Treatment of pulmonary conditions with integrin subunit alpha 1 (ITGA1) inhibitors

Abstract

The present disclosure provides methods for treating a subject having or at risk of developing a pulmonary disease, such as a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease (COPD), or asthma, and methods for identifying subjects at high risk for developing a pulmonary disease.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically as an XML file entitled 381203631SEQ, created on November 30, 2022, and is 86,876 kilobytes in size. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to treatment of subjects having or at risk of developing a pulmonary disease, such as fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease (COPD), or asthma, with integrin subunit alpha 1 (ITGA1) inhibitors, and to methods of identifying subjects at high risk for developing a pulmonary disease. [Background technology]

[0003] Fibrotic lung disease is progressive and irreversible. Standard treatments are merely palliative and do not address the underlying disease mechanism once a subject has progressed to a symptomatic stage. Thus, there is a long-felt and unmet need in the art for a method to treat asymptomatic subjects as well as subjects at risk of developing fibrotic lung disease to prevent disease onset, delay disease onset, or reduce the severity of disease symptoms. The disclosed method provides a preventative or effective treatment for asymptomatic subjects as well as subjects at risk of developing disease, unlike mere palliative treatment.

[0004] Integrin subunit α1 (ITGA1) is a component of cell surface integrin receptors that heterodimerize with the β1 subunit to form cell surface receptors for collagen and laminin. ITGA1 recognizes the proline hydroxylation sequence GFPGER in collagen and is involved in the anchorage-dependent negative regulation of EGF-stimulated cell proliferation. ITGA1 acts on cell binding to laminin and collagen and on neurite outgrowth and peripheral nerve regeneration. Proper collagen-integrin interactions are important for fracture healing, suggesting that the ITGA1 gene is involved in mesenchymal stem cell proliferation and chondrogenesis. This heterodimeric receptor is also involved in cell-cell adhesion and plays a role in inflammation and fibrosis. Summary of the Invention

[0005] The present disclosure provides a method of treating a subject having or at risk of developing a fibrotic lung disease, the method comprising administering to the subject an ITGA1 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing a fibrotic lung disease, comprising administering to the subject an ITGA1 inhibitor.

[0006] The present disclosure also provides a method of treating a subject having or at risk of developing interstitial lung disease, comprising administering to the subject an ITGA1 inhibitor.

[0007] The present disclosure also provides a method of treating a subject having or at risk of developing chronic obstructive pulmonary disease (COPD), comprising administering to the subject an ITGA1 inhibitor.

[0008] The present disclosure also provides a method of treating a subject having asthma or at risk of developing asthma, the method comprising administering to the subject an ITGA1 inhibitor. The present disclosure also provides a method of treating a subject with a pulmonary disease therapeutic agent, the subject having or at risk of developing a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma, comprising determining whether the subject has an ITGA1 variant nucleic acid molecule by obtaining or having a biological sample obtained from the subject and performing or having a sequence analysis performed on the biological sample to determine whether the subject has a genotype that includes an ITGA1 variant nucleic acid molecule; Also provided is a method comprising administering or continuing administration of a standard dose of a pulmonary disease therapeutic agent to a reference subject, and / or administering an ITGA1 inhibitor to the subject; and / or administering or continuing administration of an amount of a pulmonary disease therapeutic agent equal to or less than the standard dose to a subject who is heterozygous for an ITGA1 variant nucleic acid molecule, and administering an ITGA1 inhibitor to the subject; wherein the presence of a genotype having an ITGA1 variant nucleic acid molecule indicates that the subject has a low risk of developing a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma.

[0009] The present disclosure also provides a method for identifying a subject at high risk of developing a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma, comprising determining the presence or absence or having the presence or absence determined of an ITGA1 variant nucleic acid molecule in a biological sample obtained from a subject, where if the subject is an ITGA1 reference, the subject has a high risk of developing a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma, and if the subject is heterozygous or homozygous for the ITGA1 variant nucleic acid molecule, the subject has a low risk of developing a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma.

[0010] The present disclosure also provides a pulmonary disease therapeutic agent for use in treating a pulmonary disease in a subject identified as having an ITGA1 variant nucleic acid molecule, or a complement thereof, wherein the variant nucleic acid molecule comprises a nucleotide sequence comprising a nucleotide at a position corresponding to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule, or a complement thereof.

[0011] The present disclosure also provides an ITGA1 inhibitor for use in treating a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma in a subject, wherein the subject is a) a reference to an ITGA1 genomic nucleic acid molecule, an ITGA1 mRNA molecule, or an ITGA1 cDNA molecule, or is heterozygous for an ITGA1 variant genomic nucleic acid molecule, or its complement, an ITGA1 variant mRNA molecule, or its complement, or an ITGA1 variant cDNA molecule, or its complement.

[0012] 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]

[0013] [Figure 1] We show association of rare pLoF<1% (M1.1) in ITGA1 with various immune- and respiratory-related quantitative traits. [Diagram 2] Figure 2 shows the association of rare pLoF<1% (M1.1) in ITGA1 with various immune- and respiratory-related diseases. The red bars indicate the burden of pLoF results in good lung function and a trend towards protection against asthma and COPD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention Various terms relating to 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.

[0015] Unless expressly stated otherwise, any method or embodiment set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless specifically stated by a method claim in the claims or description that the steps should be limited to a particular order, no order is intended to be imposed in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or work flow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0016] 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 that 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.

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

[0018] As used herein, the term "isolated" refers to a nucleic acid molecule or polypeptide, meaning that the nucleic acid molecule or polypeptide is in a state other than its natural environment, e.g., apart from blood and / or animal tissue. 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 may be in a highly purified form, i.e., more than 95% pure or more 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.

[0019] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include a polymeric form 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 complementary strand.

[0020] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cattle, pigs), pets (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. In some embodiments, the subject is a smoker. In some embodiments, the subject is a smoker of tobacco products.

[0021] The present disclosure identifies rare variants of the ITGA1 gene that are associated with a low risk of developing lung disease in humans. The present disclosure further observes that loss-of-function variant nucleic acid molecules of ITGA1 (in certain subjects, these variations are homozygous or heterozygous) are associated with a low risk of developing lung disease. Furthermore, the present disclosure identifies the association of additional variants with gene burden masks, indicating that ITGA1 itself (rather than linkage disequilibrium with variants of another gene) is responsible for the protective effect in lung disease. It is believed that none of the variants of the ITGA1 gene or protein has any known association with lung disease, such as fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease, or asthma. Taken together, the genetic analysis described herein surprisingly shows that the ITGA1 gene, particularly loss-of-function variants in the ITGA1 gene, are associated with a low risk of developing fibrotic lung disease. Thus, subjects with ITGA1 references at high risk of developing fibrotic lung disease can be treated to prevent fibrotic lung disease, reduce symptoms, and / or inhibit the onset of symptoms. Thus, the present disclosure provides a method of using the identification of such variants in subjects to identify or stratify the risk in such subjects of developing fibrotic lung disease, or to diagnose a subject as having high risk of developing fibrotic lung disease, so that subjects at risk or with active disease can be treated accordingly.

[0022] For the purpose of this disclosure, any particular human can be classified as having one of three ITGA1 genotypes: i) ITGA1-referenced, ii) heterozygous for ITGA1 variant nucleic acid molecule, and iii) homozygous for ITGA1 variant nucleic acid molecule. A human is ITGA1-referenced if it does not have a copy of the ITGA1 variant nucleic acid molecule. A human is heterozygous for ITGA1 variant nucleic acid molecule if it has a single copy of the ITGA1 variant nucleic acid molecule. A human is homozygous for ITGA1 variant nucleic acid molecule if it has two copies of the ITGA1 variant nucleic acid molecule. A human having an ITGA1 polypeptide with partial loss of function (or predicted partial loss of function) is hypomorphic for ITGA1.

[0023] As used herein, ITGA1 variant nucleic acid molecule is any ITGA1 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes an ITGA1 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. ITGA1 variant nucleic acid molecule may be a missense variant, a splice site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, or an in-frame indel variant, or a variant that encodes a truncated ITGA1 predicted loss-of-function polypeptide. ITGA1 variant nucleic acid molecule may be any nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, cDNA molecule, etc.) that causes complete loss, reduced expression, or abnormal expression of ITGA1 mRNA or polypeptide. ITGA1 variant nucleic acid molecule may be any missense variant nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that causes a decrease in ITGA1 activity.

[0024] Suitable examples of ITGA1 variant nucleic acid molecules, such as ITGA1 loss-of-function variant nucleic acid molecules, include the following variations (chromosome: location (GRCh38.p13): reference allele: alternative allele): 5:52920430:C:T, 5:52944942:G:T, 5:52910243:G:T, 5:52918899:G:C, 5:52918730:A:T, 5:52952404:CA:C, 5:52861561:T:C, 5:52939 693:T:C, 5:52910201:C:T, 5:52788416:T:C, 5:52788356:G:A, 5:52893783:G:T, 5:52920332:AT:A, 5:52952436:GA:G, 5:52939945:G: T, 5:52925371:C:T, 5:52939690:TG:T, 5:52898385:T:C, 5:52897529:GT:G, 5:52937490:C:G, 5:52947412:CT:C, 5:52882022:G:T, 5:5 2939946:T:A, 5:52861503:A:AT, 5:52881929:T:G, 5:52932134:C:A, 5:52939933:AC:A, 5:52933993:C:G, 5:52927645:TCCTG:T, 5:5293 9934:CT:C, 5:52893841:G:C, 5:52881939:G:T, 5:52932134:C:G, 5:52864814:GA:G, 5:52849381:C:A, 5:52922818:CT:C, 5:52925286: TG:T, 5:52865083:G:A, 5:52939606:C:CAT, 5:52915509:C:CGTGGTGA, 5:52865083:G:T, 5:52944999:ATC:A, 5:52910194:TA:T, 5:52947 462:G:A, 5:52898323:C:T, 5:52945006:GT:G, 5:52910292:CT:C, 5:52861560:G:A, 5:52939642:G:GA, 5:52864979:GC:G, 5:52927590: CA:C, 5:52918814:G:T, 5:52788351:G:GC, 5:52920469:G:A, 5:52905808:A:AT, 5:52922876:GT:G, 5:52861501:C:CT, 5:52865771:T:A,5:52947456:TGG:T, 5:52918736:C:T, 5:52920376:C:T, 5:52915496:G:GA, 5:52898346:GTC:G, 5:52947462:G:T, 5:52920444:C:A, 5:52882022:G:A, 5:52927640:TC:T, 5:52925470:G:GT, 5:52952430:GA:G, 5:52937516:T:C, 5:52915570:G:GC, 5:52861530:C:A, 5:52932046:G:A, 5:52893841:G:T, 5:52887965:TGTAA:T, 5:52937399:AG:A, 5:52887863:AG:A, 5:52905910:T:A, 5:52925304:TA:T, 5:52910222:C:T, 5:52887906:C:T, 5:52910199:AG:A, 5:52909042:G:A, 5:52898269:TA:T, 5:52939588:A:C, 5:52937515:G:GT, 5:52865745:C:A, 5:52937474:GT:G, 5:52925299:G:T, 5:52920367:CA:C, 5:52898296:CAG:C, 5:52925283:CT:C, 5:52893817:GAGAA:G, 5:52915565:TG:T, 5:52933892:A:T, 5:52915524:G:T, 5:52887846:C:T, 5:52937515:G:A, 5:52929670:GA:G, 5:52915463:G:A, 5:52905900:G:T, 5:52865688:AG:A, 5:52933993:C:A, 5:52932137:G:A, 5:52788355:T:C, 5:52897529:G:A, 5:52947344:G:A, 5:52910417:CAAGT:C, 5:52864772:C:A, 5:52939677:C:T, 5:52918807:C:A, 5:52915502:TTTTGG:T, 5:52893693:C:T, 5:52939855:A:T, 5:52925489:T:C, 5:52939673:C:CA, 5:52887966:G:T, 5:52918877:GA:G, and 5:52947463:T:C are included. Alternative allele(s) is / areis a variant nucleotide(s), and the position is a variant position.

[0025] For subjects that are genotyped or determined to be ITGA1 reference, such subjects have high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma.For subjects that are genotyped or determined to be either ITGA1 reference or heterozygous for ITGA1 variant nucleic acid molecule, such subjects can be treated with ITGA1 inhibitors.

[0026] In any of the embodiments described throughout this disclosure, the pulmonary disease is a fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma. In any of the embodiments described throughout this disclosure, the pulmonary disease is a fibrotic lung disease. In any of the embodiments described throughout this disclosure, the pulmonary disease is pulmonary fibrosis. In any of the embodiments described throughout this disclosure, the pulmonary disease is an interstitial lung disease. In any of the embodiments described throughout this disclosure, the pulmonary disease is a chronic obstructive pulmonary disease. In any of the embodiments described throughout this disclosure, the pulmonary disease is an asthma.

[0027] Symptoms of lung diseases, such as fibrotic lung diseases, include, but are not limited to, shortness of breath (dyspnea), dry cough, fatigue, unexplained weight loss, muscle and joint pain, and widened, rounded tips of the fingers or toes (clubbing).

[0028] The present disclosure provides a method of treating a subject having or at risk of developing a fibrotic lung disease, the method comprising administering to the subject an ITGA1 inhibitor. The present disclosure also provides a method of treating a subject having or at risk of developing a fibrotic lung disease, comprising administering to the subject an ITGA1 inhibitor.

[0029] The present disclosure also provides a method of treating a subject having or at risk of developing interstitial lung disease, comprising administering to the subject an ITGA1 inhibitor.

[0030] The present disclosure also provides a method of treating a subject having or at risk of developing COPD, comprising administering to the subject an ITGA1 inhibitor. The present disclosure also provides a method of treating a subject having asthma or at risk of developing asthma, the method comprising administering to the subject an ITGA1 inhibitor.

[0031] In some embodiments, the ITGA1 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 inhibitory nucleic acid molecules can be designed to target any region of an ITGA1 nucleic acid molecule, such as an mRNA molecule. In some embodiments, the inhibitory nucleic acid molecule hybridizes to a sequence in an ITGA1 genomic nucleic acid molecule or an mRNA molecule in a subject's cell to reduce the expression of an ITGA1 polypeptide. In some embodiments, the ITGA1 inhibitor comprises an antisense molecule that hybridizes to an ITGA1 genomic nucleic acid molecule or an mRNA molecule in a subject's cell to reduce the expression of an ITGA1 polypeptide. In some embodiments, the ITGA1 inhibitor comprises an siRNA that hybridizes to an ITGA1 genomic nucleic acid molecule or an mRNA molecule in a subject's cell to reduce the expression of an ITGA1 polypeptide. In some embodiments, the ITGA1 inhibitor comprises an shRNA that hybridizes to an ITGA1 genomic nucleic acid molecule or an mRNA molecule in a subject's cells to reduce expression of an ITGA1 polypeptide.

[0032] In some embodiments, the antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 32-18299. In some embodiments, the siRNA molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 18300-69677 (sense and antisense strands set forth sequentially) (e.g., the sense strand is, e.g., SEQ ID NO: 18300 and the corresponding antisense strand is, e.g., SEQ ID NO: 18301, the sense strand is, e.g., SEQ ID NO: 18302 and the corresponding antisense strand is, e.g., SEQ ID NO: 18303, etc.).

[0033] The inhibitory nucleic acid molecule may comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule may also be linked or fused to a heterologous nucleic acid sequence or a heterologous label, for example in a vector. For example, the inhibitory nucleic acid molecule may 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 may also be linked or fused to a heterologous label. The label may be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels may 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 may also be, for example, a chemiluminescent substance, a metal-containing substance, or an enzyme, in which case enzyme-dependent secondary signal generation occurs. The term "label" may also refer to a "tag" or hapten that can be selectively attached to a conjugated molecule such that when the conjugated molecule is subsequently added with a substrate, it can be used to generate a detectable signal. For example, biotin can be used as a tag with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and test with 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.

[0034] 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.

[0035] The inhibitory nucleic acid molecule may contain one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains modifications 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 different purine or pyrimidine bases, such as 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 (pseudouracil), ), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (such as 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0036] Nucleotide analogs may also include modifications at the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of the ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10 Exemplary 2' sugar modifications also 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 CH3)]2, where n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1-10These 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 can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of a 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.

[0037] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate sites include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. These phosphate or modified phosphate linkages between two nucleotides can be through 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).

[0038] 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.

[0039] 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.

[0040] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking successive ribose nucleosides have been shown to increase the stability and bioavailability of siRNA in vivo. The non-ester group (-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.

[0041] 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.

[0042] In some embodiments, siRNA molecules are bound to lipids.Lipids can be bound to the 5'-end or 3'-end of siRNA, and can associate with serum lipoproteins to improve their bioavailability in vivo.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitic acid and tocopherol.

[0043] 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.

[0044] In any of the embodiments described herein, the inhibitory nucleic acid molecule may 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 may be administered at 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 to about 640 mg (mg / kg to mg / m2 based on an assumed body weight of 70 kg and a mg / kg dose multiplier value of 37 for humans). 2Based on conversion to dose levels, 2.5 to 9.14 mg / kg, 92.5 to 338 mg / m 2 ) may be administered at dosage levels ranging from 0.1 to 100 mg / kg.

[0045] 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.

[0046] 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.

[0047] In some embodiments, the ITGA1 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) or a DNA binding protein that binds to the recognition sequence in the ITGA1 genomic nucleic acid molecule. The recognition sequence can be located in the coding region of the ITGA1 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 ITGA1 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 the coding region between the two nuclease recognition sequences can be deleted by cleavage with these nuclease agents.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.

[0048] 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, including, for example, about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALEN proteins or TALENs, and about 20 bp for CRISPR / Cas guide RNAs.

[0049] In some embodiments, the CRISPR / Cas system can be used to modify the ITGA1 genomic nucleic acid molecule in cells. The methods and compositions disclosed herein can use the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the ITGA1 nucleic acid molecule.

[0050] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins may also contain nuclease domains (such as DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (such as FnCpf1). Cas proteins may have full cleavage activity to form double-stranded breaks in ITGA1 genomic nucleic acid molecules, or may be nickases to form single-stranded breaks in ITGA1 genomic nucleic acid molecules. Further examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (Cas B), Cse3 (CasE), 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 modified forms thereof. In some embodiments, a Cas system, for example, Cas12a, may have multiple gRNAs encoded by a single crRNA. Cas proteins may also be operably linked to heterologous polypeptides as fusion proteins. For example, the Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. The Cas protein 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 may be provided in the form of a nucleic acid molecule, such as RNA or DNA, that encodes the Cas protein.

[0051] In some embodiments, targeted genetic modification of ITGA1 genomic nucleic acid molecule can be generated by contacting a cell with Cas protein and one or more gRNAs that hybridize with one or more gRNA recognition sequences in a target genomic locus in ITGA1 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 a variant position. 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 from the variant position. The gRNA recognition sequence can include or be adjacent to the start codon of ITGA1 genomic nucleic acid molecule or the stop codon of ITGA1 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon or the stop codon.

[0052] The gRNA recognition sequence in the target genomic locus in the ITGA1 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease. A canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. The gRNA can transport Cas9 anywhere in the genome for gene editing, but editing cannot occur at sites other than the site where Cas9 recognizes the PAM. Additionally, 5'-NGA-3' can be a non-canonical PAM that is highly efficient in 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, immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide, immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0053] gRNA is an RNA molecule that binds to Cas protein and guides Cas protein to a specific location in ITGA1 genomic nucleic acid molecule. An exemplary gRNA is an effective gRNA for inducing Cas enzyme to bind to or cleave ITGA1 genomic nucleic acid molecule, where the gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence in ITGA1 genomic nucleic acid molecule that includes or is adjacent to a position corresponding to a variant position. For example, the gRNA can be selected to hybridize to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the variant position. Other exemplary gRNA comprises a DNA targeting segment that hybridizes to a gRNA recognition sequence present in ITGA1 genomic nucleic acid molecule that includes or is adjacent to a start codon or a stop codon. For example, a gRNA can be selected to hybridize to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon, or to a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the 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 can include 20 nucleotides.

[0054] Examples of suitable gRNA recognition sequences located within the ITGA1 reference gene are listed in Table 1 as SEQ ID NOs: 12-31.

[0055] [Table 1]

[0056] The Cas protein and gRNA form a complex, and the Cas protein cuts the target ITGA1 genomic nucleic acid molecule. The Cas protein can cut the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target ITGA1 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including the gRNA hybridized with the gRNA recognition sequence and complexed with the Cas protein) can cause one or both strand cuts 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 ITGA1 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0057] Such a method may result in ITGA1 genomic nucleic acid molecule with, for example, SEQ ID NO:1 region being destroyed, start codon being destroyed, stop codon being destroyed, or coding sequence being destroyed or deleted. Optionally, cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of ITGA1 genomic nucleic acid molecule. By contacting cell with one or more additional gRNAs (such as a second gRNA that hybridizes with a second gRNA recognition sequence), cleavage by Cas protein can produce two or more double-strand breaks or two or more single-strand breaks.

[0058] In some embodiments, the ITGA1 inhibitor comprises a small molecule. In some embodiments, the ITGA1 inhibitor is obtustatin. In some embodiments, the methods of treatment further comprise detecting the presence or absence of ITGA1 variant nucleic acid in a biological sample obtained from the subject.

[0059] The present disclosure also provides a method of treating a subject with a pulmonary disease therapeutic. In some embodiments, the subject has a pulmonary disease. In some embodiments, the subject has a high risk of developing a pulmonary disease. In some embodiments, the method includes obtaining or having a biological sample obtained from the subject, and determining whether the subject has an ITGA1 variant nucleic acid molecule by performing or having a sequence analysis on the biological sample to determine whether the subject has a genotype that includes an ITGA1 variant nucleic acid molecule. If the subject is ITGA1 reference, the subject is administered or continues to be administered a pulmonary disease therapeutic at a standard dose, and / or an ITGA1 inhibitor is administered to the subject. If the subject is heterozygous for the ITGA1 variant nucleic acid molecule, the subject is administered or continues to be administered a pulmonary disease therapeutic at a standard dose or at a lower dose, and / or an ITGA1 inhibitor is administered to the subject. The presence of a genotype that includes an ITGA1 variant nucleic acid molecule indicates that the subject has a low risk of developing a pulmonary disease. In some embodiments, the subject is ITGA1 reference. In some embodiments, the subject is heterozygous for the ITGA1 variant nucleic acid molecule.

[0060] In the case of subjects who have been genotyped or determined to be heterozygous for an ITGA1 reference or ITGA1 variant nucleic acid molecule, such subjects can be treated with an ITGA1 inhibitor as described herein.

[0061] Detecting the presence or absence of an ITGA1 variant nucleic acid molecule in a biological sample from a subject and / or determining whether a subject has an ITGA1 variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the ITGA1 variant nucleic acid molecule can be present in a cell obtained from the subject.

[0062] In some embodiments, if the subject is ITGA1 reference, the subject is also administered a standard dose of a pulmonary disease therapeutic agent. In some embodiments, if the subject is heterozygous for the ITGA1 variant nucleic acid molecule, the subject is also administered a dose of a pulmonary disease therapeutic agent that is equal to or less than the standard dose.

[0063] In some embodiments, the method of treatment further comprises detecting the presence or absence of predicted loss-of-function polypeptide of ITGA1 in a biological sample from the subject.In some embodiments, if the subject does not have predicted loss-of-function polypeptide of ITGA1, the subject is also administered a standard dose of a pulmonary disease therapeutic agent.In some embodiments, if the subject has predicted loss-of-function polypeptide of ITGA1, the subject is also administered a dose of a pulmonary disease therapeutic agent that is equal to or less than the standard dose.

[0064] The present disclosure also provides a method of treating a subject with a pulmonary disease therapeutic. In some embodiments, the subject has a pulmonary disease. In some embodiments, the subject has a high risk of developing a pulmonary disease. In some embodiments, the method includes obtaining or having a biological sample obtained from the subject, and performing or having an assay performed on the biological sample to determine whether the subject has a predicted loss-of-function polypeptide of ITGA1, thereby determining whether the subject has a predicted loss-of-function polypeptide of ITGA1. If the subject does not have a predicted loss-of-function polypeptide of ITGA1, the subject is administered or continues to be administered a standard dose of the pulmonary disease therapeutic, and an ITGA1 inhibitor is administered to the subject. If the subject has a predicted loss-of-function polypeptide of ITGA1, the subject is administered or continues to be administered a standard dose or a lower amount of the pulmonary disease therapeutic, and / or an ITGA1 inhibitor is administered to the subject. The presence of a predicted loss-of-function polypeptide of ITGA1 indicates that the subject has a low risk of developing a pulmonary disease. In some embodiments, the subject has a predicted loss-of-function polypeptide of ITGA1. In some embodiments, the subject does not have a predicted loss-of-function polypeptide of ITGA1.

[0065] Detecting the presence or absence of a predicted loss-of-function polypeptide of ITGA1 in a biological sample of a subject and / or determining whether a subject has a predicted loss-of-function polypeptide of ITGA1 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 predicted loss-of-function polypeptide of ITGA1 can be present in a cell obtained from the subject.

[0066] Examples of pulmonary medications include, but are not limited to, nintedanib, pirfenidone, prednisone, azathioprine, cyclophosphamide, mycophenolate mofetil, rituximab, tacrolimus, cotrimoxazole, lebrikizumab, nandrolone decanoate, sirolimus, thalidomide, and pomalidomide.

[0067] In some embodiments, the dose of the pulmonary disease therapeutic agent can be reduced (i.e., less than the standard dose) in subjects who are heterozygous for ITGA1 variant nucleic acid molecules compared to subjects who are ITGA1 reference (can be administered with the standard dose). In some embodiments, the dose of the pulmonary disease therapeutic agent can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In addition, the dose of the pulmonary disease therapeutic agent can be administered less frequently in subjects who are heterozygous for ITGA1 variant nucleic acid molecules compared to subjects who are ITGA1 reference.

[0068] The administration of the pulmonary disease therapeutic agent and / or ITGA1 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. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a certain dosing regimen, the subject can be treated for an extended period of time, for example, 6 months, 1 year, or longer. Furthermore, the pulmonary disease therapeutic agent and / or ITGA1 inhibitor can be administered sequentially or simultaneously. Furthermore, the pulmonary disease therapeutic agent and / or ITGA1 inhibitor can be administered in separate compositions or together in the same composition.

[0069] The administration of the pulmonary disease therapeutic agent and / or ITGA1 inhibitor can be by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical composition to be administered is preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a single dose for administration). The pharmaceutical composition can be formulated using one or more physiologically and pharmacologic 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 deleterious 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 the following following administration of the agent or a composition comprising the agent: a reduction / reduction in pulmonary disease, a reduction / reduction in the severity of pulmonary disease (e.g., a reduction or inhibition of the onset of pulmonary disease), a reduction / reduction in symptoms and pulmonary disease related effects, a delay in the onset of symptoms and pulmonary disease related effects, a reduction in the severity of symptoms of pulmonary disease related effects, a reduction in the severity of acute episodes, a reduction in the number of symptoms and pulmonary disease related effects, a reduction in the latency period of symptoms and pulmonary disease related effects, an improvement in symptoms and pulmonary disease related effects, a reduction in secondary symptoms, a reduction in secondary infections, a prevention of recurrence of pulmonary disease, a reduction in the number or frequency of recurrent episodes, an increase in the latency period between symptomatic episodes, an increase in the time to sustained progression, a promotion of remission, an induction of remission, an increase in remission, an acceleration of recovery, or an increase in the effectiveness of or a decrease in resistance to alternative therapies, and / or an increase in the survival time of an affected host animal. A prophylactic effect can include complete or partial avoidance / inhibition, or delay (e.g., complete or partial avoidance / inhibition or delay, etc.) of the onset / progression of pulmonary disease following implementation of a treatment protocol, and prolongation of survival of the diseased host animal. Treatment of pulmonary disease encompasses treatment of a subject already diagnosed as having any form of pulmonary disease at any clinical stage or symptom, delaying the onset or progression or exacerbation or worsening of symptoms or signs of pulmonary disease, and / or preventing and / or reducing the severity of pulmonary disease.

[0071] The present disclosure also provides a method for identifying a subject with high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma. In some embodiments, the method includes determining the presence or absence of ITGA1 variant nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, and / or cDNA molecule) in a biological sample obtained from a subject, or the presence or absence is determined. If a subject lacks ITGA1 variant nucleic acid molecule (i.e., the subject is genotypically classified as ITGA1 reference), the subject has a high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma. If a subject has ITGA1 variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for ITGA1 variant nucleic acid molecule), the subject has a lower risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma compared to the subject who is ITGA1 reference.

[0072] When a subject has a single copy of ITGA1 variant nucleic acid molecule, the subject is more protected from developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma compared to when the subject does not have a copy of ITGA1 variant nucleic acid molecule.Without intending to be limited to any particular theory or mechanism of action, it is believed that a single copy of ITGA1 variant nucleic acid molecule (i.e., heterozygous for ITGA1 variant nucleic acid molecule) protects a subject from developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma, and it is believed that by having two copies of ITGA1 variant nucleic acid molecule (i.e., homozygous for ITGA1 variant nucleic acid molecule), the subject is further protected from developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma compared to when the subject has a single copy. Therefore, in some embodiments, it is believed that a single copy of ITGA1 variant nucleic acid molecule does not completely protect a subject from developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma, but instead protects partially or incompletely.Without wishing to be bound by any particular theory, it is believed that the subject with a single copy of ITGA1 variant nucleic acid molecule still has additional factors or molecules involved in the development of fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma, and therefore is incompletely protected from developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma.

[0073] Detecting the presence or absence of an ITGA1 variant nucleic acid molecule in a biological sample from a subject and / or determining whether a subject has an ITGA1 variant nucleic acid molecule can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the ITGA1 variant nucleic acid molecule can be present in a cell obtained from the subject.

[0074] In some embodiments, if the subject is identified as having a high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma, the subject is further treated with a pulmonary disease therapeutic agent and / or an ITGA1 inhibitor as described herein. For example, if the subject is ITGA1 reference and therefore has a high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma, the subject is administered an ITGA1 inhibitor. In some embodiments, such a subject is also administered a pulmonary disease therapeutic agent. In some embodiments, if the subject is heterozygous for an ITGA1 variant nucleic acid molecule, the subject is administered a dose of a pulmonary disease therapeutic agent that is the same as or less than the standard dose, and is also administered an ITGA1 inhibitor. In some embodiments, the subject is ITGA1 reference. In some embodiments, the subject is heterozygous for an ITGA1 variant nucleic acid molecule.

[0075] The present disclosure also provides a method for detecting the presence or absence of ITGA1 mutant sense genomic variant nucleic acid molecules in a biological sample obtained from a subject, and / or ITGA1 variant mRNA molecules in a biological sample obtained from a subject, and / or ITGA1 variant cDNA molecules generated from mRNA molecules in a biological sample obtained from a subject. It should be understood that gene sequences in a population and the mRNA molecules encoded by these genes may differ due to polymorphisms such as single nucleotide polymorphisms. The sequences provided herein for ITGA1 variant genomic nucleic acid molecules, ITGA1 variant mRNA molecules, and ITGA1 variant cDNA molecules are merely exemplary sequences. Other sequences of ITGA1 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0076] The biological sample may be derived from any cell, tissue, or biological fluid from a subject. The biological sample may include any clinically significant tissue, such as, for example, a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of bodily fluid (such as 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 treated differently depending on the assay used. For example, when detecting ITGA1 variant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for ITGA1 variant nucleic acid molecules may be used. A variety of techniques may be used for this purpose. When detecting the level of any ITGA1 variant mRNA molecule, different techniques may be used to enrich the biological sample containing the mRNA molecule. A variety of methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular variant genomic DNA locus.

[0077] The present disclosure also provides a method for detecting a variant nucleic acid molecule of ITGA1 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 ITGA1 variant nucleic acid molecule.

[0078] In some embodiments, the ITGA1 variant nucleic acid molecule or its complement is a genomic nucleic acid molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement. In some embodiments, the ITGA1 variant nucleic acid molecule is an mRNA molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule, or its complement. In some embodiments, the ITGA1 variant nucleic acid molecule is a cDNA generated from an mRNA molecule in a biological sample having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule, or its complement.

[0079] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can further comprise, for example, obtaining a biological sample from a subject that comprises ITGA1 genomic nucleic acid molecule or mRNA molecule, and, in the case of mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can comprise, for example, determining the identity of these positions of a particular ITGA1 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0080] In some embodiments, the determining, detecting, or sequence analysis step includes sequencing at least a portion of the nucleotide sequence of an ITGA1 genomic nucleic acid molecule in the biological sample, an ITGA1 mRNA molecule, or an ITGA1 cDNA molecule generated from an mRNA molecule in the biological sample, wherein the sequenced portion contains one or more mutations that cause or are predicted to cause loss of function (partial or complete).

[0081] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of: i) the nucleotide sequence of an ITGA1 genomic nucleic acid molecule in a biological sample, or its complement, in which the portion to be sequenced includes a position corresponding to the variant position of an ITGA1 variant nucleic acid molecule; ii) the nucleotide sequence of an ITGA1 mRNA molecule in a biological sample, or its complement, in which the portion to be sequenced includes a position corresponding to the variant position of an ITGA1 variant nucleic acid molecule; and / or iii) the nucleotide sequence of an ITGA1 cDNA molecule in a biological sample, or its complement, in which the portion to be sequenced includes a position corresponding to the variant position of an ITGA1 variant nucleic acid molecule. If the portion to be sequenced of the ITGA1 nucleic acid molecule in a biological sample includes a nucleotide at a position corresponding to the nucleotide at the variant position of an ITGA1 variant nucleic acid molecule, the ITGA1 nucleic acid molecule in the biological sample is an ITGA1 variant nucleic acid molecule.

[0082] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the ITGA1 genomic nucleic acid molecule in the biological sample, and the portion to be sequenced comprises a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement.If the portion to be sequenced of the ITGA1 nucleic acid molecule in the biological sample comprises a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule, then the ITGA1 nucleic acid molecule in the biological sample is an ITGA1 variant genomic nucleic acid molecule.

[0083] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the ITGA1 mRNA molecule in the biological sample, and the portion to be sequenced comprises a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement.If the portion to be sequenced of the ITGA1 mRNA molecule in the biological sample comprises a nucleotide sequence comprising a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule, the ITGA1 nucleic acid molecule in the biological sample is an ITGA1 variant mRNA molecule.

[0084] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of an ITGA1 cDNA molecule generated from an mRNA molecule in a biological sample, and the portion to be sequenced comprises a position corresponding to a variant position of an ITGA1 variant nucleic acid molecule or its complement. If the portion to be sequenced of the ITGA1 cDNA molecule in the biological sample comprises a nucleotide sequence comprising a nucleotide at a position corresponding to a variant position of the ITGA1 variant nucleic acid molecule, the ITGA1 nucleic acid molecule in the biological sample is an ITGA1 variant cDNA molecule.

[0085] In some embodiments, the determining, detecting, or sequence analysis step comprises the steps of: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of i) an ITGA1 genomic nucleic acid molecule or its complement adjacent to a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement, ii) an ITGA1 mRNA molecule or its complement adjacent to a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement, and / or iii) an ITGA1 cDNA molecule or its complement adjacent to a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement; and b) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of i) an ITGA1 genomic nucleic acid molecule or its complement corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement, ii) an ITGA1 mRNA molecule or its complement corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement, and / or iii) an ITGA1 cDNA molecule or its complement adjacent to a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement. and c) 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 nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0086] In some embodiments, the determining, detecting, or sequence analysis comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an ITGA1 genomic nucleic acid molecule or its complement adjacent to a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement; b) extending the primer at least through the position of the nucleotide sequence of the ITGA1 genomic nucleic acid molecule or its complement that corresponds to the variant position of the ITGA1 variant nucleic acid molecule or its complement; and c) determining whether the extension product of the primer comprises a nucleotide at a position corresponding to the nucleotide of the variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0087] In some embodiments, the determining, detecting, or sequence analysis step comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of ITGA1 mRNA or its complement adjacent to a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement; b) extending the primer through at least the position of the nucleotide sequence of the ITGA1 mRNA molecule that corresponds to the variant position of the ITGA1 variant nucleic acid molecule or its complement; and c) determining whether the extension product of the primer comprises a nucleotide at a position corresponding to the nucleotide of the variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0088] In some embodiments, the determining, detecting, or sequence analysis step comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of ITGA1 cDNA or its complement adjacent to a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule or its complement; b) extending the primer through at least the position of the nucleotide sequence of the ITGA1 cDNA molecule that corresponds to the variant position of the ITGA1 variant nucleic acid molecule or its complement; and c) determining whether the extension product of the primer comprises a nucleotide at a position corresponding to the nucleotide of the variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0089] In some embodiments, the entire nucleic acid molecule is sequenced. In some embodiments, only the ITGA1 genomic nucleic acid molecule is analyzed. In some embodiments, only the ITGA1 mRNA is analyzed. In some embodiments, only the ITGA1 cDNA obtained from the ITGA1 mRNA is analyzed.

[0090] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of an ITGA1 nucleic acid molecule or its complement in a biological sample, wherein the amplified portion comprises a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; and c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0091] In some embodiments, the determining, detecting, or sequence analysis comprises: a) amplifying at least a portion of an ITGA1 genomic nucleic acid molecule or its complement in a biological sample, the portion comprising a nucleotide at a position corresponding to the nucleotide at the variant position of an ITGA1 variant nucleic acid molecule or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, the variant-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a nucleotide at a position corresponding to the nucleotide at the variant position of an ITGA1 variant nucleic acid molecule or its complement; and d) detecting the detectable label.

[0092] In some embodiments, the determining, detecting, or sequence analysis comprises: a) amplifying at least a portion of an ITGA1 mRNA molecule or its complement in a biological sample, wherein the portion comprises a nucleotide at a position corresponding to the nucleotide at the variant position of an ITGA1 variant nucleic acid molecule or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a nucleotide at a position corresponding to the nucleotide at the variant position of an ITGA1 variant nucleic acid molecule or its complement; and d) detecting the detectable label.

[0093] In some embodiments, the determining, detecting, or sequence analysis comprises: a) amplifying at least a portion of an ITGA1 cDNA molecule or its complement in a biological sample, wherein the portion comprises a nucleotide at a position corresponding to the nucleotide at the variant position of an ITGA1 variant nucleic acid molecule or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a variant-specific probe, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising a nucleotide at a position corresponding to the nucleotide at the variant position of an ITGA1 variant nucleic acid molecule or its complement; and d) detecting the detectable label.

[0094] In some embodiments, the nucleic acid molecule is mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step. In some embodiments, the determining, detecting, or sequence analysis step comprises contacting an ITGA1 nucleic acid molecule or its complement in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the ITGA1 nucleic acid molecule or its complement comprising a nucleotide at a position corresponding to a variant position of the ITGA1 variant nucleic acid molecule or its complement, and detecting the detectable label.

[0095] In some embodiments, the determining, detecting, or sequence analysis step comprises contacting an ITGA1 genomic nucleic acid molecule or its complement in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the ITGA1 genomic nucleic acid molecule or its complement comprising a nucleotide at a position corresponding to a variant position of the ITGA1 variant nucleic acid molecule or its complement, and detecting the detectable label.

[0096] In some embodiments, the determining, detecting, or sequence analysis step comprises contacting an ITGA1 mRNA molecule or its complement in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the ITGA1 mRNA molecule or its complement comprising a nucleotide at a position corresponding to a variant position of the ITGA1 variant nucleic acid molecule or its complement, and detecting the detectable label.

[0097] In some embodiments, the determining, detecting, or sequence analysis step comprises contacting an ITGA1 cDNA molecule or its complement generated from an mRNA molecule in a biological sample with a variant-specific probe comprising a detectable label, wherein the variant-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the ITGA1 cDNA molecule or its complement comprising a nucleotide at a position corresponding to a variant position of the ITGA1 variant nucleic acid molecule or its complement, and detecting the detectable label.

[0098] In some embodiments, the ITGA1 nucleic acid molecule is present in a cell obtained from the subject. Mutation-specific polymerase chain reaction techniques can be used to detect mutations, such as SNPs, in nucleic acid sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0099] In some embodiments, the determining, detecting, or sequence analysis step includes contacting the biological sample with a primer or probe, such as a variant-specific primer or variant-specific probe, that specifically hybridizes to an ITGA1 variant genomic sequence, variant mRNA sequence, or variant cDNA sequence under stringent conditions and does not hybridize to the corresponding ITGA1 reference sequence, and determining whether hybridization has occurred.

[0100] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing the mRNA into cDNA, for example by reverse transcriptase polymerase chain reaction (RT-PCR).

[0101] 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 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules of ITGA1. The hybridization or reaction conditions to achieve this result can be determined by the operator. The nucleotide length can be any length sufficient for use in the detection method of choice, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. The probes and primers may have complete nucleotide sequence identity of consecutive nucleotides in the target nucleotide sequence, but probes that differ from the target nucleotide sequence but retain the ability to specifically detect and / or identify the target nucleotide sequence may be designed by conventional methods. 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.

[0102] In some embodiments, to determine whether the ITGA1 genomic nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 genomic nucleic acid molecule, the biological sample can be subjected to an amplification method using a primer pair that includes a first primer derived from a 5' flanking sequence adjacent to the nucleotide at the position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule and a second primer derived from a 3' flanking sequence adjacent to the nucleotide at the position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule, thereby generating an amplicon that indicates the presence of a SNP at a position corresponding to the variant position of the ITGA1 variant nucleic acid molecule. In some embodiments, the amplicon can range from 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 to about 20,000 nucleotide base pairs. Optionally, the primer pair flanks a region that includes a position corresponding to the variant position in the ITGA1 variant nucleic acid molecule and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on either side of the position corresponding to the variant position in the ITGA1 variant nucleic acid molecule.

[0103] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be obtained from known sequences by 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 scanned and primers manually identified using known guidelines.

[0104] 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).

[0105] In hybridization techniques, stringent conditions can be employed so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target sequence to a detectably higher degree than other non-target sequences, for example, at least 2 times, at least 3 times, at least 4 times, or more than background, including more than 10 times background. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target nucleotide sequence to a detectably higher degree than other nucleotide sequences at least 2 times. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target nucleotide sequence to a detectably higher degree than other nucleotide sequences at least 3 times. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes with its target nucleotide sequence to a detectably higher degree than other nucleotide sequences at least 4 times. 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 at more than 10-fold background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0106] 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 +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 adding 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 length of the wash time will be at least long enough to reach equilibrium.

[0107] The present disclosure also provides a method for detecting the presence of predicted loss-of-function polypeptides of ITGA1, comprising performing an assay on a biological sample obtained from a subject to determine whether the ITGA1 polypeptide in the biological sample contains one or more mutations that cause the polypeptide to have loss-of-function (partial or complete) or predicted loss-of-function (partial or complete).The predicted loss-of-function polypeptides of ITGA1 can be any of the predicted loss-of-function polypeptides of ITGA1 described herein.

[0108] In some embodiments, the methods include assaying a biological sample obtained from the subject to determine whether an ITGA1 polypeptide in the biological sample comprises an amino acid sequence that includes a mutation encoded by any of the ITGA1 variant nucleic acid molecules described herein.

[0109] In some embodiments, the detecting step comprises sequencing at least a portion of an ITGA1 polypeptide that comprises an amino acid sequence that includes a mutation encoded by any of the ITGA1 variant nucleic acid molecules described herein.

[0110] In some embodiments, the detecting step comprises an immunoassay to detect the presence of an ITGA1 polypeptide comprising an amino acid sequence containing a mutation encoded by any of the ITGA1 variant nucleic acid molecules described herein.

[0111] In some embodiments, if a subject does not have a predicted loss-of-function polypeptide of ITGA1, the subject has a high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma.In some embodiments, if a subject has a predicted loss-of-function polypeptide of ITGA1, the subject has a low risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma.

[0112] The present disclosure also provides an isolated nucleic acid molecule that hybridizes to an ITGA1 variant genomic nucleic acid molecule, an ITGA1 variant mRNA molecule, and / or an ITGA1 variant cDNA molecule (e.g., any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein). In some embodiments, such an isolated nucleic acid molecule hybridizes to an ITGA1 variant nucleic acid molecule under stringent conditions. Such a nucleic acid molecule can be used, for example, as a probe, primer, variant-specific probe, or variant-specific primer described or exemplified herein.

[0113] In some embodiments, the isolated nucleic acid molecule hybridizes to a portion of the ITGA1 nucleic acid molecule that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule.

[0114] 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.

[0115] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an ITGA1 variant genomic nucleic acid molecule, an ITGA1 variant mRNA molecule, and / or an ITGA1 variant 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.

[0116] In some embodiments, the isolated variant-specific probe or variant-specific primer comprises at least about 15 nucleotides, and the variant-specific probe or variant-specific primer comprises a nucleotide sequence that is complementary to a nucleotide sequence of a portion of an ITGA1 variant nucleic acid molecule or its complement. In some embodiments, the portion comprises a position that corresponds to a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0117] In some embodiments, the variant-specific probe and the variant-specific primer comprise DNA. In some embodiments, the variant-specific probe and the variant-specific primer comprise RNA.

[0118] In some embodiments, the probes and primers described herein (including variant-specific probes and variant-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.

[0119] In some embodiments, primers, including modification-specific primers, can be used in second generation or high throughput sequencing. In some examples, primers, including modification-specific primers, can be modified. In particular, 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 generally 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. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. Adapters can include 5'-biotin tags for immobilizing DNA libraries on streptavidin-coated beads.

[0120] The probes and primers described herein can be used to detect nucleotide variations in any of the ITGA1 variant genomic nucleic acid molecules, ITGA1 variant mRNA molecules, and / or ITGA1 variant cDNA molecules disclosed herein.The primers described herein can be used to amplify the ITGA1 variant genomic nucleic acid molecules, ITGA1 variant mRNA molecules, or ITGA1 variant cDNA molecules, or fragments thereof.

[0121] The present disclosure also provides primer pairs comprising any of the primers described above. In the context of this disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleic acid sequence encoding an ITGA1 reference genomic nucleic acid molecule, an ITGA1 reference mRNA molecule, and / or an ITGA1 reference cDNA molecule.

[0122] In any of the embodiments described throughout this disclosure, the probe (e.g., the modification-specific probe) can include a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0123] 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 molecules such as any of the probes disclosed herein can be associated. 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.

[0124] In some embodiments, any of the methods described herein may further comprise determining the burden of a subject having ITGA1 variant nucleic acid molecule and / or a predicted loss-of-function variant polypeptide of ITGA1 associated with a low risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma. The burden is the sum of all variants of the ITGA1 gene that can be performed in an association analysis with a lung disease, such as fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma. In some embodiments, the subject is homozygous for one or more ITGA1 variant nucleic acid molecules associated with a low risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma. In some embodiments, the subject is heterozygous for one or more ITGA1 variant nucleic acid molecules associated with a low risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma. The results of the association analysis suggest that the ITGA1 variant nucleic acid molecule is associated with a lower risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma. If the subject has a low burden, the subject has a higher risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma compared to subjects with a high burden, and the subject is administered or continues to be administered a standard dose of a pulmonary disease treatment drug and / or an ITGA1 inhibitor. If the subject has a high burden, the subject has a lower risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma compared to subjects with a low burden, and the subject is administered or continues to be administered a pulmonary disease treatment drug at the same or lower dose. The higher the burden, the lower the risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma. The ITGA1 variant nucleic acid molecule may be any of the ITGA1 variant nucleic acid molecules having any of the mutations described herein.

[0125] In some embodiments, the burden of a subject having any one or more ITGA1 variant nucleic acid molecules represents the weighted sum of multiple ITGA1 variant nucleic acid molecules. In some embodiments, the burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about, or more than 1,000,000 genetic variants present in or surrounding (up to 10 Mb) the ITGA1 gene, and the genetic burden is the number of alleles multiplied by the estimated association with fibrotic lung disease or related prognosis for each allele (e.g., a weighted polygenic burden score). This may include any genetic variants close to the ITGA1 gene (up to 10Mb around the gene) that show non-zero association with fibrotic lung disease-related traits in genetic association analysis, regardless of genome annotation.In some embodiments, if a subject has a load higher than a desired threshold score, the subject has a low risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma.In some embodiments, if a subject has a load lower than a desired threshold score, the subject has a high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma.

[0126] In some embodiments, the burden can be divided into quintiles, for example, top quintile, middle quintile, and bottom quintile, with the top quintile of burden corresponding to the lowest risk group and the bottom quintile of burden corresponding to the highest risk group. In some embodiments, subjects with a larger burden include those with the highest weighted burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% of burdens from the subject population. In some embodiments, the genetic variants include genetic variants with association with fibrotic lung disease in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range for the association. In some embodiments, each of the identified genetic variants is about 10 -2 Below, about 10 -3 Below, about 10 -4 Below, about 10 -5 Below, about 10 -6 Below, about 10 -7 Below, about 10 -8 Below, about 10 -9 Below, about 10 -10 Below, about 10 -11 Below, about 10 -12 Below, about 10 -13 Below, about 10 -14 Less than or equal to 10 -15 In some embodiments, the identified genetic variants include those with an association with fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma at a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants having an association with fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma in high-risk subjects with an odds ratio (OR) of about 1.5 or more, about 1.75 or more, about 2.0 or more, or about 2.25 or more for the top 20% of the distribution, or about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more for the top 20% of the distribution, relative to the remainder of the reference population. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or may be greater than 7.0. In some embodiments, high-risk subjects include subjects with a burden in the bottom decile, quintile, or tertile in the reference population. The burden threshold is determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0127] In some embodiments, if the subject is identified as having a high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma, the subject is further administered a pulmonary disease therapeutic agent and / or an ITGA1 inhibitor as described herein. For example, if the subject is ITGA1 reference and therefore has a high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma, the subject is administered an ITGA1 inhibitor. In some embodiments, such a subject is also administered a pulmonary disease therapeutic agent. In some embodiments, if the subject is heterozygous for ITGA1 variant nucleic acid molecule, the subject is administered a dose of a pulmonary disease therapeutic agent that is the same as or less than the standard dose, and is also administered an ITGA1 inhibitor. In some embodiments, the subject is ITGA1 reference. In some embodiments, the subject is heterozygous for ITGA1 variant nucleic acid molecule. Furthermore, if the subject has a low burden of having ITGA1 variant nucleic acid molecule, and therefore has a low risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD or asthma, the subject is administered a pulmonary disease therapeutic drug.In some embodiments, if the subject has a low burden of having ITGA1 variant nucleic acid molecule, the subject is administered a pulmonary disease therapeutic drug at a dose equal to or higher than the standard dose administered to the subject with a high burden of having ITGA1 variant nucleic acid molecule.

[0128] The nucleotide sequence of the ITGA1 reference genomic nucleic acid molecule is set forth in SEQ ID NO:1. The nucleotide sequence of the ITGA1 reference mRNA molecule is set forth in SEQ ID NO: 2. The nucleotide sequence of another ITGA1 reference mRNA molecule is set forth in SEQ ID NO: 2. The nucleotide sequence of another ITGA1 reference mRNA molecule is set forth in SEQ ID NO: 3. The nucleotide sequence of another ITGA1 reference mRNA molecule is set forth in SEQ ID NO: 5.

[0129] The nucleotide sequence of another ITGA1 reference cDNA molecule is set forth in SEQ ID NO: 6. The nucleotide sequence of another ITGA1 reference cDNA molecule is set forth in SEQ ID NO: 7. The nucleotide sequence of another ITGA1 reference cDNA molecule is set forth in SEQ ID NO: 8. The nucleotide sequence of another ITGA1 reference cDNA molecule is set forth in SEQ ID NO: 9.

[0130] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from any organism.For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from humans, or orthologs from other organisms, such as non-human mammals, rodents, mice, or rats.It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms.The examples provided herein are merely exemplary sequences.Other sequences are also possible.

[0131] Also provided herein is a functional polynucleotide that can interact with the disclosed nucleic acid molecule.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 molecule.

[0132] The isolated nucleic acid molecule or its complement may be present in a host cell. In some embodiments, the host cell may comprise a vector comprising any of the nucleic acid molecules described herein, or their complements. In some embodiments, the nucleic acid molecule is operably linked to a promoter active in the host cell. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell.

[0133] Desired regulatory sequences for mammalian host cell expression may include, for example, viral elements that induce high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer, etc.), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer, etc.), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells (e.g., yeast cells, etc.) are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter, etc.), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter, etc.).

[0134] The percentage of identity (or complementarity) between specific stretches of nucleotide sequences in a nucleic acid molecule or amino acid sequences in a polypeptide can be routinely determined using the BLAST program (basic local alignment 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 by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings using 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.

[0135] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of the numbering of a particular nucleotide or nucleotide sequence or position, refers to the numbering of the specified reference sequence when the particular nucleotide or nucleotide sequence is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:6, etc.). In other words, the residue (e.g., nucleotide or amino acid, etc.) number or residue (e.g., nucleotide or amino acid, etc.) position of a particular polymer is specified with reference to the reference sequence, not by the actual numerical position of the residue within a 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 a particular nucleotide or nucleotide sequence is done with respect to the reference sequence to which the nucleotide or nucleotide sequence is aligned.

[0136] As described herein, for example, the position in the ITGA1 variant genomic nucleic acid molecule corresponding to the variant position can be identified by performing sequence alignment between the nucleotide sequence of a particular ITGA1 nucleic acid molecule and the nucleotide sequence of the variant nucleic acid molecule. For example, there are various computational algorithms that can be used to perform sequence alignment to identify the nucleotide position corresponding to the variant position of the ITGA1 variant nucleic acid molecule. For example, sequence alignment can be performed 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 manually aligned.

[0137] The amino acid sequences of the ITGA1 reference polypeptide are set forth in SEQ ID NO: 10 (isoform 1) and SEQ ID NO: 11 (isoform 2). With reference to SEQ ID NO: 10 (isoform 1), the ITGA1 reference polypeptide is 1,179 amino acids in length. With reference to SEQ ID NO: 11 (isoform 2), the ITGA1 reference polypeptide is 1,173 amino acids in length.

[0138] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter code for amino acids. The nucleotide sequences follow the standard convention of proceeding from a beginning at the 5'-end of the sequence 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 proceeding in the amino- to carboxy-terminus direction of the sequence (i.e., from left to right on each line).

[0139] The disclosure also provides a pulmonary disease therapeutic for use in treating fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma in a subject having any of the ITGA1 variant genomic nucleic acid molecules, variant mRNA molecules, and / or variant cDNA molecules described herein (or for use in preparing a medicament for treating fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma). The pulmonary disease therapeutic can be any of the pulmonary disease therapeutics described herein.

[0140] In some embodiments, the subject is identified as having an ITGA1 variant genomic nucleic acid molecule, and the genomic variant nucleic acid molecule has a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0141] In some embodiments, the subject is identified as having an ITGA1 variant mRNA molecule, wherein the mRNA molecule has a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0142] In some embodiments, the subject is identified as having an ITGA1 variant cDNA molecule, wherein the cDNA molecule has a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0143] In some embodiments, the subject is identified as having i) an ITGA1 variant genomic nucleic acid molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement, ii) an mRNA molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement, or iii) a cDNA molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0144] In some embodiments, the subject is identified as having an ITGA1 variant genomic nucleic acid molecule having a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0145] In some embodiments, the subject is identified as having i) an ITGA1 variant mRNA molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0146] In some embodiments, the subject is identified as having i) an ITGA1 variant cDNA molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0147] In some embodiments, the subject is identified as having a predicted loss-of-function polypeptide of ITGA1 that comprises an amino acid sequence that includes a mutation encoded by any of the ITGA1 variant nucleic acid molecules described herein.

[0148] The present disclosure also provides an ITGA1 inhibitor for use in treating fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma in a subject that is heterozygous for or references to an ITGA1 variant genomic nucleic acid molecule, a variant mRNA molecule, and / or a variant cDNA molecule (or for use in preparing a medicament for treating fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma). The ITGA1 inhibitor may be any of the ITGA1 inhibitors described herein.

[0149] In some embodiments, the subject is a reference for the ITGA1 genomic nucleic acid molecule, the ITGA1 mRNA molecule, or the ITGA1 cDNA molecule. In some embodiments, the subject is a reference for the ITGA1 genomic nucleic acid molecule. In some embodiments, the subject is a reference for the ITGA1 mRNA molecule. In some embodiments, the subject is a reference for the ITGA1 cDNA molecule.

[0150] In some embodiments, the subject is identified as heterozygous for an ITGA1 variant genomic nucleic acid molecule, and the genomic nucleic acid molecule has a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0151] In some embodiments, the subject is identified as heterozygous for an ITGA1 variant mRNA molecule, wherein the mRNA molecule has a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0152] In some embodiments, the subject is identified as heterozygous for an ITGA1 variant cDNA molecule, and the cDNA molecule has a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0153] In some embodiments, the subject is identified as being heterozygous for i) an ITGA1 variant genomic nucleic acid molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement, ii) an ITGA1 variant mRNA molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement, or iii) an ITGA1 variant cDNA molecule having a nucleotide sequence that includes a nucleotide at a position corresponding to the nucleotide at the variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0154] In some embodiments, the subject is identified as heterozygous for an ITGA1 variant genomic nucleic acid molecule having a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0155] In some embodiments, the subject is identified as heterozygous for an ITGA1 variant mRNA molecule having a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0156] In some embodiments, the subject is identified as heterozygous for an ITGA1 variant cDNA molecule having a nucleotide sequence that includes a nucleotide at a position that corresponds to a nucleotide at a variant position of the ITGA1 variant nucleic acid molecule or its complement.

[0157] 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 item 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 that refers to the accession number, if applicable. Similarly, where different versions of publications, websites, etc. are 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 made within the scope of the appended claims.

[0158] 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 ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0159] Example 1 Association between loss-of-function variants of ITGA1 and improved lung function We examined the association of common and rare variants with three lung function traits: forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and the FEV1 / FEV1 ratio, a measure of airway obstruction. We showed an association between rare (low allele frequency <1%) predicted loss-of-function variants in ITGA1 and improved lung function (higher FEV1 / FEV1 ratio, Figure 1). We also observed a small association trending toward protection from asthma and COPD, suggesting that loss of ITGA1 may improve lung function and confer protection in asthma / COPD or other fibrotic lung diseases (Figure 2).

[0160] 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 for all purposes.

Claims

1. An in vitro method for assessing a subject's risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease (COPD), or asthma, comprising: determining the presence or absence of an integrin subunit alpha 1 (ITGA1) variant nucleic acid molecule in a biological sample obtained from the subject; if the subject is ITGA1 reference, it indicates that the subject is at high risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma; The method, wherein if the subject is heterozygous or homozygous for an ITGA1 variant nucleic acid molecule, the subject is at low risk of developing fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, COPD, or asthma.

2. The ITGA1 variant nucleic acid molecule is 5:52920430:C:T, 5:52944942:G:T, 5:52910243:G:T, 5:52918899:G:C, 5:52918730:A:T, 5:52952404:CA:C, 5:52861561:T:C, 5:52939693:T:C, 5:52910201:C:T, 5:52788416:T:C, 5:52788356:G:A, 5:52893783:G:T, 5:52920332:AT:A, 5:52952436:GA:G, 5:52939945:G:T, 5:52925371:C:T, 5:52939690:TG:T, 5:52898385:T:C, 5:52897529:GT:G, 5:52937490:C:G, 5:52947412:CT:C, 5:52882022:G:T, 5:52939946:T:A, 5:52861503:A:AT, 5:52881929:T:G, 5:52932134:C:A, 5:52939933:AC:A, 5:52933993:C:G, 5:52927645:T CCT:T, 5:52939934:CT:C, 5:52893841:G:C, 5:52881939:G:T, 5:52932134:C:G, 5:52864814:GA:G, 5:52849381:C:A, 5:52922818:CT:C, 5:52925286:TG:T, 5:52865083:G:A, 5:52939606:C:CAT, 5:52915509:C:CGTGGGTGA, 5:52865083:G:T, 5:52944999:ATC:A, 5:52910194:TA:T, 5:52947462:G:A, 5:52898323:C:T, 5:52945006:GT:G, 5:52910292:CT:C, 5:52861560:G:A, 5:52939642:G:GA, 5:52864979:GC:G, 5:52927590:CA:C, 5:52918814:G:T, 5:52788351:G:GC, 5:52920469:G:A, 5:52905808:A:AT, 5:52922876:GT:G, 5:52861501:C:CT, 5:52865771:T:A, 5:52947456:TGG:T, 5:52918736:C:T, 5:52920376:C:T, 5:52915496:G:GA,5:52898346:GTC:G, 5:52947462:G:T, 5:52920444:C:A, 5:52882022:G:A, 5:52927640:TC:T, 5:52925470:G:GT, 5:52952430:GA:G, 5:52937516:T:C, 5:52915570:G:GC, 5:52861530:C:A, 5:52932046:G:A, 5:52893841:G:T, 5:52887965:TGTAA:T, 5:52937399:AG:A, 5:52887863:AG:A, 5:52905910:T:A, 5:52925304:TA:T, 5:52910222:C:T, 5:52887906:C:T, 5:52910199:AG:A, 5:52909042:G:A, 5:52898269:TA:T, 5:52939588:A:C, 5:52937515:G:GT, 5:52865745:C:A, 5:52937474:GT:G, 5:52925299:G:T, 5:52920367:CA:C, 5:52898296:CAG:C, 5:52925283:CT:C, 5:52893817:GAGAA:G, 5:52915565:TG:T, 5:52933892:A:T, 5:52915524:G:T, 5:52887846:C:T, 5:52937515:G:A, 5:52929670:GA:G, 5:52915463:G:A, 5:52905900:G:T, 5:52865688:AG:A, 5:52933993:C:A, 5:52932137:G:A, 5:52788355:T:C, 5:52897529:G:A, 5:52947344:G:A, 5:52910417:CAAGT:C, 5:52864772:C:A, 5:52939677:C:T, 5:52918807:C:A, 5:52915502:TTTTGG:T, 5:52893693:C:T, 5:52939855:A:T, 5:52925489:T:C, 5:52939673:C:CA, 5:52887966:G:T, 5:52918877:GA:G, and 5:52947463:T:C, the method according to claim 1, selected from.

3. A drug for treating pulmonary diseases, comprising: an ITGA1 variant genomic nucleic acid molecule that encodes an integrin subunit alpha 1 (ITGA1) polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function, or that results in complete loss or reduced expression of ITGA1 mRNA or polypeptide; or A pulmonary disease therapeutic agent for use in treating a pulmonary disease in a subject having an ITGA1 variant mRNA molecule that encodes an ITGA1 polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function, or that results in complete loss or reduced expression of ITGA1 mRNA or polypeptide.

4. a) refers to an integrin subunit alpha 1 (ITGA1) genomic nucleic acid molecule or an ITGA1 mRNA molecule; or b) i) an ITGA1 variant genomic nucleic acid molecule that encodes an ITGA1 polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function, or that results in complete loss or reduced expression of ITGA1 mRNA or polypeptide; or ii) An ITGA1 inhibitor for use in treating fibrotic lung disease, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease (COPD), or asthma in a subject heterozygous for an ITGA1 variant mRNA molecule that encodes an ITGA1 polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function, or that results in complete loss of or reduced expression of ITGA1 mRNA or polypeptide.

5. The ITGA1 inhibitor of claim 4, which is an inhibitory nucleic acid molecule.

6. The ITGA1 inhibitor of claim 5, 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 to an ITGA1 nucleic acid molecule.

7. The ITGA1 inhibitor of claim 4, comprising a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an ITGA1 genomic nucleic acid molecule.

8. The ITGA1 inhibitor of claim 7, wherein the Cas protein is Cas9 or Cpf1.

9. The ITGA1 inhibitor of claim 7, wherein the gRNA recognition sequence includes or is adjacent to the start codon of an ITGA1 genomic nucleic acid molecule or the stop codon of an ITGA1 genomic nucleic acid molecule.

10. The ITGA1 inhibitor of claim 7, wherein the gRNA recognition sequence is located at 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 from the position corresponding to the start codon of the ITGA1 genomic nucleic acid molecule or the stop codon of the ITGA1 genomic nucleic acid molecule.

11. The ITGA1 inhibitor of claim 7, wherein a protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

12. The ITGA1 inhibitor according to any one of claims 7 to 11, wherein the gRNA comprises about 17 to about 23 nucleotides.

13. The ITGA1 inhibitor according to any one of claims 7 to 11, wherein the gRNA recognition sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 12 to 31. **Claim 14**: The ITGA1 variant nucleic acid molecule is 5:52920430:C:T, 5:52944942:G:T, 5:52910243:G:T, 5:52918899:G:C, 5:52918730:A:T, 5:52952404:CA:C, 5:52861561:T:C, 5:52939693:T:C, 5:52910201:C:T, 5:52788416:T:C, 5:52788356:G:A, 5:52893783:G:T, 5:52920332:AT:A, 5:52952436:GA:G, 5:52939945:G:T, 5:52925371:C:T, 5:52939690:TG:T, 5:52898385:T:C, 5:52897529:GT:G, 5:52937490:C:G, 5:52947412:CT:C, 5:52882022:G:T, 5:52939946:T:A, 5:52861503:A:AT, 5:52881929:T:G, 5:52932134:C:A, 5:52939933:AC:A, 5:52933993:C:G, 5:52927645:TCCCT:T, 5:52939934:CT:C, 5:52893841:G:C, 5:52881939:G:T, 5:52932134:C:G, 5:52864814:GA:G, 5:52849381:C:A, 5:52922818:CT:C, 5:52925286:TG:T, 5:52865083:G:A, 5:52939606:C:CAT, 5:52915509:C:CGTGGGTGA, 5:52865083:G:T, 5:52944999:ATC:A, 5:52910194:TA:T, 5:52947462:G:A, 5:52898323:C:T, 5:52945006:GT:G, 5:5295:52898346:GTC:G, 5:52947462:G:T, 5:52920444:C:A, 5:52882022: G:A, 5:52927640:TC:T, 5:52925470:G:GT, 5:52952430:GA:G, 5:5293 7516:T:C, 5:52915570:G:GC, 5:52861530:C:A, 5:52932046:G:A, 5:5 2893841:G:T, 5:52887965:TGTAA:T, 5:52937399:AG:A, 5:52887863: AG:A, 5:52905910:T:A, 5:52925304:TA:T, 5:52910222:C:T, 5:52887 906:C:T, 5:52910199:AG:A, 5:52909042:G:A, 5:52898269:TA:T, 5:5 2939588:A:C, 5:52937515:G:GT, 5:52865745:C:A, 5:52937474:GT:G , 5:52925299:G:T, 5:52920367:CA:C, 5:52898296:CAG:C, 5:52925283 :CT:C, 5:52893817:GAGAA:G, 5:52915565:TG:T, 5:52933892:A:T, 5: 52915524:G:T, 5:52887846:C:T, 5:52937515:G:A, 5:52929670:GA:G , 5:52915463:G:A, 5:52905900:G:T, 5:52865688:AG:A, 5:52933993: C:A, 5:52932137:G:A, 5:52788355:T:C, 5:52897529:G:A, 5:5294734 4:G:A, 5:52910417:CAAGT:C, 5:52864772:C:A, 5:52939677:C:T, 5:52918807:C:A, 5:52915502:TTTTGG:T, 5:52893693:C:T, 5:52939855:A:T, 5:52925489:T:C, 5:52939673:C:CA, 5:52887966:G:T, 5:52918877:GA:G, or 5:52947463:T:C.

15. The pulmonary disease therapeutic agent described in claim 3 or 14, wherein the pulmonary disease therapeutic agent comprises nintedanib, pirfenidone, prednisone, azathioprine, cyclophosphamide, mycophenolate mofetil, rituximab, tacrolimus, cotrimoxazole, lebrikizumab, nandrolone decanoate, sirolimus, thalidomide, or pomalidomide.

16. The ITGA1 variant genomic nucleic acid molecule or ITGA1 variant mRNA molecule is selected from the group consisting of 5:52920430:C:T, 5:52944942:G:T, 5:52910243:G:T, 5:52918899:G:C, 5:52918730:A:T, 5:52952404:CA:C, 5:52861561:T:C, 5:52939693:T:C, 5:52910201:C:T, 5:52788416:T:C, 5:52788356:G:A, 5:52893783:G:T, 5:52920332:AT:A, 5:52952 436:GA:G, 5:52939945:G:T, 5:52925371:C:T, 5:52939690:TG:T, 5:5289 8385:T:C, 5:52897529:GT:G, 5:52937490:C:G, 5:52947412:CT:C, 5:528 82022:G:T, 5:52939946:T:A, 5:52861503:A:AT, 5:52881929:T:G, 5:529 32134:C:A, 5:52939933:AC:A, 5:52933993:C:G, 5:52927645:TCCTG:T, 5: 52939934:CT:C, 5:52893841:G:C, 5:52881939:G:T, 5:52932134:C:G, 5: 52864814:GA:G, 5:52849381:C:A, 5:52922818:CT:C, 5:52925286:TG:T, 5:52865083:G:A, 5:52939606:C:CAT, 5:52915509:C:CGTGGTGA, 5:52865 083:G:T, 5:52944999:ATC:A, 5:52910194:TA:T, 5:52947462:G:A, 5:5289 8323:C:T, 5:52945006:GT:G, 5:52910292:CT:C, 5:52861560:G:A, 5:529 39642:G:GA, 5:52864979:GC:G, 5:52927590:CA:C, 5:52918814:G:T, 5:5 2788351:G:GC, 5:52920469:G:A, 5:52905808:A:AT, 5:52922876:GT:G, 5 :52861501:C:CT, 5:52865771:T:A, 5:52947456:TGG:T, 5:52918736:C:T,The ITGA1 inhibitor according to claim 4, comprising a gene variation present in 5:52920376: C: T, 5:52915496: G: GA, 5:52898346: GTC: G, 5:52947462: G: T, 5:52920444: C: A, 5:52882022: G: A, 5:52927640: TC: T, 5:52925470: G: GT, 5:52952430: GA: G, 5:52937516: T: C, 5:52915570: G: GC, 5:52861530: C: A, 5:52932046: G: A, 5:52893841: G: T, 5:52887965: TGTA A: T, 5:52937399: AG: A, 5:52887863: AG: A, 5:52905910: T: A, 5:52925304: TA: T, 5:52910222: C: T, 5:52887906: C: T, 5:52910199: AG: A, 5:52909042: G: A, 5:52898269: TA: T, 5:52939588: A: C, 5:52937515: G: GT, 5:52865745: C: A, 5:52937474: GT: G, 5:52925299: G: T, 5:52920367: CA: C, 5:52898296: CAG: C, 5:52925283: CT: C, 5:52893817: GAGAA: G, 5:52915565: TG: T, 5:52933892: A: T, 5:52915524: G: T, 5:52887846: C: T, 5:52937515: G: A, 5:52929670: GA: G, 5:52915463: G: A, 5:52905900: G: T, 5:52865688: AG: A, 5:52933993: C: A, 5:52932137: G: A, 5:52788355: T: C, 5:52897529: G: A, 5:52947344: G: A, 5:52910417: CAAGT: C, 5:52864772: C: A, 5:52939677: C: T, 5:52918807: C: A, 5:52915502: TTTTG G: T, 5:52893693: C: T, 5:52939855: A: T, 5:52925489: T: C, 5:52939673: C: CA, 5:52887966: G: T, 5:52918877: GA: G, or 5:52947463: T: C.

17. An ITGA1 inhibitor as described in claim 4, used in combination with a drug for treating pulmonary diseases.

18. The ITGA1 inhibitor described in claim 17, wherein the pulmonary disease treatment drug comprises nintedanib, pirfenidone, prednisone, azathioprine, cyclophosphamide, mycophenolate mofetil, rituximab, tacrolimus, cotrimoxazole, lebrikizumab, nandrolone decanoate, sirolimus, thalidomide, or pomalidomide.

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