Treatment of osteoarthritis with procollagen galactosyltransferase 2 (COLGALT2) inhibitors

Abstract

The present disclosure is generally directed to treating a subject having osteoarthritis or confronting risk of developing osteoarthritis by administering to the subject a procollagen galactosyltransferase 2 (COLGALT2) inhibitor.

Description

[0001] References to sequence lists This application includes a sequence list submitted electronically as an XML file, named 381204327SEQ, created on October 24, 2024, and measuring 2,481,246 bytes. The sequence list is incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to treating subjects who have osteoarthritis or are at risk of developing osteoarthritis by administering a procollagen galactosyltransferase 2 (COLGALT2) inhibitor, and to methods for identifying subjects at increased risk of developing osteoarthritis. Background Technology

[0003] Osteoarthritis (OA) is the most common type of arthritis, affecting millions of people worldwide. Osteoarthritis is a degenerative joint disease that affects all tissues in the joint. Damage to the articular cartilage (the layer of soft tissue covering the ends of bones) occurs early in the disease and is considered an onset of irreversible joint damage. While OA can affect any joint, the most commonly affected joints are the knee, hip, hand, and spine. OA can be diagnosed through physical examination and may include imaging tests (X-rays) to assess severity, as well as laboratory tests (such as, for example, blood or urine tests and synovial fluid analysis). Symptoms of OA include: i) joint pain, ii) joint stiffness upon waking or after inactivity, iii) tenderness upon applying slight pressure to the joint or surrounding area, iv) loss of flexibility, v) a grating or clicking sensation when using the joint, vi) the potential formation of bone spurs around the affected joint, and vii) swelling (inflammation of the soft tissues around the joint). Risk factors for OA include: i) older age (e.g., the risk of OA increases with age), ii) obesity (weight gain puts more stress on weight-bearing joints, and proteins produced by adipose tissue can cause harmful inflammation in and around the joints), iii) joint injury, iv) sex (women are more likely to develop OA), v) repetitive stress on joints, vi) genetics, vii) skeletal deformities, and viii) some metabolic diseases (such as diabetes and hemochromatosis).

[0004] COLGALT2 is encoded by a 108 kb gene located at 1q25.3. The COLGALT2 protein is 626 amino acids long and is a 73 kDa β-galactosyltransferase that transfers β-galactose to hydroxylysine residues in collagen. COLGALT2 is thought to possess procollagen galactosyltransferase activity involved in collagen fiber organization. Furthermore, COLGALT2 is predicted to participate in collagen chain trimerization and extracellular matrix organization pathways. Summary of the Invention

[0005] This disclosure provides a method for treating subjects who have OA or are at risk of developing OA, the method comprising administering a COLGALT2 inhibitor to the subject.

[0006] This disclosure also provides a method for treating a subject with OA or at risk of developing OA by administering an OA therapeutic agent or OA therapy, the method comprising: determining or having determined whether the subject has a COLGALT2 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 containing a COLGALT2 variant nucleic acid molecule; administering or continuing to administer an OA therapeutic agent or OA therapy in an amount equal to or less than a standard dose to a subject as a COLGALT2 reference and / or administering a COLGALT2 inhibitor to the subject; administering or continuing to administer an OA therapeutic agent or OA therapy in an amount equal to or less than a standard dose to a subject heterozygous for a COLGALT2 variant nucleic acid molecule and / or administering a COLGALT2 inhibitor to the subject; or administering or continuing to administer a standard dose of an OA therapeutic agent or OA therapy to a subject homozygous for a COLGALT2 variant nucleic acid molecule; wherein the presence of the COLGALT2 variant nucleic acid molecule indicates a reduced risk of the subject developing OA.

[0007] 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 COLGALT2 variant nucleic acid molecule in a biological sample obtained from the subject; wherein: when the subject is a COLGALT2 reference, the subject's risk of developing OA is increased; and when the subject is heterozygous or homozygous for the COLGALT2 variant nucleic acid molecule, the subject's risk of developing OA is reduced.

[0008] This disclosure also provides OA therapeutic agents for the treatment or prevention of OA in subjects with COLGALT2 variant nucleic acid molecules.

[0009] This disclosure also provides a COLGALT2 inhibitor for the treatment or prevention of OA in subjects who are COLGALT2 reference or heterozygous for COLGALT2 variant nucleic acid molecules. Attached Figure Description

[0010] Figure 1 The study showed that rare coding variants in COLGALT2 were associated with a clinically defined reduced risk of knee and hip osteoarthritis (OA).

[0011] Figure 2The association between rare variants of COLGALT2 and mJSW derived by larger dual-energy X-ray absorptiometry (DXA) was demonstrated, confirming its protective association with OA.

[0012] Figure 3 Rare pLOF and missense variants in COLGALT2 were shown to be associated with a reduced risk of knee or hip replacement, but only the pLOF mutation was observed to have a stronger effect size. Detailed Implementation

[0013] Various terms relating to aspects of this disclosure are used throughout the specification and claims. Unless otherwise specified, 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.

[0014] 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.

[0015] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural referents.

[0016] As used herein, the term "about" means that the listed values ​​are approximate and that small variations will not significantly affect the practice of the disclosed embodiments. When using numerical values, unless the context otherwise indicates, the term "about" means that the values ​​may vary by ±10% and remain within the range of the disclosed embodiments.

[0017] As used herein, in certain embodiments, the term "comprising" may be replaced with "consisting of" or "substantially composed of" as needed.

[0018] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “polynucleotide,” or “oligonucleotide” can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. One strand of a nucleic acid also refers to its complementary sequence.

[0019] 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 pigs), 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 the care of a physician.

[0020] According to observations in this disclosure, rare COLGALT2 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, rare pLOF and missense variants in COLGALT2 are associated with a reduced risk of OA, particularly in the hip and knee joints (OR, 0.89; p = 2.3e-8). Furthermore, rare pLOF variants in COLGALT2 alone are associated with a significant protective effect against knee and hip OA (40% reduction in heterozygous carrier odds ratio: OR, 0.58; p = 0.013). In addition, analysis of knee minimum joint space width (mJSW) derived from DXA imaging validates the protective association of COLGALT2. It is believed that rare variants predicted to induce COLGALT2 function loss are not protective against OA in humans. Therefore, COLGALT2 inhibitors can be used to treat subjects who are heterozygous for COLGALT2 reference or COLGALT2 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 methods 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 COLGALT2 variant nucleic acid molecules in the subject.

[0021] For the purposes of this disclosure, any particular subject, such as a human, may be classified as having one of three COLGALT2 genotypes: i) COLGALT2 reference; ii) heterozygous for a COLGALT2 variant nucleic acid molecule; or iii) homozygous for a COLGALT2 variant nucleic acid molecule. A subject is a COLGALT2 reference when they do not have a copy of the COLGALT2 variant nucleic acid molecule. A subject is heterozygous for a COLGALT2 variant nucleic acid molecule when they have a single copy of the COLGALT2 variant nucleic acid molecule. A subject is homozygous for a COLGALT2 variant nucleic acid molecule when they have two copies of the COLGALT2 variant nucleic acid molecule.

[0022] In any of the embodiments described herein, the COLGALT2 variant nucleic acid molecule may be any nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, or cDNA molecule derived from an mRNA molecule) encoding a COLGALT2 variant polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. Subjects with a partially (or predictedly partially) lost-of-function (or lost-of-function) COLGALT2 polypeptide are sub-alleles for COLGALT2. In some embodiments, the COLGALT2 variant nucleic acid molecule results in reduced or abnormal expression or activity of COLGALT2 mRNA or polypeptide. In some embodiments, the COLGALT2 variant nucleic acid molecule is associated with a weakened in vitro response to the COLGALT2 ligand compared to a reference COLGALT2. In some embodiments, the COLGALT2 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 encoding a truncated COLGALT2 variant polypeptide. In some embodiments, the COLGALT2 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the COLGALT2 variant nucleic acid molecule contains a single nucleotide polymorphism (SNP). In some embodiments, the COLGALT2 variant nucleic acid molecule contains a variation in the coding region. In some embodiments, the COLGALT2 variant nucleic acid molecule does not contain a variation in the non-coding region but contains a variation in the splice acceptor region (two bases before the start point of any exon other than the first exon). In some embodiments, the COLGALT2 variant nucleic acid molecule causes or is predicted to cause premature truncation of the COLGALT2 polypeptide compared to a reference COLGALT2. In some embodiments, the COLGALT2 variant nucleic acid molecule is a variant that, as predicted by an in vitro prediction algorithm (such as Polyphen, SIFT, or similar algorithms), causes impairment of protein function (and thus, in this case, is protective against humans). In some embodiments, the COLGALT2 variant nucleic acid molecule is a variant that causes or is predicted to cause non-synonymous amino acid substitutions in the COLGALT2 nucleic acid molecule and whose allele frequency is less than 1 / 100 alleles in the population from which subjects are selected. In some embodiments, the COLGALT2 variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%; or 1 / 1,000 alleles), or any splice site variant, stop codon gain variant, start codon deletion variant, stop codon deletion variant, frameshift variant, or in-frame insertion / deletion variant, or other frameshift COLGALT2 variant.

[0023] In any of the embodiments described herein, the COLGALT2 variant genomic nucleic acid molecule may contain one or more variants at any location on chromosome 1 (i.e., positions 183,929,854–184,037,729), using the nucleotide sequence of the COLGALT2 reference genomic nucleic acid molecule in the GRCh38 / hg38 human genome assembly (see ENSG00000198756.12 and ENST00000361927.9 annotated in the Ensembl database (https: / / useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000198756;r=1:183929854-184037729;transcript=ENST00000361927.9) as a reference sequence. The sequences of the COLGALT2 genomic nucleic acid molecules provided in these transcripts are merely exemplary sequences. Other sequences of the COLGALT2 genome nucleic acid molecule are also possible.

[0024] In any of the embodiments described herein, the COLGALT2 variant nucleic acid molecule may comprise any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly: position), or an mRNA molecule derived from said genomic nucleic acid molecule, or a cDNA molecule derived from said mRNA molecule. In some instances, in any embodiment described in this disclosure, the COLGALT2 variant is not Tyr212Cys or Tyr212Phe.

[0025] Subjects who are genotyped or identified as COLGALT2 references have an increased risk of developing OA. Subjects who are genotyped or identified as COLGALT2 references or heterozygous for COLGALT2 variants can be treated with COLGALT2 inhibitors.

[0026] In any of the embodiments described herein, subjects who prevent OA by administering a COLGALT2 inhibitor may be any subject at risk of developing OA, including but not limited to subjects with a genetic predisposition to develop OA. In some embodiments, a COLGALT2 inhibitor may be administered to a subject with OA to prevent recurrence of OA in a subject already suffering from OA. In any of the embodiments described herein, the method may be used to improve OA.

[0027] In any of the embodiments described herein, the COLGALT2 predicted loss-of-function peptide may be any COLGALT2 peptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.

[0028] Any one or more (i.e., any combination) of the COLGALT2 variant nucleic acid molecules described herein can be used in any of the methods described herein to determine whether a subject's risk of developing OA is increased or decreased. Combinations of specific variants can form a mask for statistical analysis of a specific correlation between COLGALT2 and an increased or decreased risk of developing OA. In some embodiments, the mask used for statistical analysis of a specific correlation between COLGALT2 and an increased or decreased risk of developing OA may exclude any one or more of the COLGALT2 variant nucleic acid molecules described herein.

[0029] In any of the implementations described herein, the subject may have OA. In any of the implementations described herein, the subject may be at risk of developing OA.

[0030] This disclosure provides a method for treating subjects who have OA or are at risk of developing OA, the method comprising administering a COLGALT2 inhibitor to the subject.

[0031] This disclosure also provides methods for preventing or delaying the need for joint replacements (such as knee and / or hip replacements) in subjects, the methods comprising administering a COLGALT2 inhibitor to the subject. For example, such prolongation can range from months to years. For instance, a COLGALT2 inhibitor can be administered to subjects at increased risk of requiring joint replacements (e.g., knee and / or hip replacements) to prolong the time period prior to actual joint replacement surgery or to completely prevent actual joint replacement. In some embodiments, the need for joint replacement surgery is a consequence of osteoarthritis.

[0032] In some embodiments, the COLGALT2 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 may be designed to target any region of the COLGALT2 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within the COLGALT2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the COLGALT2 peptide in the subject's cells. In some embodiments, the COLGALT2 inhibitor comprises an antisense molecule that hybridizes to the COLGALT2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the COLGALT2 peptide in the subject's cells. In some embodiments, the COLGALT2 inhibitor comprises siRNA that hybridizes to the COLGALT2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the COLGALT2 peptide in the subject's cells. In some embodiments, the COLGALT2 inhibitor comprises shRNA that hybridizes to the COLGALT2 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the COLGALT2 peptide in the subject's cells. Table 1 lists exemplary antisense sequences, and Table 2 lists exemplary siRNA molecules.

[0033] Table 1: Antonyms Table 2: siRNA molecules

[0034] Repressive nucleic acid molecules may include RNA, DNA, or both. Repressive nucleic acid molecules may also be linked or fused with heterologous nucleic acid sequences (such as heterologous nucleic acid sequences in a vector) or heterologous markers. For example, repressive nucleic acid molecules may be contained within a vector containing a repressive nucleic acid molecule and a heterologous nucleic acid sequence, or as an exogenous donor sequence containing a repressive nucleic acid molecule and a heterologous nucleic acid sequence. Repressive nucleic acid molecules may also be linked or fused with heterologous markers. Markers may be directly detectable (such as fluorophores) or indirectly detectable (such as haptens, enzymes, or fluorophore quenchers). Such markers can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such markers include, for example, radioactive markers, pigments, dyes, chromogens, spin markers, and fluorescent markers. Markers may also be, for example, chemiluminescent substances; metal-containing substances; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term "marker" may also refer to a "tag" or hapten that selectively binds to a conjugated molecule such that the conjugated molecule is used to generate a detectable signal when subsequently added with a substrate. For example, biotin can be used as a tag, along with avidin or streptavidin conjugates of horseradish peroxidase (HRP), to bind to the tag and be checked using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorescent substrate to detect the presence of HRP. Exemplary tags 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 proteins, epitope tags, or the Fc portion of immunoglobulins. Many tags include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other tags.

[0035] Inhibitory nucleic acid molecules may 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 portions into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, amination-modified, deamination-modified, alkylated, benzylated, and fluorescently labeled nucleotides.

[0036] Inhibitory nucleic acid molecules may also contain one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides containing modifications to their base, sugar, or phosphate moieties. Modifications to the base moieties include, but are not limited to, natural and synthetic modifications to A, C, G, and T / U, as well as to various 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-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-Uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (such as, for example, 5-bromine), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deadenine, 7-deadenine, 3-deadenine and 3-deadenine.

[0037] Nucleotide analogs may also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of ribose and deoxyribose. 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 the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C- groups. 1-10 Alkyl or C 2-10 alkenyl and C 2-10 Alkyne group. 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, where n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1-10 Alkyl groups, substituted lower alkyl groups, alkylaryl groups, aralkyl groups, O-alkylaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups used to improve the pharmacokinetic properties of oligonucleotides, or groups used to improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, specifically at the 3' position of the sugar on the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in 2'-5' linked oligonucleotides. Modified sugars can also include those sugars containing modifications (e.g., CH2 and S) at the bridging epoxy. Nucleotide sugar analogs can also have sugar mimics that replace furanopentoses, such as cyclobutyl moiety.

[0038] Nucleotide analogs may also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that can be modified such that the bond between two nucleotides contains the following modified phosphate moieties: thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), thiocarbonyl aminophosphates, thiocarbonyl alkylphosphonates, thiocarbonyl alkyl phosphate triesters, and boron phosphates. These phosphate or modified phosphate bonds between the two nucleotides can be 3'-5' or 2'-5' bonds, and said bonds may contain reversed 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 substitutes also include peptide nucleic acids (PNAs).

[0039] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first one to seven nucleotides at the 5' and 3' ends are each modified with 2'-methoxyethyl (2'-MOE). In some embodiments, the first five nucleotides at the 5' and 3' ends are each modified with 2'-MOE. 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 link between nucleotides is a phosphate thioester linker.

[0040] In some embodiments, the siRNA molecule has a terminal modification. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, a non-hydrolyzable 5'-phosphate analogue, such as 5'-(E)-vinylphosphonate, is used.

[0041] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribonucleosides have been shown to enhance the stability and in vivo bioavailability of siRNA. The non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate esters to obtain thiophosphate, borate phosphate, and phosphonoacetate bonds. Additionally, replacing the phosphodiester group with a phosphate triester can promote cellular uptake of siRNA and retain it on serum components by eliminating its negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), thereby allowing the 2'-hydroxyl group to act as a nucleophile and attack adjacent phosphorus in the phosphodiester bond. Such substitutions include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0042] In some embodiments, the siRNA molecule has base modifications. In some embodiments, the bases may be substituted with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0043] In some implementations, siRNA molecules are conjugated to lipids. Lipids may conjugate to the 5' or 3' end of the siRNA to enhance its bioavailability in vivo by allowing it 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.

[0044] In some implementations, a representative siRNA has the following formula: Meaningful: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antonym: / 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 2'-F modification; “m” is 2'-O-methyl modification; “I” is an internal base; and “*” is a thiophosphate skeleton bond.

[0045] In some implementations, the siRNA molecule comprises commercially available siRNA, such as, for example, siRNA IDs: 136748, 136749, 136750, 154471, 154472, 154473, 22411, 22507, 22603, 98933, 99029, and 99125 (catalog number AM16708, ThermoFisher Scientific); siRNA IDs: HSS118219, HSS118220, and HSS118221 (catalog number 1299001, ThermoFisher Scientific); siRNA IDs: MSS218945, MSS218946, and MSS218947 (catalog number 1320001, ThermoFisher Scientific); siRNA ID numbers: s114239, s114240, s114241, s144050, s144051 and s144052 (catalog number 4390771, ThermoFisherScientific); and siRNA ID numbers: s23091, s23092 and s23093 (catalog number 4392420, ThermoFisherScientific).

[0046] In any of the embodiments described herein, the inhibitory nucleic acid molecule may be administered, for example, as an intravenous infusion or subcutaneous injection over one to two hours. In any of the embodiments described herein, the inhibitory nucleic acid molecule may range 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 / m²). 2 – Based on the assumption of a body weight of 70 kg and according to the dose multiplier value of 37 mg / kg for humans, the mg / kg dose level is shifted to mg / m 2 (Dose level conversion) dosage level administration.

[0047] This disclosure also provides vectors comprising one or more of the repressive nucleic acid molecules. In some embodiments, the vector comprises one or more of the repressive 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 granule (such as, for example, a circular double-stranded DNA in which an additional DNA segment can be linked). In some embodiments, the vector is a viral vector in which an additional DNA segment can be linked to a viral genome. Expression vectors include, but are not limited to, plasmids, granules, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), episomes derived from Epstein-Barr virus (EBV), and other expression vectors known in the art.

[0048] This 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 acid-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, and lipid microtubules. Carriers may include buffered saline solutions, such as PBS, HBSS, etc.

[0049] In some implementations, the COLGALT2 inhibitor comprises a nuclease 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 the COLGALT2 genomic nucleic acid molecule. The recognition sequence may be located within the coding region of the COLGALT2 gene or within a regulatory region affecting gene expression. The recognition sequence of the DNA-binding protein or nuclease may be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence may contain or be close to the start codon of the COLGALT2 gene. For example, the recognition sequence may be located approximately 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start codon. As another example, two or more nucleases may be used, each targeting a nuclease recognition sequence containing or close to the start codon. As another example, two nuclease agents can be used: one targeting a nuclease recognition sequence containing or near the start codon, and the other targeting a nuclease recognition sequence containing or near the stop codon, wherein cleavage by the nuclease agent results in the deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break to the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0050] Suitable nucleases and DNA-binding proteins used in this study 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 distributed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The recognition sequence length can vary and includes, for example, recognition sequences of approximately 30-36 bp for zinc finger proteins or ZFN pairs, approximately 15-18 bp for each ZFN, approximately 36 bp for TALE proteins or TALENs, and approximately 20 bp for CRISPR / Cas guide RNAs.

[0051] In some embodiments, the CRISPR / Cas system can be used to modify intracellular COLGALT2 genomic nucleic acid molecules. The methods and compositions disclosed herein employ the CRISPR-Cas system using a CRISPR complex (containing a guide RNA (gRNA) complexed with a Cas protein) for site-directed cleavage of COLGALT2 nucleic acid molecules.

[0052] Cas proteins typically contain at least one RNA recognition or binding domain that can interact with gRNA. Cas proteins may also contain nuclease domains (such as, for example, DNase or RNase domains), 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 Cpf1 proteins (such as, for example, FnCpf1). Cas proteins may have full cleavage activity to produce double-strand breaks in COLGALT2 genomic nucleic acid molecules, or they may be cleavage enzymes that produce single-strand breaks in COLGALT2 genomic nucleic acid molecules. Additional examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), and Cse4. (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and their homologs or modified forms. In some embodiments, the Cas system, such as Cas12a, may have multiple gRNAs encoding a single crRNA. Cas proteins may also be operatively linked as fusion proteins to heterologous peptides. For example, Cas proteins may be fused to cleavage domains, epigenetic modification domains, transcriptional activation domains, or transcriptional repression domains. Cas proteins may be provided in any form. For example, Cas proteins can be provided in the form of proteins, such as Cas proteins complexed with gRNA. Alternatively, Cas proteins can be provided in the form of nucleic acid molecules encoding Cas proteins, such as RNA or DNA.

[0053] In some embodiments, targeted genetic modifications to the COLGALT2 genomic nucleic acid molecule can be generated by contacting cells with a Cas protein and one or more gRNAs, wherein the one or more gRNAs hybridize to one or more gRNA recognition sequences within a target genomic locus in the COLGALT2 genomic nucleic acid molecule. The gRNA recognition sequence may include or be close to the start codon or stop codon of the COLGALT2 genomic nucleic acid molecule. For example, the gRNA recognition sequence may be located approximately 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start or stop codon.

[0054] In the COLGALT2 genome nucleic acid molecule, the gRNA recognition sequence within the target genomic locus 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 the Cas9 nuclease. A typical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleotides. The gRNA can transport Cas9 to any location in the genome for gene editing, but editing may not occur at any site other than the Cas9-recognized PAM site. Alternatively, 5'-NGA-3' can serve as a highly efficient atypical PAM for human cells. Generally, the PAM is located approximately 2-6 nucleotides downstream of the gRNA-targeted DNA sequence. The PAM can be side-attached to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence may be side-attached to the 3' end by the PAM. In some embodiments, the gRNA recognition sequence may be side-attached to the 5' end by the PAM. For example, the cleavage site of the Cas protein can be approximately 1 to 10 base pairs, approximately 2 to 5 base pairs, or three base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when using Streptococcus pyogenes...), S. pyogenes When N is a Cas9 or closely related Cas9, the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is the 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. Therefore, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide and is the 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

[0055] gRNA is an RNA molecule that binds to the Cas protein and targets the Cas protein to a specific location within the COLGALT2 genomic nucleic acid molecule. An exemplary gRNA is one that effectively guides the Cas enzyme to bind to or cleave the COLGALT2 genomic nucleic acid molecule, wherein the gRNA contains a DNA targeting region that hybridizes to a gRNA recognition sequence within the COLGALT2 genomic nucleic acid molecule. The exemplary gRNA contains a DNA targeting region that hybridizes to a gRNA recognition sequence present within the COLGALT2 genomic nucleic acid molecule, said gRNA recognition sequence including or near a start codon or stop codon. For example, the gRNA can be selected such that it hybridizes with a gRNA recognition sequence located at approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start codon or approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the stop codon. Suitable gRNAs may contain approximately 17 to approximately 25 nucleotides, approximately 17 to approximately 23 nucleotides, approximately 18 to approximately 22 nucleotides, or approximately 19 to approximately 21 nucleotides. In some implementations, gRNA may contain 20 nucleotides.

[0056] The Cas protein and gRNA form a complex, and the Cas protein cleaves the COLGALT2 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at sites within or outside the DNA target region of the gRNA present in the COLGALT2 genomic nucleic acid molecule. For example, the formation of a CRISPR complex (containing gRNA that hybridizes to the gRNA recognition sequence and complexes with the Cas protein) can result in the cleavage of one or both strands within or near the DNA target region of the gRNA present in the COLGALT2 genomic nucleic acid molecule (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs).

[0057] Such methods can produce COLGALT2 genomic nucleic acid molecules in which, for example, regions are disrupted, start codons are disrupted, stop codons are disrupted, or coding sequences are disrupted or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within target genomic loci in the COLGALT2 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as a second gRNA that hybridizes to a second gRNA recognition sequence), cleavage by the Cas protein can produce two or more double-strand breaks or two or more single-strand breaks.

[0058] In any of the treatments or preventative methods described herein, the subject receiving treatment may contain a COLGALT2 variant nucleic acid molecule. In some embodiments, the subject receiving treatment is heterozygous for the COLGALT2 variant nucleic acid molecule. In some embodiments, the subject receiving treatment is homozygous for the COLGALT2 variant nucleic acid molecule. In some embodiments, the subject receiving treatment is a COLGALT2 reference. The COLGALT2 variant nucleic acid molecule may be any COLGALT2 variant nucleic acid molecule disclosed herein. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule generated from said genomic nucleic acid molecule.

[0059] In some embodiments, the treatment or prevention method further includes detecting the presence or absence of a COLGALT2 variant nucleic acid molecule in a biological sample from a subject. In some embodiments, the COLGALT2 variant nucleic acid molecule may be any COLGALT2 variant nucleic acid molecule disclosed herein. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule generated from said genomic nucleic acid molecule.

[0060] This disclosure also provides methods for treating subjects with OA therapeutic agents or OA therapies that treat or inhibit OA, wherein the subjects have OA or are at risk of developing OA. The methods include determining whether the subject has a COLGALT2 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 containing a COLGALT2 variant nucleic acid molecule. In embodiments where the subject is a COLGALT2 reference, the methods further include administering or continuing to administer an OA therapeutic agent or OA therapy to the subject in an amount equal to or less than a standard dose and / or administering a COLGALT2 inhibitor to the subject. In embodiments where the subject is heterozygous for a COLGALT2 variant nucleic acid molecule, the methods further include administering or continuing to administer an OA therapeutic agent or OA therapy to the subject in an amount equal to or less than a standard dose and / or administering a COLGALT2 inhibitor to the subject. In embodiments where the subject is homozygous for a COLGALT2 variant nucleic acid molecule, the methods further include administering or continuing to administer a standard dose of an OA therapeutic agent or OA therapy to the subject. The presence of a COLGALT2 variant nucleic acid molecule indicates a reduced risk of developing OA in the subject. In some embodiments, the subject is a COLGALT2 reference. In some embodiments, the subject is heterozygous for the COLGALT2 variant nucleic acid molecule. In some embodiments, the subject is homozygous for the COLGALT2 variant nucleic acid molecule. In any of the embodiments described herein, a COLGALT2 inhibitor is an example of an OA therapeutic agent. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule derived from said genomic nucleic acid molecule.

[0061] For subjects who have been genotyped or identified as COLGALT2 reference or heterozygous for COLGALT2 variant nucleic acid molecules, COLGALT2 inhibitors may be administered to such subjects as described in this article.

[0062] Detecting the presence or absence of COLGALT2 variant nucleic acid molecules in biological samples from a subject and / or determining whether a subject possesses COLGALT2 variant nucleic acid molecules can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecules can be present within cells obtained from the subject.

[0063] In some implementations, when the subject is a COLGALT2 reference, the subject is administered an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor at a dose equal to or less than the standard dose. In some implementations, when the subject is heterozygous for a COLGALT2 variant nucleic acid molecule, the subject is administered an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor at a dose equal to or less than the standard dose.

[0064] In some embodiments, the treatment or prevention method includes detecting the presence or absence of reduced expression of COLGALT2 variant mRNA or peptide in a biological sample from the subject. In some embodiments, when the expression of COLGALT2 variant mRNA or peptide in the subject is not reduced, the subject is administered an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor in an amount equal to or less than a standard dose. In some embodiments, when the expression of COLGALT2 variant mRNA or peptide in the subject is reduced, the subject is administered a standard dose of an OA therapeutic agent or OA therapy.

[0065] This disclosure also provides a method of treating a subject with an OA therapeutic agent or OA therapy that treats or inhibits OA, wherein the subject has OA or is at risk of developing OA. The method includes determining whether the expression of a COLGALT2 variant mRNA or peptide in the subject is reduced by obtaining or having obtained a biological sample from the subject, and by performing or having performed a assay on the biological sample to determine whether the expression of the subject's COLGALT2 variant mRNA or peptide is reduced. In embodiments where the expression of the subject's COLGALT2 variant mRNA or peptide is not reduced, the method further includes administering or continuing to administer an OA therapeutic agent or OA therapy to the subject in an amount equal to or less than a standard dose and / or administering a COLGALT2 inhibitor to the subject. In embodiments where the expression of the subject's COLGALT2 variant mRNA or peptide is reduced, the method further includes administering or continuing to administer a standard dose of an OA therapeutic agent or OA therapy to the subject. The presence of reduced COLGALT2 variant mRNA or peptide expression indicates a reduced risk of the subject developing OA. In some embodiments, the expression of the subject's COLGALT2 variant mRNA or peptide is reduced. In some embodiments, the expression of the subject's COLGALT2 variant mRNA or peptide is not reduced. In any of the embodiments described herein, a COLGALT2 inhibitor is an example of an OA therapeutic agent. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly: position), or an mRNA molecule generated from said genomic nucleic acid molecule, or a cDNA molecule generated from said mRNA molecule.

[0066] Detection of reduced expression of COLGALT2 variant mRNA or peptide can be performed using a variety of known methods. In some embodiments, these methods are performed in vitro. In some embodiments, these methods are performed in situ. In some embodiments, these methods are performed in vivo. In any of these embodiments, the mRNA or peptide may be present within cells obtained from the subject.

[0067] In some embodiments, the treatment or prevention method includes detecting the presence or absence of the COLGALT2 variant peptide in a biological sample from the subject. In some embodiments, when the subject does not have the COLGALT2 variant peptide, the subject is administered an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor in an amount equal to or less than the standard dose. In some embodiments, when the subject has the COLGALT2 variant peptide, the subject is administered a standard dose of the OA therapeutic agent or OA therapy.

[0068] This disclosure also provides a method of treating a subject with an OA therapeutic agent or OA therapy that treats or inhibits OA, wherein the subject has OA or is at risk of developing OA. The method includes determining whether the subject has a COLGALT2 variant peptide by obtaining or having obtained a biological sample from the subject, and by performing or having performed a assay on the biological sample to determine whether the subject has a COLGALT2 variant peptide. When the subject does not have a COLGALT2 variant peptide, administering to the subject an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor in an amount equal to or less than a standard dose. When the subject has a COLGALT2 variant peptide, administering to the subject a standard dose of the OA therapeutic agent or OA therapy. The presence of the COLGALT2 variant peptide indicates a reduced risk of the subject developing OA. In some embodiments, the subject has a COLGALT2 variant peptide. In some embodiments, the subject does not have a COLGALT2 variant peptide.

[0069] This disclosure also provides a method for preventing a subject from developing OA by administering an OA therapeutic agent or OA therapy for the prevention of OA. In some embodiments, the method includes determining whether a subject has a COLGALT2 variant peptide by obtaining or having obtained a biological sample from the subject, and by performing or having performed a assay on the biological sample to determine whether the subject has a COLGALT2 variant peptide. When the subject does not have a COLGALT2 variant peptide, administering to the subject an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor in an amount equal to or less than a standard dose. When the subject has a COLGALT2 variant peptide, administering to the subject a standard dose of an OA therapeutic agent or OA therapy. The presence of the COLGALT2 variant peptide indicates a reduced risk of the subject developing OA. In some embodiments, the subject has a COLGALT2 variant peptide. In some embodiments, the subject does not have a COLGALT2 variant peptide.

[0070] Detecting the presence or absence of the COLGALT2 variant peptide in a biological sample from a subject and / or determining whether a subject possesses the COLGALT2 variant peptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the peptide may be present within cells obtained from the subject.

[0071] In some embodiments, the COLGALT2 inhibitor is a small molecule. In some embodiments, the small molecule is a low molecular weight (<900 Daltons) organic compound.

[0072] In some embodiments, the COLGALT2 inhibitor comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or its antigen-binding fragment specifically binds to human COLGALT2. In some embodiments, the antibody is a fully human monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to and neutralizes, inhibits, blocks, eliminates, reduces, or interferes with at least one activity of COLGALT2 (especially human COLGALT2). In some embodiments, the antibody or its fragment can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of COLGALT2 by binding to an epitope of COLGALT2 that is directly involved in the targeting activity of COLGALT2. In some embodiments, the antibody or its fragment can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of COLGALT2 by binding to an epitope of COLGALT2 that is not directly involved in the targeting activity of COLGALT2, but the antibody or fragment bound to said epitope spatially or conformally inhibits, blocks, eliminates, reduces, or interferes with the targeting activity of COLGALT2. In some embodiments, an antibody or fragment thereof binds to an epitope of COLGALT2 that is not directly involved in the targeting activity of COLGALT2 (i.e., a non-blocking antibody). However, the antibody or fragment bound to the epitope results in enhanced clearance of COLGALT2 from circulation compared to clearance in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, eliminating, reducing, or interfering with the activity of COLGALT2. Clearance of COLGALT2 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 COLGALT2. The antibody may be full-length (e.g., IgG1 or IgG4 antibody) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect function, e.g., to eliminate residual effector function (Reddy et al., J. Immunol., 2000, 164, 1925-1933).

[0073] In some implementations, the antibody or its antigen-binding fragment specifically binds to the equilibrium dissociation constant (K) of COLGALT2. D The values ​​are approximately 7 nM or less, approximately 6 nM or less, approximately 5 nM or less, approximately 4 nM or less, approximately 3 nM or less, approximately 2 nM or less, or approximately 1 nM or less, as determined by surface plasmon resonance (e.g., BIACORE). TM ) as measured. In some implementations, the antibody exhibits KD Approximately 800 pM or less; approximately 700 pM or less; approximately 600 pM or less; approximately 500 pM or less; approximately 400 pM or less; approximately 300 pM or less; approximately 200 pM or less; approximately 100 pM or less; or approximately 50 pM or less.

[0074] In some implementations, anti-COLGALT2 antibodies have modified glycosylation patterns. In some applications, the removal of unwanted glycosylation sites, or, for example, the removal of the fucose moiety to enhance antibody-dependent cytotoxicity (ADCC) function, may be useful (see Shield et al., J. Biol. Chem., 2002, 277, 26733). In other applications, the removal of N-glycosylation sites may reduce undesirable immune responses against therapeutic antibodies or increase antibody affinity. In still other applications, galactosylation may be modified to modify complement-dependent cytotoxicity (CDC).

[0075] In some embodiments, the anti-COLGALT2 antibody may include a complementarity-determining region (CDR) of a commercially available antibody, such as, for example, COLGALT2 rabbit pAb (catalog number A15407, Universal Biologicals), COLGALT2 polyclonal antibody (catalog number PIPA5109571, Fisher Scientific), and COLGALT2 polyclonal antibody (catalog number PA5-56845, ThermoFisher Scientific).

[0076] Antigen-binding fragments include, but are not limited to: monovalent Fab', bivalent Fab2, F(ab)'3 fragments, single-chain variable region fragments (scFv), biscFv, (scFv)2, biantibodies, mini antibodies, nanobodies, triantibodies, tetraantibodies, disulfide-stabilized Fv proteins (dsFv), single-domain antibodies (sdAb), Ig NAR, bispecific antibodies or their binding fragments, bispecific T-cell conjugates (BiTE), trispecific antibodies and their chemically modified derivatives.

[0077] This disclosure also provides compositions comprising an antibody or an antigen-binding fragment thereof in combination with an OA therapeutic agent.

[0078] In some embodiments, OA treatment agents include, but are not limited to, drugs such as, for example, acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs) such as, for example, ibuprofen and naproxen sodium, duloxetine, corticosteroids such as, for example, cortisone), lubricants such as, for example, hyaluronic acid, and analgesics such as opioids, or any combination thereof. In some embodiments, the OA treatment agent includes acetaminophen. In some embodiments, the OA treatment agent includes a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, the NSAID includes ibuprofen. In some embodiments, the NSAID includes naproxen sodium. In some embodiments, the OA treatment agent includes duloxetine. In some embodiments, the OA treatment agent includes a corticosteroid. In some embodiments, the corticosteroid includes cortisone. In some embodiments, the OA treatment agent includes a lubricant. In some embodiments, the lubricant includes hyaluronic acid. In some embodiments, the OA treatment agent includes an analgesic. In some embodiments, the analgesic includes opioids. In some embodiments, the OA treatment agent includes a nerve growth factor (NGF) inhibitor. In some embodiments, the OA treatment agent includes fasinumab.

[0079] In some implementations, OA therapeutic agents include, but are not limited to, anabolic chondrogenic therapeutic agents. Exemplary anabolic chondrogenic therapeutic agents include, but are not limited to, recombinant human FGF18 (Sprifermin) and TGF-β1 (a cell therapy called TissueGene-C).

[0080] In some implementations, OA therapy includes any therapy used to reduce or manage OA. In some implementations, OA therapy includes physical therapy, occupational therapy, transcutaneous electrical nerve stimulation (TENS), or surgery and other procedures (such as, for example, knee osteotomy, joint replacement, and osteopathic surgery) or any combination thereof. These treatments can be delayed or completely avoided by treatment with a COLGALT2 inhibitor as described herein.

[0081] In some embodiments, OA therapy includes cartilage repair techniques, such as, for example, autologous chondrocyte transplantation. In some embodiments, chondrocytes can be obtained from a specific subject to be treated. These obtained chondrocytes are cultured with a COLGALT2 inhibitor and re-implanted into the subject. In some embodiments, the cultured chondrocytes can be embedded in a matrix and re-implanted at the site of cartilage defect in the subject. In some embodiments, the re-implanted chondrocytes are cartilage stem cells. In some embodiments, the re-implanted chondrocytes have been genetically modified, such as by CRISPR technology, to contain the COLGALT2 gene, which contains one or more genetic variations (heterozygous or homozygous) in the genomic nucleic acid molecules listed in Table 4 (referring to the chromosomes listed in the GRCh38 / hg38 human genome assembly). In such cases, the re-implanted chondrocytes do not need to be cultured with a COLGALT2 inhibitor prior to re-implantation into the subject.

[0082] In some implementations, OA treatment agents or OA therapy may be combined with COLGALT2 inhibitors.

[0083] In some embodiments, the dose of the OA therapeutic agent for treating, preventing, or inhibiting OA may be reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90% for subjects heterozygous for the COLGALT2 variant nucleic acid molecule or as a COLGALT2 reference (i.e., less than the standard dose) compared to subjects homozygous for the COLGALT2 variant nucleic acid molecule (whose acceptable standard dose). In some embodiments, the dose of the OA therapeutic agent for treating, preventing, or inhibiting OA may be reduced by approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90% for subjects heterozygous for the COLGALT2 variant nucleic acid molecule or as a COLGALT2 reference compared to subjects as a COLGALT2 reference. In addition, compared to subjects who are heterozygous for COLGALT2 variant nucleic acid molecules, the dosage of OA treatment agents can be administered at a lower frequency to subjects who are heterozygous for COLGALT2 variant nucleic acid molecules or who serve as a COLGALT2 reference.

[0084] Administration of OA therapeutic agents and / or COLGALT2 inhibitors for the treatment, prevention, or inhibition of OA may be repeated, for example, 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. Repeat administration may be at the same dose or at different doses. Administration may be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, depending on certain dosing regimens, subjects may receive therapy for longer periods, such as, for example, six months, one year, or longer.

[0085] OA therapeutic agents and / or COLGALT2 inhibitors may be administered via any suitable route, including but not limited to intra-articular, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular. The pharmaceutical composition intended for administration is expected to be sterile and substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., a single-dose dose). The pharmaceutical composition may be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other components of the formulation and is substantially harmless to the recipient.

[0086] As used herein, the terms “treat,” “treating,” and “treatment,” as well as “prevent,” “preventing,” and “prevention,” refer to eliciting a desired biological response, such as a therapeutic effect and a preventive effect, respectively. In some embodiments, after administration of the agent or a composition containing the agent, the therapeutic effect includes one or more of the following: reduction / alleviation of OA, reduction / alleviation of the severity of OA (such as, for example, alleviating or inhibiting the development of OA), reduction / alleviation of symptom and disease-related effects, delay of the onset of symptom and disease-related effects, reduction of the severity of symptoms of disease-related effects, reduction of the number of symptom and disease-related effects, reduction of the latency of symptom and disease-related effects, improvement of symptom and disease-related effects, reduction of secondary symptoms, reduction of secondary infections, prevention of OA relapse, reduction of the number or frequency of relapses, increase of the latency between symptom onsets, increase of the duration of progression, accelerated recovery or increased efficacy of alternative treatments or reduction of resistance to alternative treatments and / or increased survival time of affected host animals. Preventive effects may include completely or partially avoiding / inhibiting or delaying the development / progression of OA (e.g., completely or partially avoiding / inhibiting or delaying it) after the administration of a treatment regimen, and increasing the survival time of affected host animals. Treatment of OA covers treating subjects diagnosed with any form of OA at any clinical stage or presentation, delaying the onset or evolution or exacerbation or worsening of symptoms or signs of OA, and / or preventing and / or reducing the severity of OA.

[0087] In some embodiments, a COLGALT2 inhibitor and an OA therapeutic agent are contained within a pharmaceutical composition. In some embodiments, a COLGALT2 inhibitor is contained within a first pharmaceutical composition and an OA therapeutic agent is contained 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 before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0088] In any of the embodiments described herein, imaging can be used to assess the presence and severity of OA. In some embodiments, imaging can be used to assess the current status of a subject's OA. In some embodiments, imaging can be used to assess the progression of a subject's OA. In some embodiments, imaging can be used to assess the lack of progression in a subject's OA. In some embodiments, imaging can be used to monitor the efficacy of therapeutic treatments for OA.

[0089] In any of the embodiments described herein, the imaging can be any medical imaging. Medical imaging modalities include, but are not limited to, weight-bearing X-rays, 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, hip joint, hand joints, spine, and shoulder.

[0090] In some implementations, images are obtained by measuring the absorption of X-ray radiation (weight-bearing X-rays, DXA, or CT), thereby capturing measurements of localized bone mineral density. In such implementations, two-dimensional (2D) projection or 3D reconstruction can quantify bone structure and integrity, which can be used as biomarkers for assessing OA. Several methods can be used to extract biomarkers from images, including but not limited to segmenting joint bones to measure JSW (e.g., femur and tibia), directly measuring JSW on images using electronic calipers, or having an experienced reader assess disease severity using validated clinical scores, or quantifying trabecular, subchondral, and cortical bone regions.

[0091] In some implementations, the images are obtained based on the resonant excitation principle of tissue magnetization induced by an external magnetic field. The images are obtained by encoding spatial location to the precession frequency of the magnetization using a magnetic field gradient. Images obtained in this manner provide excellent soft tissue contrast and enable assessment of all connective tissue components in the joint. Biomarkers can be extracted from the images using various methods, including, but not limited to, varying image contrast according to a specific protocol to derive quantitative measurements (e.g., measuring T2 relaxation time acquired at different echo times), using contrast agents for enhancement or quantification processes (e.g., using Gd contrast agent for contrast-enhanced MRI to detect inflammation), or quantifying joint anatomy from the images (e.g., measuring cartilage thickness).

[0092] In some implementations, a radioactive contrast agent is used to track the accumulation location of this contrast agent in the body. This contrast agent has molecularly specific components and radioactive elements for binding / uptake, and its byproducts produce particles that can be detected directly or indirectly (e.g., PET). Images acquired using this principle provide numerous biomarkers, including but not limited to standardized uptake values, tissue volume, or pharmacokinetic model parameters fitted to the measurement signal.

[0093] In any of the methods described herein, measurements derived from images can be used as biomarkers for predicting the presence, severity, progression (or lack thereof) of OA, or for assessing treatment efficacy. For example, a subject suspected of having or developing OA may have initial images (such as X-rays) and subsequent images, or a series of images of the same pattern, obtained from the affected joint. At each time point, the same imaging biomarkers are calculated using the same methods to assess changes. X-ray imaging of the affected joint may be performed, with additional imaging as needed. In some embodiments, sensitivity definitions and prospective imaging are used. In some embodiments, MRI is commonly used as a baseline and for determination, and it is superior to conventional radiographic imaging in detecting joint lesions. For example, a decrease in JSW in an affected joint over time indicates that OA in that joint is progressing. The subject may wish to receive treatment, such as through any of the methods described herein. Any of the imaging methods described herein can monitor the efficacy of treatment to determine its effectiveness.

[0094] In some implementations, patients receiving a specific therapeutic agent show a significant difference in the mean change in imaging biomarkers (e.g., an increase in JSW) compared to patients receiving a placebo, which can be interpreted as a positive indicator of improvement. This change will be interpreted as a positive efficacy of the therapy.

[0095] In some implementations, the subject may have rapidly progressive osteoarthritis type 1 (RPOA-1) (e.g., joint space narrowing), rapidly progressive osteoarthritis type 2 (RPOA-2) (e.g., limited / partial joint collapse; bone injury), primary osteonecrosis (e.g., avascular necrosis), subchondral bone incomplete fracture (SIF), or destructive arthropathy (DA) (e.g., complete joint collapse). In some implementations, the subject has RPOA-1. In some implementations, the subject has RPOA-2. In some implementations, the subject has primary osteonecrosis. In some implementations, the subject has SIF. In some implementations, the subject has DA.

[0096] In some implementations, RPOA-1 can be characterized by a rapid loss of joint space width from baseline without evidence of bone fragmentation or destruction. If a rapid loss of joint space width from baseline is observed on X-ray, MRI is available and significant focal or diffuse loss of hyaline cartilage / articular cartilage consistent with RPOA-1 can be observed. A rapid change in joint space width from baseline is defined as: a) Knee: If JSW ≥ 2 mm at baseline, then at any time point during the study, JSW decreases by ≥ 2 mm or 50% (whichever is greater) from baseline; and if JSW < 2 mm at baseline or JSW cannot be accurately measured, then JSW becomes 0 mm; b) Hip: If JSW ≥ 1.5 mm at baseline, then decreases by > 1.5 mm from baseline; and if JSW < 1.5 mm at baseline or JSW cannot be accurately measured, then JSW becomes 0 mm. RPOA-1 type cannot be determined by definition if no previous images of the same joint are available for comparison.

[0097] In some implementations, RPOA-2 may 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 primarily observed by MRI, but may also be detected by X-ray.

[0098] In some implementations, primary osteonecrosis may be characterized by focal, well-defined, or extensive mottled radiolucent 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.

[0099] In some implementations, the SIF may be characterized as a subchondral radiolucent area, which may have sclerotic linear components and flattened articular surfaces, and can be confirmed by MRI. The SIF does not include significant collapse or fragmentation.

[0100] In some implementations, DA may be characterized by abnormal bone fragmentation, destruction, or fracture within a short period of time, including near-complete collapse of the articular surfaces, and often accompanied by subluxation or dislocation. All of these features are inconsistent with the radiological findings typically observed in conventional late OA and are readily observable by X-ray.

[0101] In some implementations, specific treatment parameters may be adjusted when a subject has DA. For example, a) treatment may be limited to patients who are unresponsive to or intolerant of acetaminophen, NSAIDs, and opioids; b) patients with comorbidities that may increase the risk of destructive arthropathy may be excluded; c) concomitant use of NSAIDs may be restricted; and / or d) may include rigorous radiological monitoring by: i) excluding patients with a history of RPOA, ON, SIF, or other arthropathy that may place the patient at risk of joint destruction; ii) scheduling radiological monitoring; iii) promptly evaluating (clinical assessment / X-ray / MRI) any reports of worsening joint pain; and iv) treating RPOA / SIF / ON as AESI and discontinuing the study drug.

[0102] This disclosure also provides methods for identifying subjects at increased risk of developing OA. In some embodiments, the method includes determining, or having determined, the presence or absence of a COLGALT2 variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, and / or cDNA molecule) in a biological sample obtained from the subject. When a subject lacks a COLGALT2 variant nucleic acid molecule (i.e., the subject's genotype is classified as COLGALT2 reference), the subject's risk of developing OA is increased. When a subject has a COLGALT2 variant nucleic acid molecule that may induce loss of function in some way (i.e., the subject is heterozygous or homozygous for a COLGALT2 variant nucleic acid molecule), the subject's risk of developing OA is reduced. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule derived from said genomic nucleic acid molecule.

[0103] A single copy of the COLGALT2 variant nucleic acid molecule provides better protection against the development of OA than a copy without the COLGALT2 variant nucleic acid molecule. Without intending to be limited to any particular theory or mechanism of action, it is believed that a single copy of the COLGALT2 variant nucleic acid molecule (i.e., heterozygous for the COLGALT2 variant nucleic acid molecule) protects a subject from the development of OA, and it is also believed that two copies of the COLGALT2 variant nucleic acid molecule (i.e., homozygous for the COLGALT2 variant nucleic acid molecule) provide better protection against the development of OA compared to a subject with a single copy. Therefore, in some implementations, a single copy of the COLGALT2 variant nucleic acid molecule may not provide complete protection, but may provide partial or incomplete protection against the development of OA. While not wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of OA that are still present in subjects with a single copy of the COLGALT2 variant nucleic acid molecule, thus resulting in less complete protection against the development of OA.

[0104] Determining whether a subject possesses a COLGALT2 variant nucleic acid molecule in a sample from the subject and / or determining whether a subject possesses a COLGALT2 variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule may be present within cells obtained from the subject.

[0105] In some embodiments, when a subject is identified as having an increased risk of developing OA, an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor is administered to the subject, as described herein. For example, when a subject is a COLGALT2 reference and therefore has an increased risk of developing OA, an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor is administered to the subject in an amount equal to or less than the standard dose. In some embodiments, when a subject is heterozygous for a COLGALT2 variant nucleic acid molecule, an OA therapeutic agent or OA therapy and / or a COLGALT2 inhibitor is administered to the subject in an amount equal to or less than the standard dose. In some embodiments, when a subject is homozygous for a COLGALT2 variant nucleic acid molecule, a standard dose of an OA therapeutic agent or OA therapy is administered to the subject. In some embodiments, the subject is a COLGALT2 reference. In some embodiments, the subject is heterozygous for a COLGALT2 variant nucleic acid molecule. In some embodiments, the subject is homozygous for a COLGALT2 variant nucleic acid molecule.

[0106] This disclosure also provides a total burden or risk score for determining whether a subject has a total burden or risk score for two or more COLGALT2 variant nucleic acid molecules and / or two or more COLGALT2 variant peptides associated with a reduced risk of developing OA. Total burden is the sum of genetic variants that can be analyzed in association with OA. In some embodiments, the subject is homozygous for one or more COLGALT2 variant nucleic acid molecules associated with a reduced risk of developing OA. In some embodiments, the subject is heterozygous for one or more COLGALT2 variant nucleic acid molecules associated with a reduced risk of developing OA. When a subject has a low total burden, the risk of developing OA is increased, and the subject is given or continues to receive an OA treatment agent or OA therapy and / or a COLGALT2 inhibitor at a standard dose or less. When a subject has a high total burden, the risk of developing OA is reduced, and the subject is given or continues to receive a standard dose of an OA treatment agent or OA therapy. The higher the total burden, the lower the risk of developing OA.

[0107] In some implementations, the total load of any two or more COLGALT2 variant nucleic acid molecules in the subject represents a weighted sum of multiple COLGALT2 variant nucleic acid molecules. In some implementations, the total load 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 the COLGALT2 gene (up to 10 Mb), where the genetic load is the number of alleles multiplied by an association estimate with OA or a result associated with each allele (e.g., a weighted polygenic load score). In some implementations, the risk of developing OA is reduced when the subject's total workload is higher than the expected threshold score. In other implementations, the risk of developing OA is increased when the subject's total workload is lower than the expected threshold score.

[0108] In some implementations, the total burden may be divided into quintiles, such as the highest quintile, second quintile, middle quintile, fourth quintile, and lowest quintile, wherein the highest quintile of the total burden corresponds to the lowest risk group, and the lowest quintile of the total burden corresponds to the highest risk group. In some implementations, subjects with higher total burdens include those with the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% of the total burden of the subject population. In some implementations, the genetic variants include OA-associated variants in the top 10%, top 20%, top 30%, top 40%, or top 50% of the relevant p-value range. In some implementations, each identified genetic variant includes an OA-associated variant with a p-value no greater than about 10. -2 Approximately 10 -3 Approximately 10 -4 Approximately 10 -5 Approximately 10 -6 Approximately 10 -7 Approximately 10 -8 Approximately 10 -9 Approximately 10 -10 Approximately 10 -11 Approximately 10 -12 Approximately 10 -13 Approximately 10 -14 Or about 10 -15 In some implementations, the identified genetic variants include those with p-values ​​less than 5 × 10⁻⁶. -8 Genetic variants associated with OA. In some embodiments, the identified genetic variants include those associated with OA in high-risk subjects, having the following odds ratios (ORs) compared to the remainder of the reference population: approximately 1.5 or greater, approximately 1.75 or greater, approximately 2.0 or greater, or approximately 2.25 or greater for the top 20% of the distribution; or approximately 1.5 or greater, approximately 1.75 or greater, approximately 2.0 or greater, approximately 2.25 or greater, approximately 2.5 or greater, or approximately 2.75 or greater. In some implementations, the odds ratio (OR) may 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 implementations, high-risk subjects have the lowest decile, quintile, or tertiary total load in the reference population. The threshold for total load may be determined based on the nature of the intended practical application and the risk difference that would be considered meaningful for said practical application.

[0109] In embodiments for determining the total burden of COLGALT2 genetic variants associated with OA, the total burden represents a risk score for the subject to develop OA. In some embodiments, the total burden or risk score includes a COLGALT2 variant genomic nucleic acid molecule containing any one or more genetic variations from the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule derived from said genomic nucleic acid molecule. In some embodiments, the total burden of the subject may be determined against the COLGALT2 genetic variant associated with OA in combination with other genetic variants of other genes associated with OA (such as, for example, intercalary protein 2 (CILP2)) to generate a polygenic risk score (PRS) for developing OA. In some embodiments, the PRS includes a COLGALT2 variant genomic nucleic acid molecule containing any one or more genetic variations from the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule derived from said genomic nucleic acid molecule.

[0110] This disclosure also provides a method for detecting the presence or absence of COLGALT2 variant nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules derived from mRNA molecules) in biological samples from subjects. It should be understood that gene sequences and mRNA molecules encoded by such genes within a population can vary due to polymorphisms, such as single nucleotide polymorphisms.

[0111] Biological samples can be derived from any cells, tissues, or biological fluids from a subject. Biological samples may include any clinically relevant tissue, such as bone marrow samples, tumor biopsies, fine-needle aspiration, or bodily fluid samples, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cystic fluid, or urine. In some cases, samples include oral swabs. The biological samples used in the methods disclosed herein can vary based on the assay format, the nature of the detection method, and the tissue, cells, or extract used as the sample. Biological samples may be processed differently depending on the assay employed. For example, when detecting any COLGALT2 variant nucleic acid molecules, preliminary processing designed to isolate or enrich the genomic DNA of the biological sample may be employed. Various techniques are available for this purpose. When detecting the level of any COLGALT2 variant nucleic acid molecules, different techniques may be used to enrich the biological sample with mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA loci.

[0112] In some embodiments, detecting COLGALT2 variant nucleic acid molecules in a subject includes sequence analysis of a biological sample obtained from the subject to determine the presence of COLGALT2 genomic nucleic acid molecules and / or COLGALT2 mRNA molecules and / or COLGALT2 cDNA molecules derived from mRNA molecules in the biological sample. In some embodiments, the method detects COLGALT2 variant genomic nucleic acid molecules comprising any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or mRNA molecules derived from said genomic nucleic acid molecules, or cDNA molecules derived from said mRNA molecules.

[0113] In some implementations, methods for detecting the presence or absence of COLGALT2 variant nucleic acid molecules (such as, for example, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules derived from mRNA molecules) in a subject include assaying a biological sample obtained from the subject. The assay determines whether the nucleic acid molecules in the biological sample contain a specific nucleotide sequence.

[0114] In some embodiments, the biological sample comprises cells or cell lysates. Such methods may further include, for example, obtaining a biological sample containing a COLGALT2 genomic nucleic acid molecule or mRNA molecule from a subject, and optionally, reverse transcribing the mRNA into cDNA if it is mRNA. Such assays may include, for example, identifying these locations of a specific COLGALT2 nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0115] In some embodiments, the determining step, detection step, or sequence analysis includes sequencing at least a portion of the nucleotide sequence of a COLGALT2 genomic nucleic acid molecule, a COLGALT2 mRNA molecule, or a COLGALT2 cDNA molecule in a biological sample containing genetic variations compared to a corresponding COLGALT2 reference molecule. In some embodiments, the sequencing portion includes one or more variations that cause or are predicted to cause loss of function (partial or complete).

[0116] In some embodiments, the assay includes sequencing the entire nucleic acid molecule. In some embodiments, only the COLGALT2 genomic nucleic acid molecule is analyzed. In some embodiments, only COLGALT2 mRNA is analyzed. In some embodiments, only COLGALT2 cDNA obtained from COLGALT2 mRNA is analyzed.

[0117] Modified-specific polymerase chain reaction (MCR) techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Modified-specific primers can be used because DNA polymerase will not extend when there is a mismatch with the template.

[0118] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present within cells obtained from a subject.

[0119] In some embodiments, the assay includes contacting a biological sample with primers or probes, such as modified specific primers or modified specific probes, which under stringent conditions specifically hybridize with a COLGALT2 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence, rather than specifically hybridizing with a corresponding COLGALT2 reference sequence, and determining whether hybridization occurs.

[0120] In some implementations, the determining step, detection step, or sequence analysis includes: a) amplifying at least a portion of a COLGALT2 nucleic acid molecule encoding a COLGALT2 polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support containing a probe that modifies specificity; and d) detecting the detectable label.

[0121] In some embodiments, the assay includes RNA sequencing (RNA-Seq). In some embodiments, the assay also includes, for example, reverse transcription of mRNA into cDNA via reverse transcriptase polymerase chain reaction (RT-PCR).

[0122] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides comprising a COLGALT2 variant genomic nucleic acid molecule, a variant mRNA molecule, or a variant cDNA molecule. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for the chosen detection method (including any assay described or exemplified herein). Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. Probes and primers can have complete nucleotide sequence identity with adjacent nucleotides within the target nucleotide sequence, but can be designed using conventional methods to differ from the target nucleotide sequence while retaining the ability to specifically detect and / or identify the target nucleotide sequence. Probes and primers can have approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% sequence identity or complementarity with the nucleotide sequence of the target nucleic acid molecule.

[0123] Illustrative examples of nucleic acid sequencing technologies include, but are not limited to, Sanger sequencing and dye-terminated sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, including the use of labeled primers or probes for purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid molecule can be amplified before or simultaneously with detection. Illustrative examples of nucleic acid amplification technologies include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0124] In hybridization techniques, stringent conditions can be employed to enable probes or primers to hybridize specifically with their targets. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target sequences to a detectable degree greater than hybridization with other non-target sequences, for example, at least 2, 3, 4, or more times (relative to background), including more than 10 times (relative to background). In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree at least 2 times greater than hybridization with other nucleotide sequences. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree at least 3 times greater than hybridization with other nucleotide sequences. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree at least 4 times greater than hybridization with other nucleotide sequences. In some embodiments, under stringent conditions, polynucleotide primers or probes will hybridize with their target nucleotide sequences to a detectable degree more than 10 times greater than hybridization with other nucleotide sequences (relative to background). The stringent conditions are sequence-dependent and will vary in different environments.

[0125] Suitable stringent conditions for promoting DNA hybridization (e.g., 6X sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by washing with 2X SSC at 50°C) are known and available in [the context of DNA hybridization]. Current Protocols in Molecular Biology John Wiley & Sons, NY (1989), 6.3.1–6.3.6. Typically, the stringent conditions used for hybridization and detection will be those described below: a salt concentration of less than approximately 1.5 M Na at pH 7.0 to 8.3. + Ions, typically about 0.01 to 1.0 M Na +The ion concentration (or other salt) and temperature are at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes (e.g., greater than 50 nucleotides). Tight conditions can also be achieved by adding a destabilizing agent (e.g., formamide). Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours. The washing time will be at least long enough to reach equilibration.

[0126] In some implementations, the isolated nucleic acid molecules contain 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 5 5, 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 or composed thereof. In some embodiments, the isolated nucleic acid molecule comprises or consists 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.

[0127] In some implementations, such isolated nucleic acid molecules hybridize under stringent conditions with COLGALT2 variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules). These nucleic acid molecules can be used as probes, primers, modified-specific probes, or modified-specific primers, as described or illustrated herein, and include, but are not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each described in more detail elsewhere herein, and can be used in any of the methods described herein.

[0128] In some embodiments, the isolated nucleic acid molecule hybridizes with at least about 15 consecutive nucleotides of a nucleic acid molecule having 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% identity with a COLGALT2 variant nucleic acid molecule. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 35 nucleotides.

[0129] In some embodiments, the specific probe and specific primer are modified to contain DNA. In some embodiments, the specific probe and specific primer are modified to contain RNA.

[0130] In some embodiments, the probes and primers described herein (including modified-specific probes and modified-specific primers) have nucleotide sequences that specifically hybridize with any nucleic acid molecule disclosed herein or its complementary sequence. In some embodiments, the probes and primers specifically hybridize with any nucleic acid molecule disclosed herein under stringent conditions.

[0131] In some implementations, primers (including modified-specific primers) can be used in next-generation sequencing or high-throughput sequencing. In some cases, primers can be modified, including modified-specific primers. In particular, primers can contain various modifications used in different steps of, for example, massively parallel signature sequencing (MPSS), polymerase cloning sequencing (Polony sequencing), and 454 pyrosequencing. Modified primers can be used in several steps of the process, including the use of biotinylated primers in the cloning step and fluorescently labeled primers in the bead loading and detection steps. Polymerase cloning sequencing is typically performed using paired-end tagged libraries, where each DNA template molecule is approximately 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonameric oligonucleotides are used in the detection step. The adaptor may contain a 5'-biotin tag for immobilizing the DNA library onto streptavidin-coated beads.

[0132] The probes and primers described herein can be used to detect nucleotide variations within any of the COLGALT2 variant nucleic acid molecules disclosed herein. The primers described herein can be used to amplify any COLGALT2 variant nucleic acid molecule or fragment thereof.

[0133] In the context of this disclosure, "specific hybridization" means that a probe or primer (such as, for example, by altering a specific probe or primer) does not hybridize with the nucleic acid sequence encoding a COLGALT2 reference genome nucleic acid molecule, a COLGALT2 reference mRNA molecule, and / or a COLGALT2 reference cDNA molecule.

[0134] In some embodiments, the probe (such as, for example, a modified specific probe) contains a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0135] This disclosure also provides supports for substrates to which one or more of the probes disclosed herein are attached. A solid support is a solid substrate or support to which molecules (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 of detectors. An array of detectors is a solid support to which multiple different probes are coupled in an array, grid, or other organized pattern. One form of a solid substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a porous glass slide, typically containing one array per well, may be used.

[0136] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can originate from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or orthologs of another organism (e.g., non-human mammals, rodents, mice, or rats). It should be understood that gene sequences within a population can vary due to polymorphisms such as single nucleotide polymorphisms.

[0137] This article also provides 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, triplet-forming molecules, and external guide sequences. Functional polynucleotides can act as influencers, inhibitors, regulators, and stimulators of the specific activities of target molecules, or they can have properties independent of any other molecule. brand new active.

[0138] The isolated nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules may also be ligated or fused to heterologous nucleic acid sequences (such as in a vector) or heterologous tags. For example, the isolated nucleic acid molecules disclosed herein may 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 may also be ligated or fused to heterologous tags. Tags may be directly detectable (such as fluorophores) or indirectly detectable (such as haptens, enzymes, or fluorophore quenchers). Such tags can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such tags include, for example, radioactive tags, pigments, dyes, chromogens, spin tags, and fluorescent tags. Tags may also be, for example, chemiluminescent substances; metal-containing substances; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term “tag” may also refer to a “label” or hapten that selectively binds to a conjugated molecule such that the conjugated molecule is used to generate a detectable signal when subsequently added with a substrate. For example, biotin can be used as a tag, along with avidin or streptavidin conjugates of horseradish peroxidase (HRP), to bind to the tag and be checked using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorescent substrate to detect the presence of HRP. Exemplary tags 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 proteins, epitope tags, or the Fc portion of immunoglobulins. Many tags include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other tags.

[0139] The percentage of identity (or complementarity) between specific elongations of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or routinely using the Gap program (Wisconsin Sequence Analysis Package for Unix, version 8, Genetics Computer Group, University Research Park, Madison Wis.) with default settings (which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489)). In this document, a higher percentage of sequence identity is preferred over a lower percentage when referring to sequence identity percentages.

[0140] This disclosure also provides OA therapeutic agents for treating, preventing, or inhibiting OA in subjects having OA. Any of the OA therapeutic agents for treating, preventing, or inhibiting OA described herein may be used herein. Any of the COLGALT2 variant nucleic acid molecules disclosed herein may be used herein. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule generated from said genomic nucleic acid molecule.

[0141] This disclosure also provides OA therapeutic agents for treating, preventing, or inhibiting OA, which are used in the preparation of pharmaceutical agents for treating or preventing OA in subjects having COLGALT2 variant nucleic acid molecules. Any of the OA therapeutic agents for treating, preventing, or inhibiting OA described herein may be used herein. Any of the COLGALT2 variant nucleic acid molecules disclosed herein may be used herein. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule generated from said genomic nucleic acid molecule.

[0142] This disclosure also provides COLGALT2 inhibitors for the treatment or prevention of OA in subjects who are heterozygous for COLGALT2 or for COLGALT2 variant nucleic acid molecules. Any of the COLGALT2 inhibitors described herein may be used herein. Any of the COLGALT2 variant nucleic acid molecules disclosed herein may be used herein. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule generated from said genomic nucleic acid molecule.

[0143] This disclosure also provides a COLGALT2 inhibitor for use in the preparation of a pharmaceutical agent for the treatment or prevention of OA in a subject who is a COLGALT2 reference or heterozygous for a COLGALT2 variant nucleic acid molecule. Any of the COLGALT2 inhibitors described herein may be used herein. Any of the COLGALT2 variant nucleic acid molecules disclosed herein may be used herein. In some embodiments, the COLGALT2 variant nucleic acid molecule is a COLGALT2 variant genomic nucleic acid molecule containing one or more genetic variations of the genomic nucleic acid molecules listed in Table 4 (referring to chromosomes listed in the GRCh38 / hg38 human genome assembly), or an mRNA molecule or a cDNA molecule generated from said genomic nucleic acid molecule.

[0144] In some embodiments, a COLGALT2 inhibitor and an OA therapeutic agent are contained within a pharmaceutical composition. In some embodiments, a COLGALT2 inhibitor is contained within a first pharmaceutical composition and an OA therapeutic agent is contained 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 before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0145] All patent documents, websites, other publications, registration numbers, etc., cited above or below are incorporated herein by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated as being incorporated by reference. If different versions of a sequence are associated with registration numbers at different times, the version associated with the registration number on the effective filing date of this application is indicated. The effective filing date is the earlier of the actual filing date or the filing date of the priority application referencing the registration number (if applicable). Similarly, if different versions of publications, websites, etc., are published at different times, the version most recently published on the effective filing date of this application is indicated, unless otherwise stated. Unless otherwise specifically stated, any feature, step, element, embodiment, or aspect of this disclosure may be used in combination with any other feature, step, element, embodiment, or aspect. Although this disclosure has been described in detail by illustration and example for clarity and understanding purposes, it will be apparent that certain changes and modifications may be implemented within the scope of the appended claims.

[0146] The following examples are provided to describe the embodiments in more detail. They are intended to illustrate, but not limit, the claimed embodiments. The following examples provide a disclosure and description, for those skilled in the art, of how the compounds, compositions, articles, apparatuses, and / or methods described herein are prepared and evaluated, and are intended to be exemplary only and not to limit the scope of any claim. Efforts have been made to ensure accuracy regarding figures (such as, for example, amounts, temperatures, etc.), but some errors and deviations may be expected. Unless otherwise stated, parts are parts by weight, temperatures are in °C or at ambient temperature, and pressures are at or near atmospheric pressure.

[0147] Example Example 1: General Method Participation queue Genetic association studies were conducted in the UK Biobank (UKB) cohort, the Geisinger Health System (GHS) MyCode Community Health Advocacy DiscoverEHR cohort, the Mount Sinai BioMe Biobank cohort (SINAI), the University of Pennsylvania PennMedicine Biobank (UPENN-PMBB), and the Malmo Diet and Cancer Study (MDCS). The UKB is a population-based cohort study that recruited individuals aged 40 to 69 years from 22 testing centers in the UK between 2006 and 2010 (Sudlow et al., PLoS Med., 2015, 12, e1001779). It included over 430,000 participants of European descent from the UKB with available whole-genome sequencing and clinical phenotypic data. The GHS MyCode Research Community Health Advocacy is a health-system-based cohort of patients recruited from central and eastern Pennsylvania, USA, between 2007 and 2019 (Carey et al., Genet Med., 2016, 18, 906-13). It includes over 130,000 participants of European descent from GHS with available whole-genome sequencing and clinical phenotypic data. SINAI is a patient-based health-system-based cohort from the Mount Sinai BioMe biobank, recruited by the Mount Sinai Medical System (Abul-Husn et al., Cell, 2019, 177, 58-69). UPENN-PMBB is a patient-based health-system-based cohort from the PennMedicine biobank at the University of Pennsylvania (Park et al., Genet. Med., 2020, 22, 102-111). MDCS is a Swedish population-based prospective observational cohort study that recruited between 1991 and 1996 (Berglund et al., J. Intern. Med., 1993, 233, 45-51).

[0148] Phenotype definition In each cohort, cases of osteoarthritis were defined based on one or more of the following criteria: 1) a history of osteoarthritis in an electronic health record (using International Classification of Diseases, Tenth Revision (ICD-10) diagnostic codes M15, M16, M17, M18, M19, M47 or the corresponding ICD-9 code), with at least one hospitalization 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 an electronic health record or self-reported; 3) a self-reported physician diagnosis of osteoarthritis. Individuals who did not meet any of the criteria for osteoarthritis were used as controls. Additionally, individuals with a history of other forms of arthritis or joint-related symptoms in their electronic health records or self-reported, or who had undergone surgery involving the joint, were excluded from the control group.

[0149] The imaging phenotype was derived from dual-energy X-ray absorptiometry (DXA) images of the knee joint. DXA images are 2D projections similar to those acquired by standard X-rays. A convolutional network algorithm based on the U-Net architecture was used to segment the knee bones on the DXA images. The joint space width (JSW) was quantified after measuring the distance between the proximal tibia and distal femur. JSW serves as a separate imaging-derived biomarker for joint health.

[0150] In each cohort, cases of knee and hip replacement were defined as follows: 1) a history of knee or hip replacement surgery in the electronic health record, defined using OPCS-4, CPT-4, ICD-10-PCS, or NOMESCO surgical codes; or 2) a self-reported history of knee or hip replacement surgery. Individuals who did not meet any of the criteria for knee or hip replacement were used as controls. Additionally, individuals with a history of other forms of arthritis or joint-related symptoms in their electronic health record or self-reported, or who had undergone surgery involving the joint, were excluded from the control group.

[0151] 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 (VCRome; for a portion of the GHS cohort) or modified forms of xGen designed from Integrated DNA Technologies (IDT; for the remainder of the GHS and other cohorts) were used for exome target sequence capture. A unique 6-base-pair (bp) barcode (VCRome) or 10 bp barcode (IDT) was added to each DNA fragment during library preparation to facilitate multiplex exome capture and sequencing. Equal volumes of samples were pooled prior to exome capture. Sequencing was performed using 75 bp paired end reads on an Illumina v4HiSeq 2500 (for a portion of the GHS cohort) or NovaSeq (for the remainder of the GHS and other cohorts). The sequencing coverage depth (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 VCRome samples and 20x coverage of 90% of the target bases in 99% of IDT samples. Data processing steps included sample de-multiplexing using Illumina software, alignment with the GRCh38 human genome reference sequence, including generating binary alignment and mapping files (BAMs), and processing the BAM files (e.g., marking duplicate reads and other read mapping evaluations). Variant calls were performed using the GLNexus system (DOI: 10.1101 / 343970). Variant mapping and annotation were based on the GRCh38 human genome reference sequence and the Ensembl v85 gene definitions using snpEff software. SnpEff predictions involving the annotated initiation and termination of protein-coding transcripts were then combined into single functional effect predictions by selecting the most harmful functional effect category for each gene. The levels of these annotations (from most harmful to least harmful) are frameshift, termination gain, termination loss, splice acceptor, splice donor, termination loss, in-frame insertion / deletion, missense, and other annotations. Predicted LOF genetic variants include: a) insertions or deletions resulting in frameshifts; b) insertions, deletions, or single nucleotide variants resulting in the introduction of premature stop codons or loss of transcription start or stop sites; and c) variants of donor or acceptor splice sites.Potential functional impacts were predicted using SIFT (Adzhubei et al., Nat. Methods, 2010, 7, 248-9), 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). count computer The prediction algorithm classifies missense variants. For each gene, the alternative allele frequency (AAF) and functional annotation of each variant determine the following seven gene load exposures: 1) pLOF variants where AAF < 1%; 2) pLOF or missense variants predicted as harmful by all five of the five algorithms, where AAF < 1%; 3) pLOF or missense variants predicted as harmful by all five of the five algorithms, where AAF < 0.1%; 4) pLOF or missense variants predicted as harmful by at least one of the five algorithms, where AAF < 1%; 5) pLOF or missense variants predicted as harmful by at least one of the five algorithms, where AAF < 0.1%; 6) pLOF or any missense variant, where AAF < 1%; 7) pLOF or any missense variant, where AAF < 0.1%.

[0152] Association analysis of rare pLOF with gene load of missense variants The association between the load and phenotype of rare predicted loss-of-function or missense variants in a given gene was analyzed by fitting linear (for quantitative traits) or first-bias-corrected logistic regression (for binary traits) models, adjusted for a polygenic score using REGENIE to reduce the genomic kinship matrix (Mbatchou et al., Nat. Genet., 2021, 53, 1097-1103). This was done for age, sex, and age-related factors. 2 Age by gender and age by sex 2Interaction terms and covariates for several specific cohorts, including batch-related covariates and principal component pairs from common and rare variant sources, were stratified and adjusted by ancestry for analysis. Fixed-effects inverse variance-weighted meta-analysis was used to combine cross-cohort results for each variant-phenotype association. In gene load testing, all individuals were labeled heterozygous if they carried one or more eligible rare variants (based on frequency and functional annotation as described above), and homozygous if they carried any eligible variant in a homozygous state. This “composite genotype” was then used to test associations.

[0153] Example 2: The association between osteoarthritis and COLGALT2 Using the data and methods described in Example 1, the burden of rare pLoF and missense variants of COLGALT2 was found to be associated with a significantly reduced odds ratio for knee and / or hip osteoarthritis in the whole exome (odds ratio, 0.89; p-value, 2.3E-08; see Table 3).

[0154] Table 3: Estimates of the association between the burden of rare pLOF missense variants in COLGALT2 and the risk of hip and / or knee osteoarthritis.

[0155] The last two columns show the odds ratio (OR) and p-value (Pval) for the association between rare pLOF variants only and hip and / or knee OA. Genotype counts are shown as three numbers separated by "|", representing the number of individuals who do not carry any relevant rare coding variants in COLGALT2 (homozygous non-carriers), the number of individuals who carry the relevant rare coding variant in a single COLGALT2 allele (heterozygous carriers), and the number of individuals who carry the relevant rare coding variant in both COLGALT2 alleles (homozygous carriers). Genotype counts are shown by case / control status for hip and / or knee OA. pLOF, predicted loss of function; AAF, frequency of alternative alleles; OR, odds ratio; CI, confidence interval; Pval, p-value.

[0156] Rare coding variants in COLGALT2 were also observed to be associated with a reduced risk of knee and hip osteoarthritis (see also: Figure 1 Furthermore, the association between rare variants of COLGALT2 and mJSW confirms its protective association with OA (see [link to relevant documentation]). Figure 2 ).

[0157] Figure 1The association between rare coding variants in COLGALT2 and osteoarthritis of the hip, knee, and spine is illustrated. Specifically, three gene burden exposures are shown: 1 / 5 algorithm predicts a harmful pLOF or missense variant with an AAF <1%; at least 5 / 5 algorithm predicts a harmful pLOF or missense variant with an AAF <1%; and pLOF variant only with an AAF <1%. Genotype counts are shown as three numbers separated by "|", representing the number of individuals who do not carry any relevant rare coding variants in COLGALT2 (homozygous non-carriers), the number of individuals who carry a relevant rare coding variant in a single COLGALT2 allele (heterozygous carriers), and the number of individuals who carry a relevant rare coding variant in both COLGALT2 alleles (homozygous carriers). Genotype counts are shown by case / control status for hip and / or knee OA. pLOF, predicted loss of function; AAF, frequency of alternative alleles; OR, odds ratio; CI, confidence interval; Pval, p-value.

[0158] Figure 2 The association between rare coding variants in COLGALT2 and minimum joint space width (mJSW) derived from DXA imaging is shown. Specifically, two types of gene burden exposure are shown: those predicting harmful pLOF or missense variants with AAF < 1% using a 1 / 5 algorithm, and those predicting only pLOF variants with AAF < 1%. Genotype counts are shown as three numbers separated by "|", representing the number of individuals carrying no relevant rare coding variants in COLGALT2 (homozygous non-carriers), the number of individuals carrying relevant rare coding variants in a single COLGALT2 allele (heterozygous carriers), and the number of individuals carrying relevant rare coding variants in both COLGALT2 alleles (homozygous carriers). pLOF, predicted loss of function; AAF, frequency of alternative alleles; OR, odds ratio; CI, confidence interval; Pval, p-value.

[0159] Figure 3This study illustrates the association between rare coding variants in COLGALT2 and the risk of knee or hip replacement. Specifically, it shows two types of gene burden exposure: those predicting harmful pLOF or missense variants using a 1 / 5 algorithm with AAF <1%, and those with only pLOF variants with AAF <1%. Genotype counts are shown as three numbers separated by "|", representing the number of individuals who do not carry any relevant rare coding variants in COLGALT2 (homozygous non-carriers), the number of individuals who carry a relevant rare coding variant in a single COLGALT2 allele (heterozygous carriers), and the number of individuals who carry a relevant rare coding variant in both COLGALT2 alleles (homozygous carriers). Genotype counts are shown by case / control status for hip and / or knee replacement. pLOF, predicted loss of function; AAF, frequency of replacement alleles; OR, odds ratio; CI, confidence interval; Pval, p-value.

[0160] Table 4 includes all pLoF and missense variants detected by COLGALT2 exome sequencing, and these variants were included in the OA phenotypic analysis. The CPRA (Physical Genomic Location) indicates the genomic coordinates of each variant based on the 38th edition of the human genome sequence established by the Human Genome Reference Consortium: chromosomal, physical genomic location in base pairs, reference allele, and alternative allele. The HGVS.p and HGVS.c columns indicate protein and coding DNA variations according to the Human Genome Variation Association nomenclature. The transcript column indicates the predictive impact of variants on the classic COLGALT2 transcript (ENST00000361927.9).

[0161] Table 4

[0162] In addition to the various modifications described herein, various modifications to the subject matter will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Every 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, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. A method for treating a subject who has osteoarthritis or is at risk of developing osteoarthritis, the method comprising administering a procollagen galactosyltransferase 2 (COLGALT2) inhibitor to the subject.

2. The method of claim 1, wherein the COLGALT2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a COLGALT2 nucleic acid molecule.

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

4. The method of claim 3, wherein the inhibitory nucleic acid molecule comprises 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 osteoarthritis treatment agent or osteoarthritis therapy is further administered to the subject.

7. The method of any one of claims 1 to 6, further comprising detecting the presence or absence of a COLGALT2 variant nucleic acid molecule in a biological sample from the subject.

8. The method of claim 7, further comprising administering to the subject an amount of osteoarthritis treatment agent or osteoarthritis therapy equal to or less than the standard dose when the COLGALT2 variant nucleic acid molecule is not present in the biological sample.

9. The method of claim 7, further comprising administering to the subject an amount of osteoarthritis treatment agent or osteoarthritis therapy equal to or less than a standard dose when the subject is heterozygous for the COLGALT2 variant nucleic acid molecule.

10. The method of any one of claims 7 to 9, wherein the COLGALT2 variant nucleic acid molecule comprises a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, and / or a variant encoding a truncated COLGALT2 variant polypeptide.

11. The method of any one of claims 7 to 9, wherein the COLGALT2 variant nucleic acid molecule comprises any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4, or an mRNA molecule or a cDNA molecule generated from the genomic nucleic acid molecule.

12. A method of treating a subject suffering from or at risk of developing osteoarthritis by administering an osteoarthritis treatment agent or osteoarthritis therapy, the method comprising: Whether the subject possesses a procollagen galactosyltransferase 2 (COLGALT2) variant nucleic acid molecule was determined or has been determined by the following methods: Biological samples obtained or acquired from the subject; as well as The biological sample was subjected to or has been subjected to sequence analysis to determine whether the subject has a genotype containing a COLGALT2 variant nucleic acid molecule; as well as Administer or continue to administer the osteoarthritis treatment agent or osteoarthritis therapy and / or COLGALT2 inhibitor to subjects who are serving as a COLGALT2 reference dose, at the same or less than the standard dose. Administering or continuing to administer the osteoarthritis treatment agent or osteoarthritis therapy and / or COLGALT2 inhibitor to subjects heterozygous for the COLGALT2 variant nucleic acid molecules; or Administer or continue standard doses of the osteoarthritis treatment agent or osteoarthritis therapy to subjects homozygous for the COLGALT2 variant nucleic acid molecule; The presence of the COLGALT2 variant nucleic acid molecule indicated a reduced risk of the subject developing osteoarthritis.

13. The method of claim 12, wherein the COLGALT2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a COLGALT2 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 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, an amount equal to or less than a standard dose of the osteoarthritis treatment agent or osteoarthritis therapy and the COLGALT2 inhibitor to a subject heterozygous for the COLGALT2 variant nucleic acid molecule.

18. The method of any one of claims 12 to 16, wherein the method comprises administering, or continuing to administer, an amount equal to or less than a standard dose of the osteoarthritis treatment agent or osteoarthritis therapy and the COLGALT2 inhibitor to a subject serving as a COLGALT2 reference.

19. The method of any one of claims 12 to 18, wherein the COLGALT2 variant nucleic acid molecule comprises a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, and / or a variant encoding a truncated COLGALT2 variant polypeptide.

20. The method of any one of claims 12 to 19, wherein the COLGALT2 variant nucleic acid molecule comprises any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4, or an mRNA molecule or a cDNA molecule generated from the genomic nucleic acid molecule.

21. A method for identifying subjects at increased risk of developing osteoarthritis, the method comprising: The presence or absence of procollagen galactosyltransferase 2 (COLGALT2) variant nucleic acid molecules in biological samples obtained from the subject has been determined or has been determined. in: When the subject was a COLGALT2 reference, the subject had an increased risk of developing osteoarthritis; and When the subject is heterozygous or homozygous for the COLGALT2 variant nucleic acid molecule, the subject's risk of developing osteoarthritis is reduced.

22. The method of claim 21, wherein the COLGALT2 variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, or a variant encoding a truncated COLGALT2 variant polypeptide.

23. The method of claim 21 or claim 22, wherein the COLGALT2 variant nucleic acid molecule comprises any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4, or an mRNA molecule or a cDNA molecule generated from the genomic nucleic acid molecule.

24. The method of any one of claims 21 to 23, further comprising administering to a subject, as a COLGALT2 reference, an amount of osteoarthritis treatment agent or osteoarthritis therapy and / or COLGALT2 inhibitor equal to or less than a standard dose.

25. The method of claim 24, wherein the subject is a COLGALT2 reference, and the subject is given or continues to be given an amount of the osteoarthritis treatment agent or osteoarthritis therapy and the COLGALT2 inhibitor that is the same as or less than the standard dose.

26. The method of any one of claims 21 to 23, further comprising administering to a subject heterozygous for a COLGALT2 variant nucleic acid molecule an amount of osteoarthritis treatment agent or osteoarthritis therapy and / or a COLGALT2 inhibitor the same as or less than a standard dose.

27. The method of claim 26, wherein the subject is heterozygous for the COLGALT2 variant nucleic acid molecule, and the subject is given or continues to be given an amount of the osteoarthritis treatment agent or osteoarthritis therapy and the COLGALT2 inhibitor that is the same as or less than the standard dose.

28. The method of any one of claims 24 to 27, wherein the COLGALT2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a COLGALT2 nucleic acid molecule.

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

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

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

32. An osteoarthritis treatment agent for treating or preventing osteoarthritis in a subject with a procollagen galactosyltransferase 2 (COLGALT2) variant nucleic acid molecule.

33. The osteoarthritis treatment agent of claim 32, wherein the COLGALT2 variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, or a variant encoding a truncated COLGALT2 variant polypeptide.

34. The osteoarthritis treatment agent of claim 32 or claim 33, wherein the COLGALT2 variant nucleic acid molecule comprises any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4, or an mRNA molecule or a cDNA molecule generated from the genomic nucleic acid molecule.

35. A procollagen galactosyltransferase 2 (COLGALT2) inhibitor for the treatment or prevention of osteoarthritis in subjects who are COLGALT2 references or heterozygous for COLGALT2 variant nucleic acid molecules.

36. The COLGALT2 inhibitor of claim 35, wherein the COLGALT2 variant nucleic acid molecule is a splice site variant, a stop codon gain variant, a start codon deletion variant, a stop codon deletion variant, a frameshift variant, a missense variant, an in-frame insertion deletion variant, or a variant encoding a truncated COLGALT2 variant polypeptide.

37. The COLGALT2 inhibitor of claim 35 or claim 36, wherein the COLGALT2 variant nucleic acid molecule comprises any one or more genetic variations of the genomic nucleic acid molecules listed in Table 4, or an mRNA molecule or a cDNA molecule generated from the genomic nucleic acid molecule.

38. The COLGALT2 inhibitor according to any one of claims 35 to 37, wherein the COLGALT2 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with the COLGALT2 nucleic acid molecule.

39. The COLGALT2 inhibitor of claim 38, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

40. The COLGALT2 inhibitor of claim 39, wherein the inhibitory nucleic acid molecule comprises siRNA.

41. The COLGALT2 inhibitor of claim 39, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

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