Patched genes and uses related thereto

• US 6,551,782 B1
• Filed: 11/28/2000
• Issued: 04/22/2003
• Est. Priority Date: 10/07/1994
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Stanford University, University of California
• Inventors: Johnson, Ronald L., Goodrich, Lisa V., Epstein, Ervin Jr., Scott, Matthew P.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US09/724,631
• Time in Patent Office: 875 Days
• Field of Search: 436/94, 435/6, 435/325, 435/91.2, 536/23.5, 536/24.31, 536/24.33
• US Class Current: 435/6.13
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/40 Fish
• CPC Class Codes: C07K 14/43563 from insects
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748

Abstract

Methods for isolating patched genes, particularly mammalian 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 cancers may be familial, having as a component of risk an inherited genetic predisposition, or may be sporadic. 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 15 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

1. An assay for phenotyping the patched status of a cell, comprising detecting in vitro, in a sample of mammalian cells, the presence or absence of a genetic lesion in a patched gene characterzed by at least one of (i) aberrant modification or mutation of a patched gene, and (ii) mis-expression of the patched gene.

2. The assay of claim 1, wherein detecting said lesion includes:

i. providing one or more nucleic acid probes comprising a nucleic acid including a region of nucleotide sequence which hybridizes to a sense or antisense sequence of said patched gene, or naturally occurring mutants thereof;

or 5′

or 3′

flanking sequence naturaly associated with the gene;

ii. combining said probe with nucleic acid from said sample; and

iii. detecting, by hybridization of said probe to the nucleic acid from said sample, the existence of at least one of a deletion of one or more nucleotides from said patched gene, an addition of one or more nucleotides to said patched gene, a substitution of one or more nucleotides of said patched gene, a chromosomal rearrangement of all or a portion of said patched gene, an alteration in the level of an mRNA script of said patched gene, or a non-wild type splicing pattern of an mRNA transcript of said patched gene.

3. The assay of claim 2, wherein hybridization of said probe further comprises subjecting the probe and cellular nucleic acid to a polymerase chain reaction (PCR) and detecting abnormalities in an amplified product.

4. The assay of claim 2, wherein said probe hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid designated by SEQ ID No. 9 or 18.

5. The assay of claim 2, wherein said probe hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleic acid designated by SEQ ID No. 18.

6. The assay of claim 2, wherein said probe further comprises a label group attached to said nucleic acid, wherein said label group is detectable.

7. The assay of claim 5, wherein detecting said lesion comprises ascertaining, from a methylation pattern of said patched gene, the presence or absence of aberrant methylation of said patched gene.

8. The assay of claim 7, wherein the methylation pattern of said patched gene is determined by combining nucleic acid of said cell sample with one or more methylation-sensitive restriction endonucleases and determining the restriction digest pattern of at least a portion of said patched gene.

9. The assay of claim 1, wherein detecting said lesion comprises detecting the presence or absence of a non-wild type level of a patched protein product of said patched gene in cells of said cell sample.

10. The assay of claim 9, wherein the level of said patched protein is detected in an immunoassay.

11. The assay claim 1, wherein detecting said lesion comprises ascertaining, relative to a wild-type level of hedgehog-dependent patched signal transduction, the ability of cells in said cell sample to respond to hedgehog induction.

12. The assay of claim 1, wherein said cell sample is obtained from a human patient.

18. The method of claim 1, wherein said genetic predisposition is a predisposition for developing a fibroma.

13. A method for diagnosing a genetic predisposition of an animal for at least one of a developmental abnormality or a proliferative disorder wherein the developmental abnormality or proliferative disorder correlates with aberrant expression or activity of a patched gene or gene product, comprising detecting in vitro the presence of a predisposing mutation in a patched gene in cells of said animal, wherein the presence of said predisposing mutation indicates that said individual has a genetic predisposition for at least one of developmental abnormalities or a proliferative disorder.

14. The method of claim 13, wherein said genetic predisposition is basal cell nevus syndrome.

15. The method of claim 13, wherein said genetic predisposition is a predisposition for developing a carcinoma.

16. The method of claim 13, wherein said genetic predisposition is a predisposition for developing a meningiomas.

17. The method of claim 13, wherein said genetic predisposition is a predisposition for developing a medullomas.

19. The method of claim 13, wherein said detecting step comprises, analyzing a nucleic acid sample obtained from said animal.

20. The method of claim 13, wherein said detecting step comprises functional analysis of patched protein function.

21. The method of claim 13, wherein said detecting step comprises detecting antibody binding to abnormal patched protein.

30. The method of claim 19, wherein said detecting step comprises functional analysis of patched protein function.

31. The method of claim 19, wherein said detecting step comprises detecting antibody binding to abnormal patched protein.

22. A method for characterizing the phenotype of a tumor, comprising detecting the presence of an oncogenic patched mutation in cells of the tumor, wherein the presence of said oncogenic mutation indicates that said tumor has a patched-associated phenotype.

23. The method of claim 22, wherein said tumor is a carcinoma.

24. The method of claim 23, wherein said carcinoma is a basal cell carcinoma.

25. The method of claim 22, wherein said tumor is a meningioma.

26. The method of claim 22, wherein said tumor is a medulloma.

27. The method of claim 22, wherein said tumor is a fibroma.

28. The method of claim 22, wherein said once genic patched mutation is detected by analyzing DNA of said tumor.

29. The method of claim 22, wherein said noncogenic patched mutation is detected by analyzing mRNA of said tumor.