WO2004044202A1 - Method for the gene regulation at both transcription and post-transcription levels - Google Patents

Abstract

A method for regulating a target gene, which comprises introducing into a cell a zinc finger protein binding to a promoter of the target gene or a DNA encoding same, and a RNA molecule binding to an mRNA transcribed from the target gene to inhibit the expression of said target gene; and a composition for regulating a target gene comprising the zinc finger protein or a DNA encoding same, and the RNA molecule provide a substantially complete gene regulating effect due to the synergistic effect of the combination of ZFP and RNAi technologies.

Description

METHOD FOR THE GENE REGULATION AT BOTH TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL LEVELS

FIELD OF THE INVENTION

The present invention relates to methods and compositions for regulating a target gene at both transcriptional and post-transcriptional levels. More particularly, it includes in one embodiment, a method for regulating a target gene, which comprises introducing into a cell a zinc finger protein binding to a promoter of the target gene or a DNA encoding said protein, and a RNA molecule binding to an mRNA transcribed from the target gene to inhibit the expression of said target gene.

BACKGROUND OF THE INVENTION

Gene expression can be regulated at two different levels , which are transcriptional and post-transcriptional levels. Chromosomal DNA is used as a target for regulation at the transcriptional level; and mRNA or a protein, for regulation at the post-transcriptional level.

The zinc finger proteins of C2-H2 class constitute one of the most common DNA binding motifs found in eukaryotic transcription factors including those of human. Specificity and the modular nature of these motifs have been extensively studied by several groups of researchers (J. R. Desjarlais and J. M. Berg, 1993, PNAS, vol 90, 2256; H. A. Greisman and C. O. Pabo, Science, 1997 Jan 31; 275(5300):657-61; and D. J. Segal et al, Proc Natl Acad Sci U S A., 1999 Mar 16; 96(6):2758-63). The advance of zinc finger protein technology allowed the production of viable artificial zinc finger proteins by in vitro screening of randomized composite zinc finger proteins (Choo, Y. & Klug, A., Proc Natl Acad Sci U S A, 1994 Nov 8; 91(23):11168-72; and H. A. Greisman and C. O. Pabo, supra) or by in vivo selection using yeast one-hybrid system after mixing and matching individual zinc finger domains (Korean Patent Laid- open Publication No. 10-2001-0084880, Toolgen Inc.). Such studies allow the design of a specific ZFP which can bind with high affinity to a given target gene promoter sequences and eventually up- or down-regulate the target gene expression when the zinc finger domains are fused with a transcriptional activator domain or repressor domain, respectively. The effects of ZFPs to regulate endogenous genes have been reported and accumulated evidences clearly indicate that the zinc finger protein technology is one of the most versatile technologies for up- or down-regulating a target gene (Liu et al, J Biol Chem., 2001 Apr 6; 276(14): 11323-34; and Zhang L., et al., J Biol Chem., 2000 Oct 27; 275(43):33850-60). The zinc finger proteins successfully regulated genes in mammalian cells (Rebar E. J. et al., Nat Med., 2002 Dec; 8(12): 1427-32; Jouvenot Y. et al., Gene Then, 2003 Mar; 10(6):513-22; and Bae K. H. et al, Nat Biotechnol., 2003 Mar; 21(3):275-80), in plants (Guan X. et al., Proc Natl Acad Sci U S A, 2002 Oct 1; 99(20): 13296-301) and in yeasts (KS Park in press Nature Biotech. 2003). The modular structure of zinc finger proteins facilitated the generation of libraries that can interact with specific DNA sequences in the genome in a wide scale, and recent studies show that ZFPs are useful in the induction of particular phenotypes and identification of genes that are responsible for the phenotype (Blancafort P. et al., Nat Biotechnol., 2003 Mar;21(3):269-74; and Park K.S. et al., Nat. Biotechnol. 2003; 21(3) 1208-14).

RNAi technology is based on the change of mRNA stability induced by the formation of a transient double-strand RNA between intracellular mRNA and exogenous RNA introduced in a cell. In 1998, Fire et al. reported that double- stranded RNA inhibited target mRNAs more efficiently than individual single- stranded RNAs did, and that this may be apparently inheritable in C. elegans . This phenomenon was observed in many genes in C. elegans (Shi Y. and Mello C, Genes Dev., 1998 Apr l;12(7):943-55; Montgomery and Fire, Nature, 1998 Feb 19; 391(6669):806-11; and Tabara H. et al., Science, 1998 Oct 16; 282(5388):430-l), in Drosophila (Kennerdell and Carthew, Nat Biotechnol, 2000 Aug; 18(8):896-8), and in plants (Voinnet O., Trends Genet., 2001 Aug; 17(8):449-59; and Waterhouse P.M., Proc Natl Acad Sci U S A, 1998 Nov 10; 95(23): 13959-649). The effects of RNAi in mammalian cells were reported for the mouse oocyte (Svoboda P. et al, Development., 2000 Oct; 127(19):4147-56; and Svoboda P. et al, Biochem Biophys Res Conrmun., 2001 Oct 12; 287(5): 1099-104) and for human embryonic kidney cell line HEK239 (Elbashir S.M. et al, Nature, 2001 May 24; 411(6836):494-8) after transfection of plasmid encoding a target gene. The exogenous dsRNA is processed into 19-23 nt small interfering RNA (siRNA) by enzyme Dicer. The incorporation of 19-23 nt siRNA into RISC (RNA induced gene silencing complex) leads to the recognition and subsequent degradation of mRNA homologous to the siRNA (Hammond S.M. et al, Science, 2001 Aug 10; 293(5532): 1146-50).

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a method for efficiently regulating a gene at both transcriptional and post-transcriptional levels.

It is another object of the present invention to provide a composition for efficiently regulating a gene at both transcriptional and post-transcriptional levels. In accordance with one aspect of the present invention, there is provided a method for regulating a target gene, which comprises introducing into a cell a zinc finger protein binding to a sequence in the target gene, e.g., the promoter of the target gene or other regulatory sequence, or a nucleic acid encoding said protein, and a RNA molecule that includes a strand having a sequence complementary to an mRNA transcribed from the target gene, or a nucleic acid that can be used to produce the RNA molecule, to inhibit the expression of said target gene.

For example, a DNA molecule that includes two promoters can be used to produce a dsRNA based on the sequence between the two promoters. DNA molecules can similarly be transcribed to produce anti-sense and ribozymes in a cell or in vitro. In one embodiment, the zinc finger protein binds to the sequence in the target gene with a K_D of less than 10^"7, lO^-8 , 10^-9 , 10^-10 , 10^-11 , 10^"12 or 10^"14M.

In one embodiment, the combination of a zinc fmger protein and the RNA molecule results in greater than 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 100, or 500 fold repression relative to a reference cell that does not include the zinc fmger protein or the RNA molecule. Repression can reduce detectable mRNA or protein levels of the target gene to less than 5, 4, 3, 2, 1, 0.2, 0.01, 0.005, 0.001, or 0.0001% of the reference, or below detection.

In accordance with another aspect of the present invention, there is provided a composition for regulating a target gene comprising a zinc fmger protein binding to a promoter of the target gene or a DNA encoding said protein, and a RNA molecule binding to an mRNA transcribed from the target gene to inhibit the expression of said target gene.

In one embodiment, it is also possible to use another exogenous transcription factors, e.g., an artificial transcription factor that includes domains other than or in addition to a zinc finger domain.

In one embodiment, a zinc finger protein and an RNA molecule are used to selectively regulate an alternate transcript of a gene. For example, the zinc fmger protein, which may include a transcriptional activation domain, can be used to increase expression of the gene, and the RNA molecule can be used to inhibit a first transcript of the gene without inhibiting a second transcript of the gene, thereby increasing production of the second transcript. The RNA molecule can be made specific to the first transcript relative to the second transcript, e.g., by having a sequence complementary to a sequence that is only present in the first transcript.

In one embodiment, an RNA molecule is used to shut down expression of a target gene activated by a zinc fmger protein. The method can be used to provide limited temporal or spatial activation of the target gene. The method includes delivering the zinc finger protein (e.g., including an activation domain) or a nucleic acid encoding the protein to a cell, tissue, or subject, such that the target gene is activated, and delivering or producing (e.g., in a cell) the RNA molecule to selectively reduce expression of the target gene. The RNA molecule can include a strand with a sequence complementary to a transcript of the target gene or to a transcript that encodes the zinc finger protein. The RNA molecule can be delivered or produced in a cell after the target gene is being activated, during, or before, or can be provided in a spatially restricted manner so that activation of the target gene is limited to particular cells or a particular time. For example, the RNA molecule can be produced by tissue-specific promoters.

As used herein, a "zinc finger protein" refers to a protein that includes at least one zinc fmger domain. The term "domain" refers to a functional unit within a polypeptide. A domain's tertiary structure may be folded or unfolded. A "zinc fmger domain" is a domain that includes approximately 30 amino acid residues (e.g., 26-36) in which there are four residues, either cysteine or histidine, appropriately spaced such that they can coordinate a zinc ion, and forming a folded structure that can interact with nucleic acid. The term "exogenous" refers to an agent that is supplied from without.

The term "dissociation constant" refers to the equilibrium dissociation constant (K ) of a polypeptide for binding to a 28-basepair double-stranded DNA that includes one target site for the polypeptide being assayed. For example, if the polypeptide has a three-finger DNA binding domain, the DNA will include a 9-bp or larger target site that the polypeptide specifically recognizes. The dissociation constant is determined by gel shift analysis using a purified protein that is bound in 20 mM Tris pH 7.7, 120 mM NaCl, 5 mJVI MgCl₂, 20 μM ZnS0₄, 10% glycerol, 0.1% Nonidet P-40, 5 mM DTT, and 0.10 mg/mL BSA (bovine serum albumin) at room temperature. Additional details are provided in the example below and Rebar and Pabo (1994) Science 263:671-673. Polypeptides that bind to sites larger than 28-basepairs can be assayed using a larger double- stranded DNA. Exemplary dissociation constants include constants less than 10^"7 M, 10^"8 M, 10^-9 M, 10^"10 M, 10^-11 M, or 10^"12 M.

The terms "hybrid" and "chimera" refer to a non-naturally occurring polypeptide that comprises amino acid sequences derived from either (i) at least two different naturally occurring sequences, or non-contiguous regions of the same naturally occurring sequence, wherein the non-contiguous regions are made contiguous in the hybrid; (ii) at least one artificial sequence (i.e., a sequence that does not occur naturally) and at least one naturally occurring sequence; or (iii) at least two artificial sequences (same or different). A zinc finger protein can be a hybrid of different zinc finger domains, e.g., naturally occurring zinc finger domains.

When describing a sequence, the term "naturally occurring" refers to a sequence (e.g., a nucleic acid or amino acid sequence) which is present in a cell of a natural organism, i.e., an organism that has not been modified by molecular biological techniques. For example, a transgenic mouse is not a natural organism, but a highly inbred mouse that has not been modified by molecular biological techniques is considered natural. When describing a sequence, the term "viral" refers to a sequence of a naturally occurring virus, i.e., a virus that has not been modified by molecular biological techniques. One embodiment of the invention includes proteins that include a zinc finger domain from Homo sapiens, Mus musculus, Arabidopsis thaliana, Drosophila melanogaster, Escherichia coli, Saccharomyces cerevisiae, or Oryza sativa.

An "artificial sequence" is a sequence constructed by artificial means. Examples of artificial sequences include mutants of a naturally occurring sequence that are generated by site directed mutagenesis or random mutagenesis and de novo designed sequences.

The term "fusion" refers to a single polypeptide chain that includes the components that are fused. An exemplary fusion protein includes a DNA binding domain and a transcriptional repression domain. The fused components need not be directly linked. For example, another sequence (e.g., a linker or a functional domain) can be located between the fused elements.

The term "heterologous polypeptide" refers either to a polypeptide with a non-naturally occurring sequence (e.g., a hybrid polypeptide) or a polypeptide with a sequence identical to a naturally occurring polypeptide but present in a milieu in which it does not naturally occur. It is also possible for one element of a polypeptide to be heterologous relative to another element. For example, a fusion protein can link domains that are from different naturally occurring proteins so that the domains are heterologous to each other.

The term "isolated" describes a composition that is removed from at least 90% of at least one component of a sample (e.g., a natural sample or cell, e.g., a recombinant cell) or a synthetic reaction from which the isolated composition can be obtained. Compositions described herein produced artificially or naturally can be "compositions of at least" a certain degree of purity if the species or population of species of interest is at least 5, 10, 25, 50, 75, 80, 90, 95, 98, or 99%o pure on a weight-weight basis, respectively. Any of the compositions describe herein can be in an isolated form.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

Figs. 1A to IE: schematic diagrams showing the structures of: expression vectors for zinc fmger proteins containing CMV promoter, HA-epitope and nuclear localization signal (NLS) and zinc fmger domains (Fig. 1A); expression vectors containing zinc fmger domains and a KRAB transcriptional repressor domain (Fig. IB); luciferase reporter constructs containing TATA-minimal promoter, 5 copies of Gal4-VP16 binding sequences as enhancer sites, and a single copy of binding sequences for zinc fmger proteins(Fig. 1C); luciferase reporter constructs containing SV40 or CMV promoter and zinc finger binding sequence (Fig. ID); and luciferase reporter construct containing native human VEGF promoter(Fig. IE).

Fig 2: the repression of luciferase by zinc finger protein. Fig 3: the specific repression of luciferase by zinc fmger proteins. Fig 4: the structures of reporter constructs containing a wild type(SEQ ID NO: 20) or mutant luciferase gene(SEQ ID NO: 21).

Fig 5: the specific inhibition of luciferase by small interference RNA (siRNA).

Fig 6: the synergistic repression of luciferase induced by combining ZFP and siRNA.

Fig 7: the dose-dependent synergistic effects induced by combining ZFP and siRNA in the repression of luciferase.

Fig 8: the synergistic effect generated by combining ZFP and siRNA in the repression of luciferase by employing a luciferase reporter containing native human VEGF promoter.

Fig. 9: the structures of cloning site in an expression vector for shRNA recognizing VEGF-A mRNA(SEQ ID NOs: 24 and 25), and structures of shRNAs shVEGF+64(SEQ ID NO: 26) and shVEGF+338(SEQ ID NO: 27) having superior VEGF-A repression activities.

Fig. 10: the synergistic repression effect generated by combining ZFP and shRNA in the generation of endogenous VEGF-A mRNA. Fig. 11: the dose-dependent synergistic repression effect induced by combining ZFP and shRNA in the production of endogenous VEGF-A protein.

Fig. 12: silencing of endogenous VEGF-A gene observed when ZFP and shRNA were combined under a condition inducing VEGF-A expression.

DETAILED DESCRIPTION OF THE INVENTION

We have developed a method of modulating biological activity that uses two different modes of regulation. We have discovered, among other things, an unexpected synergistic effect from using both the zinc finger protein and siRNA technologies. This combination can be used for improved gene regulation, particularly repression or silencing.

Among several technologies, zinc fmger protein-mediated transcriptional regulation is important not only for its effectiveness in regulation per se, but also for its various applicability because zinc finger proteins can up- or down-regulate the expression of a target gene. Further, the expression of a target gene can be regulated at the post-transcriptional level by RNAi technology, which comprises introducing a heterogeneous RNA into a cell to degrade mRNA of the target gene. Blocking of transcription by zinc fmger protein may facilitate the siRNA attack at the post-transcriptional level by lowering the concentration of the target mRNA molecules. In many implementations, combining ZFP and RNAi technologies results in synergies that produce enhanced gene repression and/or silencing.

Any zinc finger protein can be used in the present invention insofar as it can specifically bind to a target DNA sequence. The zinc finger proteins may consist of 3 to 6 zinc finger domains and each zinc finger domain may be a wild type, a non-wild type or a combination thereof. Other exemplary zinc fmger proteins include more than six zinc finger domains or fewer than three zinc finger domains. In one example, a zinc finger protein includes a zinc finger domain and another DNA binding domain, e.g., a non-zinc fmger domain, such as a homeodomain.

Zinc fingers are small polypeptide domains of approximately 30 amino acid residues in which there are four residues, either cysteine or histidine, appropriately spaced such that they can coordinate a zinc ion (for reviews, see, e.g., Klug and Rhodes, (1987) Trends Biochem. Set.12:464-469(1987); Evans and Hollenberg, (1988) Cell 52:1-3; Payre and Vincent, (1988) FEBS Lett. 234:245- 250; Miller et al, (1985) EMBO J. 4: 1609-1614; Berg, (1988) Proc. Natl. Acad. Sci. U.S.A. 85:99-102; Rosenfeld and Margalit, (1993) J. Biomol. Struct. Dyn. 11:557-570). Hence, zinc finger domains can be categorized according to the identity of the residues that coordinate the zinc ion, e.g., as the Cys₂-His₂ class, the Cys₂-Cys₂ class, the Cys₂-CysHis class, and so forth. The zinc coordinating residues of Cys₂-His₂ zinc fingers are typically spaced as follows:

C-X₂.₅-C-X₃-X_a-X₅-Ψ-X₂-H-X₃.₅-H (SEQ ID NO: 33), where ψ (psi) is a hydrophobic residue (Wolfe et al, (1999) Annu. Rev.

Biophys. Biomol. Struct. 3:183-212), "X" represents any amino acid, X_a is phenylalanine or tyrosin, the subscript number indicates the number of amino acids, and a subscript with two hyphenated numbers indicates a typical range of intervening amino acids. In many zinc fmger domains, the initial cysteine is preceded by phenylalanine or tyrosine and then a non-cysteine amino acid. Typically, the intervening amino acids fold to form an anti-parallel β-sheet that packs against an α-helix, although the anti-parallel β-sheets can be short, non- ideal, or non-existent. The fold positions the zinc-coordinating side chains so they are in a tetrahedral conformation appropriate for coordinating the zinc ion. The base contacting residues are in the loop region between the pair of metal chelating residues.

Exemplary zinc fmger proteins and exemplary individual zinc fmger domains (which can be used in a zinc fmger protein) include those described in documents (US Published Applications 2002-0061512, 2003-165997 and 2003- 194727; U.S. Serial Nos. 10/669,861, 60/431,892 and 60/477,459;) Lei Zhang et al, J Biol. Chem., 2000, Vol. 275(43): 33850-33860; Delin Ren et al, Genes & Development 2002, 16:37-32; Liu PQ et al, J Biol. Chem. 2001, 376(14), 11323- 11334; and Bae KH et al, Nat. Biotechnol.2003; 21(3)275-280). Examples include zinc fmger protein ZFP-RDER(SEQ ID NO: 3) which is zinc fmger protein zif268(Kim and Pabo, J Biol Chem., 1997 Nov 21; 272(47):29795-800) substituted with human zinc finger domain RDER (Korean Patent Laid-open Publication No. 10-2001-0084880 (Toolgen, Inc.) and PCT Publication No. WO 01/60970) on its 3rd fmger position; zinc fmger protein ZFP-F121 (SEQ ID NO: 4), which is an assembled zinc finger protein consisting of 3 human zinc fmger domains designed to bind 9 bp sequences of human VEGF promoter at position +434 from the transcriptional initiation site; zinc fmger protein ZFP-F109 (SEQ ID NO: 5), which is an assembled zinc finger protein consisting of 4 human zinc fmger domains designed to bind 12 bp sequences of human VEGF promoter at position -536 from the transcriptional initiation site; and zinc finger protein ZFP- F435 (SEQ ID NO: 6), which is an assembled zinc fmger protein consisting of 3 human zinc finger domains designed to bind 9 bp sequences at -90R and - 391R(wherein, R means reverse strand) of human VEGF gene. A nucleic acid encoding the zinc finger protein can be introduced in a cell using any method. In one example, the nucleic acid is in the form of a plasmid and is introduced by a conventional transfection method. The nucleic acid can include a regulatable sequence (e.g., an inducible promoter) that can be used to selectively activate synthesis of the zinc finger protein. For example, the regulatable sequence can be operably linked to the sequence encoding the zinc fmger protein. In one embodiment, the promoter is inducible in response to a small molecule such as a steroid (e.g., doxycyclin) or antibiotic (e.g., tetracyclin), etc. In other embodiments, the method includes introducing an RNA encoding the zinc finger protein, or introducing the protein itself. For example, the protein can be physically associated with a protein transduction domain.

Further, the sequence encoding the zinc finger protein can further include a sequence encoding a transcriptional repressor domain, such that translation of the sequence produces a fusion protein that includes a zinc finger DNA binding domain and the repressor domain. The repressor domain can enhance the transcription inhibition effect of the zinc finger protein. Exemplary transcriptional repressor domains include KRAB domain having the amino acid sequence of SEQ ID NO: 9, which a minimal domain retaining the transcriptional repressor activity of KRAB-A domain of rat kidney transcription factor kid-1 (Witzgall R. et al, Proc. Natl. Acad. Sci. U.S.A., 1994 May 10;91(10): 4514-8), KRAB domain of human Koxl(NCBI protein database AAH24182; Gl: 18848329) having the amino acid sequence of SEQ ID NO: 32, and other transcriptional repressor domains known in the art. In other embodiments, the zinc fmger protein is fused to other functional domains, e.g., a histone acetylation or deacetylation domain, an activation domain, a methylase, and so forth.

The ability of zinc fmger proteins for transcriptional repression can be assayed by transfecting a suitable cell line, e.g., human embryonic kidney 293 cell (ATCC CRL 1573) with a vector for expressing a zinc fmger protein together with a luciferase reporter vector, and determining the quantity of expressed luciferase. The luciferase reporter vector contains target DNA binding sequences introduced at the proximal region (+18) from the transcriptional initiation site. The effectiveness of the binding site placement at the proximity of the transcriptional initiation site for zinc fmger protein binding assay was well studied by Kim and Pabo (supra). Further, the luciferase reporter vector may contain 5 copies of Gal4-VP16 binding sequences in the early promoter region in order to facilitate the observation of luciferase basal level expression. Examples of such luciferase reporter vector include plasmids pGL3-TATA-RDERBS, pGL3-TATA-F121BS and pGL3-TATA-F109BS, which are prepared in the preferred embodiment of the present invention based on plasmid pGL3- TATA/Inr+18(Kim and Pabo, supra). Exemplary reporter vectors also include plasmids pGL3-SV40-RDERBS and pGL3-CMV-RDERBS, which are prepared by replacing TATA promoter of plasmid pGL3-TATA-RDERBS with SV40 and CMV promoters, respectively, and plasmid pGL3-VEGFprom prepared by introducing human VEGF promoter (-950 to +450 nt from transcription initiation site) in plasmid pGL3-Basic (promega, E1751).

In one preferred embodiment of the present invention for assaying transcription inhibitory effect of zinc finger protein, 293 cells were co-transfected with plasmid pZFP-RDER, pZFP-F121 or pZFP-F109 expressing zinc finger protein RDER, F121 and F109, respectively, together with pGal4-VP16, a luciferase reporter plasmid and renila luciferase internal control reporter. The luciferase activity was measured 48 hours after the transfection and it was confirmed from the result that zinc fmger proteins specifically bound to target DNA sequences and inhibited transcription.

On the other hand, non-limiting examples of RNA molecules used in the present invention for RNAi include an anti-sense RNA, ribozyme or a double- stranded RNA mediating RNAi of the target gene. However, any RNA molecule can be employed insofar as it can modulate activity of an mRNA (e.g., stability, degradation, subcellular localization, splicing, or translation), e.g., thereby inhibiting expression. In one embodiment, the RNA includes a strand that can bind to mRNA of the target gene. In one embodiment, the RNA includes a strand that includes a sequence of at least 15, 19, 20, 21, 22, or 23 nucleotides that is at least 90, 95, 96, 97, 98, or 99% complementary, e.g., exactly complementary to an mRNA. The RNA molecule may be a chemically synthesized or naturally occurring derivative of RNA, double-stranded hairpin RNA, which can be chemically synthesized or produced from a vector (e.g., by transcription of one or more DNA vectors), or a ribozyme produced by similar methods.

The double-stranded RNA mediating RNAi preferably has the size of 21 to 23 nucleotides and may consist of a base-paired region of 19 nucleotides and 3 '-end single- stranded regions of 2 to 4 nucleotides. Such dsRNAs are referred to as "siRNAs" herein. In the preferred embodiments of the present invention, a pair of small interfering RNAs (siRNAs), which have the nucleotide sequences of SEQ ID NOs: 22 and 23 and are complementary to the coding region sequences at +153 to + 173 from the transcriptional initiation site of firefly luciferase mRNA, were chemically synthesized and used. The siRNA specifically inhibits the luciferase expression when transfected into 293 cells together with a luciferase reporter plasmid. dsRNA molecule can be produced in cells, e.g., from a nucleic acid vector that includes a promoter, e.g., two promoters that face each other. The promoters can be inducible, e.g., inducible in response to a small molecule such as a steroid (e.g., doxycyclin) or antibiotic (e.g., tetracyclin), etc.

In the other preferred embodiment of the present invention, a short hairpin RNA (shRNA) was used and it was found that, when a 293F cell(Gibco life technologies) was transfected with a plasmid containing the shRNA, the generation of endogenous VEGF mRNA was inhibited. Among the synthesized shRNAs, shVEGF+64 and shVEGF+338 having the nucleotide sequences of SEQ ID NO: 26 and 27, respectively, were most effective.

However, siRNA or shRNA useful in the present invention for RNAi technology is not limited to the specific examples as described above, and any RNA molecule capable of binding to a target mRNA and exhibiting RNAi effect can be used in the present invention.

In the inventive method using a combination of zinc finger protein and RNAi, the inhibition of gene expression was almost complete (average 60 fold repression for endogenous VEGF promoter-luciferase reporter in an optimal condition, i.e. 93.3 % inhibition), while the use of the methods separately exhibited a normal repression effect (average 5 to 10 fold repressions, i.e., 80 to 90 % inhibition, respectivelyXsee Fig. 8).

Further, the inhibitory effect of shRNA on endogenous VEGF-A mRNA generation in 293F cell was 3.5 fold for the shRNA-treated group as compared to non-treated control, but it increased dramatically to average 13.3 fold (ZFP 200 ng) and average 18.2 fold (ZFP 400 ng) when co-transfected with zinc fmger proteins (Fig. 10). The combined use of shRNA and zinc fmger protein synergistically inhibited the expression of endogenous VEGF-A at both mRNA and protein levels (Fig. 11).

Moreover, the effect of such combination was observed in in vivo systems using reporter plasmids having various promoter systems, suggesting that the present method may be effectively used in regulating any target genes.

The promoter of the target gene may be a native one or an artificially substituted exogenous one, and may be modified for containing a recognition site for a zinc finger protein. The promoter may be any one known in the art inclusive of a tissue-specific promoter, a differentiated state-dependent promoter, a developmental promoter, a cell cycle-specific promoter, VEGF promoter, TATA promoter, and a viral promoter (e.g,. SV40 promoter and CMV promoter).

The present method is useful for the regulation of gene expression in eukaryotic cells, especially in mammalian cells.

The target gene can be any gene, e.g., a gene whose activity might be regulated, e.g., increased or decreased. For example, a gene required by a pathogen can be repressed, a gene of a virus or other pathogen can be repressed, a gene required for cancerous growth can be repressed, a gene poorly expressed or encoding an unstable protein can be activated and overexpressed, and so forth. Examples of specific target genes include genes that encode: cell surface proteins (e.g., glycosylated surface proteins), cancer-associated proteins, cytokines, chemokines, peptide hormones, neurotransmitters, cell surface receptors (e.g., cell surface receptor kinases, seven transmembrane receptors, virus receptors and co-receptors, extracellular matrix binding proteins, cell-binding proteins, antigens of pathogens (e.g., bacterial antigens, malarial antigens, and so forth). Additional protein targets include enzymes such as enolases, cytochrome P450s, acyltransferases, methylases, TIM barrel enzymes, isomerases, acyl transferases, and so forth.

More specific examples include: integrins, cell attachment molecules or "CAMs" such as cadherins, selections, N-CAM, E-CAM, U-CAM, I-CAM and so forth); proteases (e.g., subtilisin, trypsin, chymotrypsin; a plasminogen activator, such as urokinase or human tissue-type plasminogen activator); bombesin; factor IX, thrombin; CD-4; platelet-derived growth factor; insulin-like growth factor-I and -II; nerve growth factor; fibroblast growth factor (e.g., aFGF and bFGF); epidermal growth factor (EGF); VEGFa; transforming growth factor (TGF, e.g., TGF-α and TGF-β ; insulin-like growth factor binding proteins; erythropoietin; thrombopoietin; mucins; human serum albumin; growth hormone (e.g., human growth hormone); proinsulin, insulin A-chain insulin B-chain; parathyroid hormone; thyroid stimulating hormone; thyroxine; follicle stimulating hormone; calcitonin; atrial natriuretic peptides A, B or C; leutinizing hormone; glucagon; factor VIII; hemopoietic growth factor; tumor necrosis factor (e.g., TNF-α and TNF-β); enkephalinase; Mullerian-inhibiting substance; gonadotropin-associated peptide; tissue factor protein; inhibin; activin; vascular endothelial growth factor; receptors for hormones or growth factors; rheumatoid factors; osteoinductive factors; an interferon, e.g., interferon-α,β,γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1, IL-2, IL-3, IL-4, etc.; decay accelerating factor; and immunoglobulins. In some embodiments, the target protein is associated with a disease, e.g., cancer, an infectious disease, inflammation, or a cardiovascular disease.

For instance, a synergistic inhibitory effect can be obtained from the combined use of zinc finger protein and RNAi technologies in an anti-caner treatment for inhibiting the expression of VEGF-A responsible for angiogenesis in tumor tissues. In this context, for instance, a zinc finger protein selected from the group consisting of the polypeptides having the amino acid sequences of SEQ ID NOs: 3 to 6, and a hairpin RNA having the nucleotide sequence of SEQ ID NO: 26 or 27 may be employed. Further, the combined use of zinc finger protein and RNAi strongly inhibited the expression of VEGF-A gene even at a hypoxia condition wherein VEGF-A production is strongly induced (Fig. 12).

The inventive method provides a new application of zinc fmger protein and RNAi technologies, and is particularly significant in that it provides an idea of improving gene regulation methods by combining differently established technologies concerning different levels of gene regulation, making it possible to reach a highly effective gene regulation. Further, the present method may be applied together with any gene regulation technologies such as anti-sense technology, Aptamer, antibody technology, small drug-mediated gene regulation, etc.

In another aspect, the present invention provides a composition for regulating a target gene comprising a zinc finger protein binding to the target gene or a DNA encoding said protein, and a RNA molecule that includes a strand that includes a sequence complementary to an mRNA transcribed from the target gene or a nucleic acid that can produce such a RNA molecule. The kinds and properties of zinc finger proteins, the DNA encoding the proteins and RNA molecules that may be contained in the inventive composition are as described in the above.

The above composition may be sterilized, and may further comprise preservatives, stabilizers, wetting agents, emulsifiers, lubricating agents, flavoring agents, additives such as salts and/or buffers for regulating osmotic pressure, and pharmaceutically acceptable carriers or excipients. The composition of the present invention may be formulated into various preparations by employing any of the procedures well known in the art. The resulting preparations may be administered through various oral or parenteral routes.

The amounts of a zinc finger protein or a DNA encoding same, and a RNA molecule may be determined within suitable ranges in light of various relevant factors including the kinds of target cells and genes, the desired extent of inhibitory effect, etc. For instance, in order to inhibit VEGF-A expression in human, 10 ng to 500 μg of DNA encoding zinc finger protein or RNA, and 10 ng to 500 mg of zinc finger protein, which is purified sufficiently to be delivered into a cell, can be administered daily in a single dose or in divided doses. The administration may be repeated for several times at one day- or one week-interval However, it should be understood that the above doses do not limit the scope of the invention in any way.

The DNA or RNA may be preferably administered by injection and can be administered in the form of naked DNA or RNA, or a vector mediating intracellular delivery such as retrovirus, adenovirus and adeno-associated virus.

For example, a nucleic acid that can be used to produce a zinc finger protein and/or RNA molecule in a cell can be prepared for delivery as naked nucleic acid, as a component of a virus, or of an inactivated virus, or as the contents of a liposome or other delivery vehicle. Alternatively, where the gene delivery agent, e.g., a viral vector, can be produced from recombinant cells which produce the gene delivery system. Appropriate viral vectors include retroviruses, e.g., Moloney retrovirus, adenoviruses, adeno-associated viruses, and lentiviruses, e.g., Herpes simplex viruses (HSV). HSV is potentially useful for infecting nervous system cells. A gene therapy vector (e.g., that can produce the zinc finger protein and/or RNA molecule) can be administered to a subject, for example, by intravenous injection, by local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The gene therapy agent can be further formulated, for example, to delay or prolong the release of the agent by means of a slow release matrix. One method of providing a recombinant therapeutic tri-domain polypeptide, is by inserting a gene therapy vector into bone marrow cells harvested from a subject. The cells are infected, for example, with a retroviral gene therapy vector, and grown in culture. Meanwhile, the subject is irradiated to deplete the subject of bone marrow cells. The bone marrow of the subject is then replenished with the infected culture cells. The subject is monitored for recovery and for production of the therapeutic polypeptide.

Purified zinc fmger proteins may be administered by injection into a desired site, intravenous injection, oral administration or topical application. Preparations of purified zinc finger proteins may include various forms designed to deliver a protein into cells, e.g., a fusion with a protein transduction domain (PTD), a mixture with a polymer facilitating the intracellular delivery, and a chemical conjugate with the PTD or polymer. Further, the purified proteins may be formulated to have a functional group for the site-specific delivery of proteins to a target tissue. A "protein transduction domain" or "PTD" is an amino acid sequence that can cross a biological membrane, particularly a cell membrane. When attached to a heterologous polypeptide, a PTD can enhance the translocation of the heterologous polypeptide across a biological membrane. The PTD is typically covalently attached (e.g., by a peptide bond) to the heterologous DNA binding domain. For example, the PTD and the heterologous DNA binding domain can be encoded by a single nucleic acid, e.g., in a common open reading frame or in one or more exons of a common gene. An exemplary PTD can include between 10-30 amino acids and may form an amphipathic helix. Many PTD's are basic in character, e.g., include at least 4, 5, 6 or 8 basic residues (e.g., arginine or lysine). A PTD may be able to enhance the translocation of a polypeptide into a cell that lacks a cell wall or a cell from a particular species, e.g., a eukaryotic cell, e.g., a vertebrate cell, e.g., a mammalian cell, such as a human, simian, murine, bovine, equine, feline, or ovine cell.

Typically a PTD is linked to a zinc fmger protein by producing the DNA binding domain of the zinc finger protein and the PTD as a single polypeptide chain, but other methods of for physically associating a PTD can be used. For example, the PTD can be associated by a non-covalent interaction (e.g., using biotin-avidin, coiled-coils, etc.). More typically, a PTD can be linked to a zinc finger protein, for example, using a flexible linker. Flexible linkers can include one or more glycine residues to allow for free rotation. For example, the PTD can be spaced from a DNA binding domain of the transcription factor by at least 10, 20, or 50 amino acids. A PTD can be located N- or C-terminal relative to a DNA binding domain.

A zinc fmger protein can also include a plurality of PTD's, e.g., a plurality of different PTD's or at least two copies of one PTD. Exemplary PTD's include the following segments from the antennapedia protein, the herpes simplex virus VP22 protein and HIV TAT protein.

Tat. The Tat protein from Human Immunodeficiency virus type I (HIV-1) has the remarkable capacity to enter cells when added exogenously (Frankel A.D. and Pabo CO. (1988) Cell 55:1189-1193, Mann D.A and Frankel A.D. (1991) EMBO J. 10:1733-1739, Fawell et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 664-668). The minimal Tat PTD includes residues 47-57 of the human immunodeficiency virus Tat protein. This peptide sequence is referred to as "TAT" herein.

Antennapedia. The antennapedia homeodomain also includes a peptide that is a PTD. Derossi et al (1994) J. Bio. Chem. 269: 10444-10450. This peptide, also referred to as "Penetratin".

VP22. The HSV VP22 protein also includes a PTD. This PTD is located at the VP22 C-terminal 34 amino acid residues. See, e.g., Elliott and OΗare (1997) Cell 88:223-234 and U.S. 6,184,038. Cell-specific PTD's. Some PTD's are specific for particular cell types or states. One exemplary cell-specific PTD is the Hnl synthetic peptide described in U.S. Published Application 2002-0102265. Hnl is internalized by human head and neck squamous carcinoma cells and can be used to target an artificial transcription factor to a carcinoma, e.g., a carcinoma of the head or neck. or closely related sequences. U.S. Published Application 2002-0102265 also describes a general method for using phage display to identify other peptides and proteins which can function as cell specific PTD's. For additional information about PTD's, see also U.S. 2003-0082561; U.S. 2002-0102265; U.S. 2003- 0040038; Schwarze et al. (1999) Science 285: 1569-1572; Derossi et al. (1996) J. Biol. Chem. 271 :18188; Hancock et al. (1991) EMBO J. 10:4033-4039; Buss et al. (1988) Mol. Cell. Biol. 8:3960-3963; Derossi et al. (1998) Trends in Cell Biology 8:84-87; Lindgren et al. (2000) Trends in Pharmacological Sciences 21 :99-103; Kilic et al. (2003) Stroke 34:1304-10; Asoh et al. (2002) Proc Natl Acad Sci USA 99(26): 17107-12; and Tanaka et al. (2003) J Immunol. 170(3): 1291-8.

However, the above exemplary forms of preparations do not limit the scope of the invention in any way. All patents, patent applications, and references cited herein are incorporated by reference in their entirety. The following patent applications: WO 01/60970 (Kim et al); US Published Applications 2002-0061512, 2003-165997, and 2003-194727, and U.S. Serial Nos. 10/669,861, 60/431,892 and 60/477,459, are expressly incorporated by reference in their entirety for all purposes.

The following Examples are intended to further illustrate the present invention without limiting its scope.

Further, percentages given below for solid in solid mixture, liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis, respectively, and all the reactions were carried out at room temperature, unless specifically indicated otherwise.

Reference Example 1 : Cell culture and transfection of cells

Human embryonic kidney 293 cells (ATCC CRL 1573) were maintained in Dulbecco's modified Eagle medium (DMEM, Life Technologies) supplemented with 100 unit/ml Penicillin, 100 ug/ml streptomycin (Life Technologies) and 10 % fetal bovine serum (FBS, Life Technologies). For the transient transfection, 10⁴ cells/well of 293 cells were pre-cultured in 100 ul of DMEM supplemented with 10 % FBS on a 96-well culture plate (Costar) for 24 hours. The 293 cells were transfected with a desired plasmid and/or RNA using

Lipofectamine plus transfection kit (Life Technologies) in accordance with the manufacturer's instructions. Cells were further incubated for 48 hours under a humid atmosphere containing 5 % C0₂ at 37°C .

Reference Example 2: Luciferase assay

48 hours after the transfection, cultured cells were washed twice with 100 ul of PBS, and 30 ul of lysis buffer (Promega, El 960) was added thereto. To 8 ul of the resulting cell lysate were added sequentially 40 ul of LAR II reagent (Promega, El 960) and 40 ul of stop and Glo reagent (Promega, El 960), and firefly- and Renila luciferase activities were measured with a TD-20/20 luminophotometer (Turner Designs, CA).

Example 1: Preparation of plasmid constructs comprising zinc finger domains and a reporter gene

(Step 1) Construction of plasmid containing zinc finger domains

Plasmid P3 prepared by modifying plasmid pcDNA3 (Invitrogen, Carlsbad, CA) containing CMV promoter was used as a parental vector for expressing ZFPs in mammalian cells. P3 was prepared by inserting an HA tag (YPYDVPDYA; SEQ ID NO: 1) and a SV40 nuclear localization signal (NLS)(PPKKKRKV; SEQ ID NO: 2) in the multi-cloning site of the plasmid pcDNA3 (Fig. 1A).

On the other hand, new zinc fmger proteins targeting a specific DNA sequence were designed by employing a simple computer algorithm ((ZFPsearch, Toolgen Inc.) that finds a match between the recognition sites of zinc fingers and the target DNA sequence. In accordance with this method using the promoter sequence of human VEGF-A gene as the target sequence, the zinc finger proteins shown in Table 1, i.e., ZFP-RDER(SEQ ID NO: 3), ZFP-F121(SEQ ID NO: 4) and ZFP-F435(SEQ ID NO: 6), which consist of three zinc fmger domains and would bind a stretch of 9 bases constituting a target site, and ZFP-F109(SEQ ID NO: 5), which consist of four zinc fmger domains and would bind a stretch of 12 bases constituting a target site, were found.

Table 1

ZFP-RDER is a derivative of well-defined mouse zinc fmger protein zif268 (Kim and Pabo, J Biol Chem., 1997 Nov 21; 272(47):29795-800) and its

3rd fmger is replaced with human zinc fmger domain designated RDER(Korean Patent Laid-open Publication No. 10-2001-0084880 (Toolgen, Inc.) and PCT

Publication No. WO 01/60970).

ZFP-F121 consisted of three human zinc fmger domains designed to bind

9 bp sequences of human VEGF promoter at position +434 from the transcription initiation site of human VEGF-A gene; ZFP-F109 consisted of four human zinc fmger domains designed to bind 12 bp sequence of human VEGF promoter at the -536 position from the transcription initiation site of human VEGF-A gene; and ZFP-F435 consisted of three human zinc finger domains designed to bind 9 bp sequences at the positions -90R and -391R(wherein R means reverse strand) of human VEGF-A gene. The zinc fmger proteins were assembled in accordance with the method described in PCT Publication No. WO 01/60970. Specifically, respective zinc fmger domains were amplified by PCR employing human chromosomal DNA (Promega, G3041) as a template, and optionally biotinylated 5'-universal primer (SEQ ID NO: 7, 5*-ATA TCG GGA TCC GAA TTC C-3*) and normal 3'- universal primer (SEQ ID NO: 8, 5*-TTT TGC GGC CGC TAT TTT TCA C-3'). The resulting DNA segments encoding individual zinc finger domains were subcloned into the P3 vector at the EcoRI/Notl site, and the resulting plasmids were used as starting materials for ZFP construction.

Two zinc fmger domains were assembled on streptavidin-coated magnetic beads (Dynal) by using Agel and Xmal restriction endonucleases capable of producing adhesive ends. The resulting plasmids containing assembled zinc fmger domains were cut with Xma I and Not I, and ligated with the Age I/Not I fragment of P3 vector containing other zinc fmger domain to obtain plasmids pZFP-RDER, pZFP-F121 and ρZFP-F435 containing three zinc fmger domains (Fig. 1A). Plasmid pZFP-F109 containing four zinc fmger domains was prepared by adding a further zinc fmger domain to a plasmid containing three zinc finger domains prepared as above.

Otherwise, the plasmids containing multiple zinc finger domains were prepared as follows. A zinc fmger domain insert was obtained by cutting P3 vector containing one zinc finger domain with Xmal and Notl, and the resulting insert was cloned into the linearized vector P3 having one zinc fmger domain, said vector being previously linearized by the treatment with Agel and Notl, to obtain a plasmid containing a zinc finger protein gene consisting of two zinc finger domains. A zinc finger domain insert consisting of two zinc fmger domains was prepared by the above method and cloned into Agel/Notl-linearized vector P3 having one or two zinc fmger domains to obtain a plasmid containing a zinc fmger protein gene consisting of three or four zinc finger domains.

All restriction endonucleases and ligases used above were obtained from New England Biolabs(Beverly, MA).

(Step 2) Construction of plasmids containing transcriptional repressor domain

In order to prepare a plasmid further containing a transcriptional repressor domain KRAB, the respective DNAs encoding the assembled zinc finger protein contained in the plasmids prepared in Step 1 was subcloned into the EcoRI/Notl restriction site of pLFD-KRAB (P3 vector containing KRAB domain (Toolgen, Inc.); Fig. IB) to obtain plasmids pLFD-RDER-KRAB, pLFD- F121-KRAB, pLFD-F109-KRAB and pLFD-F435-KRAB(Fig. IB). The repressor domain KRAB contains 63 amino acids

(VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLE NYSNLASMAGFLFTKPKVISLLQQGEDPW; SEQ ID NO: 9), which is a minimal domain retaining the transcriptional repressor activity of KRAB-A domain of rat kidney transcription factor kid-l(Witzgall R. et al, Proc. Natl. Acad. Sci. U.S.A., 1994 May 10;91(10): 4514-8), obtained from pTet-tTS vector (Clontech). On the other hand, several other plasmids were prepared by employing

KRAB domain (amino acid 2-97) of human Koxl protein (Zinc finger protein 10; NCBI protein database AAH24182; Gl: 18848329) as a transcription repressor domain instead of KRAB domain. KOX domain consisted of 96 amino acids(DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLEN YKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV) of SEQ ID NO: 32.

(Step 3) Construction of reporter plasmids containing luciferase gene

Plasmid ρGL3-TATA Inr+18 (Kim and Pabo, supra) was cut with Kpnl and Mlul to remove the sequence at position -124 to -110 from the transcriptional initiation site of luciferase gene, blunt-end ligated, and a Bglll site was introduced thereto by substituting A for G at position +12 from the transcription initiation site for the insertion of zinc fmger binding sequences (BS). Further, in order to facilitate the observation of luciferase basal level expression, five copies of Gal4-VP16 binding sequence were inserted at the early promoter region of TATA minimal promoter. The resulting plasmid was designated pGL3- TATA (Fig.lC).

In order to prepare the target DNA binding sequences shown in Table 2, respective oligonucleotides were chemically synthesized, and the resulting oligonucleotides were hybridized by dissolving them in an annealing buffer(100 mM potassium acetate, 30 mM HEPES KOH pH 7.4, 2 mM Magnesium acetate), heating at 75 °C and then gradually cooling to 25 °C .

Table 2

Each of the binding sequences thus prepared was inserted to the Bglll/Hindlll site of plasmid pGL3-TATA to obtain plasmids pGL3-TATA- RDERBS, pGL3-TATA-F121BS and pGL3-TATA-F109BS, respectively (Fig. 1C).

(Step 4) Construction of luciferase reporter plasmids containing various promoters

The CMV promoter sequence obtained from plasmid pCDNA3(Invitrogen, V790-20) was cloned into the Mlul/Hindlll site of plasmid ρGL3-Basic(Promega, E1751), and the ZFP-RDER binding sequence(ZFP- RDERBS) prepared in Step 3 was inserted into the Kpnl/Mlul site of the resulting plasmid to obtain reporter plasmid pGL3-CMV-RDERBS. Further, reporter plasmid pGL3-SV40-RDERBS was prepared by employing the SV40 promoter sequence from plasmid pRL-SV40 (Promega, E6261), instead of the CMV promoter(Fig. ID).

(Step 5) Construction of luciferase reporter plasmids containing human VEGF promoter

The native human VEGF promoter DNA (at position -950 to +450 from transcription initiation sequence) was PCR-amplified from human genomic DNA using the forward primer of SEQ ID NO: 18 (5'-CGG GGT ACC CCC TCC CAG TCA CTG ACT AAC-3') and the reverse primer of SEQ ID NO: 19 (5'- CCG CTC GAG TCC GGC GGT CAC CCC CAA AAG-3'). The DNA fragment thus prepared was cloned into the Kpnl/Xhol restriction site of plasmid pGL3(Promega, E1751), and the resulting plasmid was designated pGL3- VEGFprom (Fig. IE).

Example 2: Repression of luciferase by zinc fmger protein

293 cells were co-transfected with 15 ng of effector plasmid pZFP- RDER together with (A) 68.5 ng enhancer pGal4-VP16 + 15 ng plasmid pGL3- TATA-RDERBS + 1.5 ng pRLSV40 renila luciferase internal control reporter(Promega, E6261), (B) 15 ng plasmid pGL3-SV40-RDERBS + 1.5 ng pRLSV40, or (C) 15 ng plasmid pGL3-CMV-RDERBS + 1.5 ng pRLSV40, respectively, in accordance with the method of Reference Example 1. Total amount of DNA was adjusted to 100 ng by employing plasmid P3. As a control, 293 cells were transfected with the same amount of plasmid P3 without using the effector. The luciferase activity was measured by the method of Reference Example 2. Fold repression values were calculated by normalizing the firefly luciferase activity against the renila luciferase activity and compared the result with that of the control.

The results exhibited that zinc finger proteins specifically bind to the promoter sequence and inhibit the transcription. Specifically, when the cells were transfected with pZFP-RDER together with a reporter plasmid having a TATA promoter, the luciferase activity was inhibited by about 10 fold (i.e., 90 % inhibition) as compared to the control (Fig. 2A). When a reporter plasmid having the SV40 or CMV promoter was employed, the luciferase activity was inhibited by about 4 fold (i.e., 75 % inhibition)(Figs. 2B and 2C).

The differences in fold repression obtained using the same ZFP were considered mainly due to the differences in the strength of the promoters used, which is judged based on the observation that when the cells were transfected with a ZFP expression vector together with plasmid pGL3-TATA-ZFPBS and a various amount of enhancer Gal4-VP16, the fold repression values were slightly different according to the basal expression level of the reporter, despite the effectiveness of ZFP to regulate the target reporter gene was constant.

Example 3: Specific repression of luciferase by zinc finger protein

The specificities of zinc fmger proteins ZFP-RDER and ZFP-F121 were examined using pZFP-RDER and pZFP-F121 vis-a-vis reporter constructs pGL3- TATA-RDERBS and pGL3-TATA-F121BS in combination to see whether they match or not, as follows. (A) 15 ng of pGL3 -TATA-RDERBS was mixed with 1.5 ng of pRLSV40 renila luciferase reporter and 68.5 ng of enhancer pGal4-VP16, and 15 ng of pZFP-RDER or pZFP-F121 was added thereto. The total amount of DNA was adjusted to 100 ng by employing plasmid P3. (B) 15 ng of pGL3-TATA-F121BS was mixed with 1.5 ng of pRLSV40 renila luciferase reporter, and 15 ng of pZFP-RDER or pZFP-F121 was added thereto. The total amount of DNA was adjusted to 100 ng by employing plasmid P3. 293 cells were co-transfected with one of the above DNA mixtures in accordance with the method of Reference Example 1, and the luciferase activity was measured by the method of Reference Example 2. The fold repression values were calculated by normalizing the firefly luciferase activity against the renila luciferase activity and the result was compared with that of the control.

Consequently, the ZFP-RDER specifically inhibited the luciferase activity when co-transfected with the reporter pGL3 -TATA-RDERBS containing ZFP-RDER binding sequences, while ZFP-F121 did not show a significant difference (Fig. 3 A). Likewise, only ZFP-F121 showed significant inhibition when co-transfected with the reporter pGL3-TATA-F121BS containing ZFP- F121 binding sequences (Fig. 3B). These results clearly demonstrate the specificity of ZFPs in the recognition of target DNA sequences.

Example 4: Construction of reporter plasmid for RNAi target and preparation of effector antisense RNA

(1) Reporter construct containing wild-type or mutant luciferase gene

Reporter constructs pGL3-TATA-RDERBS and pGL3-TATA-F121BS containing wild-type luciferase gene were modified by substituting three nucleotides in the target sequence(+153 to + 173 sequence from the transcription initiation site of luciferase gene) for RNAi by site-directed mutagenesis to obtain Reporter constructs pGL3mut-TATA-RDERBS and pGL3mut-TATA-F121BS containing the mutant luciferase gene. The wild-type(SEQ ID NO: 20) and mutant target sequences(gray box, SEQ ID NO: 21) for siRNA are shown in Fig. 4.

(2) Synthesis of effector antisense RNA and hybridization

Antisense oligo RNAs complementary to the +153 to + 173 coding region sequence(target sequence) from the transcription initiation site of firefly luciferase mRNA were chemically synthesized by Synthetic Genetics(San Diego, CA). Antisense RNA contains thymines replacing two uracils at the 3 '-end of the oligoribonucleotides. The sequences of oligo RNAs GL3-F and GL3-R were 5'- CUUACGCUGAGUACUUCGATT-3'(SEQ ID NO: 22) and 5'- UCGAAGUACUCAGCGUAAGTT-3'(SEQ ID NO: 23), respectively, as was published by Elbashir S. M. et al(Narure, 2001 May 24; 411(6836):494-8)).

1 ug each of antisense oligo RNAs GL3-F and GL3-R were dissolved in an annealing buffer(100 mM potassium acetate, 30 mM HEPES KOH pH 7.4, 2 mM Magnesium acetate), heated at 95 °C for 10 minutes and then gradually cooling to room temperature to allow them to hybridize with each other. The resulting double- stranded RNA(siRNA) was stored at -80 ^°C .

Example 5: Inhibition of luciferase activity by RNA interference

The effect of RNAi on the degradation of luciferase transcripts was examined by the method of Elbashir S. M. et ah(supra).

293 cells were transfected with 50 ng of siRNA prepared in Example 4 together with reporter plasmid ρGL3-TATA-RDER, pGL3-SV40-RDER or pGL3-CMV-RDER, and the luciferase activity was measured by the method of Reference Example 2. The fold repression values were calculated by normalizing the firefly luciferase activity against the renila luciferase activity and the result was compared with that of the control, wherein 293 cells were transfected with plasmid P3 and reporter plasmid.

The results employing reporter plasmids pGL3-TATA-RDER, pGL3- SV40-RDER and pGL3-CMV-RDER showed 20 fold, 4.8 fold and 4.2 fold repression, respectively, of luciferase activities (Fig. 6; A, B and C; see the groups of RNA 50 ng and pZFP-RDER 0 ng).

Further, the following experiments were carried out in order to examine whether the supressive effect of siRNA was specific. 293 cells were transfected with reporter plasmid ρGL3 -TATA-RDERBS alone or together with 50 ng siRNA(Fig. 5, A). On the other hand, 293 cells were transfected with reporter plasmid pGL3mut-TATA-RDERBS, which contains a mutated promoter, alone or together with 50 ng siRNA(Fig. 5, B).

The luciferase activity was measured by the method of Reference Example 2. Fold repression values were calculated by normalizing the firefly luciferase activity against the renila luciferase activity and the result was compared with that of the control(non-transfected 293 cells).

As can be seen from the result in Fig. 5, the specific inhibition of luciferase activity by siRNA was observed only for the wild type promoter group(A), but not for the mutated promoter group(B). The siRNA specifically recognized the 21-bp target sequence, but hardly recognized the mutated target sequence having substitutions of three nucleotides, as reported by Elbashir et al. These results confirm that siRNA mediated gene suppression is effective and highly sequence-specific.

Example 6: Gene silencing by combination of siRNA and ZFP-RDER

293 cells were transfected with 0 or 50 ng of siRNA prepared in Example 4, 0 or 15 ng of pZFP-RDER and 15 ng of (A) reporter plasmid pGL3- TATA-RDER, (B) reporter plasmid ρGL3-SV40-RDER, or (C) reporter plasmid pGL3-CMV-RDER. The total amount of DNA was adjusted to 100 ng by employing plasmid P3.

The luciferase activity was measured by the method of Reference Example 2. Fold repression values were calculated by normalizing the firefly luciferase activity against the renila luciferase activity and the result was compared with that of the control, wherein 293 cells were transfected with plasmid P3 and reporter plasmid.

Consequently, co-transfection of pZFP-RDER with siRNA resulted in a dramatic increase in the fold repression values from 10 to 142 with pGL3 -TATA- RDERBS reporter (Fig. 6, A), from 4.3 to 20.8 with pGL3-SV40-RDERBS reporter (Fig. 6, B), and from 4.4 to 14.7 with pGL3-CMV-RDERBS reporter (Fig. 6, C), as compared with the group transfected with pZFP-RDER only. These results demonstrated the significant synergistic effect of the zinc finger protein and siRNA in gene silencing.

Example 7: Dose-dependent synergistic gene silencing effect by combination of ZFP and siRNA

In order to examine dose-dependencies of ZFP and siRNA in the repression of luciferase activity, 293 cells were transfected with 0 to 15 ng of pZFP-RDER and 0 to 50 ng of siRNA prepared in Example 4, and the luciferase activity was measured by the method of Reference Example 2. Fold repression values were calculated by normalizing the firefly luciferase activity against the renila luciferase activity and the result was compared with that of the control(non-transfected 293 cells).

Consequently, the dose-dependent synergistic effect of pZFP-RDER and RNAi on pGL3-CMV-RDERBS was observed, as shown in Fig. 7.

Example 8: Synergistic gene silencing effect of combination of ZFP and siRNA using the luciferase reporter containing native human VEGF promoter

293 cells were transfected with luciferase reporter plasmid pGL3-

VEGFprom containing native human VEGF promoter(-1500 to +500 from the transcription initiation site) and (A) 0 or 30 ng of pLFD-F121-KRAB alone or together with 0 or 50 ng of siRNA, or (B) 0 or 30 ng of pLFD-F109-KRAB alone or together with 0 or 50 ng of siRNA.

Then, the luciferase activity was measured by the method of Reference

Example 2. Fold repression values were calculated by normalizing the firefly luciferase activity against the renila luciferase activity and the result was compared with that of the control wherein 293 cells were transfected with vector pLFD and the reporter plasmid.

As can be seen from the results in Fig. 8, A, when pLFD-F 121 -KRAB (30 ng) was employed in combination with siRNA (50 ng), a dramatic increase in the fold repression values in the order of up to 60 fold was observed in a siRNA- dose dependent manner as compared to the control employing pLFD only without using siRNA, while pLFD-F121-KRAB and siRNA alone resulted in an increase in the fold repression value by 8.7 fold and 5 fold, respectively. Similar synergistic effect was observed for pLFD-F109-KRAB when combined with siRNA, as shown in Fig. 8, B.

The blocking of transcription by zinc fmger protein may facilitate the siRNA attack at the post-transcriptional level by lowering the concentration of target mRNA molecules. Accordingly, the combination of ZFP and siRNA induces a superior gene silencing effect over the additive effect of both methods.

Example 9: Repression of endogenous VEGF-A gene expression by combination of shRNA and ZFP

(1) Preparation of shRNA for RNAi in a mammalian cell

A shRNA, which is capable of recognizing and degrading endogenous VEGF-A mRNA in a 293 cell, thereby repressing the expression of VEGF-A protein, was prepared as follows. A plurality of short DNA sequences(Fig. 9, SEQ ID NOs: 24 and 25) encoding various shRNA recognizing VEGF-A mRNA were designed based on the information on the nucleotide sequence of VEGF-A mRNA(NCBI Nucleotide database: AF486837; Gl 19909064) and chemically synthesized. The DNA sequences were cloned into the BamHI/Hindlll restriction site of Generepressor vector (IMGENEX), and the resulting shRNA expression plasmids were then transfected into human embryonic kidney 293F cells (Gibco Life Technologies) that permit very high efficiency of transfection.

Specifically, 293F cells placed in the wells of a 24-well culture plate in an amount of 10⁵ cells/well were pre-cultured in 1 ml of DMEM supplemented with 10 % FBS for 24 h in a humid atmosphere containing 5 % C0₂ at 37°C. Then the cells were transfected with 0 or 100 ng of the shRNA expression plasmids using a Lipofectamine plus transfection kit (Life Technologies) according to the manufacturer's instructions. The cells were further incubated for 48 hours. The cells and culture supematants were harvested separately. The total RNAs were extracted form the cells by employing Trizol reagent (Gibco Life Technologies).

Quantification of VEGF mRNA was carried out by the following real time RT-PCR.

The reverse transcription reactions were performed with 4 μg of the total RNA using oligo-dT as the first-strand synthesis primer for mRNA, dNTP and MMLV reverse transcriptase provided in the Superscript first-strand synthesis system (Gibco Life Technologies) to obtain a first-strand cDNA. To analyze mRNA quantities, 1 μl of the first-strand cDNA thus obtained was amplified by real time PCR using VEGF-A cDNA specific primers of SEQ ID NOs: 28 and 29. Since this method is susceptible to the initial amount of RNA, the initial RNA amounts were normalized to the GAPDH mRNA quantities calculated by specific amplification using GAPDH-specific primers of SEQ ID NOs: 30 and 31. The amplifications of VEGF- and GAPDH-specific cDNA were monitored and analyzed in real-time with a Quantitect SYBR kit (Qiagen, Valencia, CA) and Rotorgene 2000 real-time cycler (Corbett, Sydney, Australia), and the cDNAs were quantified by serial dilution of the standards included in the reactions.

By the above method, two shRNAs having the nucleotide sequences of SEQ ID NOs: 26 and 27 were selected based on their highest VEGF-A repression activities and designated shVEGF+64 and shVEGF+338, respectively, since they were hybridized at positions +64 and +338 bp, respectively, from the translation initiation codon of VEGF-A mRNA(Fig. 9).

(2) Repression of VEGF-A mRNA synthesis by combination of shRNA and ZFP

293F cells were transfected with 0 or 100 ng of shVEGF+64 in combination with 0, 200 or 400 ng of pLFD-F435-KRAB, the total RNA was extracted, and VEGF-A mRNA was quantified by Real time RT-PCR, in accordance with the method of (1).

As can. be seen from the result in Fig. 10, the quantity of endogenous VEGF-A mRNA was reduced up to 3.5 fold by the treatment with shVEGF+64 shRNA alone, as compared with the non-treated control. The suppressive effect dramatically increased when co-transfected with zinc fmger proteins by, on the average, 13.3 fold(pLFD-F435-KRAB 200 ng) and 18.2 fold (pLFD-F435- KRAB 400 ng). On the other hand, since the quantity of endogenous VEGF-A mRNA was reduced by, on the average, 2 fold(pLFD-F435-KRAB 200 ng) and 4 fold (pLFD-F435-KRAB 400 ng), respectively, by the treatment with zinc fmger protein F435-KRAB alone, as compared to the non-treated control, it can be concluded that the combination of shRNA and zinc finger protein induces a significant synergistic effect.

(3) Repression of VEGF-A protein production by combination of shRNA and ZFP

In order to examine whether the repression of VEGF-A mRNA expression resulted in the reduction of VEGF-A protein secretion, 293F cells were transfected with 0 to 100 ng of shRNA expression plasmids(shVEGF+64 or shVEGF+338) in combination with 0 to 200 ng of pLFD-F435-KRAB, and cultured for 72 hours. The VEGF protein accumulated in the culture medium was quantified by enzyme linked immunosorbent assay (ELISA), wherein the supernatant of culture was reacted with anti-human VEGF antibody (R&D systems; AF-293-NA) and biotinylated anti-human VEGF antibody (R&D systems; BAF293) conjugated with streptavidin alkaline phosphatase (Chemicon;SA110) and the antigen-antibody complex was reacted with pNPP(p- Nitrophenyl phosphate) (Sigma ; N-9389) dissolved in pNPP buffer(Chemicon; ES011). The OD (optical density) at 405 nm was determined with PowerWave X340(Bio TEK Instrument). Fold repression values were calculated based on the amount of VEGF-A expression by non-transfected 293F cells(control).

The result is shown in Fig. 11, which demonstrates dose-dependent repressions of VEGF production as function of the amounts of shRNA and zinc finger protein.

In case when shVEGF+64 was used as the shRNA, the fold repression value increased when combined with zinc fmger protein(pLFD-F435-KRAB) from a maximum of 2.3 fold obtained by 100 ng shRNA alone up to an average 6 fold obtained when shVEGF+64 100 ng plus ZFP 200 ng were used. In case of shVEGF+338, the fold repression value increased when combined with zinc fmger protein(pLFD-F435-KRAB) from a maximum of 2.4 fold obtained by 100 ng shRNA alone up to an average 10 fold obtained when shVEGF+64 100 ng plus ZFP 200 ng were used.

These results shows that shRNA and zinc fmger protein effectively inhibit the expression of endogenous VEGF-A at both mRNA and protein levels, and that the combined use of sliRNA and zinc fmger can inhibit the expression of an endogenous gene very efficiently.

(4) Repression of VEGF-A gene expression by combination of ZFP and shRNA in hypoxia condition

VEGF-A gene is known as a crucial factor for inducing angiogenesis essential for the development and growth of many tumors, and a high level of VEGF-A expression is observed in tumor cells, which is known to be stimulated by hypoxia condition in cancer tissues. When the medium for culturing 293 cells is treated with 100 to 800 μM of CoCl₂ for about 7 hours, a hypoxia condition is induced and VEGF production by cells is rapidly escalated. The following experiment was carried out in order to examine whether shRNA and zinc fmger protein, alone or in combination, can inhibit the VEFG expression in the hypoxia condition.

293F cells(10⁴ cells/well, 96-well plate) were transfected with one or both of pLFD-F435-KRAB 50 ng and shVEGF+64 100 ng, and incubated for 48 hours. In order to induce the hypoxic condition, 800 μM of CoCl₂ was added to the medium at the last 7 hours stage of the culture. The amount of VEGF-A secreted in the culture medium was determined by ELISA.

As can be seen from Fig. 12, the VEGF production from the hypoxic culture with mock-transfection(Hypoxia+Mock) increased to about 1,039 pg/ml, in contrast to about 273 pg/ml of the CoC12-untreated control(Non- hypoxia+Mock), confirming strong induction of VEGF-A production in hypoxia condition. The induction of VEGF production via hypoxia was inhibited in the cells transfected with pLFD-F435-KRAB(Hypoxia+F435-KRAB), the cells transfected with shVEGF+64 shRNA(Hypoxia+shRNA) being inhibited by 3.5 fold (average 297.6 pg/ml) and 3.8 fold (average 272.4 pg/ml), respectively. On the other hand, the inhibition of VEGF-A hypoxic induction in the cells co- transfected with pLFD-F435-KRAB and shVEGF+64 increased 5.4 fold (average 192.2 pg/ml).

Zinc fmger protein and shRNA lowered the VEGF expression to a level similar to that obtained at normoxia, under the hypoxia condition wherein VEGF-A production was strongly induced. Moreover, the combined use of zinc fmger protein and shRNA proved to be very effective since it not only causes the substantially complete inhibition of VEGF-A induction at hypoxia condition but also inhibits the VEGF expression by about 30 %> as compared to that at normoxic condition, i.e., 273 pg/ml Considering the transfection rate (about 85- 90 %) in the present experiment. These results suggest that VEGF-A was probably completely knocked out in the transfected cells. Namely, it seems that the transfected cells did not produce VEGF-A at all and only the non-transfected cells (10-15 % of total cells) produced a basal level of VEGF-A in response to the hypoxia condition. The above results demonstrate that zinc finger proteins can be used in anti-cancer treatment for inhibiting the growth of a cancer owing to their ability to inhibit the expression of VEGF-A responsible for angiogenesis in tumor tissues, and can be used in combination with any technology for post- transcriptional gene regulation, e.g., shRNA technology degrading VEGF-A mRNA, for synergistic inhibitory effect. While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for regulating a target gene, which comprises introducing into a cell a zinc fmger protein that can bind to a target gene or a nucleic acid encoding said protein, and a RNA molecule comprising a strand that includes a sequence complementary to an mRNA transcribed from the target gene to regulate the expression of said target gene.

2. The method of claim 1, wherein the zinc fmger protein contains 3 to 6 zinc finger domains.

3. The method of claim 2, wherein the zinc finger domains are a wild- type, non-wild-type or a combination thereof.

4. The method of claim 2, wherein the zinc finger protein comprises a transcriptional repressor domain.

5. The method of claim 1, wherein the zinc finger protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6.

6. The method of claim 1, wherein the nucleic acid encoding the zinc fmger protein is a DNA in the form of a plasmid.

7. The method of claim 1, wherein the zinc fmger protein can bind to a regulatory sequence of the target gene.

8. The method of claim 1, wherein the zinc finger protein can bind to a promoter sequence, an enhancer sequence, a coding sequence, or an intronic sequence of the target gene.

9. The method of claim 8, wherein the promoter sequence of the target gene is a native one or an artificially substituted exogenous one.

10. The method of claim 8, wherein the promoter sequence of the target gene is modified to contain a recognition site for a zinc finger protein.

11. The method of claim 10, wherein the promoter sequence of the target gene is selected from the group consisting of VEGF promoter, TATA promoter, SV40 promoter and CMV promoter.

12. The method of claim 1, wherein the zinc finger protein binds to the sequence of the target gene with a K_D of less than 10^"7 M.

13. The method of claim 1, wherein the zinc fmger protein alters expression of fewer than 5%> of genes in the genome.

14. The method of claim 1, wherein the RNA molecule is selected from the group consisting of anti-sense RNA, ribozyme and double-stranded RNA.

15. The method of claim 14, wherein the RNA molecule is a double- stranded RNA, and each strand of the molecule has a size of 21 to 23 nucleotides.

16. The method of claim 15, wherein the double-stranded RNA has a double-stranded region of 19 nucleotides and 3 '-end single-stranded regions of 2 to 4 nucleotides.

17. The method of claim 14, wherein the double-stranded RNA comprises two complementary RNA strands.

18. The method of claim 14, wherein the double-stranded RNA is a hairpin RNA.

19. The method of claim 1, wherein the RNA molecule is a chemically synthesized or naturally occurring derivative of RNA.

20. The method of claim 1, wherein the cell is a eukaryotic cell.

21. The method of claim 20, wherein the eukaryotic cell is a mammalian cell.

22. The method of claim 1, wherein the cell is in an organism.

23. The method of claim 1, wherein the nucleic acid encoding the zinc fmger protein is introduced using a liposome, a virus, or a viral particle.

24. The method of claim 1, wherein the RNA molecule is introduced into the cell by inserting a nucleic acid that includes a sequence of a strand of the RNA molecule, or a complement thereof into the cell, and transcribing or amplifying the nucleic acid in the cell, thereby producing the RNA molecule in the cell.

25. A composition for regulating a target gene, the composition comprising

(i) a zinc finger protein that can bind to the target gene or a nucleic acid encoding said protein, and (ii) a RNA molecule that includes a strand including a sequence complementary to an mRNA transcribed from the target gene.

26. The composition of claim 25, wherein the zinc finger protein contains 3 to 6 zinc finger domains.

27. The composition of claim 26, wherein the zinc finger domains are wild-type, non-wild-type or a combination thereof.

28. The composition of claim 26, wherein zinc finger protein comprises a transcriptional repressor domain.

29. The composition of claim 25, wherein the zinc fmger protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6.

30. The composition of claim 25, wherein the nucleic acid encoding the zinc finger protein is provided in the form of a plasmid.

31. The composition of claim 25, wherein the zinc finger protein can bind to a regulatory sequence of the target gene.

32. The composition of claim 25, wherein the zinc finger protein can bind to a promoter sequence, an enhancer sequence, a coding sequence, or an intronic sequence of the target gene.

33. The composition of claim 32, wherein the promoter of the target gene is a native one or an artificially substituted exogenous one.

34. The composition of claim 32, wherein the promoter of the target gene is modified to contain a recognition site for a zinc finger protein.

35. The composition of claim 34, wherein the promoter of the target gene is selected from the group consisting of VEGF promoter, TATA promoter, SV40 promoter and CMV promoter.

36. The composition of claim 25, wherein the zinc finger protein binds to the sequence of the target gene with a K_D of less than 10^"7 M.

37. The composition of claim 25, wherein the zinc finger protein alters expression of fewer than 5% of genes in the genome.

38. The composition of claim 25, wherein the RNA molecule is selected from the group consisting of anti-sense RNA, ribozyme and double- stranded RNA.

39. The composition of claim 38, wherein the RNA molecule is a double-stranded RNA, and each strand of the molecule has a size of 21 to 23 nucleotides.

40. The composition of claim 39, wherem the double-stranded RNA has a double-stranded region of 19 nucleotides and 3 '-end single-stranded regions of 2 to 4 nucleotides.

41. The composition of claim 38, wherein the double-stranded RNA comprises two complementary RNA strands.

42. The composition of claim 38, wherein the double-stranded RNA is a hairpin RNA.

43. The composition of claim 25, wherein the RNA molecule is a chemically synthesized or a naturally occurring derivative of RNA.

44. The composition of claim 25, wherein the composition includes a zinc finger protein that comprises a protein transduction domain.

45. The composition of claim 25, wherein the target gene is VEGF-A gene and the composition is used for modulating VEGF-A gene expression.

46. The composition of claim 45, which comprises a zinc fmger protein including an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5 and 6, and a hairpin RNA having the nucleotide sequence of SEQ ID NO: 26 or 27.

47. A method of treating a neoplastic disorder in a subject, the method comprising; providing the composition of claim 45 , and administering the composition to the subject in an amount effective to modulate VEGF-A gene expression in a cell of the subject.

48. A kit that comprises : (i) a zinc finger protein that can bind to a target gene or a nucleic acid encoding said protein, and

(ii) a RNA molecule that includes a strand having a sequence complementary to an mRNA transcribed from the target gene.

49. The kit of claim 48 further comprising instructions for administering the kit components to a subject.

50. The kit of claim 48, wherein component (i) and (ii) are in separate containers.

51. A modified eukaryotic cell that comprises:

(i) a zinc fmger protein that can bind to a target gene or a nucleic acid encoding said protein, wherein the zinc fmger protein or the nucleic acid are heterologous to the cell, and (ii) a RNA molecule that includes a strand having a sequence complementary to an mRNA transcribed from the target gene.

52. The cell of claim 51 , wherein the target gene is an endogenous gene of the cell.

53. The cell of claim 51, wherein the RNA molecule is a double- stranded RNA molecule that comprises a duplex of less than 24 basepairs.

54. The cell of claim 51, wherein the cell comprises a heterologous DNA molecule that produces the RNA molecule.

55. A method of modulating expression of a target gene in a cell of a subject, the method comprising; administering (i) a first component that comprises a zinc finger protein or a nucleic acid encoding the zinc finger protein, and (ii) a second component that comprises a RNA molecule or a nucleic acid that can produce the RNA molecule, to the subject, wherein the zinc finger protein can bind to the target gene and modulate transcription of the target gene, the RNA molecule can reduce translatability of a transcript of the target gene, and the first and second components are administered in amounts effective to modulate expression of the target gene in a cell of the subject.

56. The method of claim 55, wherein the first and second components are administered separately.

57. The method of claim 55, wherein the first and second components are administered together.

58. The method of claim 55, wherein the RNA molecule is a double- stranded RNA molecule that comprises a duplex of less than 24 basepairs.

59. The method of claim 57, wherein the first and second components are formulated for delivery by a cell penetrating vehicle.

60. The method of claim 59, wherein the cell penetrating vehicle is a liposome, virus, or viral-like particle.

61. The method of claim 55, wherein the target gene promotes cell growth or proliferation.

62. The method of claim 55, wherein the target gene promotes inflammation.

63. A method of modulating expression of a target gene, the method comprising; identifying a zinc finger protein that can bind to the target gene and modulate transcription of the target gene, identifying a RNA molecule can reduce translatability of a transcript of the target gene, the molecule comprising a strand that includes a sequence complementary to the transcript, and providing, in a form suitable for introduction into a cell or administration to a subject, (i) a first component that comprises the identified zinc finger protein or a nucleic acid encoding the zinc finger protein, and (ii) a second component that comprises the identified RNA molecule or a nucleic acid that can produce the RNA molecule into a cell.

64. The method of claim 63, wherein the first and second components are combined and formulated as a pharmaceutical composition.

65. The method of claim 64, wherein the first and second components are combined by preparing a nucleic acid vector that comprises one sequence corresponding to the first component and another sequence corresponding to the second component.

66. The method of claim 63, wherein identifying the zinc finger protein comprises mixing and matching characterized zinc fmger domains.

67. The method of claim 63, wherein identifying the zinc finger protein comprises screening a library of zinc finger protein for a protein that alters the phenotype of a cell.