Antisense oligonucleotide-based antifibrotic therapeutic agents

Abstract

The present invention provides antisense oligonucleotides that can enhance the expression of antifibrotic genes or reduce the expression of profibrotic genes in target tissues.The antisense oligonucleotides can be used to treat myocardial fibrosis.The antisense oligonucleotides can include gapmers that can bind to target sequences in the mRNA of profibrotic genes and form double-stranded structures with them.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 373,796, filed August 29, 2022, which is incorporated herein by reference.

[0002] This invention was made with government support awarded by the National Institutes of Health under grants HL132899 and HL147954. The United States government has certain rights in this invention.

[0003] The present invention relates to the field of medical treatments. More specifically, the present invention provides compositions and methods useful for treating fibrosis-related diseases and conditions. [Background technology]

[0004] Fibrosis is a physiological process in which connective tissue replaces normal parenchymal tissue. Fibrosis can occur in many tissues in the body, such as the heart, lungs, liver, and kidneys, typically as a result of inflammation, tissue injury, or aging. However, if tissue injury is severe or recurring, or if the fibrotic process itself becomes dysregulated, the entire process can lead to a progressive, irreversible fibrotic response. Fibrotic diseases cause more than 800,000 deaths worldwide each year, mainly due to pulmonary and myocardial fibrosis. We will further summarize current diagnostic tools and identify preclinical or clinical therapeutic strategies for addressing myocardial fibrosis. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need to develop therapeutic strategies to address fibrosis-related diseases, such as myocardial fibrosis.

[0006] One of the key approaches to increasing the production of therapeutic proteins is via gene therapy. AAV delivery of transgenes has drawbacks regarding toxicity, and lipid nanoparticle (LNP) delivery is currently being refined within the industry. Therefore, there is a need to find a platform to potently, safely, and titratably increase protein synthesis. [Means for solving the problem]

[0007] One aspect of the present invention relates to an antisense oligonucleotide comprising 8 to 50 nucleotides, which is capable of binding to a target sequence in the mRNA of an antifibrotic gene and forming a double-stranded structure therewith, wherein the target sequence is located in the non-coding strand of the double-stranded stem structure downstream of and adjacent to the uORF start codon in the mRNA, and when the antisense oligonucleotide binds to the target sequence, the double-stranded stem structure of the uORF is disrupted and translation of the mORF of the mRNA is enhanced.

[0008] Another aspect of the present invention relates to an antisense oligonucleotide comprising 8 to 50 nucleotides, which is capable of binding to and forming a double-stranded structure with a target sequence in the mRNA of an antifibrotic gene, the target sequence being located downstream of and adjacent to the mORF start codon in the mRNA, and wherein binding of the antisense oligonucleotide to the target sequence enhances translation from the mORF start codon.

[0009] Another aspect of the present invention relates to an antisense oligonucleotide comprising 8 to 50 nucleotides, which is capable of binding to and forming a double-stranded structure with a target sequence in the mRNA of a pro-fibrosis gene, the target sequence being located downstream of and adjacent to the uORF start codon in the mRNA, and wherein binding of the antisense oligonucleotide to the target sequence reduces translation from the mORF start codon.

[0010] Another aspect of the present invention relates to an antisense oligonucleotide comprising 8 to 50 nucleotides, which is a gapmer that can bind to a target sequence in the mRNA of a pro-fibrosis gene and form a double-stranded structure therewith, wherein the target sequence is located within a region extending from 55 nucleotides upstream to 55 nucleotides downstream of the uORF start codon in the mRNA, and wherein binding of the antisense oligonucleotide to the target sequence reduces translation from the mORF start codon and results in degradation of the target mRNA.

[0011] Another aspect of the present invention is a pharmaceutical composition for antifibrotic therapy comprising an antisense oligonucleotide described herein; and a pharmaceutically acceptable carrier.

[0012] Another aspect of the present invention is a method for treating myocardial fibrosis, comprising administering in a subject in need thereof an effective amount of an antisense oligonucleotide described herein or an effective amount of a pharmaceutical composition described herein. [Brief explanation of the drawings]

[0013] [Figure 1]Figure 1 shows a model for translation activation or repression. (A) shows a model for repression of uORF translation and activation of mORF translation by type I uotASO. (B) shows a model for activation of uORF translation and repression of mORF translation targeted by type II uotASO. This design does not rely on the presence of an endogenous dsRNA stem structure and will activate the translation of uORFs that do not contain an endogenous dsRNA stem structure. (C) shows a model for activation of mORF translation targeted by type II motASO. [Figure 2] Figure 1 shows a composite image of drawings and paintings illustrating the interaction between a uORF and adjacent double-stranded RNA structural elements in regulating mORF translation. Panel A) Schematic of a dual-luciferase reporter assay. Panel B) Schematic of a FLuc reporter construct. Panel C) Dual-luciferase reporter assay using a series of constructs containing a uORF start codon and an adjacent dsRNA structure (Kan-HP1 hairpin) spaced at different distances. N=3 biological replicates. Data are presented as mean ± SEM. P values ​​were calculated by unpaired, two-tailed Student's t-test. Panel D) Dual-luciferase reporter assay using a mutant construct containing three nucleotide mutations that result in disruption of the stem structure. N=3 biological replicates. Data are presented as mean ± SEM. P values ​​were calculated by unpaired, two-tailed Student's t-test. No AUG: ATG to TTG mutation. AUG -2: The start codon is located at position -2 relative to the hairpin. Intact: A stable wild-type hairpin. Attenuated: Three mutations have been introduced into the hairpin to destabilize the structure. [Figure 3] This figure shows the localization of artificial uORF-KanHP1 mRNA mutants within the 40S ribosomal subunit or 80S monosome fraction in HEK293T lysates upon centrifugation through a 10-35% sucrose gradient. The experiment was repeated twice, and representative data are shown. [Figure 4]A composite image of drawings and drawings identifying uORFs as key translational regulatory elements within mRNAs encoding cardiac transcription factors. Panel A) illustrates the overlap of uORF-containing mRNAs in failing human and mouse hearts based on ribosome profiling (Ribo-Seq), along with gene ontology analysis for human cardiac uORFs. Multiple cardiac mRNAs, such as GATA4, and embedded uORFs are highlighted. Panel B) The GATA4 uORF is present across mammals, as shown in representative species. Panel C) Schematic of wild-type and mutant GATA4 5'UTRs cloned into a FLuc reporter construct. Panel D) dsRNA elements are required for uORF-mediated translational repression of mORFs. Dual-luciferase reporter assays with wild-type, ΔuORF, secondary structure mutants, and rescuing mutants. N=3 biological replicates. Data presented as mean ± SEM. P values ​​were calculated by unpaired two-tailed Student's t test. [Figure 5]This is a composite image of drawings and drawings showing the mechanism-based design of ASOs to regulate mORF translation. Panels A-B, left, show schematics of designed ASOs targeting the GATA4 uORF dsRNA element. ASOs that are disruptive to the formation of a double-stranded RNA structure immediately downstream of the AUG start codon of the GATA4 uORF are referred to as type I uotASOs or ASO1 (Panel A). ASOs that are capable of forming a double-stranded RNA structure immediately downstream of the AUG start codon of the GATA4 uORF are referred to as type II uotASOs or ASO2 (Panel B). Panels A-B, middle, show dual-luciferase reporter assays with wild-type GATA4 and the ΔuORF mutant GATA4 after transfection with ASO1 and ASO2 (oligo sequences are shown in Panel F). N = 3 biological replicates. Comparisons were performed using an unpaired, two-tailed Student's t-test. Panel C) Western blot analysis of the dose-response manipulation of endogenous GATA4 protein expression by ASO1 and ASO2 in the AC16 human cardiomyocyte cell line. Panels D-E) Polysome profiling of wild-type and ΔuORF AC16 cells with ASO1 / ASO2 treatment in cells. Panel F) Immunostaining of AC16 cells with b-actin after transfection with control ASO, ASO1, and ASO2. Cell surface area was measured and quantified (n ≥ 200 cells). Scale bar: 20 mm. In the violin plots, solid lines indicate the median for the group, and dashed lines represent the two quartiles within each group. P values ​​were calculated using an unpaired, two-tailed Student's t-test. [Figure 6]Development of type II ASOs to inhibit the expression of profibrotic eIF4G2 (using type II uotASOs) or enhance the expression of antifibrotic GATA4, MEF2C, and NKX2-5 (using type II motASOs). Panel A: Western blot analysis of eIF4G2 protein upon transfection of 50 nM uORF-enhanced ASOs with modifications for eIF4G2 mRNA. Panel B: GATA4 mORF-targeted ASOs enhance GATA4 protein levels. Similar to uORF-targeted ASOs, the combination of 2'-O-methyl and LNA is superior to 2'-O-methyl alone and does not alter mRNA levels (Panel C). Panel D: For MEF2C and NKX2-5, mORF-targeted ASOs (50 nM) with 2'-O-methyl and LNA increase protein levels of mRNAs with the cognate start codons. Data are expressed as mean ± SD. *P<0.05, **P<0.01, **P<0.001. For A-D, statistical significance was determined by unpaired two-tailed Student's t-test (N=3 biological replicates). [Figure 7] Figure 1 describes the design strategies for ASOs targeting different mRNAs. A: Schematic diagram of mORF-activated type II motASOs and 5'-UTR-targeting gapmer ASOs. B: Detailed information on therapeutic target mRNAs, specific ASO sequences, and chemical modifications. [Figure 8]Figure 1 shows the translation activation effects of type II motASOs targeting MYBPC3 and CRYAB. Panel A: Dose-dependent effect of ASO targeting MYBPC3 mRNA in the human AC16 ventricular cardiomyocyte cell line. Western blots were performed 24 hours after ASO transfection using increasing doses of 0.4% Lipofectamine 3000. Panel B: Dose-dependent effect of ASO targeting CRYAB mRNA in the human AC16 ventricular cardiomyocyte cell line. Panel C: Expression of MYBPC3 mRNA and CRYAB mRNA in AC16 cells transfected with increasing doses of ASO. Experiments were repeated twice (biological replicates), and representative Western blot data are shown. Densitometry analysis data are shown between blot images and calculated as the ratio of target protein normalized to α-tubulin as an internal loading control. [Figure 9] Figure 1 shows the mRNA degradation and protein expression silencing effects of 5'UTR-targeting gapmer ASOs targeting EIF4G2 mRNA. Panel A: Downregulation effect of ASOs targeting EIF4G2 mRNA in a human immortalized cardiac fibroblast (IHCF) cell line. Western blots were performed 48 hours after transfection with 100 nanomolar ASOs using 0.3% RNAiMAX. The observed molecular weight of EIF4G2 is approximately 97 kDa (theoretical MW is 102 kDa). Experiments were repeated three times (biological triplicates), and representative Western blot data are shown (technical triplicates are also included). Panel B: Quantitative densitometric analysis data were calculated as the ratio of target protein normalized to α-tubulin as an internal loading control. Panel C: Reduction of EIF4G2 mRNA expression in IHCF cells following ASO transfection. DETAILED DESCRIPTION OF THE INVENTION

[0014] Reference will now be made in detail to certain aspects and exemplary embodiments of the present invention, illustrated by way of example with the accompanying structures and figures. Aspects of the present invention will be described in conjunction with exemplary embodiments, including methods, materials, and examples; such descriptions are intended to be non-limiting, and the scope of the present invention is intended to encompass all equivalents, alternatives, and modifications that are commonly known or incorporated herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Those skilled in the art will recognize many techniques and materials similar or equivalent to those described herein that can be used in the practice of aspects and embodiments of the present invention. The aspects and embodiments described herein are not limited to the methods and materials described.

[0015] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0016] Ranges can 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 each of the endpoints of a range has significance both in relation to the other endpoint, and independently of the other endpoint. It is understood that a number of values ​​are disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if a value of "10" is disclosed, "about 10" is also disclosed. As will be appreciated by those of skill in the art, when a value is disclosed as "less than or equal to" that value, it is understood that "greater than or equal to" that value is also disclosed. For example, if a value of "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. When more than one value is disclosed, all possible ranges between any two values ​​are disclosed.

[0017] I. Definition As used herein, the following terms or phrases (those in parentheses) shall have the following meanings:

[0018] "Target protein" refers to a protein whose amount, concentration, or activity is desired to be increased or decreased. In certain embodiments, the target protein is encoded by the primary open reading frame of the target transcript.

[0019] "Major open reading frame" or "mORF" refers to the portion of a target transcript that encodes the major (or primary) protein associated with the mRNA transcript. In certain embodiments, the mORF encodes the target protein.

[0020] The terms "uORF" and "upstream open reading frame" refer to the portion of a target transcript that includes the start site and in-frame stop codon upstream (i.e., 5') of the mORF. In certain embodiments, a uORF is the portion of a target transcript that is translated when translation is initiated at the start site of the uORF. In certain embodiments, a uORF does not overlap with an mORF. In certain embodiments, a uORF overlaps with an mORF. In certain embodiments, a uORF overlaps with another uORF. In certain embodiments, a uORF is out-of-frame with an mORF.

[0021] The term "oligonucleotide" refers to a compound comprising multiple linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides and / or unmodified deoxyribonucleosides and / or one or more modified nucleosides.

[0022] The term "oligonucleoside" refers to an oligonucleotide in which none of the internucleoside linkages contain a phosphorus atom. The term "oligonucleotide" includes oligonucleosides.

[0023] The term "internucleoside linkage" refers to the covalent linkage between adjacent nucleosides within an oligonucleotide.

[0024] A "naturally occurring internucleoside linkage" refers to a 3'-5' phosphodiester linkage.

[0025] The term "modified internucleoside linkage" refers to any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0026] 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 and capable of hybridizing to a target nucleic acid, target nucleotide sequence (target sequence), target site of a nucleotide sequence (target site), or target region of a nucleotide sequence (target region), resulting in at least one antisense activity. In some embodiments, the ASO comprises a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95% complementary to the target sequence, target site, or target region. In some embodiments, the ASO comprises an antisense oligonucleotide conjugated to a conjugate group. In some embodiments, the conjugate group is a non-nucleotide conjugate group.

[0027] "Antisense activity" refers to any detectable and / or measurable change attributable to hybridization of an antisense oligonucleotide with its target nucleic acid, target sequence, target site, or target region.

[0028] The term "GATA4 transcript" refers to a naturally occurring GATA4 mRNA transcript that encodes the upstream open reading frame (uORF) and major open reading frame (mORF), which encode the GATA4 protein.

[0029] The term "nucleoside" refers to a molecule comprising a nucleobase moiety, such as a purine or pyrimidine base, covalently linked to a sugar moiety, such as a sugar ribose or deoxyribose. Nucleosides include, but are not limited to, naturally occurring nucleosides (found in DNA and RNA) and modified nucleosides. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2-N,N-dimethylguanosine (also referred to as "rare" nucleosides). Nucleosides may be linked to a phosphate moiety.

[0030] The term "modified nucleoside" refers to a nucleoside that contains at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. A modified nucleoside includes a modified sugar moiety and / or a modified nucleobase.

[0031] The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to its sugar moiety in an ester linkage. Exemplary nucleotides include nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates. The term "linked nucleosides" refers to nucleosides joined in a contiguous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0032] The term "modified nucleotide" refers to a nucleotide containing a modified nucleoside with any modification in the phosphate linker. In certain exemplary embodiments, the modified nucleotide is modified at any position to alter certain chemical properties of the nucleotide while still retaining the ability of the modified nucleotide to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, such as 6-(2-amino)propyluridine; and the 8-position for adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deazaadenosine; O-modified nucleotides and N-modified nucleotides (e.g., alkylated nucleotides, e.g., N-methyladenosine, or nucleotides otherwise known in the art); and other heterocyclic-modified nucleotides, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., August 2000, 10(4):297-310. Modified nucleotides can also include modifications to the sugar portion of the nucleotide. For example, the 2'-OH group can be replaced with a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH, NHR, NR, COOR, or OR, where R is a substituted or unsubstituted C-C alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988 and 6,291,438.The phosphate linkage of a nucleotide may also be modified, for example, by substituting sulfur for one or more of the oxygens of the phosphate group (e.g., phosphorothioate), or by other substitutions that allow the nucleotide to perform its intended function, such as those described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev., April 2000, 10(2):117-21; Rusckowski et al., Antisense Nucleic Acid Drug Dev., October 2000, 10(5):333-45; Stein, Antisense Nucleic Acid Drug Dev., October 2001, 11(5):317-25; Vorobjev et al., Antisense Nucleic Acid Drug Dev., April 2001, 11(2):77-85; and U.S. Pat. No. 5,684,143. Certain of the modifications mentioned above (eg, phosphate group modifications) reduce the rate of hydrolysis in vivo or in vitro of, for example, a polynucleotide comprising the modified nucleotide.

[0033] The term "chemical modification" refers to the chemical difference of a compound compared to its naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. When referring to oligonucleotides, chemical modifications do not include differences in nucleobase sequence alone.

[0034] The term "furanosyl" refers to a structure containing a five-membered ring containing four carbon atoms and one oxygen atom.

[0035] The phrase "naturally occurring sugar moiety" refers to a ribofuranosyl found in naturally occurring RNA or a deoxyribofuranosyl found in naturally occurring DNA.

[0036] The term "sugar moiety" refers to a naturally occurring or modified sugar moiety of a nucleoside.

[0037] The term "modified sugar moiety" refers to a substituted sugar moiety or sugar surrogate.

[0038] The term "substituted sugar moiety" refers to a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to, furanosyl that contain substituents at the 2', 3', 5', and / or 4' positions. Certain substituted sugar moieties are bicyclic sugar moieties.

[0039] The term "2'-substituted sugar moiety" refers to a furanosyl containing a substituent other than H or OH at the 2'-position. Unless otherwise indicated, a 2'-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of the 2'-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring).

[0040] The term "2'-F nucleoside" refers to a nucleoside containing a sugar that contains a fluorine at the 2' position. Unless otherwise indicated, the fluorine in a 2'-F nucleoside is at the ribo position (replacing the OH of natural ribose).

[0041] The term "2'(ara)-F" refers to a 2'-F substituted nucleoside in which the fluoro group is in the arabino position.

[0042] The term "sugar surrogate" refers to structures that do not contain furanosyl and can replace the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside subunits can be linked together and / or to other nucleosides to form oligonucleotides that can hybridize with complementary oligonucleotides. Such structures include rings containing a different number of atoms than furanosyl (e.g., 4-, 6-, or 7-membered rings); replacement of the oxygen of furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or changes in both the number of atoms and the oxygen replacement. Such structures can also contain substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates, optionally containing additional substituents). Sugar surrogates also include more complex sugar substitutions (e.g., the acyclic systems of peptide nucleic acids). Sugar surrogates include, without limitation, morpholino, cyclohexenyl, and cyclohexitol.

[0043] The term "bicyclic sugar moiety" refers to a modified sugar moiety comprising a 4- to 7-membered ring (including, but not limited to, furanosyl) that contains a bridge connecting two atoms of the 4- to 7-membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4- to 7-membered ring is a sugar ring. In certain embodiments, the 4- to 7-membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2' and 4' carbons of the furanosyl.

[0044] The term "nucleobase" refers to a group of atoms that can be linked to a sugar moiety to create a nucleoside that can be incorporated into an oligonucleotide, and which can be bonded to a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. The nucleobase can be a naturally occurring nucleobase or a modified nucleobase.

[0045] The terms "unmodified nucleobase" and "naturally occurring nucleobase" refer to the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

[0046] The term "modified nucleobase" refers to any nucleobase that is not a naturally occurring nucleobase.

[0047] The term "bicyclic nucleoside" or "BNA" refers to a nucleoside that includes a bicyclic sugar moiety.

[0048] The terms "constrained ethyl nucleoside" and "cEt" refer to a nucleoside that includes a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge.

[0049] The term "locked nucleic acid nucleoside" or "LNA" refers to a nucleoside comprising a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge.

[0050] The term "2'-substituted nucleoside" refers to a nucleoside that includes a substituent at the 2'-position other than H or OH. Unless otherwise indicated, a 2'-substituted nucleoside is not a bicyclic nucleoside.

[0051] The term "2' deoxynucleoside" refers to a nucleoside containing a 2'-H furanosyl sugar moiety found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, 2' deoxynucleosides may contain modified nucleobases or RNA nucleobases (e.g., uracil).

[0052] The term "oligomeric compound" refers to a polymeric structure comprising two or more substructures. In certain embodiments, the substructures are nucleotides or nucleosides. In certain embodiments, the oligomeric compound comprises an oligonucleotide. In certain embodiments, the oligomeric compound consists of an oligonucleotide. In certain embodiments, the oligomeric compound consists of an antisense oligonucleotide.

[0053] The term "terminal group" refers to one or more atoms attached to one or both of the 3'-end or 5'-end of an oligonucleotide. In certain embodiments, the terminal group is a conjugate group. In certain embodiments, the terminal group comprises one or more terminal nucleosides.

[0054] The term "conjugate group" refers to an atom or group of atoms attached to an oligonucleotide or oligomeric compound. Generally, conjugate groups modify one or more properties of the oligonucleotide or oligomeric compound to which they are attached, including, but not limited to, the pharmacodynamic, pharmacokinetic, binding, absorption, intracellular distribution, intracellular uptake, charge, and / or clearance properties.

[0055] The term "conjugate linking group" refers to any atom or group of atoms used to join a conjugate to an oligonucleotide or oligomeric compound.

[0056] The terms "detect" and "measure" refer to the performance of a test or assay to detect or measure. Such detection and / or measurement may result in a value of zero. Thus, if a test to detect or measure results in a finding of no activity (zero activity), a step of detecting or measuring activity has nevertheless been performed.

[0057] The phrase "detectable and / or measurable activity" refers to a measurable activity that is not zero.

[0058] The term "essentially unchanged" refers to little or no change in a particular parameter, especially compared to another parameter that changes much more. In certain embodiments, a parameter is essentially unchanged if its change is less than 5%. In certain embodiments, a parameter is essentially unchanged if its change is less than 2-fold when another parameter changes by at least 10-fold. For example, in certain embodiments, antisense activity is a change in the amount of target nucleic acid. In certain such embodiments, the amount of non-target nucleic acid is essentially unchanged if its change is much less than that of target nucleic acid, but the change is not necessarily zero.

[0059] The term "expression" refers to the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modifications (e.g., splicing, polyadenylation, 5' capping), translation, and post-translational modifications.

[0060] The term "translation" refers to the process by which a polypeptide (e.g., a protein) is translated from an mRNA. In certain embodiments, increased translation refers to an increase in the number of polypeptide (e.g., protein) molecules produced per copy of the mRNA encoding the polypeptide.

[0061] The term "target nucleic acid" refers to a nucleic acid molecule to which an antisense oligonucleotide is intended to hybridize.

[0062] The term "mRNA" refers to an RNA molecule that encodes a protein.

[0063] The term "pre-mRNA" refers to an RNA transcript that has not been fully processed into mRNA. A pre-RNA may contain one or more introns.

[0064] The terms "targeting" and "targeted to" refer to the association of an antisense oligonucleotide with a specific target nucleic acid molecule or a specific 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.

[0065] When referring to a nucleobase, the terms "nucleobase complementarity" and "complementarity" refer to a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). Complementarity can be partial or total complementarity. Partial complementarity occurs when one or more nucleobases do not match according to the base pairing rules. Total or complete complementarity between nucleic acids occurs when each and every nucleobase matches another base according to the base pairing rules. In certain embodiments, complementary nucleobases refer to nucleobases of an antisense oligonucleotide that can base pair with nucleobases of its target nucleic acid. For example, if the nucleobase at a certain position of antisense oligonucleotide can hydrogen bond with the nucleobase at a certain position of target nucleic acid, the hydrogen bond position between oligonucleotide and target nucleic acid is considered to be complementary in this nucleobase pair.The nucleobase containing a certain modification can maintain the ability to pair with the corresponding nucleobase, and thus can also be nucleobase complementary.

[0066] The term "non-complementary" when used in reference to nucleobases refers to pairs of nucleobases that do not form hydrogen bonds with one another.

[0067] The term "complementarity," when used in reference to an oligomeric compound (e.g., linked nucleosides, oligonucleotides, or nucleic acids), refers to the ability of such an oligomeric compound, or a region thereof, to hybridize to another oligomeric compound, or a region thereof, through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at every nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or complementary oligomeric regions are complementary at 70% of the nucleobases (70% complementarity). In certain embodiments, complementary oligomeric compounds or complementary oligomeric regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or complementary oligomeric regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or complementary oligomeric regions are 95% complementary. In certain embodiments, the complementary oligomeric compounds or complementary oligomeric regions are 100% complementary.

[0068] The term "mismatch" refers to a nucleotide of a first polynucleotide that is not capable of pairing with a nucleotide at a corresponding position in a second polynucleotide when the first and second polynucleotides are aligned.

[0069] The term "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense oligonucleotide and its target nucleic acid). Without being limited to a particular mechanism, the most common pairing mechanism involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen, between complementary nucleobases.

[0070] The term "specifically hybridize" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.

[0071] The term "fully complementary," when used in reference to an oligonucleotide or portion thereof, means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, the region of fully complementary does not contain any mismatched or unhybridized nucleobases in either strand.

[0072] The term "percent complementarity" refers to the percentage of nucleobases of an oligomeric compound that are complementary to an equivalent length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of an oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

[0073] The term "percent identity" refers to the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as the nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

[0074] The term "modulation" refers to a change in the quantity or quality of a molecule, function, or activity compared to the quantity or quality of the molecule, function, or activity prior to modulation. For example, modulation includes a change that increases (stimulates or induces) or decreases (inhibits or reduces) gene expression. By way of further example, modulation of expression can include a change in splice site selection in pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice variant compared to the amount in the absence of modulation.

[0075] The term "modification motif" refers to a pattern of chemical modifications within or within a region of an oligomeric compound. A motif can be defined by modifications of an oligomeric compound at certain nucleosides and / or at certain linking groups.

[0076] The term "nucleoside motif" refers to a pattern of nucleoside modifications within an oligomeric compound or region thereof. The linkages of such oligomeric compounds may or may not be modified. Unless otherwise indicated, a motif described herein in terms of nucleosides alone is intended to be a nucleoside motif. Thus, in such cases, the linkages are not limited.

[0077] The term "glycomotif" refers to the pattern of sugar modifications within or within a region of an oligomeric compound.

[0078] The term "linkage motif" refers to a pattern of linkage modifications within an oligomeric compound or region thereof. The nucleosides of such oligomeric compounds may be modified or unmodified. Unless otherwise indicated, a motif described herein with respect to linkage alone is intended to be a linkage motif. Thus, in such cases, the nucleosides are not limiting.

[0079] The term "nucleobase modification motif" refers to the pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, the nucleobase modification motif is independent of the nucleobase sequence.

[0080] The term "sequence motif" refers to a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modification and, therefore, can have any combination of chemical modifications, including no chemical modifications.

[0081] The term "modified" when referring to a nucleoside or a "type" of nucleoside refers to the chemical modification of the nucleoside and includes modified and unmodified nucleosides. Thus, unless otherwise indicated, a "nucleoside having a type 1 modification" can be an unmodified nucleoside.

[0082] The term "differentially modified" refers to chemical modifications or chemical substituents that differ from one another, including the absence of modification. Thus, for example, DNA nucleosides are unmodified, but MOE nucleosides and unmodified DNA nucleosides are "differentially modified." Similarly, DNA and RNA are both naturally occurring, unmodified nucleosides, but are "differentially modified." Nucleosides that are identical except for containing different nucleobases are not differentially modified. For example, a nucleoside containing a 2'OMe-modified sugar and an unmodified adenine nucleobase is not differentially modified, compared to a nucleoside containing a 2'OMe-modified sugar and an unmodified thymine nucleobase.

[0083] The term "same modification type" refers to modifications that are the same as each other, including the absence of a modification. Thus, for example, a DNA nucleoside is unmodified, but two unmodified DNA nucleosides have the "same modification type." Such nucleosides with the same modification type may contain different nucleobases.

[0084] The phrase "pharmaceutically acceptable carrier or diluent" refers to any substance suitable for use in administration to an animal. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0085] The terms "substituent" and "substituent group" refer to an atom or group that replaces an atom or group of a referenced parent compound. For example, a substituent of a modified nucleoside is any atom or group that is different from an atom or group found in a naturally occurring nucleoside (e.g., a modified 2' substituent is any atom or group other than H or OH at the 2' position of a nucleoside). Substituents may be protected or unprotected. In certain embodiments, compounds of the invention have substituents at one or more positions of a parent compound. Substituents may also be further substituted with other substituents and may be attached to the parent compound directly or through a linking group such as an alkyl group or a hydrocarbyl group.

[0086] When used in reference to a chemical functional group, the term "substituent" refers to an atom or group of atoms different from those normally present in the referenced functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen that replaces one of the hydrogen atoms of the unsubstituted methyl group). Unless otherwise indicated, groups suitable for use as substituents include, but are not limited to, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)Ra), carboxyl (-C(O)O-Ra), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-O-Ra), aryl, aralkyl, heterocyclic radicals, heteroaryl, heteroarylalkyl, aryl, arylsulfonyl ... amine (-N-(Rbb)(Rcc)), imino (=NRbb), amide (-C(O)N(Rbb)(Rcc) or -N-(Rbb)C(O)Raa), azido (-N3), nitro (-NO2), cyano (-CN), carbamide (-OC(O)N(Rbb)(Rcc) or -N-(Rbb)C(O)ORaa), ureido (-N-(Rbb)C(O)N(Rbb)(Rcc)), thioureido (-N-(Rb b) C(S)N(Rbb)-(Rcc)), guanidinyl (-N-(Rbb)C(=NRbb)N(Rbb)(Rcc)), amidinyl (-C(=NRbb)N(Rbb)(Rcc) or -N-(Rbb)C(=NRbbb)(Raa)), thiol (-SRbb), sulfinyl (-S(O)Rbb), sulfonyl (-S(O)Rbb), and sulfonamidyl (-S(O)N(Rbb)(Rcc) or -N-(Rbb)S-(O)Rbb), where each R a, R b, and R c is independently H, an optionally linked chemical functionality, or a further substituent including, but not limited to, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic alkyl, heterocyclic alkyl, and heteroaryl alkyl, as set forth in a non-limiting preferred list. Selected substituents may occur recursively within the compounds described herein.

[0087] The term "alkyl" refers to a saturated straight-chain or branched carbohydrate radical containing up to 24 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl, and the like. Alkyl groups typically contain 1 to about 24 carbon atoms, more typically 1 to about 12 carbon atoms (C1-C12 alkyl), with 1 to about 6 carbon atoms being more preferred.

[0088] The term "alkenyl" refers to a straight-chain or branched hydrocarbon chain radical containing up to 24 carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene, and the like. Alkenyl groups typically contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. As used herein, alkenyl groups may optionally contain one or more further substituents.

[0089] The term "alkynyl" refers to a straight-chain or branched carbohydrate radical containing up to 24 carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically contain from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms, with from 2 to about 6 carbon atoms being more preferred. As used herein, alkynyl groups may optionally include one or more further substituents.

[0090] The term "acyl" refers to a radical formed by removal of a hydroxyl group from an organic acid and has the general formula: -C(O)-X, where X is typically aliphatic, alicyclic, or aromatic. Examples include aliphatic carbonyl, aromatic carbonyl, aliphatic sulfonyl, aromatic sulfinyl, aliphatic sulfinyl, aromatic phosphate, aliphatic phosphate, and the like. As used herein, acyl groups can optionally include further substituents.

[0091] The term "alicyclic" refers to a ring system in which the ring is aliphatic. The ring system can include one or more rings, where at least one ring is aliphatic. Preferred alicyclic rings include rings having from about 5 to about 9 carbon atoms in the ring. As used herein, alicyclic rings can optionally include further substituents.

[0092] The term "aliphatic" refers to a straight-chain or branched carbohydrate radical containing up to 24 carbon atoms, with saturation between any two carbon atoms being a single, double, or triple bond. Aliphatic groups preferably contain from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms, with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of the aliphatic group can be interrupted by one or more heteroatoms, including nitrogen, oxygen, sulfur, and phosphorus. Such heteroatom-interrupted aliphatic groups include, but are not limited to, polyalkoxy groups such as polyalkylene glycols, polyamines, and polyimines. As used herein, aliphatic groups can optionally contain additional substituents.

[0093] The term "alkoxy" refers to a radical formed between an alkyl group and an oxygen atom, where the oxygen atom is used to attach the alkoxy group to the parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. Alkoxy groups as used herein can optionally include further substituent groups.

[0094] The term "aminoalkyl" refers to an amino-substituted C1-C12 alkyl radical. The alkyl portion of the radical forms a covalent bond with the parent molecule. The amino group can be located at any position, and the aminoalkyl group can be substituted with further substituents on the alkyl and / or amino portions.

[0095] The terms "aralkyl" and "arylalkyl" refer to an aromatic group covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with the parent molecule. Examples include, but are not limited to, benzyl, phenethyl, and the like. As used herein, aralkyl groups can optionally include additional substituents attached to the alkyl group, the aryl group, or both groups to form the radical group.

[0096] The terms "aryl" and "aromatic" refer to a monocyclic or polycyclic carbocyclic ring system radical having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl, and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituents.

[0097] The terms "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0098] The terms "heteroaryl" and "heteroaromatic" refer to a radical containing a monocyclic or polycyclic aromatic ring, aromatic ring system, or aromatic fused ring system, in which at least one of the rings is aromatic and contains one or more heteroatoms. Heteroaryl is also intended to include fused ring systems, including systems in which one or more of the fused rings does not contain a heteroatom. Heteroaryl groups typically contain one ring atom selected from sulfur, nitrogen, or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or a heteroatom. As used herein, heteroaryl groups can optionally contain additional substituents.

[0099] The compounds described herein include variations in which one or more atoms are replaced with non-radioactive or radioactive isotopes of the indicated elements. For example, compounds herein containing hydrogen atoms include: 1 For each H hydrogen atom, all possible deuterium substitutions are included.

[0100] Isotopic substitutions embraced by the compounds herein include, for example, 1 Replaces H 2 H or 3 H, 12 Replaces C 13 C or 14 C. 14 Replaces N 15 N, 16 Replaces O 17 O or 18 O, and 32 Replaces S 33 S, 34 S, 35 S, or 36Non-radioactive isotope substitutions include, but are not limited to, S and the like. In certain embodiments, non-radioactive isotope substitutions may impart new properties to oligomeric compounds that are beneficial for use as therapeutic or research tools. In certain embodiments, radioactive isotope substitutions may make the compounds suitable for research purposes, such as imaging.

[0101] As used herein, the term "nanoparticle" refers to any particle having an average diameter of less than 500 nanometers (nm). In some embodiments, nanoparticles have an average diameter of less than 300 nm, less than 100 nm, less than 50 nm, less than 25 nm, less than 10 nm, or less than 5 nm. In some embodiments, each nanoparticle has a diameter of less than 300 nm, less than 100 nm, less than 50 nm, less than 25 nm, less than 10 nm, or less than 5 nm.

[0102] As used herein, the term "hypertrophy" refers to an increase in the mass of an organ or structure independent of natural proliferation without tumor formation. Organ or tissue hypertrophy results from an increase in the mass of individual cells (true hypertrophy), or an increase in the number of cells that make up the tissue (hyperplasia), or both. Certain organs, such as the heart, lose the ability to undergo cell division shortly after birth.

[0103] The term "fibrosis-associated gene" refers to a gene encoding a protein involved in the fibrosis process, including proteins that directly participate in the fibrosis process, such as extracellular matrix proteins, and proteins that modulate the fibrosis process, such as GATA4 and eIF4G2 proteins. Fibrosis-associated genes include profibrotic and antifibrotic genes. Expression of a profibrotic gene promotes fibrosis, whereas expression of an antifibrotic gene inhibits fibrosis.

[0104] The term "myocardial fibrosis-related gene" refers to a fibrosis-related gene involved in the fibrosis process in the heart. Examples of myocardial fibrosis-related genes include, but are not limited to, pro-fibrotic genes such as eIF4G2 (eukaryotic translation initiation factor 4 gamma 2), EPRS (glutamyl-prolyl-tRNA synthetase), and MEOX1 (mesenchyme homeobox 1), as well as anti-fibrotic genes such as GATA4 (GATA binding protein 4), MEF2C (myocyte enhancer factor 2C), NKX2-5 (NK2 homeobox 5), TBX5 (T-box transcription factor 5), HNF4a (hepatocyte nuclear factor 4 alpha), alpha crystallin B (CRYAB), TCF21 (transcription factor 21), and myosin-binding protein C (MYBPC3).

[0105] As used herein, the terms "treating," "treatment," and the like refer to any treatment of myocardial fibrosis, including, but not limited to, preventive and therapeutic treatment. "Treating" includes any effect that results in an improvement of myocardial fibrosis, such as slowing, reducing, modulating, or eliminating myocardial fibrosis. "Treating" myocardial fibrosis or "treatment" of myocardial fibrosis includes inhibiting myocardial fibrosis, i.e., halting the development of myocardial fibrosis or its clinical symptoms; or alleviating myocardial fibrosis, i.e., causing temporary or permanent regression of the disease or its clinical symptoms. Patients in need of treatment include those already suffering from myocardial fibrosis and those in whom myocardial fibrosis is to be prevented.

[0106] As used herein, the term "subject" refers to mammals, such as humans, companion animals (e.g., dogs, cats, birds, etc.), farm animals (e.g., cattle, sheep, pigs, horses, poultry, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, birds, etc.). A "subject in need thereof" refers to a subject having, diagnosed with, suspected of having, or seeking prevention of myocardial fibrosis.

[0107] As used herein, an "effective amount" or "therapeutically effective amount" in the context of treating myocardial fibrosis refers to an amount that, when administered to a cell, tissue, or subject alone or in combination with another therapeutic agent, is effective in slowing, reducing, inhibiting, or otherwise negating the growth of myocardial fibrosis. An "effective amount" also refers to the amount of a compound sufficient to result in symptomatic improvement, e.g., treatment, cure, prevention, or amelioration of myocardial fibrosis, or to increase the rate of treatment, cure, prevention, or amelioration of cardiac hypertrophy. When applied to an individual compound (active ingredient) administered alone, an "effective amount" refers to that ingredient alone. When applied to a combination, an "effective amount" refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An "effective amount" will vary depending on the cause of myocardial fibrosis and the severity of myocardial fibrosis, as well as the age, weight, etc., of the subject being treated. In addition, the "effective amount" may vary depending on the dosage form used and the route of administration utilized. A physician or veterinarian skilled in the art can easily determine and prescribe the effective amount (e.g., ED50) of the active ingredient required. For example, the physician or veterinarian can start the administration of the compound to be administered at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0108] II. Compositions for modulating translation Translation of proteins encoded by messenger ribonucleic acid (mRNA) typically begins at the start codon of the mRNA's major open reading frame (mORF). Some mRNAs contain one or more upstream ORFs (uORFs) located within the 5' untranslated region of the mRNA. When the corresponding uORF of an mORF is translated, the uORF has been established as a negative regulatory element that represses the translation of the mORF. Antisense oligonucleotide (ASO) technology provides an effective means for modulating the expression of specific mRNAs or proteins based on Watson-Crick base pairing between a properly designed ASO and its target mRNA. ASO technology is most commonly used to reduce the amount of mRNA or alter the splicing of pre-mRNA transcripts in cells through antisense-induced RNase H cleavage. In contrast, this application primarily presents antisense oligonucleotides (ASOs) and modified antisense oligonucleotides that are not designed to induce cleavage. Specifically, the present application provides antisense oligonucleotides and modified oligonucleotides that can selectively increase or decrease translation of a desired target protein in a cell by disrupting a double-stranded region within the uORF of an mRNA (referred to later in this specification as Type I ASO or ASO1) or by forming an intermolecular double-stranded region adjacent to and downstream of the uORF start codon or mORF start codon (referred to later in this specification generally as Type II ASO or ASO2).

[0109] The present invention also provides antisense oligonucleotides and modified oligonucleotides having gapmer structures that can form a double-stranded region together with the sequence of the 5'-UTR region of an mRNA by 5'-UTR-targeting gapmer ASO, thereby reducing the translation of a desired target protein and degrading the target mRNA in cells.

[0110] The term "gapmer" refers to a chimeric oligomeric compound comprising a central region (the "gap") and regions on either side of the central region (the "wings"), where the gap comprises at least one modification difference compared to each wing. Such modifications include nucleobase, monomer linkage, and sugar modifications, as well as the absence of a modification (unmodified RNA or unmodified DNA). Thus, in certain embodiments, the nucleotide linkage in each of the wings is different from the nucleotide linkage in the gap. In certain embodiments, each wing comprises nucleotides with a high-affinity modification, and the gap comprises nucleotides without this modification. In certain embodiments, the nucleotides in the gap and the nucleotides in the wings all comprise a high-affinity modification, but the high-affinity modification in the gap is different from the high-affinity modification in each of the wings. In certain embodiments, the modifications in the wings are the same as each other. In certain embodiments, the modifications in the wings are different from each other. In certain embodiments, the nucleotides in the gap are unmodified and the nucleotides in the wings are modified. In certain embodiments, the modification(s) in each wing are the same. In certain embodiments, the modification(s) in one wing are different from the modification(s) in the other wing. Gapmers are described in U.S. Patent Nos. 9,550,988 and 9,045,754, which are incorporated herein by reference.

[0111] Without wishing to be bound by theory, the inventors hypothesize that type I ASOs (e.g., ASO1) disrupt the original double-stranded structure within the uORF by forming a double-stranded structure with the non-coding strand of the original double-stranded structure, thereby inhibiting translation of the uORF, which results in enhanced translation of the corresponding mORF (Figure 1, panel A).

[0112] In contrast, type II ASOs disrupt the original double-stranded structure within the uORF by forming a double-stranded structure with the coding strand of the original double-stranded structure, thereby enhancing the translation of the uORF, which results in a reduction in the translation of the corresponding mORF (Figure 1, panel B). Furthermore, type II ASOs can function in the absence of the original double-stranded structure within the uORF. In this case, the type II ASO is designed to form a double-stranded structure with a target sequence adjacent to and downstream of the uORF start codon, thereby enhancing the translation of the uORF, which results in a reduction in the translation of the corresponding mORF. These uORF-targeting type II ASOs are referred to as "type II uotASOs."

[0113] In addition, type II ASOs may be designed to form a double-stranded structure with a target sequence downstream of and adjacent to the start codon of an mORF, thereby enhancing translation of the mORF. These mORF-targeting type II ASOs are referred to as "type II motASOs."

[0114] 5'UTR-targeting gapmer ASOs can be designed to form double-stranded structures with target sequences within the 5'-UTR of a target gene's mRNA. In some embodiments, the target sequence is located within a region of the mRNA spanning from 55 nucleotides upstream of the uORF start codon to 55 nucleotides downstream of the uORF start codon, thereby enhancing translation of the uORF or blocking scanning by the transcription preinitiation complex, which results in reduced translation of downstream mORFs and, alternatively, triggering RNase H1-mediated mRNA degradation.

[0115] ASOs that enhance the expression of antifibrotic gene products One aspect of the present invention is directed to antisense oligonucleotides (ASOs) that can selectively increase or decrease the translation of myocardial fibrosis-related gene products. The design and use of these ASOs can be utilized to treat or prevent myocardial fibrosis.

[0116] In some embodiments, the antisense oligonucleotides (ASOs) of the present invention can bind to a target sequence located in the non-coding strand of the double-stranded stem structure adjacent to and downstream of the uORF AUG start codon of the mRNA of a myocardial fibrosis-related gene, forming a double-stranded structure with the target sequence. Upon binding, the double-stranded stem structure of the uORF is disrupted, enhancing translation of the corresponding mORF of the myocardial fibrosis-related gene (type I uotASO).

[0117] In some embodiments, the myocardial fibrosis-related gene is an anti-fibrotic gene. Examples of anti-fibrotic genes include, but are not limited to, GATA4, MEF2C, NKX2-5, TBX5, HNF4A, CRYAB, TCF21, and MYBCP3.

[0118] In some embodiments, the target sequence comprises the human GATA4 mRNA sequence of SEQ ID NO:27.

[0119] In some embodiments, the ASO comprises a sequence that is at least 50%, 60%, 70%, 80%, or 90% complementary to the target sequence. In some embodiments, the ASO comprises a sequence that is 100% complementary to the target sequence. In some embodiments, the ASO further comprises one or more modified nucleotides and / or modified internucleotide linkages.

[0120] In some embodiments, the ASO is a human GATA4 type I uot ASO comprising SEQ ID NO:8.

[0121] In some embodiments, the ASO is capable of forming a double-stranded structure with a target sequence downstream of and adjacent to the mORF start codon of the mRNA of the antifibrotic gene, thereby enhancing translation of the mORF of the antifibrotic gene (type II motASO).

[0122] In some embodiments, the target region comprises a region 2-8 nucleotides away from the adenine (A) of the AUG start codon of the mORF.

[0123] In some embodiments, the anti-fibrotic genes are GATA4, MEF2C, NKX2-5, TBX5, HNF4A, CRYAB, TCF21, and MYBPC3. In certain embodiments, the anti-fibrotic gene is MYBPC3 or CRYAB. An overview of MYBPC3 and CRYAB and their applications is shown in the table below. Suitable targets are down-regulated in human tissues involved in cardiometabolic diseases, and up-regulation is believed to be therapeutically advantageous.

[0124] [Table 1]

[0125] In some embodiments, the target sequence comprises the nucleotide sequence 5'-gcctgagccggggaag-3', which is the human MYBPC3 type II motASO target sequence of SEQ ID NO:47.

[0126] In some embodiments, the target sequence comprises the nucleotide sequence 5'-ggacatcgccatccac-3', which is the human CRYAB type II motASO target sequence of SEQ ID NO:48.

[0127] In some embodiments, the target sequence comprises the human GATA4 mRNA sequence of SEQ ID NO:28.

[0128] In some embodiments, the target sequence comprises the human MEF2C mRNA sequence of SEQ ID NO:29.

[0129] In some embodiments, the target sequence comprises the human NKX2-5 mRNA sequence of SEQ ID NO:30.

[0130] In some embodiments, the ASO comprises a sequence that is at least 50%, 60%, 70%, 80%, or 90% complementary to the target sequence. In some embodiments, the ASO comprises a sequence that is 100% complementary to the target sequence. In some embodiments, the ASO further comprises one or more modified nucleotides and / or modified internucleotide linkages.

[0131] In some embodiments, the ASO is a human MYBPC3 type II motASO comprising SEQ ID NO:45.

[0132] In some embodiments, the ASO is a human CRYAB type II motASO comprising SEQ ID NO:46.

[0133] In some embodiments, the ASO is a human GATA4 type II motASO comprising SEQ ID NO:9 or SEQ ID NO:10.

[0134] In some embodiments, the ASO is a human NKX2-5 type II motASO comprising SEQ ID NO:15.

[0135] In some embodiments, the ASO is a human MEF2C type II motASO comprising SEQ ID NO:21.

[0136] In some embodiments, the 5' end of the ASO binds between nucleotide positions +3 and +19 relative to the AUG start codon of the mORF, where +1 corresponds to the adenine in the AUG start codon. In some embodiments, the 3' end of the ASO contains at least one nucleotide that is complementary to a nucleotide in the mORF start codon. In certain embodiments, the 3' end of the ASO contains a cytosine that is complementary to the guanine in the AUG start codon.

[0137] ASOs that reduce the expression of profibrotic gene products In some embodiments, the ASO is capable of forming a double-stranded structure with a target sequence downstream and adjacent to the start codon of a uORF in the mRNA of a profibrotic gene, thereby inhibiting translation of the mORF of the profibrotic gene (type II uotASO). In some embodiments, the target region comprises a region 2-8 nucleotides away from the adenine (A) of the uORF AUG start codon.

[0138] In some embodiments, the pro-fibrotic gene is eIF4G2, EPRS, or MEOX1.

[0139] In some embodiments, the pro-fibrotic gene is eIF4G2, a pro-fibrotic stress mediator essential for the translation of extracellular matrix (ECM) mRNA. MCM Genetic knockout of eIF4G2 in cardiac myocytes (Myh6) attenuates cardiac dysfunction, pathological hypertrophy, and fibrosis. The TGFβ-eIF4G2-IGFBP7 axis is a novel translational regulatory pathway that mediates cardiac fibroblast activation and plays a key role in myocardial fibrosis. MCM ) has been shown to not cause severe heart disease within 5 months.

[0140] In some embodiments, the target sequence comprises the human EIF4G2 mRNA sequence of SEQ ID NO:31.

[0141] In some embodiments, the ASO comprises a sequence that is at least 50%, 60%, 70%, 80%, or 90% complementary to the target sequence. In some embodiments, the ASO comprises a sequence that is 100% complementary to the target sequence. In some embodiments, the ASO further comprises one or more modified nucleotides and / or modified internucleotide linkages.

[0142] In some embodiments, the 5' end of the ASO binds between nucleotide positions +3 and +19 relative to the AUG start codon of the uORF, where +1 corresponds to the adenine in the AUG start codon. In some embodiments, the 3' end of the ASO contains at least one nucleotide that is complementary to a nucleotide in the uORF start codon. In certain embodiments, the 3' end of the ASO contains a cytosine that is complementary to the guanine in the AUG start codon.

[0143] In some embodiments, the ASO is a human EIF4G2 type II uot ASO comprising SEQ ID NO: 17.

[0144] In some embodiments, the ASO is a 5'-UTR-targeting gapmer ASO that targets the 5'-UTR of the mRNA of a pro-fibrotic gene. In some embodiments, the 5'-UTR-targeting gapmer ASO is capable of binding to a target located within a region spanning 55 nucleotides upstream to 55 nucleotides downstream of the uORF start codon in the mRNA. In some embodiments, the 5'-UTR-targeting gapmer ASO is capable of binding to a target located within a region spanning 45 nucleotides upstream to 45 nucleotides downstream of the uORF start codon in the mRNA. In some embodiments, the 5'-UTR-targeting gapmer ASO is capable of binding to a target located within a region spanning 35 nucleotides upstream to 35 nucleotides downstream of the uORF start codon in the mRNA. In some embodiments, the 5'-UTR-targeting gapmer ASO is capable of binding to a target located within a region spanning 25 nucleotides upstream to 25 nucleotides downstream of the uORF start codon in the mRNA. In some embodiments, the 5'-UTR-targeting gapmer ASO is capable of binding to a target located within a region spanning 15 nucleotides upstream to 15 nucleotides downstream of the uORF start codon within the mRNA.

[0145] In some embodiments, the 5'-UTR-targeting gapmer ASO comprises a sequence that is at least 50%, 60%, 70%, 80%, or 90% complementary to the target sequence, hi some embodiments, the 5'-UTR-targeting gapmer ASO comprises a sequence that is 100% complementary to the target sequence.

[0146] In some embodiments, the 5'-UTR-targeting gapmer ASO targets the 5'-UTR of the mRNA of the human eIF4G2 gene. In some embodiments, the 5'-UTR-targeting gapmer ASO has a gap region of 5-20 nucleotides flanked by two wing regions of 3-10 nucleotides. In some embodiments, the 5'-UTR-targeting gapmer ASO has a gap region of 10 nucleotides flanked by two wing regions of 5 nucleotides.

[0147] In some embodiments, the target sequence comprises the human eIF4G2 mRNA sequence of SEQ ID NO:49.

[0148] In some embodiments, the 5'-UTR-targeting gapmer ASO has the nucleotide sequence of SEQ ID NO:41.

[0149] ASO design steps Step 1: Determine the dominant alternative splicing isoform of mRNA in the organ as an ASO target.

[0150] Step 2: Consider multiple parameters for ASO design, including the length of the 5'UTR, the presence of uORFs, the GC content of the 5'UTR, dsRNA elements, Kozak sequences near the uORF and mORF start codons, and effects from dsRNA-binding proteins or RNA helicases.

[0151] Step 3: Mechanism-based screen for ASOs: (1) Targeting the uORF start codon using a type I uotASO, which inhibits the uORF while enhancing the translation of the mORF; (2) Targeting the mORF start codon using a class II motASO, which directly enhances the translation of the mORF.

[0152] Step 4 (Hit from Target): A tiling screen is performed by shifting the ASO to the target in the upstream or downstream region 3 nucleotides (then 1 nucleotide) from the initial ASO.

[0153] Step 5 (hit to lead): After validation of ASO efficacy in human cell lines and primary mouse cells, optimal ASOs are identified through in vivo testing in animal models.

[0154] Exemplary embodiments for the design strategy and sequences for MYBPC3 and CRYAB targets are shown in the table below for mORF-activated Type II motASOs.

[0155] [Table 2]

[0156] In some embodiments, the ASOs of the invention (e.g., Type I uotASOs, Type II uotASOs, Type II motASOs, and 5'-UTR-targeting gapmer ASOs) have a length of between 8 and 50, 8 and 40, 8 and 30, 8 and 25, 8 and 20, 8 and 16, 8 and 12, 10 and 50, 10 and 40, 10 and 30, 10 and 25, 10 and 20, 10 and 16, 10 and 12, 12 and 50, 12 and 40, 12 and 30, 12 and 25, 12 and 20, 12 and 16, 15 and 50, 15 and 40, 15 and 30, 15 and 25, 15 and 20, 20 and 50, 20 and 40, 20 and 30, or 20 and 25 nucleotides.

[0157] Modified Nucleosides The ASOs of the present invention can comprise or consist of oligonucleotides containing at least one modified nucleoside. Such modified nucleosides can include modified sugar moieties, modified nucleobases, or both. In some embodiments, the ASOs contain at least 5, at least 10, at least 15, at least 20, at least 25, or more modified nucleosides relative to the total number of nucleosides in the ASO. In some embodiments, the modified ASOs contain a modified region of at least 5, at least 10, at least 15, at least 20, or at least 25, or more consecutive modified nucleosides within the ASO. In some embodiments, each of the nucleosides in the ASO is modified.

[0158] In certain preferred embodiments, one or more modified nucleotides comprise a 2'-O-methyl modified sugar moiety and / or a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.

[0159] In some embodiments, the ASO of the invention comprises one or more sugar-modified nucleotides. In some embodiments, the ASO comprises the nucleotide sequence of any one of SEQ ID NOs: 3-6 with one or more modified sugar moieties and / or modified internucleoside linkages.

[0160] In certain embodiments, the ASO comprises the nucleotide sequence of AmoCmoGmoUmoAmoUmoUmoAmoAmoAmoUmoCmoCmoAmoGmoCm (SEQ ID NO: 7), or AmoCmoGmoAmoAmoUmoUmoAmoAmoAmoUmoCmoCmoAmoGmoCm (SEQ ID NO: 8), CmoUmoUmoCmoCmoCmoCmoGmoCmoUmoCmoAmoGmoCm (SEQ ID NO: 45), or GmoUmoGmoAmoUmoGmoAmoUmoGmoGmoCmoGmoAmoUmoGmoUmoCmoCm (SEQ ID NO: 46), where "m" indicates a 2'-O-methyl modification and "o" indicates a phosphodiester or phosphorothioate internucleoside linkage. It should be noted that in any of the sequences disclosed in this application that incorporate the modifications "o" or "mo," such modifications may be replaced with any nucleoside modification described herein, or may contain no nucleoside modifications at all.

[0161] In certain embodiments, the ASO comprises the nucleotide sequence GesCesCesAesCesCdsTdsCdsCdsAdsTdsAdsGdsAdsGdsCesUesCesCesGe (SEQ ID NO: 41), where "e" indicates a 2'-O-methoxyethyl (MOE) modification, "s" indicates a phosphorothioate internucleoside linkage, and "d" indicates DNA.

[0162] sugar part ASOs of the present invention may contain nucleosides with naturally occurring sugar moieties and / or nucleosides with modified sugar moieties. ASOs containing nucleosides with modified sugar moieties may have desirable properties, such as enhanced nuclease stability or increased binding affinity to target nucleic acids, compared to ASOs containing only nucleosides with naturally occurring sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In certain embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety. In certain embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may contain one or more substitutions corresponding to the substitutions in the substituted sugar moiety.

[0163] In certain embodiments, the modified sugar moiety is a substituted sugar moiety containing one or more substituents, including, but not limited to, substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH3 ("O-methyl"), and 2'-O(CH2)2OCH3. In certain embodiments, the sugar substituent at the 2' position is selected from allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, O-C1-C10 substituted alkyl; O-C1-C10 alkoxy; O-C1-C10 substituted alkoxy, OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each of Rm and Rn is independently H or a substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5' position include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In certain embodiments, the substituted sugar comprises more than one non-bridging sugar substituent, such as a 2'-F-5'-methyl sugar moiety (see, e.g., PCT International Application Publication No. 2008 / 101157 for additional 5',2'-bis-substituted sugar moieties and 5',2'-bis-substituted nucleosides).

[0164] Nucleosides that include a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In certain embodiments, the 2'-substituted nucleoside is selected from the group consisting of halo, allyl, amino, azido, O-C1-C10 alkoxy, O-C1-C10 substituted alkoxy, SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, or N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, and O-alkylenyl- and 2' substituents selected from O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), or O-CH2-C(=O)-N(Rm)(Rn), where each of Rm and Rn is independently H, an amino protecting group, or a substituted or unsubstituted C1-C10 alkyl. These 2' substituents can be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro(NO2), thiol, thioalkoxy(S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0165] In certain embodiments, a 2'-substituted nucleoside comprises a 2'-substituent selected from F, NH, N, OCF, O-CH, O(CH)NH, CH-CH=CH, O-CH-CH=CH, OCHCHOCH, O(CH)SCH, O-(CH)-ON(R)(R), O(CH)O(CH)N(CH), and N-substituted acetamide (O-CH-C(=O)-N(R)(R), where each of R and R is independently H, an amino-protecting group, or a substituted or unsubstituted C-C alkyl.

[0166] In certain embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, OCF, O-CH, OCHCHOCH, O(CH)SCH, O-(CH)-ON(CH), -O(CH)O(CH)N(CH), and O-CH-C(=O)-N(H)CH.

[0167] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4'-furanose ring atom and the 2'-furanose ring atom. Examples of such 4'-2' sugar substituents include -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]nO-, -C(RaRb)-N(R)-O-, or -C(RaRb)-ON(R)-; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2; 4'(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2'(cEt) and 4'-CH(CH 2OCH3)-O-2', and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)-O-2', and analogs thereof (see, e.g., WO2009 / 006478, published January 8, 2009); 4'-CH2-N-(OCH3)-2', and analogs thereof (see, e.g., WO2008 / 150, published December 11, 2008). 729); 4'-CH2-ON(CH3)-2' (see, e.g., US 2004 / 0171570, published September 2, 2004); 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2'- [wherein each R is independently H, a protecting group, or a C1-C12 alkyl]; 4'-CH2-N(R)-O-2' [wherein R is H, a C1-C12 alkyl, or a protecting group] (see, e.g., US 2004 / 0171570, published September 2, 2004). 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' and analogs thereof (see, PCT International Application Publication No. 2008 / 154401, published December 8, 2008).

[0168] In certain embodiments, such 4'-2' bridges are independently -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra) 2- 1 to 4 linking groups independently selected from -S(=O)x-, -S(=O)x-, and -N-(Ra)- [In the formula, x is 0, 1, or 2; n is 1, 2, 3, or 4; Each of Ra and Rb independently represents H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, C N, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); each of J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0169] Nucleosides containing a bicyclic sugar moiety are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, as depicted below, (A) α-L-methyleneoxy (4'-CH2-O-2') BNA, (B) β-D-methyleneoxy (4'-CH2-O-2') BNA (also referred to as locked nucleic acid or LNA), (C) ethyleneoxy (4'-(CH2)2-O-2') BNA, (D) aminooxy (4'-CH2-ON(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-O-2') BNA, (F) methyl ( (M) 4'-CH-O-CH-2', (M) methylene-thio (4'-CH-S-2'), (H) methylene-amino (4'-CH-N(R)-2'), (I) methyl carbocyclic (4'-CH-CH(CH)-2'), (J) propylene carbocyclic (4'-(CH)-2'), and (M) 4'-CH-O-CH-2'.

[0170] [ka] TIFF2025530741000005.tif153170

[0171] wherein Bx is a nucleobase moiety and R is independently H, a protecting group, or C1-C12 alkyl.

[0172] Additional bicyclic sugar moieties are known in the art (e.g., Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (July 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, and WO2007 / 134181; U.S. Patent Application Publication Nos. 2004 / 0171570 and 2007 / 0287 831, and 2008 / 0039618; U.S. Patent Application Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and PCT International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922).

[0173] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by their isomeric configuration. For example, nucleosides comprising a 4'-2' methyleneoxy bridge can be in either the α-L or β-D configuration. Already, α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides and have demonstrated antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0174] In certain embodiments, the substituted sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted sugars and 4'-2'-bridging sugars) (see PCT International Application Publication No. 2007 / 134181, published November 22, 2007, in which LNAs are substituted, e.g., with 5'-methyl or 5'-vinyl groups).

[0175] In certain embodiments, the modified sugar moiety is a sugar surrogate. In certain such embodiments, the oxygen atom of the naturally occurring sugar is replaced with, for example, a sulfur atom, a carbon atom, or a nitrogen atom. In certain such embodiments, such modified sugar moieties also contain bridging and / or non-bridging substituents as described above. For example, certain sugar surrogates contain a 4' sulfur atom and substitutions at the 2' position (see, e.g., U.S. Patent Application Publication No. 2005 / 0130923, published June 16, 2005) and / or the 5' position. By way of further example, carbocyclic bicyclic nucleosides having a 4'-2' bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0176] In certain embodiments, the sugar surrogate comprises a ring having more than five atoms. For example, in certain embodiments, the sugar surrogate comprises a six-ring-membered tetrahydropyran. Such tetrahydropyrans can be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, CJ., Bioorg. & Med. Chem. (2002), 10:841-854), fluoro-HNA (F-HNA), and nucleosides of formula VII:

[0177] [ka]

[0178] wherein, independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII: Bx is a nucleobase moiety; T3 and T4 are each independently an internucleoside linking group linking tetrahydropyran nucleoside analogs of antisense oligonucleotides, or one of T3 and T4 is an internucleoside linking group linking tetrahydropyran nucleoside analogs of antisense oligonucleotides, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugate group, or a 5' or 3' terminal group; q1, q2, q3, q4, q5, q6, and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; each of R1 and R2 is independently selected from among hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where X is O, S, or NJ1, and each of J1, J2, and J3 is independently H or C1-C6 alkyl. These include, but are not limited to, compounds having the formula:

[0179] In certain embodiments, modified THP nucleosides are provided of Formula VII, where q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, THP nucleosides are provided of Formula VII, where one of R1 and R2 is F. In certain embodiments, R1 is fluoro, R2 is H, R1 is methoxy, R2 is H, or R1 is methoxyethoxy, and R2 is H.

[0180] Many other bicyclic and tricyclic sugars and sugar surrogate ring systems are known in the art that can be used to modify nucleosides (see, for example, the review article: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0181] In certain embodiments, sugar surrogates comprise rings with more than five atoms and more than one heteroatom.For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patent Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506).As used herein, the term "morpholino" refers to the following structure:

[0182] [ka]

[0183] means a sugar surrogate having the formula:

[0184] In certain embodiments, morpholinos can be modified, for example, by adding or altering various substituents derived from the morpholino structures described above. Such sugar surrogates are referred to herein as "modified morpholinos."

[0185] Combinations of modifications are also provided, including, but not limited to, 2'-F-5'-methyl substituted nucleosides (see PCT International Application Publication No. 2008 / 101157, published August 21, 2008, for other disclosed 5',2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2' position (see U.S. Patent Application Publication No. 2005 / 0130923, published June 16, 2005), or alternatively, 5' substitution of bicyclic nucleic acids (see PCT International Application Publication No. 2007 / 134181, published November 22, 2007, in which 4'-CH2-O-2' bicyclic nucleosides are further substituted at the 5' position with a 5'-methyl or 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides, along with their oligomerization and biochemical studies, have also been described (see, for example, Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

[0186] Modified Nucleobases In certain embodiments, nucleosides of the invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the invention comprise one or more modified nucleobases.

[0187] In certain embodiments, the modified nucleobases are selected from universal bases, hydrophobic bases, hybrid bases, size-extended bases, and fluorinated bases, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2-, N-6-, and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine. Alkynyl derivatives of pyrimidine bases, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyl (-C=C-CH3)uracil and 5-propynyl (-C=C-CH3)cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, 6-azocytosine, and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-haloadenine and 8-halogua uracil and cytosine, in particular 5-bromouracil and 5-trifluoromethyluracil, 5-trifluoromethyluracil and 5-trichloromethyluracil, 5-trifluoromethyluracil, 5-trichloro ... Trifluoromethylcytosine, as well as other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, hybrid bases, size-extended bases, and fluorinated bases, as defined herein.Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases can also include modified nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, edited by Kroschwitz, JI, John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, edited by Crooke, ST and Lebleu, B, CRC Press, 1993, 273-288.

[0188] Representative U.S. patents that teach the preparation of certain of the above-referenced modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367, 066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469; No. 5,594,121; No. 5,596,091; No. 5,614,617; No. 5,645,985; No. 5,681,941; No. 5,750,692; No. 5,763,588; No. 5,830,653 and No. 6,005,096.

[0189] Internucleoside linkages In certain embodiments, nucleosides can be linked together to form oligonucleotides using any internucleoside linkage. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified linkages compared to natural phosphodiester linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides. In certain embodiments, internucleoside linkages having chiral atoms can be prepared as racemic mixtures or as separate optical isomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0190] The oligonucleotides described herein contain one or more asymmetric centers, which result in optical isomers, diastereomers, and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R) or (S), α or β, such as α or β for sugar anomers, or (D) or (L), such as (D) or (L) for amino acids. The antisense oligonucleotides presented herein include all such possible stereoisomers, as well as their racemic and optically pure forms.

[0191] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide 3 (3'-CH2-C(=O)-N(H)-5'), amide 4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'S-CH2-O-5'). Additional neutral internucleoside linkages include nonionic linkages, including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, e.g., "Carbohydrate Modifications in Antisense Research," edited by Y.S. Sanghvi and P.D. Cook, ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include non-ionic linkages containing mixed moieties based on N, O, S, and CH2.

[0192] motif In some embodiments, the ASO of the invention comprises a modified oligonucleotide. In some embodiments, the modified oligonucleotide comprises one or more modified sugars. In some embodiments, the modified oligonucleotide comprises one or more modified nucleobases. In some embodiments, the modified oligonucleotide comprises one or more modified internucleoside linkages. In some embodiments, the modifications (sugar modifications, nucleobase modifications, and / or linkage modifications) define a pattern or motif. In some embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, a modified oligonucleotide can be described by its sugar modification motif, internucleoside linkage motif, and / or nucleobase modification motif (nucleobase modification motif, as used herein, describes a chemical modification to a nucleobase independent of the sequence of the nucleobases).

[0193] In certain embodiments, all sugar moieties of modified oligonucleotides of the invention are modified. In certain embodiments, modified oligonucleotides contain one or more unmodified sugar moieties.

[0194] full length In certain embodiments, the present invention provides modified oligonucleotides of any of various lengths.In certain embodiments, the present invention provides oligomeric compounds or oligonucleotides that consist of nucleosides linked from X to Y, where X represents the minimum number of nucleosides within the range, and Y represents the maximum number of nucleosides within the range.In certain such embodiments, X and Y are each independently selected from 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 and 50, with the proviso that X≦Y. For example, in certain embodiments, the present invention provides 5-6, 5-7, 5-8, 5-9, 5-5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7- 8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29 , 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19 9, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27,11~28、11~29、11~30、12~13、12~14、12~15、12~16、12~17、12~18、12~19、12~20、12~21、12~22、12~23、12~24、12~25、12~26、12~27、12~28、12~29、12~30、13~14、13~15、13~16、13~17、13~18、13~19、13~20、13~21、13~22、13~23、13~24、13~25、13~26、13~27、13~28、13~29、13~30、14~15、14~16、14~17、14~18、14~19、14~20、14~21、14~22、14~23、14~24、14~25、14~26、14~27、14~28、14~29、14~30、15~16、15~17、15~18、15~19、15~20、15~21、15~22、15~23、15~24、15~25、15~26、15~27、15~28、15~29、15~30、16~17、16~18、16~19、16~20、16~21、16~22、16~23、16~24、16~25、16~26、16~27、16~28、16~29、16~30、17~18、17~19、17~20、17~21、17~22、17~23、17~24、17~25、17~26、17~27、17~28、17~29、17~30、18~19、18~20、18~21、18~22、18~23、18~24、18~25、18~26、18~27、18~28、18~29、18~30、19~20、19~21、19~22、19~23、19~24、19~25、19~26、19~29、19~28、19~29、19~30、20~21、20~22、20~23、20~24、20~25、20~26、20~27、20~28、20~29、20~30、21~22、21~23、21~24、21~25、21~26、21~27、21~28、21~29、21~30、22~23、22~24、22~25、22~26、22~27、22~28、22~29、22~30、23~24、23~25、23~26、23~27、23~28、23~29、23~30、24~25、24~26、24~27、24~28、24~29、24~30、25~26、25~27、25~28、25~29、25~30、26~27、26~28、Modified oligonucleotides are provided, including oligonucleotides consisting of 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked nucleosides. In embodiments where the number of nucleosides in an oligomeric compound or oligonucleotide is limited to a range or specific number, the oligomeric compound or oligonucleotide may nevertheless further comprise other substituents. For example, an oligonucleotide containing 830 nucleosides excludes an oligonucleotide having 31 nucleosides, although unless otherwise indicated, such oligonucleotides may further comprise, for example, one or more conjugates, terminal groups, or other substituents. In certain embodiments, the modified oligonucleotide has any of the lengths described above.

[0195] Furthermore, when an oligonucleotide is described by a total length range and a region having a specified length, and the sum of the specified lengths of the region is less than the upper limit of the total length range, the oligonucleotide may have additional nucleosides beyond those of the specified region, provided that the total number of nucleosides does not exceed the upper limit of the total length range.

[0196] In certain embodiments, the oligonucleotides of the present application are characterized by their modification motif and overall length, and in certain embodiments, each such parameter is independent of the other.

[0197] oligomeric compounds In certain embodiments, the present invention provides oligomeric compounds consisting of an oligonucleotide (modified or unmodified), optionally consisting of one or more conjugate groups and / or terminal groups. A conjugate group consists of one or more conjugate moieties and a conjugate linker that connects the conjugate moieties to the oligonucleotide. A conjugate group may be attached to one or both termini of an oligonucleotide and / or at any internal position. In certain embodiments, a conjugate group is attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, a conjugate group attached to one or both termini of an oligonucleotide is a terminal group. In certain such embodiments, a conjugate group or terminal group is attached at the 3'- and / or 5'-terminus of an oligonucleotide. In certain such embodiments, a conjugate group (or terminal group) is attached at the 3'-terminus of an oligonucleotide. In certain embodiments, a conjugate group is attached near the 3'-terminus of an oligonucleotide. In certain embodiments, a conjugate group (or terminal group) is attached at the 5'-terminus of an oligonucleotide. In certain embodiments, the conjugate group is attached near the 5' end of the oligonucleotide.

[0198] Examples of terminal groups include, but are not limited to, a conjugate group, a capping group, a phosphate moiety, a protecting group, an abasic nucleoside, a modified or unmodified nucleoside, and two or more nucleosides that are independently modified or unmodified.

[0199] In certain embodiments, antisense oligonucleotides are provided in which the 5'-terminal group comprises a 5'-terminal stabilized phosphate. A "5'-terminal stabilized phosphate" is a 5'-terminal phosphate group that has one or more modifications that increase nuclease stability compared to the 5' phosphate.

[0200] In certain embodiments, the 5' terminal group has the formula IIe:

[0201] [ka]

[0202] [In the formula, Bx is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine; T2 is a phosphorothioate internucleoside linking group that links the compound of Formula IIe to an oligomeric compound; G is a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2N(CH3)2, or OCH2-N(H)-C(=NH)NH2] An antisense oligonucleotide is provided, having the formula:

[0203] In certain embodiments, antisense oligonucleotides are provided wherein the 5' terminal compound has Formula IIe, where G is F, OCH3, or O(CH2)2-OCH3.

[0204] In certain embodiments, the 5'-terminal group is a 5'-terminal stabilized phosphate, including a vinyl phosphonate, represented by Formula IIe above.

[0205] Conjugate Group In certain embodiments, the ASO of the present application comprises an antisense oligonucleotide modified by the covalent attachment of one or more conjugate groups (also referred to as "conjugate partners"). Generally, the conjugate group modifies one or more properties of the attached oligonucleotide, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, intracellular distribution, cellular uptake, charge, and clearance. As used herein, "conjugate group" refers to a radical group comprising an atomic group attached to an oligonucleotide or oligomeric compound. Generally, the conjugate group modifies one or more properties of the compound to which it is attached, including, but not limited to, pharmacodynamic properties, pharmacokinetic properties, binding properties, absorption properties, intracellular distribution properties, cellular uptake properties, charge properties, and / or clearance properties. Conjugate groups are commonly used in the chemical arts and may include conjugate linkers that covalently link the conjugate group to the oligonucleotide or oligomeric compound. In certain embodiments, the conjugate group comprises a cleavable moiety that covalently links the conjugate group to the oligonucleotide or oligomeric compound. In certain embodiments, the conjugate group comprises a conjugate linker and a cleavable moiety that covalently links the conjugate group to the oligonucleotide or oligomeric compound. In certain embodiments, the conjugate group has the general formula:

[0206] [ka]

[0207] wherein n is 1 to about 3, and when n is 1, m is 0, or when n is 2 or 3, m is 1, j is 1 or 0, k is 1 or 0, and the sum of j and k is at least 1. It has.

[0208] In certain embodiments, n is 1, j is 1, and k is 0. In certain embodiments, n is 1, j is 0, and k is 1. In certain embodiments, n is 1, j is 1, and k is 1. In certain embodiments, n is 1, j is 1, and k is 1. In certain embodiments, n is 2, j is 1, and k is 0. In certain embodiments, n is 2, j is 0, and k is 1. In certain embodiments, n is 2, j is 1, and k is 1. In certain embodiments, n is 3, j is 1, and k is 0. In certain embodiments, n is 3, j is 0, and k is 1. In certain embodiments, n is 3, j is 1, and k is 1.

[0209] Herein, conjugate groups are referred to as radicals that provide a bond for forming a covalent bond with an oligomeric compound, such as an oligonucleotide. In certain embodiments, the conjugation point on an oligomeric compound is at the 3'-terminal nucleoside or modified nucleoside. In certain embodiments, the conjugation point on an oligomeric compound is at the 3'-oxygen atom of the 3'-hydroxyl group of the 3'-terminal nucleoside or modified nucleoside. In certain embodiments, the conjugation point on an oligomeric compound is at the 5'-terminal nucleoside or modified nucleoside. In certain embodiments, the conjugation point on an oligomeric compound is at the 5'-oxygen atom of the 5'-hydroxyl group of the 5'-terminal nucleoside or modified nucleoside. In certain embodiments, the conjugation point on an oligomeric compound is at any reactive site on a nucleoside, modified nucleoside, or internucleoside linkage.

[0210] As used herein, "cleavable moiety" and "cleavable bond" refer to a cleavable bond or cleavable group of atoms that is capable of being split or broken under certain physiological conditions. In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety comprises a cleavable bond. In certain embodiments, the cleavable moiety is a group of atoms. In certain embodiments, the cleavable moiety is selectively cleaved inside a cell or an intracellular compartment such as a lysosome. In certain embodiments, the cleavable moiety is selectively cleaved by an endogenous enzyme such as a nuclease. In certain embodiments, the cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.

[0211] In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety covalently joins the oligomeric compound to the conjugate linker. In certain such embodiments, the cleavable moiety covalently joins the oligomeric compound to the cell-targeting moiety.

[0212] In certain embodiments, the cleavable bond is selected from among an amide bond, a polyamide bond, an ester bond, an ether bond, one or both ester bonds of a phosphodiester bond, a phosphate ester bond, a carbamate bond, a disulfide bond, or a peptide bond. In certain embodiments, the cleavable bond is one of the ester bonds of a phosphodiester bond. In certain embodiments, the cleavable bond is one or both ester bonds of a phosphodiester bond. In certain embodiments, the cleavable moiety is a phosphodiester linkage between the oligomeric compound and the remainder of the conjugate group. In certain embodiments, the cleavable moiety comprises a phosphodiester linkage located between the oligomeric compound and the remainder of the conjugate group. In certain embodiments, the cleavable moiety comprises a phosphate ester or a phosphodiester. In certain embodiments, the cleavable moiety is joined to the conjugate linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is joined to the conjugate linker by a phosphodiester linkage. In certain embodiments, the conjugate group does not comprise a cleavable moiety.

[0213] In certain embodiments, the cleavable moiety is a cleavable nucleoside or modified nucleoside. In certain embodiments, the nucleoside or modified nucleoside optionally comprises a protected heterocyclic base selected from purine, substituted purine, pyrimidine, or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine.

[0214] In certain embodiments, the cleavable moiety is a 2'-deoxynucleoside linked to the 3'- or 5'-terminal nucleoside of the oligomeric compound by a phosphodiester linkage and covalently linked to the remainder of the conjugate group by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine linked to the 3'- or 5'-terminal nucleoside of the oligomeric compound by a phosphodiester linkage and covalently linked to the remainder of the conjugate group by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine linked to the 3'-oxygen atom of the 3'-hydroxyl group of the 3'-terminal nucleoside or modified nucleoside by a phosphodiester linkage. In certain embodiments, the cleavable moiety is 2'-deoxyadenosine joined by a phosphodiester linkage to the 5' oxygen atom of the 5' hydroxyl group of the 5' terminal nucleoside or modified nucleoside. In certain embodiments, the cleavable moiety is joined to the 2' position of a nucleoside or modified nucleoside of an oligomeric compound.

[0215] As used herein in the context of a conjugate group, the term "conjugate linker" refers to any atom or group of atoms that covalently link a cell targeting moiety to an oligomeric compound, either directly or via a cleavable moiety. In certain embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether (-S-), and hydroxylamino (-ON(H)-). In certain embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus linking group. In certain embodiments, the conjugate linker comprises at least one phosphodiester group. In certain embodiments, the conjugate linker comprises at least one neutral linking group.

[0216] In certain embodiments, the conjugate linker is covalently bonded to the oligomeric compound. In certain embodiments, the conjugate linker is covalently bonded to the oligomeric compound and the branched group. In certain embodiments, the conjugate linker is covalently bonded to the oligomeric compound and the tethering ligand. In certain embodiments, the conjugate linker is covalently bonded to the cleavable moiety. In certain embodiments, the conjugate linker is covalently bonded to the cleavable moiety and the branched group. In certain embodiments, the conjugate linker is covalently bonded to the cleavable moiety and the tethering ligand. In certain embodiments, the conjugate linker comprises one or more cleavable bonds. In certain embodiments, the conjugate group does not comprise the conjugate linker.

[0217] As used herein, "branched group" refers to an atomic group having at least three positions capable of forming a covalent linkage to two or more tether-ligands and the remainder of the conjugate group. Generally, a branched group provides multiple reactive sites for connecting tethering ligands to an oligomeric compound via a conjugate linker and / or cleavable moiety. In certain embodiments, a branched group comprises a group selected from an alkyl group, an amino group, an oxo group, an amide group, a disulfide group, a polyethylene glycol group, an ether group, a thioether group, and a hydroxylamino group. In certain such embodiments, a branched group comprises a branched aliphatic group comprising a group selected from an alkyl group, an amino group, an oxo group, an amide group, a disulfide group, a polyethylene glycol group, an ether group, a thioether group, and a hydroxylamino group. In certain such embodiments, a branched aliphatic group comprises a group selected from an alkyl group, an amino group, an oxo group, an amide group, and an ether group. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl groups, amino groups, and ether groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl groups and ether groups. In certain embodiments, the branched group comprises a monocyclic or polycyclic ring system.

[0218] In certain embodiments, the branched group is covalently bonded to the conjugate linker. In certain embodiments, the branched group is covalently bonded to the cleavable moiety. In certain embodiments, the branched group is covalently bonded to each of the conjugate linker and the tethering ligand. In certain embodiments, the branched group comprises one or more cleavable bonds. In certain embodiments, the conjugate group does not comprise a branched group.

[0219] In certain embodiments, the conjugate group provided herein comprises a cell targeting moiety with at least one tethering ligand.In certain embodiments, the cell targeting moiety comprises two tethering ligands that are covalently bonded to branched group.In certain embodiments, the cell targeting moiety comprises three tethering ligands that are covalently bonded to branched group.

[0220] As used herein, "tether" refers to an atomic group connecting a ligand to the remainder of a conjugate group. In certain embodiments, each tether is a linear aliphatic group containing one or more groups selected from alkyl groups, substituted alkyl groups, ether groups, thioether groups, disulfide groups, amino groups, oxo groups, amide groups, phosphodiester groups, and polyethylene glycol groups, in any combination. In certain embodiments, each tether is a linear aliphatic group containing one or more groups selected from alkyl groups, ether groups, thioether groups, disulfide groups, amino groups, oxo groups, amide groups, and polyethylene glycol groups, in any combination. In certain embodiments, each tether is a linear aliphatic group containing one or more groups selected from alkyl groups, substituted alkyl groups, phosphodiester groups, ether groups, and amino groups, oxo groups, and amide groups, in any combination. In certain embodiments, each tether is a linear aliphatic group containing one or more groups selected from alkyl groups, ether groups, and amino groups, oxo groups, and amide groups, in any combination. In certain embodiments, each tether is a linear aliphatic group containing one or more groups selected from alkyl groups, amino groups, and oxo groups, in any combination. In certain embodiments, each tether is a linear aliphatic group containing one or more groups selected from alkyl groups and oxo groups, in any combination. In certain embodiments, each tether is a linear aliphatic group containing one or more groups selected from alkyl groups and phosphodiester groups, in any combination. In certain embodiments, each tether contains at least one phosphorus or neutral linking group.

[0221] In certain embodiments, the tether comprises one or more cleavable bonds. In certain embodiments, each tethering ligand is attached to a branched group. In certain embodiments, each tethering ligand is attached to a branched group via an amide group. In certain embodiments, each tethering ligand is attached to a branched group via an ether group. In certain embodiments, each tethering ligand is attached to a branched group via a phosphorus linking group or a neutral linking group. In certain embodiments, each tethering ligand is attached to a branched group via a phosphodiester group. In certain embodiments, each tether is attached to a ligand via an amide group or an ether group. In certain embodiments, each tether is attached to a ligand via an ether group.

[0222] In certain embodiments, each tether comprises about 8 to about 20 atoms in the chain length between the ligand and the branching group. In certain embodiments, each tether comprises about 10 to about 18 atoms in the chain length between the ligand and the branching group. In certain embodiments, each tether comprises about 13 atoms in the chain length.

[0223] In certain embodiments, the present disclosure provides ligands in which each ligand is covalently conjugated via a tether to the remainder of the conjugate group. In certain embodiments, each ligand is selected to have affinity for at least one receptor on a target cell. In certain embodiments, a ligand is selected to have affinity for at least one receptor on the surface of mammalian liver cells. In certain embodiments, a ligand is selected to have affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is independently selected from galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose. In certain embodiments, each ligand is N-acetylgalactosamine (GalNAc). In certain embodiments, the targeting moiety comprises one to three ligands. In certain embodiments, the targeting moiety comprises three ligands. In certain embodiments, the targeting moiety comprises two ligands. In certain embodiments, the targeting moiety comprises one ligand. In certain embodiments, the targeting moiety comprises the ligand 3-N-acetylgalactosamine. In certain embodiments, the targeting moiety comprises the ligand 2-N-acetylgalactosamine. In certain embodiments, the targeting moiety comprises the ligand 1-N-acetylgalactosamine.

[0224] In certain embodiments, each ligand is a carbohydrate, a carbohydrate derivative, a modified carbohydrate, a polyvalent carbohydrate cluster, a polysaccharide, a modified polysaccharide, or a polysaccharide derivative. In certain embodiments, each ligand is an amino sugar or a thio sugar. For example, the amino sugar can be selected from any number of compounds known in the art, such as glucosamine, sialic acid, α-D-galactosamine, N-acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, and N-sulfo-D-glucosamine, and N-glycoloyl-α-neuraminic acid. For example, the thiosugar may be selected from the group consisting of 5-thio-β-D-glucopyranose, methyl-2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, and ethyl-3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside.

[0225] In certain embodiments, the conjugate groups provided herein comprise carbohydrate clusters. As used herein, "carbohydrate cluster" refers to a portion of a conjugate group in which two or more carbohydrate residues are joined to a branched group via a tether group (for examples of carbohydrate conjugate clusters, see, e.g., Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting," Bioconjugate Chemistry, 2003(14):18-29; or Rensen et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor," J. Med. Chem., 2004(47):5798-5808, which are incorporated herein by reference in their entireties).

[0226] As used herein, "modified carbohydrate" means any carbohydrate that has one or more chemical modifications compared to a naturally occurring carbohydrate.

[0227] As used herein, "carbohydrate derivative" means any compound that can be synthesized using a carbohydrate as a starting material or intermediate.

[0228] As used herein, "carbohydrate" means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

[0229] In certain embodiments, the cell targeting moiety has the formula:

[0230] [ka]

[0231] A conjugate group is provided having the following structure:

[0232] In certain embodiments, the cell targeting moiety has the formula:

[0233] [ka]

[0234] A conjugate group is provided having the following structure:

[0235] In certain embodiments, the cell targeting moiety has the formula:

[0236] [ka]

[0237] A conjugate group is provided having the following structure:

[0238] In certain embodiments, the conjugate group has the formula:

[0239] [ka]

[0240] It has.

[0241] Representative U.S. patents, U.S. patent application publications, and international patent application publications that teach the preparation of certain modifications of conjugated oligomeric compounds, such as ASOs containing the above-referenced conjugate groups, conjugate groups, tethers, conjugate linkers, branching groups, ligands, cleavable moieties, as well as other modifications, include, but are not limited to, each of which is incorporated herein by reference in its entirety. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, US2006 / 0148740, US2011 / 0123520, WO2013 / 033230, and WO2012 / 037254, which are incorporated herein by reference.

[0242] Representative publications that teach the preparation of certain modifications of conjugated oligomeric compounds, such as ASOs containing the above-mentioned conjugate groups, conjugate groups, tethers, conjugate linkers, branching groups, ligands, and cleavable moieties, as well as other modifications, include, but are not limited to, BIESSEN et al., "The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent," J. Med. Chem. (1995), 38:1846-1852; BIESSEN et al., "Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor," J. Med. Chem. (1995), 38:1538-1546; LEE et al., "New and more efficient multivalent 50daman-ligands for asialoglycoprotein receptor of mammalian hepatocytes", Bioorganic & Medicinal Chemistry (2011), 19:2494~2500; RENSEN et al., "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo", J.Biol.Chem.(2001), 276(40): 37577 - 37584; RENSEN et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor", J. Med. Chem. (2004), 47: 5798 - 5808; SLIEDREGT et al., "Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor", J. Med. Chem. (1999), 42: 6096 - 618; and Valentijn et al., "Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor", Tetrahedron, 1997, 53(2), 759 - 770, including.

[0243] In certain embodiments, conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, choline acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantine, acridine, fluorescein, rhodamine, coumarin, and dyes.Certain conjugate groups, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), choline acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBOJ., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as dihexadecyl-rac-glycerol or triethyl-ammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or 1-adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylaminocarbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937) have already been described.

[0244] In certain embodiments, the conjugate group comprises an active ingredient, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethicine, barbiturates, cephalosporins, sulfa drugs, antidiabetics, antibacterial agents, or antibiotics.

[0245] Some non-limiting examples of conjugate linkers include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl, where a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0246] Conjugate groups may be attached to one or both termini of the oligonucleotide (terminal conjugate groups) and / or at any internal position.

[0247] In certain embodiments, conjugate group is at the 3'-end of the oligonucleotide of oligomeric compound.In certain embodiments, conjugate group is near the 3'-end.In certain embodiments, conjugate is attached at the 3'-end of oligomeric compound, but attached before one or more terminal nucleosides.In certain embodiments, conjugate group is located in terminal group.

[0248] III. Pharmaceutical Compositions Another aspect of the invention relates to a pharmaceutical composition comprising one or more ASOs of the present application and a pharmaceutically acceptable carrier.

[0249] In some embodiments, the pharmaceutical composition comprises one or more antifibrotic gene-enhanced ASOs.

[0250] In some embodiments, the pharmaceutical composition comprises one or more profibrotic gene-inhibiting ASOs. In some embodiments, the one or more profibrotic gene-inhibiting ASOs are selected from the group consisting of SEQ ID NOs: 17 and 41.

[0251] In some embodiments, the pharmaceutical composition comprises (1) an anti-fibrotic gene-enhancing ASO and (2) one or more pro-fibrotic gene-inhibiting ASOs.

[0252] In some embodiments, the one or more anti-fibrotic gene-enhanced ASOs are selected from the group consisting of SEQ ID NOs: 8, 9, 19, 15, 21, 45, and 46.

[0253] In some embodiments, the one or more pro-fibrotic gene-inhibiting ASOs are selected from the group consisting of SEQ ID NOs: 17 and 41.

[0254] In some embodiments, the pharmaceutical composition comprises one or more carriers suitable for delivering the therapeutic agent to cardiac tissue. Exemplary carriers for delivery include nanoparticles, lipids, liposomes, micelles, polymers, polymer micelles, emulsions, polyelectrolyte complexes, hydrogels, microcapsules, viruses, virus-like particles (VLPs), peptides, antibodies, aptamers, small molecule chemicals, exosomes, combinations thereof, and pegylated derivatives thereof. In certain embodiments, the pharmaceutical composition comprises a nanoparticle formulation comprising an ASO according to the present invention.

[0255] In certain embodiments, the carriers described above, including nanoparticles, can be linked to cardiac tissue-specific targeting peptides or antibodies to facilitate carrier-mediated delivery of the active agents described herein to cardiac tissue. For example, in certain embodiments, the pharmaceutical composition comprises nanoparticles or liposomes covalently or non-covalently coated with cardiac tissue-specific targeting peptides or antibodies.

[0256] Exemplary nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, polymeric nanoparticles, nanoworms, nanoemulsions, nanogels, fullerene-like materials, inorganic nanotubes, dendrimers (e.g., covalently conjugated with metal chelates), nanocapsules, nanospheres, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. Nanoparticles can generate detectable signals, for example, by absorption and / or emission of photons (radiofrequency and visible photons) and plasmon resonance. Nanoparticles can be biodegradable or non-biodegradable.

[0257] In certain embodiments, the nanoparticles are metal nanoparticles, metal oxide nanoparticles, or semiconductor nanocrystals. The metal of the metal nanoparticles or metal oxide nanoparticles can be 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, an element of the lanthanide series or an element of the actinide series (e.g., cerium, praseodymium, yttrium ... The metals may include chromium, 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 may be iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum, silver, gold, cerium, or samarium. The metal oxide may be an oxide of any of these materials or a combination of these materials. For example, the metal may be gold, or the metal oxide may be iron oxide, cobalt oxide, zinc oxide, cerium oxide, or titanium oxide. The preparation of metal and metal oxide nanoparticles is described, for example, in US Pat. Nos. 5,897,945 and 6,759,199.

[0258] In other embodiments, the polymeric nanoparticles are composed of synthetic biodegradable polymers, natural biodegradable polymers, or combinations thereof. Synthetic biodegradable polymers can include polyesters, such as poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone; polyorthoesters, polyanhydrides, polydioxanone, poly-alkyl-cyanoacrylates (PAC), polyoxalates, polyiminocarbonates, polyurethanes, polyphosphazenes, or combinations thereof. Natural biodegradable polymers can include starch, hyaluronic acid, heparin, gelatin, albumin, chitosan, dextran, or combinations thereof.

[0259] In some embodiments, the pharmaceutical composition comprises a delivery vehicle, such as a nanoparticle or liposome, encapsulating a pharmaceutically effective amount of an antisense oligonucleotide. In some embodiments, the pharmaceutically effective amount of the ASO is about 0.001 μg / mL to about 10 μg / mL (w / v) of a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically effective amount of the ASO is about 0.1 μg / mL to about 1 μg / mL (w / v) of a pharmaceutically acceptable carrier.

[0260] In some embodiments, the pharmaceutical composition comprises an ASO of the present invention and a lipid moiety. Lipid moieties have been used in various ways with nucleic acid drugs. In certain such methods, nucleic acids are introduced into preformed liposomes or lipoplexes composed of a mixture of cationic and neutral lipids. In certain methods, DNA complexes are formed with monocationic or polycationic lipids without the presence of neutral lipids. In certain embodiments, the lipid moiety is selected to increase the distribution of the drug to cardiac tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the drug to the myocardium.

[0261] In certain embodiments, the pharmaceutical compositions provided herein comprise one or more ASOs and one or more excipients. Exemplary excipients include water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and combinations thereof. In certain embodiments, the pharmaceutical compositions comprise one or more hydrophobic compounds, including organic solvents such as dimethyl sulfoxide.

[0262] In certain embodiments, the pharmaceutical compositions provided herein include a cosolvent system. The cosolvent system may include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such a cosolvent system is used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which is a solution of 3% w / v benzyl alcohol, 8% w / v of the nonpolar surfactant Polysorbate 80™, and 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of such a cosolvent system may be varied considerably without significantly altering their solubility and toxicity. Furthermore, the identity of the cosolvent components may be varied. For example, other surfactants may be used in place of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers, such as polyvinylpyrrolidone, may replace polyethylene glycol; and other sugars or polysaccharides may be used in place of dextrose.

[0263] In some embodiments, the pharmaceutical composition comprises sterile saline and one or more ASOs. In certain embodiments, the pharmaceutical composition consists of sterile saline and one or more ASOs. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, the pharmaceutical composition comprises one or more ASOs and sterile water. In certain embodiments, the 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 and phosphate buffered saline (PBS). In certain embodiments, the pharmaceutical composition consists of one or more ASOs and sterile phosphate buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.

[0264] In certain embodiments, the ASO is mixed with pharmaceutically acceptable active and / or inactive substances to prepare a pharmaceutical composition or formulation. The composition and method for formulation into a pharmaceutical composition may depend on several criteria, including, but not limited to, the route of administration, the extent of the disease, and / or the dose to be administered.

[0265] Pharmaceutical compositions containing ASOs can include any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions containing ASOs include one or more oligonucleotides that can produce (directly or indirectly) biologically active metabolites or residues thereof when administered to an animal, such as a human. Thus, for example, the present disclosure also covers pharmaceutically acceptable salts of ASOs, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0266] Prodrugs may include the incorporation of additional nucleosides at one or both termini of the oligomeric compound such that they are cleaved by endogenous nucleases in the body to form the active compound.

[0267] The pharmaceutical compositions of the present invention are formulated according to a specific administration route. The administration routes of the therapeutic agents of the present invention include oral administration and parenteral administration, i.e., injection, infusion, or implantation, or some other route other than the digestive tract. Specific administration modes include injection, for example, intravenous, intramyocardial, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0268] In certain preferred embodiments, the pharmaceutical composition is formulated for administration by intravenous or intramyocardial injection. In certain embodiments, the pharmaceutical composition is formulated in an aqueous solution such as water or a physiologically compatible buffer, for example, Hank's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or act as preservatives). In certain embodiments, an injectable suspension is prepared using appropriate liquid carriers, suspending agents, etc. Certain pharmaceutical compositions for injection are provided in unit dosage form, for example, in ampoules or 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 agents, stabilizers, and / or dispersing agents. Certain solvents suitable for use in injectable pharmaceutical compositions 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 injectable suspensions may contain

[0269] IV. Methods for Treating Myocardial Fibrosis Another aspect of the invention relates to a method for treating myocardial fibrosis in a subject, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising an ASO of the invention.

[0270] In some embodiments, the methods involve administration of a pharmaceutical composition comprising one or more antifibrotic gene-enhanced ASOs.

[0271] In some embodiments, the method includes administering a pharmaceutical composition comprising one or more pro-fibrotic gene-inhibiting ASOs.

[0272] In some embodiments, the method includes administering a pharmaceutical composition comprising (1) an anti-fibrotic gene-enhancing ASO and (2) one or more pro-fibrotic gene-inhibiting ASOs.

[0273] In some embodiments, the one or more anti-fibrotic gene-enhanced ASOs are selected from the group consisting of SEQ ID NOs: 8, 9, 19, 15, 21, 45, and 46.

[0274] In some embodiments, the one or more pro-fibrotic gene-inhibiting ASOs are selected from the group consisting of SEQ ID NOs: 17 and 41.

[0275] In another embodiment, the method comprises administering a pharmaceutical composition containing the ASO formulated in a nanoparticle formulation.

[0276] In one embodiment, the method comprises administering the pharmaceutical composition to the subject intravenously or intramyocardially.

[0277] In some embodiments, the ASO dosage can be expressed as the amount of compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of ASOs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., to determine 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 of LD50 to ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used to formulate a dosage or amount range for use in mammals (e.g., humans). The dosage or amount of the ASO preferably lies within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage or amount can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the patient's condition. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety sufficient to maintain the desired effect.

[0278] In certain embodiments, the ASO may be administered to a mammal having, suspected of having, or at risk for myocardial fibrosis and / or related conditions in an amount sufficient to reduce the expression or activity of a target protein. According to certain embodiments, the ASO may be administered at a dose suitable to reduce the expression of the target protein by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any range thereof.

[0279] The dosage for a particular patient can be determined by one of ordinary skill in the art using conventional considerations (e.g., using appropriate conventional pharmacological protocols). A physician may, for example, prescribe a relatively low dose initially and then increase the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to produce a beneficial therapeutic response in the patient over time, or, for example, to reduce symptoms, or for other appropriate activity depending on the application. The dose may be determined by the effectiveness of the particular formulation, as well as the activity, stability, or serum half-life of the ASO used and the patient's condition, as well as the body weight or body surface area of ​​the patient being treated. The size of the dose may also be determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular composition in a particular patient.

[0280] Optimal accuracy in achieving effective ASO concentration within the range that achieves maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the availability of pharmaceutical compositions in targeted cardiac tissue.When determining the optimal concentration for treatment regimen, the distribution, equilibrium, and excretion of pharmaceutical compositions may be considered.In general, the pharmaceutical compositions of the present invention may be administered in a manner that maximizes efficacy and minimizes toxicity.

[0281] Furthermore, the dosage of the compositions of the present invention can be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosing regimens can be selected, and a pharmacokinetic / pharmacodynamic model can be used to determine the pharmacokinetic / pharmacodynamic profile of the one or more dosing regimens. Then, based on the specific pharmacokinetic / pharmacodynamic profile, one of the dosing regimens can be selected for administration that achieves the desired pharmacokinetic / pharmacodynamic response. See, for example, U.S. Patent No. 6,747,002, which is expressly incorporated herein by reference in its entirety.

[0282] More specifically, the pharmaceutical composition may be administered in a single daily dose, or the total daily dose may be administered in divided doses two, three, or four times daily. For oral administration, the daily dosage of the composition may vary over a wide range, from about 0.1 ng to about 1,000 mg per patient per day. The range may be, more specifically, about 0.001 ng to 10 mg per kg of body weight per day, about 0.1 to 100 μg, about 1.0 to 50 μg, or about 1.0 to 20 mg per kg of body weight per day for an adult (about 60 kg).

[0283] The daily dosage of the pharmaceutical composition can vary over a wide range, from about 0.1 ng to about 1000 mg per adult human per day. For oral administration, the composition can be administered in the form of tablets containing about 0.1 ng 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, allowing for symptomatic dosage adjustment in accordance with the patient being treated. An effective amount of the pharmaceutical composition is typically supplied at a dosage level of about 0.1 ng to about 20 mg per kg of body weight per day. In one embodiment, the range is about 0.2 ng to about 10 mg per kg of body weight per day. In another embodiment, the range is from about 0.5 ng to about 10 mg per kg of body weight per day. The pharmaceutical compositions may be administered on a regimen of from about 1 to about 10 times per day.

[0284] In the case of injection, it is usually preferable to administer an amount of about 0.01 μg to 30 mg, about 0.01 μg to 20 mg, or about 0.01 to 10 mg per day to an adult (about 60 kg) via the intravenous route. For other animals, a dose calculated for a 60 kg body weight can be similarly administered.

[0285] The dosage of the pharmaceutical composition of the present invention is expressed as a serum concentration per single or multiple administration 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, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 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, 12.5, 12.9, 13.0 , 13.5, 13.9, 14, 14.5, 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, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and / or 5000 m / ml, or any range, value, or percentage thereof, optionally at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 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, 90, 1000, 1500, 2000, 2500, 3000, 39, 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, 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, or any range, value, or percentage thereof, including but not limited to 0.0001 μg to 1000 mg / kg per dose, or 0.001 μg to 100.0 mg / kg per dose, 0.01 μg to 10 mg / kg per dose, 0.1 μg to 10 mg / kg per dose.

[0286] By way of non-limiting example, treatment of humans or animals can be achieved using a single infusion or multiple doses for at least 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 days. One, or alternatively or in addition, 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 weeks, or alternatively or in addition, 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, at least one of 0.0001, 0.001, 0.01, 0.1, 0.5, 0.9, 1.0, 1.1, or It may be administered as a single dose or periodic doses of the composition of the invention ranging from 0.1 ng to 100 mg / kg, such as 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.

[0287] Specifically, the pharmaceutical composition is administered at least once per week for several weeks. In one embodiment, the pharmaceutical composition is administered at least once per week for several weeks to several months. In another embodiment, the pharmaceutical composition is administered once per week for 4 to 8 weeks. In yet another embodiment, the pharmaceutical composition is administered once per week for 4 weeks.

[0288] More specifically, the pharmaceutical composition is administered at least once per day for about 2 days, at least once per day for about 3 days, at least once per day for about 4 days, at least once per day for about 5 days, at least once per day for about 6 days, at least once per day for about 7 days, at least once per day for about 8 days, at least once per day for about 9 days, at least once per day for about 10 days, at least once per day for about 11 days, at least once per day for about 12 days, at least once per day for about 13 days, at least once per day for about 14 days, at least once per day for about 15 days, at least once per day for about 16 days, at least once per day for about It may be administered at least once per day for 17 days, at least once per day for about 18 days, at least once per day for about 19 days, at least once per day for about 20 days, at least once per day for about 21 days, at least once per day for about 22 days, at least once per day for about 23 days, at least once per day for about 24 days, at least once per day for about 25 days, at least once per day for about 26 days, at least once per day for about 27 days, at least once per day for about 28 days, at least once per day for about 29 days, at least once per day for about 30 days, or at least once per day for about 31 days.

[0289] Alternatively, the pharmaceutical composition may be administered about once daily, about once every other day, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every 7 days, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 14 days, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, about once every 30 days, about once every 31 days, about once every 32 days, about once every 33 days, about once every 34 days, about once every 35 days, about once every 36 days, about once every 37 days, about once every 38 days, about once every 39 days, about once every 40 days, about once every 41 days, about once every 42 days, about once every 43 days, about once every 44 days, about once every 45 days, about once every 46 days, about once every 47 days, about once every 48 days, about once every 49 days, about once every 50 days, about once every 51 days, about once every 52 days, about once every 53 days, about once every 54 days, about once every 55 days, about once every 56 days, about once every 57 days, about once every 58 days, about once every 59 days, about once every 60 days, about once every 61 days, about once every 62 days, about once every 63 days, about once every 64 days, about once every It may be administered about once every 7 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, about once every 30 days, or about once every 31 days. The pharmaceutical compositions of the present invention may alternatively be administered about once every week, about once every two weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about once every 9 weeks, about once every 10 weeks, about once every 11 weeks, about once every 12 weeks, about once every 13 weeks, about once every 14 weeks, about once every 15 weeks, about once every 16 weeks, about once every 17 weeks, about once every 18 weeks, about once every 19 weeks, or about once every 20 weeks.

[0290] Alternatively, the pharmaceutical composition may be administered about once every month, about once every other month, about once every three months, about once every four months, about once every five months, about once every six months, about once every seven months, about once every eight months, about once every nine months, about once every ten months, about once every eleven months, or about once every twelve months.

[0291] Alternatively, the pharmaceutical composition may be administered at least once per week for about 2 weeks, at least once per week for about 3 weeks, at least once per week for about 4 weeks, at least once per week for about 5 weeks, at least once per week for about 6 weeks, at least once per week for about 7 weeks, at least once per week for about 8 weeks, at least once per week for about 9 weeks, at least once per week for about 10 weeks, at least once per week for about 11 weeks, at least once per week for about 12 weeks, at least once per week for about 14 weeks, at least once per week for about 15 weeks, at least once per week for about 16 weeks, at least once per week for about 17 weeks, at least once per week for about 18 weeks, at least once per week for about 19 weeks, at least once per week for about 20 weeks, at least once per week for about 21 weeks, at least once per week for about 22 weeks, at least once per week for about 23 weeks, at least once per week for about 24 weeks, at least once per week for about 25 weeks, at least once per week for about 26 weeks, at least once per week for about 27 weeks, at least once per week for about 28 weeks, at least once per week for about 29 weeks, at least once per week for about 30 weeks, at least once per week for about 31 weeks, at least once per week for about 32 weeks, at least once per week for about 33 weeks, at least once per week for about 34 weeks, at least once per week for about 35 weeks, at least once per week for about 36 weeks, at least once per week for about 37 weeks, at least once per week for about 38 weeks, at least once per week for about 39 weeks, at least once per week for about 40 weeks, at least once per week for about 41 weeks, at least once per week for about 42 weeks, at least once per week for about 43 weeks, at least once per week for about 44 weeks, at least once per week for about For example, the compound may be administered at least once per week for about 12 weeks, at least once per week for about 13 weeks, at least once per week for about 14 weeks, at least once per week for about 15 weeks, at least once per week for about 16 weeks, at least once per week for about 17 weeks, at least once per week for about 18 weeks, at least once per week for about 19 weeks, at least once per week, or at least once per week for about 20 weeks.

[0292] Alternatively, the pharmaceutical composition may be administered at least once per week for about 1 month, for about 2 months, at least once per week, for about 3 months, at least once per week, for about 4 months, at least once per week, for about 5 months, at least once per week, for about 6 months, at least once per week, for about 7 months, at least once per week, for about 8 months, at least once per week, for about 9 months, at least once per week, for about 10 months, at least once per week, for about 11 months, or at least once per week for about 12 months.

[0293] Combination Therapy In some embodiments, the ASO of the present invention can be administered in combination with one or more other therapeutic agents. The ASO of the present invention and the other therapeutic agents can be administered simultaneously or sequentially by the same or different administration routes. The identification and determination of the amount of therapeutic agents for use in the methods described herein can be easily made by medical professionals using standard techniques known in the art. Generally, the ASO of the present application is administered in combination with an effective amount of another therapeutic agent to treat myocardial fibrosis and / or any cardiac disease or cardiac disease symptoms associated with myocardial fibrosis.

[0294] Other therapeutic agents include, but are not limited to, beta-blockers, antihypertensives, cardiac inotropes, antithrombotic agents, vasodilators, hormone antagonists, inotropes, diuretics, endothelin antagonists, calcium channel blockers, phosphodiesterase inhibitors, ACE inhibitors, angiotensin type 2 antagonists and cytokine blockers / inhibitors, and HDAC inhibitors.

[0295] More specifically, the ASO may be combined with another therapeutic agent, including, but not limited to, an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic / fibrinolytic agent, a coagulant, an antiarrhythmic agent, an antihypertensive agent, a treatment for congestive heart failure, an antianginal agent, an antibacterial agent, or a combination thereof.

[0296] In certain embodiments, the ASO of the invention is used in combination with an antihyperlipoproteinemic agent, including aryloxyalkanoic acid / fibric acid derivatives, resins / bile acid sequestrants, HMG CoA reductase inhibitors, nicotinic acid derivatives, thyroid hormones or thyroid hormone analogs, miscellaneous agents or combinations thereof, acifuran, azacosterol, benfluorex, β-benzalbutyramide, carnitine, chondroitin sulfate, clomestrone, detaxitran, dextran sulfate sodium, eritadenine, furazabol, meglutol, melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine, pentaerythritol tetraacetate, phenylbutyramide, pirozadil, probucol (Lorelco), β-sitosterol, piperazine sultosilate, tiadenol, triparanol, and xenbucin.

[0297] In some embodiments, the ASO of the invention is used in combination with an anti-atherosclerotic agent, such as a pyridinol carbamate. In other embodiments, the ASO is used in combination with an anticoagulant (acenocoumarol, ancrod, anisindione, bromindione, chlorindione, coumetalol, cyclocoumarol, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscum acetate, ethylidene dicumarol, fluindione, heparin, hirudin, liaporate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, thiochromarol, and warfarin); an anticoagulant antagomir (antagomir). in combination with antithrombotic / fibrinolytic agents, including, but not limited to, antiplatelet agents (aspirin, dextran, dipyridamole (Persantin), heparin, sulfinpyranone (Anturan), and ticlopidine (Ticlid)); thrombolytic agents (tissue plasminogen activator (Activase), plasmin, pro-urokinase, urokinase (Abokinase), streptokinase (Streptase), anistreplase / APSAC (Eminase)); thrombolytic agent antagonists or combinations thereof).

[0298] In other embodiments, the ASO is used in combination with blood coagulation agents, including, but not limited to, thrombolytic agent antagonists (aminocaproic acid (Amicar) and tranexamic acid (Amstat)); antithrombotic agents (anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine, and triflusal); and anticoagulant antagonists (protamine and vitamin K1).

[0299] Alternatively, ASOs may be used in combination with antiarrhythmic agents, including, but not limited to, Class I antiarrhythmic agents (sodium channel blockers), Class II antiarrhythmic agents (beta-adrenergic blockers), Class III antiarrhythmic agents (repolarization prolonging agents), Class IV antiarrhythmic agents (calcium channel blockers), and miscellaneous antiarrhythmic agents. Non-limiting examples of sodium channel blockers include Class IA (dispiramide (norpace), procainamide (pronestyl), and quinidine (quinidex)); Class IB (lidocaine (xylocaine), tocamide (tonocard), and mexiletine (mexitil)); and Class IC antiarrhythmic agents (encamide (enkaid) and flekamide (tambocor)).

[0300] Non-limiting examples of beta-blockers (also known as β-adrenergic blockers, β-adrenergic antagonists or Class II antiarrhythmic agents) include acebutolol (sectoral), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamol, lol, cloranolol, dilevalol, epanolol, esmolol (Brevibloc), indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol, propanolol (Inderal), sotalol (Betapace), sulfumalol, talinolol, tertatolol, timolol, toliprolol, and xibinolol. In certain embodiments, the beta-blocker comprises an aryloxypropanolamine derivative. Non-limiting examples of aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol, and toliprolol. Non-limiting examples of repolarization prolonging agents, also known as Class III antiarrhythmic agents, include amiodarone (cordarone) and sotalol (betapace).

[0301] Non-limiting examples of calcium channel blockers, otherwise known as Class IV antiarrhythmic agents, include arylalkylamines (e.g., bepridil, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), dihydropyridine derivatives (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine), piperazine derivatives (e.g., cinnarizine, flunarizine, lidoflazine), or various calcium channel blockers such as bencyclane, etafenone, magnesium, mibefradil, or perhexiline. In certain embodiments, the calcium channel blocker comprises a long-acting dihydropyridine (nifedipine-type) calcium antagonist.

[0302] Non-limiting examples of miscellaneous antiarrhythmic agents include adenosine (adenocard), digoxin (lanoxin), acecamide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydroquinidine, indecamide, ipatropium bromide, lidocaine, lorazimine, lorcamide, meobentine, moricizine, pirmenol, prajumaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate, and viquidil.

[0303] In other embodiments, the ASO of the invention is used in combination with antihypertensive agents, including, but not limited to, alpha / beta blockers (labetalol (normodyne, trandate)), alpha blockers, anti-angiotensin II agents, sympatholytics, beta blockers, calcium channel blockers, vasodilators, and various antihypertensive agents.

[0304] Non-limiting examples of alpha blockers, also known as alpha-adrenergic blockers or alpha-adrenergic antagonists, include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoraamine, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin, and yohimbine. In certain embodiments, the alpha blocker may comprise a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin, and trimazosin.

[0305] Non-limiting examples of anti-angiotensin II agents include angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists. Non-limiting examples of angiotensin-converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (Vasotec), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, movertpril, perindopril, quinapril, and ramipril. Non-limiting examples of angiotensin II receptor blockers, also known as angiotensin II receptor antagonists, ANG receptor blockers, or ANG-II type 1 receptor blockers (ARBS), include angiocandesartan, eprosartan, irbesartan, losartan, and valsartan. Non-limiting examples of sympatholytic agents include centrally acting sympatholytics or peripherally acting sympatholytics. Non-limiting examples of centrally acting sympatholytics, also known as central nervous system (CNS) sympatholytics, include clonidine (Catapres), guanabenz (Wytensin), guanfacine (Tenex), and methyldopa (Aldomet). Non-limiting examples of peripherally acting sympatholytics include ganglionic blocking agents, adrenergic neuron blocking agents, β-adrenergic blocking agents, or al-adrenergic blocking agents. Non-limiting examples of ganglionic blocking agents include mecamylamine (Inversine) and trimethaphan (Arfonade). Non-limiting examples of adrenergic neuron blocking agents include guanethidine (Ismelin) and reserpine (Serpasil). Non-limiting examples of beta-adrenergic blockers include acenitrol (Secetral), atenolol (Tenormin), betaxolol (Kerlon), carteolol (Cartrol), labetalol (Normodyne, Trandate), metoprolol (Lopressor), nadanol (Corgard), penbutolol (Levatol), pindolol (Visken), propranolol (Inderal), and timolol (Brocadren). Non-limiting examples of alpha-adrenergic blockers include prazosin (Minipress), doxazosin (Cardura), and terazosin (Hytrin).

[0306] In certain embodiments, the antihypertensive agent can include a vasodilator (eg, a cerebral vasodilator, a coronary vasodilator, or a peripheral vasodilator). In certain embodiments, vasodilators comprise coronary vasodilators, including but not limited to amotrifen, bendazole, benfurodil hemisuccinate, benziodarone, chloracizine, chromonal, clobenfurol, clonitrate, dilazep, dipyridamole, droprenylamine, efloxate, erythrityl tetranitrane, etafenone, fendiline, floresdil, gangrefen, herestrol bis(p-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin, lidofuranin, mannitol hexanitrane, mezibazine, nicolglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexiline, pimethylline, trapidil, tricromyl, trimetazidine, trolnitrate phosphate, and visnadine.

[0307] In certain embodiments, the vasodilator may include a long-term treatment vasodilator or a hypertensive emergency vasodilator. Non-limiting examples of long-term treatment vasodilators include hydralazine (apresoline) and minoxidil (loniten). Non-limiting examples of hypertensive emergency vasodilators include nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten), and verapamil.

[0308] Non-limiting examples of miscellaneous antihypertensive agents include ajmaline, gamma-aminobutyric acid, bufeniod, cicletanine, cyclosidomine, cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4 pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, protoveratrine, raubasine, recimetol, rilmenidine, saralasin, sodium nitrolside, ticrynafen, trimethaphan camsylate, tyrosinase, and urapidil.In certain embodiments, the antihypertensive agent is an arylethanolamine derivative (amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol, and sulfumalol); a benzothiadiazine derivative (altizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethizide, methyclothiazide, , meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazide, and trichlormethiazide; N-carboxyalkyl (peptide / lactam) derivatives (alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril, and ramipril); dihydropyridine derivatives (amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nife ... soldipine and nitrendipine; guanidine derivatives (betanidine, debrisoquine, guanabenz, guanacrine, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz, and guanoxan); hydrazine / phthalazines (budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine, and todralazine); imidazole derivatives (clonidine, lofexidine, phentolamine, tiamenidine, and todralazine) lonidine); quaternary ammonium compounds (azamethonium bromide, chlorisondamine chloride, hexamethonium, pentasinium bis(methylsulfate), pentamethonium bromide, pentolinium tartrate, phenactropinium chloride, and trimethidinium methylsulfate); reserpine derivatives (bietaserpine, deserpidine, rescinnamine, reserpine, and syrosingopine); or sulfonamide derivatives (ambuside, clopamide, farosemide, indapamide, quinethazone, tripamide, and xipamide).

[0309] In another embodiment, the ASO of the present invention is used in combination with a vasopressor.A vasopressor is generally used to increase blood pressure during shock, which may occur during surgical procedures.Non-limiting examples of vasopressors, also known as antihypertensives, include amezinium methylsulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrine, gepefrine, metaraminol, midodrine, norepinephrine, phoredrine and synephrine.

[0310] In some embodiments, the ASOs of the invention are used in combination with treatments for congestive heart failure, including, but not limited to, anti-angiotensin II agents, afterload-preload reduction treatments (hydralazine (apresoline) and isosorbide dinitrate (isodil, sorbitrate)), diuretics, and inotropic agents.

[0311] Non-limiting examples of diuretics include thiazide or benzothiadiazine derivatives (e.g., althiazide, bendroflumethiazide, bayesthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, ide), tetrachlormethiazide, trichlormethiazide), organic mercury compounds (e.g., chlormerodrin, merallide, mercamphamide, mercaptomerin sodium, mercumaryl acid, mercumatilin sodium, mercurous chloride, mersalyl), pteridines (e.g., flutellene, triamterene), purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamabrom, protheobromine, theobromine), acefylline Steroids including ludostarone antagonists (e.g., canrenone, oleandrin, spironolactone), sulfonamide derivatives (e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, chlorexolone, diphenylmethane-4,4'-disulfonamide, disulfamide, ethoxzolamide, furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone) , torasemide, tripamide, xipamide), uracil (e.g., aminometrazine, amisometrazine), potassium-sparing antagonists (e.g., amiloride, triamterene), or various diuretics such as aminozine, arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol, metochalcone, muzolimine, perhexiline, ticrnafen, and urea.

[0312] Non-limiting examples of positive inotropic agents, also known as cardiac inotropes, include acefylline, acetyldigitoxin, 2-amino-4-picoline, aminone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, and dopexamine. , enoximone, erythrophlein, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridin, resibufogenin, scillaren, scillarenin, strophanthin, sulmazole, theobromine and xamoterol.

[0313] In certain embodiments, the inotropic agent is a cardiac glycoside, a beta-adrenergic agonist, or a phosphodiesterase inhibitor. Non-limiting examples of cardiac glycosides include digoxin (lanoxin) and digitoxin (cristodigin). Non-limiting examples of beta-adrenergic agonists include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (Dobutrex), dopamine (Intropin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol, and xamoterol. Non-limiting examples of phosphodiesterase inhibitors include Aminone (Innocol).

[0314] In certain embodiments, secondary therapeutic agents may include several types of surgery, including, for example, preventative, diagnostic or staging, curative, and palliative surgery. Surgery, particularly curative surgery, may be used in conjunction with other treatments, such as the present invention and one or more other agents.

[0315] Such surgical treatments for hypertrophic, vascular, and cardiovascular diseases and disorders are well known to those skilled in the art and include, but are not limited to, performing surgery on an organism, providing a mechanical prosthetic heart valve, angiogenesis, coronary reperfusion, catheter ablation, providing a subject with an implantable cardioverter-defibrillator, mechanical circulatory support, or a combination thereof. Non-limiting examples of mechanical circulatory support that may be used in the present invention include intra-aortic balloon counterpulsation, a left ventricular assist device, or a combination thereof.

[0316] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this invention and the figures and tables are hereby incorporated by reference. Example 1

[0317] material and method Standardized procedures for ASO operations Each ASO (IDT) was reconstituted in a tube with RNase-free water.

[0318] Aliquots of 20-50 ul per tube were prepared to limit freeze-thaw cycles.

[0319] Cell seeding and transfection conditions Cells were seeded into 12-well and 6-well plates for total RNA and protein isolation, respectively. Cells were seeded to achieve 40-50% confluency prior to transfection. Lipofectamine 3000 (at a concentration of 0.2%) was used to transfect HEK293T cells, while LipoRNAiMAX (at a concentration of 0.35%) was used to transfect IHCFs. Transfection of 50 nM ASO was carried out for 24 hours.

[0320] Loading for Western Blotting A gradient curve was generated on the left side of the gel with loading control samples at 1x, 0.5x, and 0.25x concentrations. Experimental samples were loaded according to the gradient curve.

[0321] quantitative The intensity of the gradient curve bands was quantified using ImageJ. A calibration curve was plotted and an equation was derived. ImageJ was used to quantitate the intensity of experimental sample bands. The arbitrary values ​​were obtained from the calibration curve using the formula: Sample values ​​were normalized by loading controls (alpha-tubulin, beta-actin, or GAPDH).

[0322] Detailed procedures for cell culture and transfection Human HEK293T cells were propagated in Dulbecco's modified Eagle's medium (DMEM; Gibco), and AC16 cells were propagated in an equal mix of F12 and DMEM media (Gibco). Both media were supplemented with 10% fetal bovine serum. For ASO studies, 5 × 10 HEK293T or AC16 cells were cultured at 10 °C. 5 Cells were seeded in 10 cm dishes. After overnight attachment, the culture medium was replaced with OPTI-MEM (Gibco) and transfected with 50 nM ASO using RNAiMAX (Thermo Fisher Scientific) for 6 hours according to the manufacturer's guidelines. The medium was then replaced with regular culture medium. Cells were harvested 18 hours after transfection.

[0323] Western blotting Cells were lysed in RIPA buffer (Thermo Fisher Scientific), and total cellular proteins were separated in 6%–15% denaturing polyacrylamide gels, transferred to polyvinylidene difluoride membranes (PVDF; Amersham Biosciences), and probed with antibodies recognizing GATA4 (Santa Cruz), β-actin (Thermo Fisher Scientific), and DDX3X (Sigma), followed by incubation with mouse or rabbit secondary antibodies conjugated with horseradish peroxidase (GE Biosciences). Blots were quantified using ImageJ (NIH).

[0324] Dual-luciferase assay Untreated HEK293T cells were plated in a 96-well plate at 1 x 10 cells per well. 4 Cells were seeded at a density of 1000 and allowed to attach overnight. Cells were then transfected with 50 ng each of the 5'UTR-FLuc reporter plasmid and a control Renilla luciferase (RLuc) plasmid using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's guidelines. After 24 hours, cells were incubated with Dual-Glo luciferase substrate (Promega) according to the manufacturer's recommendations. Final Fluc readings were then normalized to Rluc to obtain relative luminescence readings.

[0325] RNA purification and RT-qPCR For ASO-transfected cells, the medium was aspirated from the adherent cells and then washed twice with cold PBS. Cells were then lysed by adding 1000 μl of Trizol (Qiagen) directly to the cells, which were then mixed with 200 μl of chloroform in a 1.5 ml tube and left on ice for 5 minutes. The mixture was then spun down at 16,000 g for 10 minutes. RNA was precipitated from the aqueous layer by adding 2 volumes of isopropanol and spinning down at 16,000 g for 10 minutes. The pellet was then washed twice with 70% ethanol, dried, and resuspended in nuclease-free water. For quantification of mRNA levels, cDNA was prepared using an iScript master mix RT kit (Biorad) and subsequently amplified by qPCR using a SYBR primer assay kit (Biorad). It is noteworthy that when a primer set was first used, the identity of the resulting PCR product was confirmed by cloning and sequencing. The quantitative nature of each primer was also assessed by generating a standard curve with varying amounts of cDNA. Once confirmed, melting curves were used in each subsequent PCR to verify that each primer set reproducibly and specifically yielded the same PCR product.

[0326] Polysome profiling and RNA extraction After transfection with ASO, cells were incubated with cycloheximide (100 μg / ml; Sigma) for 10 minutes and then harvested using a native lysis buffer containing 100 mM KCl, 5 mM MgCl, 10 mM HEPES, pH 7.0, 0.5% Nonidet P-40, 1 mM DTT, 100 U / ml RNasin RNase inhibitor (Promega), 2 mM vanadyl ribonucleoside complex solution (Sigma-Aldrich (Fluka BioChemika)), 25 μl / ml protease inhibitor cocktail for mammalian tissues (Sigma-Aldrich), and cycloheximide (100 μg / ml). The lysate was then spun down at 1500 g for 5 minutes to pellet the nuclei. The supernatant was then loaded onto a 10-50% sucrose gradient and spun at 150,000 g for 2 hours and 20 minutes in an ultracentrifuge. The gradient was then transferred to a splitter coupled to a UV absorbance detector, which output an electrical waveform across the gradient. Using a 60% sucrose chaser, the gradient was then pumped into the splitter and divided into 12 equal fractions. RNA was extracted by mixing 500 μl of each fraction with an equal volume of chloroform:phenol:chloroform:isoamyl alcohol (25:24:1) and 0.1 volume of 3 M sodium acetate (pH 5.2), then spinning down at 16,000 g for 10 minutes. This extraction process was then repeated using the upper aqueous layer. The final upper aqueous layer was then mixed with 2 volumes of 100% ethanol and incubated overnight at -20°C. The solution is then spun at top speed for 30 minutes to pellet the RNA, which is then washed twice with 70% ethanol and finally resuspended in nuclease-free water.

[0327] Immunofluorescence WGA (5 mg) was dissolved in 5 ml of PBS (pH 7.4). We performed deparaffinization using the following steps: (i) two 5-minute cycles of xylene (100%); (ii) two 5-minute cycles of ethanol (100%); (iii) one 5-minute cycle of ethanol (95%); and (iv) two 5-minute cycles of ddH2O. For antigen retrieval, the slides were placed in a pressure cooker with citrate buffer (10 mM, pH 6.0) for 10 minutes. We quenched the slides with 0.1 M glycine in phosphate buffer (pH 7.4) at room temperature for 1 hour. A circle was drawn with a Dako pen, and the slides were blocked with normal goat serum for 30 minutes. 10 μg / ml WGA-Alexa Fluor 488 (Sigma-Aldrich) was applied to the slides for 1 hour of incubation at room temperature. Slides were rinsed three times for 5 minutes each in PBS. For imaging, coverslips were placed on the slides with VECTASHIELD HardSet antifade mounting medium with DAPI (Vectorlabs). For AC16 cardiomyocyte cultures and ESC-derived cardiomyocyte cultures, cells were fixed for 10 minutes using 4% paraformaldehyde in PBS, washed with PBS, and permeabilized for 10 minutes using 0.2% Triton X-100. Cells were blocked for 1 hour in 2% BSA / PBS, stained with the appropriate primary antibody for 1 hour at room temperature, and then incubated with a fluorescently labeled secondary antibody. Stained cells were gently washed three times for 5 minutes each in PBS and mounted on slides using mounting medium with DAPI. When measuring cell size, which is the surface area within a myocardial section or AC16 slide, five different fields were selected and the cell size of at least 200 cardiomyocytes was measured using Image J. For ESC-derived cardiomyocytes, as with AC16 cells, it was found to be difficult to measure the cell surface area of ​​these cells individually.Therefore, we used Image J to measure the total surface area of ​​all colonies for at least five colonies per sample, and then divided these by the number of nuclei.

[0328] statistics All quantitative data are presented as mean ± SD and were analyzed using Prism 8.3.0 software (GraphPad). For comparisons between two groups, an unpaired two-tailed Student's t-test was performed for normally distributed data. For mouse echocardiographic studies, statistical significance between groups was determined using ANOVA followed by a Holm-Sidak post-hoc test. A two-tailed P value of <0.05 was considered to indicate statistical significance. Specific statistical methods were described in the figure captions.

[0329] Sequences of ASOs used in this study (5'-3'; "m" indicates 2'-O-methyl modification, "e" indicates 2'-O-methoxyethyl modification, "+" indicates LNA, "s" indicates phosphorothioate, "o" indicates phosphodiester internucleoside linkage) are shown. GATA4 human ASO control: used as a control in humans and mice; underlined: mismatch compared to GATA4 ASO1.

[0330] All ASOs were synthesized at 100 nmol and purified using a desalting column at IDT, Inc. Analytical ESI-MS confirmed the purity and quality of the ASOs. All ASOs were synthesized at 100 nmol and purified using a desalting column at IDT, Inc. Analytical ESI-MS confirmed the purity and quality of the ASOs.

[0331] FIG. 10 shows the following: human GATA4 mRNA sequence (SEQ ID NO: 22), mouse GATA4 mRNA sequence (SEQ ID NO: 23), human MEF2C mRNA sequence (SEQ ID NO: 24), human NKX2-5 mRNA sequence (SEQ ID NO: 25), and human eIF4G2 mRNA sequence (SEQ ID NO: 26), GATA4 human type I uotASO target sequence (SEQ ID NO: 27), GATA4 human type II motASO target sequence (SEQ ID NO: 28), MEF2C human type II motASO target sequence (SEQ ID NO: 29), NKX2-5 human type II motASO target sequence (SEQ ID NO: 30), eIF4G2 human type II uotASO target sequence (SEQ ID NO: 31), human, gorilla, and monkey GATA4 uORF DNA sequence (SEQ ID NO: 32), cat and dolphin GATA4 uORF DNA sequence (SEQ ID NO: 33), golden hamster GATA4 uORF DNA sequence (SEQ ID NO: 34), rat GATA4 uORF DNA sequence (SEQ ID NO: 35), mouse GATA4 The uORF DNA sequence (SEQ ID NO: 36), wild-type human GATA4 stem-loop region (SEQ ID NO: 37), ΔuORF human GATA4 stem-loop region (SEQ ID NO: 38), mutant human GATA4 stem-loop region (SEQ ID NO: 39), rescue mutant human GATA4 stem-loop region (SEQ ID NO: 40), EIF4G2-ASO1 gapmer (SEQ ID NO: 41), unmodified EIF4G2-ASO1 (SEQ ID NO: 42), human MYBPC3 mRNA (SEQ ID NO: 43), human CRYAB mRNA (SEQ ID NO: 44), MYBPC3 ASO (SEQ ID NO: 44), and CRYAB ASO (SEQ ID NO: 46) are listed. Example 2

[0332] Downstream dsRNA structures adjacent to uORFs inhibit translation of mORFs Double-stranded RNA (dsRNA) structures embedded within the 5'UTR have been reported to either inhibit or activate translation, depending on their positional and structural features. Additionally, upstream open reading frames (uORFs) are known to inhibit the translation of major open reading frames (mORFs). To explore the potential interaction between such dsRNA structures and uORFs, we constructed an artificial luciferase reporter containing both dsRNA and uORF elements for dual-luciferase reporter assays (Figure 2, panel A).

[0333] Specifically, a series of 5'UTR-firefly luciferase (FLuc) reporter fusions were generated from a 5'UTR containing a CA repeat backbone with a stable hairpin, Kan-HP1, 40 nucleotides from the 5' end and 20 nucleotides from the FLuc ORF start codon. The 5'UTRs were synthesized as oligonucleotides (both positive and negative strands) by IDT and then cloned into the FLuc construct corresponding to the mORF. The 5'UTR backbone contained a CA repeat (i.e., [CA]xn), a known linear sequence. A hairpin was added to this backbone 40 nucleotides from the 5' end and 20 nucleotides from the firefly mORF coding sequence. The hairpin was derived from a Disney template to create a uORF if it contains a G at the beginning and preceded by an AU. The AUG was then shifted back three nucleotides for every reporter, up to a maximum of 27. This backbone was then mutagenized by inserting start codons (i.e., ATG) at various positions spaced 2 to 23 nucleotides apart relative to the stem base (Figure 2, panel B). Dual-Luc assays showed that start codons at positions -2 to -5 conferred the most robust inhibition of luciferase activity, while start codons at position -8 conferred weaker inhibition (Figure 2, panel C). Start codons at positions -8 to -23 conferred no detectable inhibition.

[0334] To elucidate the role of hairpin stability on uORF activity, mismatches were introduced into the hairpin of the parental construct (containing no AUG) and the construct containing an initiation codon at position -2 (AUG -2) (Figure 2, panel D, left). Mutations in the parental sequence did not confer suppression of luciferase activity, whereas mutations in the AUG -2 mutant showed rescue of luciferase activity, suggesting that RNA structural stability is required for the AUG codon to confer suppression. Compared with the parental AUG -2 construct, the mutagenized AUG -2 mutant exhibited enhanced luciferase activity and protein levels (Figure 2, panel D, right). Enhanced luciferase activity was also observed from the parental construct in which the AUG was deleted. Together, these results support a functional link between the upstream ATG or uORF, RNA structural stability, and translation initiation of mORFs. In particular, we found that a dsRNA stem-loop RNA structure placed proximal (2-11 nucleotides away) downstream of a uORF enhanced uORF activity and reduced mORF translation. These results support the finding that a dsRNA stem-loop structure immediately downstream of a uORF can enhance uORF activity and suppress mORF translation.

[0335] To explore the mechanism underlying the dsRNA-mediated shift from uORF to mORF translation, we performed in vitro transcription of a series of mRNAs with 5' UTRs based on the constructs used in Figure 2, panel B, and then incubated them with rabbit reticulocyte lysate (RRL) (FIA). The lysates were then fractionated on a 10-35% sucrose gradient by ultracentrifugation. The hairpin-bearing 5' UTR of the AUG-free reporter with RRL resulted in co-sedimentation of the 5' UTR with the 40S ribosomal subunit (Figure 3, panel B, red), which was not observed with the control 5' UTR lacking the hairpin and start codon, suggesting a hairpin-specific co-sedimentation effect (Figure 3, panel B, cyan). In contrast, coupling of the adjacent hairpin downstream of the start codon resulted in a shift in the profile from the 40S peak to assembled 80S monosomes (Figure 3, panel B, green) to a greater extent than the start codon alone (Figure 3, panel B, yellow), suggesting enhanced translation initiation by the hairpin structure downstream of the start codon. Collectively, these results indicate that the presence of a hairpin downstream of a uORF start codon enhances the repressive ability of the uORF on mORF translation, and this synergistic effect of the start codon and hairpin dsRNA is abolished when the hairpin stem is destabilized. Example 3

[0336] Presence of uORFs in human cardiac transcription factor mRNAs To further explore the role of dsRNA stem-loop RNA structures and uORFs in repressing mORF translation, we conducted a study to identify naturally occurring mRNA transcripts containing one or more uORFs within or surrounding these structural elements of dsRNA. This was performed by data mining against an unbiased high-throughput ribosome profiling (Ribo-seq) database. Overlapping Ribo-seq hits revealed a conserved cohort of mRNAs containing uORF translation in mouse and human (Figure 4, Panel A, left, center). Gene ontology analysis of the overlapping genes revealed transcription factors including GATA4, GATA6, TBX5, TBX20, MYOCD, and NKX2-5 as a highly enriched set of genes containing translatable uORFs (Figure 4, Panel A, right).

[0337] Among these six transcription factors, GATA4 was of particular interest because its mRNA contains a single uORF that exhibits ribosomal footprints in human hearts by Ribo-seq analysis (Figure 4, panel A, right). The GATA4 uORF is conserved across diverse mammals and contains an 11-nucleotide sequence downstream of the uORF start codon that is highly conserved throughout evolution. However, the fact that the uORF protein sequence is not conserved suggests that the GATA4 uORF is likely a regulatory element rather than a bioactive peptide.

[0338] GATA4 is a key transcription factor required for cardiomyocyte proliferation and hypertrophy. Prediction of RNA structure using the TurboFold tool suggested the presence of a 10-base pair (bp) stem immediately downstream of the uORF start codon, as shown in the example of the predicted structure of the 5'UTR (Figure 4, panel C). Using the SHAPE (Selective 2' Hydroxyl Acylation Analyzed by Primer Extension) assay, we confirmed the presence of a double-stranded secondary stem structure within the 5'UTR of GATA4 mRNA. Briefly, nucleotides located within the double-stranded stem structure tend to be less modified by the electrophile N-methylisatoic anhydride (NAI), whereas single-stranded regions are exposed to more extensive modification. Confirmation of the double-stranded RNA structure was obtained by showing that the predicted 10-bp stem-loop downstream of the AUG start codon exhibited the lowest SHAPE activity (data not shown). This result was further supported by experiments using towprinting assays showing stalling of the 40S ribosomal subunit at the hairpin dsRNA region (data not shown). Example 4

[0339] GATA4-targeting ASOs regulate the translation efficiency of GATA4 mORF in cells The GATA4 5'UTR mutant studies provided an impetus to explore the potential therapeutic effects of using 5'UTR-directed agonists or antagonists to modify GATA4 expression in a therapeutic context. Such studies are based on the perturbative activity of the GATA4 uORF and GATA4 mORF relative to each other. In this regard, two hypotheses were examined: 1) disruption of the dsRNA structure results in inactivation of the uORF and increased Luc activity; and 2) blockade of the uORF results in increased translation and decreased Luc activity. The first hypothesis was tested by preventing blockade of the uORF-containing strand by the upstream strand through the design of a uORF-inhibitory 16-mer ASO (human ASO1, SEQ ID NO: 8) that mimics disruption of the upstream strand (Figure 5, panel A, left). The second hypothesis was tested by designing a uORF-enhancing ASO (human ASO2, sequence number 3) that could tightly sequester the uORF through complementary binding, thereby forming a stable 16-bp double-stranded stem (Figure 5, panel B, left).

[0340] In the dual-Luc assay, ASO1 increased Luc activity, suggesting inhibition of uORF translation, whereas ASO2 decreased Luc activity, suggesting activation of uORF translation (Figure 5, panel A, right). These effects were uORF-dependent, as indicated by the fact that ΔuORF reporter activity was unchanged by either ASO (Figure 5, panel B, right).

[0341] Targeting endogenous GATA4 mRNA in AC16 human cardiomyocytes with these ASOs resulted in observable changes in protein levels (Figure 5, panel C). The uORF-inhibitory ASO1 increased GATA4 protein levels, whereas the uORF-enhancing ASO2 reduced them. To further confirm uORF-mediated translational regulation of mORFs, polysome profiling was performed. The results of this analysis showed that the global polysome profile remained unchanged (Figure 5, panel D). Subsequent RT-qPCR analysis confirmed that treatment with ASO1 shifted FLuc mRNA carrying the wild-type 5'UTR to a highly translated fraction, whereas treatment with ASO2 resulted in a low-translatable fraction (Figure 5, panel E). Given that no significant changes in mRNA levels were observed (data not shown), it was concluded that ASO1 and ASO2 differentially affected the translation efficiency of their target mRNAs. Thus, when transfected into AC16 cells, ASO1 caused cardiomyocyte (CM) hypertrophy, whereas ASO2 caused cardiomyocyte atrophy (Fig. 5, panel F). Example 5

[0342] Optimizing and exploring the utility of type II ASO-mediated biomolecular helix formation in a wide range of applications As shown in Figure 6, panel A, type II uotASOs targeting the uORF of eIF4G2 mRNA resulted in reduced translation of the eIF4G2 mORF, resulting in reduced amounts of eIF4G2 protein as detected by Western blot. Figure 6, panel B shows that type II motASOs targeting the mORF of GATA4 mRNA induced enhanced production of GATA4 protein. Specifically, 2'-O-methyl-modified type II motASOs significantly enhanced GATA4 protein levels to 21.2 ± 2.0% of the control ASO group (Figure 6, panel A). The ASOs did not alter mRNA expression (Figure 6, panel B). In contrast, the combination of four LNA nucleotides at the 3' end of the 2'-O-methyl ASO resulted in a more potent mORF enhancement effect without affecting mRNA expression levels (Figure 6, panel C). Similar mORF-enhancing effects were also observed with type II motASOs targeting mORFs within MEF2C and NKX2-5 mRNAs.

[0343] In summary, the ASOs of the present invention can reduce harmful proteins and increase beneficial proteins. In addition, ASOs can increase protein levels in a manner simpler than viral delivery (data not shown). There is a long-standing need for overexpression of therapeutic proteins to treat diseases caused by gene haploinsufficiency or pathological depletion. Alternatively, switching cell identity is a promising approach to improving organ function and reversing disease progression, such as transdifferentiation of cardiac fibroblasts into cardiomyocytes, driven by overexpressing a cocktail of TFs including GATA4, MEF2C, TBX5, and NKX2-5. This application presents "proof-of-concept" evidence supporting the idea of ​​using type II motASOs to increase GATA4, MEF2C, and NKX2-5 protein levels by approximately 1.5- to 3-fold. Another situation in which Type II motASOs may be useful is when the mRNA desired for overexpression is too large for viral delivery methods (e.g., titin).

[0344] Switching cellular identity, such as the transdifferentiation of cardiac fibroblasts (CFs) into cardiomyocytes (CMs) driven by overexpressing a cocktail of transcription factors including GATA4, MEF2C, TBX5, and NKX2-5, is a promising approach to improving organ function and reversing disease progression. Therefore, enhancing the protein expression of GATA4, MEF2C, or NKX2-5 by type II motASOs would likely impair myocardial fibrosis as a result of the transition from cardiac fibroblasts to cardiomyocytes. On the other hand, eIF4G2 promotes the translation of profibrotic extracellular matrix proteins, significantly contributing to myocardial fibrosis (unpublished results from our laboratory). Therefore, type II uotASOs, which promote the activity of uORFs and inhibit the translation of eIF4G2 mORFs, would reduce myocardial fibrosis. Example 6

[0345] Type II motASOs against CRYAB and MYBPC3 increase protein but not mRNA expression We developed a platform that allows users to design antisense oligonucleotides (ASOs) (type II motASOs) that bind to specific regions within the major open reading frame (mORF) of mRNA and selectively increase mRNA translation and protein synthesis (Figure 7A). This study selected two mRNA targets (MYBPC3 and CRYAB) to focus on. MYBPC3 and CRYAB are myofilament and heat shock proteins, respectively, required for normal cardiomyocyte contractile function. The proteins encoded by these two target mRNAs are known to protect the heart from myocardial fibrosis when overexpressed in cardiomyocytes (Figure 7B).

[0346] MYBPC3 (myosin-binding protein C3) encodes the cardiac isoform of myosin-binding protein C, a myosin-associated protein found within the cross-bridge-bearing zone (C) of the A-band in striated muscle. MYBPC3 is expressed exclusively in cardiac muscle and is a key regulator of cardiac contraction. Heterozygous mutations in this gene are a frequent cause of familial hypertrophic cardiomyopathy caused by haploinsufficiency.

[0347] CRYAB (crystallin alpha B): Mammalian lens crystallins are divided into the alpha, beta, and gamma families. Alpha crystallins are composed of two gene products, alpha A and alpha B, which correspond to acidic and basic products, respectively. Alpha crystallins are induced by heat shock and are members of the small heat shock protein (HSP20) family. Alpha crystallins act as molecular chaperones by retaining proteins in large soluble aggregates rather than renaturing and releasing them in a bona fide chaperone manner. These heterogeneous aggregates consist of 30-40 subunits, with a 3:1 ratio of alpha A to alpha B subunits, respectively. Two additional functions of alpha crystallins are autokinase activity and participation in intracellular architecture. The encoded proteins have been identified as accessory functional proteins based on their ability to perform mechanistically distinct functions. The gene products, alpha A and alpha B, are differentially expressed; alpha A is preferentially restricted to the lens, while alpha B is widely expressed in many tissues and organs. Elevated expression of alpha B crystallin occurs in many neurological disorders; in one family, a missense mutation cosegregated with desmin-related myopathy. Alternative splicing results in multiple transcript variants.

[0348] As described herein, studies designed, generated, and assessed ASOs with the potential to selectively increase protein synthesis of these two targets (Figure 7B) (a 16-nucleotide MYBPC3 ASO for activation of mRNA translation using a type II motASO targeting the mORF: CmoUmoUmoCmoCmoCmoGmoGmoCmoUmoCmoAmoGmoGmoCm (SEQ ID NO: 45); a 16-nucleotide CRYAB ASO for activation of mRNA translation using a type II motASO targeting the mORF: GmoUmoGmoAmoUmoGmoGmoCmoGmoAmoUmoGmoUmoCmoCm (SEQ ID NO: 46)).

[0349] Following bioinformatics analysis of mRNA sequences and structures, we designed multiple candidate ASOs targeting double-stranded RNA (dsRNA) near the upstream open reading frame (uORF) and the major ORF (mORF) for in vitro testing. ASOs were designed and manufactured based on predicted mRNA 5'UTR structures or mRNA 5'UTR sequence features. The efficacy of candidate ASOs in manipulating protein expression of two targets in relevant cell lines in vitro was determined. Target gene mRNA and protein expression served as readouts for regulation. The translational activation effects of candidate ASOs were verified in AC16 human cardiomyocytes (Sigma cell line SCC109) using Western blotting (measuring steady-state protein levels) and RT-qPCR (measuring steady-state mRNA levels) for CRYAB and MYBPC3 (cardiomyocyte protection).

[0350] The study identified two 16-nucleotide ASOs (with 2'-O-methyl modifications) that target MYBPC3 and CRYAB mRNAs for translational activation. Data suggested that the two type II motASOs could dose-dependently increase MYBPC3 and CRYAB protein expression in the AC16 human cardiomyocyte cell line without affecting mRNA expression (Figure 8, panels A-C). Example 7

[0351] 5'-UTR-targeting gapmer ASO reduces eIFG4G2 protein expression in immortalized human cardiac fibroblasts eIF4G2, a translation factor essential for the synthesis of extracellular matrix proteins, was discovered as a potential anti-fibrotic target gene in cardiac fibroblasts. A 20-nucleotide-long ASO was designed to target an evolutionarily conserved region (in humans and mice) that overlaps with the upstream open reading frame (uORF) and combines features of a gapmer formulation (10 DNA nucleic acids + 5 RNA nucleic acids with phosphorothioate linkages in the center of the ASO) (Figure 8, Panels A and B; 5'-UTR-targeting gapmer ASO: GesCesCesAesCesCdsTdsCdsCdsAdsTdsAdsGdsAdsGdsCesUesCesCesGe (SEQ ID NO: 41) [where e: MOE modification; s: phosphorothioate; d: DNA; unmodified sequence: GCCACCTCCATAGAGCUCCG (SEQ ID NO: 42) = target 5'UTR of both human and mouse eIF4G2]).

[0352] Following bioinformatics analysis of mRNA sequences and structures, we designed multiple candidate ASOs targeting double-stranded RNA (dsRNA) near the upstream open reading frame (uORF) and the major ORF (mORF) for in vitro testing. ASOs were designed and manufactured based on predicted mRNA 5'UTR structures or mRNA 5'UTR sequence features. The efficacy of candidate ASOs in manipulating protein expression of two targets in relevant cell lines in vitro was determined. Target gene mRNA and protein expression served as readouts for regulation. The translational activation effects of the candidate ASOs were verified in immortalized human cardiac fibroblasts (IHCF; abm#T0446):eIF4G2 (anti-fibrotic effect) using Western blotting (measuring steady-state protein levels) and RT-qPCR (measuring steady-state mRNA levels).

[0353] Those skilled in the art will recognize that the wings and gaps discussed above can be selected and then combined in various combinations to create oligomeric compounds with gap regions, including, but not limited to, antisense oligomeric compounds with gap regions and antisense oligonucleotides with gap regions. The characteristics (length, modification, linkage) of the 5' and 3' wings can be selected independently of each other. The gap characteristics include at least one difference in modification compared to the 5' wing characteristics and at least one difference compared to the 3' wing characteristics (i.e., there must be at least one difference in modification between the adjacent regions to distinguish these adjacent regions from each other). Otherwise, the gap characteristics can be selected independently.

[0354] This ASO could significantly reduce EIF4G2 mRNA and protein expression through RNase H-mediated mRNA degradation (Figure 9, panels AC).

[0355] Taken together, the results of Examples 6 and 7 herein demonstrate that type II motASOs and 5'UTR-targeting gapmer ASOs can efficiently activate mRNA translation and silence protein expression, respectively. Using these two types of ASOs, users can enhance the translation of antifibrotic mRNAs or inhibit the expression of profibrotic proteins, thereby achieving potential antifibrotic effects in vitro or in vivo.

[0356] [Table 3] TIFF2025530741000015.tif30170TIFF2025530741000016.tif228170TIFF2025530741000017.tif46170TIFF2025530741000018.tif218170TIFF2025530741000019.tif53170TIFF2025530741000020.tif210170TIFF2025530741000021.tif37170TIFF2025530741000022.tif226170TIFF2025530741000023.tif18170TIFF2025530741000024.tif235170TIFF2025530741000025.tif255170TIFF2025530741000026.tif217170TIFF2025530741000027.tif46170TIFF2025530741000028.tif194170TIFF2025530741000029.tif69170TIFF2025530741000030.tif164170TIFF2025530741000031.tif100170TIFF2025530741000032.tif146170TIFF2025530741000033.tif118170TIFF2025530741000034.tif113170TIFF2025530741000035.tif149170TIFF2025530741000036.tif117170TIFF2025530741000037.tif146170TIFF2025530741000038.tif91170TIFF2025530741000039.tif167170TIFF2025530741000040.tif73170TIFF2025530741000041.tif184170TIFF2025530741000042.tif55170TIFF2025530741000043.tif203170TIFF2025530741000044.tif213170

[0357] "m" indicates a 2'-O-methyl modification, "e" indicates a 2'-O-methoxyethyl modification, "+" indicates LNA, "s" indicates phosphorothioate, "o" indicates a phosphodiester internucleoside linkage, and "d" indicates DNA.

[0358] This specification incorporates 1134-119 PCT.xml, a sequence listing file created on August 18, 2023, and having a size of 10,400 bytes.

[0359] While various embodiments have been described above, it should be understood that such disclosure has been presented by way of example only, and is 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.

[0360] The above description is intended to teach those skilled in the art how to carry out the present invention, and is not intended to detail all obvious modifications and variations of the present invention that will become apparent to those skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of the present invention, as defined by the claims that follow. The claims are intended to cover any sequence of elements and steps that are effective to achieve the goals intended in the claims, unless the context specifically dictates to the contrary.

Claims

1. An antisense oligonucleotide comprising 8 to 50 nucleotides, the antisense oligonucleotide is capable of binding to and forming a double-stranded structure with a target sequence in the mRNA of the antifibrotic gene; the target sequence is located within the non-coding strand of a double-stranded stem structure downstream of and adjacent to the uORF start codon in the mRNA; When the antisense oligonucleotide binds to the target sequence, the double-stranded stem structure of the uORF is disrupted, enhancing translation of the mORF into mRNA. Antisense oligonucleotides.

2. The antisense oligonucleotide of claim 1 , comprising one or more modified nucleotides.

3. The antisense oligonucleotide of claim 2 , wherein the modified nucleotide comprises one or more modified sugar moieties and / or one or more modified internucleoside linkages.

4. The antisense oligonucleotide of any one of claims 1 to 3, wherein the anti-fibrotic gene is selected from the group consisting of GATA4, MEF2C, NKX2-5, TBX5, HNF4α, CRYAB, TCF21, and MYBPC3.

5. The antisense oligonucleotide according to claim 4, wherein the myocardial fibrosis-related gene is GATA4.

6. The antisense oligonucleotide of claim 5 , wherein the target sequence comprises the nucleotide sequence of SEQ ID NO:

27.

7. The antisense oligonucleotide of claim 1 , which is RNA.

8. The antisense oligonucleotide of claim 1 comprising SEQ ID NO:

8.

9. an antisense oligonucleotide comprising 8 to 50 nucleotides, the antisense oligonucleotide is capable of binding to and forming a double-stranded structure with a target sequence in the mRNA of the antifibrotic gene; the target sequence is located downstream of and adjacent to the mORF start codon in the mRNA; When the antisense oligonucleotide binds to the target sequence, translation from the mORF start codon is enhanced. Antisense oligonucleotides.

10. The antisense oligonucleotide of claim 9, comprising one or more modified nucleotides.

11. The antisense oligonucleotide of claim 9 , wherein the modified nucleotide comprises one or more modified sugar moieties and / or one or more modified internucleoside linkages.

12. The antisense oligonucleotide of any one of claims 9 to 11, wherein the anti-fibrotic gene is selected from the group consisting of GATA4, MEF2C, NKX2-5, TBX5, HNF4α, CRYAB, TCF21, and MYBPC3.

13. The antisense oligonucleotide of claim 12, wherein the target sequence comprises SEQ ID NO:

28.

14. The antisense oligonucleotide of claim 12, wherein the target sequence comprises SEQ ID NO:

29.

15. The antisense oligonucleotide of claim 12, wherein the target sequence comprises SEQ ID NO:

30.

16. The antisense oligonucleotide of claim 12, wherein the target sequence comprises SEQ ID NO:

47.

17. The antisense oligonucleotide of claim 12, wherein the target sequence comprises SEQ ID NO:

48.

18. 18. The antisense oligonucleotide of any one of claims 9 to 17, which is RNA.

19. The antisense oligonucleotide of claim 12 comprising SEQ ID NO: 9 or 10.

20. The antisense oligonucleotide of claim 12, comprising SEQ ID NO:

15.

21. The antisense oligonucleotide of claim 12, comprising SEQ ID NO:

21.

22. The antisense oligonucleotide of claim 12, comprising SEQ ID NO:

45.

23. The antisense oligonucleotide of claim 12, comprising SEQ ID NO:

46.

24. An antisense oligonucleotide comprising 8 to 50 nucleotides, the antisense oligonucleotide is capable of binding to and forming a double-stranded structure with a target sequence within the mRNA of the pro-fibrotic gene; the target sequence is located downstream of and adjacent to the uORF start codon in the mRNA; When the antisense oligonucleotide binds to the target sequence, translation from the mORF start codon is reduced. Antisense oligonucleotides.

25. 25. The antisense oligonucleotide of claim 24, comprising one or more modified nucleotides.

26. 26. The antisense oligonucleotide of claim 25, wherein the modified nucleotide comprises one or more modified sugar moieties and / or one or more modified internucleoside linkages.

27. 27. The antisense oligonucleotide of any one of claims 24 to 26, wherein the pro-fibrotic gene is selected from the group consisting of eIF4G2, EPRS, and MEOX1.

28. 28. The antisense oligonucleotide of claim 27, wherein the target sequence comprises SEQ ID NO:

31.

29. 29. The antisense oligonucleotide of any one of claims 24 to 28, which is RNA.

30. 25. The antisense oligonucleotide of claim 24, comprising SEQ ID NO:

17.

31. An antisense oligonucleotide comprising 8 to 50 nucleotides, the antisense oligonucleotide is a gapmer capable of binding to and forming a double-stranded structure with a target sequence in the mRNA of the pro-fibrotic gene; the target sequence is located within a region spanning 55 nucleotides upstream to 55 nucleotides downstream of the uORF start codon in the mRNA; When the antisense oligonucleotide binds to the target sequence, translation from the mORF start codon is reduced, resulting in degradation of the target mRNA. Antisense oligonucleotides.

32. 32. The antisense oligonucleotide of claim 31, comprising one or more modified nucleotides.

33. 33. The antisense oligonucleotide of claim 32, wherein the modified nucleotide comprises one or more modified sugar moieties and / or one or more modified internucleoside linkages.

34. 34. The antisense oligonucleotide of any one of claims 31 to 33, wherein the pro-fibrotic gene is selected from the group consisting of eIF4G2, EPRS, and MEOX1.

35. 35. The antisense oligonucleotide of claim 34, wherein the target sequence comprises SEQ ID NO:

49.

36. 36. The antisense oligonucleotide of claim 35, comprising SEQ ID NO:

41.

37. 37. The antisense oligonucleotide of any one of claims 1 to 36; and a pharmaceutically acceptable carrier.

10. A pharmaceutical composition for antifibrotic therapy comprising:

38. A method for treating myocardial fibrosis, comprising administering an effective amount of an antisense oligonucleotide described in any one of claims 1 to 36 or an effective amount of a pharmaceutical composition described in claim 37 to a subject in need thereof.

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