Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-13
2004-09-07
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100
Reexamination Certificate
active
06787321
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not Applicable
FIELD OF THE INVENTION
This invention pertains to the fields of molecular biology and oncology. In particular, this invention provides a novel two-hybrid system to screen for agents that modulate the ability of a cell to degrade a metabolic product (e.g. certain cancer cells) or to selectively kill a cell that has a defect in its ability to degrade a metabolic product.
BACKGROUND OF THE INVENTION
A wide variety of diseases are characterized by the abnormal accumulation of one or more metabolic products. Such diseases, sometimes referred to as “storage diseases” are typically caused by the increased accumulation of metabolic products (e.g., lipids, proteins, complex carbohydrates, etc.) due to either the inactivity of an enzyme that degrades the products or the hyperactivity of an enzyme that creates the products. Storage disease include but are not limited to glycogen storage disease 1, GM1 gangliosidoses, MPS IV B (Morquio B), GM2 gangliosidoses (O, B, AB, B1 variants), Niemann-Pick disease (A, B, and C), Metachromatic leukodystrophy (arylsulfatase A and SAP-1 deficient), Krabbe disease, Fabry disease, Gaucher disease, Farber disease, Wolman disease (cholesterol ester storage disease), MPS I (Hurler and Scheie syndromes), MPS II (Hunter syndrome), MPS III A, C, and D (Sanfilippo A, C, and D), PS III B (Sanfilippo B), MPS IV A (Morquio A), MPS VI (Maroteaux-Lamy syndrome) MPS VII (beta-glucuronidase deficiency), Multiple sulfatase deficiency, Mucolipidosis I (Sialidosis), Mucolipidosis II & III, alpha-Mannosidosis, beta-Mannosidosis, Fucosidosis, Sialic acid storage disease, Galactosialidosis, and Aspartylglucosaminuria Cystinosis.
Historically, storage diseases have been treated by supplementing the “missing” enzymatic activity. Thus, for example, Gaucher's disease can be treated by use of a glucocerebrosidase targeted to spleen cells. Similarly, superoxide dismutase can be targeted to the liver as an anti-oxidant, and so forth.
Such approaches typically have seen limited success. Often the “therapeutic agent” must be specifically targeted to a particular organ or tissue. In addition consistent delivery of the targeted therapeutic agent at physiologically relevant concentrations has proven difficult. In addition, the identification of viable therapeutic agents has proven difficult.
SUMMARY OF THE INVENTION
This invention provides novel approaches to the treatment (e.g. amelioration of one or more symptoms) of pathological states characterized by the undesired accumulation of one or more metabolic products. In addiction, this invention provides effective systems in which to screen for agents that modulate the ability of a cell to accumulate and/or degrade a metabolic product.
In preferred embodiments, this invention utilizes a two-hybrid system to screen for agents that modulate the ability of a cell to degrade or to accumulate a metabolic product or to selectively kill a cell or to selectively express a gene or cDNA in a cell that has a defect in its ability to degrade or to accumulate a metabolic product. In one such embodiment, this invention provides a method of screening for an agent that modulates the ability of a cell to accumulate or to degrade a metabolic product. The method involves providing a mammalian cell comprising a nucleic acid encoding a peptide binding domain and an effector gene; a first chimeric protein comprising a nucleic acid binding domain that binds the peptide binding domain attached to the metabolic product or to a ligand that binds to the metabolic product; and a second chimeric protein comprising an expression control protein attached to the metabolic product or to the ligand that binds to the metabolic product such that when the first chimeric protein comprises the metabolic product, said second chimeric protein comprises the ligand and when the first chimeric protein comprises the ligand, said second chimeric protein comprises the metabolic product. The cell is contacted with one or more test agent(s) and alteration of expression of the effector gene is detected wherein a difference in the expression of the effector gene in the test cell, as compared to a control cell contacted with a lower concentration of test agent or no test agent indicates that the test agent(s) modulate the ability of said cell to accumulate or degrade the metabolic product.
In particularly preferred embodiments, the expression control protein is a transactivator (e.g. VP16) or a repressor. Particularly preferred nucleic acid binding proteins include, but are not limited to GAL-4, and GAL-4-Y. In certain embodiments, preferred effectors include, but are not limited to, a reporter gene (e.g., chloramphenicol acetyl transferase (CAT), luciferase, b-galactosidase (b-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), green fluorescent protein (GFP), etc.), a cytotoxin (e.g., thymidine kinase, pseudomonas exotoxin, diphtheria toxin, ricin, abrin, etc.), and/or an apoptosis inducing (e.g. P53, P73, Bax, Bad, FADD, a caspase gene, (ect). In various embodiments, the ligand and metabolic product respectively include, but are not limited to a beta-catenin and a Tcf, a NF-kB and I-kB, a P53 and MDM2, a receptor and its heteromelic receptor partner.
The first and/or the second chimeric protein(s) are expressed from a nucleic acid in the cell (e.g. under control of an inducible (e.g. ecdysone promoter), tissue-specific, or constitutive promoter), or the first and/or the second chimeric protein is a protein transported into the cell, e.g. by using a protein comprising an HIV TAT domain.
Preferred cells for the practice of the methods of this invention include, but are not limited to SW480, SW48, DLD-1, HCT-116, HT29, 293, U-20S, T-47D, MCF-7, HeLa, A549, Hep G2, and/or Jarkat cell.
In one particularly preferred embodiment the nucleic acid encodes a GAL-4 binding site, the effector gene is a reporter gene, the first chimeric protein comprises a GAL-4 nucleic acid binding protein and a beta catenin or a Tcf; and the second chimeric protein comprises a VP-16 and beta catenin or a Tcf. One particularly preferred Tcf is Tcf4.
In certain embodiments, the cell further comprises a second nucleic acid encoding the ligand or metabolic product operably linked to an inducible promoter (e.g. an ecdysone promoter).
In another embodiment, this invention provides a method of selectively expressing an effector gene in a cell that accumulates or degrades a metabolic product. The method involves providing a cell comprising a nucleic acid encoding a peptide binding site and an effector gene, a first chimeric protein comprising a nucleic acid binding protein that binds the peptide binding domain where the nucleic acid binding protein is attached to the metabolic product or to a ligand that binds to said metabolic product, and a second chimeric protein comprising an expression control protein attached to the metabolic product or to a ligand that binds to the metabolic product such that when said first chimeric protein comprises the metabolic product, said second chimeric protein comprises the ligand and when the first chimeric protein comprises the ligand, said second chimeric protein comprises the metabolic product; whereby the cell, in the absence of the ability to degrade the metabolic product or the ligand that binds the metabolic product activates or represses expression of said effector gene. A preferred expression control protein is a transactivator (e.g. VP16) or a repressor. Preferred nucleic acid binding proteins, effector genes, ligands and metabolic products include, but are not limited to those described above.
The first and/or the second chimeric protein(s) are expressed from a nucleic acid in the cell (e.g., under control of an inducible (e.g. ecdysone promoter), tissue-specific, or constitutive promoter), or the first and/or the second chimeric protein is a protein transported into the cell, e.g. b
McCormick Frank
Tetsu Osamu
Wakita Kenichi
Katcheves Konstantina
Ketter James
Quine Intellectual Property Law Group P.C.
The Regents of the University of California
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