WO2025080939A1 - Compositions and methods for treating conditions associated with cartilage oligomeric matrix protein (comp) mutations - Google Patents

Abstract

This application provides antisense nucleic acid compounds that diminish COMP mRNA levels and/or COMP polypeptide levels in chondrocytes, thereby reducing accumulation of mutant COMP and restoring chondrocyte health. The application further provides compositions comprising antisense nucleic acid compounds and their use in methods of preventing or treating conditions associated with COMP accumulation in chondrocytes of a subject.

Description

COMPOSITIONS AND METHODS FOR TREATING CONDITIONS ASSOCIATED WITH

CARTILAGE OLIGOMERIC MATRIX PROTEIN (COMP) MUTATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/590.222, filed 13 October 2023, the entire disclosure of which is hereby incorporated herein by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING XML

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. Said XML file, created 08 October 2024, is named UXN045-01WO_SL, and is 10,176,132 bytes in size.

TECHNICAL FIELD OF THE DISCLOSURE

[0003] This disclosure relates to antisense nucleic acid compounds for inhibiting expression of cartilage oligomeric matrix protein (COMP) in chondrocytes and compositions comprising the same for use in the treatment of conditions associated with COMP accumulation arising from COMP mutations such as in pseudoachondroplasia and multiple epiphyseal dysplasia.

BACKGROUND OF THE INVENTION

[0004] Cartilage oligomeric matrix protein (COMP), encoded by NCBI Reference Sequence: NM_000095.3, is a homopentameric non-collagenous extracellular matrix (ECM) protein consisting of five identical glycoprotein subunits, each with EGF-like and calcium-binding (thrombospondin-like) domains. The protein is expressed in cartilage, ligament, and tendon.

[0005] Pseudoachondroplasia (PSACH), an autosomal dominant skeletal disorder, and other skeletal dysplasias can be caused by mutations in the COMP gene. For instance, contraction or expansion of a 5 amino acid aspartate repeat and other mutations in the COMP gene can cause skeletal dysplasias including PSACH and multiple epiphyseal dysplasia (MED). (See Briggs MD, et al. Pseudoachondroplasia and multiple epiphyseal dysplasia due to mutations in the cartilage oligomeric matrix protein gene. Nat Genet 10 (3), 330-336 (1995) PMID 7670472.) Mutations in the COMP gene may interfere with calcium binding and/or disturb protein folding and trafficking to the ECM. Consequently, mutant COMP protein accumulates in the endoplasmic reticulum, leading to massive intracellular retention and concomitant ER stress, inflammation, and oxidative stress. Affected chondrocytes exhibit cytotoxicity and premature death, constraining long-bone growth. (See Posey, K.L. and Hecht, J.T., Bone. 102:60-68, 2017.)

[0006] Individuals with PSACH typically have a decreased growth rate and, subsequently, disproportionate short stature, premature joint degeneration and osteoarthritis. MED affects the epiphyses, the ends of the long bones in the arms and legs. Symptoms of MED include joint pain, early-onset arthritis, and in some cases mild short stature and/or a waddling walk. Both PSACH and MED stem from the intracellular retention and accumulation of cartilage oligomeric matrix protein in the enlarged rough endoplasmic reticulum of chondrocytes, causing cell stress and death in grow th plate and articular cartilage. COMP accumulation in chondrocytes can also reduce the stability of the ECM. causing abnormalities in the ECM and promoting erosion of the ECM during normal physical activity. Additionally, the circulating level of COMP in the serum is found to be lower in PSACH and MED patients with COMP mutations than healthy individuals. (See Mabuchi A, Momohara S, Ohashi H, Takatori Y, HagaN, Nishimura G, Ikegawa S. Circulating COMP is decreased in pseudoachondroplasia and multiple epiphyseal dysplasia patients carrying COMP mutations. Am J Med Genet A. 2004 Aug 15;129A(l):35-8. doi: 10.1002/ajmg.a.30164. PMID: 15266613.)

[0007] Reliable statistics are limited but it is estimated that PSACH affects 1 in 100,000 to 1 in 30,000 individuals, yet no approved therapy exists. Thus, there remains a high unmet need for therapeutic approaches to treating PSACH and other inherited skeletal dysplasias stemming from dysfunction of the COMP gene. The present disclosure addresses this need by providing novel nucleic acid molecules that knockdown COMP mRNA in chondrocytes (in growth plate and articular cartilage), thereby reducing accumulation of mutant COMP and restoring chondrocyte health.

BRIEF SUMMARY OF THE INVENTION

[0008] This disclosure provides compositions comprising novel nucleic acid molecules that can be used to inhibit expression of COMP mRNA in chondrocytes. The disclosure further provides methods of using these compositions for the prevention or treatment of skeletal dysplasias stemming from COMP accumulation in chondrocytes, including, e.g., PSACH and MED. More specifically, embodiments of this disclosure provide antisense nucleic acid compounds (i.e., “antisense compounds⁷’) targeting the human COMP mRNA, and methods of their use for the treatment of PSACH.

[0009] Provided herein are antisense compounds such as antisense oligonucleotides (ASOs) comprising 14 to 20 linked nucleosides having a nucleobase sequence that is complementary to a target nucleic acid sequence in SEQ ID NO: 1. wherein the target nucleic acid sequence comprises 14 to 20 contiguous nucleotides and has a start position of 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1340, 1341, 1342, 1343, or 1344 of SEQ ID NO: 1, and wherein the ASO comprises one or more of: a gap segment consisting of linked deoxynucleosides; a 5’ segment consisting of at least 2 linked nucleosides; a 3’ segment consisting of at least 2 linked nucleosides; at least one phosphorothioate intemucleoside linkage; at least one nucleoside comprising a modified sugar; and at least one nucleoside comprising a modified nucleobase.

[0010] Where a gap segment is present in an antisense compound or ASO, it may be positioned between the 5’ segment and the 3’ segment and may comprise 5 to 15 linked nucleosides.

[0011] Where present, the 3‘ segment may comprise 2-5 linked nucleosides and/or the 5’ segment may comprise 2-5 linked nucleosides. One or all of the nucleosides of the 3’ and/or the 5’ segment may comprise a modified sugar, such as a bicyclic sugar, e.g., 4’-(CH2) — O-2’ (LNA); 4’-(CH₂)₂— O-2’ (ENA); or 4’-CH(CH₃)— 0-2’ (cEt).

[0012] In some embodiments, each intemucleoside linkage of the antisense compounds provided herein is a phosphorothioate intemucleoside linkage.

[0013] In some embodiments, the antisense compounds provided herein comprise a 5- methylcytosine nucleobase in place of a non-5 -methyl cytosine residue.

[0014] In some embodiments, an ASO is provided comprising a 2-11-2 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1310, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0015] In some embodiments, an ASO is provided comprising a 3-10-2 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1311, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0016] In some embodiments, an ASO is provided comprising a 2-11-2 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1811, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0017] In some embodiments, an ASO is provided comprising a 3-10-3 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1302, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages. All cytosine nucleosides may be replaced with 5-methylcytosine nucleosides. The ASO may comprise a 2’OMe-modified nucleoside at position 2 in the DNA gap.

[0018] In some embodiments, an ASO is provided comprising a 3-10-3 MOE-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1299, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0019] In some embodiments, an ASO is provided comprising a 3-10-3 LNA-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1300, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0020] In some embodiments, an ASO is provided comprising a 3-10-3 MOE-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1301, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0021] In some embodiments, an ASO is provided comprising a 3-10-3 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1459, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages. All cytosine nucleosides may be replaced with 5-methylcytosine nucleosides. The ASO may comprise a 2’OMe-modified nucleoside at position 2 in the DNA gap.

[0022] In some embodiments, an ASO is provided comprising a 3-10-3 LNA-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1457, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0023] In some embodiments, an ASO is provided comprising a 3-10-3 MOE-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1458, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0024] In some embodiments, an ASO is provided comprising a 3-10-3 MOE-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1460, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0025] Also provided herein are compositions comprising antisense compounds including ASOs described herein, or salts thereof, and a pharmaceutically acceptable carrier. In some embodiments, the compositions are provided as pharmaceutical formulations.

[0026] Also provided are methods of reducing the level of a COMP RNA in a cell, comprising contacting the cell with the antisense compounds including ASOs described herein, or compositions thereof, thereby reducing the level of the COMP RNA.

[0027] Also provided are methods of inhibiting expression of cartilage oligomeric matrix protein in a cell, comprising contacting the cell with the antisense compounds including ASOs described herein, or compositions thereof, thereby reducing expression of cartilage oligomeric matrix protein in the cell.

[0028] Also provided are methods of inhibiting accumulation of cartilage oligomeric matrix protein in a cell comprising a mutant COMP allele, the method comprising contacting the cell the antisense compounds including ASOs described herein, or compositions thereof, thereby reducing expression of cartilage oligomeric matrix protein in the cell.

[0029] In some embodiments, the cell is a growth plate cell, a tendon cell, or a cartilage cell. In some embodiments, the methods are carried out in vitro. In some embodiments, the methods are carried out in vivo and the cell is in a subject. For instance, the subject may be a human and the antisense compound, e.g., ASO, or composition may be administered to the subject.

[0030] Also provided are methods of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject, comprising administering to the subject the antisense compounds including ASOs described herein, or compositions thereof, thereby treating, preventing, or ameliorating the disease.

[0031] In some embodiments, the disease is pseudoachondroplasia (PSACH). In some embodiments, the disease is multiple epiphyseal dysplasia (MED).

[0032] The disclosure further provides, in some embodiments. ASOs or compositions thereof for use in a method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject. Thus, the disclosure provides for use of an ASO or composition thereof for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject, and/or for the preparation of a medicament for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells of a subject.

[0033] These and other aspects and features of the disclosure are described in the following sections of the application. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. For example, those skilled in the art reading the specification will appreciate that the present disclosure demonstrates usefulness of certain oligonucleotide sequences to impact expression of COMP, and furthermore teaches usefulness of oligonucleotide formats that are, or target (e.g., are complementary to), such sequences. Those skilled in the art will appreciate that the present disclosure is not limited to any particular mechanism of action; the provided oligonucleotides may be useful regardless of whether they act via an antisense mechanism, for example, involving RNase H activity, and other therapeutic formats (e.g., siRNA, shRNA, nuclease gRNA, etc.). Analogously, those skilled in the art will appreciate that the present disclosure, by defining useful oligonucleotide sequences, also describes a variety of formats for such sequences (e.g., as part of a nucleic acid vector such as a vector from which they may be expressed (e.g., in vivo, in vitro, or both, etc.). Thus, those skilled in the art, reading the present disclosure, will appreciate that reference to “ASOs” is exemplary, and appropriate nucleic acids (e.g., oligonucleotides) may be utilized regardless of mechanism of action; those skilled in the art are aware of extensive literature regarding appropriate format and structure of nucleic acids (e.g., oligonucleotides) that operate via any of a variety of mechanisms (e.g., siRNA, shRNA, nuclease gRNA, etc.). In some embodiments, the provided nucleic acids incorporate format and/or structural features known in the art to be useful in one or more mechanistic contexts (e.g.. involving RNase H, RISC, a nucleic-acid-directed nuclease such as a Cas, etc.).

DETAILED DESCRIPTION OF THE INVENTION

[0034] This disclosure provides antisense nucleic acid compounds including, e.g.. antisense oligonucleotides (ASOs), for use in therapeutic applications. In some embodiments, the antisense compounds including ASOs and compositions or formulations thereof of this disclosure can be used for ameliorating, preventing or treating pseudoachondroplasia (PSACH), and/or any diseases associated with accumulation of COMP protein and/or expression of a mutant COMP allele. Such a mutant COMP allele, when expressed in a subject, may give rise to autosomal dominant phenotypic aberrant accumulation or dysfunction of COMP protein in the tissue(s) of a subject. In some embodiments, the antisense compounds of the disclosure reduce levels of human COMP mRNA in a subject upon administration to the subject. In some embodiments, the antisense compounds of the disclosure reduce levels of COMP protein in a subject upon administration to the subject. The antisense compounds may reduce expression of an autosomal dominant mutant COMP allele in chondrocytes of a subject upon administration to the subject. In these manners, the antisense compounds provided herein may slow, ameliorate, or reverse the effects of COMP protein accumulation in chondrocytes.

[0035] Thus, this disclosure encompasses synthetic, purified, antisense nucleic acid compounds such as antisense oligonucleotides for therapeutic use in reducing COMP mRNA and/or reducing COMP protein expression upon administration to a subject. The antisense compounds may contain natural and modified nucleotides, natural and modified intemucleoside linkages, varying nucleotide lengths, and/or varying nucleoside (RNA and DNA) compositions, as provided herein.

I. Definitions

[0036] Throughout the present specification, the terms ‘'about” and/or ‘'approximately” may be used in conjunction with numerical values and/or ranges. The term “about” is understood to mean those values near to a recited value. Furthermore, the phrases “less than about [a value]” or “greater than about [a value]” should be understood in view of the definition of the term “about” provided herein. The terms '‘about” and “approximately” may be used interchangeably. Unless otherwise noted, the term “about” when immediately preceding a numerical value means ± 10% of the numerical value. [0037] Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise endpoints and all subranges therein, including each integer in and between a disclosed range. Thus, the range “from 50 to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.) as well as each individual integer from 50 to 80 (e.g.. 50. 51. 52. 53. 54. etc.). Where ranges are provided in the form of fractions, percentages, decimals, and the like, such ranges likewise include all possible subranges therein and each individual fraction, percentage, decimal, etc. in and between the disclosed range. For example, the range “from 0.1 to 1.0” includes all possible ranges therein (e.g., 0.2 to 0.9, etc.) and each individual l/10th decimal from 0.1 to 1.0 (e.g., 0.1, 0.2, 0.3. 0.4, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.).

[0038] The term “a” or “an” refers to one or more of that entity; for example, “an antisense oligonucleotide targeting COMP” refers to one or more such antisense oligonucleotides or at least one such antisense oligonucleotide. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein.

[0039] The terms “comprise,” “comprising,” and the like, as used in this specification and in the claims are used in the non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The present invention may suitably “comprise”, “consist of’, or “consist essentially of’, the steps, elements, and/or reagents described in the claims. [0040] “Antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. Antisense activity includes knockdown or elimination of expression of a gene targeted by a compound having antisense activity.

[0041] “Antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity. An antisense oligonucleotide (ASO) is an “antisense compound”.

[0042] “Antisense oligonucleotide” or “ASO” means short (typically 12 to 25 nucleotides), single-stranded, synthetic DNA or RNA or hybrid DNA/RNA molecules designed to target either a coding or a non-coding nucleic acid such as an mRNA sequence by complementary base-pairing in order to modulate its expression level.

[0043] “Bicyclic nucleoside” or “BN A” means a nucleoside comprising a bicyclic sugar moiety. [0044] “Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In some 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’ -carbon and the 4’ -carbon of the furanosyl.

[0045] “Cartilage oligomeric matrix protein” or “COMP” means any protein encoded by a COMP nucleic acid. In some embodiments, the COMP nucleic acid has the sequence set forth in GENBANK Accession No. NM_000095.3 (incorporated herein as SEQ ID NO: 1). In some instances throughout the specification, the term COMP is used as shorthand to refer to the COMP gene and/or the protein expressed from a COMP nucleic acid such as COMP mRNA or human COMP mRNA (i.e., NM 000095.3).

[0046] “Complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In some embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary' oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary' oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary' oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary' oligomeric compounds or regions are 100% complementary'.

[0047] “Conjugate” means a compound comprising two molecules that are covalently linked. In some embodiments, a conjugate comprises an antibody or other targeting agent and a modified oligonucleotide.

[0048] “Constrained ethyl nucleoside” or “cEf ’ means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH(CH3) — O-2’ bridge.

[0049] “Expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of 5 ’-cap), and translation.

[0050] “Hybridization” or “base-pairing” means the pairing of complementary' oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary' nucleobases.

[0051] “Intemucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide. [0052] '‘Locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH2 — 0-2’ bridge.

[0053] “Mismatch” means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.

[0054] “Modified intemucleoside linkage” means any intemucleoside linkage other than a naturally occurring intemucleoside linkage.

[0055] “Modified nucleobases” include universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Particular examples of modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine. 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl denvatives of adenine and guanine, 2-thiouraciL 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl ( — C=C — CEL) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil. 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo. 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7 -methyl adenine, 2-F-adenine, 2-amino- adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3 -deazaguanine and 3 -deazaadenine. 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][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3‘,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.

[0056] “Modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides may comprise a modified sugar moiety and/or a modified nucleobase.

[0057] '‘Modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified intemucleoside linkage. Examples of modified oligonucleotides include single-stranded and double-stranded compounds, such as, antisense oligonucleotides. siRNAs, shRNAs, ssRNAs, and occupancy-based compounds. [0058] '‘Modified sugar” means a substituted sugar moiety or a sugar surrogate present in a modified nucleoside. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2‘-position, the 3’-position, the 5’-position, and/or the 4’-position. Certain substituted sugar moieties are bicyclic sugar moieties. Examples of modified sugars include T -substituted sugar moieties (a furanosyl comprising a substituent at the 2’-position other than H or OH). Sugar surrogates are structures that do not comprise a furanosyl but are capable of substituting for the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols. Bicyclic sugar moieties comprise a 4 to 7 membered ring (including but not limited to a furanosyl) comprising 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’ -carbon and the 4^?-carbon of the furanosyl.

[0059] '‘Modulation” means a change of amount or quality of a molecule, function, or activity following an intervention when compared to the amount or quality of the molecule, function, or activity prior to or without the intervention. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of 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.

[0060] “MOE” means — OCH2CH2OCH3

[0061] “mRNA” means “messenger RNA”, i.e.. an RNA molecule that encodes a protein. [0062] '‘Naturally occurring intemucleoside linkage” means a 3’ to 5’ phosphodiester linkage. [0063] “Naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.

[0064] “Nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified. “Unmodified nucleobase” or “naturally occurring nucleobase” means 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 -methylcytosine), and uracil (U).

[0065] “Nucleoside” refers to a compound comprising a nucleobase moiety and a sugar moiety'. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

[0066] “Nucleotide” means a nucleoside further comprising a phosphate linking group. “Linked nucleosides” may or may not be linked by phosphate linkages and thus include “linked nucleotides.” “Linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are present between those that are linked).

[0067] “Oligomeric compound” means a polymeric structure comprising two or more substructures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound consists of an oligonucleotide.

[0068] “Oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides. [0069] “Pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline. [0070] “Polynucleotide” refers to an “oligomer,” i.e., a molecule comprising at least two monomers and includes oligonucleotides such as DNA oligonucleotides, RNA oligonucleotides, and mixed DNA/RNA oligonucleotides, as well as polynucleotides including DNA polynucleotides, RNA polynucleotides, and mixed DNA/RNA polynucleotides, as well as polynucleotides bearing synthetic or modified monomers such as modified nucleotides described herein.

[0071] “Pre-mRNA” means an RNA transcript that has not been fully processed into mRNA. Pre-mRNA includes one or more intron.

[0072] “Subject” refers to a human or any non-human animal (e.g.. mouse, rat. rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient” unless the context requires otherwise. A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0073] “Substituted sugar moiety" means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosy Is comprising substituents at the 2’-position, the 3^?-position, the 5’-position and/or the ^-position. Certain substituted sugar moieties are bicyclic sugar moieties.

[0074] “Sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

[0075] “Sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary' oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

[0076] “Targeting” or “targeted to” means the association of an antisense compound to a particular sequence or region of a nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.

[0077] “2’-(ara)-F” refers to a 2’-F substituted nucleoside, wherein the fluoro group is in the arabino position.

[0078] “2’ -deoxynucleoside” means a nucleoside comprising 2’-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2’- deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

[0079] “2’-F nucleoside” refers to a nucleoside comprising a sugar comprising fluorine at the 2^? position. Unless otherwise indicated, the fluorine in a 2’-F nucleoside is in the ribo position (replacing the OH of a natural ribose).

[0080] “2’ -substituted nucleoside” means a nucleoside comprising a substituent at the 2’- position other than H or OH. Unless otherwise indicated, a 2’ -substituted nucleoside is not a bicyclic nucleoside.

[0081] “2’ -substituted sugar moiety” means a furanosyl comprising a substituent at the 2’- position other than H or OH. Unless otherwise indicated, a 2’ -substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of a 2'-substituted sugar moiety’ does not form a bridge to another atom of the furanosyl ring.

[0082] “3’-endo-furanosyl nucleoside” means an RNA-like nucleoside that comprises a substituted sugar moiety’ that has a 3’-endo conformation. 3’-endo-furanosyl nucleosides include, but are not limited to: 2’-M0E (“MOE”), 2’-F, 2’-OMe (“OMe”), locked nucleic acids (LNA), 2'- O,4'-C-ethylene-bridged nucleic acids (“ENA”), and 2'-0-Ethyl (cEt) nucleosides.

[0083] “Acyl,” means 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 carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

[0084] “Alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

[0085] “Aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more ty pically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines Aliphatic groups as used herein may optionally include further substituent groups.

[0086] “Alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more ty pically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

[0087] “Alkoxy” means a radical formed between an alkyd group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

[0088] '‘Alkyl/’ means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the tike. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.

[0089] “Alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the tike. Alkynyl groups typically include 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. Alkynyl groups as used herein may optionally include one or more further substituent groups.

[0090] “Aminoalkyl” means an amino substituted C1-C12 alkyd radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyd group can be substituted with a further substituent group at the alkyl and/or amino portions.

[0091] “Aralky l” and “ary lalkyl” mean an aromatic group that is covalently linked to a C 1-C 12 alkyd radical. The alkyl radical portion of the resulting aralkyl (or arylalky 1) group forms a covalent bond with a parent molecule. Examples include without limitation, benzy l, phenethyl and the tike. Aralky l groups as used herein may optionally include further substituent groups attached to the alkyl, the ary 1 or both groups that form the radical group.

[0092] “Aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, 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 substituent groups.

[0093] “Furanosy 1” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

[0094] ■‘Halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.

[0095] “Heteroary l,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl. thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, 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 hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

II. Overview

[0096] The antisense compounds targeting human COMP mRNA described herein and compositions or formulations comprising the same may be used to ameliorate, prevent, or treat any symptom or disorder associated with aberrant COMP accumulation or expression in a subject, such as symptoms or disorders arising from an autosomal dominant mutation in the COMP gene of a subject. For example, the antisense compounds targeting human COMP mRNA may be used to ameliorate, prevent, or treat any symptom or disease associated with aberrant accumulation of COMP protein in chondrocytes of a subject or in specific sub-cellular compartments of chondrocytes in a subject such as endoplasmic reticulum. In some embodiments, the subject may present with PSACH disorder.

[0097] The antisense compounds targeting human COMP mRNA described herein may be any antisense compound useful for inhibiting expression of the target gene, i.e., COMP. Example sequences for portions of antisense compounds targeting human COMP mRNA are provided in Table 1. For example, antisense compounds include single and double stranded nucleic acid compounds and may suppress target gene expression via RNaseH-mediated mRNA decay, steric hindrance-based mechanisms, RNA-induced silencing complex (RISC), or guide strand-directed nuclease-mediated mRNA decay mechanisms. Thus, antisense compounds targeting human COMP mRNA described herein may comprise antisense oligonucleotides (ASOs), RNAi compounds such as small interfering RNA (siRNA) and short hairpin RNA (shRNA), or nuclease guide RNA (gRNA) compounds for use with nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Cas9, and similar nucleases. In general, persons skilled in the art are aw are of modifications needed to adapt the antisense compounds disclosed herein, such as those in Table 1. to be suitable for use as, e.g., an ASO, an siRNA, or a gRNA. Such modifications include, e.g.. incorporation of sense and/or antisense strands, preparation of single or double stranded compositions, use of DNA or RNA nucleosides, and oligonucleotide length, among others.

[0098] In some embodiments, the antisense compounds of the disclosure are antisense oligonucleotides or “ASOs”. The ASOs of the disclosure are designed to target human COMP mRNA and thereby modulate its expression. The ASOs can be administered to a subject in need (e.g.. a patient with PSACH) and reduce functionally active COMP mRNA or COMP protein levels in the subject. Thus, the ASOs described herein and compositions or formulations comprising the same can be used for preventing, treating, ameliorating, or reversing any symptoms of aberrant COMP protein accumulation or activity' in a subject. In an exemplary' embodiment, the subject is a human patient presenting with an autosomal dominant mutation in the COMP gene, resulting in aberrant expression, activity, or accumulation of COMP protein in chondrocytes. In some embodiments, a composition or formulation comprising an ASO of the disclosure can be delivered to chondrocyte cells of the subject.

III. Antisense Compounds Targeting Human COMP mRNA

[0099] In certain embodiments, the present invention provides antisense nucleic acid compounds targeting human COMP mRNA to suppress expression of the COMP gene. The antisense compounds may comprise oligonucleotides. In some embodiments, oligonucleotides of the disclosure comprise one or more chemical modifications such as one or more nucleoside modification (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more intemucleoside linkage.

A. Modified Nucleosides

[0100] The antisense compounds of the disclosure may comprise or consist of oligonucleotides comprising at least one modified nucleoside. Such modified nucleosides may comprise a modified sugar moiety, a modified nucleobase, or both a modified sugar moiety and a modified nucleobase. i. Sugar Modifications

[0101] The antisense compounds described herein may comprise one or more nucleosides comprising a modified sugar to confer desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to antisense compounds comprising non-modified sugar moieties. Modified sugars may be substituted sugar moieties, bicyclic or tricyclic sugar moieties, or sugar surrogates.

[0102] Substituted sugar moieties may comprise one or more substituent, including but not limited to substituents at the 2' and/or 5' positions. Examples of sugar substituents suitable for the 2'-position include, but are not limited to: 2'-F, 2'-OCHs (“OMe” or “O-methyl”), and 2'- O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents at the 2' position are selected from allyl, amino, azido, thio, O-allyl substitutions. Examples of sugar substituents at the 5'- position, include, but are not limited to: 5'-methyl (R or S); 5'-vinyl, 5’-vinylphosphonate, and 5'- methoxy.

[0103] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In certain embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido. O — C1-C10 alkoxy; O — C1-C10 substituted alkoxy. SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(R_m)-alkyl; O-alkenyl, S-alkenyl, or N(R_m)-alkenyl; O- alkynyl, S-alkynyl, N(R_m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralky l, O-alkaryl, O- aralkyl. O(CH₂)₂SCH₃, O— (CH₂)₂— O— N(R_m)(Rn) or O— CH₂— C(=O)— N(R_m)(Rn), where each R_m and Rn is. independently. H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.

[0104] In certain embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from F, NH₂, Ns, OCF3, O— CH3, O(CH₂)₃NH2, CH₂— CH=CH₂, O— CH₂— CH=CH₂, OCH₂CH₂OCH₃. O(CH₂)₂SCH₃. O— (CH₂)₂— O— N(R_m)(Rn), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N- substituted acetamide (O — CH₂ — C(=O) — N(R_m)(Rn) where each R_m and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.

[0105] Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2’ furanose ring atoms. Examples of such 4' to 2' sugar substituents, include, but are not limited to: — [C(R_a)(Rb)]_n — , — [C(Ra)(Rb)]n — O — , — C(RaRb)— N(R)— O— or, — C(R_aRb)— O— N(R)— ; 4'-CH₂-2', 4'-(CH₂)₂-2',4'-(CH₂)₃-2', 4'- (CH₂)— O-2' (LNA); 4'-(CH₂)— S-2; 4'-(CH₂)₂— 0-2' (ENA); 4'-CH(CH₃)— 0-2' (cEt) and 4'- CH(CH₂OCH3) — 0-2', and analogs thereof (see, e.g.. U.S. Pat. No. 7,399,845. issued on Jul. 15,

2008); 4'-C(CH3)(CH3) — 0-2' and analogs thereof, (see, e.g., W02009/006478, published Jan. 8,

2009); 4'-CH₂ — N(OCH3)-2' and analogs thereof (see, e.g., W02008/150729, published Dec. 11, 2008); 4'-CH₂— O— N(CH₃)-2' (see, e.g., US2004/0171570, published Sep. 2, 2004); 4'-CH₂— O — N(R)-2', and 4'-CH₂ — N(R) — 0-2'-. wherein each R is, independently, H, a protecting group, or Ci-Ci₂ alkyl; 4'-CH₂ — N(R) — 0-2', wherein R is H. Ci-Ci₂ alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4'-CH₂— C(H)(CHs)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH₂ — C(=CH₂)-2' and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).

[0106] Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) a-L-Methyleneoxy (4'-CH₂ — 0-2') BNA, (B) 0-D-Methyleneoxy (4'-CH₂ — 0-2') BNA (also referred to as locked nucleic acid or LNA). (C) Ethyleneoxy (4'-(CH₂)₂— 0-2') BNA, (D) Aminooxy (4'-CH₂— O— N(R)-2') BNA, (E) Oxyamino (4'-CH2 — N(R) — 0-2') BNA, (F) Methyl (methyleneoxy) (4'-CH(CH?) — 0-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH2 — S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2’) BNA, (I) methyl carbocyclic (4'-CH2 — CH(CH3)-2') BNA, (J) propylene carbocyclic (4'-(CH2)3-2') BNA, and (M) 4'-CH2 — O — CH2-2'.

[0107] Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 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; Snvastava et al., J. Am. Chem. Soc.. 129(26) 8362-8379 (Jul. 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. Then, 2001, 3, 239-243; U.S. Pat. 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; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. 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 Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and

PCT/US2008/068922.

[0108] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge, may be in the a-L configuration or in the (J-D configuration. Previously, a-L-methyleneoxy (4’-CH2 — O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity’ (Frieden et al.. Nucleic Acids Research, 2003, 21, 6365-6372).

[0109] In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5'-substituted and 4'-2' bridged sugars). (See, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).

[0110] In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety' also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4'-sulfur atom and a substitution at the 2'-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5' position.

[0111] In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C J. Bioorg. &Med. Chem. (2002) 10:841-854), and fluoro HNA (F-HNA).

[0112] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used to modify nucleosides (see, e g., review article: Leumann, J. C, Bioorganic &Medicinal Chemistry, 2002, 10, 841-854).

[0113] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in antisense compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0114] In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

[0115] Combinations of modifications are also provided without limitation, such as 2'-F-5'- methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 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 published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5 '-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4'-CH2 — O-2' bicyclic nucleoside is further substituted at the 5' position with a 5 '-methyl or a 5 '-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379). ii. Modified Nucleobases

[0116] The antisense compounds described herein may comprise one or more nucleosides comprising modified nucleobases.

[0117] Modified nucleobases may include universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Particular examples of modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine. 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil. 2- thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl ( — C=C — CH?) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino- adenine, 8-azaguanme and 8-azaadenine. 7-deazaguanine and 7-deazaadenine, 3 -deazaguanine and 3 -deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5.4-b][l,4]benzoxazin-2(3H)-one). carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.

B. Modified Intemucleoside Linkages

[0118] Individual nucleosides of the antisense compounds described herein may be linked together using any intemucleoside linkage to form oligonucleotides. Intemucleoside linkages include phosphorus containing intemucleoside linkages including, but not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Non-phosphorus containing intemucleoside linkages include, but are not limited to, methylenemethylimino ( — CH2 — N(CH?) — O — CH2 — ), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ); siloxane ( — O — Si(H)2 — O — ); and N,N'-dimethylhydrazine ( — CH2 — N(CHs) — N(CHj) — ). Modified linkages, compared to natural phosphodiester linkages, may improve nuclease resistance of the oligonucleotide. In some embodiments, intemucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing intemucleoside linkages are known to those skilled in the art.

C. Oligonucleotide Motifs

[0119] Antisense compounds described herein may comprise oligonucleotides having one or more chemical modification, i.e.. one or more modified sugars and/or one or more modified nucleobases and/or one or more modified intemucleoside linkages. These chemical modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) may be present in a given oligonucleotide according to a defined pattern or motif. The patterns of chemical modifications of sugar moieties. intemucleoside linkages, and nucleobases may be independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, intemucleoside linkage motif and/or nucleobase modification motif (independent of the sequence of nucleobases). i. Sugar Motifs

[0120] Oligonucleotides described herein may comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern.

[0121] For example, the oligonucleotides may comprise or consist of a region having a gapmer sugar motif, which comprises two external regions or segments and a central or internal region or gap. The three regions of a gapmer sugar motif (the 5'-segment, the gap segment, and the 3'- segment) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the 5’ and 3’ segments differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each 5’ and 3’ segment that are closest to the gap (the 3'-most nucleoside of the 5'-segment and the 5'- most nucleoside of the 3 '-segment) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the 3’ and 5’ segments and the gap. In some embodiments, the sugar moieties within the gap are the same as one another. In some embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety' of one or more other nucleosides of the gap. In some embodiments, the sugar motifs of the 5‘ and the 3‘ segments are the same as one another (symmetric sugar gapmer). In certain embodiments, the sugar motifs of the 5'-segment differs from the sugar motif of the 3'-segment (asymmetric sugar gapmer).

[0122] Segments of a gapmer may be different lengths or equal lengths. For example, in some instances, a gapmer consists of a 5 nucleoside 5’ segment, a 5 nucleoside gap segment, and a 5 nucleoside 3’ segment. In other instances, a gapmer consists of, e g., a 3 nucleoside 5’ segment, a 9 nucleoside gap segment, and a 3 nucleoside 3’ segment. In still other instances, a gapmer consists of, e.g., a 2 nucleoside 5’ segment, a 10 nucleoside gap segment, and a 3 nucleoside 3’ segment. Thus, each of the segments may be of differing lengths compared to the other segments. The lengths of 5’ and 3’ segments can vary, typically from 2 nucleosides to 5 nucleosides in length. The length of the gap segment can also vary, typically from 5 nucleosides to 15 nucleosides in length.

[0123] Gapmers can be referred to according to their segment lengths in the 5’ to 3 ‘ direction. For instance, a gapmer consisting of a 3 nucleoside 5^? segment, a 10 nucleoside gap. and a 3 nucleoside 3' segment can be referred to as a 3-10-3 gapmer, more specifically a 3-10-3 16mer. For specific gapmers where sugar modifications are defined, the gapmers can be more specifically referred to by defining the sugar modifications of each segment. For example, a gapmer consisting of a 3 nucleoside 5’ segment having 4'-CH2-2', 4'-(CH2)2-2',4'-(CH2)3-2', or 4'-(CH2) — 0-2' (LN A) sugar modifications, a gap having 10 linked deoxynucleosides, and a 3 nucleoside 3’ segment having 2'-O(CH2)2OCH3 (“MOE”) sugar modifications can be referred to as a 3-10-3 LNA-DNA-MOE gapmer, more specifically a 3-10-3 16mer.

[0124] Gapmers may comprise one or more modified sugar at a specific position in one or more segment. That is, gapmers may comprise a sugar modification at a specific nucleoside position within the 5’ segment, the gap segment, and/or the 3’ segment. In some embodiments, gapmers described herein comprise a 2'OMe modified sugar at a specific position within the DNA gap. For example, gapmers may comprise a 2'OMe modified sugar at the nucleoside of position 2 in the DNA gap. ii. Nucleobase Modification Motifs

[0125] Oligonucleotides described herein may comprise chemical modifications to nucleobases arranged along the oligonucleotide or region/segment thereof in a defined pattern or nucleobase modification motif. In certain embodiments, each nucleobase is modified.

[0126] Oligonucleotides may comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3'-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3'-end of the oligonucleotide. In certain such embodiments, the block is at the 5 '-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5 '-end of the oligonucleotide.

[0127] Nucleobase modifications can depend on the base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified. [0128] In some embodiments, oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, oligonucleotides having a gapmer sugar motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer sugar motif. In certain embodiments, the sugar is an unmodified 2' deoxynucleoside. In certain embodiments, the modified nucleobase is selected from: a 2-thio pyrimidine and a 5- propyne pyrimidine.

[0129] In some embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methylcytosine. 5-methylcytosine is not a ‘'modified nucleobase.’’ Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5-methyl. In some embodiments, the methylation state of all or some cytosine nucleobases is specified.

D. Length of Antisense Compounds

[0130] The present invention provides antisense nucleic acid compounds including oligonucleotides of differing nucleotide lengths. For example, the antisense compounds may consist of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20. 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 10 to 11, 10 to 12, 10 to 13. 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19. 10 to 20. 10 to 21. 10 to 22. 10 to 23, 10 to 24, 10 to 25, 1 1 to 12, 1 1 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23. 12 to 24, 12 to 25, 13 to 14, 13 to 15, 13 to 16. 13 to 17. 13 to 18. 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23. 17 to 24, 17 to 25, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 22 to 23, 22 to 24, 22 to 25, 23 to 24, 23 to 25 or 24 to 25 linked nucleosides. In some embodiments, an antisense oligonucleotide of the disclosure can be from about 12 nucleotides to about 25 nucleotides in length, such as 12, 13, 14, 15, 16, 17, 18, 19, 20. 21. 22. 23, 24, or 25 nucleotides in length. Generally, oligonucleotides having a length of n linked nucleosides are referred to as '‘nmers” such that an oligonucleotide of 16 linked nucleosides is referred to as a 16mer.

E. Conjugations for Improved Biodistribution to Chondrocytes

[0131] Antisense compounds described herein may be modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached antisense compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, biodistribution, charge, and clearance. Conjugate groups are used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an antisense compound, such as an oligonucleotide. Conjugate groups include without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.

[0132] In some embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an antisense compound. In general, a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e g., double or triple bonds), and the like.

[0133] Some nonlimiting examples of conjugate linking moieties 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 linking groups include, but are not limited to, substituted Ci-Cio alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, ary l, alkenyl and alkynyl.

[0134] Conjugate groups may be attached to either or both ends (that is. 5’ and/or 3’ ends) of an oligonucleotide (terminal conjugate groups) and/or at any internal position. In some embodiments, conjugate groups are at the 5'-end of an antisense compound described herein, or near the 5'-end. In some embodiments, conjugate groups are at the 3'-end of an antisense compound described herein, or near the 3 '-end.

[0135] In some embodiments, conjugate groups may interact with the lipid components of the cell membrane, bind to specific cell surface proteins or receptors, and/or penetrate the cell through endogenous transport mechanisms carrying the antisense compound with them.

[0136] The conjugate can be attached at the 5'- and/or the 3'-end of a sense and/or antisense strand of an antisense compound via a covalent attachment such as a nucleic acid or non-nucleic acid linker. The conjugate can be attached through a carbamate group or other linking group (see, e.g., U.S. Patent Publication Nos. 20050074771, 20050043219, and 20050158727).

[0137] In some embodiments, the conjugate group may be a molecular entity that facilitates the delivery of the antisense compound into a cell or may be a molecule that comprises a drug or label. Examples of conjugate molecules suitable for attachment to antisense compounds of the present disclosure include, without limitation, lipophilic molecules (e.g., fatty⁷ acids), cholesterols, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, antibodies, aptamers, and combinations thereof (see, e.g., U.S. Patent Publication Nos. 20030130186, 20040110296, and 20040249178; and U.S. Pat. No. 6,753,423).

[0138] The conjugate group used and the extent of conjugation to the antisense compound can be evaluated for improved pharmacokinetic profiles, bioavailability, biodistribution, and/or stability⁷ of the antisense compound. As such, one skilled in the art can screen antisense compounds having various conjugates attached thereto to identify conjugates having improved properties using any of a variety of well-known in vitro cell culture techniques or in vivo animal models.

[0139] In some embodiments, an antisense compound disclosed herein may be conjugated to a lipophilic molecule (for example, a long chain fatty acid or LCFA), an antibody (for example, anti-transferrin receptor antibody), an aptamer, a ligand, a peptide, or a polymer.

[0140] In some embodiments, an antisense compound disclosed herein may be conjugated to a ligand or receptor having specific binding activity to a cognate receptor or ligand of a chondrocyte. Thus, the antisense compounds may be specifically⁷ targeted to chondrocyte cells by' binding receptors or ligands expressed thereon.

[0141] In some embodiments, an antisense compound disclosed herein may be conjugated to an antibody. In some embodiments, the antibody is a chondrocyte-targeting antibody, e.g., an antibody having specificity for a chondrocyte-specific cell surface marker. Examples of such chondrocyte-specific cell surface markers may include cartilage-specific membrane antigen (CH65), human cartilage glycoprotein-39 (HC gp-39), hyaluronan binding adhesion molecule CD44, thymocyte antigen-1 (Thy-1) - CD90, signal transducer - CD24, lymphocyte function- associated antigen-3 (LFA-3) - CD58, and ty pe I transmembrane protein Tmp21 (see, e.g., Osiecka-Iwan A, et al. Antigenic and immunogenic properties of chondrocytes. Implications for chondrocyte therapeutic transplantation and pathogenesis of inflammatory⁷ and degenerative joint diseases. Cent Eur J Immunol. 2018;43(2):209-219. doi: 10.5114/ceji.2018.77392. Epub 2018 Jun 30. PMID: 30135635; PMCID: PMC610261 1; and Salter DM, et al. Integrin expression by human articular chondrocytes. Br J Rheumatol. 1992 Apr;31(4):231-4. doi:

10.1093/rheumatology/31.4.231. PMID: 1372838; both incorporated herein by reference).

IV. Pharmaceutical Formulations

[0142] In some aspects, this application provides compositions containing antisense nucleic acid compounds described herein and a suitable carrier such as a buffer, solvent, or diluent. The compositions may be liquid, wherein the antisense compound is dissolved in a suitable solvent or diluent, or may be solid or lyophilized, wherein the antisense compound is present in dry form with a suitable carrier or buffer. Such compositions containing antisense compounds can further be formulated for particular uses, such as for achieving a pharmacological effect in a cell based system and/or for pharmaceutical application. Thus, the disclosure provides pharmaceutical formulations containing a polynucleotide such as an ASO of the disclosure and a pharmaceutically acceptable carrier.

[0143] A pharmaceutical formulation can be capable of local or systemic administration. In some aspects, a pharmaceutical formulation can be capable of any mode of administration. In certain aspects, the administration can be by any route, including intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, inhalation or nasal administration.

[0144] The present disclosure provides pharmaceutical formulations comprising one or more antisense compound described herein. Such pharmaceutical formulations may comprise a suitable pharmaceutically acceptable diluent or carrier, such as a pharmaceutical grade diluent or carrier. In some embodiments, a pharmaceutical formulation comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical formulations may consist of a sterile saline solution and one or more antisense compound. In some embodiments, a pharmaceutical formulation comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical formulation consists of one or more antisense compound and sterile water. In some embodiments, a pharmaceutical formulation comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical formulation consists of one or more antisense compound and sterile phosphate- buffered saline (PBS).

[0145] The antisense compounds of the disclosure may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical formulations or formulations depending on intended route of administration, extent of disease, or dose to be administered.

[0146] Pharmaceutical formulations provided herein comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical formulations comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0147] A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense nucleic acid compound.

[0148] In certain embodiments, a pharmaceutical formulation provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical formulations including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

[0149] Lipid moieties have been used in nucleic acid therapies and delivery systems therefor in a variety of compositions and methods. For example, the nucleic acid may be introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

[0150] In some embodiments, an antisense compound may be delivered via liposomes, nanoparticles, lipid nanoparticles (LNPs). polymers, microparticles, microcapsules, micelles, or extracellular vesicles and formulations comprising the same.

[0151] In some embodiments, an antisense compound may be delivered via a lipid nanoparticle (LNP). Examples of LNPs capable of delivering antisense compounds are described in International Patent Publication Nos. WO/2015/074085, WO/2016/081029, WO/2017/117530, WO/2018/118102, WO/2018/119163. WO/2018/222926. WO/2019/191780. and

WO/2020/154746. In some embodiments, an LNP may be decorated with targeting moiety, e.g., an antibody, a receptor, or a fragment thereof capable of binding to a target ligand such as a chondrocyte-specific cell surface marker described herein.

[0152] In some embodiments, a lipid nanoparticle comprises (a) a nucleic acid (e.g., an antisense compound described herein), (b) a cationic lipid, (c) an aggregation reducing agent (such as a PEG-lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally a sterol. In one embodiment, the lipid nanoparticle comprises (i) at least one cationic lipid; (ii) a neutral lipid, e.g., DSPC; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, in a molar ratio of about 20- 65% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid. In some embodiments, the cationic lipid is selected from ATX-002, ATX-081, ATX-095, or ATX-126, as described in WO/2018/222926.

[0153] In some embodiments, an antisense compound is delivered via a nanocarrier comprising a molecule enabling specific receptor-mediated endosomal uptake. In one embodiment, said molecule can enable receptor binding, endosomal uptake, controlled breakdown of the endosomal membrane, and release of the antisense compound into a target cell. Examples of nanocarriers capable of delivering antisense compounds are described in WO/2009/141257. In some embodiments, the nanocarrier is a lipid-based nanocarrier, e.g., a lipid nanoparticle (LNP).

[0154] Pharmaceutical formulations provided herein may comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0155] Pharmaceutical formulations provided herein may comprise a co-solvent system comprising, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol. 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such cosolvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied; for example: other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0156] Pharmaceutical formulations as described herein may be formulated for. i.e., prepared for purposes of, oral administration, nasal administration, buccal administration, or administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). For example, a pharmaceutical formulation may comprise a carrier and be formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical formulations for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical formulations for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical formulations for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

V. Methods of Administration

[0157] An effective dose of an antisense compound or pharmaceutical formulation comprising an antisense compound of this disclosure can be an amount that is sufficient to reduce the amount of COMP mRNA or cartilage oligomeric matrix protein in a cell or tissue that expresses the COMP gene under physiological conditions. An effective dose may eliminate COMP mRNA or may eliminate cartilage oligomeric matrix protein in a cell or tissue that otherwise expresses the COMP gene under physiological conditions. In some embodiments, an effective dose reduces the amount of COMP mRNA or cartilage oligomeric matrix protein by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a cell or tissue that expresses the COMP gene under physiological conditions in the absence of the effective dose of an antisense compound or pharmaceutical formulation thereof described herein.

[0158] A therapeutically effective dose can be an amount of an antisense compound or formulation thereof that is sufficient to reduce, slow, halt, or reverse aberrant accumulation of cartilage oligomeric matrix protein in cells or tissue of a subject. In some embodiments, a therapeutically effective dose reduces the amount of COMP mRNA or cartilage oligomeric matrix protein by about 10%. 20%. 30%. 40%. 50%. 60%, 70%, 80%, 90%, or more compared to cells or tissues of the subject absent the therapeutically effective dose of an antisense compound or pharmaceutical formulation thereof described herein.

[0159] A therapeutically effective dose can be administered to a subject in one or more separate administrations, and by different routes. As will be appreciated in the art. a therapeutically effective dose or a therapeutically effective amount is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical formulations of the present disclosure. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g.. treating, modulating, curing, preventing and/or ameliorating a disease or condition associated with aberrant COMP activity, expression, or accumulation in chondrocytes). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and/or prophylactic effect. Generally, the amount of a therapeutic agent (e.g., an ASO described herein) administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art armed with the present disclosure may be able to determine appropriate dosages depending on these and other related factors such as pharmacodynamic and pharmacokinetic properties of the therapeutic agents described herein. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.

[0160] Therapeutically effective doses of the antisense compounds described herein can be administered to subjects by appropriate administration routes. For example, doses may be administered via local or systemic administration. In some aspects, doses of the antisense compounds are administered to a subj ect via intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, inhalation or nasal administration.

[0161] Methods provided herein contemplate single as well as multiple administrations of a therapeutically effective amount of the polynucleotide (e.g., an ASO targeting COMP mRNA) described herein. Pharmaceutical formulations comprising a polynucleotide described herein can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition (e.g., the severity of a subject’s disease state and the associated symptoms). In some embodiments, a therapeutically effective amount of the polynucleotide of the present disclosure may be administered periodically at regular intervals, e.g. : once every year, once every’ six months, once every four months, once every three months, once every two months, once a month, once every two weeks, once a week, or more frequently than once a week. For example, a therapeutically effective amount of an ASO of the present disclosure may be administered weekly, once every two weeks, or monthly.

[0162] In some embodiments, the pharmaceutical formulations of the present disclosure are formulated such that they are suitable for extended-release of the polynucleotide contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. Also contemplated herein are pharmaceutical formulations which are formulated for depot administration (e.g., subcutaneously, intramuscularly) to either deliver or release a polynucleotide of the disclosure over extended periods of time. The extended-release means employed may be combined with modifications made to the polynucleotide to enhance stability. [0163] In some embodiments, administering a therapeutically effective dose of a composition or formulation comprising a polynucleotide of the disclosure can result in decreased levels of COMP protein in chondrocytes of a treated subject. In some embodiments, administering a composition or formulation comprising a polynucleotide of the disclosure results in a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% decrease in levels of COMP protein in chondrocytes of the subject relative to a baseline COMP protein level in the subject prior to treatment or relative to a baseline COMP protein level in age-matched or disease-state-matched subjects having PSACH disorder. In certain embodiments, administering a therapeutically effective dose of a composition or formulation comprising a polynucleotide of the disclosure will result in a decrease in levels of COMP protein relative to baseline COMP protein levels in tissues of the subject prior to treatment.

[0164] In some embodiments, a therapeutically effective dose, when administered regularly, results in reduced expression of COMP in chondrocytes as compared to baseline levels prior to treatment. In some embodiments, administering a therapeutically effective dose of a compound or formulation comprising an antisense compound of the disclosure results in the expression of COMP in chondrocytes of the subject at a level at or below' about 10%, about 20%, about 30%, about 40%. about 50%, about 60%, about 70%, about 80%, or about 90% compared to expression in chondrocytes of the subject prior to treatment.

[0165] A therapeutically effective dose of an antisense compound described herein or formulation thereof may comprise about 0.01 to about 50 mg of the antisense compound per kg body weight of the subject. For instance, the therapeutically effective dose may be about 0.1 to about 50 mg/kg body weight, about 0. 1-25 mg/kg. about 0. 1-20 mg/kg, about 0.1-15 mg/kg, about 0.1-10 mg/kg, about 0.1-5 mg/kg, about 0.1-1 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 1-50 mg/kg, about 5-50 mg/kg, about 10-50 mg/kg, about 15-50 mg/kg, about 20-50 mg/kg, or about 25-50 mg/kg body weight of the subject.

[0166] The antisense compounds and formulations thereof as described herein may be administered parenterally. Parenteral administration may be by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump. In certain embodiments, parenteral administration is by injection, e.g., subcutaneous or intramuscular injection.

[0167] The antisense compounds and formulations thereof as described herein may be administered to a subject by injection or infusion once every day, once every' other day, once every week, once every' other week, once every 4 weeks, once every month, once every 6 w eeks, once every two months, once every 90 days, once every' 3 months, once every 6 months, twice a year, or once a year. VI. Methods of Treatment

[0168] The present disclosure provides antisense compounds and formulations thereof for use in ameliorating, preventing, delaying onset of, or treating conditions associated with aberrant COMP accumulation or expression in a subject, such as symptoms or disorders arising from an autosomal dominant mutation in the COMP gene of a subject. Thus, also provided are methods for ameliorating, preventing, delaying onset of, or treating conditions associated with aberrant COMP accumulation or expression in a subject, such as symptoms or disorders arising from an autosomal dominant mutation in the COMP gene of a subject. In some embodiments, the subject is diagnosed with PSACH disorder. In some embodiments, the subject is diagnosed with MED disorder. In some embodiments, the subject has a mutation in at least one allele of the COMP gene. In some embodiments, the subject has an autosomal dominant mutation in at least one allele of the COMP gene. In some embodiments, the methods comprise administering to the subject an antisense compound or a pharmaceutical formulation thereof. In some embodiments, the methods may further comprise the administration of one or more additional therapeutics (i.e., a “second pharmaceutical agents”) before, simultaneous with, or after administration of the antisense compound or pharmaceutical formulation thereof.

[0169] Pharmaceutical agents that may be co-administered according to the methods described herein include anti-inflammatory and/or antioxidant agents, such as, e.g., non-steroidal antiinflammatory’ drugs (including diclofenac, indomethacin, ibuprofen, aspirin, ketoprofen, naproxen), turmeric, resveratrol, grape seed extract, grapefruit seed extract, coenzymeQIO, vitamin E, cucurmin, fish oil, omega-3 fatty acids, fucoidan, indomethacin, omega-3 -acid ethyl esters, cordycepin, and diacerein.

[0170] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially⁷ of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0171] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it will be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0172] Further, it will be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and/or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and disclosure(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the disclosure(s) described and depicted herein. All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

[0173] It will be understood that the expression "at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects will be understood to have the same meaning unless otherwise understood from the context.

[0174] The use of the term “include.” “includes,” “including,” “have,” “has.” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, will be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0175] The use of any and all examples, or exemplary language herein, for example, “for instance”, “such as”, “for example”, “e.g.,”, or “including” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of the disclosure unless claimed. No language in the specification will be construed as indicating any non-claimed element as essential to the practice of the subject matter of the present disclosure.

[0176] It is understood that this disclosure is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which will be encompassed by the appended claims.

[0177] While antisense nucleic acid compounds, compositions, formulations, and methods described herein have been described with specificity according to certain embodiments, the following examples serve only to illustrate examples of these compounds, compositions, formulations, and methods, and are not intended to limit the same. [0178] Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2'-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2'-OH for the natural 2'-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).

[0179] Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an antisense nucleic acid compound having the nucleobase sequence “ATCGATCG” encompasses any nucleic acid compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and nucleic acid compounds having other modified or naturally occurring bases, such as “AT^meCGAUCG,” wherein^meC indicates a cytosine base comprising a methyl group at the 5- position.

ENUMERATED EMBODIMENTS

[0180] The scope of this disclosure is indicated by the appended claims and the following enumerated embodiments, and all changes that come within the meaning and range of equivalency of the claims and enumerated embodiments are intended to be embraced therein. Thus, the disclosure provides:

[0181] El. An antisense compound comprising 14 to 20 linked nucleosides having anucleobase sequence that is complimentary to a target nucleic acid sequence in SEQ ID NO: 1, wherein the target nucleic acid sequence comprises 14 to 20 contiguous nucleotides and has a start position of 1044, 1045, 1046, 1047. 1048, 1049, 1050, 1340, 1341, 1342, 1343, or 1344 of SEQ ID NO: 1, and wherein the antisense compound comprises one or more of: a gap segment consisting of linked deoxynucleosides; a 5’ segment consisting of at least 2 linked nucleosides; a 3’ segment consisting of at least 2 linked nucleosides; at least one phosphorothioate intemucleoside linkage; at least one nucleoside comprising a modified sugar; and at least one nucleoside comprising a modified nucleobase.

[0182] E2. The antisense compound of El, wherein the gap segment is positioned between the 5’ segment and the 3’ segment.

[0183] E3. The antisense compound of El or E2, wherein the gap segment comprises 5 to 15 linked nucleosides.

[0184] E4. The antisense compound of any one of E1-E3. wherein the 3’ segment comprises 2- 5 linked nucleosides.

[0185] E5. The antisense compound of any one of E1-E4, wherein the 5’ segment comprises 2- 5 linked nucleosides.

[0186] E6. The antisense compound of any one of E1-E5, wherein at least one nucleoside of the 5’ segment and at least one nucleoside of the 3’ segment comprises a modified sugar.

[0187] E7. The antisense compound of any one of E1-E6, wherein each nucleoside of the 5’ segment and each nucleoside of the 3’ segment comprises a modified sugar.

[0188] E8. The antisense compound of any one of E6 or E7, wherein the modified sugar comprises a bicyclic sugar.

[0189] E9. The antisense compound of E8, wherein the bicyclic sugar is selected from the group consisting of: 4’-(CH₂)— O-2’ (LNA); 4’-(CH₂)₂— 0-2’ (ENA); and 4’-CH(CH₃)— 0-2’ (cEt).

[0190] E10. The antisense compound of any one of E1-E9, wherein each intemucleoside linkage is a phosphorothioate intemucleoside linkage.

[0191] El l. The antisense compound of any one of E1-E10, comprising a 5-methylcytosine nucleobase in place of a non-5-methyl cytosine residue.

[0192] E12. The antisense compound of any one of El-El l, comprising a 2-11-2 LNA-DNA- LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1310, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0193] E13. The antisense compound of any one of El-El l, comprising a 3-10-2 LNA-DNA- LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1311, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0194] E14. The antisense compound of any one of El-El l, comprising a 2-11-2 LNA-DNA- LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1811, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0195] E15. The antisense compound of any one of El-El l, comprising a 3-10-3 LNA-DNA- LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1297, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0196] E16. The antisense compound of E15, wherein all cytosine nucleosides are replaced with 5-methylcytosine nucleosides and comprising a 2’OMe-modified nucleoside at position 2 in the DNA gap.

[0197] E17. The antisense compound of any one of El-El 1, comprising a 3-10-3 MOE-DNA- MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1299, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5 -methylcytosine nucleosides.

[0198] E18. The antisense compound of any one of El-El l, comprising a 3-10-3 LNA-DNA- MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1300, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5 -methylcytosine nucleosides.

[0199] E19. The antisense compound of any one of El-El l, comprising a 3-10-3 MOE-DNA- LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1301, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0200] E20. The antisense compound of any one of El-El l, comprising a 3-10-3 LNA-DNA- LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1456, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0201] E21. The antisense compound of E20, wherein all cytosine nucleosides are replaced with 5-methylcytosine nucleosides and comprising a 2’OMe-modified nucleoside at position 2 in the DNA gap.

[0202] E22. The antisense compound of any one of El-El l, comprising a 3-10-3 LNA-DNA- MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1457, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0203] E23. The antisense compound of any one of El-El 1, comprising a 3-10-3 MOE-DNA- LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1458, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0204] E24. The antisense compound of any one of El-El l, comprising a 3-10-3 MOE-DNA- MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1460, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0205] E25. A composition comprising the antisense compound of any one of E1-E24 or salt thereof and a pharmaceutically acceptable carrier.

[0206] E26. The composition of E25, wherein the composition is a pharmaceutical formulation. [0207] E27. A method of reducing the level of a COMP RNA in a cell, comprising contacting the cell with the antisense compound of any one of E1-E24 or the composition of E25 or E26, thereby reducing the level of the COMP RNA. [0208] E28. A method of inhibiting expression of cartilage oligomeric matrix protein in a cell, comprising contacting the cell with the antisense compound of any one of E1-E24 or the composition of E25 or E26, thereby reducing expression of cartilage oligomeric matrix protein in the cell.

[0209] E29. A method of inhibiting accumulation of cartilage oligomeric matrix protein in a cell comprising a mutant COMP allele, the method comprising contacting the cell with the antisense compound of any one of E1-E24 or the composition of E25 or E26, thereby reducing expression of cartilage oligomeric matrix protein in the cell.

[0210] E30. The method of any one of E27-E29, wherein the cell is a growth plate cell.

[0211] E31. The method of any one of E27-E29, wherein the cell is a tendon cell.

[0212] E32. The method of any one of E27-E29, wherein the cell is a cartilage cell.

[0213] E33. The method of any one of E27-E32, wherein the cell is in vitro.

[0214] E34. The method of any one of E27-E32, wherein the cell is in a subject.

[0215] E35. The method of E3 L wherein the subject is a human and the antisense compound or composition is administered to the subject.

[0216] E36. A method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject, comprising administering to the subject an antisense compound of any one of E1-E24 or the composition of E25 or E26, thereby treating, preventing, or ameliorating the disease.

[0217] E37. The method of E36, wherein the disease is pseudoachondroplasia (PSACEI).

[0218] E38. The method of E36. wherein the disease is multiple epiphyseal dy splasia (MED).

[0219] E39. An antisense compound according to any one of E1-E24 or the composition of E25 or E26 for use in a method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject.

[0220] E40. The antisense compound for use of E39, wherein the disease is pseudoachondroplasia.

[0221] E41. The antisense compound for use of E39, wherein the disease is multiple epiphyseal dysplasia.

[0222] E42. Use of an antisense compound of any one of E1-E24 or the composition of E25 or E26 for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject.

[0223] E43. Use of an antisense compound of any one of E1-E24 or the composition of E25 or E26 for the preparation of a medicament for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells of a subject.

[0224] E44. The use of E42 or E43, wherein the disease is pseudoachondroplasia. [0225] E45. The use of E42 or E43, wherein the disease is multiple epiphyseal dysplasia.

[0226] E46. The method of any one of E35-E38, wherein the administration is intramuscular. [0227] E47. The method of any one of E35-E38, wherein the administration is subcutaneous.

[0228] E48. The method of any one of E35-E38, wherein the administration is intravenous or intraperitoneal.

[0229] E49. The method of any one of E35-E38 or E46-E47, comprising administering to the subject a second pharmaceutical agent for treating, preventing, or ameliorating one or more symptoms of PSACH or MED.

[0230] E50. The method of E49, wherein the second pharmaceutical agent comprises an antiinflammatory compound.

[0231] E51. The method of E50, wherein the anti-inflammatory compound is a non-steroidal anti-inflammatory (NS AID) compound.

[0232] E52. The method of E50 or E51, wherein the second pharmaceutical agent comprises indomethacin, ibuprofen, or naproxen.

[0233] E53. The method of E49, wherein the second pharmaceutical agent comprises an antioxidant compound.

[0234] E54. The method of E53. wherein the second pharmaceutical agent comprises omega-3- acid ethyl esters.

[0235] E55. The method of E53, wherein the second pharmaceutical agent comprises resveratrol. [0236] E56. The method of E49, wherein the second pharmaceutical agent comprises diacerein or cordycepin.

[0237] E57. An antisense oligonucleotide comprising 14 to 20 linked nucleosides having a nucleobase sequence that is complimentary to a target nucleic acid sequence in SEQ ID NO: 1, wherein the target nucleic acid sequence comprises 14 to 20 contiguous nucleotides and has a start position of 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1340, 1341, 1342, 1343, or 1344 of SEQ ID NO: 1, and wherein the antisense oligonucleotide comprises one or more of: a gap segment consisting of linked deoxynucleosides; a 5’ segment consisting of at least 2 linked nucleosides; a 3’ segment consisting of at least 2 linked nucleosides; at least one phosphorothioate intemucleoside linkage; at least one nucleoside comprising a modified sugar; and at least one nucleoside comprising a modified nucleobase.

[0238] E58. The antisense oligonucleotide of E57, wherein the gap segment is positioned between the 5’ segment and the 3’ segment.

[0239] E59. The antisense oligonucleotide of E57 or E58, wherein the gap segment comprises 5 to 15 linked nucleosides.

[0240] E60. The antisense oligonucleotide of any one of E57-E59, wherein the 3’ segment comprises 2-5 linked nucleosides.

[0241] E61. The antisense oligonucleotide of any one of E57-E60, wherein the 5’ segment comprises 2-5 linked nucleosides.

[0242] E62. The antisense oligonucleotide of any one of E57-E61, wherein at least one nucleoside of the 5’ segment and at least one nucleoside of the 3’ segment comprises a modified sugar.

[0243] E63. The antisense oligonucleotide of any one of E57-E62, wherein each nucleoside of the 5’ segment and each nucleoside of the 3’ segment comprises a modified sugar.

[0244] E64. The antisense oligonucleotide of any one of E62 or E63, wherein the modified sugar comprises a bicyclic sugar.

[0245] E65. The antisense oligonucleotide of E64, wherein the bicyclic sugar is selected from the group consisting of: 4’-(CH2) — O-2’ (LNA); 4’-(CH2)2 — 0-2’ (ENA); and 4’-CH(CH3) — O- 2’ (cEt).

[0246] E66. The antisense oligonucleotide of any one of E57-E65, wherein each intemucleoside linkage is a phosphorothioate intemucleoside linkage.

[0247] E67. The antisense oligonucleotide of any one of E57-E66, comprising a 5- methylcytosine nucleobase in place of a non-5-methyl cytosine residue.

[0248] E68. The antisense oligonucleotide of any one of E57-E67. comprising a 2-11-2 LNA- DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1310, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0249] E69. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-2 LNA- DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1311. wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0250] E70. The antisense oligonucleotide of any one of E57-E67, comprising a 2-11-2 LNA- DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1811, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0251] E71. The antisense oligonucleotide of any one of E57-E67. comprising a 3-10-3 LNA- DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1297, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0252] E72. The antisense oligonucleotide of E15, wherein all cytosine nucleosides are replaced with 5-methylcytosine nucleosides and comprising a 2’OMe-modified nucleoside at position 2 in the DNA gap.

[0253] E73. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-3 MOE- DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1299, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0254] E74. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-3 LNA- DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1300, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0255] E75. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-3 MOE- DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1301, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0256] E76. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-3 LNA- DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1456, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

[0257] E77. The antisense oligonucleotide of E76. wherein all cytosine nucleosides are replaced with 5-methylcytosine nucleosides and comprising a 2’OMe-modified nucleoside at position 2 in the DNA gap.

[0258] E78. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-3 LNA- DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1457, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0259] E79. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-3 MOE- DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1458. wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0260] E80. The antisense oligonucleotide of any one of E57-E67, comprising a 3-10-3 MOE- DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1460, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

[0261] E81. A composition comprising the antisense oligonucleotide of any one of E57-E80 or salt thereof and a pharmaceutically acceptable carrier.

[0262] E82. The composition of E81, wherein the composition is a pharmaceutical formulation. [0263] E83. A method of reducing the level of a COMP RNA in a cell, comprising contacting the cell with the antisense oligonucleotide of any one of E57-E80 or the composition of E81 or E26, thereby reducing the level of the COMP RNA.

[0264] E84. A method of inhibiting expression of cartilage oligomeric matrix protein in a cell, comprising contacting the cell with the antisense oligonucleotide of any one of E57-E80 or the composition of E81 or E82, thereby reducing expression of cartilage oligomeric matrix protein in the cell.

[0265] E85. A method of inhibiting accumulation of cartilage oligomeric matrix protein in a cell comprising a mutant COMP allele, the method comprising contacting the cell with the antisense oligonucleotide of any one of E57-E80 or the composition of E81 or E82. thereby reducing expression of cartilage oligomeric matrix protein in the cell.

[0266] E86. The method of any one of E83-E85, wherein the cell is a growth plate cell.

[0267] E87. The method of any one of E83-E85, wherein the cell is a tendon cell.

[0268] E88. The method of any one of E83-E85, wherein the cell is a cartilage cell.

[0269] E89. The method of any one of E83-E88, wherein the cell is in vitro.

[0270] E90. The method of any one of E83-E88, wherein the cell is in a subject.

[0271] E91. The method of E87, wherein the subject is a human and the antisense oligonucleotide or composition is administered to the subject.

[0272] E92. A method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject, comprising administering to the subject an antisense oligonucleotide of any one of E57-E80 or the composition of E81 or E82, thereby treating, preventing, or ameliorating the disease.

[0273] E93. The method of E92, wherein the disease is pseudoachondroplasia (PSACEI).

[0274] E94. The method of E92, wherein the disease is multiple epiphyseal dysplasia (MED).

[0275] E85. An antisense oligonucleotide according to any one of E57-E80 or the composition of E81 or E82 for use in a method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject.

[0276] E96. The antisense oligonucleotide for use of E85, wherein the disease is pseudoachondroplasia.

[0277] E97. The antisense oligonucleotide for use of E85, wherein the disease is multiple epiphyseal dysplasia.

[0278] E98. Use of an antisense oligonucleotide of any one of E57-E80 or the composition of E81 or E82 for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject.

[0279] E99. Use of an antisense oligonucleotide of any one of E57-E80 or the composition of E81 or E82 for the preparation of a medicament for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells of a subject.

[0280] El 00. The use of E98 or E99, wherein the disease is pseudoachondroplasia.

[0281] E101. The use of E98 or E99, wherein the disease is multiple epiphyseal dysplasia.

[0282] El 02. The method of any one of E91-E94. wherein the administration is intramuscular. [0283] E103. The method of any one of E91-E94, wherein the administration is subcutaneous.

[0284] El 04. The method of any one of E91-E94, wherein the administration is intravenous or intraperitoneal.

[0285] E105. The method of any one of E91-E94 or E102-E103, comprising administering to the subject a second pharmaceutical agent for treating, preventing, or ameliorating one or more symptoms of PSACH or MED.

[0286] E106. The method of El 05, wherein the second pharmaceutical agent comprises an antiinflammatory compound.

[0287] E107. The method of E106, wherein the anti-inflammatory compound is a non-steroidal anti-inflammatory (NSAID) compound.

[0288] E108. The method of E106 or E107, wherein the second pharmaceutical agent comprises indomethacin, ibuprofen, or naproxen.

[0289] El 09. The method of El 05, wherein the second pharmaceutical agent comprises an antioxidant compound.

[0290] E110. The method of El 09, wherein the second pharmaceutical agent comprises omega- 3-acid ethyl esters.

[0291] El 11. The method of E109, wherein the second pharmaceutical agent comprises resveratrol.

[0292] El 12. The method of E105, wherein the second pharmaceutical agent comprises diacerein or cordycepin.

[0293] El 13. An antisense oligonucleotide targeting human COMP mRNA set forth in Table 1 herein.

[0294] El 14. Use of an antisense oligonucleotide targeting human COMP mRNA set forth in Table 1 in a method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject.

EXAMPLES

[0295] The disclosure now being generally described will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.

Example 1 : Design and synthesis of antisense oligonucleotides targeting human COMP mRNA

[0296] Antisense oligonucleotides of 14, 15, and 16 nucleotide lengths (i.e., 14-, 15-, and 16- mers, respectively) were designed targeting the human COMP mRNA sequence of GENBANK Accession No. NM_000095.3 (SEQ ID NO: 1). The coding region of the human COMP mRNA (nucleotides 37 - 2310 of SEQ ID NO: 1) was targeted for ASO design to ensure that ASOs identified in the screen could be tested in a transgenic animal model in which the human COMP mRNA coding sequence is expressed under control of a tetracycline-inducible promoter (the “h- MT-COMP” mouse model described, e.g., in Posey, K.L., et al. Am J Pathol 175(4): 1555-63, 2009. See also Posey, K.L. et al. Mol Therapy 25(3):705-14, 2017 and Coustry. F. and Posey, K.L. et al. Matrix Biol 67:75-89, 2018).

[0297] Regions of the COMP mRNA deemphasized in the initial ASO design process included the 5’ untranslated region (UTR) and the 3’ UTR, nucleotides 1 - 36 and nucleotides 2311 - 2452 of SEQ ID NO: 1, respectively. Design of ASOs took into account melting temperature, G/C content, off-target hits with <2bp mismatch within humans and model species (i.e., M. musculus and M. fascicularis), and ASO target sequence conservation across human and model species.

[0298] All 14-, 15-, and 16-mer ASOs were synthesized with 3 locked nucleic acid (LNA) modified nucleosides on both 5' and 3' ends with DNA nucleosides in between (i.e., 3-8-3, 3-9-3, or 3-10-3 LNA-DNA-LNA gapmers) and all ASOs were fully phosphorothioate-modified (i.e., all phosphorothioate intemucleoside linkages). Table 1 shows example ASO sequences designed and/or synthesized according to the present example. In Table 1. the value in the ‘"Start Pos” column indicates the nucleotide position of the 5’ base of the target sequence of each ASO with respect to the human COMP mRNA sequence of SEQ ID NO: 1.

Table 1. Human COMP ASO design

Example 2: Screening select human COMP ASOs for potency in vitro

[0299] Briefly, human fibroblasts were cultured in Eagle's Minimum Essential Medium supplemented with 15% fetal bovine serum. On day 1, antisense oligonucleotides (ASOs) synthesized according to Example 1 were mixed with approximately 25,000 resuspended human fibroblasts per well in 96 well plates to obtain a target concentration of 1 pM. All ASOs were synthesized with 3 locked nucleic acid (LNA) modified nucleosides on both 5' and 3' ends with DNA nucleosides in between and all ASOs were fully phosphorothioate-modified. Following 72 hours of gynmosis incubation, cells were harvested for ASO potency evaluation by QuantiGene RNA assay to determine the reduction in human COMP mRNA relative to untreated controls. Human PPIB mRNA was also measured to monitor cell viability and used to normalized human COMP mRNA value from each well. Table 2 shows potency results for select ASOs tested in the initial potency screen (n = 2 for 14-mer and 15-mer, n = 3 for 16-mers). As in Table 1 above, the value in the “Start Position” column indicates the nucleotide position of the 5’ base of the target sequence of each ASO with respect to the human COMP mRNA sequence of SEQ ID NO: 1. The value in the “Avg %KD” column indicates the average percent knockdown of human COMP mRNA observed relative to untreated controls and “SD” refers to standard deviation. The nucleotide sequence of each ASO evaluated is provided by reference to the unique number provided in the “SEQ ID NO” column. Scramble ASO and a non-related ASO targeting human GAPDH were included as negative controls.

Table 2. Initial in vitro ASO potency screen results

Example 3: Dose-response study for select human COMP ASOs

[0300] A follow-up screen was carried out to re-confirm potential ASO compounds identified in Example 2 with a dose-response analysis. Briefly, doses of 1.1 pM, 3.3 pM, and 10 pM were tested in human fibroblasts as described in Example 2. All ASOs were synthesized with 3 locked nucleic acid (LNA) modified nucleosides on both 5' and 3' ends with DNA nucleosides in between and all ASOs were fully phosphorothioate-modified. Table 3 provides results in average % knockdown of human COMP mRNA for each ASO dosage (i.e., ASO concentrations of 1.1 pM, 3.3 pM, and 10 pM) for each ASO relative to untreated cells.

Table 3. Dose-response study results

Example 4: Screening additional human COMP ASOs

[0301] Additional 18mer and 20mer ASOs were designed to target the human COMP mRNA region surrounding start position 1047 relative to SEQ ID NO: 1. Table 4 provides sequence and modification information for all additional 18mer and 20mer ASOs designed. All ASOs were synthesized with locked nucleic acid (LN A) modified nucleosides on both 5' and 3' ends with remaining DNA nucleosides positioned in between. Specifically, the “ASO ID” column of Table 4 provides the length of each ASO (e.g., “20mer”) followed by the nucleotide position of the 5’ base of the target sequence of each ASO with respect to the human COMP mRNA sequence of SEQ ID NO: 1 (i.e., the start position, e.g., “1044”), followed by the LNA-DNA-LNA modification patern for each ASO (e.g., 3-14-3, meaning 3 LNA nucleosides in the 5’ segment, 14 DNA nucleosides in the middle gap region, and 3 LNA nucleosides in the 3’ segment). The ASO sequence is provide in Table 4 with LNA nucleosides in lowercase script and DNA nucleosides in uppercase. All ASOs were fully phosphorothioate-modified (i.e., all phosphorothioate intemucleoside linkages). Briefly, doses of 10 pM, 3 pM. and 1 pM were tested in human fibroblasts as described in Example 2 and 3. 18mer and 20mer scramble ASOs with identical modification patern were synthesized for negative controls. Table 4 provides results in average % knockdow n (Avg. % KD) of human COMP mRNA relative to untreated cells for each additional 18mer and 20mer ASO tested at the given concentration in duplicates from 2 independent experiments.

Table 4. Dose-response of additional 18mer and 20mer ASOs

Example 5: IC50 evaluation of select human COMP ASOs

[0302] Two sites within the human COMP mRNA coding region centered around nucleotide positions 1047 and 1342 demonstrated high potency in previous screens and were selected as ASO target regions for further evaluation. ASOs targeting these regions were selected for additional studies to determine half maximal inhibitory concentrations (IC50) and to confirm potency. ASOs targeting these sites were incubated at concentrations from 0.003 pM to 10 pM with human fibroblasts for 72 hours (gymnosis) and human COMP mRNA was measured as in Example 2. The results from 5 independent experiments are provided in Table 5, which provides sequence and modification information for each ASO tested. All ASOs were synthesized with locked nucleic acid (LNA) modified nucleosides on both 5' and 3' ends with remaining DNA nucleosides positioned in between. Specifically, the "‘ASO ID” column of Table 5 provides the length of each ASO (e.g., “20mer”) followed by the nucleotide position of the 5’ base of the target sequence of each ASO with respect to the human COMP mRNA sequence of SEQ ID NO: 1 (i.e., the start position, e.g., “1044”), followed by the LNA-DNA-LNA modification pattern for each ASO (e.g., 3-14-3. meaning 3 LNA nucleosides in the 5’ segment, 14 DNA nucleosides in the middle gap region, and 3 LNA nucleosides in the 3’ segment). The ASO sequence is provide in Table 5 with LNA nucleosides in lowercase script and DNA nucleosides in uppercase. All ASOs were fully phosphorothioate-modified (i.e., all phosphorothioate intemucleoside linkages).

Table 5. IC50 evaluation of select human COMP ASOs

[0303] Top candidate ASOs 20mer_1047_3-14-3, 18mer_1047_3-12-3, and 16mer_1342_3- 10-3 were identified as having favorable in vitro potency /ICso profiles. Having ASO candidates targeting two different regions of human COMP mRNA potentially enables the evaluation of target specificity in downstream in vivo pharmacology study. These candidates were selected for further evaluation.

Example 6: In vivo biodistribution and tolerability study

[0304] Three top candidate ASOs described and identified in previous examples (20mer_1047_3-14-3, 18mer_1047_3-12-3, and 16mer_1342_3-10-3) were evaluated in vivo in wild ripe mice for biodistribution to chondrocytes and tolerability/toxicity. ASOs were administered subcutaneously at 30 mg/kg twice weekly for 3 weeks beginning around post-natal day 7. PBS vehicle and scramble ASO were included as negative control groups. Mice were sacrificed 24 hours post the last dose. Blood was collected and processed for serum chemistry analysis. Hindlimb, liver, and kidney were fixed in 10% NBF and processed into FFPE blocks for histology and in situ hybridization detection of ASO.

[0305] All three ASOs showed distribution to chondrocytes by in situ hybridization analysis, albeit with stronger signal in articular cartilage chondrocytes and relatively weaker distribution to grow th plate chondrocytes (data not shown). Nevertheless, staining w as apparent in articular and growth plate cells, confirming adequate distribution of ASOs to these target cells.

[0306] All three ASOs demonstrated significant liver toxicity with heightened liver enzyme alanine transaminase (ALT - 23-fold to 64-fold increase) and aspartate aminotransferase (AST - 15-fold to 36-fold increase) and significant reductions in body weight observed after 2 w eeks of ASO administration as compared to PBS vehicle and scrambled ASO negative controls (data not shown). Early unscheduled euthanasia was carried out after two weeks of dosing for 6 out of 7 animals receiving 20mer_1047_3-14-3 and 18mer_1047_3-12-3, as these animals were determined to be moribund. On the other hand, 6 out of 7 animals receiving vehicle or scramble control and 6 out of 7 animals receiving 16mer_1342_3-10-3 completed 3 weeks of dosing.

Example 7: ASO modifications for improving tolerability (in vitro tolerability screen)

[0307] A series of additional further modified ASOs targeting human COMP mRNA were designed to improve tolerability and liver toxicity profiles relative to the ASOs tested in Example 6. ASO modification strategies tested for improving tolerability included: replacement of locked nucleic acid (LNA) nucleosides with 2'-methoxyethoxy (2'MOE) modified nucleosides; incorporation of 2'0Me modified residues at position 2 in the DNA gap; reduction of guanine nucleosides and CG dinucleotides in the ASO sequence; and/or inclusion of 5'-methylcytosine nucleosides in place of all cytosine residues. A total of 41 additional ASOs were designed and synthesized to be screened for improved tolerability and toxicity profiles.

[0308] An in vitro cytotoxicity assay was developed to screen for modified ASOs with improved tolerability/toxicity profiles. Briefly, human HepG2 cells were cultured in 96 well plate and transfected with 0.1 pM modified ASOs via lipofectamine RNAiMAX. Following 24-hour incubation, caspase activation was measured by Caspase-Gio 3/7 assay. 1 pM staurosporine was used as the positive control while lipofectamine reagent only was the negative control. Fold increase in caspase signal was calculated relative to lipofectamine reagent only control. The in vitro assay demonstrated caspase signal elevation following incubation with 20mer_1047_3-14- 3, 18mer_1047_3-12-3, and 16mer_1342_3-10-3 ASO candidates while minimal change was observed with scramble ASO, which reproduced the in vivo toxicity observation. Potency of all modified ASOs was screened at doses of 1 pM, 3 pM, and 10 pM in human fibroblasts as described in Examples 2, 3, and 4.

[0309] Table 6 shows in vitro potency in terms of average % knockdown of human COMP mRNA at each ASO concentration tested (“Avg %KD”, n = 2) and in vitro tolerability in terms of fold change relative to control conditions (“Avg Tox”, n = 4) for 41 modified ASOs designed according to the present example. In Table 6, the “ASO ID"’ column provides sequence and modification information for each ASO tested. Specifically, the “ASO ID” column of Table 6 provides the length of each ASO (e.g., “15mef ’) followed by the nucleotide position of the 5’ base of the target sequence of each ASO with respect to the human COMP mRNA sequence of SEQ ID NO: 1 (i.e., the start position, e.g., “1048”), followed by the modification pattern for each ASO, also shown in the “ASO Sequence” column, where uppercase script denotes DNA nucleosides, lowercase script denotes LNA nucleosides, italicized script denotes MOE nucleosides, and bold script denotes OMe nucleosides. All ASOs shown in Table 6 were synthesized with 5- methylcytosine in place of all cytosine residues except for toxicity' control ASOs (Scramble ASO, 20mer_1047_3-14-3, 16mer_1342_3-10-3, and 18mer_1047_3-12-3), and all ASOs were fully phosphorothioate modified.

Table 6. In vitro tolerability screen results

[0310] 15 modified ASOs with improved in vitro tolerability were further selected to determine half maximal inhibitory concentrations (ICso) and to confirm potency. ICso was measured as described in example 5. The results from 3 independent experiments are provided in Table 7. In Table 7, the “ASO ID” column provides the length of each ASO (e.g., “15mer”) followed by the nucleotide position of the 5' base of the target sequence of each ASO with respect to the human COMP mRNA sequence of SEQ ID NO: 1 (i.e., the start position, e.g.. “1048”). followed by the modification pattern for each ASO, also shown in the “ASO Sequence” column, where uppercase script denotes DNA nucleosides, lowercase script denotes LNA nucleosides, italicized script denotes MOE nucleosides, and bold script denotes OMe nucleosides. All ASOs shown in Table 7 were synthesized with 5 -methylcytosine in place of all cytosine residues except for control/comparator ASOs (16mer_1342_3-10-3 and 18mer_1047_3-12-3), and all ASOs were fully phosphorothioate modified.

Table 7. Potency evaluation of further modified ASOs

[0311] The screen identified 6 ASOs with improved in vitro cytotoxicity without affecting in vitro potency (i.e., less than 2-fold reduction in potency compared to parental ASO prior to present modification and cytotoxicity signal lower than parent form and between 1 to 1.5 fold increase relative to vehicle control).

Example 8: In vivo tolerability of modified ASOs

[0312] The ASOs with improved tolerability and comparable potency identified in Example 7 were further evaluated to confirm in vivo tolerability following repeat dosing in wild type mice. Animals were administered subcutaneously at 25 mg/kg ASO twice weekly for 5 doses. Mice were sacrificed 24 hours post the last dose. Blood was collected and processed for serum chemistry analysis. Hindlimb, liver, and kidney were fixed in 10% NBF and processed into FFPE blocks for potential histology and detection of ASO. Three out of six ASO treatments were tolerated following 2 weeks of subcutaneous dosing beginning at around post-natal day 14 (i.e., absence of unscheduled death/early euthanasia and/or dose discontinuation). Table 8 shows results of each of six ASOs tested according the present example, with in vitro potency (ICso). in vitro tolerability (‘Tol”), 2-week in-life tolerability observation (”2Wk Tol”), and serum chemistry results (ALT = alanine aminotransferase, a common indicator of liver stress or disease; AST = aspartate aminotransferase, a common indicator of liver stress or disease; ALP = alkaline phosphatase, a common indicator of liver stress or disease; and Creatinine = a common indicator of kidney stress or disease) for each ASO.

Table 8. In vivo tolerability of modified ASOs

[0313] Modified ASO 16mer_1047_3-10-3_MOE5’ demonstrated a favorable tolerability profile with minimal elevation in serum chemistry⁷ following repeat dosing in WT mice compared to scramble ASO control.

Example 9. Knockdown of human COMP mRNA in PSACH mouse model

[0314] The heterozygous human mutant COMP (“h-MT-COMP”) mouse model expresses h- MT-COMP and wild type mouse COMP to mimic the autosomal dominant phenotype in the PSACH disorder. The mutation used in this model is del469D (in the human mutant COMP^c'h- MT-COMP”). This is the most common mutation in PSACH patients, occurring in approximately 30% of PSACH patients. The mouse model employs tetracycline-inducible expression in tissues expressing type II collagen. Human mutant COMP (h-MT-COMP) expression is driven by a tetracycline responsive element promoter, providing tissue selective expression controlled by type II collagen promoter-regulated TET-On tetracycline transactivator expression. This model provides exaggerated expression with up to 6-fold higher levels of h-MT-COMP compared to wild ty pe mouse COMP.

[0315] A pharmacological investigation of 16mer 1047 3-10-3 MOE5’ and/or other candidate modified ASOs described herein will be conducted to evaluate whether unconjugated ASO at physiologically meaningful and supraphysiological dosing and frequency can achieve adequate knockdown of human COMP expression in the h-MT-COMP PSACH mouse model. Subcutaneous doses of ASO may be administered at a low dose, e.g., 10 mg/kg to 30 mg/kg twice weekly for 3 weeks and at one or more higher doses, e.g.. 30 mg/kg to 60 mg/kg twice weekly or three times weekly for 3 weeks beginning, e.g., at post-natal day 7. Animals may be sacrificed approximately 48 hours after the final dose and evaluated for changes in human MT-COMP mRNA level and protein level, and ASO distribution in chondrocytes may be evaluated. Additional phenotypic assessments by immunohistochemistry may be performed to evaluate modulations of PSACH cellular markers in chondrocytes including apoptosis, inflammation, proliferation, and autophagy⁷. ANTISENSE COMPOUND SEQUENCES

[0316] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0317] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing Examples are therefore to be considered in all respects illustrative rather than limiting the disclosure and additional embodiments described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the disclosure. Scope of the disclosure is thus indicated by the appended claims and enumerated embodiments, and all changes that come within the meaning and range of equivalency of the claims and enumerated embodiments are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. An antisense oligonucleotide (ASO) comprising 14 to 20 linked nucleosides having a nucleobase sequence that is complimentary to a target nucleic acid sequence in SEQ ID NO: 1, wherein the target nucleic acid sequence comprises 14 to 20 contiguous nucleotides and has a start position of 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1340, 1341, 1342, 1343, or 1344 of SEQ ID NO: 1, and wherein the ASO comprises one or more of: a. a gap segment consisting of linked deoxynucleosides; b. a 5’ segment consisting of at least 2 linked nucleosides; c. a 3’ segment consisting of at least 2 linked nucleosides; d. at least one phosphorothioate intemucleoside linkage; e. at least one nucleoside comprising a modified sugar: and f. at least one nucleoside comprising a modified nucleobase.

2. The ASO of claim 1, wherein the gap segment is positioned between the 5’ segment and the 3’ segment.

3. The ASO of claim 1 or 2, wherein the gap segment comprises 5 to 15 linked nucleosides.

4. The ASO of any one of claims 1-3, wherein the 3’ segment comprises 2-5 linked nucleosides.

5. The ASO of any one of claims 1-4, wherein the 5’ segment comprises 2-5 linked nucleosides.

6. The ASO of any one of claims 1-5, wherein at least one nucleoside of the 5’ segment and at least one nucleoside of the 3’ segment comprises a modified sugar.

7. The ASO of any one of claims 1-6, wherein each nucleoside of the 5’ segment and each nucleoside of the 3’ segment comprises a modified sugar.

8. The ASO of any one of claims 6 or 7, wherein the modified sugar comprises a bicyclic sugar.

9. The ASO of claim 8, wherein the bicyclic sugar is selected from the group consisting of: 4’-(CH₂)— O-2’ (LNA); 4’-(CH₂)2— O-2’ (ENA); and 4’-CH(CH₃)— O-2’ (cEt).

10. The ASO of any one of claims 1-9, wherein each intemucleoside linkage is a phosphorothioate intemucleoside linkage.

11. The ASO of any one of claims 1-10, comprising a 5 -methylcytosine nucleobase in place of a non-5-methyl cytosine residue.

12. The ASO of any one of claims 1-11, comprising a 2-11-2 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1310, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

13. The ASO of any one of claims 1-11, comprising a 3-10-2 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1311, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

14. The ASO of any one of claims 1-11, comprising a 2-11-2 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1811, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

15. The ASO of any one of claims 1-11, comprising a 3-10-3 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1297, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

16. The ASO of claim 15, wherein all cytosine nucleosides are replaced with 5-methylcytosine nucleosides and comprising a 2’OMe-modified nucleoside at position 2 in the DNA gap.

17. The ASO of any one of claims 1-11, comprising a 3-10-3 MOE-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1299, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

18. The ASO of any one of claims 1-11, comprising a 3-10-3 LNA-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1300, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

19. The ASO of any one of claims 1-11, comprising a 3-10-3 MOE-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1301, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

20. The ASO of any one of claims 1-11, comprising a 3-10-3 LNA-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1456, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages.

21. The ASO of claim 20, wherein all cytosine nucleosides are replaced with 5-methylcytosine nucleosides and comprising a 2’OMe-modified nucleoside at position 2 in the DNA gap.

22. The ASO of any one of claims 1-11, comprising a 3-10-3 LNA-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1457, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

23. The ASO of any one of claims 1-11, comprising a 3-10-3 MOE-DNA-LNA gapmer having a nucleic acid sequence according to SEQ ID NO: 1458, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

24. The ASO of any one of claims 1-11, comprising a 3-10-3 MOE-DNA-MOE gapmer having a nucleic acid sequence according to SEQ ID NO: 1460, wherein all intemucleoside linkages are phosphorothioate intemucleoside linkages, and all cytosine nucleosides are replaced with 5-methylcytosine nucleosides.

25. A composition comprising the ASO of any one of claims 1-24 or salt thereof and a pharmaceutically acceptable carrier.

26. The composition of claim 25, wherein the composition is a pharmaceutical formulation.

27. A method of reducing the level of a COMP RNA in a cell, comprising contacting the cell with the ASO of any one of claims 1-24 or the composition of claim 25 or 26, thereby reducing the level of the COMP RNA.

28. A method of inhibiting expression of cartilage oligomeric matrix protein in a cell, comprising contacting the cell with the ASO of any one of claims 1-24 or the composition of claim 25 or 26, thereby reducing expression of cartilage oligomeric matrix protein in the cell.

29. A method of inhibiting accumulation of cartilage oligomeric matrix protein in a cell comprising a mutant COMP allele, the method comprising contacting the cell with the ASO of any one of claims 1-24 or the composition of claim 25 or 26, thereby reducing expression of cartilage oligomeric matrix protein in the cell.

30. The method of any one of claims 27-29, wherein the cell is a growth plate cell.

31. The method of any one of claims 27-29, wherein the cell is a tendon cell.

32. The method of any one of claims 27-29, wherein the cell is a cartilage cell.

33. The method of any one of claims 27-32, wherein the cell is in vitro.

34. The method of any one of claims 27-32, wherein the cell is in a subject.

35. The method of claim 31. wherein the subject is a human and the ASO or composition is administered to the subject.

36. A method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject, comprising administering to the subject an ASO of any one of claims 1-24 or the composition of claim 25 or 26, thereby treating, preventing, or ameliorating the disease.

37. The method of claim 36, wherein the disease is pseudoachondroplasia (PSACH).

38. The method of claim 36, wherein the disease is multiple epiphyseal dysplasia (MED).

39. An ASO according to any one of claims 1-24 or the composition of claim 25 or 26 for use in a method of treating, preventing, or ameliorating a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject.

40. The ASO for use of claim 39, wherein the disease is pseudoachondroplasia.

41. The ASO for use of claim 39, wherein the disease is multiple epiphyseal dysplasia.

42. Use of an ASO of any one of claims 1-24 or the composition of claim 25 or 26 for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells in a subject.

43. Use of an ASO of any one of claims 1-24 or the composition of claim 25 or 26 for the preparation of a medicament for the treatment of a disease associated with retention of cartilage oligomeric matrix protein in chondrocyte cells of a subject.

44. The use of claim 42 or 43, wherein the disease is pseudoachondroplasia.

45. The use of claim42 or 43, wherein the disease is multiple epiphyseal dysplasia.

46. The method of any one of claims 35-38, wherein the administration is intramuscular.

47. The method of any one of claims 35-38, wherein the administration is subcutaneous.

48. The method of any one of claims 35-38, wherein the administration is intravenous or intraperitoneal.

49. The method of any one of claims 35-38 or 46-47, comprising administering to the subject a second pharmaceutical agent for treating, preventing, or ameliorating one or more symptoms of PSACH or MED.

50. The method of claim 49, wherein the second pharmaceutical agent comprises an antiinflammatory compound.

51. The method of claim 50, wherein the anti-inflammatory compound is a non-steroidal antiinflammatory (NSAID) compound.

52. The method of claim 50 or 51, wherein the second pharmaceutical agent comprises indomethacin, ibuprofen, or naproxen.

53. The method of claim 49, wherein the second pharmaceutical agent comprises an antioxidant compound.

54. The method of claim 53, wherein the second pharmaceutical agent comprises omega-3- acid ethyl esters.

55. The method of claim 53, wherein the second pharmaceutical agent comprises resveratrol.

56. The method of claim 49, wherein the second pharmaceutical agent comprises diacerein or cordycepin.