Transgenic animals and lats genes

Details Transgenic animals and lats genes Transgenic animals and lats genes

1999-11-17

2003-10-07

Guzo, David (Department: 1636)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S013000, C800S014000, C435S320100, C435S455000, C435S463000, C536S023100, C536S023500

Reexamination Certificate

active

06630613

ABSTRACT:

1. INTRODUCTION
The present invention relates to tumor suppressor genes, in particular to “lats” genes (large tumor suppressor) and their encoded protein products, as well as derivatives and analogs thereof. Production of lats proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.
2. BACKGROUND OF THE INVENTION
Tumorigenesis in humans is a complex process involving activation of oncogenes and inactivation of tumor suppressor genes (Bishop, 1991, Cell 64:235-248). Tumor suppressor genes in humans have been identified through studies of genetic changes occurring in cancer cells (Ponder, 1990, Trends Genet. 6:213-218; Weinberg, 1991, Science 254:1138-1146). In Drosophila, tumor suppressor genes have been previously identified by recessive overproliferation mutations that cause late larval and pupal lethality (Gateff, 1978, Science 200:1448-1459; Gateff and Mechler, 1989, CRC Crit. Rev. Oncogen 1:221-245; Bryant, 1993, Trends Cell Biol. 3:31-35; Török et al., 1993, Genetics 135:71-80). Mutations of interest were identified when dissection of dead larvae and pupae revealed certain overproliferated tissues. Several genes identified in homozygous mutants have been cloned including l(1) discs large-1(dlg; Woods and Bryant, 1991, Cell 66:451-464; Woods and Bryant, 1993, Mechanisms of Development 44:85-89), fat (Mahoney et al., 1991, Cell 67:853-868), 1(2) giant larvae (lgl. Lützelschwab et al., 1987, EMBO J. 6:1791-1797; Jacob et al., 1987, Cell 50:215-225), expanded (ex; Boedigheimer and Laughon, 1993, Development 118:1291-1301; Boedigheimer et al., 1993, Mechanisms of Development 44:83-84), hyperplastic discs (hyd; Mansfield et al., 1994, Developmental Biology 165:507-526) and the gene encoding the S6 ribosomal protein (Watson et al., 1992, Proc. Natl. Acad. Sci. USA 89:11302-11306; Stewart and Denell, 1993, Mol. Cell. Biol. 13:2524-2535).
Although examining homozygous mutant animals has allowed the successful identification of overproliferation mutations that cause late larval and pupal lethality, mutations that cause lethality at early developmental stages are unlikely to be recovered by this approach. The present invention solves this problem by providing a method for identifying tumor suppressor genes that does not exclude genes that when mutated cause lethality in early developmental stages, and provides genes thus identified with a fundamental role in regulation of cell proliferation.
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention relates to nucleotide sequences of lats genes (Drosophila, human, and mouse lats and lats homologs of other species), and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences are also provided. In a specific embodiment, the lats protein is a human protein.
The invention also relates to a method of identifying tumor suppressor genes that does not exclude from identification genes that cause lethality at early developmental stages, thus overcoming the limitations of prior art methods. The method thus allows the identification of genes that regulate cell proliferation and that act at early developmental stages. The genes which thus can be identified play a fundamental role in regulation of cell proliferation such that their dysfunction (e.g., by lack of expression or mutation) leads to overproliferation and cancer.
Lats is a gene provided by the present invention, identified by the method of the invention, that acts to inhibit cell proliferation, and that plays a crucial role throughout development.
The invention also relates to lats derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) lats protein. Such functional activities include but are not limited to kinase activity, antigenicity [ability to bind (or compete with lats for binding) to an anti-lats antibody], immunogenicity (ability to generate antibody which binds to lats), and ability to bind (or compete with lats for binding) to a receptor/ligand for lats (e.g., a SH3 domain-containing protein).
The invention further relates to fragments (and derivatives and analogs thereof) of lats which comprise one or more domains of a lats protein.
Antibodies to lats, and lats derivatives and analogs, are additionally provided.
Methods of production of the lats proteins, derivatives and analogs, e.g., by recombinant means, are also provided.
The present invention also relates to therapeutic and diagnostic methods and compositions based on lats proteins and nucleic acids. Therapeutic compounds of the invention include but are not limited to lats proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the lats proteins, analogs, or derivatives; and lats antisense nucleic acids.
The invention provides for treatment of disorders of overproliferation (e.g., cancer and hyperproliferative disorders) by administering compounds that promote lats activity (e.g., lats, an agonist of lats; nucleic acids that encode lats).
The invention also provides methods of treatment of disorders involving deficient cell proliferation (growth) or in which cell proliferation is otherwise desired (e.g., degenerative disorders, growth deficiencies, lesions, physical trauma) by administering compounds that antagonize, (inhibit) lats function (e.g., antibodies, antisense nucleic acids).
Antagonizing lats function can also be done to grow larger animals and plants, e.g., those used as food or material sources.
Animal models, diagnostic methods and screening methods for predisposition to disorders, and methods to identify lats agonists and antagonists, are also provided by the invention.
3.1. Definitions
As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, “lats” shall mean the lats gene, whereas “lats” shall indicate the protein product of the lats gene.
4. DESCRIPTION OF THE FIGURES
FIG. 1A-B
. Identifying overproliferation mutations in mosaic flies. (A) Although animals that are homozygous for a lethal mutation could die at an early developmental stage, mosaic flies carrying clones of cells that are homozygous for the same mutation could live. One can identify potential tumor suppressors by generating and examining clones of overproliferated mutant cells in mosaic animals. The genetic constitution of these mosaic flies is similar to the mosaicism of the tumor patients. (B) Genetic scheme. The P-element insertions carrying the FLP recombinase (hsFLP; Golic and Lindquist, 1989, Cell 59:499-509), its target site, FRT (solid arrows, Xu and Rubin, 1993, Development 117:1223-1237), the yellow
−
and mini-white
+
marker genes (y
+
and mini-w
+
, open arrows) are indicated. Mutagenized males were crossed to females to produce heterozygous embryos. Clones of cells homozygous for the induced mutations were generated in developing first-instar larvae by mitotic recombination at the FRT sites induced with the FLP recombinase. Mosaic adults were examined for overproliferated mutant patches (w
−
, y
−
). Individuals carrying clones of interest were then mated to recover the mutations of interest in the next generation (Xu and Rubin, 1993, Development 11 7:1223-1237; Xu and Harrison, 1994; Methods in Cell Biology 44:655-682). Clones of ommatidia derived from fast proliferating mutant cells were identified since they were larger than their darkly pigmented wt (wild-type) twin-spot clones (mini-w
+
/mini-w
+
). Clones,of cells homozygous for the induced mutatio

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