Treatment of uveitis with endoplasmic reticulum aminopeptidase 1 (ERAP1) inhibitors

Abstract

The present disclosure provides methods of treating a subject with uveitis, and methods of identifying subjects at high risk for developing uveitis.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as an XML file of 4,801 kilobytes in size under the title 381203620SEQ, created on October 11, 2022. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to the treatment of subjects having uveitis, such as anterior uveitis, with an endoplasmic reticulum aminopeptidase 1 (ERAP1) inhibitor, and to methods for identifying subjects at high risk for developing uveitis. [Background technology]

[0003] Anterior uveitis (AU) is an inflammation of the middle layer of the eye. This middle layer includes the iris (the colored part of the eye) and the adjacent tissue known as the ciliary body. Anterior uveitis can result from trauma to the eye, such as being hit on the eye or having a foreign body placed in the eye. It can also be associated with common health problems such as rheumatoid arthritis, syphilis, tuberculosis, sarcoid, viruses (herpes simplex, shingles, cytomegalovirus) or idiopathic with no apparent underlying cause. Acute anterior uveitis (AAU) involves inflammation of the iris and ciliary body of the eye. AAU can occur alone or as an extra-articular feature of ankylosing spondylitis (AS). It can be vision threatening and can lead to ophthalmic sequelae such as cataracts, posterior iris synechiae, and glaucoma. Uveitis accounts for approximately 10% of individuals with severe visual impairment and blindness. The prevalence of anterior uveitis is approximately 0.1-0.3% (1-3:1,000). Approximately 300,000-350,000 cases of uveitis occur in the United States. AAU occurs in 30-40% of individuals with AS and increases in frequency with disease duration, such that the prevalence approaches 60% in individuals with AS beyond 50 years. AAU is known to be associated with HLA-B*27 independently of the association of this allele with AS (approximately 50% of affected individuals are HLA-B*27 carriers).

[0004] Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an aminopeptidase involved in trimming HLA class I-binding precursors, a necessary step for most HLA class I-binding peptides to be generated so that they can be presented on MHC class I molecules. Trimming of peptides is essential for customizing long precursor peptides to the correct length required for presentation on MHC class I molecules. ERAP1 acts as a monomer or as a heterodimer with ERAP2. ERAP1 has a strong preference for substrates 9-16 residues long, and rapidly degrades 13-mers to 9-mers and then terminates. ERAP1 preferentially hydrolyzes peptides with the residue Leu and a hydrophobic C-terminus, but is less active against peptides with a charged C-terminus. Summary of the Invention

[0005] The present disclosure provides a method for treating a subject having uveitis, the method comprising administering an ERAP1 inhibitor to the subject. The present disclosure also provides a method for treating a subject having iridocyclitis, the method comprising administering an ERAP1 inhibitor to the subject.

[0006] The present disclosure also provides a method for treating a subject having iritis, the method comprising administering an ERAP1 inhibitor to the subject. The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or inhibiting uveitis, wherein the subject has uveitis, the method comprising: obtaining or obtaining a biological sample from the subject; determining whether the subject has an ERAP1 mutant nucleic acid molecule by performing or performing sequence analysis on the biological sample to determine whether the subject has a genotype including the ERAP1 mutant nucleic acid molecule; administering or continuing to administer a therapeutic agent for treating or inhibiting uveitis at a standard dosage to the subject who is an ERAP1 standard, and / or administering an ERAP1 inhibitor to the subject; administering or continuing to administer a therapeutic agent for treating or inhibiting uveitis at the same or a smaller dosage to the subject who is heterozygous for the ERAP1 mutant nucleic acid molecule, and / or administering an ERAP1 inhibitor to the subject; wherein the presence of a genotype having the ERAP1 mutant nucleic acid molecule indicates that the subject has a low risk of developing uveitis.

[0007] The present disclosure also provides a method for identifying a subject at high risk of developing uveitis, the method comprising determining or having determined the presence or absence of an ERAP1 mutant nucleic acid molecule in a biological sample obtained from the subject; wherein, if the subject is an ERAP1 criterion, the subject has a high risk of developing uveitis; and, if the subject is heterozygous or homozygous for the ERAP1 mutant nucleic acid molecule, the subject has a low risk of developing uveitis.

[0008] The present disclosure also relates to a therapeutic agent for treating or inhibiting uveitis, comprising: i) an ERAP1 mutant genomic nucleic acid molecule or a complement thereof having a nucleotide sequence including guanine or a complement thereof at a position corresponding to position 19,474 set forth in SEQ ID NO: 2, adenine or a complement thereof at a position corresponding to position 21,595 set forth in SEQ ID NO: 3, cytosine or a complement thereof at a position corresponding to position 21,811 set forth in SEQ ID NO: 4, or thymine or a complement thereof at a position corresponding to position 42,579 set forth in SEQ ID NO: 5; ii) an ERAP1 mutant genomic nucleic acid molecule or a complement thereof having a nucleotide sequence including guanine or a complement thereof at a position corresponding to position 19,474 set forth in SEQ ID NO: 2, adenine or a complement thereof at a position corresponding to position 21,595 set forth in SEQ ID NO: 3, cytosine or a complement thereof at a position corresponding to position 21,811 set forth in SEQ ID NO: 4, or thymine or a complement thereof at a position corresponding to position 42,579 set forth in SEQ ID NO: The present invention provides a therapeutic agent for use in treating uveitis in a subject identified as having an ERAP1 mutant mRNA molecule or its complement, which has a nucleotide sequence including guanine or its complement, or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; or iii) an ERAP1 mutant cDNA molecule or its complement, which has a nucleotide sequence including guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10, or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:11.

[0009] The present disclosure also relates to an ERAP1 inhibitor, comprising: a) an ERAP1 genomic nucleic acid molecule, an ERAP1 mRNA molecule, or an ERAP1 cDNA molecule, or b) an ERAP1 mutant genomic nucleic acid molecule or a complement thereof, which has a nucleotide sequence including i) a guanine or a complement thereof at a position corresponding to position 19,474 set forth in SEQ ID NO: 2, an adenine or a complement thereof at a position corresponding to position 21,595 set forth in SEQ ID NO: 3, a cytosine or a complement thereof at a position corresponding to position 21,811 set forth in SEQ ID NO: 4, or a thymine or a complement thereof at a position corresponding to position 42,579 set forth in SEQ ID NO: 5; ii) a guanine or a complement thereof at a position corresponding to position 1,841 set forth in SEQ ID NO: 7, or a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10, or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO: 11, wherein the ERAP1 inhibitor is for use in treating uveitis in a subject that is heterozygous for an ERAP1 mutant cDNA molecule or its complement having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10, or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO: 11.

[0010] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several features of the present disclosure. [Brief description of the drawings]

[0011] [Figure 1A] A forest plot of the top single nucleotide polymorphisms (SNPs) in the HLA-B27 locus is shown. [Figure 1B] 1 shows a forest plot of top single nucleotide polymorphisms (SNPs) in the ERAP1 gene locus. [Diagram 2] 3 shows ERAP1 protection across seven B27-positive cohorts. [Diagram 3] 1 shows combinations of the risk of anterior uveitis due to ERAP1 and HLA-B27 / HLA-B40. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0013] Unless expressly stated otherwise, no method or embodiment set forth herein is intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically specifies in the claim or description that the steps are to be limited to a particular order, it is not intended to dictate order in any respect. This includes any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or workflow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0014] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that a cited numerical value is approximate, and small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0015] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0016] As used herein, with respect to a nucleic acid molecule or polypeptide, the term "isolated" means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., away from blood and / or other tissues. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0017] 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 multistranded. A strand of a nucleic acid also refers to its complement.

[0018] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates (e.g., apes and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0019] Mutations in the ERAP1 gene that result in reduced expression of ERAP1 or result in loss-of-function polypeptides predicted by ERAP1 that are associated with a reduced risk of developing uveitis in humans, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis, have been identified according to the present disclosure. For example, it has been observed that genetic mutations that change the adenine at position 19,474 of the ERAP1 reference genomic nucleic acid molecule (see SEQ ID NO: 1) to guanine, or change the guanine at position 21,595 of the ERAP1 reference genomic nucleic acid molecule (see SEQ ID NO: 1) to adenine, or change the thymine at position 21,811 of the ERAP1 reference genomic nucleic acid molecule (see SEQ ID NO: 1) to cytosine, or change the cytosine at position 42,579 of the ERAP1 reference genomic nucleic acid molecule (see SEQ ID NO: 1) to thymine indicate that subjects with such mutations may have a low risk of developing uveitis. In summary, the genetic analysis described herein surprisingly shows that the ERAP1 gene and, in particular, variants of the ERAP1 gene are associated with a reduced risk of developing uveitis. In some embodiments, this association is independent of HLA-B*27. In some embodiments, this association is synergistic with HLA-B*27. Thus, subjects with ERAP1 criteria at high risk of developing uveitis, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis, can be treated to prevent uveitis, alleviate its symptoms, and / or inhibit the onset of symptoms. Thus, the present disclosure provides methods that utilize the identification of such variants in a subject to identify or stratify the risk in such a subject of developing uveitis, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis, or to diagnose a subject as having an elevated risk of developing uveitis, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis, such that subjects at risk or with active disease can be treated accordingly.

[0020] For the purpose of this disclosure, any particular subject can be classified as having one of three ERAP1 genotypes: i) ERAP1 standard; ii) heterozygous for ERAP1 mutant nucleic acid molecule; or iii) homozygous for ERAP1 mutant nucleic acid molecule. If the subject does not have a copy of ERAP1 mutant nucleic acid molecule, the subject is ERAP1 standard. If the subject has a single copy of ERAP1 mutant nucleic acid molecule, the subject is heterozygous for ERAP1 mutant nucleic acid molecule. As used herein, ERAP1 mutant nucleic acid molecule is any ERAP1 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes an ERAP1 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function, or any ERAP1 nucleic acid molecule that reduces the expression of ERAP1 polypeptide. A subject having an ERAP1 mutant nucleic acid molecule is hypomorphic for ERAP1. The ERAP1 mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted for ERAP1 can be any nucleic acid molecule encoding ERAP1 Lys528Arg or Asp575Asn. In some embodiments, the ERAP1 mutant nucleic acid molecule encodes ERAP1 Lys528Arg. In some embodiments, the ERAP1 mutant nucleic acid molecule encodes ERAP1 Asp575Asn. When the subject has two copies of the ERAP1 mutant nucleic acid molecule, the subject is homozygous for the ERAP1 mutant nucleic acid molecule.

[0021] For subjects who are genotyped or determined to be ERAP1-based, such subjects are at high risk of developing uveitis, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis. For subjects who are genotyped or determined to be ERAP1-based or heterozygous for an ERAP1 variant nucleic acid molecule, such subjects can be treated with an ERAP1 inhibitor.

[0022] In some embodiments, a subject who has been genotyped or determined to be ERAP1 standard is at a higher risk of developing uveitis, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis, if the subject also has an increased number of HLA-B27 alleles (e.g., one or two HLA-B27 alleles compared to no HLA-27 alleles). In some embodiments, a subject who has been genotyped or determined to be heterozygous for an ERAP1 variant nucleic acid molecule is at a higher risk of developing uveitis, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis, if the subject also has an increased number of HLA-B27 alleles (e.g., one or two HLA-B27 alleles compared to no HLA-27 alleles). In some embodiments, a subject may have one HLA-B27 allele and one HLA-B40 allele (and be heterozygous for the ERAP1 reference or ERAP1 variant nucleic acid molecule) and be at a higher risk of developing uveitis, such as anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, and panuveitis (compared to a similar subject not having the HLA-B27 allele).

[0023] In any of the embodiments described throughout this disclosure, the ERAP1 mutant nucleic acid molecule can be any ERAP1 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes an ERAP1 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function, or any ERAP1 nucleic acid molecule that reduces the expression of an ERAP1 polypeptide. In any of the embodiments described herein, the ERAP1 mutant nucleic acid molecule can be any nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that is a missense mutant, a splice site mutant, a stop-gain mutant, a start-loss mutant, a stop-loss mutant, a frameshift mutant, an in-frame indel mutant, or a mutant that encodes a truncated ERAP1 polypeptide. For example, the ERAP1 mutant nucleic acid molecule can be any nucleic acid molecule that encodes ERAP1 Lys528Arg. In some embodiments, the ERAP1 mutant nucleic acid molecule encodes ERAP1 Asp575Asn. In some embodiments, the ERAP1 mutant nucleic acid molecule is not rs30187.

[0024] In any of the embodiments described throughout this disclosure, the predicted loss-of-function polypeptide in ERAP1 can be any ERAP1 polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In any of the embodiments described throughout this disclosure, the predicted loss-of-function polypeptide in ERAP1 can be any of the ERAP1 polypeptides described herein, including, for example, ERAP1 Lys528Arg or Asp575Asn. In some embodiments, the predicted loss-of-function polypeptide in ERAP1 is ERAP1 Lys528Arg. In some embodiments, the predicted loss-of-function polypeptide in ERAP1 is ERAP1 Asp575Asn.

[0025] In any of the embodiments described throughout this disclosure, the uveitis is anterior uveitis, acute anterior uveitis, iridocyclitis, iritis, or panuveitis. In any of the embodiments described throughout this disclosure, the uveitis is anterior uveitis. In any of the embodiments described throughout this disclosure, the uveitis is acute anterior uveitis. In any of the embodiments described throughout this disclosure, the anterior uveitis includes iridocyclitis. In any of the embodiments described throughout this disclosure, the anterior uveitis includes iritis. In any of the embodiments described throughout this disclosure, the uveitis is panuveitis. In any of the embodiments described throughout this disclosure, the subject may have ankylosing spondylitis. In any of the embodiments described throughout this disclosure, the subject does not have ankylosing spondylitis.

[0026] Symptoms of anterior uveitis include, but are not limited to, severe redness of the eye, pain, dark spots in the field of vision (also known as "floaters"), sensitivity to light, and / or blurred vision.

[0027] The present disclosure provides a method for treating a subject having uveitis, the method comprising administering an ERAP1 inhibitor to the subject. In some embodiments, the uveitis is anterior uveitis. In some embodiments, the uveitis is acute anterior uveitis. In some embodiments, the uveitis is panuveitis.

[0028] The present disclosure also provides a method for treating a subject having iridocyclitis, the method comprising administering an ERAP1 inhibitor to the subject. The present disclosure also provides a method for treating a subject having iritis, the method comprising administering an ERAP1 inhibitor to the subject.

[0029] In some embodiments, the ERAP1 inhibitor comprises an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule, a small interfering RNA (siRNA) molecule, or a short hairpin RNA (shRNA) molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an shRNA molecule. Such an inhibitory nucleic acid molecule can be designed to target any region of an ERAP1 nucleic acid molecule, such as an mRNA molecule. In some embodiments, the inhibitory nucleic acid molecule hybridizes with a sequence within an ERAP1 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of an ERAP1 polypeptide in a cell of a subject. In some embodiments, the ERAP1 inhibitor comprises an antisense RNA that hybridizes with an ERAP1 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of an ERAP1 polypeptide in a cell of a subject. In some embodiments, the ERAP1 inhibitor comprises an siRNA that hybridizes with an ERAP1 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of an ERAP1 polypeptide in a cell of a subject. In some embodiments, the ERAP1 inhibitor comprises an shRNA that hybridizes with an ERAP1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the ERAP1 polypeptide in a subject cell.

[0030] In some embodiments, the ERAP1 antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 35 to 784. In some embodiments, the ERAP1 siRNA molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 785 to 2578 (sense strand and antisense strand are presented consecutively) (e.g., the sense strand is, for example, SEQ ID NO: 785 and the corresponding antisense strand is SEQ ID NO: 786; the sense strand is, for example, SEQ ID NO: 787 and the corresponding antisense strand is SEQ ID NO: 788, etc.).

[0031] The inhibitory nucleic acid molecule can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous nucleic acid sequence, for example in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecule can be present as an exogenous donor sequence in or containing a vector that contains the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, whereby an enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0032] Inhibitory nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0033] The inhibitory nucleic acid molecule can also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains a modification to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include 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 (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0034] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10 Exemplary 2' sugar modifications include -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 Other modifications at the 2' position include, but are not limited to, C 1~10Examples of suitable substituents include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0035] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.

[0036] Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse 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).

[0037] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first 1-7 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-MOE modification. In some embodiments, the first 1-7 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0038] In some embodiments, the siRNA molecule has terminal modification.In some embodiments, the 5'-end of the antisense strand is phosphorylated.In some embodiments, a 5'-phosphate analog that cannot be hydrolyzed, such as 5'-(E)-vinyl-phosphonate, is used.

[0039] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to increase the stability and bioavailability of siRNA in vivo. Non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate to obtain phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, the phosphodiester group can be replaced with a phosphotriester to facilitate cellular uptake of the siRNA and retention in serum components by removing 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), allowing the 2'-hydroxyl to act as a nucleophile and attack the adjacent phosphorus of the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

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

[0041] In some embodiments, siRNA molecules are bound to lipid.Lipid can be bound to 5'-end or 3'-end of siRNA, and can improve their bioavailability in vivo by associating with serum lipoprotein.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitic acid and tocopherol.

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

[0043] In any of the embodiments described herein, the inhibitory nucleic acid molecule can be administered, for example, as a 1-2 hour intravenous infusion or subcutaneous injection. In any of the embodiments described herein, the inhibitory nucleic acid molecule can be administered in a dose of 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 (2.5-9.14 mg / kg; 92.5-338 mg / m 2 , i.e., based on an assumption of a body weight of 70 kg, and based on a mg / kg dose multiplier of 37 for humans, the dose is calculated from mg / kg to mg / m 2 The drug may be administered at dosage levels ranging from 0.1 mg / kg to 100 mg / kg (calculated as a dose level of 0.1 mg / kg).

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

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

[0046] In some embodiments, the ERAP1 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in a recognition sequence(s) in an ERAP1 genomic nucleic acid molecule or a DNA binding protein that binds to the recognition sequence. The recognition sequence can be located in the coding region of the ERAP1 gene or in a regulatory region that affects the expression of the gene. The recognition sequence of the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence can include or be adjacent to the start codon of the ERAP1 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each of which targets a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence containing or adjacent to a start codon, and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and cleavage by these nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences.Any nuclease agent that induces a nick or double-strand break at 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.

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

[0048] In some embodiments, the CRISPR / Cas system can be used to modify the ERAP1 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can employ the CRISPR-Cas system by utilizing a CRISPR complex (including a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the ERAP1 nucleic acid molecule.

[0049] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins can also contain nuclease domains (e.g., 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 (e.g., FnCpf1). Cas proteins can have full cleavage activity to create double-stranded breaks in ERAP1 genomic nucleic acid molecules, or can be nickases that create single-stranded breaks in ERAP1 genomic nucleic acid molecules. Additional examples of Cas proteins include 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), Cas proteins include, but are not limited to, 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, as well as homologs or variants thereof. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Cas proteins can be provided in any form. For example, the Cas protein can be provided in the form of a protein, e.g., a Cas protein complexed with a gRNA.Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule, e.g., RNA or DNA, encoding the Cas protein.

[0050] In some embodiments, targeted genetic recombination of the ERAP1 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize with one or more gRNA recognition sequences in a target genomic locus in the ERAP1 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within the region of SEQ ID NO: 1. The gRNA recognition sequence can also include or be adjacent to a position corresponding to position 19,474, position 21,595, position 21,811, or position 42,579. For example, the gRNA recognition sequence can be located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides away from a position corresponding to position 19,474, position 21,595, position 21,811, or position 42,579. The gRNA recognition sequence can include or be adjacent to the start codon or stop codon of the ERAP1 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from the start codon or stop codon.

[0051] The gRNA recognition sequence in the target genomic locus in the ERAP1 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. A standard PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. Although the gRNA can transport Cas9 anywhere in the genome for gene editing, it cannot edit at sites other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-standard PAM for human cells. In general, the PAM is about 2 to about 6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 3' end. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10 base pairs, about 2 to about 5 base pairs, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (e.g., when using Cas9 from S. pyogenes or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand is 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0052] gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific position in the ERAP1 genomic nucleic acid molecule. An exemplary gRNA is a gRNA that is effective for inducing a Cas enzyme to bind to or cleave an ERAP1 genomic nucleic acid molecule, in which the gRNA comprises a DNA targeting segment that hybridizes with a gRNA recognition sequence in the ERAP1 genomic nucleic acid molecule that includes or is adjacent to a position corresponding to position 19,474, 21,595, 21,811, or 42,579. For example, the gRNA can be selected to hybridize with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a position corresponding to 19,474, 21,595, 21,811, or 42,579. Other exemplary gRNAs include a DNA targeting segment that hybridizes with a gRNA recognition sequence present in the ERAP1 genomic nucleic acid molecule that includes or is adjacent to the start codon or stop codon. For example, a gRNA can be selected to hybridize with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a start codon, or with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a stop codon. Suitable gRNAs can include about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA includes 20 nucleotides.

[0053] Examples of suitable gRNA recognition sequences located in the ERAP1 reference gene are described in Table 1 as SEQ ID NOs: 15 to 34.

[0054] [Table 1]

[0055] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target ERAP1 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target ERAP1 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including a gRNA that hybridizes with a gRNA recognition sequence and forms a complex with a Cas protein) can result in the cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 or more base pairs) the nucleic acid sequence present in the ERAP1 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0056] Such a method can produce an ERAP1 genomic nucleic acid molecule in which, for example, a region of SEQ ID NO:1 is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the ERAP1 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes with a second gRNA recognition sequence), cleavage by the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0057] In some embodiments, the ERAP1 inhibitor comprises a small molecule degrader, a proteolysis-inducing chimera, or an immunomodulator. In some embodiments, the ERAP1 inhibitor comprises an anti-ERAP1 antibody. In some embodiments, the ERAP1 inhibitor is DG002 and DG013 (see Zervoudi et al., Proc. Nat'l Acad. Sci. USA, 2013, 110, 19890-19895). In some embodiments, the ERAP1 inhibitor is a phosphinic acid dipeptide analog or a phosphinic acid tripeptide analog (see Weglarz-Tomczak et al., Bioorg. Med. Chem. Lett., 2016, 26, 4122-4126). In some embodiments, the ERAP1 inhibitor is (N-(N-(2-(1H-indol-3-yl)ethyl)carbamimidoyl)-2,5-difluorobenzenesulfonamide), (1-(1-(4-acetylpiperazine-1-carbonyl)cyclohexyl)-3-(p-tolyl)urea), or (4-methoxy-3-(N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)sulfamoyl)benzoic acid (Maben et al. al., J. Med. Chem., 2020, 63, 103-121). In some embodiments, the ERAP1 inhibitor is (4aR,5S,6R,8S,8aR)-5-(2-(furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid (see Liddle et al., J. Med. Chem., 2020, 63, 3348-3358). In some embodiments, the ERAP1 inhibitor is DG013A or a phosphinic acid tripeptide or dipeptide or an aminophosphonic acid derivative, or a 3,4-diaminobenzoic acid (DABA) derivative, or a derivative of thimerosal (Georgiadis et al., Cur. (See Med. Chem., 2019, 26, 2715-2729).In some embodiments, the ERAP1 inhibitor is a benzofuran or 7-benzofuran amide variant (see Deddouche-Grass et al., ACS Med. Chem. Lett., 2021, 12, 1137-1142).

[0058] In some embodiments, the treatment method further comprises detecting the presence or absence of an ERAP1 mutant nucleic acid molecule in a biological sample obtained from a subject.As used throughout this disclosure, "ERAP1 mutant nucleic acid molecule" is any ERAP1 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes an ERAP1 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function, or any ERAP1 nucleic acid molecule that reduces the expression of an ERAP1 polypeptide.

[0059] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits uveitis. In some embodiments, the subject has uveitis, e.g., anterior uveitis. In some embodiments, the subject is at risk of developing uveitis, e.g., anterior uveitis. In some embodiments, the method includes determining whether the subject has an ERAP1 variant nucleic acid molecule by obtaining or obtaining a biological sample obtained from the subject and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes the ERAP1 variant nucleic acid molecule. If the subject is an ERAP1 standard, a therapeutic agent that treats or inhibits uveitis is administered or continues to be administered to the subject at a standard dosage, and / or an ERAP1 inhibitor is administered to the subject. If the subject is heterozygous for the ERAP1 variant nucleic acid molecule, a therapeutic agent that treats or inhibits uveitis is administered or continues to be administered to the subject at the same or less than the standard dosage, and / or an ERAP1 inhibitor is administered to the subject. The presence of a genotype having an ERAP1 variant nucleic acid molecule indicates that the subject has a low risk of developing uveitis. In some embodiments, the subject is ERAP1 standard. In some embodiments, the subject is heterozygous for the ERAP1 variant nucleic acid molecule.

[0060] For subjects who are genotyped or determined to be heterozygous for an ERAP1 reference or ERAP1 mutant nucleic acid molecule, such subjects can be treated with an ERAP1 inhibitor as described herein.

[0061] Detecting the presence or absence of an ERAP1 mutant nucleic acid molecule in a biological sample obtained from a subject and / or determining whether the subject has an ERAP1 mutant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in a test tube. 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 ERAP1 mutant nucleic acid molecule can be present in a cell obtained from the subject.

[0062] In some embodiments, if the subject is an ERAP1 standard, the subject is also administered a therapeutic agent for treating or inhibiting uveitis at a standard dose. In some embodiments, if the subject is heterozygous for the ERAP1 variant nucleic acid molecule, the subject is also administered a therapeutic agent for treating or inhibiting uveitis at a dose equal to or less than the standard dose.

[0063] In some embodiments, the treatment method further comprises detecting the presence or absence of a loss-of-function polypeptide predicted by ERAP1 in a biological sample obtained from the subject. In some embodiments, if the subject does not have a loss-of-function polypeptide predicted by ERAP1, the subject is also administered a therapeutic agent that treats or inhibits uveitis at a standard dose. In some embodiments, if the subject has a loss-of-function polypeptide predicted by ERAP1, the subject is also administered a therapeutic agent that treats or inhibits uveitis at a dose equal to or less than the standard dose.

[0064] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits uveitis. In some embodiments, the subject has uveitis, e.g., anterior uveitis. In some embodiments, the subject is at risk of developing uveitis, e.g., anterior uveitis. In some embodiments, the method includes determining whether the subject has a loss-of-function polypeptide predicted in ERAP1 by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a loss-of-function polypeptide predicted in ERAP1. If the subject does not have a loss-of-function polypeptide predicted in ERAP1, a therapeutic agent that treats or inhibits uveitis is administered or continues to be administered to the subject at a standard dose, and / or an ERAP1 inhibitor is administered to the subject. If the subject has a loss-of-function polypeptide predicted by ERAP1, a therapeutic agent for treating or inhibiting uveitis is administered or continues to be administered to the subject at the same or a smaller amount than the standard dose, and / or an ERAP1 inhibitor is administered to the subject. The presence of a loss-of-function polypeptide predicted by ERAP1 indicates that the subject is at a low risk of developing uveitis. In some embodiments, the subject has a loss-of-function polypeptide predicted by ERAP1. In some embodiments, the subject does not have a loss-of-function polypeptide predicted by ERAP1.

[0065] Detecting the presence or absence of a loss-of-function polypeptide predicted by ERAP1 in a biological sample obtained from a subject and / or determining whether a subject has a loss-of-function polypeptide predicted by ERAP1 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 loss-of-function polypeptide predicted by ERAP1 can be present in a cell obtained from a subject.

[0066] Examples of therapeutic agents that treat or inhibit uveitis, such as anterior uveitis, include, but are not limited to, ophthalmic steroids, such as prednisolone, prednisone, difluprednate, triamcinolone acetonide, fluoromethol, fluocinolone, or dexamethasone; immunosuppressants, such as azathioprine and cyclophosphamide; glucocorticoids, such as cortisone; and antirheumatic drugs, such as adalimumab. Additional therapeutic agents that treat or inhibit uveitis include, but are not limited to, cyclopentolate, atropine, homatropine, corticotropin, gentamicin, corticotropin, loteprednol, tobramycin, atropine, sulfasalazine, hydrocortisone, neomycin, polymyxin b, bacitracin, and sodium sulfacetamide.

[0067] In some embodiments, the dose of a therapeutic agent for treating or inhibiting uveitis can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject who is heterozygous for an ERAP1 variant nucleic acid molecule (i.e., less than the standard dose) compared to a subject who is the ERAP1 standard (which may receive the standard dose). In some embodiments, the dose of a therapeutic agent for treating or inhibiting uveitis can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dose of a therapeutic agent for treating or inhibiting uveitis in a subject who is heterozygous for an ERAP1 variant nucleic acid molecule can be administered less frequently compared to a subject who is the ERAP1 standard.

[0068] Administration of the therapeutic agent for treating or inhibiting uveitis and / or the ERAP1 inhibitor can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. The repeated administration can be the same dose or different doses. Administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, according to a particular dosing regimen, a subject can be treated for an extended period of time, such as, for example, six months, one year, or more. Furthermore, the therapeutic agent for treating or inhibiting uveitis and / or the ERAP1 inhibitor can be administered sequentially or simultaneously. Furthermore, the therapeutic agent for treating or inhibiting uveitis and / or the ERAP1 inhibitor can be administered in separate compositions or together in the same composition.

[0069] Administration of the therapeutic agent and / or ERAP1 inhibitor for treating or inhibiting uveitis can occur by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical composition for administration is desirably sterile, substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition can be provided in a unit dosage form (i.e., a single dose for administration). The pharmaceutical composition can be formulated using one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially harmful to the recipient thereof.

[0070] 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 prophylactic effect, respectively. In some embodiments, the therapeutic effect comprises one or more of: reduction / alleviation of uveitis, reduction / alleviation of the severity of uveitis (e.g., reduction or inhibition of the onset of anterior uveitis), reduction / alleviation of symptoms and uveitis-related effects, delaying the onset of symptoms of uveitis-related effects, reducing the severity of symptoms of uveitis-related effects, reducing the severity of acute episodes, reducing the number of symptoms of uveitis-related effects, reducing the latency period of symptoms of uveitis-related effects, improving symptoms of uveitis-related effects, reducing secondary symptoms, reducing secondary infections, preventing recurrence of uveitis, reducing the number or frequency of recurrent episodes, increasing the latency period between symptomatic episodes, increasing the time to sustained progression, promoting remission, inducing remission, enhancing remission, accelerating recovery, or increasing the effectiveness of or reducing resistance to alternative therapeutic agents, and / or increasing the survival time of the affected host animal following administration of any therapeutic agent or composition. A prophylactic effect may include complete or partial avoidance / inhibition or delay (e.g., complete or partial avoidance / inhibition or delay) of the onset / progression of uveitis following administration of a treatment protocol, and increasing the survival time of an affected host animal. Treatment of uveitis includes treatment of subjects already diagnosed with some form of uveitis, either at a clinical stage or clinical symptoms, delaying the onset or progression or progression or worsening of symptoms or signs of uveitis, and / or preventing and / or reducing the severity of uveitis.

[0071] The present disclosure also provides a method for identifying a subject at high risk of developing uveitis, for example, anterior uveitis. In some embodiments, the method includes determining or having determined the presence or absence of an ERAP1 variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule) in a biological sample obtained from a subject. If a subject lacks an ERAP1 variant nucleic acid molecule (i.e., the subject is classified as ERAP1 standard by genotyping), the subject has a high risk of developing uveitis, for example, anterior uveitis. If a subject has an ERAP1 variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for the ERAP1 variant nucleic acid molecule), the subject has a lower risk of developing uveitis compared to a subject who is ERAP1 standard.

[0072] Having a single copy of the ERAP1 mutant nucleic acid molecule further protects the subject from developing uveitis than not having a copy of the ERAP1 mutant nucleic acid molecule. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of the ERAP1 mutant nucleic acid molecule (i.e., heterozygous for the ERAP1 mutant nucleic acid molecule) protects the subject from developing uveitis, e.g., anterior uveitis, and it is also believed that having two copies of the ERAP1 mutant nucleic acid molecule (i.e., homozygous for the ERAP1 mutant nucleic acid molecule) may further protect the subject from developing uveitis, e.g., anterior uveitis, compared to a subject with a single copy. Thus, in some embodiments, a single copy of the ERAP1 mutant nucleic acid molecule may not completely protect the subject from developing uveitis, e.g., anterior uveitis, but may instead provide partial or incomplete protection. Without wishing to be bound by a particular theory, there may be additional factors or molecules involved in the development of uveitis that are still present in a subject having a single copy of the ERAP1 mutant nucleic acid molecule, which may result in incomplete protection from the development of uveitis.

[0073] Detecting the presence or absence of an ERAP1 mutant nucleic acid molecule in a biological sample obtained from a subject and / or determining whether the subject has an ERAP1 mutant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in a test tube. 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 ERAP1 mutant nucleic acid molecule can be present in a cell obtained from the subject.

[0074] In some embodiments, when a subject is identified as having a high risk of developing uveitis, the subject is further treated with a therapeutic agent for treating or inhibiting uveitis and / or an ERAP1 inhibitor as described herein. For example, if a subject is an ERAP1 criterion and therefore has a high risk of developing uveitis, the subject is administered an ERAP1 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or inhibiting uveitis. In some embodiments, if a subject is heterozygous for an ERAP1 variant nucleic acid molecule, the subject is administered a therapeutic agent for treating or inhibiting uveitis at a dose equal to or less than the standard dose, and an ERAP1 inhibitor is also administered. In some embodiments, the subject is an ERAP1 criterion. In some embodiments, the subject is heterozygous for an ERAP1 variant nucleic acid molecule.

[0075] In any of the methods described herein, the method may further include detecting the presence or absence of HLA-B27 and / or HLA-B40 in a biological sample obtained from the subject. In some embodiments, the subject has HLA-B27 + In some embodiments, the subject is HLA-B40 +In some embodiments, the method further comprises determining whether the subject has one or two copies of HLA-B27 and / or HLA-B40. In some embodiments, the subject has a single copy of HLA-B27 or HLA-B40. In some embodiments, the subject has two copies of HLA-B27 or HLA-B40. In some embodiments, the subject has a single copy of HLA-B27 and a single copy of HLA-B40. In some embodiments, the method further comprises administering an HLA-B27 inhibitor or an HLA-B40 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor is an antibody. In some embodiments, the antibody is an anti-HLA-B27 antibody or an anti-HLA-B40 antibody. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27 or HLA-B40.

[0076] In some embodiments, the B27 antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 2579-2923. In some embodiments, the B27 siRNA molecule comprises or consists of any of the nucleotide sequences represented by SEQ ID NOs: 2924-3661 (sense and antisense strands are presented consecutively) (e.g., the sense strand is, e.g., SEQ ID NO: 2924 and the corresponding antisense strand is SEQ ID NO: 2925; the sense strand is, e.g., SEQ ID NO: 2926 and the corresponding antisense strand is SEQ ID NO: 2927, etc.).

[0077] It has been reported that HLA class I antibodies can be produced by a number of methodologies to achieve different degrees of antigen / allele specificity and are used for in vitro assays. HLA-B27 antibodies can be produced by a number of methodologies. In addition, three commercially available antibodies for flow cytometric screening of HLA-B27 include monoclonal mouse anti-human ABC-m3, FD705, and GS145.2, each of which has been shown to have different levels of cross-reactivity to other HLA-B antigens / alleles (Levering et al., Cytometry B Clin. Cytom., 2003, 54, 28-38).

[0078] The present disclosure also provides a method for detecting the presence or absence of an ERAP1 mutant genomic nucleic acid molecule in a biological sample obtained from a subject, and / or an ERAP1 mutant mRNA molecule in a biological sample obtained from a subject, and / or an ERAP1 mutant cDNA molecule generated from an mRNA molecule in a biological sample obtained from a subject. It is understood that gene sequences in a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms such as SNPs. The sequences provided herein for the ERAP1 mutant genomic nucleic acid molecule, the ERAP1 mutant mRNA molecule, and the ERAP1 mutant cDNA molecule are merely exemplary sequences. Other sequences are possible for the ERAP1 mutant genomic nucleic acid molecule, the mutant mRNA molecule, and the mutant cDNA molecule.

[0079] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The biological sample may include any clinically relevant tissue, such as, for example, a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as, for example, blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the biological sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be subjected to different treatments depending on the assay employed. For example, when detecting ERAP1 mutant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for ERAP1 mutant nucleic acid molecules may be employed. For this purpose, various techniques may be used. When detecting the level of ERAP1 mutant mRNA molecules, various techniques may be used to enrich the biological sample containing mRNA molecules. Various methods can be used to detect the presence or level of mRNA molecules, or the presence of a particular mutant genomic DNA locus.

[0080] The present disclosure also provides a method for detecting an ERAP1 mutant nucleic acid molecule or its complement in a subject. The method includes assaying a biological sample obtained from a subject to determine whether the nucleic acid molecule in the biological sample is an ERAP1 mutant nucleic acid molecule.

[0081] In some embodiments, the ERAP1 mutant nucleic acid molecule or its complement is a genomic nucleic acid molecule having a nucleotide sequence including: a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3; a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4; or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5.

[0082] In some embodiments, the ERAP1 mutant nucleic acid molecule or its complement is an mRNA molecule having a nucleotide sequence including a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8.

[0083] In some embodiments, the ERAP1 mutant nucleic acid molecule or its complement is a cDNA molecule generated from an mRNA molecule in a biological sample, having a nucleotide sequence including a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO: 11.

[0084] In some embodiments, the ERAP1 mutant nucleic acid molecule has a nucleotide sequence that includes a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2 (for a genomic nucleic acid molecule); a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 (for an mRNA molecule); or a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10 (for a cDNA molecule obtained from an mRNA molecule).

[0085] In some embodiments, the ERAP1 mutant nucleic acid molecule has a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3 (for a genomic nucleic acid molecule); an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8 (for an mRNA molecule); or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:11 (for a cDNA molecule obtained from an mRNA molecule).

[0086] In some embodiments, the ERAP1 mutant nucleic acid molecule has a nucleotide sequence including a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4. In some embodiments, the ERAP1 mutant nucleic acid molecule has a nucleotide sequence comprising a thymine or its complement at a position corresponding to position 42,579.

[0087] In some embodiments, the biological sample includes cells or cell lysates. Such a method can further include, for example, obtaining a biological sample containing an ERAP1 genomic nucleic acid molecule or an mRNA molecule from a subject, and, if it is mRNA, optionally reverse transcribing the mRNA into cDNA. Such an assay can include, for example, determining the identity of these positions of a specific ERAP1 nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0088] In some embodiments, the assay comprises sequencing at least a part of the nucleotide sequence of the ERAP1 nucleic acid molecule or its complement in a biological sample. In some embodiments, the assay comprises sequencing at least a part of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule in a biological sample, the sequenced part of which includes a position corresponding to position 19,474 of SEQ ID NO: 2 or its complement; sequencing at least a part of the nucleotide sequence of the ERAP1 mRNA molecule in a biological sample, the sequenced part of which includes a position corresponding to position 1,841 of SEQ ID NO: 7 or its complement; and / or sequencing at least a part of the nucleotide sequence of the ERAP1 cDNA molecule generated from the mRNA in a biological sample, the sequenced part of which includes a position corresponding to position 1,841 of SEQ ID NO: 10 or its complement. When the sequenced portion of the ERAP1 nucleic acid molecule in the biological sample contains guanine or its complement at a position corresponding to position 19,474 described in SEQ ID NO:2, guanine or its complement at a position corresponding to position 1,841 described in SEQ ID NO:7, or guanine or its complement at a position corresponding to position 1,841 described in SEQ ID NO:10, the ERAP1 nucleic acid molecule in the biological sample is an ERAP1 mutant nucleic acid molecule.

[0089] In some embodiments, the assay comprises: sequencing at least a portion of the nucleotide sequence of an ERAP1 genomic nucleic acid molecule in a biological sample, the sequenced portion of which includes a position corresponding to position 21,595 of SEQ ID NO: 3 or its complement; sequencing at least a portion of the nucleotide sequence of an ERAP1 mRNA molecule in a biological sample, the sequenced portion of which includes a position corresponding to position 1,981 of SEQ ID NO: 8 or its complement; and / or sequencing at least a portion of the nucleotide sequence of an ERAP1 cDNA molecule generated from mRNA in a biological sample, the sequenced portion of which includes a position corresponding to position 1,981 of SEQ ID NO: 11 or its complement. When the sequenced portion of the ERAP1 nucleic acid molecule in the biological sample includes an adenine or its complement at a position corresponding to position 21,595 of SEQ ID NO: 3, an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO: 8, or an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO: 11, the ERAP1 nucleic acid molecule in the biological sample is an ERAP1 mutant nucleic acid molecule.

[0090] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 21,811 set forth in SEQ ID NO:4 or its complement; in which case the ERAP1 nucleic acid molecule in the biological sample is an ERAP1 mutant nucleic acid molecule.

[0091] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 42,579 set forth in SEQ ID NO:5 or its complement; in which case the ERAP1 nucleic acid molecule in the biological sample is an ERAP1 mutant nucleic acid molecule.

[0092] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement in the biological sample, wherein the sequenced portion comprises a position corresponding to position 19,474 or its complement as set forth in SEQ ID NO:2; position 21,595 or its complement as set forth in SEQ ID NO:3; position 21,811 or its complement as set forth in SEQ ID NO:4; or position 42,579 or its complement as set forth in SEQ ID NO:5. If the sequenced portion of the ERAP1 genomic nucleic acid molecule in the biological sample comprises a guanine or its complement at a position corresponding to position 19,474 as set forth in SEQ ID NO:2; an adenine or its complement at a position corresponding to position 21,595 as set forth in SEQ ID NO:3; a cytosine or its complement at a position corresponding to position 21,811 as set forth in SEQ ID NO:4; or a thymine or its complement at a position corresponding to position 42,579 as set forth in SEQ ID NO:5, the ERAP1 genomic nucleic acid molecule in the biological sample is an ERAP1 mutant genomic nucleic acid molecule.

[0093] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of an ERAP1 mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 1,841 set forth in SEQ ID NO:7 or its complement; or position 1,981 set forth in SEQ ID NO:8 or its complement; in which case the ERAP1 mRNA molecule in the biological sample is an ERAP1 mutant mRNA molecule.

[0094] In some embodiments, the assay comprises sequencing at least a portion of the nucleotide sequence of an ERAP1 cDNA molecule generated from an mRNA molecule in a biological sample, wherein the sequenced portion comprises a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or its complement; or position 1,981 set forth in SEQ ID NO: 11 or its complement; in which case the ERAP1 cDNA molecule generated from an mRNA molecule in a biological sample is an ERAP1 mutant cDNA molecule.

[0095] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes with a part of the nucleotide sequence of an ERAP1 genomic nucleic acid molecule adjacent to a position corresponding to position 19,474 or its complement set forth in SEQ ID NO: 2 or a complement thereof; an ERAP1 mRNA molecule adjacent to a position corresponding to position 1,841 or its complement set forth in SEQ ID NO: 7 or a complement thereof; and / or an ERAP1 cDNA molecule adjacent to a position corresponding to position 1,841 or its complement set forth in SEQ ID NO: 10 or a complement thereof; and b) contacting a biological sample with a primer that hybridizes with a part of the nucleotide sequence of an ERAP1 genomic nucleic acid molecule corresponding to position 19,474 or its complement set forth in SEQ ID NO: 2 or a complement thereof; an ERAP1 mRNA molecule corresponding to position 1,841 or its complement set forth in SEQ ID NO: 7 or a complement thereof; and / or an ERAP1 cDNA molecule adjacent to a position corresponding to position 1,841 or its complement set forth in SEQ ID NO: 10 or a complement thereof. extending the primer at least through a position in the nucleotide sequence of the cDNA molecule or its complement; and c) determining whether the extension product of the primer comprises: a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7; and / or a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10.

[0096] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes with a part of the nucleotide sequence of an ERAP1 genomic nucleic acid molecule adjacent to a position corresponding to position 21,595 or its complement set forth in SEQ ID NO: 3 or a complement thereof; an ERAP1 mRNA molecule adjacent to a position corresponding to position 1,981 or its complement set forth in SEQ ID NO: 8 or a complement thereof; and / or an ERAP1 cDNA molecule adjacent to a position corresponding to position 1,981 or its complement set forth in SEQ ID NO: 11 or a complement thereof; and b) contacting a biological sample with a primer that hybridizes with a part of the nucleotide sequence of an ERAP1 genomic nucleic acid molecule corresponding to position 21,595 or its complement set forth in SEQ ID NO: 3 or a complement thereof; an ERAP1 mRNA molecule corresponding to position 1,981 or its complement set forth in SEQ ID NO: 8 or a complement thereof; and / or an ERAP1 cDNA molecule adjacent to a position corresponding to position 1,981 or its complement set forth in SEQ ID NO: 11 or a complement thereof. and c) determining whether an extension product of the primer comprises: an adenine or its complement at a position corresponding to position 21,595 of SEQ ID NO:3; an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO:8; and / or an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO:11.

[0097] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 21,811 or its complement as set forth in SEQ ID NO:4; b) extending the primer at least through the position of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement corresponding to position 21,811 or its complement as set forth in SEQ ID NO:4; and c) determining whether the extension product of the primer contains a cytosine or its complement at the position corresponding to position 21,811 as set forth in SEQ ID NO:4.

[0098] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 42,579 or its complement set forth in SEQ ID NO:5; b) extending the primer at least through the position of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement corresponding to position 42,579 or its complement set forth in SEQ ID NO:5; and c) determining whether the extension product of the primer contains thymine at the position corresponding to position 42,579 or its complement set forth in SEQ ID NO:5.

[0099] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 19,474 or its complement set forth in SEQ ID NO: 2, position 21,595 or its complement set forth in SEQ ID NO: 3, position 21,811 or its complement set forth in SEQ ID NO: 4, or position 42,579 or its complement set forth in SEQ ID NO: 5; and b) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 19,474 or its complement set forth in SEQ ID NO: 2, position 21,595 or its complement set forth in SEQ ID NO: 3, position 21,811 or its complement set forth in SEQ ID NO: 4. or extending the primer at least through a position of the nucleotide sequence of the ERAP1 genomic nucleic acid molecule or its complement corresponding to position 42,579 set forth in SEQ ID NO:5 or its complement; and c) determining whether the extension product of the primer contains: a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2, an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3, a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5.

[0100] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an ERAP1 mRNA molecule or its complement adjacent to a position corresponding to position 1,841 or its complement set forth in SEQ ID NO:7 or position 1,981 or its complement set forth in SEQ ID NO:8; b) extending the primer at least through the position of the nucleotide sequence of the ERAP1 mRNA molecule or its complement corresponding to position 1,841 or its complement set forth in SEQ ID NO:7 or position 1,981 or its complement set forth in SEQ ID NO:8; and c) determining whether the extension product of the primer contains a guanine or its complement at the position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at the position corresponding to position 1,981 set forth in SEQ ID NO:8.

[0101] In some embodiments, the assay comprises: a) contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an ERAP1 cDNA molecule or its complement adjacent to a position corresponding to position 1,841 or its complement set forth in SEQ ID NO: 10 or position 1,981 or its complement set forth in SEQ ID NO: 11; b) extending the primer at least through the position of the nucleotide sequence of the ERAP1 cDNA molecule or its complement corresponding to position 1,841 or its complement set forth in SEQ ID NO: 10 or position 1,981 or its complement set forth in SEQ ID NO: 11; and c) determining whether the extension product of the primer contains a guanine or its complement at the position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at the position corresponding to position 1,981 set forth in SEQ ID NO: 11.

[0102] In some embodiments, the assay includes sequencing the entire nucleic acid molecule. In some embodiments, only the ERAP1 genomic nucleic acid molecule is analyzed. In some embodiments, only the ERAP1 mRNA is analyzed. In some embodiments, only the ERAP1 cDNA obtained from the ERAP1 mRNA is analyzed.

[0103] In some embodiments, the assay comprises: a) amplifying at least a portion of an ERAP1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising a guanine or its complement at a position corresponding to position 19,474 as set forth in SEQ ID NO:2; a guanine or its complement at a position corresponding to position 1,841 as set forth in SEQ ID NO:7; and / or a guanine or its complement at a position corresponding to position 1,841 as set forth in SEQ ID NO:10; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the amplified nucleic acid molecule comprising a guanine or its complement at a position corresponding to position 19,474 as set forth in SEQ ID NO:2; a guanine or its complement at a position corresponding to position 1,841 as set forth in SEQ ID NO:7; and / or a guanine or its complement at a position corresponding to position 1,841 as set forth in SEQ ID NO:10; and d) detecting the detectable label.

[0104] In some embodiments, the assay comprises: a) amplifying at least a portion of an ERAP1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising an adenine or its complement at a position corresponding to position 21,595 of SEQ ID NO: 3; an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO: 8; and / or an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO: 11; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the amplified nucleic acid molecule comprising an adenine or its complement at a position corresponding to position 21,595 of SEQ ID NO: 3; an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO: 8; and / or an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO: 11; and d) detecting the detectable label.

[0105] In some embodiments, the assay comprises: a) amplifying at least a portion of an ERAP1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO: 4; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the amplified nucleic acid molecule comprising a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO: 4; and d) detecting the detectable label.

[0106] In some embodiments, the assay comprises: a) amplifying at least a portion of an ERAP1 nucleic acid molecule or its complement in a biological sample, the amplified portion comprising a thymine or its complement at a position corresponding to position 42,579 of SEQ ID NO:5; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the amplified nucleic acid molecule comprising a thymine or its complement at a position corresponding to position 42,579 of SEQ ID NO:5; and d) detecting the detectable label.

[0107] In some embodiments, the assay comprises: a) amplifying at least a portion of an ERAP1 genomic nucleic acid molecule or a complement thereof in a biological sample, the portion comprising a guanine or a complement thereof at a position corresponding to position 19,474 set forth in SEQ ID NO: 2, an adenine or a complement thereof at a position corresponding to position 21,595 set forth in SEQ ID NO: 3, a cytosine or a complement thereof at a position corresponding to position 21,811 set forth in SEQ ID NO: 4, or a thymine or a complement thereof at a position corresponding to position 42,579 set forth in SEQ ID NO: 5; b) labeling the amplified nucleic acid molecule with a detectable label; and c) labeling the labeled nucleic acid molecule. 2, an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3, a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5; and d) detecting the detectable label.

[0108] In some embodiments, the assay comprises: a) amplifying at least a portion of an ERAP1 mRNA molecule or its complement in a biological sample, the portion comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the amplified nucleic acid molecule comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; and d) detecting the detectable label.

[0109] In some embodiments, the assay comprises: a) amplifying at least a portion of an ERAP1 cDNA molecule or its complement generated from an mRNA molecule in a biological sample, the portion comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO: 11; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the amplified nucleic acid molecule comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO: 11; and d) detecting the detectable label.

[0110] 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 assay comprises contacting an ERAP1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the ERAP1 nucleic acid molecule or its complement comprising a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7; and / or a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10, and detecting the detectable label.

[0111] In some embodiments, the assay comprises contacting an ERAP1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises an adenine or its complement at a position corresponding to position 21,595 of SEQ ID NO:3, an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO:8, and / or an adenine or its complement at a position corresponding to position 1,981 of SEQ ID NO:11, and a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the ERAP1 nucleic acid molecule or its complement, and detecting the detectable label.

[0112] In some embodiments, the assay comprises contacting an ERAP1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the ERAP1 nucleic acid molecule or its complement comprising a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, and detecting the detectable label.

[0113] In some embodiments, the assay comprises contacting an ERAP1 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of the ERAP1 nucleic acid molecule or its complement comprising thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5, and detecting the detectable label.

[0114] In some embodiments, the assay includes contacting an ERAP1 genomic nucleic acid molecule or its complement in a biological sample with a mutation-specific probe containing a detectable label, wherein the mutation-specific probe contains a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2, an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3, a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5. The contacting includes detecting the detectable label.

[0115] In some embodiments, the assay comprises contacting an ERAP1 mRNA molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the ERAP1 mRNA molecule or its complement comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8, and detecting the detectable label.

[0116] In some embodiments, the assay comprises contacting an ERAP1 cDNA molecule or its complement generated from an mRNA molecule in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the ERAP1 cDNA molecule or its complement comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO: 11, and detecting the detectable label.

[0117] In some embodiments, the ERAP1 nucleic acid molecule is present in a cell obtained from a subject. Mutation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleotide sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0118] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0119] 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 including ERAP1 mutant genomic nucleic acid molecules, mutant mRNA molecules, or mutant cDNA molecules. Hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length may be any length that is sufficient for use in an optimal 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. Although a probe that is different from the target nucleotide sequence and retains the ability to specifically detect and / or identify the target nucleotide sequence may be designed by conventional methods, the probes and primers may have a complete nucleotide sequence identity of consecutive nucleotides in the target nucleotide sequence. The probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

[0120] In some embodiments, in order to determine whether an ERAP1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence containing a guanine at a position corresponding to position 19,474 set forth in SEQ ID NO: 2 (genomic nucleic acid molecule), a guanine at a position corresponding to position 1,841 set forth in SEQ ID NO: 7 (mRNA molecule), or a guanine at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 (cDNA molecule), a guanine at a position corresponding to position 19,474 set forth in SEQ ID NO: 2, a guanine at a position corresponding to position 1,841 set forth in SEQ ID NO: 7, or a guanine at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 is detected. The biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to a SNP at a position corresponding to position 19,474 in SEQ ID NO:2, a guanine at a position corresponding to position 1,841 in SEQ ID NO:7, or a guanine at a position corresponding to position 1,841 in SEQ ID NO:10, to generate an amplicon indicative of the presence of a SNP at a position encoding a guanine at a position corresponding to position 19,474 in SEQ ID NO:2, a guanine at a position corresponding to position 1,841 in SEQ ID NO:7, or a guanine at a position corresponding to position 1,841 in SEQ ID NO:10. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region including a position that includes a guanine at a position corresponding to position 19,474 in SEQ ID NO:2, a guanine at a position corresponding to position 1,841 in SEQ ID NO:7, or a guanine at a position corresponding to position 1,841 in SEQ ID NO:10, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position that includes a guanine at a position corresponding to position 19,474 in SEQ ID NO:2, a guanine at a position corresponding to position 1,841 in SEQ ID NO:7, or a guanine at a position corresponding to position 1,841 in SEQ ID NO:10.

[0121] In some embodiments, in order to determine whether an ERAP1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence containing an adenine at a position corresponding to position 21,595 set forth in SEQ ID NO: 3 (genomic nucleic acid molecule), an adenine at a position corresponding to position 1,981 set forth in SEQ ID NO: 8 (mRNA molecule), or an adenine at a position corresponding to position 1,981 set forth in SEQ ID NO: 11 (cDNA molecule), an adenine at a position corresponding to position 21,595 set forth in SEQ ID NO: 3, an adenine at a position corresponding to position 1,981 set forth in SEQ ID NO: 8, or an adenine at a position corresponding to position 1,981 set forth in SEQ ID NO: 11, The biological sample can be subjected to an amplification method using a primer pair comprising a first primer derived from a 5' flanking sequence adjacent to an adenine at a position corresponding to position 21,595 in SEQ ID NO:3, an adenine at a position corresponding to position 1,981 in SEQ ID NO:8, or an adenine at a position corresponding to position 1,981 in SEQ ID NO:11, to generate an amplicon indicative of the presence of a SNP at a position encoding an adenine at a position corresponding to position 21,595 in SEQ ID NO:3, an adenine at a position corresponding to position 1,981 in SEQ ID NO:8, or an adenine at a position corresponding to position 1,981 in SEQ ID NO:11. In some embodiments, the length of the amplicon can range from the combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be generated by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region including an adenine at a position corresponding to position 21,595 of SEQ ID NO:3, an adenine at a position corresponding to position 1,981 of SEQ ID NO:8, or an adenine at a position corresponding to position 1,981 of SEQ ID NO:11, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position including an adenine at a position corresponding to position 21,595 of SEQ ID NO:3, an adenine at a position corresponding to position 1,981 of SEQ ID NO:8, or an adenine at a position corresponding to position 1,981 of SEQ ID NO:11.

[0122] In some embodiments, in order to determine whether an ERAP1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence containing a cytosine (genomic nucleic acid molecule) at a position corresponding to position 21,811 as set forth in SEQ ID NO: 4, the biological sample is subjected to an amplification method using a primer pair including a first primer derived from a 5' flanking sequence adjacent to the cytosine at a position corresponding to position 21,811 as set forth in SEQ ID NO: 4 and a second primer derived from a 3' flanking sequence adjacent to the cytosine at a position corresponding to position 21,811 as set forth in SEQ ID NO: 4, and an amplicon indicating the presence of a SNP at a position encoding a cytosine at a position corresponding to position 21,811 as set forth in SEQ ID NO: 4 can be created. In some embodiments, the length of the amplicon can range from a combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be created by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position that includes a cytosine at a position corresponding to position 21,811 in SEQ ID NO:4 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position that includes a cytosine at a position corresponding to position 21,811 in SEQ ID NO:4.

[0123] In some embodiments, in order to determine whether an ERAP1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence containing thymine (genomic nucleic acid molecule) at a position corresponding to position 42,579 of SEQ ID NO:5, the biological sample is subjected to an amplification method using a primer pair including a first primer derived from a 5' flanking sequence adjacent to the thymine at a position corresponding to position 42,579 of SEQ ID NO:5 and a second primer derived from a 3' flanking sequence adjacent to the thymine at a position corresponding to position 42,579 of SEQ ID NO:5, and an amplicon indicating the presence of a SNP at a position encoding the thymine at a position corresponding to position 42,579 of SEQ ID NO:5 can be created. In some embodiments, the length of the amplicon can range from a combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be created by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a thymine at a position corresponding to position 42,579 of SEQ ID NO:5 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the position that includes a thymine at a position corresponding to position 42,579 of SEQ ID NO:5.

[0124] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be derived from known sequences, for example, using computer programs designed for that purpose, such as the PCR primer analysis tools in Vector NTI version 10 (Informax Inc., Bethesda Md.), PrimerSelect (DNASTAR Inc., Madison, Wis.), and Primer3 (Version 0.4.0.COPYRGT., 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, sequences can be visually inspected and primers manually specified using known guidelines.

[0125] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including the use of labeled primers or labeled probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (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).

[0126] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold or more above background, including more than 10-fold above background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0127] Suitable stringent conditions that promote DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a 2x SSC wash at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. + ion, usually about 0.01 to 1.0 M Na+ The conditions will be ionic concentration (or other salts) and temperature of at least about 30° C. for short probes (e.g., 10-50 nucleotides) and at least about 60° C. for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0128] The present disclosure also provides a method for detecting the presence of a predicted loss-of-function polypeptide in ERAP1, comprising performing an assay on a biological sample obtained from a subject to determine whether the ERAP1 polypeptide in the biological sample contains one or more mutations that cause the polypeptide to have loss of function (partial or complete) or a predicted loss of function (partial or complete). The predicted loss-of-function polypeptide in ERAP1 can be any of the predicted loss-of-function polypeptides in ERAP1 described herein. In some embodiments, the method detects the presence of ERAP1 Lys528Arg or Asp575Asn. In some embodiments, the method detects the presence of ERAP1 Lys528Arg. In some embodiments, the method detects the presence of ERAP1 Asp575Asn.

[0129] In some embodiments, the method includes performing an assay on a biological sample obtained from a subject to determine whether the ERAP1 polypeptide in the biological sample contains arginine at a position corresponding to position 528 set forth in SEQ ID NO: 13 or asparagine at a position corresponding to position 575 set forth in SEQ ID NO: 14.

[0130] In some embodiments, the assay comprises sequencing at least a portion of the ERAP1 polypeptide including a position corresponding to position 528 set forth in SEQ ID NO: 13 or SEQ ID NO: 12, or position 575 set forth in SEQ ID NO: 14 or SEQ ID NO: 12.

[0131] In some embodiments, the assay is an immunoassay that detects the presence of an ERAP1 polypeptide comprising a position corresponding to position 528 set forth in SEQ ID NO: 13 or SEQ ID NO: 12, or position 575 set forth in SEQ ID NO: 14 or SEQ ID NO: 12.

[0132] In some embodiments, if a subject does not have a loss-of-function polypeptide predicted by ERAP1, the subject has a high risk of developing uveitis. In some embodiments, if a subject has a loss-of-function polypeptide predicted by ERAP1, the subject has a low risk of developing uveitis.

[0133] The present disclosure also provides an isolated nucleic acid molecule that hybridizes with an ERAP1 mutant genomic nucleic acid molecule, an ERAP1 mutant mRNA molecule, and / or an ERAP1 mutant cDNA molecule (e.g., any of the genomic mutant nucleic acid molecules, mRNA mutant molecules, and cDNA mutant molecules disclosed herein). In some embodiments, such an isolated nucleic acid molecule hybridizes with an ERAP1 mutant nucleic acid molecule under stringent conditions. Such a nucleic acid molecule can be used, for example, as a probe, primer, mutation-specific probe, or mutation-specific primer as described or exemplified herein.

[0134] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ERAP1 nucleic acid molecule comprising a position corresponding to position 19,474 set forth in SEQ ID NO:2, position 1,841 set forth in SEQ ID NO:7, or position 1,841 set forth in SEQ ID NO:10. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ERAP1 nucleic acid molecule comprising a position corresponding to position 21,595 set forth in SEQ ID NO:3, position 1,981 set forth in SEQ ID NO:8, or position 1,981 set forth in SEQ ID NO:11. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ERAP1 nucleic acid molecule comprising a position corresponding to position 21,811 set forth in SEQ ID NO:4. In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ERAP1 nucleic acid molecule comprising a position corresponding to position 42,579 set forth in SEQ ID NO:5.

[0135] In some embodiments, such isolated nucleic acid molecules comprise at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55 , at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0136] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a nucleotide sequence of a portion of an ERAP1 nucleic acid molecule or its complement. In some embodiments, the portion comprises a position corresponding to position 19,474 or its complement as set forth in SEQ ID NO:2; position 1,841 or its complement as set forth in SEQ ID NO:7; or position 1,841 or its complement as set forth in SEQ ID NO:10. In some embodiments, the portion comprises a position corresponding to position 21,595 or its complement as set forth in SEQ ID NO:3; position 1,981 or its complement as set forth in SEQ ID NO:8; or position 1,981 or its complement as set forth in SEQ ID NO:11. In some embodiments, the portion comprises a position corresponding to position 21,811 or its complement as set forth in SEQ ID NO:4. In some embodiments, the portion comprises a position corresponding to position 42,579 or its complement as set forth in SEQ ID NO:5.

[0137] In some embodiments, the isolated nucleic acid molecule hybridizes with at least about 15 consecutive nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an ERAP1 mutant genomic nucleic acid molecule, an ERAP1 mutant mRNA molecule, and / or an ERAP1 mutant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0138] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of the ERAP1 mutant nucleic acid molecule, and the portion comprises a position corresponding to position 19,474 or its complement set forth in SEQ ID NO: 2; position 1,841 or its complement set forth in SEQ ID NO: 7; or position 1,841 or its complement set forth in SEQ ID NO: 10. In some embodiments, the portion comprises positions 19,473 to 19,475 or their complement set forth in SEQ ID NO: 2; positions 1,840 to 1,842 or their complement set forth in SEQ ID NO: 7; and / or positions 1,840 to 1,842 or their complement set forth in SEQ ID NO: 10.

[0139] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of the ERAP1 mutant nucleic acid molecule, the portion comprising a position corresponding to position 21,595 or its complement set forth in SEQ ID NO: 3; position 1,981 or its complement set forth in SEQ ID NO: 8; or position 1,981 or its complement set forth in SEQ ID NO: 11. In some embodiments, the portion comprises positions 21,595 to 21,597 or their complement set forth in SEQ ID NO: 3; positions 1,981 to 1,983 or their complement set forth in SEQ ID NO: 8; and / or positions 1,981 to 1,983 or their complement set forth in SEQ ID NO: 11.

[0140] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of the ERAP1 mutant nucleic acid molecule, the portion comprising a position corresponding to position 21,811 set forth in SEQ ID NO:4 or its complement.

[0141] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a portion of the nucleotide sequence of the ERAP1 mutant nucleic acid molecule, the portion comprising a position corresponding to position 42,579 set forth in SEQ ID NO:5 or its complement.

[0142] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a nucleotide sequence of a portion of an ERAP1 nucleic acid molecule or its complement. In some embodiments, the portion comprises a position corresponding to position 19,474 or its complement as set forth in SEQ ID NO:2; position 21,595 or its complement as set forth in SEQ ID NO:3; position 21,811 or its complement as set forth in SEQ ID NO:4; or position 42,579 or its complement as set forth in SEQ ID NO:5.

[0143] In some embodiments, the portion includes a position corresponding to positions 19,473 to 19,475 set forth in SEQ ID NO:2, or a complement thereof, or positions 21,595 to 21,597 set forth in SEQ ID NO:3, or a complement thereof.

[0144] In some embodiments, the portion includes a position corresponding to position 1,841 set forth in SEQ ID NO:7, or its complement, or position 1,981 set forth in SEQ ID NO:8, or its complement. In some embodiments, the portion includes a position corresponding to positions 1,840 to 1,842 set forth in SEQ ID NO:7, or a complement thereof, or positions 1,981 to 1,983 set forth in SEQ ID NO:8, or a complement thereof.

[0145] In some embodiments, the portion includes a position corresponding to position 1,841 set forth in SEQ ID NO:10, or its complement, or position 1,981 set forth in SEQ ID NO:11, or its complement.

[0146] In some embodiments, the portion includes a position corresponding to positions 1,840 to 1,842 set forth in SEQ ID NO: 10, or a complement thereof, or positions 1,981 to 1,983 set forth in SEQ ID NO: 11, or a complement thereof.

[0147] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0148] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0149] In some embodiments, the primers can be used in second generation or high throughput sequencing, including mutation specific primers. Sometimes the primers can be modified, including mutation specific primers. In particular, the primers can include various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is typically performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading and emulsion PCR steps. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain 5'-biotin tags for immobilizing the DNA library on streptavidin-coated beads.

[0150] The probes and primers described herein can be used to detect nucleotide mutations within any of the ERAP1 mutant genomic nucleic acid molecules, ERAP1 mutant mRNA molecules, and / or ERAP1 mutant cDNA molecules disclosed herein. The primers described herein can be used to amplify the ERAP1 mutant genomic nucleic acid molecules, ERAP1 mutant mRNA molecules, or ERAP1 mutant cDNA molecules, or fragments thereof.

[0151] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 19,474 of SEQ ID NO: 1 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of the ERAP1 reference genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 19,474 of SEQ ID NO: 2 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of the ERAP1 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 19,474 of SEQ ID NO: 2 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 1,841 of SEQ ID NO: 6 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of the ERAP1 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 1,841 of SEQ ID NO: 7 in a specific ERAP1 mRNA molecule, the presence of the amplified fragment indicates the presence of an ERAP1 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at a position corresponding to position 1,841 of SEQ ID NO: 7 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 1,841 of SEQ ID NO: 9 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of an ERAP1 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 1,841 of SEQ ID NO: 10 in a specific ERAP1 cDNA molecule, the presence of the amplified fragment indicates the presence of an ERAP1 mutant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the guanine at the position corresponding to position 1,841 set forth in SEQ ID NO:10 can be at the 3' end of the primer.

[0152] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 21,595 of SEQ ID NO: 1 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of an ERAP1-based genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 21,595 of SEQ ID NO: 3 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of an ERAP1 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at the position corresponding to position 21,595 of SEQ ID NO: 3 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 1,981 of SEQ ID NO: 6 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of an ERAP1-based mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 1,981 of SEQ ID NO: 8 in a specific ERAP1 mRNA molecule, the presence of the amplified fragment indicates the presence of an ERAP1 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 1,981 of SEQ ID NO: 8 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 1,981 of SEQ ID NO: 9 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of an ERAP1 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 1,981 of SEQ ID NO: 11 in a specific ERAP1 cDNA molecule, the presence of the amplified fragment indicates the presence of an ERAP1 mutant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 1,981 set forth in SEQ ID NO:11 can be at the 3' end of the primer.

[0153] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with thymine (rather than cytosine) at a position corresponding to position 21,811 of SEQ ID NO: 1 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of the ERAP1 reference genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with cytosine (rather than thymine) at a position corresponding to position 21,811 of SEQ ID NO: 4 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of the ERAP1 mutant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to the cytosine at the position corresponding to position 21,811 of SEQ ID NO: 4 can be at the 3' end of the primer.

[0154] The present disclosure also provides a pair of primers comprising any of the above-mentioned primers. For example, if one of the 3' ends of the primer hybridizes with cytosine (rather than thymine) at a position corresponding to position 42,579 of SEQ ID NO: 1 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of the ERAP1 reference genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with thymine (rather than cytosine) at a position corresponding to position 42,579 of SEQ ID NO: 5 in a specific ERAP1 nucleic acid molecule, the presence of the amplified fragment indicates the presence of the ERAP1 mutant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer complementary to thymine at a position corresponding to position 42,579 of SEQ ID NO: 5 can be at the 3' end of the primer.

[0155] In the context of the present disclosure, "specifically hybridize" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleotide sequence encoding an ERAP1-based genomic nucleic acid molecule, an ERAP1-based mRNA molecule, and / or an ERAP1-based cDNA molecule.

[0156] In any of the embodiments described throughout this disclosure, the probe (e.g., the mutation-specific probe) can include a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0157] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which a molecule, such as any of the probes disclosed herein, can associate. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which a plurality of different probes are attached in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish, such as a standard 96-well format. In some embodiments, a multi-well glass slide can be used, usually containing one array per well. In some embodiments, the support is a microarray.

[0158] The present disclosure also provides a molecular complex comprising or consisting of any of the ERAP1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific primers or mutation-specific probes described herein. In some embodiments, the ERAP1 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in the molecular complex, or their complements, is single-stranded. In some embodiments, the ERAP1 nucleic acid molecule is any of the genomic nucleic acid molecules described herein. In some embodiments, the ERAP1 nucleic acid molecule is any of the mRNA molecules described herein. In some embodiments, the ERAP1 nucleic acid molecule is any of the cDNA molecules described herein. In some embodiments, the molecular complex comprises or consists of any of the ERAP1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific primers described herein. In some embodiments, the molecular complex comprises or consists of any of the ERAP1 nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or their complements, and any of the mutation-specific probes described herein.

[0159] In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to the ERAP1 genomic nucleic acid molecule, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the ERAP1 genomic nucleic acid molecule at a position corresponding to position 19,474 or its complement set forth in SEQ ID NO:2; position 21,595 or its complement set forth in SEQ ID NO:3; position 21,811 or its complement set forth in SEQ ID NO:4; or position 42,579 or its complement set forth in SEQ ID NO:5.

[0160] In some embodiments, the mutation-specific primer or mutation-specific probe of the molecular complex hybridizes to an AGG codon at a position corresponding to positions 19,473 to 19,475 of SEQ ID NO:2 or an AAC codon at a position corresponding to positions 21,595 to 21,597 of SEQ ID NO:3.

[0161] In some embodiments, the genomic nucleic acid molecule of the molecular complex comprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to the ERAP1 mRNA molecule, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the ERAP1 mRNA molecule at a position corresponding to position 1,841 or its complement set forth in SEQ ID NO:7 or position 1,981 or its complement set forth in SEQ ID NO:8.

[0162] In some embodiments, the mutation-specific primer or mutation-specific probe of the molecular complex hybridizes to an AGG codon at a position corresponding to positions 1,840 to 1,842 of SEQ ID NO:7 or an AAC codon at a position corresponding to positions 1,981 to 1,983 of SEQ ID NO:8.

[0163] In some embodiments, the mRNA molecule of the molecular complex comprises SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the molecular complex comprises a mutation-specific primer or a mutation-specific probe hybridized to the ERAP1 cDNA molecule, wherein the mutation-specific primer or the mutation-specific probe hybridizes to the ERAP1 cDNA molecule at a position corresponding to position 1,841 or its complement set forth in SEQ ID NO: 10 or position 1,981 or its complement set forth in SEQ ID NO: 11.

[0164] In some embodiments, the mutation-specific primer or mutation-specific probe of the molecular complex hybridizes to an AGG codon at a position corresponding to positions 1,840 to 1,842 of SEQ ID NO:10 or an AAC codon at a position corresponding to positions 1,981 to 1,983 of SEQ ID NO:1.

[0165] In some embodiments, the cDNA molecule of the molecular complex comprises SEQ ID NO:10, SEQ ID NO:11. In some embodiments, the molecular complex comprises a mutation-specific probe or a mutation-specific primer that comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. In some embodiments, the molecular complex further comprises a non-human polymerase.

[0166] The nucleotide sequence of the ERAP1 reference genomic nucleic acid molecule is set forth in SEQ ID NO: 1. With reference to SEQ ID NO: 1, position 19,474 is adenine. With reference to SEQ ID NO: 1, position 21,595 is guanine. With reference to SEQ ID NO: 1, position 21,811 is thymine. With reference to SEQ ID NO: 1, position 42,579 is cytosine.

[0167] There is an ERAP1 mutant genomic nucleic acid molecule, in which adenine at position 19,474 is replaced with guanine. The nucleotide sequence of this ERAP1 mutant genomic nucleic acid molecule is set forth in SEQ ID NO:2.

[0168] There is another ERAP1 mutant genomic nucleic acid molecule, in which guanine at position 21,595 is replaced with adenine. The nucleotide sequence of this ERAP1 mutant genomic nucleic acid molecule is set forth in SEQ ID NO:3.

[0169] There is another ERAP1 mutant genomic nucleic acid molecule, in which thymine at position 21,811 is replaced with cytosine. The nucleotide sequence of this ERAP1 mutant genomic nucleic acid molecule is set forth in SEQ ID NO:4.

[0170] There is another ERAP1 mutant genomic nucleic acid molecule, in which cytosine at position 42,579 is replaced with thymine. The nucleotide sequence of this ERAP1 mutant genomic nucleic acid molecule is set forth in SEQ ID NO:5.

[0171] The nucleotide sequence of the ERAP1 reference mRNA molecule is set forth in SEQ ID NO: 6. With reference to SEQ ID NO: 6, position 1,841 is adenine. With reference to SEQ ID NO: 6, position 1,981 is guanine.

[0172] There is an ERAP1 mutant mRNA molecule, in which adenine at position 1,841 is replaced with guanine. The nucleotide sequence of this ERAP1 mutant mRNA molecule is set forth in SEQ ID NO:7.

[0173] Another ERAP1 mutant mRNA molecule exists, in which guanine at position 1,981 is replaced with adenine. The nucleotide sequence of this ERAP1 mutant mRNA molecule is set forth in SEQ ID NO:8.

[0174] The nucleotide sequence of the ERAP1 reference cDNA molecule is set forth in SEQ ID NO: 9. With reference to SEQ ID NO: 9, position 1,841 is adenine. With reference to SEQ ID NO: 9, position 1,981 is guanine.

[0175] There is an ERAP1 mutant cDNA molecule, in which adenine at position 1,841 is replaced with guanine. The nucleotide sequence of this ERAP1 mutant cDNA molecule is set forth in SEQ ID NO:10.

[0176] Another ERAP1 mutant cDNA molecule exists, in which guanine at position 1,981 is replaced with adenine. The nucleotide sequence of this ERAP1 mutant cDNA molecule is set forth in SEQ ID NO:11.

[0177] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be derived from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be an ortholog from human or another organism (e.g., non-human mammal, rodent, mouse, or rat). It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are only exemplary sequences. Other sequences are also possible.

[0178] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecules.

[0179] Percent identity (%) (or percent complementarity) between specific stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (basic sequence comparison search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that use the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percent sequence identity is preferred over a lower one.

[0180] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of a specified reference sequence when the particular nucleotide or sequence of nucleotides is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:6, or SEQ ID NO:9). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to a reference sequence, not by the actual position number of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of the residues in the particular nucleotide or nucleotide sequence is done with reference to the reference sequence to which it is aligned.

[0181] For example, an ERAP1 nucleic acid molecule containing a nucleotide sequence containing guanine at a position corresponding to position 19,474 of SEQ ID NO:2 means that, if the nucleotide sequence of the ERAP1 genomic nucleic acid molecule is aligned with the sequence of SEQ ID NO:2, the sequence of ERAP1 has a guanine residue at a position corresponding to position 19,474 of SEQ ID NO:2. The same applies to an ERAP1 mRNA molecule containing a nucleotide sequence containing guanine at a position corresponding to position 1,841 of SEQ ID NO:7 and an ERAP1 cDNA molecule containing a nucleotide sequence containing guanine at a position corresponding to position 1,841 of SEQ ID NO:10. These expressions refer to an ERAP1 nucleic acid molecule, in which the genomic nucleic acid molecule has a nucleotide sequence containing a guanine residue that is homologous to the guanine residue at position 19,474 of SEQ ID NO:2 (or the mRNA molecule has a nucleotide sequence containing a guanine residue that is homologous to the guanine residue at position 1,841 of SEQ ID NO:7, or the cDNA molecule has a nucleotide sequence containing a guanine residue that is homologous to the guanine residue at position 1,841 of SEQ ID NO:10).

[0182] As described herein, the position in the ERAP1 genomic nucleic acid molecule corresponding to position 19,474 of SEQ ID NO:2 can be identified, for example, by performing sequence comparison between the nucleotide sequence of a specific ERAP1 nucleic acid molecule and the nucleotide sequence of SEQ ID NO:2. For example, there are various computer algorithms that can be used to perform sequence comparison to identify the position of the nucleotide corresponding to position 19,474 of SEQ ID NO:2. For example, sequence comparison may be performed by using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). However, sequences can also be aligned manually.

[0183] The amino acid sequence of the ERAP1 reference polypeptide is set forth in SEQ ID NO: 12. With reference to SEQ ID NO: 12, the ERAP1 reference polypeptide is 948 amino acids in length. With reference to SEQ ID NO: 12, position 528 is lysine. With reference to SEQ ID NO: 12, position 575 is aspartic acid.

[0184] There is a loss-of-function polypeptide predicted in ERAP1 (Lys528Arg), and its amino acid sequence is described in SEQ ID NO: 13. With reference to SEQ ID NO: 13, the loss-of-function polypeptide predicted in ERAP1 is 948 amino acids long. With reference to SEQ ID NO: 13, position 528 is arginine.

[0185] There is another loss-of-function polypeptide predicted by ERAP1 (Asp575Asn), and its amino acid sequence is described in SEQ ID NO: 14. With reference to SEQ ID NO: 14, the loss-of-function polypeptide predicted by ERAP1 is 948 amino acids long. With reference to SEQ ID NO: 14, position 575 is asparagine.

[0186] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding toward the 3'-end (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to the shown strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each sequence).

[0187] The present disclosure also provides a therapeutic agent for treating or inhibiting uveitis, e.g., anterior uveitis, for use in treating uveitis in a subject (or for use in preparing a medicament for treating uveitis), wherein the subject has any of the ERAP1 mutant genomic nucleic acid molecules, mutant mRNA molecules, and / or mutant cDNA molecules described herein. The therapeutic agent for treating or inhibiting uveitis can be any of the therapeutic agents for treating or inhibiting uveitis described herein.

[0188] In some embodiments, the subject is identified as having an ERAP1 mutant genomic nucleic acid molecule or its complement having a nucleotide sequence including: a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3; a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4; or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5.

[0189] In some embodiments, the subject is identified as having an ERAP1 mutant mRNA molecule or its complement having a nucleotide sequence including a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8.

[0190] In some embodiments, the subject is identified as having an ERAP1 mutant cDNA molecule or its complement having a nucleotide sequence including a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO: 11.

[0191] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence that includes a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence that includes a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7; a cDNA molecule having a nucleotide sequence that includes a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10; or an ERAP1 predicted loss-of-function polypeptide that includes an arginine at a position corresponding to position 528 set forth in SEQ ID NO:13.

[0192] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3; an mRNA molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; a cDNA molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:11; or an ERAP1 predicted loss-of-function polypeptide that includes an asparagine at a position corresponding to position 575 set forth in SEQ ID NO:14.

[0193] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence that includes a cytosine at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or its complement.

[0194] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence that includes a thymine at a position corresponding to position 42,579 set forth in SEQ ID NO:5, or its complement.

[0195] The present disclosure also provides an ERAP1 inhibitor for use in treating uveitis, e.g., anterior uveitis, in a subject (or for use in preparing a medicament for treating uveitis), wherein the subject is heterozygous for any of the ERAP1 mutant genomic nucleic acid molecules, mutant mRNA molecules, and / or mutant cDNA molecules described herein, or the subject is a reference for the ERAP1 genomic nucleic acid molecule, mRNA molecule, or cDNA molecule.

[0196] In some embodiments, the subject is a reference for an ERAP1 genomic nucleic acid molecule, an ERAP1 mRNA molecule, or an ERAP1 cDNA molecule. In some embodiments, the subject is heterozygous for a genomic nucleic acid molecule or its complement having a nucleotide sequence comprising: a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3; a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4; or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5.

[0197] In some embodiments, the subject is heterozygous for an mRNA molecule, or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8.

[0198] In some embodiments, the subject is heterozygous for a cDNA molecule or its complement having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:11.

[0199] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7; a cDNA molecule having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10; or an ERAP1 predicted loss-of-function polypeptide comprising an arginine at a position corresponding to position 528 set forth in SEQ ID NO:13. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0200] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3; an mRNA molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; a cDNA molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:11; or an ERAP1 predicted loss-of-function polypeptide that includes an asparagine at a position corresponding to position 575 set forth in SEQ ID NO:14. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0201] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence comprising a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO: 4. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0202] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO: 5. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0203] In some embodiments, the subject is identified as having an ERAP1-based genomic nucleic acid molecule comprising SEQ ID NO: 1, an ERAP1-based mRNA molecule comprising SEQ ID NO: 6, an ERAP1-based cDNA molecule comprising SEQ ID NO: 9, or an ERAP1-based polypeptide comprising SEQ ID NO: 12. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0204] The present disclosure also provides a combination of one or more ERAP1 inhibitors and one or more HLA-B27 inhibitors for use in treating uveitis, e.g., anterior uveitis, in a subject (or for use in preparing a medicament for treating uveitis), wherein the subject is heterozygous for any of the ERAP1 mutant genomic nucleic acid molecules, mutant mRNA molecules, and / or mutant cDNA molecules described herein, or the subject is a reference for the ERAP1 genomic nucleic acid molecule, mRNA molecule, or cDNA molecule.

[0205] In some embodiments, the subject is a reference for an ERAP1 genomic nucleic acid molecule, an ERAP1 mRNA molecule, or an ERAP1 cDNA molecule. In some embodiments, the subject is heterozygous for a genomic nucleic acid molecule or its complement having a nucleotide sequence comprising: a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3; a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4; or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5.

[0206] In some embodiments, the subject is heterozygous for an mRNA molecule, or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8.

[0207] In some embodiments, the subject is heterozygous for a cDNA molecule or its complement having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:11.

[0208] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2; an mRNA molecule having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7; a cDNA molecule having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:10; or an ERAP1 predicted loss-of-function polypeptide comprising an arginine at a position corresponding to position 528 set forth in SEQ ID NO:13. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0209] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3; an mRNA molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; a cDNA molecule having a nucleotide sequence that includes an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:11; or an ERAP1 predicted loss-of-function polypeptide that includes an asparagine at a position corresponding to position 575 set forth in SEQ ID NO:14. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0210] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence comprising a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO: 4. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0211] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence comprising a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO: 5. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0212] In some embodiments, the subject is identified as having an ERAP1-based genomic nucleic acid molecule comprising SEQ ID NO: 1, an ERAP1-based mRNA molecule comprising SEQ ID NO: 6, an ERAP1-based cDNA molecule comprising SEQ ID NO: 9, or an ERAP1-based polypeptide comprising SEQ ID NO: 12. The ERAP1 inhibitor can be any of the ERAP1 inhibitors described herein.

[0213] The present disclosure also provides a pharmaceutical composition comprising one or more ERAP1 inhibitors in combination with one or more HLA-B27 inhibitors. All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual document was specifically and individually indicated to be so incorporated by reference. Where various versions of a sequence are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application to which the accession number refers, if applicable. Similarly, where different versions of publications, websites, etc. have been published at different times, the version last published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0214] The following examples are provided to further illustrate the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0215] Example 1: General Methodology Sequencing and genotyping Genotyping and exome sequencing were performed as previously described (Verweij et al., N. Engl. J. Med., 2022, 387, 332-344). Briefly, array genotyping data were used for common variant analysis, and imputation was performed with the TOPMed panel (Taliun et al., Nature, 2021, 590, 290-299) in all analyzed cohorts. Exome sequencing for rare variant analysis was performed with 75 bp paired-end reads using an Illumina HiSeq 2500-v4 or Illumina NovaSeq instrument (Taliun et al., Nature, 2021, 590, 290-299). Deleterious variants were considered if annotated as frameshift, stop-gain, stop-loss, splice acceptor, splice donor, in-frame insertion or deletion (indel), missense, and other annotations. Frameshift, stop-gain, stop-loss, splice acceptor, and splice donor alleles were classified as predicted loss-of-function variants. Using the alternative allele frequency and functional annotation of each variant, seven genotypes were generated based on combinations of variant burden: predicted loss-of-function variants with alternative allele frequency thresholds of 1% and 0.1%, predicted loss-of-function variants + missense variants predicted to be deleterious and with alternative allele frequency thresholds of 1% and 0.1%.

[0216] sample Genome-wide association analyses were performed on the population-based cohorts of the UK Biobank and the Geisinger Health System MyCode cohort.Other datasets included: 29,237 participants from the Malmo Diet and Cancer Study, 41,400 participants from the University of Pennsylvania Penn Medicine biobank, 29,845 participants from the Mount Sinai BioMe biobank, 49,004 participants from the Colorado cohort, 40,197 participants from the UCLA cohort, and 115,418 participants from the MAYO clinic cohort.

[0217] Exome sequencing and whole genome genotyping Array genotyping data were used for analysis of common variants, and imputation was performed using the TOPMed reference panel (Taliun et al., bioRxiv, 563866, doi:10.1101 / 563866, 2019; and Das et al., Nat. Genet., 2016, 48, 1284-1287). High-coverage exome sequencing was performed with 75 bp paired-end reads using Illumina HiSeq 2500-v4 or Illumina NovaSeq instruments. The GRCh38 human genome reference sequence and Ensembl version 85 gene definitions were used for variant identification and annotation. Variants were classified in the following order from most to least deleterious: frameshift, stop gain, stop loss, splice acceptor, splice donor, in-frame insertion or deletion (indel), missense, and other annotations. Frameshift, stop-gain, stop-loss, splice acceptor, and splice donor alleles were classified as predicted loss-of-function variants. Missense variants were classified using computer modeling and predicted functional effects with five algorithms: SIFT, Polyphen-2 HDIV, Polyphen-2 HVAR, LRT, and MutationTaster. To account for the fact that different genes have different types and frequencies of potentially causative variants, the alternative allele frequencies and functional annotation of each variant were used to create seven genotypes based on combined variant burden: predicted loss-of-function variants with alternative allele frequencies <1%, predicted loss-of-function variants + missense variants predicted to be deleterious by five of the five algorithms and with alternative allele frequencies <1% or <0.1%, predicted loss-of-function variants + missense variants predicted to be deleterious by at least one of the five algorithms and with alternative allele frequencies <1% or <0.1%, and predicted loss-of-function variants + any missense variants with alternative allele frequencies <1% or <0.1%.

[0218] statistical analysis Genotype-phenotype associations were estimated by fitting linear regression models (for quantitative traits) or Firth bias-corrected logistic regression models (for dichotomous traits) using REGENIE software version 2+. Analyses were stratified by cohort and ancestry and adjusted for age, age squared, sex, age-sex, and age-squared-sex interaction terms; experimental batch-related covariates; gene principal components derived from the first 10 common variants, principal components derived from the first 20 rare variants; and polygenic scores generated by REGENIE (which robustly adjusts for relatedness and population structure). Meta-analyses of association results across cohorts and ancestry were performed using a fixed-effects inverse variance weighting approach. A two-sided P value of 0.05 was used in other analyses.

[0219] Example 2: Decreased expression of ERAP1 is protective against B27-anterior uveitis For the largest AU case cohort to date, eight large EHR-based populations (Table 2 ) were sequenced, reaching 3,850 AU cases and 916,325 controls.

[0220] [Table 2]

[0221] When examining the association of common variants, two genome-wide significant signals were found: a risk signal of rs543685299 at the HLA-B locus (OR(95% CI)=3.37(3.12-3.65), p=7.82E-197) and a signal of rs3198304 at the ERAP1 locus (OR(95% CI)=0.84(0.79-0.89), p=5.02e-9), indicating protection from AU (data not shown). The top ERAP1 SNP showed a direction of protection that was replicated in the 6 / 8 cohort (Figure 1), with a notable protection seen in the two larger cohorts, UKB and GHS.

[0222] The analysis was replicated using a EUR-only cohort consisting of 3,180 EUR cases and 826,348 controls. Although this underpowered analysis excluded 17% of overall cases and 10% of controls, similar signals were found for both HLA-B (OR=3.4, p=9.7e-186) and ERAP1 (OR=0.83, p=3.2e-09). ERAP1 is an ER-aminopeptidase that trims peptides to be loaded and presented by MHC class I proteins. Thus, modulating ERAP1 expression alters the peptidome available for presentation by HLA class I alleles (in this case HLA-B27). This has a direct impact on which antigens are presented by HLA-B27 and the subsequent activation of immune responses in AU.

[0223] With the exception of two significant common loci, rare variants with allele frequency (AF) <0.5% did not show genome-wide significance. Similarly, we investigated several gene burden analyses using various AF thresholds, but found no significant gene burden results.

[0224] Example 3: In the analysis of B27 carriers, the ERAP1 signal was enhanced and became the only genome-wide significant signal The above results indicate that the strongest genetic risk for AU is due to HLA-B, a known signal due to the HLA-B27 allele (Linssen et al., Invest. Ophthalmol. Vis. Sci., 1991, 32, 2568-2578; and Sheehan, JR Soc. Med., 2004, 97, 10-14). This risk was observed to vary within each cohort, ranging from OR=2.4 in the GHS cohort to OR=4.3 in the UKB cohort. To correct for this difference between cohorts and to investigate the genetic signal underlying the HLA-B27 association, we stratified the cohorts by HLA-B27 carrier status using the HLA-B27 tagging SNP rs4349859. Stratification for HLA-B27 resulted in two cohorts: 1) a B27-positive cohort containing samples carrying one or two copies of the tag SNP, and 2) a B27-negative cohort containing samples carrying zero copies of the tag SNP. The B27-positive cohort consisted of 856 AU cases and 70,198 controls, suggesting that 22.23% of AU cases carried the B27 allele, significantly enriched over controls, of which 7.7% were B27 carriers, as expected from the B27 frequency in the general population. The final analysis of all ancestries included 837 B27-positive AU cases and 67,755 B27-positive controls, and the meta-analysis included the seven primary cohorts, excluding the UPENN cohort, which was underpowered for a valid analysis with 19 cases and 2,434 controls.

[0225] The HLA-B signal was completely reduced in the B27 stratified analysis, whereas the ERAP1 signal remained the only significant locus genome-wide. Moreover, the observed protective effect was stronger when examining the smaller B27 stratified cohort (rs27529, OR=0.74, p=1.3e-9), even though this cohort was much less powered, with only 22% of cases and 7.7% of controls (data not shown). Furthermore, the top ERAP1 variant was part of a significant haplotype that included the ERAP1 missense variant K528R-rs30187 (R2=1, D'=1). This haplotype is an eQTL that significantly reduces ERAP1 expression and has been shown to be associated with other HLA class I-related diseases such as ankylosing spondylitis and psoriatic arthritis (data not shown).

[0226] The above analysis was replicated in a EUR-only cohort consisting of 795 AU cases and 65,628 controls. Of note, the proportion of B27-positive AU cases was higher in the EUR than in the full all-ancestries analysis, reaching 25% of cases, which is still much lower than the previously reported rate of approximately 50% B27-AU carriers. The proportion of B27 carriers in controls remained at 8%, as expected in the EUR population.

[0227] The EUR-only B27-positive analysis also showed more significant results for the protective ERAP1 locus, with a slightly better OR (95% CI) = 0.73 (1.78-3.76) and a strong p = 5.2e-10 (Figure 2). When examining the effect of ERAP1 haplotypes across the remaining seven B27-positive cohorts, the protective effect of common ERAP1 haplotypes was evident in all cohorts, ranging from OR = 0.47 in the small Sinai cohort to OR = 0.84 in the MALMO cohort. This analysis was very underpowered given the exclusion of most cases and controls, and nominal p values ​​were obtained in the two larger cohorts, GHS (184 cases) and UKB (420 cases).

[0228] AU is a commonly observed symptom in other class I opaphias, such as AS (approximately 20–50%). In addition, a significant ERAP1 signal was previously observed for AS, which is also a B27-associated disease. Psoriatic arthritis is another MHC class I disease with a similar association of ERAP1 with AU. Therefore, a rigorous analysis was designed to exclude all samples diagnosed with either AS (ICD10-M45) or Ps (ICD10-L40) from the already underpowered B27-positive cohort. Considering only B27 carriers who were not diagnosed with either AS or Ps, 618 AU cases and 67,343 controls were identified in all eight cohorts, including all ancestries. This makes the proportion of AU cases also diagnosed with AS or Ps 28%. Among the common B27 controls, 4% were found to have been diagnosed with AS or Ps.

[0229] In the final analysis of AU without AS or Ps, the six well-powered cohorts were combined and 605 AU cases and 64,991 controls of all ancestries were considered. In this analysis, the ERAP1 locus showed a similar protection with OR=0.75, with a slightly lower p=8.2e-6. A similar signal (OR=0.74 and p=9.6e-6) was observed in the EUR-only analysis, consisting of 569 EUR AU cases and 62,360 controls.

[0230] Additive effect of B27-AU risk due to the combined effect of having two copies of HLA risk alleles and the ERAP1 risk haplotype The effect of the ERAP1 risk haplotype (tagged with rs30187) was protective in subjects carrying at least one copy of the HLA-B27 allele and was further increased in subjects carrying a second HLA-B27 allele or an HLA-B40 risk allele (Figure 3). Risk was assessed in AU samples from the combined GHS+UKB cohort, and HLA typing was performed by imputation to increase accuracy (compared to tag SNPs). Zero HLA-B and ERAP1 risk alleles were defined as the reference risk genotype (i.e., OR=1), and the risk of the ERAP1 risk allele in a B27-negative background (left panel, Figure 3) was assessed and compared with subjects with one HLA-B27 allele (center panel), and subjects with two copies of HLA-B27 or carrying HLA-B27 and HLA-B40 risk alleles (right panel). A moderate increased risk of OR=4.5 was observed in those carrying one HLA-B27 allele, which nearly doubled to OR=7.5 in the ERAP1 risk genotype. The highest risk was observed in those carrying two HLA risk alleles and the complete ERAP1 risk genotype, where the risk was approximately tripled to OR=12.5. Taken together, these results suggest that reduced expression of ERAP1 reduces the risk of AU, and this effect is strongest in the HLA-B risk background.

[0231] In addition to those described herein, various modifications of the described subject matter will become 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. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. An endoplasmic reticulum aminopeptidase 1 (ERAP1) inhibitor for use in treating a subject with uveitis, iridocyclitis, or iritis.

2. The ERAP1 inhibitor according to claim 1, wherein the uveitis is anterior uveitis, acute anterior uveitis, or panuveitis.

3. The ERAP1 inhibitor according to claim 1, wherein the ERAP1 inhibitor comprises an inhibitory nucleic acid molecule.

4. The ERAP1 inhibitor of claim 3, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with the ERAP1 nucleic acid molecule.

5. The ERAP1 inhibitor according to any one of claims 1 to 4, which is used in combination with an HLA-B27 inhibitor or an HLA-B40 inhibitor.

6. The ERAP1 inhibitor according to claim 5, wherein the HLA-B27 inhibitor or the HLA-B40 inhibitor comprises an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

7. The ERAP1 inhibitor according to claim 6, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA that hybridizes with HLA-B27 or HLA-B40.

8. A therapeutic agent for treating or inhibiting uveitis and / or an endoplasmic reticulum aminopeptidase 1 (ERAP1) inhibitor for use in treating a subject having uveitis, iridocyclitis, or iritis, wherein the treatment comprises: determining whether the subject has an endoplasmic reticulum aminopeptidase 1 (ERAP1) variant nucleic acid molecule; obtaining or having obtained a biological sample from said subject; determining by performing, or having performed, a sequence analysis on the biological sample to determine whether the subject has a genotype that includes the ERAP1 variant nucleic acid molecule; administering or continuing to administer said therapeutic agent treating or inhibiting uveitis at a standard dose to a subject meeting the ERAP1 criteria, and / or administering an ERAP1 inhibitor to said subject; administering or continuing to administer the therapeutic agent for treating or inhibiting uveitis to a subject who is heterozygous for the ERAP1 variant nucleic acid molecule at a dose equal to or less than a standard dose, and / or administering an ERAP1 inhibitor to the subject; In this case, the presence of a genotype having the ERAP1 mutant nucleic acid molecule indicates that the subject has a low risk of developing uveitis, and / or the therapeutic agent for treating or inhibiting uveitis and / or the ERAP1 inhibitor.

9. The therapeutic agent and / or ERAP1 inhibitor for treating or inhibiting uveitis according to claim 8, wherein the uveitis is anterior uveitis, acute anterior uveitis, or panuveitis.

10. A therapeutic agent and / or ERAP1 inhibitor for treating or inhibiting uveitis according to claim 8 or 9, wherein the ERAP1 mutant nucleic acid molecule encodes Lys528Arg or Asp575Asn.

11. the ERAP1 mutant nucleic acid molecule comprising: a genomic nucleic acid molecule having a nucleotide sequence comprising a guanine at a position corresponding to position 19,474 set forth in SEQ ID NO:2, an adenine at a position corresponding to position 21,595 set forth in SEQ ID NO:3, a cytosine at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or a thymine at a position corresponding to position 42,579 set forth in SEQ ID NO:5; an mRNA molecule having a nucleotide sequence comprising a guanine at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine at a position corresponding to position 1,981 set forth in SEQ ID NO:8; or a cDNA molecule generated from an mRNA molecule having a nucleotide sequence containing a guanine at a position corresponding to position 1,841 of SEQ ID NO: 10 or an adenine at a position corresponding to position 1,981 of SEQ ID NO: 11; A therapeutic agent and / or ERAP1 inhibitor for treating or inhibiting uveitis according to claim 8 or 9.

12. The therapeutic agent and / or ERAP1 inhibitor for treating or inhibiting uveitis according to claim 8 or 9, wherein the ERAP1 inhibitor comprises an inhibitory nucleic acid molecule.

13. A therapeutic agent and / or ERAP1 inhibitor for treating or inhibiting uveitis as described in claim 12, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with an ERAP1 nucleic acid molecule.

14. The therapeutic agent for treating or inhibiting uveitis and / or an ERAP1 inhibitor according to claim 8 or 9, which is used in combination with an HLA-B27 inhibitor or an HLA-B40 inhibitor.

15. The therapeutic agent for treating or inhibiting uveitis and / or ERAP1 inhibitor according to claim 14, wherein the HLA-B27 inhibitor or the HLA-B40 inhibitor comprises an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

16. The therapeutic agent for treating or inhibiting uveitis and / or ERAP1 inhibitor according to claim 15, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA that hybridizes with HLA-B27 or HLA-B40.

17. The subject is HLA-B27 ＋ or HLA-B40 ＋ The ERAP1 inhibitor according to claim 1,

18. A method performed in vitro for assessing a subject's risk of developing uveitis, iridocyclitis, or iritis, said method comprising determining, or having determined, the presence or absence of an endoplasmic reticulum aminopeptidase 1 (ERAP1) mutant nucleic acid molecule in a biological sample obtained from said subject; wherein if said subject meets the ERAP1 criteria, it indicates that said subject has a high risk of developing uveitis, iridocyclitis, or iritis; and if said subject is heterozygous or homozygous for the ERAP1 mutant nucleic acid molecule, it indicates that said subject has a low risk of developing uveitis, iridocyclitis, or iritis.

19. 19. The method of claim 18, wherein the uveitis is anterior uveitis, acute anterior uveitis, or panuveitis.

20. 20. The method of claim 18 or 19, wherein the ERAP1 mutant nucleic acid molecule encodes Lys528Arg or Asp575Asn.

21. A therapeutic agent for treating or inhibiting uveitis for use in the treatment of uveitis, comprising: an endoplasmic reticulum aminopeptidase 1 (ERAP1) variant genomic nucleic acid molecule or its complement, having a nucleotide sequence comprising guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2, adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3, cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5; an ERAP1 mutant mRNA molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; or The therapeutic agent for use in treating uveitis in a subject identified as having an ERAP1 mutant cDNA molecule or its complement, which has a nucleotide sequence including a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10, or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:

11.

22. 1. An endoplasmic reticulum aminopeptidase 1 (ERAP1) inhibitor for use in treating uveitis in a subject, wherein the subject is a) is based on an ERAP1 genomic nucleic acid molecule, an ERAP1 mRNA molecule, or an ERAP1 cDNA molecule; or b) i) an ERAP1 variant genomic nucleic acid molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2, an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3, a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5; ii) an ERAP1 mutant mRNA molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; or iii) the ERAP1 inhibitor for use in treating uveitis in the subject who is heterozygous for an ERAP1 variant cDNA molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:

11.

23. The ERAP1 inhibitor of claim 22, which is an inhibitory nucleic acid molecule.

24. The ERAP1 inhibitor according to claim 23, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with the ERAP1 nucleic acid molecule.

25. 1. An HLA-B27 inhibitor for use in treating uveitis in a subject, said subject comprising: a) is based on an ERAP1 genomic nucleic acid molecule, an ERAP1 mRNA molecule, or an ERAP1 cDNA molecule; or b) i) an ERAP1 variant genomic nucleic acid molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2, an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3, a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5; ii) an ERAP1 mutant mRNA molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; or iii) the HLA-B27 inhibitor for use in treating uveitis in the subject who is heterozygous for an ERAP1 variant cDNA molecule, or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10, or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:

11.

26. The HLA-B27 inhibitor of claim 25, wherein the HLA-B27 inhibitor is an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

27. The HLA-B27 inhibitor of claim 26, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA that hybridizes with HLA-B27.

28. 1. A combination of an endoplasmic reticulum aminopeptidase 1 (ERAP1) inhibitor and an HLA-B27 inhibitor for use in treating uveitis in a subject, wherein the subject is a) is based on an ERAP1 genomic nucleic acid molecule, an ERAP1 mRNA molecule, or an ERAP1 cDNA molecule; or b) i) an ERAP1 variant genomic nucleic acid molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 19,474 set forth in SEQ ID NO:2, an adenine or its complement at a position corresponding to position 21,595 set forth in SEQ ID NO:3, a cytosine or its complement at a position corresponding to position 21,811 set forth in SEQ ID NO:4, or a thymine or its complement at a position corresponding to position 42,579 set forth in SEQ ID NO:5; ii) an ERAP1 mutant mRNA molecule or its complement, having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO:7 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:8; or iii) said combination for use in treating said uveitis in said subject who is heterozygous for an ERAP1 variant cDNA molecule or its complement, said ERAP1 variant cDNA molecule having a nucleotide sequence comprising a guanine or its complement at a position corresponding to position 1,841 set forth in SEQ ID NO: 10 or an adenine or its complement at a position corresponding to position 1,981 set forth in SEQ ID NO:

11.

29. 29. The combination of claim 28, wherein the ERAP1 inhibitor comprises an inhibitory nucleic acid molecule.

30. The combination of claim 29, wherein the ERAP1 inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with an ERAP1 nucleic acid molecule.

31. The combination of any one of claims 28 to 30, wherein the HLA-B27 inhibitor comprises an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

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