Keratin K1 expression vectors and methods of use

• US 5,914,265 A
• Filed: 11/01/1993
• Issued: 06/22/1999
• Est. Priority Date: 04/30/1992
• Status: Expired due to Fees

• Current Assignee: Baylor College of Medicine (Baylor College), United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: Baylor College of Medicine (Baylor College), United States Department Of Health And Human Services (Government of the United States of America)
• Inventors: Yuspa, Stuart H., Greenhalgh, David A., Rothnagel, Joseph A., Roop, Dennis R.
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): HAUDA, KAREN MARIE
• Application Number:
  US08/147,777
• Time in Patent Office: 2,059 Days
• Field of Search: 435/240.2, 435/172.3, 435/320.1, 435/69.1, 536/23.1, 935/22, 935/6, 935/70, 935/71
• US Class Current: 435/320.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/20 Animal model comprising regulated expression system
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 2267/035 Animal model for multifactorial diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 49/0008 Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/495 Transforming growth factor [TGF]
• CPC Class Codes: C07K 14/81 Protease inhibitors
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2710/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2740/16322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/005 : repressible enhancer/promoter combination, e.g. KRAB
• CPC Class Codes: C12N 2830/008 : cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/42 : being an intron or intervening sequence for splicing and/or stability of RNA

Abstract

A keratin K1 vector for expression of a nucleic acid sequence in an epidermal cell. The vector includes a 5'"'"' flanking region which includes necessary sequences for expression of a nucleic acid cassette, a keratin K1 3'"'"' flanking region which regulates expression of a nucleic acid sequence, predominantly in the epidermis, and a linker which connects the 5'"'"' flanking region to a nucleic acid. The linker has a position for inserting a nucleic acid cassette. The linker does not contain the coding sequence of a gene that the linker is naturally associated with. That is, the linker is not the normal gene associated with the 5'"'"' and 3'"'"' regions.

Claims

1. A vector for expression of a nucleic acid sequence in an epidermal cell, wherein said nucleic acid sequence is other than a mammalian K1 keratin gene, said vector comprising, in operable association:

a 5'"'"' flanking region from a mammalian gene including necessary sequences for expression of said nucleic acid sequence;

a 3'"'"' flanking region from a mammalian K1 keratin gene which regulates expression predominantly in an epidermal cell of said nucleic acid sequence with which it is associated; and

a linker connecting said 5'"'"' flanking region to said nucleic acid sequence, said linker having a position for inserting said nucleic acid sequence, wherein said linker lacks the coding sequences of a gene with which it is naturally associated.

2. The vector of claim 1, wherein said 3'"'"' flanking region is 3'"'"' to said position for inserting said nucleic acid sequence.

3. The vector of claim 1, wherein said 3'"'"' flanking region is 5'"'"' to said linker.

4. The vector of claim 1, wherein said vector further comprises a nucleic acid sequence.

5. The vector of claim 1 or 4, wherein said 5'"'"' flanking region includes a promoter, 5'"'"' UTR, and a first intron and intron/exon boundary in appropriate relationship for expression of said nucleic acid sequence.

6. The vector of claim 1 or 4, wherein said 3'"'"' flanking region includes a 3'"'"' UTR and a 3'"'"' NCR containing a transcriptional termination region.

7. The vector of claim 1 or 4, wherein said 3'"'"' flanking region is from a human keratin K1 gene.

8. The vector of claim 1 or 4, wherein said 5'"'"' flanking region is from a keratin K1 gene.

9. The vector of claim 5, wherein said 5'"'"' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3'"'"' flanking region is approximately 3.9 kb of a keratin K1 gene.

10. The vector of claim 5, wherein said 5'"'"' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3'"'"' flanking region is approximately 4.3 kb of a keratin K1 gene.

11. The vector of claim 1 or 4, wherein said 5'"'"' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;

said 3'"'"' flanking region comprises nucleotides 6891 to 10747 of Sequence ID No. 1; and

said linker comprising nucleotide 2351 to 2376 of Sequence ID No. 2.

12. The vector of claim 1 or 4, wherein said 5'"'"' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;

said 3'"'"' flanking region comprises nucleotides 7895 to 7921 of Sequence ID No. 1; and

said linker comprising nucleotide 2351 to 2376 of Sequence ID No. 2.

13. The vector of claim 1 or 4, wherein said 5'"'"' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;

said 3'"'"' flanking region comprises nucleotides 7924 to 7948 of Sequence ID No. 1; and

said linker comprising nucleotide 2351 to 2376 of Sequence ID No. 2.

14. The vector of claim 4, wherein said nucleic acid sequence comprises genetic material coding for a protein, or a polypeptide.

15. The vector of claim 14, wherein said genetic material codes for a hormone, a growth factor, an enzyme, a drug, a tumor suppressor, a receptor, an apolipoprotein, a clotting factor, an antigen, an oncogene or a transforming gene.

16. The vector of claim 15, wherein said oncogene is selected from the group consisting of ras, fos, myc, erb, src, sis or jun.

17. The vector of claim 14, wherein said genetic material of said nucleic acid sequence contains the E6 or E7 transforming sequence of human papilloma virus.

18. The vector of claim 14, wherein said genetic material of said nucleic acid sequence encodes for a protein or polypeptide selected from the group consisting of a TGF-α

sequence, proinsulin, insulin, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding protein, epidermal growth factor, dermal growth factor, an angiogenesis growth factor Type IV collagen, laminin, nidogen, Type VII collagen, TGF-β

, acidic fibroblast growth factor, angiogenin, cytokine receptor, and IL-1, IL-6 and IL-8, a viral capsid protein.

19. The vector of claim 1 or 4, wherein said vector further comprises a modulator sequence associated with said 3'"'"' flanking region or said 5'"'"' flanking region.

20. The vector of claim 19, wherein said modulator sequence comprises sequences responsive to calcium, Vitamin D or its metabolite, Vitamin A or its metabolite, or progesterone.

21. An isolated or cultured mammalian epidermal cell transformed with a vector for expression of a nucleic acid sequence, wherein said nucleic acid sequence is other than a mammalian K1 keratin gene, said vector comprising, in operable association:

a 5'"'"' flanking region from a mammalian gene including necessary sequences for expression of said nucleic acid sequence;

a 3'"'"' flanking region from a mammalian K1 keratin gene which regulates expression predominantly in an epidermal cell of said nucleic acid sequence with which it is associated; and

a linker connecting said 5'"'"' flanking region to said nucleic acid sequence, said linker having a position for inserting said nucleic acid sequence, wherein said linker lacks the coding sequences of a gene with which it is naturally associated.

22. The epidermal cell of claim 21, wherein said 5'"'"' flanking region includes a promoter, 5'"'"' UTR, and a first intron and intron/exon boundary in appropriate relationship for expression of said nucleic acid sequence.

23. The epidermal cell in claim 21, wherein said 5'"'"' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3'"'"' flanking region is approximately 3.9 kb of a keratin K1 gene.

24. The epidermal cell of claim 21, wherein said 5'"'"' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3'"'"' flanking region is approximately 4.3 kb of a keratin K1 gene.

25. The epidermal cell of claim 21, wherein said nucleic acid sequence comprises genetic material coding for a protein, or a polypeptide.

26. The epidermal cell of claim 25, wherein said genetic material codes for a hormone, a growth factor, an enzyme, a drug, a tumor suppressor, a receptor, an apolipoprotein, and an antigen.

27. The epidermal cell of claim 21, wherein said vector further comprises a modulator sequence in said 5'"'"' flanking region or said 3'"'"' flanking region.

28. The epidermal cell of claim 21, wherein said 3'"'"' flanking region includes a 3'"'"' UTR and a 3'"'"' NCR containing a transcriptional termination region.

29. The epidermal cell of claim 22, wherein said 5'"'"' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;

said 3'"'"' flanking region comprises nucleotides 6891 to 10747 of Sequence ID No. 1; and

said linker comprising nucleotide 2351 to 2376 of Sequence ID No. 2.

30. The epidermal cell of claim 22, wherein said 5'"'"' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;

said 3'"'"' flanking region comprises nucleotides 7895 to 7921 of Sequence ID No. 1; and

said linker comprising nucleotide 2351 to 2376 of Sequence ID No. 2.

31. The epidermal cell of claim 22, wherein said 5'"'"' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;

said 3'"'"' flanking region comprises nucleotides 7924 to 7948 of Sequence ID No. 1; and

said linker comprising nucleotide 2351 to 2376 of Sequence ID No. 2.

32. The epidermal cell of claim 27, wherein said modulator sequence is responsive to calcium, vitamin D or its metabolite, vitamin A or its metabolite, or progesterone.

33. The epidermal cell of claim 27, wherein said 3'"'"' flanking region further includes an 18 KB EcoRV fragment.

34. The epidermal cell of claim 25, wherein said nucleic acid sequence encodes for proinsulin, insulin, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding protein, epidermal growth factor, dermal growth factor, an angiogenesis growth factor Type IV collagen, laminin, nidogen, Type VII collagen, TGF-β

, acidic fibroblast growth factor, angiogenin, cytokine receptor, and IL-1, IL-6 and IL-8, a viral capsid protein.