# US 2018/0023140 A1 · Articles for diagnosis of liver fibrosis

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- Number: US 2018/0023140 A1
- Document ID: US20180023140A1
- Status: Published application
- Kind: A1
- Application number: 15/547,168
- Filed: 2016-01-29
- Published: 2018-01-25
- Earliest claimed: 2016-01-29
- Assignees: The Government of the United States, as represented by the Secretary of the Army
- Applicants: THE GOVERNMENT OF THE UNITED STATES, as represented by THE SECRETARY OF THE ARMY
- Inventors: Mohamed Diwan M. ABDULHAMEED; Gregory J. TAWA; Danielle L. IPPOLITO; John A. LEWIS; Anders WALLQVIST; Jonathan D. STALLINGS; Matthew G. PERMENTER
- Web page: https://patentgrep.com/patent/US20180023140A1

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/68: involving nucleic acids
- G01N 33/5014: {for testing toxicity}
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/6893: {related to diseases not provided for elsewhere}
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q 2600/142: Toxicological screening, e.g. expression profiles which identify toxicity
- C12Q 2600/158: Expression markers
- G01N 2800/085: Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
- G01N 2800/56: Staging of a disease; Further complications associated with the disease
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Disclosed are methods and articles (e.g., gene arrays or antibodies) for determining the progression or regression of liver fibrosis, for the diagnosis of liver disease, and for screening compounds for hepatotoxicity and efficacy against liver fibrosis. Related therapeutic methods also are disclosed.

## Claims

### Claim 1

An in vitro method for detecting early stage liver disease in a mammalian subject, comprising: assaying a biological sample from a subject for differential expression of at least twenty-five genes selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Col1a1; Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-DMd; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; It gal; Plod2; Ctgf Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP; Slc25a24; Fbn1; Col4al; Sod2; Cyp2cl1; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vin; Vh1, and orthologs thereof corresponding to the subject, and orthologs thereof corresponding to the subject, wherein the differential expression of the at least twenty-five genes is indicative of early stage liver disease in the subject.

### Claim 2 (depends on 1)

The method of claim 1, comprising assaying the biological sample from the subject for differential expression of at least thirty genes.

### Claim 3 (depends on 1)

The method of claim 1, wherein the subject is a rat, mouse, guinea pig, pig, rabbit, dog, cat, cow, horse, or human.

### Claim 4 (depends on 3)

The method of claim 3, wherein the subject is a human.

### Claim 5 (depends on 1)

The method of claim 1, wherein increased expression of each of the at least twenty-five genes by at least +0.5-fold (log2) as compared to a control is indicative of early stage liver disease.

### Claim 6 (depends on 5)

The method of claim 5, wherein differential expression of at least one of the at least twenty-five genes by at least +0.6 fold (log2) as compared to a control is indicative of early stage liver disease.

### Claim 7 (depends on 1)

The method of claim 1, wherein increased expression of a gene selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Col1a1; Cxc116; Timp1; Plat RT1-D1Ma; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Cgf; Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP; Slc25a24, and orthologs thereof corresponding to the subject, is indicative of early stage liver disease.

### Claim 8 (depends on 1)

The method of claim 1, wherein increased expression of a gene selected from the group consisting of Lcn2; Gpnmb; Lgals3 S100A11; Anxa2; Lbp; Col1a1; Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Ctgf; Col1a2; Igfbp2, and orthologs thereof corresponding to the subject, is indicative of early stage liver disease.

### Claim 9 (depends on 1)

The method of claim 1, wherein increased expression of a gene selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Col1a1, Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce, and orthologs thereof corresponding to the subject, is indicative of early stage liver disease.

### Claim 10 (depends on 1)

The method of claim 1, wherein the method further comprises assaying one or more genes selected from the group consisting of: Cyp2c11; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, and orthologs thereof corresponding to the subject, for which decreased expression of the one or more genes is indicative of early stage liver disease.

### Claim 11 (depends on 1)

The method of claim 1, wherein the method detects early stage liver disease with a sensitivity of at least 70%.

### Claim 12 (depends on 1)

The method of claim 1, wherein the method detects early stage liver disease with at least 70% specificity.

### Claim 13 (depends on 1)

The method of claim 1, wherein the biological sample is selected from the group consisting of blood, plasma, serum, urine, or a liver biopsy.

### Claim 14 (depends on 1)

The method of claim 1, wherein differential expression is determined by quantification of the levels of the proteins encoded by the genes recited in claim 1.

### Claim 15 (depends on 14)

The method of claim 14, wherein quantification of the levels of the proteins comprises assaying a sample by Western blotting, ELISA or mass spectrometry.

### Claim 16 (depends on 1)

The method of claim 1, wherein the differential expression is determined by quantification of the levels of the gene regulator which correspond to the genes recited in claim 1.

### Claim 17 (depends on 1)

The method of claim 1, wherein differential expression is determined by quantification of the levels of mRNA corresponding to the at least twenty-five genes.

### Claim 18 (depends on 17)

The method of claim 17, wherein quantification of the levels of the mRNA comprises incubating the mRNA, or corresponding cDNA thereof, with an oligonucleotide array.

### Claim 19 (depends on 18)

The method of claim 18, wherein the mRNA or corresponding cDNA thereof, is hybridized to the oligonucleotide array by interaction with a capture probe, wherein the hybridization results in a target:capture probe pair.

### Claim 20 (depends on 19)

The method of claim 19, wherein the target:capture probe pair is labeled with a biotinylated label probe.

### Claim 21 (depends on 20)

The method of claim 20, wherein streptavidin-conjugated phycoerythrin (SAPE) is bound to the biotinylated label probe.

### Claim 22 (depends on 1)

The method of claim 1, wherein differential expression is determined by quantification of the levels of miRNA corresponding to the at least twenty-five genes.

### Claim 23

A method for detecting progression or regression of liver fibrosis in a mammalian subject, comprising detecting differential expression of at least twenty-five genes selected from the group consisting of: Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Col1a1; Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Ctgf; Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP; Slc25a24; Fbn1; Col4a1; Sod2; Cyp2cl1; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, and orthologs thereof corresponding to the subject, in a biological sample from the subject.

### Claim 23 (depends on 22)

The method of claim 22, comprising detecting differential expression of at least 30 genes selected from the group of genes recited in claim 22.

### Claim 24 (depends on 23)

The method of claim 23, wherein differential expression is determined by quantification of the levels of the proteins encoded by the genes recited in claim 23.

### Claim 24

A method of identifying a compound that increases or decreases the differential expression of at least one gene associated with hepatotoxicity, comprising
(i) providing a cell expressing at least one gene selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11: Anxa2; Lbp; Col1a1; Cxc116; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkrn2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Ctgf; Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP, Slc25a24; Fbn1; Col4a1; Sod2; Cyp2c11; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, Cyp2c11; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, and mammalian orthologs thereof;
(ii) contacting the cell with a test compound; and
(iii) determining whether the differential expression of the at least one gene is increased or decreased in the presence of the test compound.

### Claim 25 (depends on 24)

The method of claim 24, wherein quantification of the levels of the proteins comprises assaying a sample by Western blotting, ELISA or mass spectrometry.

### Claim 25 (depends on 24)

The method of claim 24, wherein differential expression is determined by quantification of the levels of the proteins encoded by the genes recited in claim 24.

### Claim 26 (depends on 23)

The method of claim 23, wherein the differential expression is determined by quantification of the levels of the gene regulator which correspond to the genes recited in claim 23.

### Claim 26 (depends on 25)

The method of claim 25, wherein quantification of the levels of the proteins comprises assaying a sample by Western blotting, ELISA or mass spectrometry.

### Claim 27 (depends on 24)

The method of claim 24, wherein the differential expression is determined by quantification of the levels of the gene regulator which correspond to the genes recited in claim 24

### Claim 28 (depends on 24)

The method of claim 24, wherein the at least one gene is selected from the group consisting of: Dsc2; Fam102b; Fam105a; Gpnmb; Pcolce; RT1-Da; RT1-DMa; Slc25a24; Tagin2; Arpc1b; Angptl3; Anxa2; Cd53; Cd9; Cp; C1qb; Fxyd5; Igfbp2; Igfbp3; Igfals; Lamac2; Lgals1; Lgals3; Lrp1; Plod2; Pkm; S100a11; S00a6; Serpine1; Serping1; Vim, and mammalian orthologs thereof.

### Claim 29 (depends on 24)

A method of treating liver disease, comprising administering to a mammalian subject in need thereof, a therapeutically effective amount of a compound identified by the method of claim 24 to decrease expression of at least one gene selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Col1a1; Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkm2; Ce12; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Ctgf; Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP; Slc25a24; Fbn1; Col4a1; Sod2; Cyp2c11; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, and orthologs thereof corresponding to the subject.

### Claim 30

An array comprising at least twenty-five target oligonucleotides immobilized on a substrate, wherein each target oligonucleotide comprises a sequence that is specifically hybridizable to mRNA, cDNA or miRNA corresponding to one of at least twenty-five separate genes selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Col1a1; Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Ctgf, Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP; Slc25a24; Fbn1; Col4al; Sod2; Cyp2c11; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, and mammalian orthologs thereof, such that the array comprises at least one target oligonucleotide specifically hybridizable to each of the at least twenty-five genes.

### Claim 31 (depends on 30)

The array of claim 30, wherein the target oligonucleotides are labelled with a detectable label.

### Claim 32 (depends on 31)

The array of claim 31, wherein the target oligonucleotides comprise cDNA-specific sequences that each comprise at least one nucleotide difference from corresponding genomic DNA.

### Claim 33 (depends on 31)

The array of claim 31, wherein the target oligonucleotides are labelled directly or indirectly with a detectable label.

### Claim 34 (depends on 31)

The array of claim 31, wherein the detectable label comprises biotin.

### Claim 35 (depends on 34)

The array of claim 34, wherein streptavidin-conjugated phycoerythrin (SAPE) is bound to the biotin.

### Claim 36 (depends on 30)

The array of claim 30, wherein the target oligonucleotide is immobilized on the substrate due to binding with a capture probe.

### Claim 37

A kit for the diagnosis of liver disease, comprising at least twenty-five detectably labelled oligonucleotides, wherein each oligonucleotides comprises a sequence that is specifically hybridizable to mRNA, cDNA or miRNA corresponding to one of at least twenty-five separate genes selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Col1a1; Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-DMd; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Ctgf; Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP; Slc25a24; Fbn1; Col4a1; Sod2; Cyp2cl1; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, and mammalian orthologs thereof, such that the kit comprises at least one detectably labelled oligonucleotide specifically hybridizable to mRNA, cDNA or miRNA corresponding to each of the at least twenty-five genes.

### Claim 38

A kit for the diagnosis of liver disease, comprising at least twenty-five antibodies, or antigen-binding fragments thereof, wherein each antibody or fragment thereof is capable of specifically binding to one of at least twenty-five separate proteins encoded by genes selected from the group consisting of Lcn2; Gpnmb; Lgals3; S100A11; Anxa2; Lbp; Collal; Cxcl16; Timp1; Plat; RT1-DMa; CD9; Lgals1; Tagin2; RT1-Da; Capg; Pkm2; Ccl2; Serpine1; Cyba; Fam105a; Cxcl1; Vim; C1qb; Pcolce; Fxyd5; Arpc1b; S100A6; Plau; Itgal; Plod2; Ctgf; Col1a2; Igfbp2; Lox; Fam102b; CD53; Itgb2; Lamc2; CP; Slc25a24; Fbn1; Col4a1; Sod2; Cyp2c11; Igfbp3; Cxcl12; Angptl3; Igfals; Tgfbi; Vtn; Vh1, and mammalian orthologs thereof, such that the kit comprises at least one antibody or antigen-binding fragment thereof that specifically binds to each of the at least twenty-five separate proteins.

### Claim 39

A method of detecting expression of at least one gene in a biological sample from a mammalian subject suspected of having or determined to be at risk of developing liver fibrosis or liver disease, comprising quantifying levels of mRNA, cDNA or miRNA corresponding to at least one gene selected from the group consisting of Dsc2; Fam102b; Fam105a; Gpnmb; Pcolce; RT1-Da; RT1-DMA; Slc25a24; Tagin2; Arpc1b; Angptl3; Anxa2; Cd53; Cd9; Cp; C1qb; Fxyd5; Igfbp2; Igfbp3; Igfals; Lamac2; Lgals1; Lgals3; Lrp1; Plod2; Pkm; S100a11; S00a6; Serpine1; Serping′; Vim, and orthologs thereof corresponding to the subject.