Treatment of osteoarthritis using cartilage interlayer protein 2 (CILP2) inhibitors

Abstract

The present disclosure is generally directed to treating a subject having osteoarthritis or confronting risk of developing osteoarthritis by administering an inhibitor of cartilage interlayer protein 2 (CILP2) to the subject.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to treating a subject having osteoarthritis or at risk of developing osteoarthritis by administering a Cartilage Intermediate Layer Protein 2 (CILP2) inhibitor to the subject, and to methods of identifying a subject having an increased risk of developing osteoarthritis. BACKGROUND

[0002] Osteoarthritis (OA) is the most common type of arthritis and affects millions of people worldwide. OA is a degenerative joint disease that affects all tissues in the joint. Damage to the articular cartilage, a soft tissue layer that covers the ends of the bones, occurs early in the disease and is thought to be the onset of irreversible joint damage. While OA can affect any joint, the most commonly affected joints are the knee, hip, hand joints, and spine. OA can be diagnosed through physical examination and can include imaging tests (X-rays) to assess severity, as well as laboratory tests such as, for example, blood or urine tests and joint fluid analysis. Symptoms of OA include: i) joint pain, ii) stiffness in the joint upon waking or after inactivity, iii) tenderness to light pressure on the joint or near the joint, iv) loss of flexibility, v) a feeling of grating or catching when using the joint, vi) bony spurs can form around the affected joint, and vii) swelling (inflammation of the soft tissue around the joint). Risk factors for OA include: i) being older (e.g., the risk of OA increases with age), ii) being obese (increased body weight puts increased pressure on weight-bearing joints, and proteins produced by fat tissue can cause harmful inflammation in the joints and around them), iii) joint injury, iv) gender (women are more likely to develop OA), v) repeated stress on the joint, vi) genetics, vii) skeletal deformities, and viii) some metabolic diseases such as diabetes and hemochromatosis.

[0003] Cartilage intermediate layer protein 2 (CILP2) is expressed by articular chondrocytes. CILP2 is highly homologous to cartilage intermediate layer protein 1 (CILP1), which is expressed in the intermediate zone of articular cartilage and is associated with cartilage degenerative disease. CILP2 is expressed on the surface of mouse articular cartilage during development. With maturation, CILP2 expression localizes around the central zone of the articular cartilage and meniscus. In humans, CILP2 is demonstrated to be proteolytically processed, N-glycosylated, and present in articular cartilage. In a surgical-induced OA mouse model, CILP1 expression is upregulated, but CILP2 gene expression is downregulated, demonstrating a differential response to joint instability leading to a reduction in CILP2 protein. Ultrastructural analysis also suggests that CILP2 can be associated with collagen VI microfibrils and can mediate interactions between matrix components in the regional and interregional extracellular matrix. mRNA expression analysis indicates that CILP1 and CILP2 are most abundantly expressed in cartilage tissue, with expression also detected in muscle and heart. (Bernardo et al., J. Biol. Chem., 2011, 286, 37758-37767). SUMMARY

[0004] The present disclosure provides methods of treating a subject having OA or at risk of developing OA, the methods comprising administering to the subject a CILP2 inhibitor.

[0005] The present disclosure also provides methods of treating a subject having OA or at risk of developing OA by administering an OA therapeutic or OA therapy, the methods comprising: determining or having determined whether the subject has a CILP2 variant nucleic acid molecule by: obtaining or having obtained a biological sample from the subject; and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype comprising a CILP2 variant nucleic acid molecule; and administering or continuing to administer to the subject an amount of the OA therapeutic or OA therapy that is the same as or less than a standard dosage amount and / or administering to the subject a CILP2 inhibitor to the subject that is a CILP2 reference; administering or continuing to administer to the subject an amount of the OA therapeutic or OA therapy that is the same as or less than a standard dosage amount and / or administering to the subject a CILP2 inhibitor to the subject that is heterozygous for the CILP2 variant nucleic acid molecule; or administering or continuing to administer to the subject a standard dosage amount of the OA therapeutic or OA therapy to the subject that is homozygous for the CILP2 variant nucleic acid molecule; wherein the presence of the CILP2 variant nucleic acid molecule indicates a reduced risk of developing OA in the subject.

[0006] This disclosure also provides a method for identifying subjects at increased risk of developing OA, the method comprising: determining or having determined the presence or absence of a CILP2 variant nucleic acid molecule in a biological sample obtained from the subject; wherein: when the subject is a CILP2 reference, the subject's risk of developing OA is increased; and when the subject is heterozygous or homozygous for the CILP2 variant nucleic acid molecule, the subject's risk of developing OA is reduced.

[0007] This disclosure also provides OA therapeutic agents for treating or preventing OA in subjects with CILP2 variant nucleic acid molecules.

[0008] This disclosure also provides CILP2 inhibitors for the treatment or prevention of OA in subjects who are CILP2 reference or heterozygous for CILP2 variant nucleic acid molecules. Attached Figure Description

[0009] Figure 1 The representative associations between CILP2 pLoF and missense variants and the minimum joint space width (mJSW) phenotype derived from DXA images are shown (Fig. A), as well as the representative association between CILP2 pLoF variants and knee osteoarthritis (OA) (Fig. B).

[0010] Figure 2 The association between the CILP2 pLoF variant and OA in various joints is shown.

[0011] Figure 3 The association between the pLoF variant in CILP2 and a reduced risk of knee replacement was demonstrated. Detailed Implementation

[0012] Various terms relating to aspects of this disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0013] Unless otherwise expressly stated, it is never intended that any method or aspect stated herein require its steps to be performed in a particular order. Therefore, in the claims or description, where a method claim does not explicitly state that the steps are limited to a particular order, it is never intended in any way to infer the order. This applies to any possible non-expressive basis of interpretation, including logical matters relative to the arrangement of steps or the flow of operations, the general meaning derived from grammatical organization or punctuation, or the numbering or type of aspects described in the description.

[0014] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural referents.

[0015] As used herein, the term“about” means that the recited value is an approximation and that slight variations do not significantly impact the practice of the disclosed embodiments. In the case of using numerical values, unless the context indicates otherwise, the term“about” means that the value can vary by ±10% and still be within the scope of the disclosed embodiments.

[0016] As used herein, the term“comprising” can be replaced with“consisting of’ or“consisting essentially of’ as desired in a particular embodiment.

[0017] As used herein, the term“nucleic acid,”“nucleic acid molecule,”“nucleic acid sequence,”“polynucleotide,” or“oligonucleotide” can include a polymeric form of nucleotides of any length, can include both DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. A strand of nucleic acid also refers to its complementary sequence.

[0018] As used herein, the term“subject” includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horses, cattle, and swine), companion animals (such as, for example, dogs and cats), laboratory animals (such as, for example, mice, rats, and rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under medical care.

[0019] According to observations in this disclosure, rare CILP2 variant nucleic acid molecules (regardless of whether these variants are homozygous or heterozygous in a given subject) are associated with a reduced risk of developing OA. Specifically, the minimum joint space width (mJSW) phenotype of the knee joint was derived from UK Biobank DXA imaging (N=51,327) and used for target discovery and validation. mJSW showed expected associations with sex, height, age, and body mass index (BMI), and also showed associations between the left and right knees and between baseline and follow-up scans (n=4,000). mJSW was reduced in both male (p=2.1e-4, N=24,567) and female (p=1.9e-4, N=25,673) OA cases. CILP2 pLoF burden (AAF < 1%) was correlated with an increase in mJSW of 0.78 standard deviations (SD) units (0.5 mm; p = 5.7e-9), and a protective effect against OA was observed (odds ratio = 0.56; p = 8.3e-4). CILP2 variant nucleic acid molecules are believed to be independent of human OA. Therefore, CILP2 inhibitors can be used to treat subjects who are heterozygous for CILP2 as a reference or against CILP2 variant nucleic acid molecules, thereby inhibiting or preventing OA, reducing or preventing its symptoms, and / or blocking or preventing the development of symptoms. It is also believed that such subjects with OA can be further treated with one or more OA therapeutic agents or OA therapies that treat or inhibit OA. Additionally, this disclosure provides a method for identifying or stratifying the risk of developing OA in such subjects, or diagnosing an increased risk of developing OA, using the presence or absence of CILP2 variant nucleic acid molecules in subjects.

[0020] For the purposes of this disclosure, any particular subject, such as a human, may be classified as having one of three CILP2 genotypes: i) CILP2 reference; ii) heterozygous for a CILP2 variant nucleic acid molecule; or iii) homozygous for a CILP2 variant nucleic acid molecule. A subject is a CILP2 reference when it does not have a copy of a CILP2 variant nucleic acid molecule. A subject is heterozygous for a CILP2 variant nucleic acid molecule when it has a single copy of a CILP2 variant nucleic acid molecule. A subject is homozygous for a CILP2 variant nucleic acid molecule when it has two copies of a CILP2 variant nucleic acid molecule.

[0021] In any of the embodiments described herein, the CILP2 variant nucleic acid molecule can be any nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule produced from an mRNA molecule) that encodes a CILP2 variant polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. A subject having a CILP2 polypeptide with partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for CILP2. In some embodiments, the CILP2 variant nucleic acid molecule results in reduced or aberrant expression or activity of a CILP2 mRNA or polypeptide. In some embodiments, the CILP2 variant nucleic acid molecule results in a CILP2 polypeptide that has reduced or aberrant activity compared to a reference CILP2. In some embodiments, the CILP2 variant nucleic acid molecule results in a CILP2 polypeptide that has reduced or aberrant activity compared to a reference CILP2. In vitro In some embodiments, the CILP2 variant nucleic acid molecule is a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant that encodes a truncated CILP2 variant polypeptide. In some embodiments, the CILP2 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the CILP2 variant nucleic acid molecule comprises a single nucleotide polymorphism (SNP). In some embodiments, the CILP2 variant nucleic acid molecule comprises a variation in the coding region. In some embodiments, the CILP2 variant nucleic acid molecule does not comprise a variation in the non-coding region, but does comprise a variation in the splice acceptor region (two bases before the start of any exon other than the first exon). In some embodiments, the CILP2 variant nucleic acid molecule results in or is predicted to result in premature truncation of the CILP2 polypeptide compared to a reference CILP2. In some embodiments, the CILP2 variant nucleic acid molecule is a variant that is predicted to impair protein function as by a prediction algorithm such as Polyphen, SIFT, or similar algorithm (and thus, in this case, is protective for humans). In some embodiments, the CILP2 variant nucleic acid molecule is a variant that results in or is predicted to result in a non-synonymous amino acid substitution in the CILP2 nucleic acid molecule and has an allelic frequency of less than 1 per 100 alleles in the population from which the subject is selected. In some embodiments, the CILP2 variant nucleic acid molecule is any rare missense variant (allelic frequency < 0.1%; or 1 per 1,000 alleles), or any splice-site variant, stop-gain variant, start-loss variant, stop-loss variant, frameshift variant, or in-frame indel variant, or other frameshift CILP2 variant. In vitro In some embodiments, the CILP2 variant nucleic acid molecule is a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, or a variant that encodes a truncated CILP2 variant polypeptide. In some embodiments, the CILP2 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the CILP2 variant nucleic acid molecule comprises a single nucleotide polymorphism (SNP). In some embodiments, the CILP2 variant nucleic acid molecule comprises a variation in the coding region. In some embodiments, the CILP2 variant nucleic acid molecule does not comprise a variation in the non-coding region, but does comprise a variation in the splice acceptor region (two bases before the start of any exon other than the first exon). In some embodiments, the CILP2 variant nucleic acid molecule results in or is predicted to result in premature truncation of the CILP2 polypeptide compared to a reference CILP2. In some embodiments, the CILP2 variant nucleic acid molecule is a variant that is predicted to impair protein function as by a prediction algorithm such as Polyphen, SIFT, or similar algorithm (and thus, in this case, is protective for humans). In some embodiments, the CILP2 variant nucleic acid molecule is a variant that results in or is predicted to result in a non-synonymous amino acid substitution in the CILP2 nucleic acid molecule and has an allelic frequency of less than 1 per 100 alleles in the population from which the subject is selected. In some embodiments, the CILP2 variant nucleic acid molecule is any rare missense variant (allelic frequency < 0.1%; or 1 per 1,000 alleles), or any splice-site variant, stop-gain variant, start-loss variant, stop-loss variant, frameshift variant, or in-frame indel variant, or other frameshift CILP2 variant.

[0022] In any of the embodiments described herein, a CILP2 variant genomic nucleic acid molecule can comprise one or more variants at any position of chromosome 19 (i.e., positions 19,538,248-19,546,659) using the nucleotide sequence of the CILP2 reference genomic nucleic acid molecule in the GRCh38 / hg38 human genome assembly (see ENSG00000160161.9, ENST ENST00000291495 annotated in the Ensembl database (website: World Wide Web "http: / / useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000160161;r=19:19538248-19546659) as the reference sequence. The sequences of the CILP2 genomic nucleic acid molecules provided in these transcripts are merely exemplary sequences. Other sequences of CILP2 genomic nucleic acid molecules are possible.

[0023] In any of the embodiments described herein, a CILP2 variant nucleic acid molecule can comprise one or more genetic variations in any of the genomic nucleic acid molecules listed in Table 1 (referring to chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0024] For a subject genotyped or determined to be CILP2 reference, such subject has an increased risk of developing OA. For a subject genotyped or determined to be CILP2 reference or heterozygous for a CILP2 variant nucleic acid molecule, such subject can be treated with a CILP2 inhibitor.

[0025] In any of the embodiments described herein, a subject being prevented from developing OA by administration of a CILP2 inhibitor can be any subject at risk of developing OA, including but not limited to a subject with a genetic predisposition to developing OA. In some embodiments, a CILP2 inhibitor can be administered to a subject with OA to prevent the development of OA in a subject already with OA. In any of the embodiments described herein, the methods can be used to improve OA.

[0026] In any of the embodiments described herein, a CILP2 predicted loss-of-function polypeptide can be any CILP2 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0027] Any one or more (i.e., any combination) of the CILP2 variant nucleic acid molecules described herein can be used in any of the methods described herein to determine whether a subject has an increased or decreased risk of developing OA. Combinations of particular variants can form a mask used to statistically analyze the particular correlation of CILP2 to an increased or decreased risk of developing OA. In some embodiments, a mask used to statistically analyze the particular correlation of CILP2 to an increased or decreased risk of developing OA can exclude any one or more of these CILP2 variant nucleic acid molecules described herein.

[0028] In any of the embodiments described herein, the subject can have OA. In any of the embodiments described herein, the subject can be at risk of developing OA.

[0029] The present disclosure provides methods of treating a subject having OA or at risk of developing OA, the methods comprising administering to the subject a CILP2 inhibitor.

[0030] The present disclosure also provides methods of preventing or delaying the need for a joint replacement, such as a knee replacement and / or a hip replacement, in a subject, the methods comprising administering to the subject a CILP2 inhibitor. For example, such a delay can be months to years. For example, a CILP2 inhibitor can be administered to a subject at increased risk of needing a joint replacement (e.g., knee and / or hip) to delay the time period before the actual joint replacement surgery or to prevent the actual joint replacement altogether. In some embodiments, the need for a joint replacement surgery is a result of osteoarthritis.

[0031] In some embodiments, the CILP2 inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of a CILP2 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within a CILP2 genomic nucleic acid molecule or mRNA molecule and reduces expression of a CILP2 polypeptide in a cell of a subject. In some embodiments, the CILP2 inhibitor comprises an antisense molecule that hybridizes to a CILP2 genomic nucleic acid molecule or mRNA molecule and reduces expression of a CILP2 polypeptide in a cell of a subject. In some embodiments, the CILP2 inhibitor comprises an siRNA that hybridizes to a CILP2 genomic nucleic acid molecule or mRNA molecule and reduces expression of a CILP2 polypeptide in a cell of a subject. In some embodiments, the CILP2 inhibitor comprises an shRNA that hybridizes to a CILP2 genomic nucleic acid molecule or mRNA molecule and reduces expression of a CILP2 polypeptide in a cell of a subject. An exemplary siRNA is sc-60386 (Santa Cruz Biotechnology, Inc.).

[0032] Inhibitory nucleic acid molecules can include RNA, DNA, or both RNA and DNA. Inhibitory nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence, such as a heterologous nucleic acid sequence in a vector, or a heterologous label. For example, an inhibitory nucleic acid molecule can be within a vector comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence, or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. Inhibitory nucleic acid molecules can also be linked or fused to a heterologous label. Labels can be directly detectable, such as, for example, fluorophores, or indirectly detectable, such as, for example, haptens, enzymes, or fluorophore quenchers. Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. Labels can also be, for example, chemiluminescent species; metal-containing species; or enzymes, where enzyme-dependent secondary signal generation occurs. The term "label" can also refer to a "tag" or hapten, which can be selectively bound to a conjugate molecule, such that the conjugate molecule, when subsequently added with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag to bind to an avidin or streptavidin conjugate of horseradish peroxidase (HRP) for detection using calorimetric substrates, such as, for example, tetramethylbenzidine (TMB), or fluorescent substrates for examination to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG, or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or Fc portions of immunoglobulins. A variety of labels include, for example, particles, fluorophores, haptens, enzymes and calorimetric, fluorescent and chemiluminescent substrates thereof, and other labels.

[0033] Inhibitory nucleic acid molecules can include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified bases, sugars, or phosphate groups, or nucleotides incorporating non-natural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

[0034] Inhibitory nucleic acid molecules can also comprise one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides containing modifications to the base, sugar, or phosphate moieties. Modifications to the base moiety include, but are not limited to, the natural and synthetic modifications of A, C, G, and T / U and different purine or pyrimidine bases such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2- aminoadenine-9-yl. Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 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 uracil and cytosine, 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 (such as, for example, 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0035] Nucleotide analogs can also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose and synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted C 1-10 alkyl or C 2-10 alkenyl and C 2-10 alkynyl. Exemplary 2' sugar modifications also include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2, and -O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are independently between 1 and about 10. Other modifications at the 2' position include, but are not limited to, C 1-10alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl, or O-arylalkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of an oligonucleotide, or groups for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A similar modification of the sugar moiety, can occur at the 3' position of an anomer of a sugar at the 3' terminus of the oligonucleotide, or when a 2'-5' linkage is involved, the 5' position of the 5' terminal nucleotide. The modified sugar can also include those sugars that contain a modification including, for example, CH2and S, at the bridge oxo. The nucleotide sugar analogs can also have sugar mimics in place of the furanose sugar.

[0036] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, modified phosphate moieties in which the linkage between two nucleotides contains a phosphorothioate, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkyl phosphotriester, a methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, a phosphinothioate, an amino-phosphoramidate, a thiono-carbapenam, a sulfhydryl, an alkyl phosphonate, a boranophosphate, and the like. These phosphate or modified phosphate linkages between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain an inverted polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Nucleotide replacements also include peptide nucleic acids (PNAs).

[0037] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first one to seven nucleotides at the 5' and 3' ends each have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first five nucleotides at the 5' and 3' ends each have a 2'-MOE modification. In some embodiments, the first one to seven nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, each backbone linkage between nucleotides is a phosphorothioate linkage.

[0038] In some embodiments, the siRNA molecule has terminal modifications. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, a non-hydrolysable 5'-phosphate ester analog, such as 5'-(E)-vinylphosphonate, is used.

[0039] In some embodiments, the siRNA molecule has backbone modifications. In some embodiments, modified phosphodiester groups linking consecutive ribonucleosides have been shown to enhance the stability and In vivo bioavailability of siRNAs. Non-ester groups of the phosphodiester linkage (-OH, =0) can be replaced by sulfur, boron, or acetate, resulting in phosphorothioate, boranophosphate, and phosphonooxyacetate linkages. Additionally, substitution of the phosphodiester groups with phosphotriesters can facilitate cellular uptake of siRNAs and remain in serum components by eliminating their negative charge. In some embodiments, the siRNA molecule has sugar modifications. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), whereby the 2'-hydroxyl group can act as a nucleophile and attack the adjacent phosphodiester bond. Such alternatives include 2'-0-methyl, 2'-0-methoxyethyl, and 2'-fluoro modifications.

[0040] In some embodiments, the siRNA molecule has base modifications. In some embodiments, the bases can be substituted with modified bases, such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0041] In some embodiments, the siRNA molecule is conjugated to a lipid. The lipid can be conjugated to the 5' or 3' end of the siRNA to improve their In vivo bioavailability by allowing them to associate with serum lipoproteins. Representative lipids include, but are not limited to, cholesterol and vitamin E, as well as fatty acids such as palmitate and tocopherol.

[0042] In some embodiments, a representative siRNA has the following formula: sense: mN*mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN / i2FN / 32FN / antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N wherein: "N" is a base; "2F" is a 2'-F modification; "m" is a 2'-0-methyl modification, "I" is an internal base; and "*" is a phosphorothioate backbone linkage.

[0043] In any of the embodiments described herein, the inhibitory nucleic acid molecule can be administered, for example, as a one to two hour intravenous infusion or subcutaneous injection. In any of the embodiments described herein, the inhibitory nucleic acid molecule can be administered at a dose level ranging from about 50 mg to about 900 mg, about 100 mg to about 800 mg, about 150 mg to about 700 mg, or about 175 mg to about 640 mg (2.5 mg / kg to 9.14 mg / kg; 92.5 to 338 mg / m 2 mg / kg dose level to mg / m 2 dose level based on an assumption of a body weight of 70 kg and a mg / kg dose level to mg / m

[0044] The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral or non-viral vector capable of transporting nucleic acid molecules. In some embodiments, the vector is a plasmid or a cosmid (such as, for example, a circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art.

[0045] The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or an excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipidic helices, and lipidic microtubes. The carrier can include a buffered salt solution, such as PBS, HBSS, and the like.

[0046] In some embodiments, the CILP2 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks at one or more recognition sequences or a DNA binding protein that binds to a recognition sequence within a CILP2 genomic nucleic acid molecule. The recognition sequence can be located within the coding region of the CILP2 gene, or within a regulatory region that affects gene expression. The recognition sequence for the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence can comprise or be proximal to the start codon of the CILP2 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each targeting a nuclease recognition sequence comprising or proximal to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence comprising or proximal to the start codon and one targeting a nuclease recognition sequence comprising or proximal to the stop codon, wherein cleavage by the nuclease agents can result in a deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break into a desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.

[0047] Suitable nuclease agents and DNA binding proteins for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary and include, for example, recognition sequences of about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALENs, and about 20 bp for CRISPR / Cas guide RNAs.

[0048] In some embodiments, a CRISPR / Cas system can be used to modify a CILP2 genomic nucleic acid molecule within a cell. The methods and compositions disclosed herein can employ a CRISPR-Cas system for site-directed cleavage of a CILP2 nucleic acid molecule by utilizing a CRISPR complex comprising a guide RNA (gRNA) complexed with a Cas protein.

[0049] Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with a gRNA. Cas proteins can also comprise nuclease domains (such as, for example, a DNAse domain or an RNAse domain), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpfl proteins (such as, for example, FnCpfl). Cas proteins can have full cleavage activity to generate a double-stranded break in a CILP2 genomic nucleic acid molecule, or they can be nickases that generate a single-stranded break in a CILP2 genomic nucleic acid molecule. Additional examples of Cas proteins include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csy l, Csy2, Csy3, Cse l (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc l, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr l, Cmr3, Cmr4, Cmr5, Cmr6, Csb l, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csx l, Csxl5, Csf l, Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof. In some embodiments, Cas systems, for example Casl2a, can have multiple gRNAs encoded as a single crRNA. Cas proteins can also be operably linked as a fusion protein with a heterologous polypeptide. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. Cas proteins can be provided in any form. For example, Cas proteins can be provided in the form of a protein (such as a Cas protein complexed with a gRNA). Alternatively, Cas proteins can be provided in the form of a nucleic acid molecule, such as an RNA or DNA, encoding a Cas protein.

[0050] In some embodiments, a targeted genetic modification of a CILP2 genomic nucleic acid molecule can be made by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in the CILP2 genomic nucleic acid molecule. The gRNA recognition sequence can include or be proximal to a start codon of the CILP2 genomic nucleic acid molecule or a stop codon of the CILP2 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon or the stop codon.

[0051] A gRNA recognition sequence within a target genomic locus in a CILP2 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by a Cas9 nuclease. A typical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. A gRNA can transport Cas9 to any location in the genome for gene editing, but editing can not occur at any site other than one where Cas9 recognizes a PAM. Additionally, 5'-NGA-3' can serve as an efficient non-canonical PAM in human cells. Generally, the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can flank the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be flanked at the 3' end by a PAM. In some embodiments, the gRNA recognition sequence can be flanked at the 5' end by a PAM. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5, or three base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when using Cas9 from Streptococcus pyogenes (SpCas9) or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and immediately 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide and immediately 5' of the gRNA recognition sequence of the complementary strand of the target DNA. S. pyogenes ) from Streptococcus pyogenes (SpCas9) or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and immediately 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide and immediately 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

[0052] A gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a particular location within a CILP2 genomic nucleic acid molecule. An exemplary gRNA is a gRNA that is effective to direct a Cas enzyme to bind to or cleave a CILP2 genomic nucleic acid molecule, wherein the gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence within the CILP2 genomic nucleic acid molecule. An exemplary gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence present within a CILP2 genomic nucleic acid molecule, the gRNA recognition sequence comprising or being proximal to a start codon or a stop codon. For example, a gRNA can be selected such that it hybridizes to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a start codon or located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from a stop codon. A suitable gRNA can comprise about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, a gRNA can comprise 20 nucleotides.

[0053] A Cas protein and a gRNA form a complex, and the Cas protein cleaves a CILP2 genomic nucleic acid molecule. A Cas protein can cleave a nucleic acid molecule at a site within or outside of a nucleic acid sequence present in a CILP2 genomic nucleic acid molecule that will bind to a DNA targeting segment of a gRNA. For example, formation of a CRISPR complex (comprising a gRNA hybridized to a gRNA recognition sequence and complexed with a Cas protein) can result in cleavage of one or both strands in or near (such as, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) a nucleic acid sequence present in a CILP2 genomic nucleic acid molecule that will bind to a DNA targeting segment of a gRNA.

[0054] Such methods can produce, for example, a CILP2 genomic nucleic acid molecule in which a region of the CILP2 genomic nucleic acid molecule is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus in the CILP2 genomic nucleic acid molecule. Cleavage by the Cas protein can produce two or more double-stranded breaks or two or more single-stranded breaks by contacting the cell with one or more additional gRNAs, such as, for example, a second gRNA that hybridizes to a second gRNA recognition sequence.

[0055] In any of the treatment or prevention methods described herein, the subject receiving treatment can comprise a CILP2 variant nucleic acid molecule. In some embodiments, the subject receiving treatment is heterozygous for a CILP2 variant nucleic acid molecule. In some embodiments, the subject receiving treatment is homozygous for a CILP2 variant nucleic acid molecule. In some embodiments, the subject receiving treatment is CILP2 reference. The CILP2 variant nucleic acid molecule can be any of the CILP2 variant nucleic acid molecules disclosed herein. In some embodiments, the CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (refer to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0056] In some embodiments, the treatment or prevention method further comprises detecting the presence or absence of a CILP2 variant nucleic acid molecule in a biological sample from the subject. In some embodiments, the CILP2 variant nucleic acid molecule can be any of the CILP2 variant nucleic acid molecules disclosed herein. In some embodiments, the CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (refer to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0057] The present disclosure also provides methods of treating a subject with an OA treatment or OA therapy to treat or inhibit OA, wherein the subject has OA or is at risk of developing OA. The methods include determining whether the subject has a CILP2 variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype comprising a CILP2 variant nucleic acid molecule. In embodiments in which the subject is CILP2 reference, the methods further include administering or continuing to administer to the subject an OA treatment or OA therapy in an amount that is the same as or less than a standard dosage amount and / or administering to the subject a CILP2 inhibitor. In embodiments in which the subject is heterozygous for a CILP2 variant nucleic acid molecule, the methods further include administering or continuing to administer to the subject an OA treatment or OA therapy in an amount that is the same as or less than a standard dosage amount and / or administering to the subject a CILP2 inhibitor. In embodiments in which the subject is homozygous for a CILP2 variant nucleic acid molecule, the methods further include administering or continuing to administer to the subject an OA treatment or OA therapy in a standard dosage amount. The presence of a CILP2 variant nucleic acid molecule indicates that the subject is at a reduced risk of developing OA. In some embodiments, the subject is CILP2 reference. In some embodiments, the subject is heterozygous for a CILP2 variant nucleic acid molecule. In some embodiments, the subject is homozygous for a CILP2 variant nucleic acid molecule. In any of the embodiments described herein, a CILP2 inhibitor is an example of an OA treatment. In some embodiments, a CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (refer to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0058] For subjects who are genotyped or determined to be CILP2 reference or heterozygous for a CILP2 variant nucleic acid molecule, a CILP2 inhibitor can be administered to such subjects, as described herein.

[0059] Detecting the presence or absence of a CILP2 variant nucleic acid molecule in a biological sample from a subject and / or determining whether a subject has a CILP2 variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed on a biological sample obtained from a subject. In vitro In some embodiments, these methods can be performed on a biological sample obtained from a subject. In situ In some embodiments, these methods can be performed on a biological sample obtained from a subject. In vivo In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject.

[0060] In some embodiments, when the subject is CILP2 reference, the subject is administered an amount of an OA therapeutic or OA therapy and / or CILP2 inhibitor that is the same as or less than a standard dosage amount. In some embodiments, when the subject is heterozygous for a CILP2 variant nucleic acid molecule, the subject is administered an amount of an OA therapeutic or OA therapy and / or CILP2 inhibitor that is the same as or less than a standard dosage amount.

[0061] In some embodiments, the method of treatment or prevention comprises detecting the presence or absence of a decrease in CILP2 variant mRNA or polypeptide expression in a biological sample from the subject. In some embodiments, when the subject does not have a decrease in CILP2 variant mRNA or polypeptide expression, the subject is administered an amount of an OA therapeutic or OA therapy and / or CILP2 inhibitor that is the same as or less than a standard dosage amount. In some embodiments, when the subject has a decrease in CILP2 variant mRNA or polypeptide expression, the subject is administered a standard dosage amount of an OA therapeutic or OA therapy.

[0062] The present disclosure also provides methods of treating a subject with an OA treatment or OA therapy to treat or inhibit OA, wherein the subject has OA or is at risk of developing OA. The methods include determining whether the subject has reduced expression of a CILP2 variant mRNA or polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the subject has reduced expression of a CILP2 variant mRNA or polypeptide. In embodiments in which the subject does not have reduced expression of a CILP2 variant mRNA or polypeptide, the methods further include administering or continuing to administer to the subject an OA treatment or OA therapy in an amount that is the same as or less than a standard dosage amount and / or administering to the subject a CILP2 inhibitor. In embodiments in which the subject has reduced expression of a CILP2 variant mRNA or polypeptide, the methods further include administering or continuing to administer to the subject an OA treatment or OA therapy in a standard dosage amount. The presence of reduced expression of a CILP2 variant mRNA or polypeptide indicates that the subject is at reduced risk of developing OA. In some embodiments, the subject has reduced expression of a CILP2 variant mRNA or polypeptide. In some embodiments, the subject does not have reduced expression of a CILP2 variant mRNA or polypeptide. In any of the embodiments described herein, a CILP2 inhibitor is an example of an OA treatment. In some embodiments, a CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (refer to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0063] Reduced expression of a CILP2 variant mRNA or polypeptide can be detected by a variety of known methods. In some embodiments, these methods can be performed on a biological sample obtained from a subject. In vitro In some embodiments, these methods can be performed on a biological sample obtained from a subject. In situ In some embodiments, these methods can be performed on a biological sample obtained from a subject. In vivo In any of these embodiments, the mRNA or polypeptide can be present within a cell obtained from the subject.

[0064] In some embodiments, the methods of treatment or prevention include detecting the presence or absence of a CILP2 variant polypeptide in a biological sample from a subject. In some embodiments, when the subject does not have a CILP2 variant polypeptide, the subject is administered an OA treatment or OA therapy in an amount that is the same as or less than a standard dosage amount and / or a CILP2 inhibitor. In some embodiments, when the subject has a CILP2 variant polypeptide, the subject is administered an OA treatment or OA therapy in a standard dosage amount.

[0065] The present disclosure also provides methods of treating a subject with an OA treatment or OA therapy to treat or inhibit OA, wherein the subject has OA or is at risk of developing OA. The methods include determining whether the subject has a CILP2 variant polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the subject has a CILP2 variant polypeptide. When the subject does not have a CILP2 variant polypeptide, the subject is administered an OA treatment or OA therapy and / or a CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount. When the subject has a CILP2 variant polypeptide, the subject is administered a standard dosage amount of the OA treatment or OA therapy. The presence of a CILP2 variant polypeptide indicates that the subject is at a reduced risk of developing OA. In some embodiments, the subject has a CILP2 variant polypeptide. In some embodiments, the subject does not have a CILP2 variant polypeptide.

[0066] The present disclosure also provides methods of preventing a subject from developing OA by administering an OA treatment or OA therapy that prevents OA. In some embodiments, the methods include determining whether the subject has a CILP2 variant polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the subject has a CILP2 variant polypeptide. When the subject does not have a CILP2 variant polypeptide, the subject is administered an OA treatment or OA therapy and / or a CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount. When the subject has a CILP2 variant polypeptide, the subject is administered a standard dosage amount of the OA treatment or OA therapy. The presence of a CILP2 variant polypeptide indicates that the subject is at a reduced risk of developing OA. In some embodiments, the subject has a CILP2 variant polypeptide. In some embodiments, the subject does not have a CILP2 variant polypeptide.

[0067] Detecting the presence or absence of a CILP2 variant polypeptide in a biological sample from a subject and / or determining whether a subject has a CILP2 variant polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In vitro In some embodiments, these methods can be performed in vivo. In situ In some embodiments, these methods can be performed in vitro. In vivo In any of these embodiments, the polypeptide can be present within a cell obtained from the subject.

[0068] In some embodiments, the CILP2 inhibitor is a small molecule. In some embodiments, the small molecule is a low molecular weight (<900 daltons) organic compound.

[0069] In some embodiments, the CILP2 inhibitor comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CILP2. In some embodiments, the antibody is a fully human monoclonal antibody (mAb) or antigen-binding fragment thereof that specifically binds to and neutralizes, inhibits, blocks, abrogates, reduces, or interferes with at least one activity of CILP2, particularly human CILP2. In some embodiments, the antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with the activity of CILP2 by binding to an epitope of CILP2 that is directly involved in the targeted activity of CILP2. In some embodiments, the antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with the activity of CILP2 by binding to an epitope of CILP2 that is not directly involved in the targeted activity of CILP2, but the antibody or fragment that binds to the epitope sterically or conformationally inhibits, blocks, abrogates, reduces, or interferes with the targeted activity of CILP2. In some embodiments, the antibody or fragment thereof binds to an epitope of CILP2 that is not directly involved in the targeted activity of CILP2 (i.e., a non-blocking antibody), but the antibody or fragment that binds to the epitope results in enhanced clearance of CILP2 from circulation compared to clearance of CILP2 in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, abrogating, reducing, or interfering with the activity of CILP2. Clearance of CILP2 from circulation can be particularly enhanced by combining two or more different non-blocking antibodies that do not compete with each other for specific binding to CILP2. The antibody can be full-length (e.g., an IgGl or IgG4 antibody), or can comprise only an antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), and can be modified to affect function, e.g., to abrogate residual effector function (Reddy et al., J. Immunol., 2000, 164, 1925-1933).

[0070] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to CILP2 with an equilibrium dissociation constant (K D ) of about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less, as measured by surface plasmon resonance assay (e.g., BIACORE TM ) In some embodiments, the antibody exhibits a K D of about 800 pM or less, about 700 pM or less; about 600 pM or less; about 500 pM or less; about 400 pM or less; about 300 pM or less; about 200 pM or less; about 100 pM or less; or about 50 pM or less.

[0071] In some embodiments, the anti-CILP2 antibody has a modified glycosylation pattern. In some applications, modification to remove an undesirable glycosylation site, or, for example, to remove a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function, can be useful (see Shield et al., J. Biol. Chem., 2002, 277, 26733). In other applications, removal of N-glycosylation sites can reduce undesirable immunological reactions or increase affinity of the antibody. In yet other applications, galactosylation can be modified to modify complement dependent cytotoxicity (CDC).

[0072] The present disclosure also provides compositions comprising an antibody or antigen binding fragment thereof in combination with an OA therapeutic.

[0073] In some embodiments, the OA therapeutic includes, but is not limited to, a drug (such as, for example, acetaminophen, a non-steroidal anti-inflammatory drug (NSAID) (such as, for example, ibuprofen and naproxen sodium), duloxetine, a corticosteroid (such as, for example, cortisone), a lubricant (such as, for example, hyaluronic acid), and an analgesic (such as an opioid) or any combination thereof. In some embodiments, the OA therapeutic includes acetaminophen. In some embodiments, the OA therapeutic includes a non-steroidal anti-inflammatory drug (NSAID). In some embodiments, the NSAID includes ibuprofen. In some embodiments, the NSAID includes naproxen sodium. In some embodiments, the OA therapeutic includes duloxetine. In some embodiments, the OA therapeutic includes a corticosteroid. In some embodiments, the corticosteroid includes cortisone. In some embodiments, the OA therapeutic includes a lubricant. In some embodiments, the lubricant includes hyaluronic acid. In some embodiments, the OA therapeutic includes an analgesic. In some embodiments, the analgesic includes an opioid. In some embodiments, the OA therapeutic includes a nerve growth factor (NGF) inhibitor. In some embodiments, the OA therapeutic includes fasinumab.

[0074] In some embodiments, the OA therapy includes any therapy for reducing or managing OA. In some embodiments, the OA therapy includes physical therapy, occupational therapy, transcutaneous electrical nerve stimulation (TENS), or surgical and other procedures (such as, for example, knee osteotomy, joint replacement (e.g., knee and / or hip replacement), and bone realignment) or any combination thereof. These therapeutic therapies can be delayed or completely avoided by treatment with a CILP2 inhibitor as described herein.

[0075] In some embodiments, the OA therapy comprises a cartilage repair technique, such as, for example, autologous chondrocyte implantation. In some embodiments, the chondrocytes can be obtained from the particular subject to be treated. These obtained chondrocytes are cultured with a CILP2 inhibitor and reimplanted into the subject. In some embodiments, the cultured chondrocytes can be embedded in a matrix and reimplanted into the subject at the site of a cartilage defect. In some embodiments, the reimplanted chondrocytes are chondrocyte stem cells. In some embodiments, the reimplanted chondrocytes have been genetically modified, such as by CRISPR technology, to contain a CILP2 gene comprising one or more genetic variations (heterozygous or homozygous) in any one or more of the genomic nucleic acid molecules listed in Table 1 (referring to the chromosome: location listed in the GRCh38 / hg38 human genome assembly). In such cases, the reimplanted chondrocytes need not be cultured with a CILP2 inhibitor prior to reimplantation into the subject.

[0076] In some embodiments, the OA therapeutic or OA therapy can be combined with a CILP2 inhibitor.

[0077] In some embodiments, the dosage of the OA therapeutic to treat, prevent, or inhibit OA can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject who is heterozygous for a CILP2 variant nucleic acid molecule or who is a CILP2 reference as compared to a subject who is homozygous for a CILP2 variant nucleic acid molecule (who can receive a standard dosage amount). In some embodiments, the dosage of the OA therapeutic to treat, prevent, or inhibit OA can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In some embodiments, the dosage of the OA therapeutic to treat, prevent, or inhibit OA can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject who is heterozygous for a CILP2 variant nucleic acid molecule or who is a CILP2 reference as compared to a subject who is a CILP2 reference. Additionally, the OA therapeutic can be administered to a subject who is heterozygous for a CILP2 variant nucleic acid molecule or who is a CILP2 reference at a lower frequency as compared to a subject who is heterozygous for a CILP2 variant nucleic acid molecule.

[0078] Administration of the OA therapeutic agent and / or CILP2 inhibitor to treat, prevent, or inhibit OA can be repeated, e.g., after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months. The repeated administration can be at the same dose or at a different dose. The administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, according to certain dosage regimens, a subject can receive therapy for a longer period of time, such as, e.g., 6 months, 1 year, or more.

[0079] Administration of the OA therapeutic agent and / or CILP2 inhibitor can be by any suitable route, including, but not limited to, intra-articular, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical composition for administration is desirably sterile and substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., the dose for a single administration). The pharmaceutical composition can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or auxiliaries. The formulation depends on the route of administration chosen. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and is not substantially harmful to the recipient thereof.

[0080] As used herein, the terms "treat," "treating," and "treatment" and "prevent," "preventing," and "prevention" refer to eliciting a desired biological response, such as therapeutic and prophylactic effects, respectively. In some embodiments, a therapeutic effect, following administration of the agent or a composition comprising the agent, includes one or more of the following: reduction / alleviation of OA, reduction / alleviation of the severity of OA (such as, for example, reducing or inhibiting the development of OA), reduction / alleviation of symptoms and disease-related effects, delaying the onset of symptoms and disease-related effects, lessening the severity of symptoms of disease-related effects, reducing the number of symptoms and disease-related effects, reducing the latency of symptoms and disease-related effects, ameliorating symptoms and disease-related effects, reducing secondary symptoms, reducing secondary infections, preventing recurrence of OA, reducing the number or frequency of recurrence onset, increasing the latency between symptom onset, increasing the time of sustained progression, accelerating recovery, or increasing the efficacy of or reducing resistance to alternative therapeutic agents, and / or increasing the survival time of the affected host animal. A prophylactic effect can include completely or partially avoiding / inhibiting or delaying the development / progression of OA (such as, for example, completely or partially avoiding / inhibiting or delaying), as well as increasing the survival time of the affected host animal, following administration of a treatment regimen. Treatment of OA encompasses treating a subject who has been diagnosed with OA in any clinical stage or manifestation of any form of OA, delaying the onset or evolution or aggravation or worsening of symptoms or signs of OA, and / or preventing and / or alleviating the severity of OA.

[0081] In some embodiments, the CILP2 inhibitor and the OA therapeutic are placed within a pharmaceutical composition. In some embodiments, the CILP2 inhibitor is placed within a first pharmaceutical composition and the OA therapeutic is placed within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered prior to the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0082] In any of the embodiments described herein, the presence and severity of OA can be assessed by imaging. In some embodiments, the current status of OA in a subject can be assessed by imaging. In some embodiments, the progression of OA in a subject can be assessed by imaging. In some embodiments, the non-progression of OA in a subject can be assessed by imaging. In some embodiments, the efficacy of a therapeutic treatment of OA can be monitored by imaging.

[0083] In any of the embodiments described herein, the imaging can be any medical imaging. Medical imaging modalities include, but are not limited to, dual-energy X-ray absorptiometry (DXA), magnetic resonance imaging (MRI), computed tomography (CT), or positron emission tomography (PET). Image sites include, but are not limited to, the knee joint, the hip joint, the hand joint, the spine, and the shoulder.

[0084] In some embodiments, the images are obtained by measuring the absorption of X-ray radiation (dual-energy X-ray absorptiometry, DXA, or CT), capturing a measure of local bone mineral density. In such embodiments, two-dimensional (2D) projections or 3D reconstructions can quantify the bone structure and integrity, which can be used as a biomarker to assess OA. Several methods can be used to extract biomarkers from the images, including but not limited to segmenting the joint bones to measure JSW (e.g., femur and tibia), using electronic calipers to directly measure JSW on the image, or assessing the severity of the disease by an experienced reader using validated clinical scores, or quantifying trabecular, subchondral, and cortical bone regions.

[0085] In some embodiments, the images are obtained based on the principle of resonance excitation of tissue magnetization induced by an external magnetic field. The images are obtained by using magnetic field gradients to encode spatial positions into the precession frequency of the magnetization. Images obtained in this way provide excellent soft tissue contrast and can assess all connective tissue components in the joint. Biomarkers can be extracted from the images by different methods, including but not limited to altering the image contrast according to a specific protocol to derive quantitative measures (e.g., measuring T2 relaxation times acquired by different echo times), or using contrast agent enhancement or quantification procedures (e.g., contrast-enhanced MRI using Gd contrast agents to detect inflammation), or by quantifying joint anatomical structures from the images (e.g., measuring cartilage thickness).

[0086] In some embodiments, a radioactive contrast agent is used to track the accumulation of this contrast agent in the body. This contrast agent has a component with molecular specificity for binding / uptake and a radioactive element, whose byproducts produce particles that can be detected directly or indirectly (e.g., PET). Images acquired using this principle provide a number of biomarkers, including but not limited to standardized uptake value, tissue volume, or pharmacokinetic model parameters fitted to the measured signal.

[0087] In any of the methods described herein, the measurements derived from the images can be used as a marker of the presence of OA, the severity of OA, the progression (or lack thereof) of OA, or the assessment of the efficacy of treatment. For example, a subject suspected of having or developing OA can have an initial image (such as an X-ray) and a subsequent image or series of images of the same modality taken from the affected joint. At each time point, the same imaging marker will be calculated using the same method to assess the change. X-ray imaging of the affected joint can be performed and additional imaging can be performed as needed. In some embodiments, a sensitive definition and prospective imaging is used. In some embodiments, MRI is commonly used at baseline and for adjudication, which is superior to conventional radiographic imaging in detecting joint pathology. For example, a decrease in JSW of the affected joint over time indicates that OA in the joint is progressing. The subject can desire to receive a treatment, such as by any of the methods described herein. Any of the imaging methods described herein can monitor the efficacy of the treatment to determine the efficacy of the treatment.

[0088] In some embodiments, the average change shown in the imaging marker (e.g., JSW increase) in patients receiving a particular therapeutic treatment is significantly different from the average change in patients receiving placebo treatment, which can be interpreted as a positive improvement indicator. Such a change will be interpreted as a positive efficacy of the therapy.

[0089] In some embodiments, the subject can have rapidly progressing osteoarthritis type 1 (RPOA-1) (e.g., joint space narrowing), rapidly progressing osteoarthritis type 2 (RPOA-2) (e.g., joint space limited / partial collapse; bone lesion), primary osteonecrosis (e.g., avascular necrosis), subchondral insufficiency fracture (SIF), or destructive joint disease (DA) (e.g., joint space complete collapse). In some embodiments, the subject has RPOA-1. In some embodiments, the subject has RPOA-2. In some embodiments, the subject has primary osteonecrosis. In some embodiments, the subject has SIF. In some embodiments, the subject has DA.

[0090] In some embodiments, RPOA-1 can be characterized by rapid loss of joint space width from baseline without evidence of bone fragmentation or destruction. If rapid loss of JSW from baseline is observed by X-ray, an MRI can be obtained and can observe substantial focal or diffuse loss of hyaline / articular cartilage consistent with RPOA 1. Rapid change in joint space width from baseline is defined as: a) knee: if JSW is > 2 mm at baseline, a decrease of > 2 mm or 50% (whichever is greater) from baseline at any time point during the study; and if JSW is < 2 mm at baseline or JSW cannot be accurately measured, JSW becomes 0 mm; b) hip: if JSW is > 1.5 mm at baseline, a decrease of > 1.5 mm from baseline; and if JSW is < 1.5 mm at baseline or JSW cannot be accurately measured, JSW becomes 0 mm. If no prior image of the same joint is available for comparison, RPOA type 1 cannot be determined by definition.

[0091] In some embodiments, RPOA-2 can be characterized by abnormal bone fragmentation or destruction over a short period of time, including limited collapse of at least one articular surface, and is observed primarily by MRI, but can also be detected by X-ray.

[0092] In some embodiments, primary osteonecrosis can be characterized by focal, well-defined or extensive patchy lucent areas and sclerosis (infarcted bone), which can be confirmed by MRI. There is no evidence of subchondral collapse or bone fragmentation prior to or concurrent with the diagnosis of primary osteonecrosis.

[0093] In some embodiments, SIF can be characterized by subchondral lucent areas, which can have a sclerotic linear component and flattening of the articular surface, and is confirmed by MRI. Significant collapse or fragmentation is not included.

[0094] In some embodiments, DA can be characterized by abnormal bone fragmentation, destruction, or fracture over a short period of time, including near complete collapse of the articular surface, and is often accompanied by subluxation or dislocation, all of which are inconsistent with the radiographic findings typically observed in traditional late-stage OA, and are easily observed by x-ray.

[0095] In some embodiments, when the subject has DA, certain treatment parameters can be adjusted. For example, a) treatment can be limited to patient populations that are either unresponsive or intolerant to acetaminophen, NSAIDs, and opioids; b) patients with comorbidities that can increase the risk of destructive arthropathy can be excluded; c) concurrent use of NSAIDs can be restricted; and / or d) strict radiological monitoring can be included by i) excluding patients with a history of RPOA, ON, SIF, or other arthropathy that can place the patient at risk for joint destruction; ii) scheduling radiological monitoring; iii) timely evaluation (clinical assessment / X-ray / MRI) of any reports of worsening joint pain; and iv) considering RPOA / SIF / ON as AESI and stopping study drug.

[0096] The present disclosure also provides methods of identifying a subject at increased risk of developing OA. In some embodiments, the methods comprise determining or having determined the presence or absence of a CILP2 variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) in a biological sample obtained from the subject. When the subject lacks a CILP2 variant nucleic acid molecule (i.e., the subject genotype is classified as CILP2 reference), then the subject is at increased risk of developing OA. When the subject has a CILP2 variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for a CILP2 variant nucleic acid molecule), then the subject is at decreased risk of developing OA. In some embodiments, the CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (referring to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0097] A single copy of a CILP2 variant nucleic acid molecule protects a subject from developing OA more than a copy that does not have a CILP2 variant nucleic acid molecule. Without intending to be bound by any particular theory or mechanism of action, it is believed that a single copy of a CILP2 variant nucleic acid molecule (i.e., heterozygous for a CILP2 variant nucleic acid molecule) protects a subject from developing OA, and it is further believed that having two copies of a CILP2 variant nucleic acid molecule (i.e., homozygous for a CILP2 variant nucleic acid molecule) can protect a subject from developing OA more than a subject having a single copy. Thus, in some embodiments, a single copy of a CILP2 variant nucleic acid molecule can not be fully protective, but can partially or incompletely protect a subject from developing OA. While not wishing to be bound by any particular theory, there can be additional factors or molecules involved in the development of OA that are still present in a subject having a single copy of a CILP2 variant nucleic acid molecule, thus resulting in less complete protection from the development of OA.

[0098] Determining whether a subject has a CILP2 variant nucleic acid molecule and / or determining whether a subject has a CILP2 variant nucleic acid molecule in a sample from the subject can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In vitro In some embodiments, these methods can be performed in vitro. In situ In some embodiments, these methods can be performed in vitro. In vivo In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject.

[0099] In some embodiments, when a subject is identified as having an increased risk of developing OA, the subject is administered an OA therapeutic or OA therapy and / or a CILP2 inhibitor, as described herein. For example, when a subject is CILP2 reference, and thus has an increased risk of developing OA, the subject is administered an OA therapeutic or OA therapy in an amount that is the same as or less than a standard dosage amount and / or a CILP2 inhibitor. In some embodiments, when a subject is heterozygous for a CILP2 variant nucleic acid molecule, the subject is administered an OA therapeutic or OA therapy in an amount that is the same as or less than a standard dosage amount and / or a CILP2 inhibitor. In some embodiments, when a subject is homozygous for a CILP2 variant nucleic acid molecule, the subject is administered a standard dosage amount of an OA therapeutic or OA therapy. In some embodiments, the subject is CILP2 reference. In some embodiments, the subject is heterozygous for a CILP2 variant nucleic acid molecule. In some embodiments, the subject is homozygous for a CILP2 variant nucleic acid molecule.

[0100] The present disclosure also provides determining that a subject has a total burden or risk score of two or more CILP2 variant nucleic acid molecules and / or two or more CILP2 variant polypeptides that is associated with a decreased risk of developing OA. The total burden is a sum of two or more genetic variants that can be analyzed in association with OA. In some embodiments, the subject is homozygous for one or more CILP2 variant nucleic acid molecules that is associated with a decreased risk of developing OA. In some embodiments, the subject is heterozygous for one or more CILP2 variant nucleic acid molecules that is associated with a decreased risk of developing OA. When a subject has a lower total burden, the subject has an increased risk of developing OA, and the subject is administered or continues to be administered an OA therapeutic or OA therapy and / or a CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount. When a subject has a higher total burden, the subject has a decreased risk of developing OA, and the subject is administered or continues to be administered a standard dosage amount of an OA therapeutic or OA therapy. The higher the total burden, the lower the risk of developing OA.

[0101] In some embodiments, the total burden of any two or more CILP2 variant nucleic acid molecules in a subject represents a weighted sum of the plurality of any CILP2 variant nucleic acid molecules. In some embodiments, the total burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about or greater than 1,000,000 genetic variants present in or around (up to 10 Mb) the CILP2 gene, where the genetic burden is the number of alleles multiplied by an estimate of association with OA or a relevant outcome per allele (e.g., a weighted polygenic burden score). In some embodiments, a subject has a reduced risk of developing OA when the subject’s total burden is above a desired threshold score. In some embodiments, a subject has an increased risk of developing OA when the subject’s total burden is below a desired threshold score.

[0102] In some embodiments, the total burden can be divided into quintiles, such as the top quintile, the second quintile, the middle quintile, the fourth quintile, and the bottom quintile, where the top quintile of total burden corresponds to the lowest risk group, and the bottom quintile of total burden corresponds to the highest risk group. In some embodiments, subjects with higher total burden include the top weighted total burden, including but not limited to the top 10%, the top 20%, the top 30%, the top 40%, or the top 50% of total burden of a subject population. In some embodiments, the genetic variants include genetic variants associated with OA in the top 10%, the top 20%, the top 30%, the top 40%, or the top 50% of the range of p-values for association. In some embodiments, each identified genetic variant includes a genetic variant associated with OA having a p-value of no greater than about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 , about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 , or about 10 -15 In some embodiments, the identified genetic variants include a p-value of less than 5 x 10 -8In some embodiments, the identified genetic variants include genetic variants associated with OA in high risk subjects having an odds ratio (OR) of about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, or about 2.25 or greater for the top 20% of the distribution; or about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, about 2.25 or greater, about 2.5 or greater, or about 2.75 or greater for the top 20% of the distribution compared to the remainder of the reference population. In some embodiments, the odds ratio (OR) can range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or greater than 7.0. In some embodiments, the high risk subject has a total burden in the lowest decile, quintile, or tertile of the reference population. The threshold for total burden can be determined based on the nature of the intended practical application and the risk differential that would be considered meaningful for that practical application.

[0103] In embodiments where the total burden of CILP2 genetic variants associated with OA is determined, then the total burden represents a risk score for the subject developing OA. In some embodiments, the total burden or risk score includes CILP2 variant genomic nucleic acid molecules comprising any one or more of the genetic variations in the genomic nucleic acid molecules listed in Table 1 (referenced to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or mRNA molecules produced from the genomic nucleic acid molecules or cDNA molecules produced from the mRNA molecules. In some embodiments, the subject total burden can be determined in combination with other genetic variants of other genes associated with OA to produce a polygenic risk score (PRS) for developing OA for CILP2 genetic variants associated with OA. In some embodiments, the PRS includes CILP2 variant genomic nucleic acid molecules comprising any one or more of the genetic variations in the genomic nucleic acid molecules listed in Table 1 (referenced to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or mRNA molecules produced from the genomic nucleic acid molecules or cDNA molecules produced from the mRNA molecules.

[0104] The present disclosure also provides methods of detecting the presence or absence of CILP2 variant nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules produced from mRNA molecules) in a biological sample from a subject. It is understood that genetic sequences within a population and mRNA molecules encoded by such genes can vary due to polymorphisms, such as single nucleotide polymorphisms.

[0105] A biological sample can be derived from any cell, tissue, or biological fluid from a subject. A biological sample can include any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymphatic fluid, ascites fluid, cyst fluid, or urine. In some cases, the sample includes a buccal swab. The biological sample used in the methods disclosed herein can vary based on the format of the assay, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample can be treated differently depending on the assay employed. For example, when detecting any CILP2 variant nucleic acid molecule, a preliminary treatment designed to isolate or enrich the genomic DNA of the biological sample can be employed. A variety of techniques can be used for this purpose. When detecting the level of any CILP2 variant nucleic acid molecule, the biological sample can be enriched for mRNA molecules using different techniques. Various methods can be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA loci.

[0106] In some embodiments, detecting a CILP2 variant nucleic acid molecule in a subject includes performing a sequence analysis on a biological sample obtained from the subject to determine whether a CILP2 genomic nucleic acid molecule in the biological sample and / or a CILP2 mRNA molecule in the biological sample and / or a CILP2 cDNA molecule produced from an mRNA molecule in the biological sample is present in the sample. In some embodiments, the method detects a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (referring to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0107] In some embodiments, a method of detecting the presence or absence of a CILP2 variant nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule produced from an mRNA molecule) in a subject includes performing an assay on a biological sample obtained from the subject. The assay determines whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0108] In some embodiments, the biological sample comprises a cell or a cell lysate. Such methods can further include, for example, obtaining a biological sample comprising a CILP2 genomic nucleic acid molecule or an mRNA molecule from a subject, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can include, for example, determining the identity of these positions for a particular CILP2 nucleic acid molecule. In some embodiments, the method is In vitro a method.

[0109] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a CILP2 genomic nucleic acid molecule, a CILP2 mRNA molecule, or a CILP2 cDNA molecule in a biological sample that comprises a genetic variation compared to a corresponding CILP2 reference molecule. In some embodiments, the sequenced portion comprises one or more variants that cause loss of function (partially or completely) or are predicted to cause loss of function (partially or completely).

[0110] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only a CILP2 genomic nucleic acid molecule is analyzed. In some embodiments, only a CILP2 mRNA is analyzed. In some embodiments, only a CILP2 cDNA obtained from a CILP2 mRNA is analyzed.

[0111] Alteration-specific polymerase chain reaction techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Alteration-specific primers can be used because DNA polymerase will not extend when there is a mismatch to the template.

[0112] In some embodiments, the nucleic acid molecule in the sample is an mRNA, and the mRNA is reverse transcribed into a cDNA prior to the amplifying step. In some embodiments, the nucleic acid molecule is present within a cell obtained from a subject.

[0113] In some embodiments, the assay comprises contacting a biological sample with a primer or probe, such as an alteration-specific primer or alteration-specific probe, that specifically hybridizes to a CILP2 variant genomic sequence, a variant mRNA sequence, or a variant cDNA sequence under stringent conditions, but not to a corresponding CILP2 reference sequence, and determining whether hybridization occurs.

[0114] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of a CILP2 nucleic acid molecule that encodes a CILP2 polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe; and d) detecting the detectable label.

[0115] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay further comprises reverse transcribing an mRNA into a cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0116] In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to a target nucleotide sequence, and specifically detect and / or identify polynucleotides comprising CILP2 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for the selected detection method, including any of the assays described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. The probes and primers can have perfect nucleotide sequence identity to contiguous nucleotides within the target nucleotide sequence, but can be designed differently from the target nucleotide sequence by routine methods and retain the ability to specifically detect and / or identify the target nucleotide sequence. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0117] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, which include the use of labeled primers or probes against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid molecule can be amplified prior to detection or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification reaction (SDA), and nucleic acid sequence-based amplification reaction (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification reaction, and thermophilic SDA (tSDA).

[0118] In hybridization techniques, stringent conditions can be employed that allow specific hybridization of the probe or primer to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence with a degree of detectable greater than, for example, at least 2-fold, at least 3-fold, at least 4-fold, or more (over background), including greater than 10-fold (over background) than hybridization to other non-target sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence with a degree of detectable greater than at least 2-fold than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence with a degree of detectable greater than at least 3-fold than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence with a degree of detectable greater than at least 4-fold than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence with a degree of detectable greater than 10-fold (over background) than hybridization to other nucleotide sequences. Stringency conditions are sequence dependent and will be different in different circumstances.

[0119] Appropriate stringency conditions that promote DNA hybridization, for example, 6X sodium chloride / sodium citrate (SSC) at about 45 °C, followed by a wash in 2X SSC at 50 °C, are known and can be found in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), Chapters 2 and 3. Current Protocols in Molecular Biology , John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Generally, stringency conditions for hybridization and detection will be those in which, as + cations, typically about 0.01 to 1.0 M Na + ion concentration (or other salts), and the temperature is at least about 30 °C for short probes (such as, for example, 10 to 50 nucleotides) and at least about 60 °C for longer probes (such as, for example, greater than 50 nucleotides). Stringency conditions can also be achieved using the addition of destabilizing agents such as formamide. Optionally, the wash buffer can comprise about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, often about 4 to about 12 hours. The duration of the wash time will be at least long enough to achieve equilibrium.

[0120] In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0121] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to a CILP2 variant nucleic acid molecule, such as a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule. Such nucleic acid molecules can be used, for example, as probes, primers, alteration-specific probes, or alteration-specific primers described or exemplified herein, and include, but are not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in greater detail elsewhere herein, and can be used in any of the methods described herein.

[0122] In some embodiments, an isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a CILP2 variant nucleic acid molecule. In some embodiments, an isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, an isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides. In some embodiments, an isolated nucleic acid molecule comprises or consists of about 15 to about 35 nucleotides.

[0123] In some embodiments, alteration-specific probes and alteration-specific primers comprise DNA. In some embodiments, alteration-specific probes and alteration-specific primers comprise RNA.

[0124] In some embodiments, probes and primers described herein, including alteration-specific probes and alteration-specific primers, have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or a complement thereof. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0125] In some embodiments, primers, including altered specificity primers, can be used in second generation sequencing or high-throughput sequencing. In some cases, primers, including altered specificity primers, can be modified. In particular, primers can comprise various modifications used in different steps of, for example, massively parallel signature sequencing (MPSS), polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including the use of biotinylated primers in the clonal step, and fluorescently labeled primers in the bead loading step and detection step. Polony sequencing is typically performed using paired-end tag libraries, where each DNA template molecule is about 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can contain a 5'-biotin tag for immobilization of the DNA library to streptavidin-coated beads.

[0126] The probes and primers described herein can be used to detect nucleotide variations within any of the CILP2 variant nucleic acid molecules disclosed herein. The primers described herein can be used to amplify any CILP2 variant nucleic acid molecule or fragment thereof.

[0127] In the context of the present disclosure, "specifically hybridizes" means that a probe or primer, such as, for example, an altered specificity probe or an altered specificity primer, does not hybridize to a nucleic acid sequence encoding a CILP2 reference genomic nucleic acid molecule, a CILP2 reference mRNA molecule, and / or a CILP2 reference cDNA molecule.

[0128] In some embodiments, a probe, such as, for example, an altered specificity probe, comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0129] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid state substrate or support with which a molecule, such as any of the probes disclosed herein, can associate. One form of a solid support is an array. Another form of a solid support is an array detector. An array detector is a solid support to which a plurality of different probes are coupled in an array, grid, or other organized pattern. One form of a solid state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multi-well glass slide can be employed, typically containing one array per well.

[0130] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or an ortholog from another organism (e.g., a non-human mammal, a rodent, a mouse, or a rat). It will be appreciated that genetic sequences within a population can vary due to polymorphisms, such as single nucleotide polymorphisms.

[0131] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of a particular activity that a target molecule possesses, or the functional polynucleotide can have an activity independent of any other molecule. de novo

[0132] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence (such as in a vector) or a heterologous label. For example, the isolated nucleic acid molecules disclosed herein can be in a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (such as, for example, a fluorophore) or indirectly detectable (such as, for example, a hapten, an enzyme, or a fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent species; a metal-containing species; or an enzyme, where an enzyme-dependent secondary signal generation occurs. The term “label” can also refer to a “tag” or a hapten, which can selectively bind to a conjugate molecule such that the conjugate molecule, when subsequently added with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag to bind to an avidin or streptavidin conjugate of horseradish peroxidase (HRP) and examined using calorimetric substrates (such as, for example, tetramethylbenzidine (TMB)) or fluorescent substrates to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG, or 3XFLAG, 6Xhis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or Fc portions of immunoglobulins. A variety of labels include, for example, particles, fluorophores, haptens, enzymes, and calorimetric, fluorescent, and chemiluminescent substrates thereof, and other labels.

[0133] ​The percent identity (or percent complementarity) between a specified stretch of nucleotides within a nucleic acid molecule or a specified stretch of amino acids within a polypeptide can be determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al. et al. , J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings which use the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In this context, a higher percent sequence identity is preferred over a lower percent sequence identity, if reference is made to percent sequence identity.

[0134] The present disclosure also provides an OA therapeutic that treats, prevents, or inhibits OA for use in treating or preventing OA in a subject having a CILP2 variant nucleic acid molecule. Any of the OA therapeutics that treat, prevent, or inhibit OA described herein can be used herein. Any of the CILP2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (refer to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0135] The present disclosure also provides an OA therapeutic that treats, prevents, or inhibits OA for use in the manufacture of a medicament for treating or preventing OA in a subject having a CILP2 variant nucleic acid molecule. Any of the OA therapeutics that treat, prevent, or inhibit OA described herein can be used herein. Any of the CILP2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (refer to the chromosome: location listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0136] The present disclosure also provides CILP2 inhibitors for use in treating or preventing OA in a subject that is CILP2 reference or heterozygous for a CILP2 variant nucleic acid molecule. Any of the CILP2 inhibitors described herein can be used herein. Any of the CILP2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (referring to the chromosomes: locations listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0137] The present disclosure also provides CILP2 inhibitors for use in the preparation of a medicament for treating or preventing OA in a subject that is CILP2 reference or heterozygous for a CILP2 variant nucleic acid molecule. Any of the CILP2 inhibitors described herein can be used herein. Any of the CILP2 variant nucleic acid molecules disclosed herein can be used herein. In some embodiments, the CILP2 variant nucleic acid molecule is a CILP2 variant genomic nucleic acid molecule comprising any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1 (referring to the chromosomes: locations listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

[0138] In some embodiments, the CILP2 inhibitor and the OA therapeutic are placed in a pharmaceutical composition. In some embodiments, the CILP2 inhibitor is placed in a first pharmaceutical composition and the OA therapeutic is placed in a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered prior to the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0139] All patent documents, websites, other publications, accession numbers, and the like referenced herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. If different versions of a sequence are associated with different accession numbers at different times, the version associated with the accession number at the effective filing date of the present application is intended. The effective filing date means the earlier of the actual filing date or the filing date of the priority application to which the accession number refers, if applicable. Likewise, if different versions of a publication, website, and the like are published at different times, the version published most recently at the effective filing date of the present application is intended, unless otherwise indicated. Unless specifically indicated otherwise, any feature, step, element, embodiment or aspect of the disclosure can be used in combination with any other feature, step, element, embodiment or aspect. While the disclosure has been described in detail and with specificity, it will be apparent to those skilled in the art that certain changes and modifications can be made to the embodiments described without departing from the scope of the appended claims.

[0140] The following examples are provided to describe the embodiments in greater detail. They are intended to illustrate but not limit the claimed embodiments. The following examples provide a disclosure and description of how the compounds, compositions, articles, devices, and / or methods described herein can be made and evaluated, and are merely intended to be illustrative and not limiting to the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.); however, some errors and deviations can be contemplated. Unless otherwise indicated, parts are parts by weight, temperature is in °C or at ambient temperature, and pressure is at or near atmospheric pressure.

[0141] Example Example 1: General Methods participating cohort Genetic association studies were performed in the UK Biobank (UKB) cohort, the Geisinger Health System (GHS) MyCode community health initiative DiscoverEHR cohort, the Mount Sinai BioMe Biobank cohort (SINAI), the University of Pennsylvania Penn Medicine Biobank (UPENN-PMBB), and the Malmo Diet and Cancer Study (MDCS). UKB is a population-based cohort study of people aged 40-69 years old recruited through 22 testing centers in the United Kingdom between 2006-2010 (Sudlow et al., PLoS Med., 2015, 12, e1001779). It includes over 430,000 European ancestry participants from UKB with available whole genome sequencing and clinical phenotype data. The GHS MyCode community health initiative is a health system-based cohort of patients recruited from central and eastern Pennsylvania between 2007-2019 (Carey et al., Genet Med., 2016, 18, 906-13). It includes over 130,000 European ancestry participants from GHS with available whole genome sequencing and clinical phenotype data. SINAI is a patient-based health system cohort from the Mount Sinai BioMe Biobank, recruited by the Mount Sinai Health System (Abul-Husn et al., Cell, 2019, 177, 58-69). UPENN-PMBB is a patient-based health system cohort from the University of Pennsylvania Penn Medicine Biobank (Park et al., Genet. Med., 2020, 22, 102-111). MDCS is a Swedish population-based prospective observational cohort study, recruited between 1991-1996 (Berglund et al., J. Intern. Med., 1993, 233, 45-51).

[0142] phenotype definition In each cohort, the determination of osteoarthritis cases was based on one or more of the following criteria: 1) a history of osteoarthritis in the electronic health record (using International Classification of Diseases, Tenth Revision [ICD-10] diagnosis codes M15, M16, M17, M18, M19, M47 or corresponding Ninth Revision [ICD-9] codes), at least one hospital visit or at least two outpatient visits, or if recorded as a cause of death; 2) a history of surgery related to knee and / or hip replacement in the electronic health record or self-report; 3) a self-reported physician diagnosis of osteoarthritis. Individuals who did not meet any of the criteria for osteoarthritis were used as controls. In addition, individuals were excluded from the control group if they had a history of other forms of arthritis or joint-related symptoms in the electronic health record or self-report, or had undergone surgery involving the joints.

[0143] Imaging phenotypes were derived from dual-energy x-ray absorptiometry (DXA) knee images. DXA images are 2D projections similar to standard x-ray acquisitions. Convolutional network algorithms based on the U-Net architecture were used to segment the knee bones on the DXA images. Joint space width (JSW) was quantified after measuring the distance between the proximal tibia and distal femur. JSW was used as an independent imaging-derived marker of joint health.

[0144] genotype data High-coverage whole exome sequencing was performed as previously described (Dewey et al., Science, 2016, 354, aaf6814; Van Hout et al., Nature, 2020, 586, 749-756) and summarized below. NimbleGen probes (VC Rome; for a portion of the GHS cohort) or xGen designed modified versions available from Integrated DNA Technologies (IDT; for the remainder of GHS and other cohorts) were used for target sequence capture of the exome. Unique 6 base pair (bp) barcodes (VC Rome) or 10 bp barcodes (IDT) were added to each DNA fragment during library preparation to facilitate multiplexed exome capture and sequencing. Equal amounts of samples were pooled prior to exome capture. Sequencing was performed on Illumina v4 HiSeq 2500 (for a portion of the GHS cohort) or NovaSeq (for the remainder of GHS and other cohorts) instruments using 75 bp paired-end reads. The depth of coverage of sequencing (i.e., the number of sequence reads covering each nucleotide in the genomic target region) was sufficient to provide greater than 20x coverage of 85% of the target bases in 96% of VC Rome samples and 20x coverage of 90% of the target bases in 99% of IDT samples. Data processing steps included de-multiplexing using Illumina software, alignment to the GRCh38 human genome reference sequence, including generation of binary alignment and mapping files (BAM), processing of BAM files (e.g., flagging of duplicate reads and other read mapping evaluation). Variant calling was performed using the GLNexus system (DOI: 10.1101 / 343970). Variant mapping and annotation was based on the GRCh38 human genome reference sequence and Ensembl v85 gene definitions using snpEff software. The snpEff predictions involving protein-coding transcripts with annotated start and stop were then combined into a single functional impact prediction by selecting the most deleterious functional effect class for each gene. The ranks of these annotations (from most deleterious to least deleterious) are frame shift, stop codon gain, stop codon loss, splice acceptor, splice donor, stop-lost, in-frame indel, missense, other annotation. Predicted LOF genetic variants include: a) insertions or deletions that result in a frame shift, b) insertions, deletions or single nucleotide variants that result in the introduction of a premature stop codon or loss of a transcription start site or stop site and c) variants of donor or acceptor splice sites.For potential functional impact, deleteriousness was predicted using SIFT (Adzhubei et al., Nat. Methods, 2010, 7, 248-9) and Polyphen2_HVAR (Adzhubei et al., Nat. Methods., 2010, 7, 248-9), LRT (Chun et al., Genome Res., 2009, 19, 1553-61), and MutationTaster (Schwarz et al., Nat. Methods., 2010, 7, 575-6). computer The prediction algorithms classify missense variants. For each gene, the alternative allele frequency (AAF) and functional annotation of each variant determine the inclusion in the following 7 gene burden exposures: 1) pLOF variants with AAF < 1%; 2) pLOF or missense variants predicted deleterious by all 5 algorithms with AAF < 1%; 3) pLOF or missense variants predicted deleterious by all 5 algorithms with AAF < 0.1%; 4) pLOF or missense variants predicted deleterious by at least 1 of 5 algorithms with AAF < 1%; 5) pLOF or missense variants predicted deleterious by at least 1 of 5 algorithms with AAF < 0.1%; 6) pLOF or any missense variants with AAF < 1%; 7) pLOF or any missense variants with AAF < 0.1%.

[0145] association analysis of gene burden of rare pLOF and missense variants The association between the burden of rare predicted loss-of-function or missense variants in a given gene and the phenotype was analyzed by fitting a linear (for quantitative traits) or firth bias-corrected logistic (for binary traits) regression model adjusted for polygenic scores that were imputed using REGENIE to account for genomic kinship matrix (Mbatchou et al., Nat. Genet., 2021, 53, 1097-1103). The association was adjusted for age, sex, age 2 , age stratified by sex, and age 2The interaction term and several covariates specific to the cohort, including batch- related covariates and principal components for common variant sources and principal components for rare variant sources, were used to stratify and adjust the analysis by ancestry. Fixed-effect inverse-variance weighted meta-analysis was used to combine the cross-cohort results for each variant-phenotype association. In the genetic burden test, all individuals were labeled as heterozygous if they carried one or more eligible rare variants (as described above, based on frequency and functional annotation), and homozygous if they carried any eligible variant in a homozygous state. This “composite genotype” was then used to test for association.

[0146] Example 2: Association of osteoarthritis with CILP2 Rare (alternate allele frequency <1%) predicted loss-of-function (pLOF) variants in CILP2 were observed to be associated with an increase of 0.78 standard deviation (SD) units in the minimum joint space width (mJSW) of the knee joint (p-value = 5.7e-9; Figure 1 A). Rare pLOF variants in CILP2 were also associated with a 44% reduction in the risk of osteoarthritis involving the knee joint (odds ratio 0.56; p-value = 8.3e-4; Figure 1 B). Analyses including both pLOF and missense variants were consistent with analyses including only pLOF. These data indicate that inhibition or loss of CILP2 can lead to increased mJSW and reduced risk of OA.

[0147] Rare pLOF variants in CILP2 were also observed to be associated with a reduced risk of knee osteoarthritis (odds ratio 0.57; p-value = 7.5e-4) and a reduced risk of osteoarthritis of the knee and / or hip joint (odds ratio 0.62; p-value = 9.1e-4) (see Figure 2 ).

[0148] Table 1 includes pLoF and missense variants detected via CILP2 exome sequencing. CPRA indicates the genomic coordinates for each variant: chromosome, physical genomic location in base pairs, reference allele, and alternate allele, according to the Human Genome Sequence, version 38, established by the Human Genome Reference Consortium. HGVS.p and HGVS.c indicate changes in protein and coding DNA according to the nomenclature of the Human Genome Variation Society. Effect in the transcript column indicates the predicted impact of the variant.

[0149] Table 1

[0150] Table 2 contains statistical data for joint space analysis and the strongest diagnostic-based OA association analysis for CILP2 (knee). In particular, shown here are all pLoF and missense variants detected via CILP2 exome sequencing. CPRA indicates the genomic coordinates for each variant: chromosome, physical genomic location in base pairs, reference allele, and alternate allele according to the Human Genome Sequence, Version 38, established by the Human Genome Reference Consortium (see Table 1). Single variant association analysis results are reported for each variant based on meta-analysis across ancestors; if sample size was insufficient to estimate (e.g., the variant was too rare), the result is listed as “NA”. For columns where effect sizes are reported, OR indicates the odds ratio, and SD indicates the standard difference units, with the corresponding 95% confidence interval attached. AAF indicates the proportion of the alternate allele frequency out of 1. In all cases, the predicted impact of the variation is based on the canonical CILP2 transcript annotated in the Ensembl database (ENST00000291495; see world wide web “useast.ensembl.org / index.html”).

[0151] Table 2

[0152] Example 3: Association of reduced risk of knee replacement with CILP2 phenotype definition: In each cohort, the determination of knee replacement cases was based on 1) a history of knee replacement surgery in electronic health records, defined using OPCS-4, CPT-4, ICD-10-PCS, or NOMESCO procedure codes; or 2) self-reported history of knee replacement surgery. Individuals who did not meet any criteria for knee replacement were used as controls. In addition, individuals were excluded from the controls if they had a history of other forms of arthritis or joint-related symptoms in electronic health records or self-reports, or had undergone surgery involving the joints. It was observed that rare pLoF variants in CILP2 were associated with reduced risk of knee replacement (odds ratio = 0.54; p-value = 0.0058) (see Figure 3 ).

[0153] Various modifications of the described subject matter, in addition to those described herein, will be apparent to those of ordinary skill in the art in view of the foregoing description. Such modifications are also intended to fall within the scope of the claims. Each reference cited in this application, including but not limited to journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, GENBANK® Accession Nos., and the like, are each incorporated herein by reference in their entirety and for all purposes.

Claims

1. A method of treating a subject having or at risk of developing osteoarthritis, the method comprising administering to the subject a cartilage intermediate layer protein 2 (CILP2) inhibitor.

2. The method of claim 1, wherein the CILP2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to a CILP2 nucleic acid molecule.

3. The method of claim 2, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

4. The method of claim 3, wherein the inhibitory nucleic acid molecule comprises an siRNA.

5. The method of claim 3, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

6. The method of any one of claims 1 to 5, wherein the subject is further administered an osteoarthritis therapeutic or osteoarthritis therapy.

7. The method of any one of claims 1 to 6, further comprising detecting the presence or absence of a CILP2 variant nucleic acid molecule in a biological sample from the subject.

8. The method of claim 7, further comprising administering to the subject an osteoarthritis therapeutic or osteoarthritis therapy in an amount that is the same as or less than a standard dosage amount when the CILP2 variant nucleic acid molecule is not present in the biological sample.

9. The method of claim 7, further comprising administering to the subject an osteoarthritis therapeutic or osteoarthritis therapy in an amount that is the same as or less than a standard dosage amount when the subject is heterozygous for the CILP2 variant nucleic acid molecule.

10. The method of any one of claims 7 to 9, wherein the CILP2 variant nucleic acid molecule comprises a splice site variant, a stop codon gain variant, a start codon loss variant, a stop codon loss variant, a frameshift variant, a missense variant, an in-frame insertion-deletion variant, and / or a variant that encodes a truncated CILP2 variant polypeptide.

11. The method of any one of claims 7 to 9, wherein the CILP2 variant nucleic acid molecule comprises any one or more genetic variations in a genomic nucleic acid molecule listed in Table 1, or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

12. A method of treating a subject having or at risk of developing osteoarthritis by administering an osteoarthritis therapeutic or osteoarthritis therapy, the method comprising: determining or having determined whether the subject has a cartilage intermediate layer protein 2 (CILP2) variant nucleic acid molecule by: obtaining or having obtained a biological sample from the subject; and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype comprising a CILP2 variant nucleic acid molecule; and administering or continuing to administer to the subject having the CILP2 reference the osteoarthritis therapeutic or osteoarthritis therapy and / or a CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount. ​ administering or continuing to administer to a subject heterozygous for the CILP2 variant nucleic acid molecule an amount of the osteoarthritis therapeutic or osteoarthritis therapy and / or CILP2 inhibitor that is the same as or less than a standard dose amount; or administering or continuing to administer to a subject homozygous for the CILP2 variant nucleic acid molecule a standard dose amount of the osteoarthritis therapeutic or osteoarthritis therapy; wherein the presence of the CILP2 variant nucleic acid molecule indicates that the subject has a reduced risk of developing osteoarthritis.

13. The method of claim 12, wherein the CILP2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to a CILP2 nucleic acid molecule.

14. The method of claim 13, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

15. The method of claim 14, wherein the inhibitory nucleic acid molecule comprises an siRNA.

16. The method of claim 14, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

17. The method of any one of claims 12 to 16, wherein the method comprises administering or continuing to administer to a subject heterozygous for the CILP2 variant nucleic acid molecule an amount of the osteoarthritis therapeutic or osteoarthritis therapy and the CILP2 inhibitor that is the same as or less than a standard dose amount.

18. The method of any one of claims 12 to 16, wherein the method comprises administering or continuing to administer to a subject that is a CILP2 reference an amount of the osteoarthritis therapeutic or osteoarthritis therapy and the CILP2 inhibitor that is the same as or less than a standard dose amount.

19. The method of any one of claims 12 to 18, wherein the CILP2 variant nucleic acid molecule comprises a splice site variant, a stop codon gain variant, a start codon loss variant, a stop codon loss variant, a frameshift variant, a missense variant, an in-frame indel variant, and / or a variant that encodes a truncated CILP2 variant polypeptide.

20. The method of any one of claims 12 to 19, wherein the CILP2 variant nucleic acid molecule comprises any one or more genetic variations in a genomic nucleic acid molecule listed in Table 1, or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

21. A method of identifying a subject having an increased risk of developing osteoarthritis, the method comprising: determining or having determined the presence or absence of a cartilage intermediate layer protein 2 (CILP2) variant nucleic acid molecule in a biological sample obtained from the subject; wherein: when the subject is a CILP2 reference, then the subject has an increased risk of developing osteoarthritis; and when the subject is heterozygous or homozygous for the CILP2 variant nucleic acid molecule, then the subject has a reduced risk of developing osteoarthritis.

22. The method of claim 21, wherein the CILP2 variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon loss variant, a stop codon loss variant, a frameshift variant, a missense variant, an in-frame indel variant, or a variant that encodes a truncated CILP2 variant polypeptide.

23. The method of claim 21 or claim 22, wherein the CILP2 variant nucleic acid molecule comprises any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1, or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

24. The method of any one of claims 21 to 23, further comprising administering to a subject that is CILP2 reference an osteoarthritis therapeutic or osteoarthritis therapy and / or a CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount.

25. The method of claim 24, wherein the subject is CILP2 reference and is administered or continues to be administered the osteoarthritis therapeutic or osteoarthritis therapy and the CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount.

26. The method of any one of claims 21 to 23, further comprising administering to a subject that is heterozygous for a CILP2 variant nucleic acid molecule an osteoarthritis therapeutic or osteoarthritis therapy and / or a CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount.

27. The method of claim 26, wherein the subject is heterozygous for the CILP2 variant nucleic acid molecule and is administered or continues to be administered the osteoarthritis therapeutic or osteoarthritis therapy and the CILP2 inhibitor in an amount that is the same as or less than a standard dosage amount.

28. The method of any one of claims 24 to 27, wherein the CILP2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to a CILP2 nucleic acid molecule.

29. The method of claim 28, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

30. The method of claim 29, wherein the inhibitory nucleic acid molecule comprises an siRNA.

31. The method of claim 29, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

32. An osteoarthritis therapeutic for use in treating or preventing osteoarthritis in a subject having a cartilage intermediate layer protein 2 (CILP2) variant nucleic acid molecule.

33. The osteoarthritis therapeutic of claim 32, wherein the CILP2 variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon loss variant, a stop codon loss variant, a frameshift variant, a missense variant, an in-frame indel variant, or a variant that encodes a truncated CILP2 variant polypeptide.

34. The osteoarthritis therapeutic of claim 32 or claim 33, wherein the CILP2 variant nucleic acid molecule comprises any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1, or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

35. A cartilage intermediate layer protein 2 (CILP2) inhibitor for use in treating or preventing osteoarthritis in a subject who is CILP2 reference or heterozygous for a CILP2 variant nucleic acid molecule.

36. The CILP2 inhibitor of claim 35, wherein the CILP2 variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon loss variant, a stop codon loss variant, a frameshift variant, a missense variant, an in-frame indel variant, or a variant encoding a truncated CILP2 variant polypeptide.

37. The CILP2 inhibitor of claim 35 or claim 36, wherein the CILP2 variant nucleic acid molecule comprises any one or more genetic variations in the genomic nucleic acid molecules listed in Table 1, or an mRNA molecule produced from the genomic nucleic acid molecule or a cDNA molecule produced from the mRNA molecule.

38. The CILP2 inhibitor of any one of claims 35 to 37, wherein the CILP2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to a CILP2 nucleic acid molecule.

39. The CILP2 inhibitor of claim 38, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

40. The CILP2 inhibitor of claim 39, wherein the inhibitory nucleic acid molecule comprises an siRNA.

41. The CILP2 inhibitor of claim 39, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

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