Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1999-12-20
2003-02-11
Nguyen, Dave T. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S018000, C435S320100, C435S007100, C435S006120, C435S455000, C435S325000
Reexamination Certificate
active
06518480
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of pharmacological control of cells expressing a protein or other molecule of interest.
BACKGROUND OF THE INVENTION
Despite the tremendous advances in the field of genetic engineering and expression of a DNA of interest in a desired cell, few tools exist in the art for the direct, selective manipulation of a specific physiological response in a cell. For example, although cell growth can be facilitated in in vitro culture by inclusion of nutrients in the culture medium, there are few adequate means available to stimulate cell growth directly and selectively.
The problem is further complicated where the cell culture contains a desired cell type as well as a variety of undesired, contaminating cells. While the nutrients in the culture medium may enhance growth of the desired cell type, most often these nutrients also enhance growth of the undesired, contaminating cells, thus interfering with attempts to enrich the culture for the desired cell type. Some of these complicating factors might be overcome by transforming the desired cell type with an antibiotic resistance gene or other selective marker. However, the desired cells may be sensitive to the selective agent and grow more slowly in its presence, or the undesired, contaminating cells may be resistant to the selective agent and grow in the culture despite the presence of the agent.
In addition, there are many settings both in vitro and in vivo in which one wishes to enrich a cell population for a specific target cell, or elicit a specific physiological response in a target cell, without substantially affecting nontarget cells in the population. Gene therapy is an example of this latter situation. The basic concept in gene therapy involves the introduction of a DNA sequence encoding a protein or other molecule that, when expressed, can overcome a genetic defect associated with the disease, or produce some other therapeutic protein or other molecule that will either cure or ameliorate symptoms of the disease. The DNA introduced by gene therapy stably integrates into genome of host target cells, thus producing genetically altered cells expressing the desired therapeutic protein and thus treating the disease.
A critical limitation of current in vivo and ex vivo gene therapy efforts is the inability to amplify the number of transfected cells. In several instances, DNA encoding the therapeutic protein of interest is successfully delivered to and expressed in the target cells, but too few target cells are transformed to provide a measurable therapeutic response. If these few transformed cells could be specifically activated at the cellular level in vivo, e.g., to proliferate or to secrete their therapeutic product, the desired therapeutic effect could be achieved. This invention addresses this problem.
SUMMARY OF THE INVENTION
The invention features methods and compositions for selective cellular activation of a target cell. Targeted, transformed cells expressing a modified G protein-coupled receptor that is activated superiorly by a synthetic ligand (RASSL) are selectively activated by synthetic small molecule binding to the RASSL. A RASSL is a modified G protein-coupled receptor having decreased binding affinity for a selected natural (i.e., endogenous) ligand (relative to binding of the selected ligand by a wild-type G protein-coupled receptor), but having normal, near normal, or enhanced binding affinity for a synthetic small molecule. Thus, RASSL-mediated activation of RASSL-expressing cells does not occur to a significant extent in the presence of the selected natural ligand, but responds significantly upon exposure to a synthetic small molecule.
In one embodiment, the RASSL-encoding DNA is introduced into the target cells to be activated, and can be cotransformed with DNA encoding a therapeutic protein of interest (e.g., cotransformed with a DNA of interest to be used in a gene therapy regimen). Cells expressing a RASSL are selectively activated by administration of an appropriate synthetic small molecule, which in turn binds the RASSL and facilitates activation of the G protein cascade and a selected physiological, cellular response (e.g., cellular proliferation, cellular secretion) in the RASSL-expressing cell.
The invention also features specific G protein-coupled receptors modified such that the receptor retains small molecule binding affinity, but is decreased in binding affinity of a selected natural ligand relative to a native G protein-coupled receptor from which the modified receptor is derived.
The invention further features a cellular implant comprising RASSL-expressing target cells that, upon binding of an appropriate synthetic small molecule ligand, exhibit a desired G protein-mediated cellular response (e.g., cellular proliferation or cellular secretion).
The invention additionally features a transgenic, reversible disease animal model. The transgenic animal expresses a RASSL that upon stimulation causes a disease- or condition-associated symptom in the animal.
A primary object of the invention is to provide a method to activate target cells selectively, particularly target cells expressing a desired protein or other molecule of interest. The desired protein or other molecule of interest can be encoded by genetic material that is either endogenous (e.g., naturally occurring) or heterologous (e.g., introduced by transformation or transfection) to the target cell genome.
Another object of the invention is to selectively activate target cells by amplifying cellular proliferation and/or cellular secretion.
Another object of the invention is to provide a method for enhancing the efficacy of gene therapy techniques by allowing controlled, targeted activation of cells transformed with a DNA of interest.
An advantage of the present invention is that targeted cellular activation can be controlled by exposing the target cells to varying amounts of the synthetic small molecule RASSL agonist.
Another advantage of the present invention is that, where the target cells are present in a mammalian host, the synthetic small molecule used to elicit a response in RASSL-expressing cells can be administered systemically (orally or intravenously), and need not be administered directly to the target cell site(s).
Yet another advantage of the invention is that the synthetic small molecules can cross the blood-brain barrier of a mammalian subject, thus allowing for activation of RASSL-expressing cells located in the mammalian host's brain tissue by oral or intravenous administration of the drug.
Another advantage of the invention is that RASSLs can be designed from G protein-coupled receptors which bind synthetic small molecule that have been developed previously for use in mammalian subjects (e.g., for uses as such as pain relief, reduction of depression and weight reduction).
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the vectors, cell lines and methodology as more fully set forth below.
REFERENCES:
patent: 5364791 (1994-11-01), Vegeto et al.
patent: 5464758 (1995-11-01), Gossen et al.
patent: 0 351 921 (1990-01-01), None
patent: 0 453 119 (1991-10-01), None
Mastrangelo et. al., Gene Therapy for Human Cancer: An Essay for Clinicians, Feb. 1996, Seminars in Oncology, vol. 23, No. 1, pp. 4-21.*
Verma et al., Gene therapy-promises, problems and prospects, Sep. 18, 1997, Nature, vol. 389, pp. 239-242.*
Anderson, Human gene therapy, Apr. 30, 1998, Nature, vol. 392, pp. 25-30.*
Heistad et al., Gene Therapy for Cerbral Vascular Disease, Sep. 1996, Stroke, vol. 27, pp. 1688-1693.*
Bork et al., Predicting functions from protein sequences-whrer are the bottlenecks?, Apr. 1998, Nature Genetics, vol. 18, pp. 313-318.*
Eck et al., Gene-Based Therapy, 1996, Goodman & Gilman's, Ninth Edition, Chapter 5, pp. 77-101.*
Ngo et al., Computational Complexity, Protein Structure Prediction and the Levinthal Paradox, 1994, Birkhauser Boston, pp. 491-495.*
Pa
Borden Paula A.
Bozicevic Field & Francis LLP
Francis Carol L.
Nguyen Dave T.
The Regents of the University of California
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