AU2005234684B2 - Regulation of gene expression using single-chain, monomeric, ligand dependent polypeptide switches - Google Patents

Description

P/00/01I Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

(ORIGINAL)

Name of Applicant(s): Actual Inventor(s): The Scripps Research Institute, of 10550 North Torrey Pines Road, La Jolla, California 92037, UNITED STATES OF

AMERICA

BEERLI, Roger SCHOPFER, Ulrich BARBAS, Carlos, F Address for Service: DAVIES COLLISON CAVE, Patent Trademark Attorneys, of 1 Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM "Regulation of gene expression using single-chain, monomeric, ligand dependent polypeptide switches" Invention Title: The following statement is a full description of this invention, including the best method of performing it known to us:- 0 Regulation of Gene Expression Using Single-Chain, Monomeric, C Ligand Dependent Polypeptide Switches

O

3Z SThis application is a divisional of Australian Patent Application No.

00 2001272544, the entire contents of which are incorporated herein by reference.

S 5 Technical Field of the Invention 00 The field of this invention is regulation of transcription. More particularly, the present invention pertains to polypeptides that can activate or repress transcription in a small molecule ligand-dependent manner.

Background of the Invention Designed transcription factors with defined target specificity and regulatory function provide invaluable tools for basic and applied research, and for gene therapy. Accordingly, the design of sequence-specific DNA binding domains has been the subject of intense interest for the last two decades. Of the many classes of DNA binding proteins studied, the modular Cys2 -Hia zinc finger DNA binding motif has shown the most promise for the production of proteins with tailored DNA binding specificity. The novel architecture of this class of proteins provides for the rapid construction of gene-specific targeting devices.

Polydactyl zinc finger proteins are most readily prepared by assembly of modular zinc finger domains recognizing predefined three-nucleotide sequences (See. eg., Segal, D. Dreier, Beerli, R. and Barbas, C. II (1999) -me. att Acad Sc. USi96, 2758-2763; Beerli, R. Segal, D. Dreier, and Barbas, C. F., HI (1998) roe. tL Acad Scd S 1995, 14628-14633; and Beerli, R. L, Dreier, B., and Barbas, C. M (2000) Pac. 9jC4cadl ScL. lUS97, 1495-1500). Polydactyl proteins can be assembled using variable numbers of zinc finger domains of varied specificity providing DNA binding proteins that not only recognize novel sequences but also sequences of varied length. By combining six zinc finger domains, proteins have been produced that recognize 18 contiguous base pairs of DNA sequence, a DNA address sufficiently complex to specify any locus in the 4 billion-base pair human genome (or any other genome). Fusion of polydactyl zinc finger proteins of this type to activation or repression domains provides c transcription factors that efficiently and specifically modulate the expression of O both transgenes and endogenous genes (Beerli, R Segal, D. Dreier, and Z Barbas, C. M (1998) Proc. Nat9.Acrad ScSd U 95, 14628-14633; and Beerli, R.

Dreier, and Barbas, C. IM (2000) roc. NatL Acad. Sci. U 97, 1495- 1500).

00 While the availability of designed transcription factors with tailored DNA binding specificities provides novel opportunities in transcriptional regulation, C additional applications would be available to ligand-dependent transcription Sfactors. Designer zinc finger proteins dependent on small molecule inducers C- 10 would have a number of applications, both for the regulation of endogenous genes, and for the development of inducible expression systems for the regulation of transgenes. Natural transcription factors are regulated by a number of different mechanisms, including postranslational modification such as phosphorylation (Janknecht, and Hunter, T. (1997) 'EMBOJI6, 1620-1627; Darnell, J. Jr.

(1997) Sdence277, 1630-1635), or by ligand binding. The prototype ligandactivated transcription factors are members of the nuclear hormone receptor family, including the receptors for sex steroids or adrenocorticoids (Carson- Jurica, M. Schrader, W. and OMalley, W. (1990) Endocrine piews 11, 201-220; Evans, R. M. (1988) Science240 889-895). These receptors are held inactive in the absence of hormone, by association with a number of inactivating factors including hsp90 (Pratt, W. and Toft, D. 0. (1997) Endurine So. 18, 306-360). Upon ligand binding, nuclear hormone receptors dissociate from the inactivating complex, dimerize, and become able to bind DNA and activate transcription (Carson-Jurica, M. Schrader, W. and O'Malley, W. (1990) 'Endoaine SYews11, 201-220; Evans, R. M. (1988) Sience240, 889-89512-14; and Pratt, W. and Toft, D. 0. (1997) Endocrine 18, 306-360). Significantly, not only hormone binding but also inactivation and dimerization functions reside within the ligand binding domain (LBD) of these proteins (Beato, M. (1989) CeK 56, 335-344). This fact has been exploited experimentally and steroid hormone receptor LBDs have found wide use as tools to render heterologous proteins hormone-dependent Cl In particular, the estrogen receptor (ER) LBD has been used to render the o functions of c-Myc (Eilers, Picard, Yamnamoto, K. and Bishop, J. M.

Z (1989) NAature 340, 66-68), c-Fos (Superti-Furga, Bergers, Picard, and 00 Busslinger, M. (1991) !Proc. NtI .Acad Sd~ US$7188, 5114-5118), and even the cytoplasmic kinase c-Raf Samuels, M. Weber, M. Bishop, J. and McMahon, M. (1993) !Mot CeIZ Diot 13, 6241-6252) hormone-dependent. To 00 develop an inducible expression system for use in basic research and gene therapy, the availability of ligand-dependent transcriptional regulators is a prerequisite. Preferentially, these regulators would be activated by a small molecule inducer with no other biological activity, bind specific sequences present only in the target promoter, and have low inimunogenicity. A number of ligand-regulated artificial transcription factors have been generated by various means, using functional domains derived from either prokaryotes (Gossen, M., and Bujard, H. (1 992) !Pro. a LtC iAcai .ScL 'US 89, 5547-5551 20. Gossen, M., Freundlieb, Bender, Mfiller, Hillen, and Bujard, H. (1995) Scienc 268, 1766-1769 21. Labow, M. Bairn, S. Shenkc, and Levine, A. J.

(1990) Mot Ce(Z !Biot 10, 3343-3356 22. Baim, S. Labow, M. Levine, A. J., and Shenk, T. (1991) Twc. NajtC Acadf SL 'USA488, 5072-5076) or eukaryotes (Christopherson, K Mark, M. Bajaj, and Godowski, P. J. (1992) !Proc.

Nzt.LcAwd ScL. ULq 89, 6314-6318 24. No, Yao, and Evans, R. M.

(1996) iPrc. NajtLt4cad Sci 'USq93p 3346-3351 25. Wang, O'Malley, B. W., Jr., Tsai, and O'Malley, B. W. (1994) !Proc Nt4 A:icad 3d. VLS$791, 8180-8184 Beerli et at -3 5-26. Wang, Xu, Pierson, O'Malley, B. and Tsai, S.

Y. (1997) Gene 27ierap!,4, 432-441 27. Braselmann, Graninger, and Busslinger, M. (1993) !Proc. Ntt Acad Sd US,%90, 1657-1661 28. Louvion, J. F., Havaux-Copf, and Picard, D. (1993) Gene 131, 129-134 29. Rivera, V. M., Clackson, Natesan, Pollock, Amara, 3. Keenan, Magari, S. R., Phillips, Courage, N. Cerasoli, Jr., Holt, DA and Gilman, M. (1996) Nture. Med 2, 1028-1032).

Of the functional domains derived from eukaryotic proteins, nuclear hormone receptor LBDs have been the most widely used. in particular, regulators based on the Gal4 DNA binding domain (DBD) fused to a human ER 0(Braselmann, Graninger, and Busslinger, M.L (1993) !Thvc. 9gtC Aca,4Sd 1657-1661; Louvion, J. Havaux-Copf, and Picard, D. (1993) Gen 131, 129-134 or progesterone receptor (PR) LBD; (Wang, Omalley, B. W., Jr., Tsai, and O'Malley, B. W. (1994) Tivc. NatjtL4cad 3d USA,91, 8180-8184; 00 Wang, Xu, Pierson, O'Malley, B. and Tsai, S. Y. (1997) Geme '7hcapy 4, 432-44 as well as the ecdysone-inducible system based on the ~Drosophfia ecdysone receptor (EcR) and the mamnnalian retinoid X receptor (RXR) (Christopherson, K. Mark, M. Bajaj, and Godowski, P. J. (1992) Proc.

jztt:Acad 3d 'U.SA89, 6314-6318; No, Yao, and Evans, R. M. (1996) Troc. Nof~tC Aca4 S. 'USAZ 93, 3346-335 1) have been described. Compared to the heterodimeric EcR/RXR system, regulators based on the ER and PR LBDs have the important advantage that they function as homodimers and require the delivery of only one cDNA. However, while ecdysone has no known biological effect on maninialian cells, estrogen and progesterone will elicit a biological response in cells or tissues that express the endogenous steroid receptors. With the availability of a mutated ER and a truncated PR LBDs that have lost responsiveness to their natural ligands but not to synthetic antagonists such as 4hydroxytanioxifen (4-O1T) (Littlewood, T. Hancock D. Danielian, P. S., Parker, M. and Evan, (1995) Nyd cds 23, 1686-1690 or RU486 (Vegeto, Allan, G. Schrader, W. Tsai, McDonnell, D. and O Malley, B. W. (1992) CeM6, 703-713), respectively, this is no longer of great concern. Thus, steroid hormone receptor LBD-based inducible expression systems can be developed that function independently of the endogenous steroid receptors.

To date, this has been shown for the PR LBD through the development of an RU486-inducible expression system based on the Gal4 DBD (Wang, Y., O'Malley, B. Jr., Tsai, and O7Valley, B. W. (1994) -wiv.octAcad. ci, 'USql 91, 8180-8.184; Wang, Xu, Pierson, O'Malley, B. and Tsai, S.

Y. (1997) Gme Tiaeapy 4, 432-44 An inducible expression system based on a point-mutated (G525R) ER LBD (Littlewood, T. Hancock, D. Danielian, P. Parker, M. and Evan, G. L (1995) N~ cidds!R94m 23, 1686-1690) that c N has lost the responsiveness to estrogen but not the antagonist 4-OHT has not been O described to date. Designed zinc finger proteins have a number of advantages as Z compared to other DBDs, including the one derived from Gal4, since the ability to 00 engineer DNA binding specificities allows ligand-dependent regulators to be directed to any desired artificial or natural promoter. Here we explore the utility 0 of fusion proteins between designed zinc finger proteins and nuclear hormone receptor LBDs for the inducible control of gene expression.

Brief Summary of the Invention In one embodiment, the present invention provides a non-naturally occurring polypeptide that contains two ligand binding domains operatively linked to each other and a first functional domain operatively linked to one of the ligand binding domains. The ligand binding domains are preferably covalently linked to each other. More preferably, the two binding domains are covalently linked by means of a peptide linker that contains from about 10 to about 40 amino acid residues, preferably from about 15 to about 35 amino acid residues and, more preferably from about 18 to about 30 amino acid residues.

In one embodiment, the ligand binding domains are derived from nuclear hormone receptors. The ligand binding domains can be derived from the same or different nuclear hormone receptors. Exemplary and preferred nuclear hormone receptors are steroid hormone receptors such as an estrogen receptor, a progesterone receptor, an ecdysone receptor and a retinoid X receptor.

The first functional domain can be any domain that alters the function or activity of a target nucleotide. In one embodiment, the first functional domain is a nucleotide binding domain. Preferably, the nucleotide binding domain is a DNA binding domain. The DNA binding domain preferably contains at least one zinc finger DNA binding motif, more preferably from two to twelve zinc finger DNA binding motifs and, even more preferably from three to six zinc finger DNA binding motifs. In one embodiment, the zinc finger DNA binding motifs specifically bind to a nucleotide sequence of the formula (GNN)I-6. where G is guanidine and N is any nucleotide. In another embodiment, the first functional C domain is a transcriptional regulating domain such as a transcription activation O domain or a transcription repression domain.

Z In still another embodiment, the polypeptide gene switch contains a 00 second functional domain. In accordance with this embodiment, a preferred first functional domain is a nucleotide binding domain and the second functional 00 domain is a transcriptional regulating domain.

In one embodiment, a polypeptide of this invention includes a DNA C- binding domain having from three to six zinc finger DNA binding motifs; a Sfirst ligand binding domain derived from a retinoid X receptor operative linked to CN 10 the DNA binding domain, a second ligand binding domain derived from an ecdyzone receptor linked to the first ligand binding domain with a peptide spacer of from 18 to 36 amino acid residues; and a transcription regulating domain operatively linked to the second binding domain.

In still another embodiment, a polypeptide gene switch includes a DNA binding domain having from three to six zinc finger DNA binding motifs; a first ligand binding domain derived from a progesterone receptor operatively linked to the DNA binding domain, a second ligand binding domain derived from a progesterone receptor linked to the first ligand binding domain with a peptide spacer of from 18 to 36 amino acid residues; and a transcription regulating domain operatively linked to the second ligand binding domain.

In another aspect, the present invention provides polynucleotides that encode a polypeptide gene switch of the invention, expression vectors containing such polynucleotides and cells containing such nucleotides.

Another aspect of this invention provides a process of regulating the function of a target nucleotide that contains a defined sequence. The process includes the step of exposing the target nucleotide to a polypeptide of this invention in the presence of a ligand that binds at least one of the ligand binding domains of the polypeptide. In a related aspect, the present invention provides a process for regulating transcription expression) of a target nucleotide gene). In accordance with that process a target nucleotide that contains a defined sequence is exposed to a polypeptide of this invention in the presence of a ligand c' that binds to at least one of the ligand binding domains of that polypeptide. The Spolypeptide contains a nucleotide binding domain that specifically binds to the Z defined sequence in the target nucleotide. Where the polypeptide gene switch 00 O contains a transcription repression domain, regulating is repression. Where the polypeptide gene switch contains a transcription activation domain, regulating is activation.

oO M Brief Description of the Drawings In the drawings that form a portion of the specification Figure 1 shows generation of designed zinc finger proteins with novel DNA binding specificity. A, amino acid sequence of the three-finger proteins B3 and N1. DNA recognition helix positions -2 to 6, shown in bold print, were grafted into the framework of the three finger protein SplC. The location of the antiparallel P sheets and the a helices, structural hallmarks of zinc finger domains, are as indicated. DNA binding specificity of each finger is show on the left. F1-3, Finger 1-3. B, ELISA analysis of DNA binding specificity. Zinc finger proteins were expressed in E. coli as MBP fusions and purified. Specificity of binding was analyzed by measuring binding to immobilized biotinylated hairpin oligonucleotides containing the indicated 5'-(GNN)3-3'sequences. Black bars, B3; gray bars, N1. The maximal signals were normalized to 1. The KD value for binding to the specific target sequence was measured by electrophoretic mobility shift assay and is labeled on top of the corresponding bars.

Figure 2 shows regulation of gene expression by hormone-dependent, single-chain ER fusion constructs. A, structure of ER fusion proteins. E2C, six finger protein; L, flexible peptide linker. B, fusion proteins with a single ER-LBD bind as dimers. HeLa cells were cotransfected with a C7-ER-VP64 expression vector, and the indicated TATA luciferase reporter plasmids carrying either one or two C7 binding sites. 24 h after transfection, cells were either left untreated or 100 nM 4-OHT was added Luciferase activity in total cell extracts was measured 48 h after transfection. Each bar represents the mean value SD) of duplicate measurements. C, control plasmid pcDNA3 that does not express a

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fusion protein. C, D,-regulation of transcription through a single binding site by z fusion proteins with two ER-LBDs. HeLa cells were cotransfected with the QO indicated expression vectors and the E2C-TATA-luciferase reporter plasmid, carrying a single E2C binding site upstream of a TATA box. 4-OHT induction and measurement of luciferase activity was carried out as described in B.

0O Figure 3 shows regulation of gene expression by hormone-dependent, single-chain RXR/EcR fusion constructs. A, structure of single-chain RXR/EcR fusion proteins. B, regulation of transcription through a single binding site. HeLa cells were cotransfected with the indicated expression vectors and the E2C- TATA-luciferase reporter plasmid, carrying a single E2C binding site: 24 h after transfection, cells were either left untreated or 5 pM Ponasterone A was added Luciferase activity in total cell extracts was measured 48 h after transfection.

Each bar represents the mean value SD) of duplicate measurements.

pcDNA3.1, control plasmid that does not express a fusion protein.

Figure 4 shows the nucleotide (SEQ ID NO: 31) and amino acid residue sequence (SEQ ID NO: 32) of zinc finger binding domain B3B.

Figure 5 shows the nucleotide (SEQ ID NO: 33) and amino acid residue sequence (SEQ ID NO: 34) of zinc finger binding domain 2C7.

Figure 6 shows the nucleotide (SEQ ID NO: 35) and amino acid residue sequence (SEQ ID NO: 36) of zinc finger binding domain B3C2.

Figure 7 shows the nucleotide (SEQ ID NO: 37) sequence of repression domain (KRAB-A) 2 Figure 8 shows the nucleotide (SEQ ID NO: 38) sequence of repression domain (SID) 2 Figure 9 shows the nucleotide (SEQ ID NO: 39) and amino acid residue sequence (SEQ ID NO: 40) ofpolypeptide E2C-ER-L-ER-VP64.

Figure 10 shows the nucleotide (SEQ ID NO: 41) and amino acid residue sequence (SEQ ID NO: 42) of polypeptide E2C-ER-LL-ER-VP64.

o Detailed Description of the Invention ZI. The Invention 00 The present invention provides polypeptide gene switches, polynucleotides that encode such polypeptides, expression vectors that contain 0 such polynucleotides, cells that contain such expression vectors or polynucleotides and processes for regulating target nucleotide function using such polypeptides, polynucleotides and expression vectors. Unlike existing gene Sswitches that contain a single ligand binding domain together with a DNA binding 10 domain and/or a transcriptional regulating domain, polypeptide gene switches of the present invention contain two ligand binding domains. Upon binding of the ligand, an intramolecular configuration change occurs that allows for alignment of the functional domains to the target gene of interest. An advantage of the present gene switches, therefore, over existing gene switches is the need for only a single molecular switch and a single expression vector for production of that switch.

II. Polvpeptides A polypeptide gene switch of the present invention includes at least three components: two ligand binding domains (LBDs) and a first functional domain The ligand binding domains are operatively linked to the first functional domain such that the polypeptide, in the presence of a defined ligand that binds to at least one of the ligand binding domains, can alter the function of nucleotide.

The domains can be arranged in any order. As shown below, the ligand binding domains can be situated in either the amino-or carboxyl-terminal direction from the first functional domain.

LBDs FD-1 FD-1 LBDs A polypeptide of this invention is non-naturally occurring. As used herein, the term "non-naturally occurring" means, for example, one or more of the c1 following: a peptide comprised of a non-naturally occurring amino acid O sequence; a peptide having a non-naturally occurring secondary structure not Z associated with the peptide as it occurs in nature; a peptide which includes one 00 or more amino acids not normally associated with the species of organism in which that peptide occurs in nature; a peptide which includes a stereoisomer oO of one or more of the amino acids comprising the peptide, which stereoisomer is 00 Snot associated with the peptide as it occurs in nature; a peptide which includes "M one or more chemical moieties other than one of the natural amino acids; or an isolated portion of a naturally occurring amino acid sequence a truncated N 10 sequence). A polypeptide of this invention exists in an isolated form and purified to be substantially free of contaminating substances. A polypeptide is synthetic in nature. That is, the polypeptide is isolated and purified from natural sources or made de novo using techniques well known in the art.

A. Ligand Binding Domain (LBD) Each LBD is an amino acid residue sequence that is capable of and binds a particular ligand. Binding of the ligand to the LBD alters the conformation/function of the polypeptide and allows for regulating a function of a target nucleotide. In the absence of ligand, the gene switch does not work to alter nucleotide function. At least one of the LBDs is capable of binding and binds a particular ligand. Both LBDs can bind a particular ligand. Thus, the LBDs can be the same or different. Preferred LBDs are derived from nuclear hormone receptors such as steroid hormone receptors.

Exemplary and preferred steroid receptors that can serve as the source of ligand binding domains include the estrogen receptor progesterone receptor glucocorticoid-a receptor, glucocorticoid-P receptor, mineralocorticoid receptor, androgen receptor, thyroid hormone receptor, retinoic acid receptor (RAR), retinoid X receptor (RXR), Vitamin D receptor, COUP-TF receptor, ecdysone receptor (EcR), Nurr-1 receptor and orphan receptors. A preferred EcR is derived either from Drosophila melanogaster (DE) or Bombyx (BE).

As is well known in the art, steroid hormone are composed of a DNA binding domain and a ligand binding domain. The DNA binding domain contains c-i the receptor regulating sequence and binds DNA and the ligand binding domain binds the specific biological compound (ligand) to activate the receptor. The term Z "lgand" refers to any compound which Activates the receptor, usually by OC) interaction with (binding) the ligand binding domain of the receptor. However, ligands also include compounds that activate the receptor without binding. Where us) used in a polypeptide gene switch of the present invention, it is preferred that the ligand receptor domain be modified from its naturally occurring ligand, a ligand other than the naturally occurring ligand (e.g.steroid hormone). Means of altering or derivatizing naturally occurring nuclear hormone receptor ligand binding domains to alter the binding specificity are well known in the art (See, e.g. United States Patent Nos. 5,874,534 and 5,599,904 the disclosures of which are incorporated herein by reference). Similarly, means for altering the estrogen receptor to change its bind affinity have reported [See. e.g. Littlewood et al., Nucleic Acids Res., 3(1 0):1 686-1690,1995].

The term "naturally occurring ligand" refers to compounds that are normally not found in animals or humans and which bind to the ligand binding domain of a receptor. The ligand can also be a "non-native ligand", a ligand that is not naturally found in the specific organism (man or animal) in which gene therapy is contemplated. For example, certain insect hormones such as ecdysone are not found in humans. This is an example of a non-native hormone to the animal or human.

Examples of non-natural ligands, and-hormones and non-native ligands include the following: I1 I -(4-dimetiylanminophenyl)- I 7J3-hydroxy-17 7c-propinyl- 4,9-e stradiene-3-one (Ru3 8486 or Mfepestone); I lIo-(4-dimethylaminophenyl)- 1 7c-hydroxy- 17 71-(3-hydroxypropyl)- I 3 cc-methyl-4,9-gonadiene-3 -one (ZK98299 or Onapristone); 11 P-(4-acetylphenyl)- I 7J3-hydroxy- 1 7cc-(I -propinyl)- 4,9-estradiene-3-one (ZK 112993); 11 1-(4-dimenthylaminophenyl)-l 7p-hydroxy- I 7a-(3-hydroxy- 1 (Z)-propenyl-estra-4,9-diene-3 -one (ZK9873 (70311 P 1 7J)- 11 -(4-dimethylaminophenyl)-7-methyl-4' dihydrospiroy'ester-4,9-diene- 17,2'(3'H)-furan !-3-one (Org3 1806); (1 1I,4,7a)A4',5'-dihydro-l 1-(4dimethylaniinophenyl)y' spi rostra-4,9-diene-17,2'(3'H}.furan !-3-one (Org3 1376); N 5-alpha-pregnane-3,2-dione. Additional non-natural ligands include, in general, synthetic non-steroidal estrogenic or anti-estrogenic compounds, broadly defined 00 as selective estrogen receptor modulators (SERMS). Exemplary compounds include, but are not limited to, tamoxifen and raloxifen, Exemplary and preferred ligands for use with various ligand binding 0 domains are EcR: Ponasterone a, Muristerone A, GS-E (Invitrogen), Tebufenocide; ER: estrogen antagonists such as 4 -hydroxy-tamoxifen,

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164384, RU 54876, Raloxifene; and PR: progesterone antagonists such as RU 0 486, RU 38486, and Onapristone.

N 10 An especially preferred LBD derived from a progesterone receptor comprises amino acid residues 645-914 from the human progesterone receptor.

An exemplary LBD derived from an estrogen receptor comprises amino acid residues 282-599 from the mouse G225R mutant.

The two LBDs are separated be an amino acid residue sequence linker that contains from about 10 to about 50 amino acid residues. Preferably, the spacer contains from about 15 to about 40 amino acid residues and, more preferably, from about 18 to about 35 amino acid residues. Exemplary and preferred spacers contain 18 30 or 36 (LLL) amino acid residues.

B. Functional Domains A second component of a present polypeptide is a functional domain. As used herein, the term "functional domain" and it's grammatical equivalents, means an amino acid residue sequence that binds to, alter the structure of, and/or alters the function of, a nucleotide. Exemplary such functional domains include nucleotide binding domains, transcriptional regulating domains transcription activation domains and transcription repression domains) and domains having nuclease activity. Such domains are well known in the art.

1. Nucleotide Binding Domains A functional domain of a polypeptide can be a nucleotide binding domain: a sequence of amino acid residues that recognize and bind to a defined nucleotide sequence. The target nucleotide sequence can be an RNA sequence or, preferably, a DNA sequence. Amino acid residue sequences that recognize and C bind to defined DNA sequences are well known in the art GAL4). Any such o DNA binding peptide can be used as a DNA binding domain of a polypeptide gene switch of this invention. It is preferred, however, that the DNA binding 00 domain of a present gene switch be one or more DNA binding zinc finger motifs.

Such zinc finger DNA binding motifs are well known in the art (See. PCT o0 Patent Application Nos. W095/19421 and WO 98/54311, the disclosures of which are incorporated herein by reference). A DNA binding domain of a C polypeptide gene switch of this invention, thus, preferably includes a multiple 0 finger, polydactyl, zinc finger peptide that is designed to bind specific nucleotide N 10 target sequences.

The present disclosure is based on the recognition of the structural features unique to the Cys 2 -His 2 zinc finger domain consist of a simple 3pot fold of approximately 30 amino acids in length. Structural stability of this fold is achieved by hydrophobic interactions and by chelation of a single zinc ion by the conserved Cys2-His 2 residues (Lee, Gippert, Soman, Case, D.A.

Wright, P.E. (1989) Science 245, 635-637). Nucleic acid recognition is achieved through specific amino acid side chain contacts originating from the orhelix of the domain, which typically binds three base pairs of DNA sequence (Pavletich, N. P. Pabo, C.O. (1991) Science 252, 809-17, Elrod-Erickson,

M.,

Rould, Nekludova, L. Pabo, C.O. (1996) Structure 4, 1171-1180).

Unlike other nucleic acid recognition motifs, simple covalent linkage of multiple zinc finger domains allows the recognition of extended asymmetric sequences of

DNA.

Studies of natural zinc finger proteins have shown that three zinc finger domains can bind 9 bp of contiguous DNA sequence (Pavletich, N.P. Pabo, C.O. (1991) Science 252, 809-17., Swimoff, A.H. Milbrandt, J. (1995) Mol.

Cell. Biol. 15, 2275-87). Whereas recognition of 9 bp of sequence is insufficient to specify a unique site within even the small genome of E.coli, polydactyl proteins containing six zinc fingers domains can specify 18-bp recognition (Liu, Segal, DJ., Ghiara, J.B. Barbas I, C.F. (1997) Proc. Natl. Acad. Sci. USA 94, 5525-5530). With respect to the development of a universal system for gene control, and 18-bp address can be sufficient to specify a single site within all 0 known genomes. And their efficacy in gene activation and repression within oO living human cells has recently been shown (Liu, Segal, DJ., Ghiara, J.B. Barbas l, C.F. (1997) Proc. Natl. Acad. Sci. USA 94, 5525-5530).

The zinc finger-nucleotide binding peptide domain can be derived or o0 produced from a wild type zinc finger protein by truncation or expansion, or as a variant of the wild type-derived polypeptide by a process of site directed I mutagenesis, or by a combination of the procedures. The term "truncated" refers to a zinc finger-nucleotide binding polypeptide that contains less that the full N1 10 number of zinc fingers found in the native zinc finger binding protein or that has been deleted of non-desired sequences. For example, truncation of the zinc fingernucleotide binding protein TFIIIA, which naturally contains nine zinc fingers, might be a polypeptide with only zinc fingers one through three. Expansion refers to a zinc finger polypeptide to which additional zinc finger modules have been added. For example, TFIIIA may be extended to 12 fingers by adding 3 zinc finger modules from more than one wild type polypeptide, thus resulting in a "hybrid" zinc finger-nucleotide binding polypeptide.

The term "mutagenized" refers to a zinc finger derived-nucleotide binding polypeptide that has been obtained by performing any of the known methods for accomplishing random or site-directed mutagenesis of the DNA encoding proteins. For instance, in TFIIIA, mutagenesis can be preformed to replace nonconserved residues in one or more of the repeats of the consensus sequence.

Truncated zinc finger-nucleotide binding proteins can also be mutagenized.

Examples of known zinc finger-nucleotide binding proteins can also be mutagenized. Examples of known zinc finger-nucleotide binding polypeptides that can be truncated, expanded, and/or mutagenized according to the present invention in order to inhibit the function of a nucleotide sequence containing a zinc finger-nucleotide binding motif includes TFfllA and zif268. Other zinc finger-nucleotide binding proteins will be known to those of skill in the art.

A zinc finger DNA binding domain can be make using a variety of standard techniques well known in the art. Phage display libraries of zinc finger proteins were created and selected under conditions that favored enrichment of O sequence specific proteins. Zinc finger domains recognizing a number of Z sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information.

A DNA binding domain used in a polypeptide of this invention is o preferably a zinc finger-nucleotide binding peptide that binds to a (GNN) 1 6 nucleotide sequence. Zinc fingers that bind specifically to (GNN)I.

6 have been Sdescribed in United States Patent Application Serial Number 09/173,941, filed 0 October 16,1998 (the disclosure of which is incorporated herein by reference).

C 10 Exemplary and preferred zinc finger DNA binding domains are designated herein as E2C, C7, B3B, 2C7, B3C2 and N1. A detailed description of the preparation of polypeptide gene switches containing zinc finger DNA binding domains can be found hereinafter in the Examples. The amino acid residue and encoding nucleotide sequences for B3B, 2C7 and B3C2 are shown in FIGs. 4-6, respectively.

2. Transcription Regulating Domains A transcription regulating domain refers to a peptide, which acts to activate or repress transcription of a target nucleotide gene). Transcriptional activation domains are well known in the art (See. Seipel et al., (1992) EMBO 11:4961-4968). Exemplary and preferred transcription activation domains include VP16, TA2, VP64, STAT6, relA, TAF-1, TAF-2, TAU-1 and TAU-2. Especially preferred activation domains for use in the present invention are VP16 and VP64. Means for linking VP16 and VP64 to ligand binding domains are set forth hereinafter in the Examples.

Transcriptional repressor domains are also well known in the art.

Exemplary and preferred such transcriptional repressors are ERD, KRAB, SID, histone deacetylase, DNA, methylase, and derivatives, multimers and combinations thereof such as KRAB-ERD, SID-ERD, (KRAB) 2

(KRAB)

3 KRAB-A, (KRAB-A) 2 (SID)2, (KRAB-A)-SID and SID-(KRAB-A). A first repressor domain can be prepared using the Krmppel-associated box (KRAB) domain (Margolin et al., 1994). This repressor domain is commonly found at the N-terminus of zinc finger proteins and presumably exerts its repressive activity on 0 TATA-dependent transcription in a distance- and orientation-independent manner, OO by interacting with the RING finger protein KAP-1. One can utilize the KRAB domain found between amino acids 1 and 97 of the zinc finger protein KOXI.

Finally, to explore the utility of histone deacetylation for repression, amino acids 00 1 to 36 of the Mad mSIN2 interaction domain (SID) can be fused to another domain (Ayer et al., 1996). This small domain is found at the N-terminus of the I transcription factor Mad and is responsible for mediating its transcriptional repression by interacting with mSIN3, which in turn interacts the co-repressor N- CI 10 CoR and with the histone deacetylase mRPD1.

The amino acid residue and nucleotide encoding sequences of preferred transcriptional repression domains (KRAB-A) 2 and (SID) 2 are shown in FIGs 7 and 8, respectively. Means for linking repression domains to ligand binding domains as well as exemplary polypeptide gene switches containing repression domains are set forth hereinafter in the Examples.

3. Polypeptide Gene Switches A polypeptide of this invention, in one embodiment, comprises two ligand binding domains and a first functional domain. In another embodiment, a polypeptide gene switch comprises two ligand binding domains, a first functional domain and a second functional domain. These domains can exist in any order as shown below.

In a preferred embodiment the two ligand binding domains (LBDs) are located directly adjacent to one another, ie. they are "serially connected" within the monomeric polypeptide gene switch of the invention and are not separated by a functional domain of the invention. The serially connected LBDs may be separated from one another by a linker molecule, such as for example a polypeptide linker molecule.

In a preferred embodiment the two LBDs are located between two functional domains (FDs) of the invention, wherein one functional domain is a Transcription Regulating Domain (TRD) and the other functional domain is a Nucleotide Binding Domain (NBD).

N1 In one particularly preferred embodiment the monomeric polypeptide gene switch of the invention consists of two FDs and two LBDs in the sequential order Z FD-I LBD-1 LBD-2 FD-2. Preferredly, in this embodiment, one 00 functional domain is a TRD and the other functional domain is a NBD.

Preferredly, the NBD employed in the monomeric polypeptide gene 00 switch of the invention includes 6 zinc finger binding motifs. As further described in the examples hereinbelow, a 6 zinc finger NBD employed in a monomeric polypeptide gene switch allows for the recognition of a unique I Sbp nucleic acid sequence, which may be syrrmetric or asymmetric.

LBDs FD-I FD-2 FD-l FD-2 LBDs ED-i LBDs FD-2 FD-2 LBDs

ED-I

FD-2 ED-I LBDs A wide variety of polypeptide gene switches have been made. Exemplary such gene switches include (see above for definition of terms): Gene Switches Using RXR. E2C. and Activation Domains E2C-RXR-L-DE-VP64, E2C-RXR-LL-DE-VP64, E2C-RXR-LLL-DE- VP64, E2C-RXR-L-BE-VP64, E2C-RXR-LL-BE-VP64, E2C-RXR-LLL-BE- VP64, E2C-RXR-L-DE-VPI 6, E2C-KXR-LL-DE-VPl 6, E2C-RXR-LLL-DE- VPI 6, E2C-RXR-L-BE-Vpl 6, E2C-RXR-LL-BE-VP1 6, E2C-RXR-LLL-BE- VPI 6; Gene Switches Using RXR. 2C7, and Activation Domains 2C7-RXR-L-DE-VP64, 2C7-RLXR-LL-DE-VP64, 2C7-R.XR-LLL-DE- VP64, 2C7-RXR-L-BE-VP64, 2C7-RXR-LL-BE-VP64, 2C7-RXR-LLL-BE- VP64, 2C7-RXR-L-DE-VP16, 2C7-RXR-LL-DE-VP16, 2C7-RXR-LLL-DE.

o VP16, 2C7-RXR-L..BE..VP16,2C7-RXR-LL-BE-.VP16, E2C-RXR-LLL-BE.

VP16; Gene Switches Using MX. 3. and-Activation Domains B3B-RXR-L-DE-VP64, B3B-RXR-LL-DE-VP64, B3B-RXR-LLL-DE.

00 VP64, B3B 7-RXR-L-BE-VP64, B3B 7-RXR-LL-BE-VP64, B3B-RXR-LLL-BE.

VP64, B3B-RXR-L-DE..VP16, B3B-RXR-LL-DE-VPI6, B3B-RXR-LLL-DE- N- VPI 6, B3B-RXR-L-BE-VPI6, B3B-RXR-LL-BE-VP16, B3B-RX-LLLBE.

VPI6; c-I 10 Gene Switches Using RXR. B3C2) anmctvtinDoan B3C2-RXR-L-DE-VP64, B3C2-RXR-LL-DE-VP64, B 3C2-RXR-LLL- DE-VP64, B3C2-RXR.-L-BE-VP64, B3C2-RXR-LL-BE..VP64, B3C2-RXR- LLL-BE-VP64, B3C2-RXR-L-DE..VPI 6, B3C2-RXR-LL-DE- VPI 6, B3C2- RXR-LLL-DE-VP 16, B3C2-RXR-L-BE-Vp1 6, B 3C2 B-RXR-LL-B E-VP1 6, B3C2-RXR-LLL-BE-Vp16; -Gene Switches Using RXR. E2C. and Repression Domains E2C-RXR-L-DE-(KJRAB E2C-RXR-LL-DE-(KRAB-A)2, E2C- RXR-LLL-DE-(KRAB-A)2, E2C-RXR-L-BE-(KRABh.A)2, E2C-RXR-LL-BE.

(KRAB-A)2, E2C-RXR-LLL-BE..{KRAB-A)2, E2C-RXR-L-DE-(KR&.B.A)2, E2C-RR-LLDEJABA)2, E2C-RXR-LLL-DE-{IKRA]3A)2, E2C-RXR-L- BE-(KRAB-A)2, E2C-RXR-LL-BE-{KRAB..A)2, E2C-RXR-LLL-BE-(KRAB- A)2, E2C-RXR-L-DE-(SID)2, E2C-RXR-LL-DE-(SLD)2, E2C-RX-LLL-DE.

(SID)2, E2C-RXR-L-BE..(SID)2, E2C-RXR-LL-BE-(SID)2, E2C-RXR-LLL-BE- (SID)2, E2C-RXR-L-DE-(SID)2, E2C-RXR-LL-DE-(SID)2, E2C-RXR-LLL-DE.

(SLD)2, E2C-RXR-L-BE-(SID)2, E2C-RXR-LL-BE-(SID)2, E2C-RXR-LLL-BE.

(SID)2; Gene SwiteUsg X. 2C7 an Repression Domains 2C7-RXR-L-DE-{KRAB-A)2,2C7-RXR-LL-DE-(KRAB-A)2, 2C7-RXR- LLL-DE-(KRAB-A)2, 2C7-RXR-L-BE-(ICAB..A)2, 2 C7-RXR-LL-BE.QCRAB.

A)2, 2C7-RXR-LLL-BE-{KRAB..A)2, 2C7-RXR-L-DE-(IKRAB-.A)2,2C7-RXR- LL-DE-(KRAB-A)2, 2C7-RXR-LL-L-DE-{KRAB..A)2, 2 C7-RXR-L-BE-(KAB-~ NI A)2, 2C7-RXR-LL-BE-(KRABA)2, E2C-RXR-LLL-BE(KRB-A)2, 207- >o RXR-L-DE-(S]ID)2, 2C7-RXR-LL-DE-(SID)2, 2C7-RXR-LLL-DE-(SID)2,2C7- Z RXR-L-BE-(SID)2, 2C7-RXR-LL-BE-(SID)2, 2C7-RXR-LLL-BE-(SID)2, 2C7- 00 RXR-L-DE-(SID)2, 2C7-RXR-L.DE-(SID)2, 2C7-RXR-LLL-DE-(SID)2, 2C7- RXR-L-BE-(SID)2, 2C7-RXR-LL-BE-(SID)2, E2C-RXR-LLL-BE-(SID)2,n; 00 ~~Gene Switces Using W210 B.AndRpeso oan B3B-RXR-L-DE-(KL&JB-A)2, B3B-RXR-LL-DE-(KRAB..A)2, B3B- RXR-LLL-DE-(KRAB-A)2, B3B 7-RXR-L-BE-(KRAB-A)2, B3B 7-RXR-LL- BE-(KRAB-A)2, B3B-RXR-LLL-BE-QcRAB..A)2, B3B-RXR-L-DE-(KRAB N- 10 A)2, B3B-RXR-LL-DE-(KRAB..A)2, B3B-RXR-LLL-DE-(KRAB..A)2, B3B- R.XR-L-BE-(KRAB-A)2, B3B-RXR-LL-BE-KJZAB..A)2, B 3B-RXR-LLL-BE- (KR.AB-A)2, B3B-RXR-L-DE-(SID)2, B3B-RXR-LL-DE-(SID)2, B3B-RXR- LLL-DE-(SID)2, B3B 7-RXR-L-BE-(SID)2, B3B 7-RXR-LL-BE-(SID)2, B3B- RXR-LLL-BE-(SID)2, B3B-RXR-L-DE-(SID)2, B3B-RXR-LL-DE-(SID)2, B3 B-RXR-LLL-DE-(SID)2, B3B-RXR-L-BE-(SID)2, B3B-RXR-LL-BE-(SID)2, B3B-RXR-LLL-BE..(SID)2; Gene Switches Using RXR. B3C2. and Repession Domains, B3C2-RXR-L-DE-(KRAB..A)2, B3C2-RXR-LL-DE-{KRAB.A)2, B3C2- RXR-LLL-DE-(KRAB-A)2, B3C2-RXR-L-BE-(KRAB..A)2, B3C2-RXR-LL- BE-(KRAB-A)2, B3C2-RXR-LLL-BB-QKRAB-A)2, B3C2-RXR-L-DE-KL& 1 n- A)2, B3C2-RXR-LL-DE-(KYAB..A)2, B3C2-RXR-LLL..DE-(KRAB..A)2, B3C2- RXR-L-BE-(KRAB-A)2, B3C2 B-RXR-LL-BE-(KRAB-A)2, B3C2-RXR-LLL- BE-(KRAB-A)2, B3C2-RXR-L-DE.{SID)2, B3C2-RXR-LL-DE-(SID)2, B3C2- RXR-LLL-DE-(SID)2, B 3C2-RXR-L-BE-(SID)2, B3C2-RXR-LL-BE-(SID)2, B3C2-RXR-LLL-BE..(SID)2, B3C2-RXR-L-DE-(SID)2, B3C2-RXR-LL-DE- (SID)2, B3C2-RXR-LLL-DE-(SfD)2, B3C2-RXR-L-BE-(SBD)2, B3C2 B-RXR- LL-BE-(SID)2, B3C2-RR-LLL-BE.(SID)2; Gene Switches Using PR.E2C. and Activation Domains E2C-PR-LPR-TP64, E2C-PR-LL-PR-VP64, E2C-PR-LLL-PR-VP64, E2C-PR-L-PR-VP64, E2C-PR-LL-PR-VP64, E2C-PR-LLL-PR-VP64, E2C-PR- L-PR-VP1 6, E2C-PR-LL-PR-Vp1 6, E2C-PR-LLL,.PR-Vp16, E2C-PR-L-PR- CI VP16, E2C-PR-LL-PR-YP16, E2C-PR-LLL-PR-VP16; O ~Gene Switches Usg PR-27 n Actvto oan Z 2C7-PR-L-PR-VP64, 2C7-PR-LL-PR-VP64, 2C7-PR-LLL-PR-VP64, 00 2C7-PR-L-PR-VP64, 2C7-PR-LL-PR-Vp64, 2C7-PR-LLL-PR-VP64, 2C7-PR-L- PR-VP16, 2C7-PR-LL-PR-\Tp16, 2C7-PR-LLL-PR..VP16, 2C7-PR-L-PR-Vp16, 00 2C7-PR-LL-PR-VPI 6, E2C-PR-LLL-PR-ypl 6; Gene Switches Using P]R WAR. andi Actvai noains c-i B3B-PR-L-PR-VP64, B3B-PR-LL-PR-VP64, B3B-PR-LLL-PR-VP64, B3B 7-PR-L-PR-VP64, B3B 7-PR-LL-PR-VP64, B3B-PR-LLL-PR-VP64, B3B- N- 10 PR-L-PR-Vp16, B3B-.PR-LL-PR-VP16, B3B-PR-LLL.PR..VPI6, B3B-PR-L-pR- VPI 6, B 3B-PR-LL-PR-Vpl 6, B3B-PR-LLL-PR-yp1 6; Gene Switches Using PR. B3IC2. and Activation Domains B3 C2-PR-L-PR-VP64, B3C2-PR-LL-PR-VP64, B3C2-PR-LLL-PR- VP64, B3C2-PR-L-PR-VP64, B3C2-PR-LL-PR-VP64, B3C2-PR-LLL-PR-VP64, B3C2-PR-L-PR-VP1 6, B3C2-PR-LL-PR-Vp1 6, B3C2-PR-LLL-PR-VP 16, B3C2-PR-L-PR-Vpl 6, B3C2 B-PR-LL-.PR-Vpl 6, B3C2-PR-LLL-PR-VP1 6; Gene Switches Using PR. E2C. and epression Domains E2C-PR-L-PR-(KRAB..A)2, E2C-PR-LL-PR-(KRAB..A)2, E2C-PR-LLL- PR-(KRAB-A)2, E2C-PR-L-PR-{KRAB..A)2, E2C-PR-LL-PR{KRAB..A)2,

E

2 C-PR-LLL-PR-{KRIAB..A)2, E2C-PR-L-PR-(KRAB E2C-PR-LL-PR- (KRAB-A)2, E2C-PR-LLL-PR-(KRAB..A)2, E2C-PR-L-PR-{KRAB..A)2, E2C- PR-LL-PR-(KRAB..A)2, E2C-PR-LLL-PR..(KRAB-.A)2, E2C-PR-L-PR-(SID)2, E2C-PR-LL-PR-(SID)2, E2C-PR-LLL-PR-(SID)2, E2C-PR-L-PR-(SID)2, E2C- PR-LL-PR-(SID)2, E2C-PR-LLUPR-(SID)2, E2C-PR-L-PR-(SLD)2, E2C-PR- LL-PR-(SID)2, E2C-PR-LLL-PR-(SID)2, E2C-PR-L-PR-(S]m)2, E2C-PR-LL- PR-(SID)2, E2C-PR-LLL-PR-(SID)2; Gene Switches Using PR, C7 and Repression Domains 2C-RLP-(RBA2 2 C7-PR-LL-PR-(KRAB-.A)2,2C7-PR-LLL- PR-(KRAB..A)2,2C7-PR-L-PR-(KRAB.A)2,2C7-PR-LL-PR-(KRAB-A)2, 2C7- PR-LLL-PR.-(KRAB..A)2, 2C7-PR-L-PR-(KRAB..A)2,2C7-PR-LL-PR-(KRAB-.

A)2, 2

C

7 -PR-LLL-PR-(KRAB..A)2, 2 C7-PR-L-PR-(KRAB..A)2, 2C7-PR-LLc-I PR-(KRAB-A)2, E2C-pR-LLL-PR-(KRAB-A)2, 2C7-PR-L-PR-(SID)2, 2C7- 0 P-LP-SD227P-L-R(I),C-RLP-SD227P-L zPR-LL-PR,2-(SD), 2-PR-(S22C7-R(SD), C-PR-L-PR)22-(SD)2,

R

0R(0),27P-L-R(1),2C-RLP-SD2

C-RL-R

(SID)2, 2C7-PR-LLL-PR-(SJD)2, 2C7-PR-L-PR-(SIOD)2, 2C7-PR-LL-PR-(SID)2, E2C-PR-LLL-PR-(SID)2,n; Gene Switches Using PR. B3BR. and Repression Domains B3B-PR-L-PR-(KRAB-A)2, B3B -PR-LL-PR-(KRAB-A)2, B 3B-PR-LLL- N- PR-(KRAB-A)2, B3B 7-PR-L-PR-(KRAB-A)2, B3B 7-PR-LL-PR-(KRAB-A)2, B3B -PR-LLL-PR-(KRAB-A)2, B 3B-PR-L-PR-(KRAB-A)2, B3B-PR-LL-PR- N- 10 (KRAB-A)2, B3B-PR-LLL-PR-(KRAB-A)2, B3B-PR-L-PR-(KRAB-A)2, B3B- PR-LL-PR-(KRAB-A)2, B3B-PR-LLL-PR-(KRAB-A)2, B3B-PR-L-PR-(SID)2, B3B -PR-LL-PR-(SID)2, B 3B-PR-LL-L-PR-(SID)2, B3B 7-PR-L-PR-(SID)2, B3B 7-PR-LL-PR-(SID)2, B3B-PR-LLL-PR-(SID)2, B3B-PR-L-PR-(SID)2, B 3B-PR- LL-PR-(SID)2, B3B-PR-LLL-PR-(SID)2, B3B-PR-L-PR-(SID)2, B3B-PR-LL- PR-(SID)2, B3B-PR-LLL-PR-(SID)2; Gene Switches Using PR. B3C2. and Rpression Domains B3 C2-PR-L-PR-(XRAB-A)2, B3C2-PR-LL-PR-(KRAB-A)2, B3C2-PR- LLL-PR-(KRAB-A)2, B3C2-PR-L-PR-(KRAB-A)2, B3C2-PR-LL-PR-(KRAB- A)2, B 3C2-PR-.LLL-PR-(KRAB-A)2, B 3C2-PR-L-PR-(KRAB-A)2, B3C2-PR- LL-PR-(KRAB-A)2, B3C2-PR-LLL-PR-(KRAB-A)2, B 3C2-PR-L-PR-(KRAB- A)2, B 3C2 B-PR-LL-PR-(KRAB-A)2, B3C2-PR-LLL-PR-(KRAB..A)2, B3C2- PR-L-PR-(SID)2, B3C2-PR-LL-PR-(SID)2, B3C2-PR-LLL-PR-(SID)2, B3C2- PR-L-PR-(SID)2, B3C2-PR-LL-PR-(SID)2, B3C2-PR-LLL-PR-(SID)2, B 3C2- PR-L-PR-(SID)2, B3C2-PR-LL-PR-(SID)2, B3 C2-PR-LLL-PR-(SID)2, B 3C2- PR-L-PR-(SID)2, B3C2 B-PR-LL-PR-(SID)2, B3C2-PR-LLL-PR-(SLID)2; Gene Swithes Using ER. CadAciaonD aIs E2C-ER-L-ER-VP64, E2C-ER-LL-ER-yP64, E2C-ER-LLL-ER-VP64, E2C-ER-L-ER-VP64, E2C-ER-LL-ER-VP64, E2C-ER-LLL-ER-VP64, E2C-ER- L-ER-VP 16, E2C-ER-LL-ER-VP 16, E2C-ER-LLL-ER-VPI 6, E2C-ER-L-ER- VP16, E2C-ER-LU-ER-VP16, E2C-ER-LLL-ER-Vp16; Gene Switches Using ER. 2CZ. and Activation Domains c-I2C7-ER-L-ER-VP64, 2C7-ER-LL-ER-yP64,2C7-ER-LLL-ERVP64 0 2C7-ER-L-ER-VP64,2C7-ER-LL-ER-VP64 ,C-R-LLL-ER-VP64,2C7-ER- Z ~L-ER-VP16, 2C7-ER-LL-ER..VP16,2C7-ER-LLLER-VP16, 2C7-ER-L-ER- 00 VPI 6, 2C7-ER-LL-ER-VP16, E2C-ER-LLL-ER-VP16; Gene Switches Using ER. M3. and Activaion Domains -L E P 6 3 R L R p 4 3 E R L L E p 4 00B3B-ER-L-ER-VP64, B3B-ER-LL-ER-VP64, B3B-ER-LLL-ERVP64,B N- ER-L-ER-VPl 6, B3B-ER-LL-ER-Vp16, B3B-ER-LLL-R-yp 16, B3B -ER-L- 0 ER-VPj 6, B3B-ER-LL..ER-WPI6, B3B-ER-LLL-ER-VPI 6; N1 10 Gene Switches Usng ER, B3C2. and Activation Domains B3 C2-ER-L-ER-VP64, B3C2-ER-LL-ER-VP64, B3C2-ER-LLL-ER-.

VP64, B 3C2-ER-L-ER-VP64 B3C2-ER-LL-ER-VP64, B3C2-ER-LLL-ER- VP64, B3C2-ER.L-ERVp1 6, B3C2-ER-LL-ER-VP16, B3C2-ER-LLL-ER.

VP16, B3C2-ER-L..ER-YP16, B3C2 B-ER-LL-ER-VP16, B3C2-ER-LLL.ER.

VP16; Gene Swths ing pR E2.adRpeso oais E2C-ER-L-ER-(KRAB-A)2, E2C-ER-LL-ER-(KRAB..A)2, E2C-ER- LLL-ER-(KRAB-A)2, E2C-ER-L-ER-(KAB-A)2, E2C-ER-LL-ER-(KRAB..

A)2, E2C-ER-LLL-ER(KRAB..A)2, E2C-ER-L-ER-(KRAB3.A)2, E2C-ER-LL- ER-(KRAB..A)2, E2C-ER-LLL-ER(KRAB-A)2, E2C-ER-L-ER-(KRAB-A)2, E2C-ER-LLER.(KRA&B.A)2, E2C-ER-LLL-ER- KRAB-A)2, E2C-ER-L-ER.

(S]JD)2, E2C-ER-LL-ER-<SWD)2, E2C-ER-LLL-ER..{SID)2, E2C-ER-L-ER- (SID)2, E2C-ER-LL-ER-(SIID)2, E2C-ER-LLL-ER-(SID)2, E2C-ER-L-ER- (SLD)2, E2C-ER-LL-ER-(SID)2, E2C-ER-LLL-ER-(SID)2, E2C-ER-L-ER- (SID)2, E2C-ER-LL..ER..(SID)2, E2C-ER-LLL-ER..(SID)2; Gene Swiche Usig ER. 207. and Reressio n Domains 2C7-ER-L-ER-{KRAB..A)2, 2 C7-ER-LL-ER-(KJXB-A)2,2C7-ER-LLL- ER-(KRZAB-A)2, 2C7-ER-L-ER-(KRZAB.A)2,2C7-ER-LL-ER..(KRAB-A)2, 2

C

7 -ER-LLL-ER{KRAB.3A)2, 2C7-ER-L-ER-{KRABA)2,2C7-ER-LL-ER- (KRAIB.A)2, 2C7-ER-LLL.ER-(KLABA)2,2C7-ER-L-ER-(KRAB-A)2, 2C7- ER-LL-ER-(KIRA.B.A)2, E2C-ER-LLL-ER.{KR&.B.A)2, 2C7-ER-L-ER-(SID)2, Cl 2C7-ER-LL-ER-(SID)2, 2C7-ER-LLL-ER-(SID)2, 2C7-ER-L-ER-(SLID)2, 2C7o ER-LL-ER-(SID)2, 2C7-ER-.LLL-ER-(SLID)2, 2C7-ER-L-ER-(SIV)2, 2C7-ER- Z LL-ER-(SID)2, 2C7-ER-LLL-ER-(S1ID)2, 2C7-ER-L-ER-(SID)2, 2C7--ER-LL- 00 ER-(SID)2, E2C-ER-LLL-ER-(SID)2,n; Gene Switches Using ER. M3. and Repression Domains 00 B3B-ER-L-ER-(KRAB-A)2, B3B-ER-LL-ER-(KRAB-A)2, B3B-ER- 0L-R(RBA203 -RLE-(RBA2 3 -RL-R(RB A),B-RLLL-ER-(KRAB-A)2, B3B -ER-L-ER-(KRAB-A)2, 33 3-ER-LL-RB ER(BA)2, B3B-ER-LLL-ER-{KRAB-A)2, B3B-ER-L-ER-(KRAB-A)2,

BE-L

c-i 10 B3BELLER-(KRAB A)2, B3B-ER-LLL-ER-(KRAB-A)2, B3B-ER-L-ER-A2 10I2 B3B-ER-LL-ER-(SIDB-)2, B3B-ER-LLL-ER-(SIDB-)2, B3B-ER-L-ER- (SID)2, B3B-ER-LL-ER-(SID)2, B 3B-ER-LLL-ER-(SJ)2, B3B -ER-L-ER- (SID)2, B3B -ER-LL-ER-(SID)2, B3B-ER-LLL-ER-(SD)2, B3B-ER-L-ER- (SID)2, B3B-ER-LL-ER-(SJD)2, B3B-ER-LLL-ER-(SID)2; 3-R--R Gene Switches Using ER. 1336, and Repression Domains B3C2-ER-L-ER-(KRAB-A)2, B3C2-ER-LL-ER-(KRAB-A)2,- B3C2-ER- LLL-ER-(KRAB-A)2, B3C2-ER-L-ER-(KRAB-A)2, B3C2-ER-LL-ER-(KRAB- A)2, B3C2-ER-LLL-ER-(KRAB B3C2-ER-L-ER-(KRAB-A)2, B3C2-ER- LL-ER.-(KRAB-A)2, B3C2-ER-LLL-ER-(KRAB-A)2, B3C2-ER-L-ER-(KRAB- A)2, B3C2 B-ER-LL-ER-(KRAB-A)2, B3C2-ER-LLL-ER-(KRAB-A)2, B3C2- ER-L-ER-(SID)2, B 3C2-ER-LL-ER-(SJD)2, B3C2-ER-LLL-ER-(S1D)2, B3C2- ER-L-ER-(SIID)2, B3C2-ER-LL-ER-(SID)2, B3C2-ER-LLL-ER-(SID)2, B3C2- ER-L-ER-(SID)2, B3C2-ER-LL-ER-(SID)2, B3C2-ER-LLL-ER-(SLD)2, B3C2- ER-L-ER-(SID)2, B3C2 B-ER-LL-ER-(SID)2, B3C2-ER-LLL-ER-(SJID)2.

The nucleotide (SEQ ID NO: 39) and amino acid residue sequence (SEQ ID NO: 40) of polypeptide E2C-ER-L-ER-VP64 are shown in FIG. 9. The nucleofide (SEQ ID NO: 41) and amino acid residue sequence (SEQ ID NO: 42) of polypeptide E2C-ER-LL-ER-VP64 are shown in Fig. MI. Polynucleotides. Expression Vectors and Host Cells In a related aspect, the present invention provides polynucleotides that encode a polypeptide gene switch of this invention, expression vectors containing 0 those polynucleotides, cells containing those polynucleotides and transformed o cells containing those expression vectors. Vectors of primary utility for gene therapy include, but are not limited to human adenovirus vectors, adenoassociated vectors, murine or lenti virus derived retroviral vectors, or a variety of 00 non-viral compositions including liposomes, polymers, and other DNA containing conjugates. Such vector systems can be used o deliver the gene switches either in vitro or in vivo, depending on the vector system. With adenovirus, for instance, 0 vectors can be administered intravenously to transduce the liver and other organs, N1 10 introduced directly into the lung, or into vascular compartments temporarily localized by ligation or other methods. Methods for constructing such vectors, and methods and uses for the described invention are known to those skilled in the field of gene therapy.

IV. Methods of Regulating Nucleotide Function The present invention further provides a process for regulating the expression of a desired nucleotide sequence such as a gene. In accordance with the process, the target nucleotide sequence is exposed to an effective amount of a gene switch and a ligand, wherein the nucleotide binding domain of the gene switch binds to a portion of the target nucleotide and wherein the ligand binds to at least one of the ligand binding domains of the gene switch. Exposure can occur in vitro, in situ or in vivo. The term "effective amount" means that amount that regulates transcription of a nucleotide structural gene or translation of RNA).

The term "regulating" refers to the suppression, enhancement, or induction of a function. For example, a polypeptide of the invention may modulate a promoter sequence by binding to a motif within the promoter, thereby enhancing or suppressing transcription of a gene operatively linked to the promoter nucleotide sequence. Alternatively, modulation may include inhibition of a gene where the polypeptide binds to the structural gene and blocks DNA dependent RNA polymerase from reading through the gene, thus inhibiting transcription of the gene. Alternatively, modulation may include inhibition of translation of a cN transcript.

O The promoter region of a gene includes the regulatory elements that Z typically lie 5' to a structural gene. If a gene is to be activated, proteins known as 00 transcription factors attach to the promoter region of the gene. This assembly resembles an "on switch" by enabling an enzyme to transcribe a second genetic 00 segment from DNA to RNA. In most cases the resulting RNA molecule serves as a template for synthesis of a specific protein; sometimes RNA itself is the final Sproduct.

O Regulation of gene expression or transcription can be accomplished both by exposing the target gene to a polypeptide switch of this invention or, preferably by transforming a cell that contains the target gene with an expression vector that contains a polynucleotide sequence that encodes a gene switch.

The Examples that follow illustrate particular embodiments of the present invention and are not limiting of the specification or claims in any way.

EXAMPLE 1: General Methods Construction of zinc finger proteins. For the construction of the B3 and N1 zinc finger proteins, DNA recognition helices from the Zif268 Finger 2 variants pmGAA, pmGAC, pmGGA, pmGGG, and pGTA were utilized [Segal, D. Dreier, Beerli, R. and Barbas, C. m (1999) Proc. 9NatL Acad Sci LSA 96, 2758-2763]. Three finger proteins binding the respective 9-bp target-sites were constructed by grafting the appropriate DNA recognition helices into the framework of the three finger protein SplC [Desjarlais, J. and Berg, J. M.

(1993) Proc. NatVtcad Sc L 90, 2256-2260]; DNA fragments encoding the two 3 finger proteins were assembled from 6 overlapping oligonucleotides as described [Beerli, R. Segal, D. Dreier, and Barbas, C. II (1998) Trot.

Nat£.ca. Sc. U'1 95, 14628-14633]. The three finger protein coding regions were then cloned into the bacterial expression vector pMal-CSS via Sfil digestion.

Protein purification. Moltose binding protein (MBP) fusion proteins were purified to >90% homogeneity using the Protein Fusion and Purification i System (New England Biolabs), except that Zinc Buffer A (ZBA; 10 mM Tris, 0 pH7.5/90 mM KCI, 1 mM MgCI 2 90 pM ZnCI 2 BSA /5 mM DTT) was used Z as the column buffer. Protein purity and concentration were determined from Coomassie blue-stained 15% SDS-PAGE gels by comparison to BSA standards.

ELISA analysis. In 96-well ELISA plates, 0.2 pg of streptavidin (Pierce) 0O was applied to each well for 1 hour at 37 0 C, then washed twice with water.

Biotinylated target oligonucleotide (0.025 pg) was applied in the same manner.

.I ZBA/3% BSA was applied for blocking, but the wells were not washed after Sincubation. All subsequent incubations were at room temperature. Starting with 2 gg purified MBP fusion protein in the top wells, 2-fold serial dilutions were applied in lx binding buffer (ZBA/1% BSA/5 mM DTT/0.12 pg/pl sheared herring sperm DNA). The samples were incubated 1 hour at room temperature, followed by 10 washes with water. Mouse anti-maltose binding protein mAb (Sigma) in ZBA/1% BSA was applied to the wells for 30 minutes, followed by washes with water. Goat anti -mouse IgG mAb conjugated to alkaline phosphatase (Sigma) was applied to the wells for 30 minutes, followed by washes with water. Alkaline phosphatase substrate (Sigma) was applied, and the

OD

4 0 5 was quantitated with SOFTmax 235 (Molecular Devices).

Gel mobility shift assays. Target oligonucleotides were labeled at their 3' ends with and gel purified. Eleven 3-fold serial dilutions of protein were incubated in 20 pl binding reactions (lx Binding Buffer/10% glycerol/'l pM target oligonucleotide) for three hours at room temperature, then resolved on a polyacrlyamide gel in 0.5x TBE buffer. Quantitation of dried gels was performed using a PhosphorImager and ImageQuant software (Molecular Dynamics), and the KD was determined by Scatchard analysis.

Reporter constructs for determining the optimal spacing and orientation of the two half-sites. C7 dimer-TATA fragments were generated by PCR amplification with C7 dimer-TATA primers (5'-GAG GGT ACC GCGTGG GCG Ao-5 GCG TGG GCG AGT CGA CTC TAG AGG GTA TAT AAT GG-3' (SEQ ID NO: 1) for direct repeats; 5'-GAG GGT ACC GCG TGG GCG CGC CCA CGC AGT CGA CTC TAG AGO GTA TAT AAT GG-3' (SEQ ID o NO: 2) for inverted repeats; 5'-GAG GOT ACC CGC CCA CGC AO-5 GICLT(IQ Z GCG AGT CGA CTC TAG AGG GTA TAT AAT GG-3' (SEQ IlD NO: 3) for 00 everted repeats) and GLprimer2 (5 t-C1 TAT GTT TIT GGC GTC TFTC C-3' (SEQ ID NO: Promega), using p17x4TATA-luc (gift from S. Y. Tsai) as a template. PCR products were cloned into pGL3-Basic (Promega) via digestion 00 with the restriction endonucleases Kpnl and Ncol.

CI RU486- and Tamoifen-inducible promoter constructs. I10xC7-TATA, l1xB3-TATA, and lOxNl-TATA fragments were assembled from two pairs of complementary oligonucleotides each and cloned into Sacl-Xmal linearized pGL3-Basic (Promega), upstream of the firefly luciferase coding region, creating the plasn-ids l0xC7-TATA-luc, 10xB3-TATA-Juc, and l~xNI -TATA-luc. To generate the 1 OxNl -TATA-lacZ reporter construct, the lacZ coding region was excised from pfogal-Basic (Clontech) and Used to replace the luciferase coding region of lOxNl-TATA-luc via Hfind3-BamHl digestion.

Luciferase and 13-gal reporter assays. For all transfections, HeLa cells were plated in 24-well dishes and used at a confluency'of 40.-60%. For luciferase reporter assays, 175 ng reporter plasinid (promnotor constructs -in pGL3 or, as negative control, pGL3-Basic) and 25 ng effector plasmid (zinc finger-steroid receptor fusions in pcDNA3 or, as negative control, empty pcDNA3) were transfected using the Lipofectamnine reagent (Gibco BRL). After approximately 24 h, expression was induced by the addition of IlOnM RU486 (Biomol), 100 nM 4- OHT (Sigma), or 5 mM Ponasterone A (Invitrogen). Cell extracts were prepared approximately 48 hours after transfection and assayed for luciferase activity using the Promega luciferase assay reagent in a MicroLumat LB96P luminometer (EG&G Berthold, Gaithersburg, MD). For dual reporter assays, 85ng luciferase reporter plasmid, 85ng b-gal reporter plasmid, and l5ng of each of the two effector plasmids were transfected. b-gal activity was measured using the luminescent b-galactosidase detection kit U (Clontech).

c-I Zinc inger-steroid receptor fusion constructs with N-terminal effector domains. The VP16 coding region was PCR amplified from pcDNA3/C7-VPI 6 using the primers VPNhe-F (5'-GAG GAG GAG GAG GCT AGC GCC ACC ATG GGG CGC GCC GGC GCT CCC CCG ACC GAT GTC AGC CTG-3') (SEQ 11) NO: and VPHind-B (5'-GAG GAG GAG GAG AAG 00 CIT Gfl AAT TAA ACC GTA CTC OG AAT TCC AAG GOC ATC G-3') (SEQ ID NO: 6) or VPNLSI-ind-B (5'-GAG GAG GAG GAG AAG CTT AAC N- TI T GCG TTf C'IT TIT CGG OTT AAT TAA ACC GTA CTC GTC AAT 0 TCC AAG GGC ATC (SEQ ID NO: The C7 coding region was CI 10 amplified from the same plasmidd, using the priners C7Hind-F (5'-GAG GAG GAG GAG AAG CTT GOG GCC ACG GCG GCC CTC GAG CCC TAT GC- (SEQ DD NO: and C7Eam-B (5'-GAG GAG GGA TCC CCC TGG CCG GCC TOG CCA CTA GTr CTA GAG TC-3') (SEQ ID NO: 9) or C7NLSBam- B (5 '-GAG GAG GGA TCC CCA ACT TTG CGT TTC T TC GOC TGG CCG GCC TOG CCA CTA OnT CTA GAG TC-3') (SEQ ID NO: 10). The human PR truncated LBD (aa645-9 14) was amplified from PAPCMVGL9I4VPc'-SV [Wang, Xu, Pierson, O'Malley, B. and Tsai, S. Y. (1997) Gme 'rierapy4, 432-441 using the primers PRBam-F GAG GAG GAG GGA TCC AGT CAG AGT TGT GAG AGC ACT OGA TG TG-3') (SEQ ID NO: 11) and PREco-B (5'-GAG GAG GAA TTC TCA AGC AAT AAC 'ITC AGA CAT CAT TrC TGG AAA TITC-3') (SEQ R) NO: 12).

The VP16-C7-PR, VP16-NLS-C7-PR, and VP16-C7-NLS-PR coding regions were then assembled in pcDNA3.I (-i)Zeo (Invitrogen) using the NheI, Hind3, BainHI, and EcoRi restriction sites incorporated in the PCR primers. In the resulting constructs, the C7 coding regions were flanked by two Sf1 sites, and the VP16 coding regions by Asc I and Pac sites. These restriction sites were introduced to facilitate the exchange of DBDs and effector domains, respectively.

To generate the VP16-G7-ER, VP16-NLS-C7-ER, and VP16-C7-NLS-ER constructs, the point-mutated murine ER LBD coding region (aa281-599, G525R) was excised from pBabe/Myc-ER [Littlewood, T. Hancock, D. Danielian, P. Parker, M. and Evan, G. I. (1995) Nuct Aci Rjes. 23, 1686-1690], and N ,used to replace the PR LBD coding region via BamHI-EcoRl restriction O digestion.

Z To generate fusion constructs with B3 or N1 DBDs, C7 was replaced by 00 the B3 or N1 coding regions via Sfil digestion. Fusion constructs containing a VP64 effector domain were produced by replacing VP16 by the VP64 coding 00 region via Asci-Pacl digestion.

Zinc finger-steroid receptor fusion constructs with C-terminal effector domains. The truncated human PR LBD was amplified from PAPCMVGL914VPc'-SV [Wang, Xu, Pierson, O'Malley, B. and Tsai, S. Y. (1997) gene '27erapy4, 432-441] using the primers PRFse-F GAG GAG GAG GAG GAG GGC CGG CCG CGT CGA CCA GGT CAG AGT TGT GAG AGC ACT GGA TGC-3) (SEQ ID NO: 13) and PRAsc-B GAG GAG GAG GAG GAG GGC GCG CCC CGT CGA CCC AGC AAT AAC TTC AGA CAT CAT TTC TGG-3) (SEQ ID NO: 14). The point-mutated mouse ER LBD was amplified from pBabe/Myc-ER [Littlewood, T. Hancock, D. C., Danielian, P. Parker, M. and Evan, G. I. (1995) Nuc Acids es. 23, 1686- 1690] using the primers ERFse-F GAG GAG GAG GAG GAG GGC CGG CCG CCG AAA TGA AAT GGG TGC TTC AGG AGA C-3) (SEQ ID NO: and ERAsc-B GAG GAG GAG GAG GAG GGC GCG CCC GAT CGT GTT GGG GAA GCC CTC TGC TTC-3) (SEQ ID NO: 16). The resulting PCR products were then inserted into pcDNA3/E2C-VP16 [Beerli, R. Segal, D. J., Dreier, and Barbas, C. l (1998) Troc. NatL .Aa Sci. 'US 95, 14628- 14633], in between the E2C and VP16 coding regions, via digestion with the restriction endonucleases Fsel and Asci.

To generate fusion constructs with B3 or N1 DBDs, E2C was replaced by the B3 or N1 coding regions via Sfil digestion. Fusion constructs containing a VP64 effector domain were produced by replacing VP16 by the VP64 coding region via Asci-Pad digestion.

Heterodimeric switch constructs. For construction of the E2C-ER fusion, the point-mutated mouse ER LBD was amplified from pBabe/Myc-ER (Littlewood,.T. Hancock, D. Danielian, P. Parker, M. and Evan, G.

1. (1995) uc Aids Res. 23, 1686-1690] using the primers ERFse-F and ERPac-B GAG GAG GAG GAG ITA Afl AAG ATC GTG 1TG GGG AAG CCC TCT GCI' TC-3') (SEQ ID NO: 17). The PCR product was then inserted into the construct pcDNA3IE2C-Vp64, replacing the VP64 coding region, via 00 FselI-Pac I digestion. To generate the ER-VP64 fusion, the ER LBD was amplified using the primers ERATGBani-F (5'-GAG GAG GAG GAG OGA TCC GCC ACC ATG CGA AAT GAA ATO GOT GCT TCA GGA GAC-3) (SEQ ED NO: 18) and ERAsc-B. The PCR product was then inserted into pcDNA3/fE2C-VP64, [Beerli, R. Segal, D. Dreier, and Barbas, C. M (1998) .Proc. Nat/Acad ScL 'U-vS95, 14628-14633] replacing the E2C coding region, via BamHI -Ascl digestion.

Single-chain switch constructs. For construction of single-chain fusions with two ER LBDs, the point-mutated mouse ER LBD was amplified from pBabe/Myc-ER [Littlewood, T. Hancock, D. Danielian, P. Parker, M.

and Evan, G. 1. (1995) Nuc Acids ARg. 23, 1686-1690] either using the primers ERFse-F and ERSpe I-B (5 t-GAG GAG GAG GAG GAG GAG ACT AGT GGA ACC ACC CCC ACC ACC GCC CGA 0CC ACC 0CC ACC AGA OGA OAT COT GTr G GAA 0CC CTC TGC-3') (SEQ ID) NO: 19), or using the primers ERNhel-FI (for l8aa linker construct; 5'-GAG GAG GAG GAG GAG GAG OCT AGC GGC GGT GGC GOT GGC TCC TCT GOT GOC OGT GGC GGT TCT TCC AAT GAA ATG GOT GCT TCA GGA OAC-3') (SEQ ID NO: 20) or ERNbeI -F2 (for 30aa linker construct; GAG GAG GAG GAG GAG GAG GCT AOC TCT TCC AAT OAA ATG GGT OCT TCA OGA GAC (SEQ ID NO: 21), and ERAsc-B. The PCR products were then digested with, respectively, FseI and Spel, or Nbel and Asci, and inserted into Fsel-Ascl linearized pcDNA3IE2C-VP64 [Beerli, R. Segal, D. Dreier, and. B arbas, C. Inl (1998) Proc. N7attAcad Sc4 UjA 9S, 14628-14633].

For construction of RXR-EcR single-chain fusions, the ligand binding domain of the human retinoid X receptor (bRXR04 aa373-654) was PCR amplified from pVgRXR (Invitrogen) using tfie primers RXRFse-F (5 '.GAG c-I GAG GAG GGC CGG CCG GGA AGC CGT GCA GGA GGA GCG GC-3') o (SEQ ID NO: 22) and RXRSpe-B (5Y-GAG GAG GAG GAG GAG ACT AGT Z OGA ACC ACC CCC ACC ACC GCC CGA GCC ACC 0CC ACC AGA GGA 00 AGT CAT TTG GTG CGG CGC CTC GAG (SEQ ID NO: 23). The ligand binding domain of the ecdysone receptor (EcR, aa202-462, drosophila me,'aIr-wsPRapiie rmpgXRuigtepimr c~eF 00 eaoatrwa CamlfefrmpgXusnthprmr c heF (for l8aa linker construct; 5'-GAG GAG GAG GAG OCT AGC TCT TCC GGT GGC GGC CAA GAC TIlT GTT AAG AAG (SEQ ID, NO: 24), or EcRNhe-F2 (for 3Oaa linker construct; 5'-GAG GAG GAG GAG OCT AGC c-i 10 GGC GOT GGC GOT GGC TCC TCT GOT GGC GGT GGC GOT TCT TCC GGT GGC GCC CAA GAG TTf G'T AAG AAG (SEQ lID NO: 25), and EcRAsc-B -GAG GAG GAG OGC GCG CCC GGC ATG AAC CrC CCA GAT CTC CTC GAG-3) (SEQ EID NO: 26). The PCR products were then digested with, respectively, FselI and Spel1, or NhelI and Asci1, and inserted into FselI-AsclI linearized pcDNA3/E2C-VP64 [Beerli, R. Segal, D. Dreier, B., and Barbas, C. IIH (1998) Proc. Na~[t(.4Al ScL U7S5! 95, 14628-14633].

DNA

binding domains were exchanged via Sf1 digestion, effector domains via Asc I- Pacl digestion.

To generate the 36aa linker, E2C-RLLE-VP64 fusion construct, the RXR LBD was PCR amplified from pcDNA3/E2C-RE-VP64 using the primers RXRFse-F and RXRSpeLL-B (5 -GAG GAG GAG GAG GAG ACT AGT AGA GCC ACC GCC CCC '1TC AGA ACC GCC CCA 0CC ACC GCC ACC AGA GG-3') (SEQ ID NO: 27). The EcR LBD was amplified from the same plasmid, using the primers EcRNheLL-F (5 '-GAG GAG GAG GAG OCT AGC GGG GGT TCG GAG GOT GGC 000 TCT GAG GGT GOG GOT GGT TCC ACT AGC TCT TCC-3') (SEQ ID NO: 28) and EcRAsc-B. The PCR products were inserted into pcDNA3IE2C-VP64 as described above.

EXAMPLE 2:GeeS tc s Generation of hormnone-regulated zinc finger-steroid receptor fusion proteins. Previous studies have shown the potential of engineered C2-H2 zinc finger proteins .for the regulation of target gene expression Liu, Segal, D. J., z Ghiara, J. and Barbas, C. M11(1997) Proc. Nfatt Arad SCL ZISAq 94, 5525- 5530; Kim, and Pabo, C. 0. (1997) Jyio(C~em 272, 29795-29800; Beerli, R. Segal, D. Dreier, and Barbas, C. HI1(1998) Tmc. NaKgt iAcad ScL 'USA 95, 14628-14633; Beerli, R. Dreier, and Barbas, C. 111 (2000) !Proe.

00 NKAa c.Uq9,19-50.Hwvr oflyraieteptnilo INDlcdS.'S 9,19-50.Hweet ul elieteptnilo engineered zinc finger proteins, it is desirable that their otherwise constitutive DNA binding activity be rendered ligand-dependent. The ligand binding domains (LBDs) of the human progesterone receptor (hPR) and the murine estrogen receptor (mER) have previously been used for the regulation of heterologous proteins, after having been modified to lack binding to the natural hormones while retaining binding to synthetic antagonists [Littlewood, T. Hancock, D. C., Danielian, P. Parker, M. and Evan, G. 1. (1995) N~ud Acids es. 23, 1686- 1690; Wang, Xu, Pierson, O'Malley, B. and Tsai, S. Y. (1997) Gene '2lierapy4, 432-441]. Thus, the Zif268 variant C7 [Wu, Yang, and Barbas, C. H11(1995) Troc. N'at Acad ScL USAL 92, 344-348] was fused to a transcriptional activation domain plus the LBD of either of the two nuclear hormone receptors. The VP64-C7-PR fusion protein contains an N-terminal VP64 activation domain [Beerli, R. Segal, D. Dreier, and Barbas, C. H11 (1998) Troc. Nat Acad cL 'USA95, 14628-14633], and a C-terminal hPR LBD (aa645-9 14) lacking amino acids 9 15-933, responsive to the progesteroneantagonist RU486fMifepristone but not to progesterone [Wang, Xu, J., Pierson, O'Malley, B. and Tsai, S. Y. (1997) Gene gaap4, 432-441]. The VP64-C7-ER fusion protein contains a C-terminal mER LBD (aa282-599) with a single amino acid substitution (G525R), and is responsive to the estrogen antagonist 4 -hydroxy-tamnoxifen (4-OHT) but not to estrogen [Littlewood, T. D., Hancock, D. Danielian, P. Parker, M. and Evan, G. I. (1995) WNucjAcids es. 23, 1686-1690].

Determination of the optimal response element for zinc finger-steroid receptor fusion proteins. Naturally occuning steroid receptors bind DNA as

O

dimers and typically recognize response elements consisting of palindromic Ssequences [Evans, R. M. (1988) Science 240, 889-895; Carson-Jurica, M. A., 00 Schrader, W. and O'Malley, W. (1990) IEndocrine Reviews 11,201-220].

Moreover, it was demonstrated that in some cases also direct repeats can serve as binding sites for receptor dimers [Aumais, J. Lee, H. DeGannes, C., 0 Horsford, and White, J. H. (1996) BioL Chem. 271, 12568-12577]. Given this obvious flexibility in DNA recognition by naturally occurring receptor dimers, the n optimal structure of a response element for an artificial, zinc finger based Stranscriptional switch was not known. However, to develop an efficient, hormoneinducible system for the regulation of target gene expression, a detailed knowledge of the binding site architecture is required.

To determine the optimal orientation and spacing of the two half-sites of a response element for a zinc finger-LBD fusion protein, a series of reporter plasmids was constructed. Each contains two C7 binding sites upstream of a TATA box and a firefly luciferase coding region. The two C7 binding sites were introduced in different orientations (direct, inverted, or everted repeat) and with various spacings (no spacing or 1 to 5 bp spacing). Plasmids directing expression of VP64-C-PR or VP64-C7-ER fusion constructs were then co-transfected with the various reporter plasmids and assayed for hormone-induced luciferase expression. Significantly, each of the C7 dimer binding sites was able to act as a response element for both PR and ER based proteins, albeit at variing efficiency.

In contrast, a reporter plasmid with a single C7 binding site was not activated, indicating that hormone-induced activation of transcription was mediated by dimers.

Optimal spacing depended on the orientation of the two half-sites. In the case of the PR fusion protein, optimal spacing seemed to be at 2-3 bp for inverted repeats and 3 bp for everted repeats. Response elements consisting of direct repeats had no single optimal spacing; the best response was obtained with 4-5 bp, or no spacing at all. For the ER fusion protein, optimal spacing was at 3-4 bp for direct repeats, 1-2 bp for inverted repeats, and 3 bp for everted repeats It should be noted that there were significant variations in the basal, i.e. ligand-independent I activity of PR and ER fusion proteins, depending on the response element tested.

o Most notably, increasing the spacing of direct repeats from 3 to 4 bp led to a 1.9- Z fold higher basal activity of VP64-C7-PR, and even a 3.7-fold increase in the case of VP64-C7-ER. High basal activity is extremely undesirable for an inducible promoter system, where tight control over the expression levels of a particular 0 gene of interest is often required, especially if the gene product is toxic. Thus, in choosing appropriate response elements, particular attention must be paid not only i to hormone inducibility but also to its basal activity in the presence of the regulatory protein. The response element consisting of direct repeats with a CN 10 spacing of three nucleotides was considered to be a good choice for use in a hormone-inducible artificial promoter, since it was compatible with both PR and ER fusion proteins. Significantly, its basal acticity in the presence of either PR or ER fusion proteins was among the lowest of all response elements tested.

Furthermore, good hormone induced activation of transcription was observed with both VP64-C7-PR (3.9-fold) and VP64-C7-ER Generation of novel DNA binding domains. While the use of the C7 DNA binding domain was well suited for the preliminary studies described above, it may not be a good choice for incorporation into an inducible transcriptional regulator. The C7 protein is a variant of the mouse transcription factor Zif268 [Pavletich, N. and Pabo, C. 0. (1991) Sdnce252, 809-817], with increased affinity but unchanged specificity [Wu, Yang, and Barbas, C. III (1995) froc. Natt Acad Sc USA 92, 344-348]. We reasoned that the use of alternate DNA binding domains would minimize potential pleiotropic effects of the chimeric regulators. Previously, we described a strategy for the rapid assembly of zinc finger proteins from a family of predefined zinc finger domains specific for each of the sixteen 5'-GNN-3'DNA triplets [Beerli, R. Segal, D.

Dreier, and Barbas, C. El (1998) Proc. Nat Lcad Sci. Z1S,95, 14628- 14633; Segal, D. Dreier, Beerli, R. and Barbas, C. m (1999) Proc.

NatL Acad SL A96, 2758-2763]. Three finger proteins binding any desired (GNN)3-3'sequence can be rapidly prepared by grafting the amino acid residues involved in base-specific DNA recognition into the framework of the consensus three finger protein SplC [Desjarlais, J. and Berg, J. M. (1993) Proc. Nat[ S.ad. Sci. U'S90, 2256-2260]. To date, well over 100 three finger proteins have Z been produced in our laboratory. Two of these, B3 and N1, were chosen to be 00 used in inducible transcriptional regulators (Figure 1A). The B3 and N1 proteins are designed to bind the sequences 5'-GGA GGG GAC-3'or 5'-GGG GTA GAA- 00 respectively. To verify their DNA binding specificity, these proteins were purified as MBP-fusions and tested by ELISA analysis using an arbitrary c. selection of oligonucleotides containing 5'-(GNN)3-3'sequences (Fig. 1B).

Significantly, both proteins recognized their target sequence and showed no CN 10 crossreactivity to any of the other 5'-(GNN) 3 -3'sequences tested. However, as judged by ELISA, binding of N1 was much weaker than binding of B3.

Therefore, affinities were determined by electrophoretic mobility-shift analysis.

The B3 protein bound its target sequence with a KD value of 15nM, similar to the KD values we previously reported for other three finger proteins [Beerli, R. R., Segal, D. Dreier, and Barbas, C. II (1998) Proc. NatAca dSci. US 14628-14633]. In contrast, N1 affinity for its target was dramatically lower and we estimate its KD value to be in the range of 5-10 pM. The fact that the two proteins had very different affinities for their respective target sequences was considered positive, since it allows to investigate the influence of affinity on the functionality of an inducible expression system.

RU486- and 4-OHT-inducible systems for the control of gene expression. To allow for a comparative analysis, a series of RU486- or 4-OHTinducible transcriptional regulators were constructed containing either the B3 or the N1 DNA binding domain. The role of placement of the activation domain was investigated, by fusing it either to the N- or the C-terminus of the protein. Two different activation domains were compared: the herpes simplex virus VP16 transactivation domain [Sadowski, Ma, Triezenberg, and Ptashne, M.

(1988) Nature335, 563-564], and the synthetic VP64 activation domain, which consists of 4 tandem repeats of VP16's minimal activation domain [Beerli, R. R., Segal, D. Dreier, and Barbas, C. II (1998) Proc. at Acal Sci.L UZ 14628-14633].

Synthetic promoters were constructed based on the B3 and N1 DNA target O sequences, and the optimal response element structure defined above. The 10xB3oQ TATA-luc and O1xNl -TATA-luc plasmids each contain five response elements, consisting of direct repeats spaced by three nucleotides, upstream of a TATA box and a firefly luciferase coding region. The response elements are separated from 00 each other by six nucleotides, which should allow the concomitant binding of five Sdimers and thus maximize the promoter activity. The activity of the various fusion constructs was assessed by transient cotransfection studies with the cognate TATA reporter plasmids in HeLa cells (Table 1).

CI Table 1 LBD=PR

LBD=ER

exp.1 exp.2 exp.1 exp. 2 VP16-B3-LBD 34x 36x 37x 26x VP64-B3-LBD 37x 24x 26x 27x B3-LBD-VP16 115x 116x 47x 58x B3-LBD-VP64 110x 85x 62x 99x VP16-N1-LBD 188x 159x 101x 39x VP64-N1-LBD 206x 390x 49x 58x N1-LBD-VP16 282x 203x 24x N1-LBD-VP64 151x 129x 1319x 464x In general, the ER fusion proteins turned out to be the stronger transactivators, and 4-OHT-induced luciferase activity was usually 3 to 6 times higher than RU486-induced luciferase activity mediated by PR fusion proteins.

However, since the basal, i. e. ligand independent, activity of ER chimeras was often somewhat higher, their hormone-induced fold-stimulation was not generally better. Hormone-dependent gene activation in excess of 2 orders of magnitude was commonly observed with both PR and ER fusion proteins, values that are significantly better than what was previously reported for the Gal4-PR fusion protein GLVPc' [Wang, Xu, Pierson, O'Malley, B. and Tsai, S. Y.

0 (1997) Gene hierapy 4, 432-441].

OC The placement of the activation domain had a significant influence on the activity of the chimeric regulators. However, favored placement was dependent on the nature of the activation domain. Whereas the VP16 domain yielded the 00 more potent activators when placed at the C-terminus, the VP64 was more active I at the N-terminus. Accordingly, direct comparisons showed that an N-terminal VP64 was more potent than a N-terminal VP16 domain, and a C-terminal VP16 was more potent than a C-terminal VP64 domain. The nature and placement of C'I 10 the activation domain was also found to have an influence on the basal activity of the chimeric regulators. In particular, a relatively high basal activity was observed in the case of regulators with N-terminal VP64 domain.

The nature of the DNA binding domain had a major influence on the extent of ligand-dependence of the chimeras. Use of the N1 protein as DNA binding domain led to more tightly regulated fusion constructs with significantly better fold-stimulation of promoter activities than the use of B3, likely due to the dramatic affinity differences between N1 and B3. In particular, the N1-ER-VP64 regulator had no significant basal activity and was capable of mediating a 464- to 1319-fold 4-OHT-induced activation of the 10xNI-TATA minimal promoter (Table The extent of ligand-induced activation of'gene expression over a range of 3 orders of magnitude is particularly remarkable, since it has thus far only been reported for the tetracycline controlled system of gene regulation [Gossen, M., and Bujard, H. (1992) !roc. NatL Acad d. UlS 89, 5547-5551; Gossen, M., Freundlieb, Bender, Miller, Hillen, and Bujard, H.(1995) Scence 268,1766-1769].

Concomitant regulation of multiple promoters. Zinc finger technology has made a large repertoire of DNA binding specificities available for use in protein engineering [Beerli, R. Segal, D. Dreier, and Barbas, C. II (1998) Proc. NatLtcad. Sci U&S95, 14628-14633; Segal, D. Dreier, Beerli, R. and Barbas, C. m (1999) Proc. (tLjcad ScL. '1A96, 2758-2763; Beerli, R. Dreier, and Barbas, C. m (2000)

O

o Natocl. Acad Sci. U997, 1495-1500]. The availability of different steroid o0 hormone receptor-derived regulatory domains [Littlewood, T. Hancock, D. C., Danielian, P. Parker, M. and Evan, G. I. (1995) Nucd Acis s. 23, 1686- 1690; Wang, Xu, Pierson, O'Malley, B. and Tsai, S. Y. (1997) Gen 00 fwiapy4, 432-441], and the ability to redirect chimeric regulators to virtually any m desired target sequence should make it possible to independently regulate the expression of multiple genes at the same time. To examine this possibility, a O reporter plasmid was constructed directing expression of P-galactosidase (p-gal) under the control of the 10xN1-TATA minimal promoter. The chimeric regulators B3-PR-VP16 and N1-ER-VP64 were then transiently expressed in HeLa cells along with the 10xB3-TATA-luc and 10xNI-TATA-0-gal reporter plasmids. The transfected cells were treated with either RU486 or 4-OHT and the luciferase and p-gal activities were monitored. Significantly, RU486 induced expression of luciferase while having no effect on p-gal reporter gene activity. 4-OHT, on the other hand, did not affect luciferase expression but efficiently activated p-gal expression. These results demonstrate that the two regulator/promoter combinations act independently from one another, and that multiple genes can efficiently and independently regulated by the selective addition of the desired hormone.

Development of a monomeric hormone-dependent gene-switch. The ability to engineer DNA binding proteins with desired specificities makes it possible to generate artificial transcription factors capable of imposing dominant regulatory effects on endogenous genes [Beerli, R. Dreier, and Barbas, C.

m (2000) Proc. Nattcad Scd. QS97, 1495-1500]. For many applications of this technology it may be desirable that the effect on endogenous gene expression is reversible. The use of steroid hormone receptor LBDs has the potential to render regulation of endogenous gene expression reversible. However, one major drawback is the fact that steroid hormone receptors, as well as the chimeric regulators described herein, bind DNA as dimers. Thus, when the fusion protein

O

c C7-ER-VP64 was transiently expressed in HeLa cells it was unable to regulate a O reporter construct carrying a single C7 binding site, while it readily regulated a _Z reporter that had two C7 binding sites and therefore accommodated binding of a 00 dimer (Fig. 2B). An additional problem was encountered when the C7 DBD was replaced by E2C, which contains six zinc finger domains and recognizes the 18o bp sequence 5'-GGG GCC GGA GCC GCA GTG-3'(SEQ ID NO; 29) in the 00 SUTR of the proto-oncogene c-erbB-2 [Yamamoto, Ikawa, Akiyama, T., SSemba, Nomura, Miyajima, Saito, and Toyoshima, K. (1986) SjNature319, 230-234; Beerli, R. Segal, D. Dreier, and Barbas, C. m cN 10 (1998) froc. Nat£Acad Sci. 1A 95, 14628-14633]. The E2C-ER-VP64 fusion protein was constitutively active on a reporter carrying a single E2C binding site, almost as active as an E2C-VP64 fusion without an ER LBD, and did not respond well to hormone. Apparently, the use of a large DNA binding domain recognizing an extended stretch of DNA with high affinity renders the chimera hormone- and dimerization-independent.

To overcome these problems, we produced two types of ER-based chimeric regulators, designed to be capable of regulating gene expression through a single binding site in a hormone-dependent manner. In the first strategy, a heterodimeric regulator was generated consisting of the engineered zinc finger protein E 2 C fused to an ER LBD, as well as an ER LBD fused to a VP64 activation domain (Fig. 2A). When this heterodimeric regulator was expressed in HeLa cells, it had no significant activity on the E2C-TATA-luc reporter plasmid in the absence of 4-OHT. Addition of hormone led to a 3- to 5-fold stimulation of luciferase expression, indicating the formation of functional heterodimers.

However, hormone-induced reporter gene activation was significantly lower than that induced by an E2C-VP64 fusion protein, presumably at least in part due to the formation of E2C-ER and ER-VP64 homodimers. Homodimers were inactive, since neither E2C-ER nor ER-VP64 alone induced luciferase expression. In the second strategy, fusion proteins were generated by combining the dimerization partners E2C-ER and ER-VP64 in one single polypeptide, through a flexible polypeptide linker. Two linkers were tested, 18 and 30 amino acids in length, creating the proteins E2C-scER/1 8-VP64 and E2C-scERI3O-VP64 (Fig. 2A).

z These proteins were expected to be activated via intramolecular, rather than OC) intermolecular, dimerization and therefore functional as monomers. Combination of two ER LBDs into one single-chain fusion con struct should allow a more efficient hormone-induced dimerization and therefore yield more efficient 00 activators. Indeed, when E2C-scER/1l8-VP64 and E2C-scERI3O-VP64 were transiently expressed in HeLa cells, they efficiently activated the E2C-TATA-Juc NI reporter in a largely hormone-dependent manner (Fig. 2B, 2C and 2D3). Thus, dimeric regulators requiring response elements similar to those of natural steroid NI 10 hormone receptors were successfully converted into monomeric, liganddependent transcription factors.

Monomeric gene-switch based on EcR and RXR LBDs. To show that the production of a ligand-dependent monomeric gene switch by fusion with two LBIs is a generally applicable strategy, the utility of other nuclear hormone receptors was tested. In particular, utility of the LBIs of the Drosophila ecdysone receptor (EcR) was investigated. In Drosophila, this receptor functions as a heterodimer between EcR and the product of the ultraspiracle (USP) gene [Yao, Forman, B. Jiang, Cherbas, Chen, McKeown, Cherbas, and Evans, R. M. (1993) Natiture 366, 476-479]. However, it has been shown that EcR also efficiently heterodimerizes with USP's vertebrate homologue retinoid X receptor (RXR) in response to the ecdysone agonists Muristerone A or Ponasterone A (PonA) [Nakanishi, K. (1992) Steroid 57, 649-657; Yao, Forman, B. Jiang, Cherbas, Chen, McKeown,

M.,

Cherbas, and Evans, R. M. (1993) Wit=n366, 476-479; No, Yao, and Evans, R. M. (1996) Troc- Natcad Sd U1Sq93,3346-335lJ. The EcR and RXR LBDs were therefore used to prepare a monomeric gene switch analogou's to the scER chimeras described above (Fig. 3A). Thus, the human RXRa LBD (aa373-654) and the Drosophila EcR LBD (Wa22-462) were inserted in between the 132C DBD and the VP64 activation domain, creating E2C-RE-VP64. In this fusion construct, the two LBDs are connected by an 18 amino acid flexible linker, the same that was used in E2C-scER/1l8-VP64. When this chimeric regulator was CI transiently expressed in HeLa cells along with the E2C-TATA-luc reporter O plasmid, significant basal activity was observed. However, activity could be Z increased 3-fold by PonA, showing that this artificial construct was hormoneresponsive. To improve the ligand dependence, the length of the linker connecting the RXR and EcR LBDs was increased, a measure that seemed beneficial in the 00 case of the single-chain ER constructs. A longer linker should allow the LBDs to optimize their contact and add to the conformational disorder in the unliganded tC state. Indeed, when the linker was elongated to 30 aa (in E2C-RLE-VP64) or 36 aa (in E2C-RLLE-VP64), basal activity was significantly reduced and PonA led N 10 to a 9- to 10-fold activation, an extent of responsiveness comparable to the one of the single-chain ER fusion constructs (Fig. 3B). Thus, serial connection of pairs of nuclear hormone receptor LBDs appears to be a generally applicable strategy to render monomeric DNA binding proteins ligand-dependent.

The hPR and mER LBDs used for the fusion proteins did not encompass their natural SV40-like nuclear localization signals (NLS), located between amino acids 637 and 644 in hPR, and between amino acids 260 and 267 in mER [Carson-Jurica, M. Schrader, W. and OMalley, W. (1990) 'Endocrine Reviews 11,201-220]. While it has been shown that this NLS is not required for hormone-dependent nuclear localization of hPR, regulation of the subcellular localization of steroid receptors appears to be complex, and it was not a priori clear whether the presence of the SV40-like NLS was required for proper function of the chimeric proteins. Thus, additional constructs were prepared that incorporated an SV40 NLS (PKKKRKV) (SEQ ID NO: 30) in single letter amino acid code), either between VP16 and C7, or between C7 and LBD.

The chimeric transcriptional regulators were then tested for their ability to regulate the 10xC7-TATA-luc reporter plasmid in a hormone dependent manner.

10xC7-TATA-luc contains ten C7 binding sites [5'-GCG TGG GCG-31 spaced by nucleotides, and a TATA box, upstream of the firefly luciferase coding region.

Each of the fusion proteins upregulated expression of luciferase in a largely hormone dependent manner. RU486 stimulated the activity of VP16-C7-PR 26fold, while 4-ORT led to a 43-fold activation of VP16-C7-ER. There was no

IO

CI detectable crossreactivity between RU486 and ER, or between 4-OHT and PR.

O The presence of a NLS in either position was not only not required, but even undesirable, since it led to an increased basal hormone-independent) activity O of the fusion constructs, presumably through increased nuclear localization. Thus, the hPR (aa645-914) and mER (aa281-599, G525R) LBDs are able to confer 00 hormone-dependence onto the zinc finger protein C7.

The ability to reversibly control the expression of multiple genes, or Calleles of a gene, could prove very useful for many basic research applications. In Sparticular, selective and independent expression of one gene, but not another (and l 10 vice versa), by small and nontoxic ligands would allow for a comparative analysis of gene function, both in vitro and in vivo. We have shown that our modular system for controlling target gene expression is indeed able to independently control the expression of two genes within the same transfected cell, as evidenced by RU486-dependent luciferase induction and 4-OHT-induced P-gal expression.

The lack of P-gal induction by RU486, and luciferase induction by 4-OHT convincingly demonstrates the specificity of the chimeric regulators described here. Not only is the exquisite specificity of the utilized DNA binding domains retained, but also there is no detectable crossreaction between RU486 and the ER LBD, or between 4-OHT and the PR LBD.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (60)

1. A non-naturally occurring polypeptide comprising two ligand binding domains derived from nuclear hormone receptors operatively linked to a first functional domain, wherein the first functional domain is a zinc finger nucleotide binding domain.

2. The polypeptide of claim 1 wherein the two ligand binding domains are covalently linked by means of a peptide linker.

3. The polypeptide about 40 amino acid residues.

4. The polypeptide about 35 amino acid residues. The polypeptide about 30 amino acid residues. of claim 2 wherein the linker contains from about 10 to of claim 2 wherein the linker contains from about 15 to of claim 2 wherein the linker contains from about 18 to

6. The polypeptide of claim 1 wherein the first and second ligand binding domains are derived from different nuclear hormone receptors.

7. The polypeptide of claim 1 wherein the first and second ligand binding domains are derived from the same nuclear hormone receptor.

8. The polypeptide of claim 1 wherein the nuclear hormone receptor is an estrogen receptor, a progesterone receptor, an ecdysone receptor or a retinoic acid receptor or a retinoid X receptor.

9. The polypeptide of claim 1 wherein at least one of the ligand binding domains is derived from a retinoid X receptor. P \OPER\Kbm\(W)5234.64 5SPA doc-30V1)/ ()7 0 -44-

10. The polypeptide of claim 1 wherein the zinc finger nucleotide binding Sdomain comprises at least one zinc finger DNA binding motif.

11. The polypeptide of claim 10 that comprises from two to twelve zinc finger 00 5 DNA binding motifs. t 12. The polypeptide of claim 10 that comprises from two to six zinc finger 0 DNA binding motifs.

13. The polypeptide of claim 10 wherein the zinc finger DNA binding motifs specifically bind to a nucleotide sequence of the formula (GNN)I-6, where GNN specifies the base sequence of the nucleotide such that G is guanine and N is any base.

14. The polypeptide of claim 10 wherein the at least one zinc finger DNA binding motif is derived from a Cys 2 -His 2 zinc finger. The polypeptide of claim 1 further comprising a second functional domain, wherein the second functional domain is a transcription regulating domain.

16. The polypeptide of claim 15 wherein the second functional domain is operatively linked to either one of the ligand binding domains or the first functional domain.

17. The polypeptide of claim 15 wherein the transcription regulating domain is an activation domain.

18. The polypeptide of claim 15 wherein the transcription regulating domain is a repression domain.

19. A non-naturally occurring polypeptide comprising two ligand binding domains operatively linked to a first functional domain, wherein the first functional P \OPER\KbmU2(05234.W84 SSPA dcc-.30/i2(X)7 oo O 0 domain is selected from the group consisting of a zinc finger DNA binding domain and a nuclease domain.

20. The polypeptide of claim 19 wherein the two ligand binding domains are covalently linked by means of a peptide linker.

21. The polypeptide of claim 20 wherein the linker contains from about 10 to about 40 amino acid residues.

22. The polypeptide of claim 20 wherein the linker contains from about 15 to about 35 amino acid residues.

23. The polypeptide of claim 20 wherein the linker contains from about 18 to about 30 amino acid residues.

24. The polypeptide of claim 19 wherein the first functional domain is a zinc finger DNA binding domain. The polypeptide of claim 24 wherein the zinc finger DNA binding domain comprises at least one zinc finger DNA binding motif.

26. The polypeptide of claim 25 that comprises from two to twelve DNA binding motifs.

27. The polypeptide of claim 25 that comprises from two to six DNA binding motifs.

28. The polypeptide of claim 25 wherein the zinc finger DNA binding motifs specifically bind to a nucleotide sequence of the formula (GNN)1- 6 where GNN specifies the base sequence of the nucleotide such that G is guanine and N is any base. P \OPERKhm1lX)521346g4 SSPA do.-3(0 W2(X)7 0 -46-

29. The polypeptide of claim 25 wherein the at least one zinc finger DNA binding motif is derived from a Cys 2 -His 2 zinc finger. The polypeptide of claim 19 further comprising a second functional domain 00 5 operatively linked to either one of the ligand binding domains or the first functional domain. S31. The polypeptide of claim 30 wherein the first functional domain is a zinc C( finger DNA binding domain and the second functional domain is a transcriptional regulating domain.

32. The polypeptide of claim 31 wherein the zinc finger DNA binding domain comprises at least one zinc finger DNA binding motif.

33. The polypeptide of claim 32 that comprises from two to twelve zinc finger DNA binding motifs.

34. The polypeptide of claim 32 that comprises from two to six zinc finger DNA binding motifs. The polypeptide of claim 32 wherein the zinc finger DNA binding motifs specifically bind to a nucleotide sequence of the formula (GNN)I. 6 where GNN specifies the base sequence of the nucleotide such that G is guanine and N is any base.

36. The polypeptide of claim 32 wherein the at least one zinc finger DNA binding motif is derived from a Cys 2 -His 2 zinc finger.

37. The polypeptide of claim 31 wherein the transcription regulating domain is an activation domain.

38. The polypeptide of claim 31 wherein the transcription regulating domain is a repression domain. P\OPER\Kbm\20052()4' M 4SPA doc3/05/2007 0 -47- Ct

39. The polypeptide of claim 1 or 25 wherein the zinc finger nucleotide binding domain is selected from the group consisting of: a zinc finger nucleotide binding domain selected from the group consisting oO 5 of E2C, C7, B3B, 2C7, B3C2, and NI; a zinc finger nucleotide binding domain derived from a zinc finger tI nucleotide binding domain selected from the group consisting of E2C, C7, B3B, 2C7, B3C2, and N1 by truncation; a zinc finger nucleotide binding domain derived from a zinc finger nucleotide binding domain selected from the group consisting of E2C, C7, B3B, 2C7, B3C2, and N1 by expansion; and a zinc finger nucleotide binding domain derived from a zinc finger nucleotide binding domain selected from the group consisting of E2C, C7, B3B, 2C7, B3C2, and N1 by mutagenesis. The polypeptide of claim 17 or 37 wherein the activation domain is selected from the group consisting of VP16, TA2, VP64, STAT6, relA, TAF-1, TAF-2, TAU-1, and TAU-2.

41. The polypeptide of claim 18 or 38 wherein the repression domain is selected from the group consisting of ERD, KRAB, SID, histone deacetylase, DNA methylase, and a derivative or multimer of KRAB, SID, of ERD selected from the group consisting of KRAB-ERD, SID-ERD, (KRAB) 2 (KRAB) 3 KRAB-A, (KRAB-A) 2 (SID) 2 (KRAB-A)-SID, and SID-KRAB-A.

42. The polypeptide of claim 1 or 19 wherein at least one of the ligand binding domains binds a ligand selected from the group consisting of: l1 dimethylaminophenyl)- 17p-hydroxy- 7a-propinyl-4,9-estradiene-3-one; 11 dimethylaminophenyl)- 17a-hydroxy- 17p-(3-hydroxypropyl)- 1 3a-methyl-4,9-gonadiene- 3-one; 11 1-(4-acetylphenyl)-17p-hydroxy-17ca-(1-propinyl)-4,9-estradiene-3-one; 111-(4- dimethylaminophenyl)-17p-hydroxy-17 a-(3-hydroxy-l(Z)-propenyl-estra-4,9-diene-3- P \OPERU b2cO05234684 4SPA dm.3/05/2lOO7 -48- one; (7p3,11 P3, 1 7P) 1 1-(4-dimethylaminophenyl)-7-methyl-4' -dihydrospiro[ester-4,9- diene-1I 7,2'(3 'H)-furan]-3 -one; (11I1P,1 4P3,1 7c)-4',5'-dihydro-1 11-(4- dimethylaminophenyl) [spiroestra-4,9-diene- 1 7,2' H)-furan] -3 -one; and pregnane-3,2-dione. 00 M43. The polypeptide of claim 1 wherein at least one of the nuclear receptors is In selected from the group consisting of estrogen receptor progesterone receptor (PR), glucocorticoid-cc receptor, glucocorticoid-3 receptor, mineralocorticoid receptor, androgen receptor, thyroid hormone receptor, retinoic acid receptor (RAR), retinoid X receptor (RXR), Vitamin D receptor, COUP-TF receptor, ecdysone receptor (EcR), Nurr- 1 receptor, and orphan receptors.

44. The polypeptide of claim 1 wherein the polypeptide is a gene switch using RXR, E2C, and activation domains wherein the gene switch is selected from the group consisting of E2C-RXR-L-DE-VP64, E2C-RXR-LL-DE-VP64, E2C-RXR-LLL-DE- VP64, E2C-RXR-L-BE-VP64, E2C-RXR-LL-BE-VP64, E2C-RXR-LLL-BE-VP64, E2C- RXR-L-DE-VP 16, E2C-RXR-LL-DE-VP 16, E2C-RXR-LLL-DE-VP 16, E2C-RXR-L-BE- VP 16, E2C-RXR-LL-BE-VP 16, and E2C-RXR-LLL-BE-VP 16.

45. The polypeptide of claim 1 wherein the polypeptide is a gene switch using RXR, 2C7, and activation domains wherein the gene switch is selected from the group consisting of 2C7-RXR-L-DE-VP64, 2C7-RXR-LL-DE-VP64, 2C7-RXR-LLL-DE-VP64, 2C7-RXR-L-BE-VP64, 2C7-RXR-LL-BE-VP64, 2C7-RXR-LLL-BE-VP64, 2C7-RXR-L- DE-VP16, 2C7-RXR-LL-DE-VP 16, 2C7-RXR-LLL-DE-VP 16, 2C7-RXR-L-BE-VP 16, 2C7-RXR-LL-BE-VP 16, and E2C-RXR-LLL-BE-VP 16.

46. The polypeptide of claim 1 wherein the polypeptide is a gene switch using RXR, 3B1, and activation domains wherein the gene switch is selected from the group consisting of B3B-RXR-L-DE-VP64, B3B-RXR-LL-DE-VP64, B3B-RXR-LLL-DE- VP64, 133B 7-RXR-L-BE-VP64, 133B 7-RXR-LL-BE-VP64, B3B-RXR-LLL-BE-VP64, P kOPERtUO5234694 4SPA dom.3/05/2007 -49- B3B-RXR-L-DE-VP 16, B3B-RXR-LL-DE-VP 16, B3B-RXR-LLL-DE-VP 16, B3B-RXR- L-BE-VP 16, B3B-RXR-LL-BE-VP 16, and B3B-RXR-LLL-BE-VP 16.

47. The polypeptide of claim 1 wherein the polypeptide is a gene switch using 00 5 RXR, B3C2, and activation domains wherein the gene switch is selected from the group consisting of B3C2-RXR-L-DE-VP64, B3C2-RXR-LL-DE-VP64, B3C2-RXR-LLL-DE- (Ni VP64, B3C2-RXR-L-BE-VP64, B3C2-RXR-LL-BE-VP64, B3C2-RXR-LLL-BE-VP64, B3C2-RXR-L-DE-VP 16, B3C2-RXR-LL-DE-VP 16, B3C2-RXR-LLL-DE-VP 16, B3C2- NRXR-L-13E-VP 16, B3C2 B-RXR-LL-BE-VP 16, and B3C2-RXR-LLL-BE-VP 16.

48. The polypeptide of claim 1 wherein the polypeptide is a gene switch using RXR, E2C, and repression domains wherein the gene switch is selected from the group consisting of E2C-RXR-L-DE-(KRAB-A) 2 E2C-RXR-LL-DE-(KRAB-A) 2 E2C-RXR- LLL-DE-(KRAB-A) 2 E2C-RXR-L-BE-(KRAB-A) 2 E2C-RXR-LL-BE-(KRAB-A) 2 E2C-RXR-LLL-BE-(KRAB-A) 2 E2C-RXR-L-DE-(KRAB-A) 2 E2C-RXR-LL-DE- (KRAB-A) 2 E2C-RXR-LLL-DE-(KRAB-A) 2 E2C-RXR-L-BE-(KRAB-A) 2 E2C-RXR- LL-BE-(KRAB-A) 2 E2C-RXR-LLL-BE-(KRAB-A) 2 E2C-RXR-L-DE-(SID) 2 E2C- RXR-LL-DE-(SID) 2 E2C-RXR-LLL-DE-(SID) 2 E2C-RXR-L-BE-(SID) 2 E2C-RXR-LL- BE-(SID) 2 E2C-RXR-LLL-BE-(SID) 2 E2C-RXR-L-DE-(SID) 2 E2C-RXR-LL-DE- (SID) 2 E2C-RXR-LLL-DE-(SID) 2 E2C-RXR-L-BE-(SID) 2 E2C-RXR-LL-BE-(S ID) 2 and E2C-RXR-LLL-BE-(SID) 2

49. The polypeptide of claim I wherein the polypeptide is a gene switch using RXR, 2C7, and repression domains wherein the gene switch is selected from the group consisting of 2C7-RXR-L-DE-(KRAB-A) 2 2C7-RXR-LL-DE-(KRAB-A) 2 2C7-RXR- LLL-DE-(KRAB-A) 2 2C7-RXR-L-BE-(KRAB-A) 2 2C7-RXR-LL-BE-(KRAB-A) 2 2C7- RXR-LLL-BE-(KRAB-A) 2 2C7-RXR-L-DE-(KRAB-A) 2 2C7-RXR-LL-DE-(KRAB-A) 2 2C7-RXR-LLL-DE-(KRAB-A) 2 2C7-RXR-L-BE-(KRAB-A) 2 2C7-RXR-LL-BE- (KRAB-A) 2 E2C-RXR-LLL-BE-(KRAB-A) 2 2C7-RXR-L-DE-(SID) 2 2C7-RXR-LL- DE-(SID) 2 2C7-RXR-LLL-DE-(SID) 2 2C7-RXR-L-BE-(SID) 2 2C7-RXR-LL-BE-(SID) 2 2C7-RXR-LLL-BE-(SID) 2 2C7-RXR-L-DE-(SID) 2 2C7-RXR-LL-DE-(SID) 2 2C7-RXR- P XOPERM.t,,\2O523468A 4SPA dm-105/2007 LLL-DE-(SID) 2 2C7-RXR-L-BE-(SID) 2 2C7-RXR-LL-BE-(SID) 2 and E2C-RXR-LLL- BE-(S ID) 2 The polypeptide of claim I wherein the polypeptide is a gene switch using 00 5 RXR, 131, and repression domains wherein the gene switch is selected from the group consisting of B3B-RXR-L-DE-(KRAB-A) 2 B3B-RXR-LL-DE-(KRAB-A) 2 B3B-RXR- LLL-DE-(KRAB-A) 2 B3B 7-RXR-L-BE-(KRAB-A) 2 B3B 7-RXR-LL-BE-(KRAB-A) 2 B3B-RXR-LLL-BE-(KRAB-A) 2 B3B-RXR-L-DE-(KRAB-A) 2 B3B-RXR-LL-DE- (KRAB-A) 2 B3B-RXR-LLL-DE-(KRAB-A) 2 B3B-RXR-L-BE-(KRAB-A) 2 B3B-RXR- LL-BE-(KRAB-A) 2 B3B-RXR-LLL-BE-(KRAB-A) 2 B3B-RXR-L-DE-(SID) 2 B3B- RXR-LL-DE-(SID) 2 B3B-RXR-LLL-DE-(SID) 2 B3B 7-RXR-L-BE-(SID) 2 B3B 7-RXR- LL-BE-(SID) 2 B3B-RXR-LLL-BE-(SID) 2 B3B-RXR-L-DE-(SID) 2 B3B-RXR-LL-DE- (SID) 2 B3B-RXR-LLL-DE-(SID) 2 B3B-RXR-L-BE-(SID) 2 B3B-RXR-LL-BE-(SID) 2 and B3B-RXR-LLL-BE-(SID) 2

51. The polypeptide of claim 1 wherein the polypeptide is a gene switch using RXR, B3C2, and repression domains wherein the gene switch is selected from the group consisting of B3C2-RXR-L-DE-(KRAB-A) 2 B3C2-RXR-LL-D-(KRAB-A) 2 B3C2-RXR- LLL-DE-(KRAB-A) 2 B3C2-RXR-L-BE-(KRAB-A) 2 B3C2-RXR-LL-BE-(KRAB-A) 2 B3C2.-RXR-LLL-BE-(KRAB-A) 2 B3C2-RXR-L-DE-(KRAB-A) 2 B3C2-RXR-LL-DE- (KRAB-A) 2 B3C2-RXR-LLL-DE-(KRAB-A) 2 B3C2-R-XR-L-BE-(KRAB-A) 2 B3C2 B- RXR-LL-BE-(KRAB-A) 2 B3C2-RXR-LLL-BE-(KRAB-A) 2 B3C2-RXR-L-DE-(SID) 2 B3C2-RXR-LL-DE-(SID) 2 B3C2-RXR-LLL-DE-(SID) 2 B3C2-RXR-L-BE-(SID) 2 B3C2-RXR-LL-BE-(SID) 2 B3C2-RXR-LLL-BE-(SID) 2 B3C2-RXR-L-DE-(SID) 2 B3C2-RXR-LL-DE-(SID) 2 B3C2-RXR-LLL-DE-(SID) 2 B3C2-RXR-L-BE-(SID) 2 B3C2 B-RXR-LL-BE-(SID) 2 and B3C2-RXR-LLL-BE-(SID) 2

52. The polypeptide of claim 1 wherein the polypeptide is a gene switch using PR, E2C, and activation domains wherein the gene switch is selected from the group consisting of E2C-PR-L-PR-VP64, E2C-PR-LL-PR-VP64, E2C-PR-LLL-PR-VP64, E2C- PR-L-PR-VP64, E2C-PR-LL-PR-VP64, E2C-PR-LLL-PR-VP64, E2C-PR-L-PR-VP 16, P WpER\Kbn\2CID2346&4 4SPA doc.3M2O07 -51 E2C-PR-LL-PR-VP 16, E2C-PR-LLL-PR-VP 16, E2C-PR-L-PR-VP 16, E2C-PR-LL-PR- VP 16, and E2C-PR-LLL-PR-VP 16.

53. The polypeptide of claim 1 wherein the polypeptide is a gene switch using 00 5 PR, 2C7, and activation domains wherein the gene switch is selected from the group consisting of 2C7-PR-L-PR-VP64, 2C7-PR-LL-PR-VP64, 2C7-PR-LLL-PR-VP64, 2C7- N PR-L-PR-VP64, 2C7-PR-LL-PR-VP64, 2C7-PR-LLL-PR-VP64, 2C7-PR-L-PR-VP 16, 2C7-PR-LL-PR-VP 16, 2C7-PR-LLL-PR-VP 16, 2C7-PR-L-PR-VP 16, 2C7-PR-LL-PR- VP 16, and E2C-PR-LLL-PR-VP 16.

54. The polypeptide of claim I wherein the polypeptide is a gene switch using PR, 131, and activation domains wherein the gene switch is selected from the group consisting of B3B-PR-L-PR-VP64, B3B-PR-LL-PR-VP64, B3B-PR-LLL-PR-VP64, B3B 7-PR-L-PR-VP64, B3B 7-PR-LL-PR-VP64, B3B-PR-LLL-PR-VP64, B3B-PR-L-PR- VP 16, B3B-PR-LL-PR-VP 16, B3B-PR-LLL-PR-VP 16, B3 B-PR-L-PR-VP 16, B3B-PR- LL-PR-VP 16, and B3 B-PR-LLL-PR-VP 16. The polypeptide of claim 1 wherein the polypeptide is a gene switch using PR, B33C2, and activation domains wherein the gene switch is selected from the group consisting of B3C2-PR-L-PR-VP64, B3C2-PR-LL-PR-VP64, B3C2-PR-LLL-PR-VP64, B3C2-PR-L-PR-VP64, B3C2-PR-LL-PR-VP64, B3C2-PR-LLL-PR-VP64, B3C2-PR-L- PR-VP 16, B3C2-PR-LL-PR-VP 16, B3C2-PR-LLL-PR-VP 16, B3C2-PR-L-PR-VP 16, B3C2 B-PR-LL-PR-VP 16, and B3C2-PR-LLL-PR-VP 16.

56. The polypeptide of claim 1 wherein the polypeptide is a gene switch using PR, E2C, and repression domains wherein the gene switch is selected from the group consisting of E2C-PR-L-PR-(KRAB-A) 2 E2C-PR-LL-PR-(KRAB-A) 2 E2C-PR-LLL-PR- (KRAB-A) 2 E2C-PR-L-PR-(KRAB-A) 2 E2C-PR-LL-PR-(KRAB-A) 2 E2C-PR-LLL-PR- (KRAB-A) 2 E2C-PR-L-PR-(KRAB-A) 2 E2C-PR-LL-PR-(KRAB-A) 2 E2C-PR-LLL-PR- (KRAB-A) 2 E2C-PR-L-PR-(KRAB-A) 2 E2C-PR-LL-PR-(KRAB-A) 2 E2C-PR-LLL-PR- (KRAB-A) 2 E2C-PR-L-PR-(SID) 2 E2C-PR-LL-PR-(SID) 2 E2C-PR-LLL-PR-(SID) 2 P OPERkX.1,2005234684 4SPA d..3052007 -52- E2C-PR-L-PR-(SID) 2 E2C-PR-LL-PR-(SID) 2 E2C-PR-LLL-PR-(SID) 2 E2C-PR-L-PR- (SID) 2 E2C-PR-LL-PR-(SID) 2 E2C-PR-LLL-PR-(SID) 2 E2C-PR-L-PR-(SID) 2 E2C-PR- LL-PR-(SID) 2 and E2C-PR-LLL-PR-(SID) 2 00 5 57. The polypeptide of claim 1 wherein the polypeptide is a gene switch using PR, 2C7, and repression domains wherein the gene switch is selected from the group N consisting of 2C7-PR-L-PR-(KRAB-A) 2 2C7-PR-LL-PR-(KRAB-A) 2 2C7-PR-LLL-PR- (KRAB-A) 2 2C7-PR-L-PR-(KRAB-A) 2 2C7-PR-LL-PR-(KRAB-A) 2 2C7-PR-LLL-PR- (KRAB-A) 2 2C7-PR-L-PR-(KRAB-A) 2 2C7-PR-LL-PR-(KRAB-A) 2 2C7-PR-LLL-PR- (KRAB-A) 2 2C7-PR-L-PR-(KRAB-A) 2 2C7-PR-LL-PR-(KRAB-A) 2 E2C-PR-LLL-PR- (KRAB-A) 2 2C7-PR-L-PR-(SID) 2 2C7-PR-LL-PR-(SID) 2 2C7-PR-LLL-PR-(SID) 2 2C7-PR-L-PR-(SID) 2 2C7-PR-LL-PR-(SID) 2 2C7-PR-LLL-PR-(SID) 2 2C7-PR-L-PR- (SID) 2 2C7-PR-LL-PR-(SID) 2 2C7-PR-LLL-PR-(SID) 2 2C7-PR-L-PR-(SID) 2 2C7-PR- LL-PR-(SID) 2 and E2C-PR-LLL-PR-(SID) 2

58. The polypeptide of claim 1 wherein the polypeptide is a gene switch using PR, B33B, and repression domains wherein the gene switch is selected from the group consisting of B3B-PR-L-PR-(KRAB-A) 2 B3B-PR-LL-PR-(KRAB-A) 2 B3B-PR-LLL-PR- (KRAB-A) 2 B3B 7-PR-L-PR-(KRAB-A) 2 B33B 7-PR-LL-PR-(KRAB-A) 2 B3B-PR-LLL- PR-(KRAB-A) 2 B3B-PR-L-PR-(KRAB-A) 2 B3B-PR-LL-PR-(KRAB-A) 2 B3B-PR-LLL- PR-(KRAB-A) 2 B3B-PR-L-PR-(KRAB-A) 2 B3B-PR-LL-PR-(KRAB-A) 2 B3B-PR-LLL- PR-(KRAB-A) 2 B3B-PR-L-PR-(SID) 2 B3B-PR-LL-PR-(SID) 2 B3B-PR-LLL-PR-(SID) 2 133B 7-PR-L-PR-(SLD) 2 133B 7-PR-LL-PR-(SID) 2 B3B-PR-LLL-PR-(SID) 2 B3B-PR-L- PR-(SID) 2 B3B-PR-LL-PR-(SID) 2 B3B-PR-LLL-PR-(SID) 2 B3B-PR-L-PR-(SID) 2 B3B-PR-LL-PR-(SID) 2 and B3B-PR-LLL-PR-(SID) 2

59. The polypeptide of claim I wherein the polypeptide is a gene switch using PR, B3C2, and repression domains wherein the gene switch is selected from the group consisting of B3C2-PR-L-PR-(KRAB-A) 2 B3C2-PR-LL-PR-(KRAB-A) 2 B3C2-PR-LLL- PR-(KRAB-A) 2 B3C2-PR-L-PR-(KRAB-A) 2 B3C2-PR-LL-PR-(KRAB-A) 2 B3C2-PR- LLL-PR-(KRAB-A) 2 B3C2-PR-L-PR-(KRAB-A) 2 B3C2-PR-LL-PR-(KRAB-A) 2 B3C2- P OPER\Kt,,,\O05234684 4SPA do.-3M/2007 -53- PR-LLL-PR-(KRAB-A) 2 B3C2-PR-L-PR-(KRAB-A) 2 B3C2 B-PR-LL-PR-(KRAB-A) 2 B3C2-PR-LLL-PR-(KRAB-A) 2 B3C2-PR-L-PR-(SID) 2 B3C2-PR-LL-PR-(SLD) 2 B3C2- PR-LLL-PR-(SID) 2 B3C2-PR-L-PR-(SID) 2 B3C2-PR-LL-PR-(SID) 2 B3C2-PR-LLL- PR-(SID) 2 B3C2-PR-L-PR-(SID) 2 B3C2-PR-LL-PR-(SID) 2 B3C2-PR-LLL-PR-(S ID) 2 00 5 B3C2-PR-L-PR-(SID) 2 B33C2 B-PR-LL-PR-(SID) 2 and B3C2-PR-LLL-PR-(SID) 2 The polypeptide of claim 1 wherein the polypeptide is a gene switch using ER, E2C, and activation domains wherein the gene switch is selected from the group consisting of E2C-ER-L-ER-VP64, E2C-ER-LL-ER-VP64, E2C-ER-LLL-ER-VP64, E2C- ER-L-ER-VP64, E2C-ER-LL-ER-VP64, E2C-ER-LLL-ER-VP64, E2C-ER-L-ER-VP 16, E2C-ER-LL-ER-VP 16, E2C-ER-LLL-ER-VP 16, E2C-ER-L-ER-VP 16, E2C-ER-LL-ER- VP 16, and E2C-ER-LLL-ER-VP 16.

61. The polypeptide of claim 1 wherein the polypeptide is a gene switch using ER, 2C7, and activation domains wherein the gene switch is selected from the group consisting of 2C7-ER-L-ER-VP64, 2C7-ER-LL-ER-VP64, 2C7-ER-LLL-ER-VP64, 2C7- ER-L-ER-VP64, 2C7-ER-LL-ER-VP64, 2C7-ER-LLL-ER-VP64, 2C7-ER-L-ER-VP 16, 2C7-ER-LL-ER-VP 16, 2C7-ER-LLL-ER-VP 16, 2C7-ER-L-ER-VP 16, 2C7-ER-LL-ER- VP 16, and E2C-ER-LLL-ER-VP 16.

62. The polypeptide of claim 1 wherein the polypeptide is a gene switch using ER, 3B1, and activation domains wherein the gene switch is selected from the group consisting of B3B-ER-L-ER-VP64, B3B-ER-LL-ER-VP64, B3B-ER-LLL-ER-VP64, B3B 7-ER-L-ER-VP64, 3B1 7-ER-LL-ER-VP64, B3B-ER-LLL-ER-VP64, B3B-ER-L-ER- VP 16, B3B-ER-LL-ER-VP 16, B3B-ER-LLL-ER-VP 16, B3B-ER-L-ER-VP 16, B3B-ER- LL-ER-VP 16, and B3B-ER-LLL-ER-VP 16.

63. The polypeptide of claim 1 wherein the polypeptide is a gene switch using ER, B33C2, and activation domains wherein the gene switch is selected from the group consisting of B3C2-ER-L-ER-VP64, B3C2-ER-LL-ER-VP64, B3G2-ER-LLL-ER-VP64, B3C2-ER-L-ER-VP64, B3C2-ER-LL-ER-VP64, B3C2-ER-LLL-ER-VP64, B3C2-ER-L- P \OPER\Kb.\205234684 4SPA dm-3M~/2007 -54- ER-VP16, B3C2-ER-LL-ER-VP 16, B3C2-ER-LLL-ER-VP 16, B3C2-ER-L-ER-VP 16, B3C2 B-ER-LL-ER-VP 16, and B3C2-ER-LLL-ER-VP16.

64. The polypeptide of claim 1 wherein the polypeptide is a gene switch using 00 5 ER, E2C, and repression domains wherein the gene switch is selected from the group co s s i goI'DE R R B A 2 E C E L E K A B A 2 2 R L L (cnsstn ER( of-A 2 E2C-ER-L-ER-(KRAB-A) 2 E2C-ER-LL-ER-(KRAB-A) 2 E2C-ER-- LER-(KRAB-A) 2 E2C-ER-L-ER-(KRAB-A) 2 E2C-ER-LL-ER-(KRAB-A) 2 E2C-R ELLL-ER-(KRAB-A) 2 E2C-ER-L-ER-(KRAB-A) 2 E2C-ER-LL-ER-(KRAB-A) 2 C 2 E2C-ER-L-ER-(SIDB-) 2 E2C-ER-LL-ER-(D B-) 2 ,-R 1 2RLLL-ER-(B-) 2 E2C-ER-L-ER-(SID) 2 E2C-ER-LL-ER-(SID) 2 E2C-ER--R LLR(SID) 2 E2C-ER-L-ER-(SID) 2 E2C-ER-LL-ER-(SID) 2 E2C-ER-LLL-ER-EC (I)ECER-L-ER-(SID) 2 E2C-ER-LL-ER-(SID) 2 ,an E2C-ER-LLL-ER-(SID) 2 2C

65. The polypeptide of claim I wherein the polypeptide is a gene switch using ER, 2C7, and repression domains wherein the gene switch is selected from the group consisting of 2C7-ER-L-ER-(KRAB-A) 2 2C7-ER-LL-ER-(KRAB-A) 2 2C7-ER-LLL-ER- (KRAB-A) 2 2C7-ER-L-ER-(KRAB-A) 2 2C7-ER-LL-ER-(KRAB-A) 2 2C7-ER-LLL-ER- (KRAB-A) 2 2C7-ER-L-ER-(KRAB-A) 2 2C7-ER-LL-ER-(KRAB-A) 2 2C7-ER-LLL-ER- (KRAB-A) 2 2C7-ER-L-ER-(KR-AB-A) 2 2C7-ER-LL-ER-(KRAB-A) 2 E2C-ER-LLL-ER- (KRAB-A) 2 2C7-ER-L-ER-(SID) 2 2C7-ER-LL-ER-(SID) 2 2C7-ER-LLL-ER-(SID) 2 2C7-ER-L-ER-(SID) 2 2C7-ER-LL-ER-(SID) 2 2C7-ER-LLL-ER-(SID) 2 2C7-ER-L-ER- (S ID) 2 2C7-ER-LL-ER-(SID) 2 2C7-ER-LLL-ER-(SID) 2 2C7-ER-L-ER-(SID) 2 2C7-ER- LL-ER-(SID) 2 and E2C-ER-LLL-ER-(SID) 2

66. The polypeptide of claim 1 wherein the polypeptide is a gene switch using ER, 131, and repression domains wherein the gene switch is selected from the group consisting of B3B-ER-L-ER-(KRAB-A) 2 B3B-ER-LL-ER-(KRAB-A) 2 B3B-ER-LLL- ER-(KRAB-A) 2 B33B 7-ER-L-ER-(KRAB-A) 2 133B 7-ER-LL-ER-(KRAB-A) 2 B3B-ER- LLL-ER,-(KRAB-A) 2 B3B-ER-L-ER-(KRAB-A) 2 B3B-ER-LL-ER-(KRAB-A) 2 B3B- ER-LLL-ER-(KRAB-A) 2 B3B-ER-L-ER-(KRAB-A) 2 B3B-ER-LL-ER-(KRAB-A) 2 P \OPERK,-'2052346M4 4SPA do.3)52(X)7 B3B-ER-LLL-ER-(KRAB-A) 2 B3B-ER-L-ER-(SID) 2 B3B-ER-LL-ER-(SID) 2 B3B-ER- LLL-ER-(SID) 2 B3B 7-ER-L-ER-(SID) 2 B3B 7-ER-LL-ER-(SID) 2 B3B-ER-LLL-ER- (SID) 2 B3B-ER-L-ER-(SID) 2 B3B-ER-LL-ER-(SID) 2 B3B-ER-LLL-ER-(SID) 2 B3B- ER-L-ER-(SID) 2 B3B-ER-LL-ER-(SID) 2 and B3B-ER-LLL-ER-(SID) 2 00

67. The polypeptide of claim 1 wherein the polypeptide is a gene switch using ER, B3C2, and repression domains wherein the gene switch is selected from the group consisting of B3C2-ER-L-ER-(KRAB-A) 2 B3C2-ER-LL-ER-(KRAB-A) 2 B3C2-ER- LLL-ER-(KRAB-A) 2 B3C2-ER-L-ER-(KRAB-A) 2 B3C2-ER-LL-ER-(KRAB-A) 2 B3C2-ER-LLL-ER-(KRAB-A) 2 B3C2-ER-L-ER-(KRAB-A) 2 B3C2-ER-LL-ER-(KRAB- A) 2 B3C2-ER-LLL-ER-(KRAB-A) 2 B3C2-ER-L-ER-(KRAB-A) 2 B3C2 B-ER-LL-ER- (KRAB-A) 2 B3C2-ER-LLL-ER-(KRAB-A) 2 B3C2-ER-L-ER-(SID) 2 B3C2-ER-LL-ER- (SID) 2 B3C2-ER-LLL-ER-(SID) 2 B3C2-ER-L-ER-(SID) 2 B3C2-ER-LL-ER-(SID) 2 B3C2-ER-LLL-ER-(SID) 2 B3C2-ER-L-ER-(SID) 2 B3C2-ER-LL-ER-(SID) 2 B3C2-ER- LLL-ER-(SID) 2 B3C2-ER-L-ER-(SID) 2 B3C2 B-ER-LL-ER-(SID) 2 and B3C2-ER-LLL- ER-(SID) 2

68. The polypeptide of claim 1 or 19 wherein the polypeptide has activation or repression activity at multiple promoters.

69. A polynucleotide that encodes the polypeptide of claim I or claim 19. An expression vector comprising the polynucleotide of claim 69.

71. A cell containing the polynucleotide of claim 69.

72. A host cell transformed with the expression vector of claim

73. A process of regulating the function of a target nucleotide that contains a defined sequence, the process comprising exposing the target nucleotide to the polypeptide of claim 1 or claim 19 in the presence of a ligand that binds one of the ligand binding P \OPER\Kbm\2005234684 4SPA doc-3/5/2007 S-56- domains of the polypeptide, wherein the DNA binding domain of the polypeptide binds the c defined sequence.

74. The polypeptide of claim 1 or claim 19, the polynucleotide of claim 69, the 00 5 expression vector of claim 70, the cell of claim 71, the host cell of claim 72, the process of l" claim 73, substantially as hereinbefore described and/or exemplified. o- tt (-N