Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-10-29
2001-11-06
Clark, Deborah J. R. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S025000, C800S010000, C435S320100, C435S325000, C435S455000, C536S023100
Reexamination Certificate
active
06313373
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to the identification and characterization of a nucleic acid sequence of the human involucrin gene which targets expression of any desired nucleic acid sequence to specific tissues and specific cells. In particular, this invention relates to nucleic acid sequences which target expression of nucleic acid sequences to suprabasal cells in stratifying squamous epithelial tissue and to uroepithelial cells. In another aspect, this invention pertains to transgenic animals which exhibit certain cancers and hyperplasias. In yet another aspect, this invention pertains to methods of screening for therapeutics for epithelial neoplasia.
BACKGROUND OF THE INVENTION
Diseases of epithelial cells are the single most common cause of morbidity and mortality of humans. Foremost among these diseases is cancer. Other diseases which are epithelial in origin include, for example, blistering disease (e.g., epidermolytic hyperkeratosis, and Dowling-Meara disease) proliferative disease (e.g., psoriasis, epidermal lysis, and Bulosa simplex) and Ichthyosis disease (e.g. Ichthyosis bullosa Simens, and recessive X-linked ichthyosis). The location of the epithelium as the lining of tissue surfaces in the body places it at a particularly high risk for repeated damage from a variety of agents in the environment. For example, most of the prevalent epithelial cancers (e.g., cancer of the lung, breast, colon, liver, cervix, etc.) are associated with exposure to carcinogens such as cigarette smoke, hydrocarbons in grilled foods, toxic molds, and infection with genital DNA tumor viruses.
The evaluation of candidate therapeutics directed at the treatment of epithelial disease has traditionally focused on animal models in which the animal is repeatedly exposed to one or a combination of chemicals. For example, models for cancer development and treatment rely on administration of carcinogenic and co-carcinogenic compounds. However, one drawback to such a model is that animals treated with chemicals exhibit a multitude of genetic and metabolic alterations. The multiplicity of genetic and metabolic changes makes it difficult to determine which of this multitude of changes is causally related to the resulting disease state, and hence makes it also difficult, if not impossible, to identify candidate therapeutics which target only relevant genetic and/or metabolic lesions. The further problems of unpredictability and variability of genetic and metabolic changes in response to chemical treatment make such animals poor models for the evaluation of therapeutics.
More recently, trangenic animals which harbor known genetic alterations and which express epithelial disease have been used. In particular, transgenic animal models which develop cancer and in which selected genes are expressed in epithelial cells in general (e.g., U.S. Pat. No. 5,550,316; Griep et al. (1994) Proc. Soc. Exp. Biol. Med. 206:24-34; Kondoh et al. (1995) Intervirology 38:181-186; Yang et al. (1995) Am J. Pathol. 147:68-78; Greenhalgh et al. (1994) Cell Growth Differ. 5:667-675; Tinsley et al. (1992) J. Gen. Virol. 73:1251-1260) have been described.
For example, the involvement of human papillomavirus (HPV) in cancer development has been investigated in model transgenic animals. Mice transgenic with HPV16 oncogenes express a number of malignancies (Table 1).
TABLE 1
Transgenic Animals Containing HPV-16-Oncogenes
Sites of mRNA or
Promoter
Gene
Protein Expression
Major Phenotype
References
Human keratin 14
HPV-16 E7
Epidermis; hair
Epidermal hyperplasia in
Herber et al.
follicles;
skin, mouth palate,
(1996) J. Virol.
sebaceous glands.
esophagus, forestomach,
70:1873-1881
and exocervix; skin rumor.
Human &bgr;-actin
HPV-16 E6
Epidermis; cervix;
Epidermal hyperplasia;
Arbeit et al.
and E7
vagina.
cervical dysplasia; vaginal
(1996) Proc. Natl.
and cervical dysplasia and
Acad. Sci. USA
carcinoma in situ (17-&bgr;-
93:2930-2935.
estradiol).
Bovine
HPV-16 E7
Thyroid.
Differentiated goiters;
Ledent et al.
thyroglobulin
invasive undifferentiated
(1995) Oncogene
goiters.
10:1789-1797
&agr;A-crystallin
HPV-16 E6
Skin; eyes.
Squamous cell carcinoma;
Frazer et al.
and E7
lenticular tumor.
(1995) Cancer
Res. 55:2635-
2639.
MMTV
HPV-16 E6
Cervix; vagina;
Cervical and vaginal
Sasagawa et al.
and E7
salivary gland.
dysplasia and hyperplasia;
(1994) J. Gen.
salivary gland carcinoma;
Virol. 75:3057-
lymphoma; skin
3065.
histiocytoma.
Bovine Keratin 6
HPV-16
Tongue; stomach;
Metastatic stomach tumors.
Searle et al.
early
female
(1994) J. Gen.
region
reproductive tract;
Virol. 75:1125-
tail skin.
1137.
Human Keratin
HPV-16
Skin.
Ear epidermal hyperplasia
Arbeit et al.
14
early
and dysplasia; facial
(1994) J. Virol.
region
epidermal hyperplasia and
68:4358-4368.
papillamatosis; anal
papilioma; truncal ulcers,
diffuse epidermal
hyperplasia; cataracts;
lenticular hyperplasia.
Human keratin 14
HPV-16 B6
Skin.
Ear epidermal hyperplasia
Arbeit et al.
and E7
and dysplasia; facial
(1994)
epiedermal hyperplasia and
WO/95/33820;
papillomatosis; anal
Arbeit et al,
papillomas, truncal ulcers;
(1994) WO
cataracts; cervico-vaginal
95/33826.
carinoma (17-&bgr;-estradiol).
Human &agr;-actin
HPV-16 E6
Neuroepithelial
Neuroepithelial carcinoma.
Arbeit et al.
and E7.
tumors
(1993) Am. J.
Pathol. 142:1187-
97.
&agr;A-crystallin
HPV-16 E6
Ocular lens; skin.
Bilateral microphthalmia
Lambert et al.
and E7
and lens tumor; skin
(1993) Proc. Natl.
preneoplastic lesion and
Acad, Sci. USA
carcinoma.
90:5583-5587.
&agr;A-crystallin
HPV-16 E6
Ocular lens; eye
Lens tumor.
Griep et al. (1993)
and E7
without lens; brain,
J. Virol. 67: 1373-
intestine, tail.
84.
MMTV-LTR
HPV-16 E6
Testicular tumor;
Testicular tumor
Kondoh et al.
and E7
submandibular
(seminoma).
(1991) J. Virol.
gland.
65:3335-3339.
While there exist transgenic animals which develop epithelial cell disease in general, and neoplastic and/or preneoplastic lesions in particular, there is no transgenic model for some epithelial diseases (e.g., blistering disease, proliferative disease, and Ichthyosis disease) or for certain cancers (e.g., colon cancer, anal cancer, etc.). Furthermore, because the development of a single cancer phenotype may be caused by more than one genetic alteration, even those cancers for which there is available a transgenic animal model having a defined genetic lesion, such a single transgenic animal model is potentially of limited use in comprehensive screening of therapeutics. This is because a compound which is not therapeutic in a transgenic animal that has a particular genetic alteration, may nevertheless be therapeutic in a transgenic animal which develops the same disease as a result of a different genetic alteration.
Thus, there is a need for a better model of epithelial cell disease. This model should be amenable to identifying therapeutic compounds.
SUMMARY OF THE INVENTION
The present invention provides methods for selective expression of a nucleic acid sequence of interest in epithelial cells of a non-human transgenic animal, and in particular to suprabasal epithelial cells. This invention further relates to methods for producing a non-human transgenic animal wherein a nucleotide sequence of interest is selectively expressed in epithelial cells of the non-human animal, and more particularly in suprabasal epithelial cells. The present invention also relates to the use of the transgenic animals for screening anti-neoplastic compounds. Further provided by this invention are oligonucleotide sequences which selectively target expression of a nucleotide sequence of interest to epithelial cells, and in particular to suprabasal epithelial cells.
The present invention provides a purified oligonucleotide comprising at least a portion of the nucleotide sequence of
FIG. 6
(SEQ ID NO:1) from nucleotide −2473 to −1953. While it is not intended that the present invention be limited to a particular type of activity of the portion of the nucleotide sequence of
FIG. 6
(SEQ ID NO:1) from nucleotide −2473 to −1953, in one embodiment, the portion of olig
Crish James F.
Eckert Richard L.
Case Western Reserve University
Clark Deborah J. R.
Drabik Christopher
Medlen & Carroll LLP
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