells and SIM-A9 cells were cultured with DMEM and DMEM F-12 medium with 10% FBS (fetal bovine serum), respectively. They were seeded into each well of 12-well plates with 3X105 / ml / well cell’s density. After 24 hours, ASO’s mixture was prepared by adding an appropriate amount of ASO to lOOul media and mixed with lOOul the same media containing 5ul of Endo-porter. After incubated at RT for 20min, the original media were removed from each well of the plate, 800ul fresh media and 200ul mixture of each indicated ASOs were then added to each well. After 24 hours (or times indicated in each figure legend), cells were harvested using 0.25% Trypsin-EDTA. The harvested cells were washed with 1XPBS, resuspended with 100-150ul lysis buffer (50mM Tris / PH7.5; 150mM NaCl; 0.2% TristonX-100; and 0.5mMEDTA and Benzonase, AscentGene, Inc). Protein concentration was determined with a Protein Assay Kit from Bio-Rad (#5000001).

[0160] THP-1 cells were cultured with RPMI 1640 medium with 10% FBS. Cells were seeded with 6X105 / 0.8ml / well into each well of 24-well plate on the day of assay. 200ul of ASO’s mixture containing 5ul of Endo-porter was then added into each well. Cells were harvested by centrifugation post 24- or 48-hours treatment, then resuspended with 100-150ul lysis buffer after washing with 1XPBS.

[0161] Western-blot:

[0162] A total 10-20 pg / per sample was analyzed on SDS page gel (SurePAGE, GeneScript, Cat# M00654) (175 Voltage for 25min). After transferred the proteins onto the nitrocellulose blotting membrane with semi -dry Trans-Blot (15 Voltage for 35min), the membrane was then blocked with 3% milk / TBST, detected with the primary antibody of human anti-OPN and / or mouse anti-OPN ab, and an appropriate 2nd antibody. Typically, membranes were incubated with the primary antibody at room temperature for 1 hour or at 4C overnight and then with the secondary antibody for one hour at room temperature or 4C overnight.

[0163] ELISA:

[0164] All reagents, standard dilutions, control, and samples were prepared as directed in the manufacture’s manuals. lOOul Assay Diluent RD1-6 (Human OPN Elisa) or 50ul RD1W (Mouse OPN Elisa) was added to each well and mixed with 50ul of Standard, control, or samples per well. After incubating at RT for 2 hours, the reaction mix was removed and washed with 200ul IX Wash Buffer for a total of five times. To detect the signal, 200ul of Human OPN Conjugate or lOOul of Mouse / Rat OPN Conjugate was added to each well and incubated at RT for 2 hours. After washed five times with the Wash Buffer, 200ul (human) or lOOul (mouse) of Substrate Solution was added to each well, incubated at RT for 15-30 min. Then 50ul (human) or lOOul (mouse) of Stop Solution was added to each well, gently tapping the plate to ensure thorough mixing. The optical density of each well was determined within 30min, using a microplate reader set to 450nm.

[0165] EXAMPLE 2: ASO activities in human U-251 cells

[0166] ASO activities in human U-251 cells were measured using Hu-01, Hu-02, Hu-03, PMO-Control, and Cell only control. FIG. 4 shows antisense oligo (ASO) activities (westernblotting) on regulating OPN (Osteopontin, or SPP1) production in some embodiments.Referring to FIG. 4, U-251 cells were treated with 1 pM or 10 pM Hu-01, Hu-02, and Hu-03, and a control (PMO-CTRL) for 24 hours. 10 pg of total protein from cell lyses of each reaction was loaded into the indicated well, detected with anti-OPN antibody.

[0167] EXAMPLE 3: Hu-01 and Hu-03 activities in human U-251 cells

[0168] Hu-01 and Hu-03 activities in human U-251 cells were measured using Hu-01, Hu-03, PMO-Control, and Cell only control, along with markers.

[0169] FIG. 5 shows antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production in some embodiments. Referring to FIG. 5A, U-251 cells were treated with 3 pM of Hu-01, Hu-03, and a control (PMO-CTRL) for 24H. 15 pg of total protein (cell lysis) was loaded into the indicated well, then detected with anti-OPN antibody. The same blot was then detected with anti -beta-actin to confirm that the same amount of samples were loaded per lane (see FIG. 5B).

[0170] EXAMPLE 4: Quantification of Hu-01 and Hu-03 Activities

[0171] Hu-01 and Hu-03 Activities were quantified along with PMO control and cell alone control.

[0172] FIG. 6 shows antisense oligo (ASO) activities on regulating OPN (Osteopontin, or SPP1) production in some embodiments. Referring to FIG. 6, U251 cells were treated as described in FIG. 5 legend. 5 pg cell lysis per well was loaded to a 96-well plate (duplicated each sample). OPN production was quantified by using a Human OPN detection Elisa Kit and then normalized to cell alone control. Mean of duplicate shown.

[0173] EXAMPLE 5: ASO activities in human THP-1 cells

[0174] ASO activities in human THP-1 cells were measured.

[0175] FIG. 7 shows antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production in THP-1 cells in some embodiments. Referring to FIG. 7A, THP-1 cells were treated with 3 pM of Hu-01, Hu-02, and Hu-03, and a control(PMO-CTRL) for 48h. Cell lyses were detected with anti-OPN antibody. The same blot was then detected with anti -beta-actin . H2O represents that THP-1 cells treated with Endo-porter without ASO (delivery reagent only control) (see FIG. 7B).

[0176] FIG. 8 shows antisense oligo (ASO) activities on regulating OPN (Osteopontin, or SPP1) production in THP-1 cells by normalizing with the production of beta actin in some embodiments. Referring to FIG. 8, THP-1 cells were treated as described in FIG. 7 legend.

[0177] EXAMPLE 6: Dose-dependent ASO activities in human U-251 cells

[0178] Dose-dependent ASO activities in human U-251 cells were measured.

[0179] FIG. 9 shows dose dependent antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production in U251 cells using Hu-01 (24 hours treatment) in some embodiments.

[0180] EXAMPLE 7: Time course of ASO activities in human U-251 cells

[0181] Time course of ASO activities in human U-251 cells was measured.

[0182] FIG. 10 shows time course of antisense oligo (ASO) activities (western-blotting) on regulating OPN (Osteopontin, or SPP1) production in U251 cells in some embodiments. Referring to FIG. 10, U-251 cells were treated with Hu-01 at concentrations of 1 pM and 3 pM for 24, 48, 96, and 144 hours. Cell lyses were detected with anti-OPN antibody. H2O represents Endo-porter without ASO (delivery reagent only control). NC: non-treated control. PMO-CTRL: Irrelevant ASO control.

[0183] EXAMPLE 8: ASO activities in mouse cells

[0184] ASO activities in mouse cells were measured.

[0185] FIG. 11 shows antisense oligo (ASO) activities (western-blotting) on regulating mouse OPN (Osteopontin, or SPP1) production in mouse microglial SIM-A9 cells in some embodiments. Referring to FIG. 11, SIM-A9 cells were treated with Mouse-01, Mouse-02, and a control at concentrations of 1 pM and 10 pM for 24 hours before harvesting. OPN production in cell lysis was detected with anti-mouse-OPN antibody. Cells were treated with either 5ul or 2.5ul endo-porter (Delivery reagent) as controls.

[0186] EXAMPLE 9: Dose-dependent ASO activities in mouse cells

[0187] Dose-dependent ASO activities in mouse cells were measured.

[0188] FIG. 12 shows dose dependent antisense oligo (ASO) activities (western-blotting) on regulating mouse OPN (Osteopontin, or SPP1) production in mouse microglial SIM-A9 cells in some embodiments. Referring to FIG. 12, SIM-A9 cells were treated with Mouse-01, Mouse-02, and a control at indicated concentrations for 24 hours before harvesting. OPN production in cell lysis was detected with anti-mouse-OPN antibody. Cells were treated with either 5ul or 2.5ul endo-porter (Delivery reagent) as controls.

[0189] EXAMPLE 10: Quantification of ASO activities in mouse cells

[0190] Quantification of ASO activities in mouse cells was performed.

[0191] FIG. 13 shows dose-dependent antisense oligo (ASO) activities (western-blotting) on regulating mouse OPN (Osteopontin, or SPP1) production in mouse microglial SIM-A9 cells in some embodiments. Referring to FIG. 13, cells were treated as described in Figure 12. Mouse OPN production in cell lysis was quantified by using a mouse OPN detection kit.5 pg protein from cell lysis was loaded per well in 96 well plate. Mean of duplicated samples shown.

[0192] EXAMPLE 11 : PPMO activities in human cellsReagents

[0193] Peptide-morpholino conjugates (PPMOs). The conjugation and purification of SPP1 -targeted PMOs to the P7, R6B or RGD peptide was performed using published methods (Abes et al., J. Control Release (2006) 1;116(3):304-l 3). The identity of each PPMO was confirmed by electrospray mass spectrometry.

[0194] Three PPMO candidates were used, each conjugated to a specific peptide (P7, R6B, or RGD), corresponding to PPMO#1, PPMO#2, and PPMO#3, respectively. The agents were shipped as dry lyophilized powder and stored at -20°C. The working solution was prepared at ImM concentration by adding PBS and then stored at 4C. U-251 cells were cultured with DMEM medium with 10% FBS, and 1U penicillin / streptomycin (Gibco 15070-063).Western-blot

[0195] U251 cells were seeded into each well of 12-well plates with 250,000 cells / ml / well cell’s density one day before assay. After 24h, cell culture medium was removed and 800ul fresh medium was added per well. 5X PPMO was prepared by adding appropriate volume of PPMO stock solution. 200ul 5X PPMO was added per well. After another 24 hours, cells were harvested using 0.25% Trypsin-EDTA. The harvested cells were washed with 1XPBS, resuspended with lOOul lysis buffer (Pierce RIPA lysis buffer, Cat: 89901). Following the instruction of Lysis collection, then protein concentration of each sample was measured by BCA test (Pierce BCA Protein Assay, Cat: 23227) and read by a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.

[0196] A total 20ug / per sample was analyzed by SDS page (Invitrogen, NuPage 4-12% Bis-tris Gel, Cat: NP0322BOX) (120V lOmin and 175V 30min). After transferred the proteins onto the nitrocellulose blotting membrane with semi-dry Trans-Blot (15 Voltage for 35min, Invitrogen iBlot2 PVDF Cat: IB24002) by iBlot2 (invitrogen), the membrane was then blocked with 5% milk / TBST for 1.5h. Then the membrane was detected with the primary human anti-OPN antibody (1:2500 at 4C overnight, Abcam / #ab214050). After 4x TBST wash, membrane was detected with anti-rabbit IgGHRP (1:5000, RT 1 hour, RPL / #95059-086). After developing the image with substrate from Pierce ECL WB Substrate (Cat: 32209), membrane was washed 4x TBST and detected with anti-Actin (1:5000, 40 min RT, Santa Cruz Cat: sc-47778 HRP). Images were taken by using Azure 280.ELISA

[0197] 20,000 U251 cells per well were seeded into 96 well cell culture plates. After 24 hours, cell culture medium was removed and 80ul fresh medium was added per well. 5X PPMO was prepared by adding appropriate volume of PPMO stock solution. 20ul 5X PPMOwas added per well. After another 24 hours, supernatants of cell culture were collected for ELISA (R&D system, Cat: DOSTOO). ELISA procedures followed the instruction of the manufacturer. ELISA plates were read by a Perkin Elmer Wallac 1420 Victor2 Microplate Reader or a Tecan Infinite M200 reader.Results

[0198] Referring to FIG. 17 and FIG. 18, lOuM P7-PPMO (PPMO#1) reduced the production of SPP1 in both cell lysis (FIG. 17) and cell culture medium (FIG. 18). Under this testing condition, rest PPMOs did not show obvious reduction of SPP1 protein production in human U-251 cells, which is consistent with cell uptake results (see Fig. 14).

[0199] Since the study shown in FIG. 17 and FIG. 18 demonstrated the knockdown effects on SPP1 production by PPMOs in 12-well cell culture system, a semi-high throughput method by ELISA in a 96-well cell culture system was used to quantify the knockdown effects. Referring to FIG. 19, 5uM P7-PPMO (PPMO#1) reduced 60% of SPP1 secretion in cell culture medium. R6B-PPMO (PPMO#2), which is less effective, reduced 35% of SPP1 secretion in U-251 cells.

[0200] Cell Uptake: In in vitro cellular uptake assays, PPMO#1 exhibited relatively stronger uptake efficiency when compared to PPMO#2 and PPMO#3. FIG. 14 and FIG. 15 shows the cellular internalization of peptide-conjugated phosphorodiamidate morpholino oligonucleotide (PPMO) Hu-01 across three peptide constructs by U-251 cells and Hela cells, respectively. The PMO Hu-01 was covalently linked to fluorescein and one of three targeting moieties: PPMO #1 (P7 peptide), PPMO #2 (R6B peptide), or PPMO #3 (RGD peptide). Following a 24-hour incubation, the cell-associated fluorescence of the unconjugated (fluorescein-labeled) Hu-01 and the three PPMOs was measured via flow cytometry. Mean Fluorescence Intensity (MFI) was used to evaluate cellular uptake, with results for 5pM and lOpM concentrations presented for comparative analysis.

[0201] FIG. 16 shows the cytotoxicity of different PPMOs in U-251 cells. 5000 U-251 cells / well were seeded into a 96-well cell culture plate. After 24 hours, cells were incubated with indicated concentrations of PMO Hu-01 and three different peptide conjugated PMOs (PPMO #l-#3). After another 24 hours, cell viability was examined with a CellTiter-Glo kit (commercial kit (Promega).

[0202] FIG. 17 shows the biological effect of three peptide-conjugated PMOs (PPMOs) on OPN production in U-251 cells. U251 cells were seeded into each well of 12-well plates with 250,000 cells / lml medium per well. After 24 hours, U-251 cells were treated with indicated concentrations of PPMO #1, #2, and #3, and three corresponding scrambled PMO controls (conjugated with P7, R6B, and RGD, respectively), along with the unconjugated PMO Hu-01. Following 24-hour incubation, cell lysates were collected and assayed for OPN production using a Human OPN detection ELISA Kit (top). Results were quantified using 5 pg of cell lysate per well (measured in duplicate, n=2). The data shown represent the mean of the duplicate measurements.

[0203] FIG. 18 shows the biological effect of three peptide-conjugated PMOs (PPMOs) on OPN production in U-251 cells. U251 cells were seeded into each well of 12-well plates with 250,000 cells / lml medium per well. After 24 hours, U-251 cells were treated with indicated concentrations of PPMO #1, #2, and #3, and three corresponding scrambled PMO controls (conjugated with P7, R6B, and RGD, respectively), along with the unconjugatedPMO Hu-01. Following 24-hour incubation, cell culture media were collected and assayed for OPN production using a Human OPN detection ELISA Kit (top). Results were quantified using 1:20 diluted cell culture supernatants (measured in duplicate, n=2). The data shown represent the mean of the duplicate measurements.

[0204] FIG. 19 shows the biological effect of PPMO #1 (P7 peptide conjugated PMO Hu-01) and PPMO #2 (R6B peptide conjugated PMO Hu-01) on OPN production in U-251 cells.20,000 U-251 cells per well were seeded into 96 well cell culture plates. After 24 hours, cell culture medium was removed, and fresh medium containing indicated concentrations of PPMOs was added. Following 24-hour incubation, cell culture media were collected and assayed for OPN production using a Human OPN detection ELISA Kit. Results were quantified using 1:20 diluted cell culture supernatants. Each condition was quadruplicated and data represented as mean ± SEM.

[0205] In summary, these results indicate that peptide conjugation represents an effective approach to enhance intracellular delivery of oligonucleotides. The data further show an association between the extent of cellular uptake and the resulting biological activity, as reflected by reduced target protein production. Among the tested PPMOs, PPMO#1 and PPMO#2 demonstrated excellent inhibition of target protein expression at the translational level, which is consistent with their higher intracellular uptake observed under the experimental conditions. These findings also show that the delivery-enhancing effect of peptide conjugation is not limited to the particular peptide sequences evaluated here and may extend to other peptide architectures or variants.

[0206] EXAMPLE 12: Oligo Activity EnhancerReagents

[0207] Two PPMO candidates (PPMO #1 and PPMO #2) recognizing human SPP1 were generated. They were shipped as dry lyophilized powder and stored at -20C. The working solution was prepared at ImM concentration by adding PBS and then stored at 4C. OAEs were prepared and shipped as dry lyophilized powder and stored at -20C.

[0208] U-251 cells were cultured with DMEM medium with 10% FBS, and 1U penicillin / streptomycin (Gibco 15070-063).ELISA

[0209] 5 ,000-20,000 U251 cells per well were seeded into 96 well cell culture plates. After 24 hours, cell culture medium was removed and 90ul fresh medium was added per well.5ul 20X PPMO and / or 5ul 20X OAE or 5ul PBS were added per well to make a final volume lOOul. After another 24 hours, supernatants of cell culture were collected for ELISA (R&D system, Cat: DOST00).Results

[0210] Small molecules can enhance nucleic acid (NA) delivery by promoting endocytosis and facilitating endosomal escape, allowing NAs to enter the cytosol and function more efficiently. Such small molecules can be defined as Oligo Activity Enhancer (OAE). The study tested lonafarnib (OAE2) with the SPP1 -targeted PPMO candidate(PPMO#1). OAE2 demonstrated a synergistic effect with PPMO#1 in disrupting / reducing target protein production. (FIG. 20).

[0211] FIG. 21 shows the cytotoxicity of the combination of fixed concentration of PPMO #1 and a variety of concentrations of Oligo Activity Enhancer (OAE 2) in Hela cells (left) and U-251 cells (right). 15 000 Hela cells / well or 5000 U-251 cells / well were seeded in 96-well cell culture plates After 24 hours, cells were treated with a combination of 10 uM PPMO #1 and indicated concentrations of OAE 2. After 24 hours, cell viability was examined with a CellTiter-Glo kit (commercial kit (Promega) Cell treatment were triplicated and data represented as mean ± SD.

[0212] In summary, these results indicate that small molecules which have RNA activity-enhancing functions, such as OAE2 (lonafamib), can enhance the activity of the candidate PPMOs.

[0213] EXAMPLE 13Formulation

[0214] A PPMO comprising a phosphorodiamidate morpholino oligomer covalently linked to a delivery peptide is formulated in a sterile, isotonic aqueous vehicle suitable for intratracheal delivery. The formulation is adjusted to a concentration such that the intended deposited dose is administered in a small bolus volume (for mice typically 50-100 pL).Animal Model

[0215] Adult C57BL / 6 mice (8-10 weeks old) are used. Pulmonary fibrosis is induced by a single intratracheal administration or oropharyngeal instillation of bleomycin (e.g., 1-2 mg / kg) on Day 0. Animals are randomized to treatment or control groups.Administration

[0216] The PPMO is delivered directly to the lower respiratory tract by intratracheal administration under brief anesthesia, using standard techniques such as nebulization (via a specialized device or needle) or orotracheal intubation (with a fine catheter or specialized microsprayer).Single-dose regimen

[0217] One intratracheal bolus of PPMO is administered on Day 3, or day 7 postbleomycin. The deposited dose is an estimated 1-10 mg / kg (mouse equivalent), delivered in a single bolus volume (50-100 pL). Control animals receive vehicle or non-targeting PPMO.Two-dose regimen

[0218] Two intratracheal boluses of PPMO are administered. The first dose is given on Day 3 (or Day 7) post-bleomycin challenge, and the second dose is administered on Day 10 post-bleomycin challenge. Each administration delivers an estimated deposited dose of 0.5-5 mg / kg per dose (total cumulative dose 1-10 mg / kg). Control animals receive two administrations of vehicle or non-targeting PPMO according to the same schedule.Assessment / Endpoints

[0219] At Day 21 (or another selected terminal time point), animals are euthanized and lungs harvested for analysis. Example endpoints include, but are not limited to:

[0220] 1. Target engagement: Reduction of OPN mRNA or protein in lung tissue (qRT- PCR, western blot, or hybridization assay).

[0221] 2. Histopathology: H&E and Masson’s tri chrome staining with semi-quantitative fibrosis scoring (e.g., modified Ashcroft score).

[0222] 3. Collagen content: Hydroxyproline assay to quantify total lung collagen.

[0223] 4. Inflammation / fibrosis biomarkers: Measurement of markers such as a-SMA, COL1A1, TGF-pi by IHC, ELISA, or qPCR.

[0224] 5. Clinical parameters: Body weight, lung weight, and clinical observations recorded during the study.

[0225] 6. Biodistribution / toxicity (optional): Assessment of PPMO distribution in major organs and basic toxicology endpoints (serum chemistry, organ histology).

[0226] Outcome

[0227] Intratracheal delivery of the disclosed PPMO (single or two administrations) results in efficient delivery to lung parenchymal cells, reduction of the designated target, and an observable decrease in fibrotic markers and collagen deposition relative to controls. The two-dose regimen provides extended target suppression in comparing with a single bolus, though single administration is sufficient depending on compound potency and pharmacodynamics.

[0228] Using alternative intratracheal devices (specialized needles, nebulization devices, microsprayers, fine catheters), alternative species (e.g. rat), varied dose ranges, administration schedules (e.g., repeated dosing beyond two doses), and alternative fibrosis induction models is also effective.

[0229] While various embodiments have been described above, it should be understood that such disclosures have been presented by way of example only and are not limiting. Thus, the breadth and scope of the subject compositions and methods should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0230] The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence which is effective to meet the objectives there intended unless the context specifically indicates the contrary.APPENDIXSEQUENCE LISTINGB=Beta-alanineX=6-aminohexanoic aid

Claims

ClaimsWhat is claimed is:

1. An antisense oligonucleotide (ASO) comprising 8-50 nucleotides,wherein the ASO is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene,wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN,wherein the target sequence comprises the start codon in the open reading frame of the mRNA, andwherein binding of the ASO to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN.

2. An ASO comprising 8-50 nucleotides,wherein the ASO is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene,wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises a partial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, andwherein binding of the ASO to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN.

3. The ASO of any one of claims 1-2, wherein the nucleotide sequence comprises at least about 85% identity to any one of the sequences:(i) SEQ ID NO 1, or(ii) SEQ ID NO 3.

4. The ASO of claim 3, wherein the nucleotide sequence comprises any one of the sequences:(i) SEQ ID NO 1, or(ii) SEQ ID NO 3.

5. The ASO of claim 4, wherein the nucleotide sequence is SEQ ID NO 1.

6. The ASO of claim 4, wherein the nucleotide sequence is SEQ ID NO 3.

7. The ASO of any one of claims 1-6, wherein the ASO is a molecule comprising modifications selected from one or more from the group comprising:a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), a 2’-O-methyl phosphorothioate (2’OMe PS) oligomer, a 2’-O-methoxyethyl (2 ’-MOE) oligomer, a 2’-O-methoxyethyl phosphorothioate (2’ -MOE PS) oligomer, a peptide nucleic acid (PNA), a locked nucleic acid (LN A), a LNA and 2’OMe PS gapmer, a LNA and 2 ’MOE gapmer, a gapmer of 2’OMe PS and natural nucleic acids, a gapmer of 2’MOE PS and natural nucleic acids, or unmodified or modified nucleic acids, a cell penetrating peptide (CPP), or a combination thereof.

8. The ASO of any one of claims 1-7, wherein the ASO is conjugated with a CPP.

9. The ASO of claim 7, wherein the ASO is a PMO.

10. The ASO of claim 7, wherein the ASO is a PPMO, wherein the ASO comprises a CPP conjugated with a PMO.

11. The ASO of claim 10, wherein the PMO is conjugated to a CPP selected from one of the group consisting of: P7, R6B, and RGD.

12. The ASO of claim 11, wherein the CPP is P7.

13. The ASO of claim 11, wherein the CPP is R6B.

14. The ASO of any one of claims 1-12, wherein the target sequence comprises at least about 85% identity to any one of the sequences:(i) SEQ ID NO 9, or(ii) SEQ ID NO 11.

15. The ASO of claim 14, wherein the target sequence comprises any one of the sequences: (i) SEQ ID NO 9, or(ii) SEQ ID NO 11.

16. The ASO of claim 15, wherein the target sequence is SEQ ID NO 9.

17. The ASO of claim 15, wherein the target sequence is SEQ ID NO 11.

18. The ASO of any one of claims 1-17, wherein the target sequence comprises at least about 85% identity to any one of the sequences:(i) SEQ ID NO 6, or(ii) SEQ ID NO 8.

19. The ASO of claim 18, wherein the target sequence comprises any one of the sequences: (i) SEQ ID NO 6, or(ii) SEQ ID NO 8.

20. The ASO of claim 19, wherein the target sequence is SEQ ID NO 6.

21. The ASO of claim 19, wherein the target sequence is SEQ ID NO 8.

22. The ASO of any one of claims 1-21, wherein the SPP1 or OPN gene is selected from one of the group consisting of SPP1, OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

23. A double-stranded ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5 ’-3’ strand,wherein the molecule is capable of binding to a target sequence in a mRNA of a secreted phosphoprotein 1 (SPP1) or osteopontin (OPN) gene,wherein the mRNA comprises a start codon in an open reading frame of the mRNA encoding the SPP1 or OPN,wherein the target sequence comprises the start codon in the open reading frame of the mRNA, andwherein binding of the molecule to the target sequence disrupts translation of the ORF encoding the SPP1 or OPN.

24. A double-stranded RNA or DNA molecule comprising a nucleotide sequence of 8-50 nucleotides on a 5 ’-3’ strand,wherein the molecule is capable of binding to a target sequence in a pre-mRNA of a SPP1 or OPN gene,wherein the pre-mRNA comprises introns and exons of the SPP1 or OPN, wherein the target sequence comprises a contiguous exon / intron junction nucleotide sequence, wherein the contiguous exon / intron junction nucleotide sequence comprises apartial nucleotide sequence of an exon and a partial nucleotide sequence of an intron that is contiguous with the exon, andwherein binding of the molecule to the target sequence disrupts splicing of the mRNA of the SPP1 or OPN.

25. The double-stranded RNA or DNA molecule of any one of claims 23-24, wherein the nucleotide sequence on the 5’-3’ strand comprises at least about 85% identity to any one of the sequences:(i) SEQ ID NO 1, or(ii) SEQ ID NO 3.

26. The double-stranded RNA or DNA molecule of claim 25, wherein the nucleotide sequence on the 5 ’-3’ strand comprises any one of the sequences:(i) SEQ ID NO 1, or(ii) SEQ ID NO 3.

27. The double-stranded RNA or DNA molecule of claim 26, wherein the nucleotide sequence on the 5’-3’ strand is SEQ ID NO 1.

28. The double-stranded RNA or DNA molecule of claim 26, wherein the nucleotide sequence on the 5’-3’ strand is SEQ ID NO 3.

29. The double-stranded RNA or DNA molecule of any one of claims 23-28, wherein the target sequence comprises at least about 85% identity to any one of the sequences:(i) SEQ ID NO 9, or(ii) SEQ ID NO 11.

30. The double-stranded RNA or DNA molecule of claim 29, wherein the target sequence comprises any one of the sequences:(i) SEQ ID NO 9, or(ii) SEQ ID NO 11.

31. The double-stranded RNA or DNA molecule of claim 30, wherein the target sequence is SEQ ID NO 9.

32. The double-stranded RNA or DNA molecule of claim 30, wherein the target sequence is SEQ ID NO 11.

33. The double-stranded RNA or DNA molecule of any one of claims 23-32, wherein the target sequence comprises at least about 85% identity to any one of the sequences:(i) SEQ ID NO 6, or(ii) SEQ ID NO 8.

34. The double-stranded RNA or DNA molecule of claim 33, wherein the target sequence comprises any one of the sequences:(i) SEQ ID NO 6, or(ii) SEQ ID NO 8.

35. The double-stranded RNA or DNA molecule of claim 34, wherein the target sequence is SEQ ID NO 6.

36. The double-stranded RNA or DNA molecule of claim 34, wherein the target sequence is SEQ ID NO 8.

37. The double-stranded RNA or DNA molecule of any one of claims 23-36, wherein the molecule is selected from one or more of the group comprising: a small interfering RNA (siRNA), a small segmented siRNA (sisiRNA), a short hairpin RNA (shRNA), Double strand RNA (dsRNA), a microRNA, an Antagomirs (anti-miRs), a Circular RNA (circRNA), a Phosphorothioate-linked DNA (S-DNA), or a combination thereof.

38. The double-stranded RNA or DNA molecule of any one of claims 23-37, wherein the molecule is conjugated with a CPP.

39. The double-stranded RNA or DNA molecule of any one of claims 23-38, wherein the SPP1 or OPN gene is selected from one of the group consisting of SPP1, OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

40. A pharmaceutical composition comprising:the ASO of any one of claims 1-22, or the double-stranded RNA or DNA molecule of any one of claims 23-39, anda pharmaceutically acceptable carrier.

41. The pharmaceutical composition of claim 40, further comprising an oligonucleotide activity enhancer (OAE).

42. The pharmaceutical composition of claim 41, wherein the OAE is selected from one of the group consisting of febuxostat, lonafamib, nitazoxanide, CGS-15943, and resveratrol.

43. The pharmaceutical composition of claim 42, wherein the OAE is lonafamib.

44. The pharmaceutical composition of any one of claims 40-43, wherein the pharmaceutical composition is a medicament used for treatment of pulmonary fibrosis.

45. A method for treating a disease in a subject in need thereof, comprising the steps of: administering an effective amount of the ASO of any one of claims 1-22, or the double-stranded RNA or DNA molecule of any one of claims 23-39, oradministering an effective amount of the pharmaceutical composition of any one of claims 40-44.

46. The method of claim 45, wherein the disease is pulmonary fibrosis.

47. The method of any of one of claims 45-46, wherein the administration step is to a cell in the subject in need thereof.

48. The method of any of one of claims 45-46, wherein the administration step is to an ex vivo cell from the subject in need thereof.

49. The method of any of one of claims 45-48, wherein mRNA translation levels of an SPP1 gene or an OPN gene are reduced in the subject in need thereof compared to a baseline for mRNA translation levels of an SPP1 gene or an OPN gene established in the subject, or compared to a known baseline mRNA translation levels of SPP1 or OPN in a human population.

50. The method of any of one of claims 45-49, wherein protein activity levels of SPP1 or OPN are reduced in the subject in need thereof compared to a baseline for protein activity levels of SPP1 or OPN established in the subject, or compared to a known baseline protein activity levels of SPP1 or OPN in a human population.

51. The method of any of one of claims 45-50, wherein the SPP1 gene or OPN gene is selected from one of the group consisting of SPP1, OPN-a, OPN-b, OPN-c, OPN-4, and OPN-5.

Images