# US 2013/0160150 A1 · Methods for identifying compounds that modulate lisch-like protein or c1orf32 protein activity and methods of use

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- Number: US 2013/0160150 A1
- Document ID: US20130160150A1
- Status: Published application
- Kind: A1
- Application number: 12/747,866
- Filed: 2008-12-12
- Published: 2013-06-20
- Earliest claimed: 2007-12-12
- Assignees: The Trustees of Columbia University in the City of New York
- Inventors: Rudolph L. Leibel; Marija Dokmanovic-Chouinard; Wendy K. Chung; Charles LeDuc; Stuart G. Fischer
- Web page: https://patentgrep.com/patent/US20130160150A1

## CPC

- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 2310/11: Antisense
- C12N 2310/3233: Morpholino-type ring
- C12N 2330/10: naturally occurring
- G01N 2800/042: Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides methods for reducing diabetes susceptibility in a subject and methods for increasing the expression of LL or CLORF32 in a subject. The invention further provides a method for identifying an agent which modulates expression of an Ll RNA or Clorf32 RNA comprising contacting a cell with an agent; determining expression of the Ll RNA or Clorf32 RNA in the presence and the absence of the agent; and comparing expression of the Ll RNA or Clorf32 RNA in the presence and the absence of the agent, wherein a change in the expression of the Ll RNA or Clorf32 RNA in the presence of the agent is indicative of an agent which modulates the level of expression of the RNA.

## Claims

### Claim 1

A method for identifying an agent which modulates expression of a murine Ll RNA , the method comprising:
a) contacting a murine cell with an agent, wherein the cell contains an L1 gene;
b) determining expression of Ll RNA in the cell in the presence and absence of the agent; and
c) comparing expression of Ll RNA in the cell in the presence and absence of the agent, wherein a change in the expression of the Ll RNA in the presence of the agent is indicative of an agent which modulates the level of expression of the RNA.

### Claim 2

A method for identifying an agent which modulates expression of a human C1orf32 RNA , the method comprising:
a) contacting a human cell with an agent, wherein the cell contains a C1orf32 gene;
b) determining expression of the C1orf32 RNA in the cell in the presence and the absence of the agent; and
c) comparing expression of the C1orf32 RNA in the cell in the presence and the absence of the agent, wherein a change in the expression of the C1orf32 RNA in the presence of the agent is indicative of an agent which modulates the level of expression of the RNA.

### Claim 3

A method for identifying an agent which modulates expression of an mRNA encoding a murine LL protein, or a fragment or an isoform thereof, the method comprising:
a) contacting a cell with an agent;
b) determining expression of the mRNA in the presence and the absence of the agent, and
c) comparing the expression of the mRNA in the presence or the absence of the agent, wherein a change in the expression of the mRNA encoding LL protein in the presence of the agent is indicative of an agent which modulates the expression of the mRNA.

### Claim 4

A method for identifying an agent which modulates expression level of an mRNA encoding the protein encoded by human C1ORF32 gene, the method comprising:
a) contacting a cell expressing the C1ORF32 gene with an agent;
b) determining expression levels of mRNA encoded by C1ORF32 in the presence and the absence of the agent; and
c) comparing the expression level of the mRNA in the presence and the absence of the agent, wherein a change in the level of expression of the mRNA encoding C1ORF32 in the presence of the agent is indicative of an agent which modulates the expression level of the mRNA.

### Claim 5

A method for identifying an agent which modulates expression of murine Ll RNA , the method comprising:
a) contacting a cell expressing L1 RNA with an agent;
b) determining expression of an antisense RNA in the presence and the absence of the agent, wherein the antisense RNA comprises the sequence shown in SEQ ID NO: 18, 19 or 20; and
c) comparing the expression of the antisense RNA in the presence and the absence of the agent, wherein a change in the expression of the antisense RNA is indicative of an agent which modulates the expression of the Ll RNA.

### Claim 6

A method for identifying an agent which modulates expression of C1orf32 RNA, the method comprising:
a) contacting a cell expressing C1ORF32 RNA with an agent;
b) determining expression of an antisense RNA in the presence and the absence of the agent, wherein the antisense RNA comprises the sequence shown in SEQ ID NO: 68, 73 or 74; and
c) comparing the expression of the antisense RNA in the presence and the absence of the agent, wherein an a change in the expression of the antisense RNA is indicative of an agent which modulates the of expression of the C1orf32 RNA.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The method of any of claims 1-6, wherein determining the expression comprises determining stability of RNA, determining level of RNA expression, determining level of expression of a type of C1ORF32 or LL RNA isoform or any combination thereof.

### Claim 8

A method for identifying an agent which modulates expression of an LL murine protein, the method comprising:
a) contacting a cell expressing the LL protein with an agent;
b) determining expression of the LL protein in the presence and the absence of the agent; and
c) comparing the expression of the LL protein in the presence or the absence of the agent, wherein a change in the expression of the LL protein in the presence of the agent is indicative of an agent which modulates the expression of the LL protein.

### Claim 9

A method for identifying an agent which modulates expression of human C1ORF32 protein, the method comprising:
a) contacting a cell expressing human C1ORF32, with an agent;
b) determining expression of the human C1ORF32 protein in the presence and absence of the agent; and
c) comparing the expression of the human C1ORF32 protein in the presence and absence of the agent, wherein a change in the expression of the C1ORF32 protein in the presence of the agent is indicative of an agent which modulates the expression of the human C1ORF32 protein.

### Claim 10 (depends on 8, 9)

The method of claim 8 or 9, wherein the LL protein or the C1ORF32 protein comprises a label.

### Claim 11 (depends on 10)

The method of claim 10, wherein the label comprises a fluorescent label.

### Claim 12 (depends on 11)

The method of claim 11, wherein the fluorescent label comprises a Green, Yellow, Cyanne, Cherry, Fluorescent Protein or any variant thereof.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 1-9, wherein the change is an increase.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 1-9, wherein the change is a decrease.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 1-9, wherein the change is transient.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 1-9, wherein the change is in localization, stability, modification, processing, posttranslational modification, or any combination thereof.

### Claim 17 (depends on 1, 2, 5, 6)

The method of any one of claims 1, 2, 5 and 6, wherein the Ll RNA or the C1orf32 RNA is endogenous.

### Claim 18 (depends on 3, 4, 8, 9)

The method of any of claim 3, 4, 8 or 9, wherein the LL RNA or protein or the C1ORF3 RNA or protein is endogenous.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 1-9, wherein the cell is transfected with a nucleic acid comprising the nucleic acid of any of SEQ ID NO: 10-13, 15-20 or a nucleic acid which is at least 75% homologous to any of SEQ ID NO: 10-13, 15-20.

### Claim 20 (depends on 19)

The method of claim 19, wherein the cell comprises a fluorescently labeled C1ORF32.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any of claims 1-9, wherein the cell is transfected with a nucleic acid comprising the nucleic acid of C1orf32 cDNA sequence or genomic sequence, with regulatory elements or a nucleic acid which is at least 75% homologous to same.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any of claims 1-9, wherein the cell is derived from a diabetes-relevant tissue.

### Claim 23 (depends on 22)

The method of claim 22, wherein the tissue comprises liver, pancreatic islet, skeletal muscle, brain, adipose tissue, or combination thereof.

### Claim 24 (depends on 22)

The method of claim 22, wherein the cell comprises a pancreatic cell, a β-cell or an islet of Langerhans cell.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any of claims 1-9, wherein the cell comprises an insulin producing beta cell, a hepatocyte cell, or a hypothalamic cell.

### Claim 26 (depends on 22)

The method of claim 22, wherein the cell comprises a murine cell, a rat cell, or a human cell.

### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any of claims 1-9, wherein the method is performed in vivo or in vitro.

### Claim 28

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO: 6 or an isolated peptide which is at least 75% identical to SEQ ID NO: 6.

### Claim 29

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO: 7 or an isolated peptide which is at least 75% identical to SEQ ID NO: 7.

### Claim 30

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO: 8 or an isolated peptide which is at least 75% identical to SEQ ID NO: 8.

### Claim 31

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO: 9 or an isolated peptide which is at least 75% identical to SEQ ID NO: 9.

### Claim 32

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO: 70 or an isolated peptide which is at least 75% identical to SEQ ID NO: 70.

### Claim 33

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO: 71 or an isolated peptide which is at least 75% identical to SEQ ID NO: 71.

### Claim 34

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO:72 or an isolated peptide which is at least 75% identical to SEQ ID NO: 72.

### Claim 35

An isolated peptide consisting essentially of the amino acid sequence of SEQ ID NO: 69 or an isolated peptide which is at least 75% identical to SEQ ID NO: 69.

### Claim 36 (depends on 28, 29, 30, 31, 32, 33, 34, 35)

A mixture comprising at least two peptides of any of claims 28-35.

### Claim 37 (depends on 28, 29, 30, 31, 32, 33, 34, 35)

An antibody which specifically binds to a polypeptide comprising the peptide of any of claims 28-35.

### Claim 38 (depends on 28, 29, 30, 31, 32, 33, 34, 35)

An antibody which specifically binds to the peptide of any of claims 28-35.

### Claim 39 (depends on 37, 38)

The antibody of claim 37 or 38, wherein the antibody is a polyclonal antibody.

### Claim 40 (depends on 37, 38)

The antibody of claim 37 or 38, wherein the antibody is a monoclonal antibody.

### Claim 41 (depends on 37, 38)

The antibody of claim 37 or 38, wherein the antibody is a soluble antibody fragment.

### Claim 42

An isolated nucleic acid consisting essentially of SEQ ID NO: 18, 19 or 20 or an isolated nucleic acid which is at least 75% homologous to the nucleic acid of SEQ ID NO: 18, 19 or 20.

### Claim 43

An isolated nucleic acid consisting essentially of SEQ ID NO: 68, 73 or 74 or an isolated nucleic acid which is at least 75% homologous to the nucleic acid of SEQ ID NO: 68, 73, or 74.

### Claim 44 (depends on 42, 43)

A composition comprising the nucleic acid of claim 42 or 43.

### Claim 45

A method for detecting a predisposition to type 2 diabetes in a subject, the method comprising determining expression of C1orf32 RNA or C1ORF32 protein in a sample obtained from a subject, wherein decreased expression, compared to expression in a control sample from a subject known not to have type 2 diabetes, indicates that the subject is susceptible to type II diabetes.

### Claim 46 (depends on 45)

The method of claim 45, wherein determining comprises measuring expression level of C1orf32 RNA or C1ORF32 protein in the sample, or determining C1ORF32 protein localization or determining post-translational modification of C1ORF32 protein.

### Claim 47 (depends on 45)

The method of claim 45, wherein determining expression level of C1ORF32 protein comprises immunohistochemistry or Western blotting using an antibody which specifically binds to C1ORF32 protein.

### Claim 48 (depends on 45)

The method of claim 45, wherein the sample from the subject and the control sample are from a diabetes-relevant tissue or cell.

### Claim 49 (depends on 45)

The method of claim 45, wherein the diabetes-relevant tissue or cell comprises liver , pancreatic islet, skeletal muscle, brain, adipose tissue, adipose cell, or any combination thereof.

### Claim 50 (depends on 45)

The method of claim 45, wherein determining comprises quantifying RNA encoding the C1Orf32 polypeptide, a variant thereof, a fragment thereof, or any combination thereof.

### Claim 51

A method for manipulating beta cell mass to treat a biological condition in a subject, comprising contacting a beta cell precursor with an agent which increases expression of C1orf32 mRNA or C1ORF32 protein, thereby manipulating beta cell mass in the subject.

### Claim 52

A method for treating a biological condition associated with reduced beta cell mass in a subject, comprising administering to the subject an agent which increases expression of C1orf32 mRNA or C1ORF32, so as to increase beta cell mass in the subject thereby treating the biological condition.

### Claim 53

A method for treating a biological condition associated with reduced levels of C1orf32 mRNA or C1ORF32 in a subject, comprising administering an agent which increases expression of C1orf32 mRNA or C1ORF32, thereby treating the biological condition.

### Claim 54 (depends on 51, 52, 53)

The method of any of claims 51-53 , wherein the biological condition is type II diabetes.

### Claim 55 (depends on 51, 52, 53)

The method of any of claims 51-53, wherein the expression of C1orf32 mRNA or C1ORF32 protein is increased in pancreas, in skeletal muscle, in adipose tissue, in brain hypothalamus, or any combination thereof.

### Claim 56 (depends on 51, 52, 53)

The method of any of claims 51-53, wherein the expression of C1orf32 mRNA or C1ORF32 protein is increased in beta cells.

### Claim 57

A method for increasing expression of C1orf32 RNA or C1ORF32 protein in a pancreatic cell, the method comprising contacting the cell with an agent which increases the levels of the C1orf32 RNA or C1ORF32 protein.

### Claim 58 (depends on 57)

The method of claim 57, wherein the pancreatic cell is a β-cell or an islet of Langerhans cell.

### Claim 59

A method of modulating beta cell development, the method comprising contacting a pancreatic cell with an agent which increases the levels of C1orf32 mRNA or C1ORF32 protein.

### Claim 60

A method for increasing beta cell mass, beta cell numbers or beta cell proliferation, the method comprising contacting a pancreatic cell with an agent which increases expression of C1orf32 mRNA or C1ORF32 protein.

### Claim 61 (depends on 59, 60)

The method of claim 59 or 60, wherein the method is performed in vivo.

### Claim 62 (depends on 59, 60)

The method of claim 59 or 60, wherein the method is performed ex vivo.

### Claim 63

A method for treating a pre-diabetic or a diabetic subject, the method comprising administering to the subject a therapeutically effective amount of an agent which increases the expression of C1orf32 mRNA or C1ORF32 protein.

### Claim 64 (depends on 51, 52, 53, 63)

The method of any of claim 51, 52, 53 or 63, wherein the subject is suspected to have or has type2 diabetes (T2DM).

### Claim 65

A method for treating a subject suffering from a disease or disorder associated with defects in beta cell mass, beta cell proliferation or beta cell activity, the method comprising:
(a) isolating a pancreatic (beta cell) cell from a donor,
(b) introducing a nucleic acid which comprises a nucleic acid sequence encoding C1ORF32 polypeptide into the pancreatic cell;
(c) transferring the pancreatic cell of (b) in the subject, wherein the pancreatic cell grows, and differentiates into insulin producing beta cell.

### Claim 66 (depends on 65)

The method of claim 65, wherein the donor is the subject.

### Claim 67 (depends on 65)

The method of claim 65, optionally comprising a step of ex vivo expanding of the pancreatic cell of step (b).

### Claim 68 (depends on 67)

The method of claim 67, wherein the step of expanding is performed in the presence of growth factors.

### Claim 69 (depends on 51, 52, 53, 57, 59, 60, 63)

The method of any one of claim 51, 52, 53, 57, 59, 60 or 63, wherein the agent is a nucleic acid which comprises a nucleic acid sequence encoding a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment.

### Claim 70 (depends on 51, 52, 53, 57, 59, 60, 63)

The method of any one of claim 51, 52, 53, 57, 59, 60 or 63, wherein the agent is a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment.

### Claim 71

A method for manipulating beta cell mass to treat a biological condition in a subject, comprising contacting a beta cell precursor with a peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment, or any combination thereof, thereby manipulating beta cell mass in the subject.

### Claim 72

A method for treating a biological condition associated with reduced beta cell mass in a subject, comprising administering to the subject a peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment, or any combination thereof, so as to increase beta cell mass in the subject thereby treating the biological condition.

### Claim 73 (depends on 71, 72)

The method of any of claims 71-72 , wherein the biological condition is type II diabetes, obesity, a dyslipidemia, or any combination thereof.

### Claim 74

A method for treating a pre-diabetic or a diabetic subject, the method comprising administering to the subject a therapeutically effective amount of a peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment, or any combination thereof.

### Claim 75

A peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment for use in treating a pre-diabetic or a diabetic condition in a subject.

### Claim 76

A peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment for use in treating a biological condition associated with reduced beta cell mass in a subject.

### Claim 77

A peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or a C1ORF32 functional fragment for use in manipulating beta cell mass to treat a biological condition in a subject.

### Claim 78

An antibody that specifically binds to a peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein, a C1ORF32 polypeptide, a C1ORF32 isoform, or any fragment thereof.

### Claim 79 (depends on 78)

A method for diagnosing type 2 diabetes in a subject, the method comprising (a) detecting expression of a peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein or a C1ORF32 polypeptide in a sample blood or tissue from a subject, wherein the antibody of claim 78 is used to determine expression, and (b) comparing expression the expression in (a) to the expression of a peptide having SEQ ID NO:1-9 or 69-72, or a C1ORF32 protein or a C1ORF32 polypeptide in a control sample.