Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-06
2003-01-07
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S455000, C435S320100, C536S023500
Reexamination Certificate
active
06503717
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
The present invention relates to methods of using libraries of randomized zinc finger proteins to identify genes associated with selected phenotypes.
BACKGROUND OF THE INVENTION
A. Using Libraries to Identify Genes Associated with a Selected Phenotype
Identification of gene function is a critical step in the selection of new molecular targets for drug discovery, gene therapy, clinical diagnostics, agrochemical discovery, engineering of transgenic plants, e.g., with novel resistance traits or enhanced nutritional characteristics, and genetic engineering of prokaryotes and higher organisms for the production of industrial chemicals, biochemicals, and chemical intermediates. Historically, library screening methods have been used to screen large numbers of uncharacterized genes to identify a gene or genes associated with a particular phenotype, e.g., hybridization screening of nucleic acid libraries, antibody screening of expression libraries, and phenotypic screening of libraries.
For example, molecular markers that co-segregate with a disease trait in a segment of patients can be used as nucleic acid probes to identify, in a library, the gene associated with the disease. In another method, differential gene expression in cells and nucleic acid subtraction can be used to identify and clone genes associated with a phenotype in the test cells, where the control cells do not display the phenotype. However, these methods are laborious because the screening step relies heavily on conventional nucleic acid cloning and sequencing techniques. Development of high throughput screening assays using these methods would therefore be cumbersome.
An example of phenotypic screening of libraries is discovery of transforming oncogenes (see, e.g., Goldfarb et al.,
Nature
296:404 (1982)). Oncogenic transformation can be observed in NIH 3T3 cells by assaying for loss of contact inhibition and foci formation. cDNA expression libraries from transformed cells are introduced into untransformed cells, and the cells were examined for foci formation. The gene associated with transformation is isolated by clonal propagation and rescue of the expression vector. Unfortunately, this method is limited by phenotype and can only be used to assay for transdominant genes.
Advances in the field of high throughput screening have increased the cell types and phenotypes that can be investigated using library screening methods. Viral vectors such as retroviral, adenoviral, and adenoviral associated vectors have been developed for efficient nucleic acid delivery to cells (see, e.g., U.S. Pat. No. 5,173,414; Tratschin et al.,
Mol. Cell. Biol.
5:3251-3260 (1985); Tratschin, et al.,
Mol. Cell. Biol.
4:2072-2081 (1984); Hermonat & Muzyczka,
Proc. Nat'l Acad. Sci. USA
81:6466-6470 (1984); and Samulski et al.,
J. Virol.
63:03822-3828 (1989); Buchscher et al.,
J. Virol.
66:2731-2739 (1992); Johann et al.,
J. Virol.
66:1635-1640 (1992); Sommerfelt et al.,
Virol.
176:58-59 (1990); Wilson et al.,
J. Virol.
63:2374-2378 (1989); Miller et al.,
J. Virol.
65:2220-2224 (1991); and PCT/US94/05700). Cells can be phenotypically analyzed either one at a time, using flow cytometry, or in arrayed clonal populations, using liquid handling robots. These techniques allow a sufficient number of library members to be tested for a wide range of potential phenotypes.
Currently, libraries of random molecules are being used with phenotypic screening for the discovery of genes associated with a particular phenotype. For example, random peptide or protein expression libraries are being used to block specific protein-protein interactions and produce a particular phenotype (see, e.g., Caponigro et al.,
Proc. Nat'l Acad. Sci USA
95:7508-7513 (1998); WO 97/27213; and WO 97 27212). In another method, random antisense nucleic acids or ribozymes are used to inactivate a gene and produce a desired phenotype (see, e.g., WO 99/41371 and Hannon et al.,
Science
283:1125-1126 (1999)).
The main shortcoming of these methods is the inherent inefficiency of the random molecules, which vastly increases the size of the library to be screened. Even with a known target nucleic acid or protein, literally hundreds of antisense, ribozyme, or peptide molecules must be empirically tested before identifying one that will inhibit gene expression or protein-protein interactions. Since the random library must be enormous to produce sufficient numbers of active molecules, huge numbers of cells must be screened for phenotypic changes. For unknown gene and protein targets, the rarity of effective, bioactive peptides, antisense molecules, or ribozyme molecules imposes significant constraints on high throughput screening assays. Furthermore, these methods can be used only for inhibition of gene expression, but not for activation of gene expression. This feature limits identification of gene function to phenotypes present only in the absence of gene expression.
Therefore, efficient high throughput library screening methods allowing random inhibition or activation of uncharacterized genes would be of great utility to the scientific community. These methods would find widespread use in academic laboratories, pharmaceutical companies, genomics companies, agricultural companies, chemical companies, and in the biotechnology industry.
B. Zinc Finger Proteins as Transcriptional Regulators
Zinc finger proteins (“ZFPs”) are proteins that bind to DNA in a sequence-specific manner and are typically involved in transcription regulation. Zinc finger proteins are widespread in eukaryotic cells. An exemplary motif characterizing one class of these proteins (the Cys
2
His
2
class) is -Cys-(X)
2-4
-Cys-(X)
12
-His-(X)
3-5
-His (SEQ ID NO:1) (where X is any amino acid). A single finger domain is about 30 amino acids in length and several structural studies have demonstrated that it contains an alpha helix containing the two invariant histidine residues co-ordinated through zinc with the two cysteines of a single beta turn. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger proteins are involved not only in DNA-recognition, but also in RNA binding and protein-protein binding. Current estimates are that this class of molecules will constitute the products of about 2% of all human genes.
The X-ray crystal structure of Zif268, a three-finger domain from a murine transcription factor, has been solved in complex with its cognate DNA-sequence and shows that each finger can be superimposed on the next by a periodic rotation and translation of the finger along the main DNA axis. The structure suggests that each finger interacts independently with DNA over 3 base-pair intervals, with side-chains at positions-1, 2, 3 and 6 on each recognition helix making contacts with respective DNA triplet sub-site.
The structure of the Zif268-DNA complex also suggested that the DNA sequence specificity of a zinc finger protein could be altered by making amino acid substitutions at the four helix positions (-1, 2, 3 and 6) on a zinc finger recognition helix, using, e.g., phage display experiments (see, e.g., Rebar et al.,
Science
263:671-673 (1994); Jamieson et al.,
Biochemistry
33:5689-5695 (1994); Choo et al.,
Proc. Natl. Acad. Sci. U.S.A.
91:11163-11167 (1994); Greisman & Pabo,
Science
275:657-661 (1997)). For example, combinatorial libraries were constructed with zinc finger proteins randomized in either the first or middle finger. The randomized zinc finger proteins were then isolated with altered target sites in which the appropriate DNA sub-site was replaced by an altered DNA triplet. Correlation between the nature of introduced mutations and the resulting alteration in binding specificity gave rise to a set of substitution rules for rational design of zinc finger proteins with altered binding specificity. These experiments thus demonstrated that randomized zinc finge
Case Casey C.
Liu Qiang
Rebar Edward J.
Wolffe Alan P.
Brennan Sean M.
Brusca John S.
Robins & Pasternak LLP
Sangamo Biosciences, Inc.
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