# US 7,465,552 B2 · Patched genes and uses related thereto

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- Number: US 7,465,552 B2
- Document ID: US7465552B2
- Status: Granted
- Kind: B2
- Application number: 10/421,446
- Filed: 2003-04-22
- Published: 2003-10-02
- Granted: 2008-12-16
- Earliest claimed: 1994-10-07
- Assignees: The Board of Trustees of the Leland Stanford Junior University; The Regents of the University of California
- Inventors: Matthew P. Scott; Lisa V. Goodrich; Ronald L. Johnson; Ervin Epstein, Jr.
- Web page: https://patentgrep.com/patent/US7465552B2

## CPC

- C07K 14/43563: {from insects}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2227/40: Fish
- 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)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

Methods for isolating patched genes, including the mouse and human patched genes, as well as invertebrate patched genes and sequences, are provided. Decreased expression of patched is associated with the occurrence of human cancers, particularly basal cell carcinomas of the skin. The patched and hedgehog genes are useful in creating transgenic animal models for these human cancers. The patched nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of cancer, identification of cell type based on expression, and the like. The DNA is further used as a diagnostic for a genetic predisposition to cancer, and to identify specific cancers having mutations in this gene.

## Claims

### Claim 1

A method of identifying an agent that mimics hedgehog inhibitory activity of patched, comprising:
(i) providing a culture of mammalian test cells;
(ii) contacting the mammalian test cells with one or more test agents, wherein the hedgehog/patched signaling pathway is active in said mammalian test cells, but said cells are not responsive to hedgehog induction, and wherein said mammalian test cells express a reporter gene that is operatively linked to a transcriptional regulatory sequence regulated by patched signal transduction, and wherein said mammalian test cells are not also contacted with exogenously provided hedgehog protein, and
(iii) identifying the one or more test agents that mimic hedgehog inhibitory activity of patched relative to that present in the absence of the identified test agents.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mammalian test cells are characterized by loss of function of a patched gene.

### Claim 3 (depends on 2)

The method of claim 2, wherein the mammalian test cells characterized by loss of function of a patched gene are basal cell carcinoma cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the hedgehog inhibitory activity is assayed by examining cell phenotype.

### Claim 5 (depends on 1)

The method of claim 1, wherein the hedgehog inhibitory activity is assayed by examining expression of a reporter gene operatively linked to a transcriptional regulatory sequence from patched, Gli, or hedgehog.

### Claim 6 (depends on 5)

The method of claim 5, wherein examining expression further comprises examining mRNA expression.

### Claim 7 (depends on 5)

The method of claim 5, wherein examining expression further comprises examining protein expression.

### Claim 8 (depends on 2)

The method of claim 2, wherein the hedgehog inhibitory activity is assayed by examining cell phenotype.

### Claim 9 (depends on 2)

The method of claim 2, wherein the hedgehog inhibitory activity is assayed by examining expression of a reporter gene operatively linked to a transcriptional regulatory sequence from patched, Gli, or hedgehog.

### Claim 10 (depends on 9)

The method of claim 9, wherein examining expression further comprises examining mRNA expression.

### Claim 11 (depends on 9)

The method of claim 9, wherein examining expression further comprises examining protein expression.

### Claim 12 (depends on 1)

The method of claim 1, wherein the mammalian test cells are characterized by loss-of-function of a patched protein.

### Claim 13 (depends on 12)

The method of claim 12, wherein the hedgehog inhibitory activity is assayed by examining cell phenotype.

### Claim 14 (depends on 12)

The method of claim 12, wherein the hedgehog inhibitory activity is assayed by examining expression of a reporter gene operatively linked to a transcriptional regulatory sequence from patched, Gli, or hedgehog.

### Claim 15 (depends on 14)

The method of claim 14, wherein examining expression further comprises examining mRNA expression.

### Claim 16 (depends on 14)

The method of claim 14, wherein examining expression further comprises examining protein expression.

### Claim 17

A method of identifying an agent that mimics hedgehog inhibitory activity of patched, comprising:
(i) providing a culture of mammalian test cells;
(ii) contacting the mammalian test cells with one or more test agents, wherein the hedgehog/patched signaling pathway is active in said mammalian test cells, but said cells are not responsive to hedgehog induction, and wherein said mammalian test cells express a reporter gene that is operatively linked to a transcriptional regulatory sequence regulated by patched signal transduction, and
(iii) identifying the one or more test agents that mimic hedgehog inhibitory activity of patched relative to that present in the absence of the identified test agents.

### Claim 18 (depends on 17)

The method of claim 17, wherein the mammalian test cells are characterized by loss of function of a patched gene.

### Claim 19 (depends on 18)

The method of claim 18, wherein the mammalian test cells characterized by loss of function of a patched gene are basal cell carcinoma cells.

### Claim 20 (depends on 17)

The method of claim 17, wherein the mammalian test cells are characterized by loss-of-function of a patched protein.

### Claim 21 (depends on 17)

The method of claim 17, wherein the hedgehog inhibitory activity is assayed by examining cell phenotype.

### Claim 22 (depends on 17)

The method of claim 17, wherein the hedgehog inhibitory activity is assayed by examining expression of a reporter gene operatively linked to a transcriptional regulatory sequence from patched, Gli, or hedgehog.

### Claim 23 (depends on 22)

The method of claim 22, wherein examining expression further comprises examining mRNA expression.

### Claim 24 (depends on 22)

The method of claim 22, wherein examining expression further comprises examining protein expression.

### Claim 25

A method of identifying an agent that mimics hedgehog inhibitory activity of patched, comprising:
(i) providing a culture of mammalian test cells characterized by loss of function of a patched gene;
(ii) contacting the mammalian test cells with one or more test agents; and
(iii) identifying the one or more test agents that mimic hedgehog inhibitory activity of patched relative to that present in the absence of the identified test agents.

### Claim 26 (depends on 25)

The method of claim 25, wherein the mammalian test cells express a reporter gene that is operatively linked to a transcriptional regulatory sequence regulated by hedgehog/patched signaling.

### Claim 27 (depends on 25)

The method of claim 25, wherein the mammalian test cells characterized by loss of function of a patched gene are basal cell carcinoma cells.

### Claim 28 (depends on 25)

The method of claim 25, wherein the mammalian test cells are characterized by loss of function of a patched protein.

### Claim 29 (depends on 25)

The method of claim 25, wherein the hedgehog inhibitory activity is assayed by examining cell phenotype.

### Claim 30 (depends on 26)

The method of claim 26, wherein the hedgehog inhibitory activity is assayed by examining expression of a reporter gene operatively linked to a transcriptional regulatory sequence from patched, Gli, or hedgehog.

### Claim 31 (depends on 30)

The method of claim 30, wherein examining expression further comprises examining mRNA expression.

### Claim 32 (depends on 30)

The method of claim 30, wherein examining expression further comprises examining protein expression.

### Claim 33 (depends on 25)

The method of claim 25, wherein the hedgehog inhibitory activity is assayed by examining expression of a reporter gene operatively linked to a transcriptional regulatory sequence from patched, Gli, or hedgehog.

### Claim 34 (depends on 33)

The method of claim 33, wherein examining expression further comprises examining mRNA expression.

### Claim 35 (depends on 33)

The method of claim 33, wherein examining expression further comprises examining protein expression.

### Claim 36 (depends on 26)

The method of claim 26, wherein the mammalian test cells characterized by loss of function of a patched gene are basal cell carcinoma cells.