WO2005068496A1 - Modulating viral transcription - Google Patents

Abstract

A protein comprising a DNA binding domain including a plurality of zinc finger domains, binds to a target sequence in a viral nucleic acid and can modulate transcription of a viral gene. Also disclosed is a method of treating or preventing a retroviral disorder, comprising providing an agent that can regulate viral gene transcription to cells of a subject, wherein the agent includes (1) a protein having a DNA binding domain and a protein transduction domain or (2) a nucleic acid that encodes a protein comprising a DNA binding domain that contains a plurality of zinc finger domains.

Description

MODULATING VIRAL TRANSCRIPTION

BACKGROUND

AIDS is a disease of global importance. In 2002, 60 million individuals were reportedly infected with the HIV (human immunodeficiency virus. One third of this population has succumbed to the disease (Anthony S. Fauci, Nature Med. 2003, 9:839-843). New therapeutic methods would greatly benefit AIDS patients.

SUMMARY

In one aspect, the present invention features a protein (e.g., an isolated or recombinant protein) that has a DNA binding domain that includes a plurality of zinc finger domains. The protein can bind to a target sequence in a viral nucleic acid, e.g., in a viral promoter, e.g., .the LTR of a retrovirus, e.g., a human immunodeficiency virus, e.g., HIV-l , e.g., HIV-1 subtype B, A, and/or E. The protein can reduce transcription (e.g., the activity of LTR promoter), e.g., in the absence of a transcriptional repression domain such as KRAB. For example, the protein reduces transcription (e.g., activity of LTR promoter, particularly the transcription activated by TAT) in a reporter assay at least 4, 5, 7, 8, 10, 12, 15, or 16-fold in the absence of any transcriptional repression domain. The protein may also reduce transcription (e.g., activity of LTR promoter, particularly transcription activated by TAT), by at least 10, 15, 20, 25, 30, 40, or 50 fold in the presence of a transcriptional repression domain, e.g., as a fusion to a KRAB domain. In one embodiment, the DNA binding domain comprises three zinc finger domains that have the DNA contacting residues of sets of motifs shown in Table 2 from N- to C-terminus or at least 8, 9, 10, or 11 of the twelve DNA contacting residues of a set of three zinc finger domains of a DNA binding domain described herein. In many embodiments, the DNA binding domains are non-naturally occurring or include at least two or three zinc finger domains derived from at least two different mammalian (e.g., human) proteins. DNA binding domains can also include one or more domains that are obtained by modifying mammalian (e.g., human) zinc finger domains. In one embodiment, the DNA binding domain binds to a target sequence in the LTR of a human immunodeficiency virus, e.g., of subtype B, A, or E. The DNA binding domain may preferably bind to a sequence in the LTR of one subtype relative to another subtype, e.g., by at least 2, 5, or 10 fold. In other cases, the protein is effective at modulating (e.g., inhibiting) the transcriptional activity of LTR for a plurality of viral subtypes, e.g., at least two or four fold for at least two subtypes, h one embodiment, the DNA binding domain binds to a target sequence that includes or overlaps with one of the target sequences shown in Table 2. In one embodiment, the DNA binding domains binds to a target sequence in a viral gene, and thereby reduces the affinity of a viral or host cell transcription factor for a binding site in the viral gene, but does not affect the binding of the viral or host cell transcription factor for a binding site in a cellular gene to the same extent. For example, the inliibitory effect on the viral gene is at least 2, 5 or 10 times greater. In one embodiment, the target sequence includes one or more of the following nucleotides: (i) G at position +42, (ii) G at position -50, (iii) T at position -49, (iv) G at position -48, (v) G at position -47, (vi) C at position -46, (vii) G at position -45, (viii) T at position -25, (ix) A at position -24, and (x) A at position -23 of the LTR sequence shown in FIG. 1. The protein can include a protein transduction domain (PTD), e.g., the PTD from TAT or another PTD, e.g., another PTD described herein. The protein can include a nuclear localization signal (NLS), e.g., an NLS from a human protein, e.g., an NLS described herein. Exemplary NLS sequences include: MyoD- NLS: CKRKTTNADRRKA (SEQ ID NO: 1); ppoI-NLS: KRKGDENDGNDENAKKKSKK (SEQ ID NO: 2); rrgl-NLS: DRNKKKKE (SEQ ID NO: 3), or fragments thereof that function to deliver a polypeptide into the nucleus. In one embodiment, the zinc finger protein includes one or more sequences that stabilize the protein, increase protein expression, and/or facilitate protein purification. Such sequences can be N- or C- terminal to the DNA binding domain, e.g., at the N- or C- terminus of the protein, or between the PTD and the NLS, between the NLS and the DNA binding domain, or between the PTD and the DNA binding domain. In one embodiment, the zinc finger protein does not include any extraneous sequences. The protein can include one or more features described herein. An isolated protein can be, for example, at least 10, 20, 30, 40, 80, 90, 95, 99, or 99.9% pure, free of other proteins originating from the source. In another aspect, the present invention features a nucleic acid that includes a coding sequence that encodes a protein described herein. The nucleic acid can further include regulatory sequences, e.g., a promoter, operably linked to the coding sequence. The coding sequence can be interrupted by one or more introns. The disclosure also features a host cell that includes the nucleic acid (e.g., in an integrated form or as a vector), and a method of preparing a protein that includes expressing the nucleic acid in a cell, e.g., a prokaryotic or eukaryotic cell. The disclosure also features modified non-human animals such as transgenic animals that include one or more cells that express a protein described herein or animals that include one or more cells, e.g., transplanted or implanted cells, that express a protein described herein. Methods for making transgenic animals, implants, and transplants are known. In another aspect, the disclosure features a pharmaceutical composition that comprises an agent described herein(e.g., a protein that includes a protein transduction domain, or an expressible nucleic acid that encodes a protein described herein) and a pharmaceutically acceptable carrier. hi still another aspect, the disclosure features a method of treating or preventing a retroviral disorder, e.g., a disorder mediated by a human immunodeficiency virus. The method comprises an agent that can regulate viral gene transcription to cells of a subject in a therapeutically effective or prophylactically effective amount, hi one embodiment, the agent includes a protein that has (i) a DNA binding domain that binds to a target sequence in the LTR of a human immunodeficiency virus, and (ii) a protein transduction domain. The protein may include one or more features described herein. In another embodiment, the agent includes a nucleic acid that encodes a protein comprising a DNA binding domain that includes a plurality of zinc finger domains. The DNA binding domain binds to a target sequence in the LTR of a human immunodeficiency virus. In some cases, the protein can reduce the transcriptional activity of LTR in the absence of a transcriptional repression domain. The protein may include one or more features described herein. The method of treatment can include evaluating the subject, e.g., to determine if they are infected with a retrovirus, e.g., a retrovirus of the a particular subtype or designation. The method can also include selecting an agent based on the results of the evaluating, e.g., to provide an agent that targets the appropriate subtype or other designation of the infecting virus. In another aspect, the present invention features a protein, e.g., an isolated or recombinant protein that includes a human NLS physically associated with an amino acid sequence that is not normally associated with the human NLS. For example, a human NLS can be fused to another protein or a fragment thereof, e.g., another human or artificial protein, e.g., a nucleic acid binding protein that includes one or more human domains. For example, a human NLS is fused to a chimeric zinc finger protein, e.g., one that includes one or more human zinc finger domains. Exemplary human NLS sequences include: MyoD-NLS (SEQ ID NO: 1), ppoI-NLS (SEQ ID NO: 2), rrgl-NLS (SEQ ID NO: 3), or fragments thereof that function to deliver a polypeptide into the nucleus. For example, these NLS sequences can be fused to a protein other than a human MyoD protein or a region thereof, other than a poly(ADP-ribose) polymerase or a region thereof, or other than a human retinoic acid receptor or a region thereof, respectively. The present invention also includes nucleic acids encoding such proteins, and cells (e.g., recombinant host cells) including a vector that contains the nucleic acid.

DESCRIPTION OF THE DRAWINGS

Fig.l lists the nucleic acid sequence (SEQ ID NO: 4) of an LTR promoter sequence found in HIV-l subtype B used in a reporter construct. Also noted are the binding sites of transcription factors SP1 and NF-kB, and the target sequences of chimeric zinc finger proteins that recognize the LTR region. The mark, +1, indicates the transcriptional initiation site.

Fig. 2 lists nucleic acids sequences of LTR promoters of HIN-1 subtype A (SEQ ID NO: 5) and E (SEQ ID NO: 6) which are compared to the LTR promoter of HIV-l subtype B (sequence 1-208 of SEQ ID NO: 4). Fig. 3 is a schematic of a plasmid encoding PTD_TA_T-ZFPKOX fusion protein.

Fig. 4 lists amino acid sequences of (a) PTD_TAT -LTR-65KOX (SEQ ID NO: 7), and (b) PTD_TA_T -LTR-62KOX (SEQ ID NO: 8). These proteins include a hexa-histidine sequence for purification (boxed), sequences for Tat-peptide (boxed, bold letters), HA-NLS-ZFP (bold letters) and a KOX domain (underlined) (SEQ ID NO: 23). Other configurations can be used, for example, ones that remove extraneous sequences or that contain alternative linker sequences.

Fig. 5 A shows the inhibition of LTR promoter activity by Tat-LTR-65-KOX protein measured at 24h post-transduction. The activity of Firefly luciferase was normalized to an internal control, Renilla luciferase (left). The LTR-activity is represented by fold-repression relative to the control (right).

- A - Fig. 5B shows the inhibition of LTR promoter activity by Tat-LTR-65-KOX protein measured at 48h post-transduction. Fig. 5C shows the inhibition of LTR promoter activity by Tat-LTR-62-KOX protein measured at 24h post-transduction. Fig. 5D shows the inhibition of LTR promoter activity by Tat-LTR-62-KOX protein measured at 48h post-transduction.

DETAILED DESCRIPTION

Retroviral replication depends on transcription from LTR (long terminal repeat). The sequence of an exemplary LTR from HTV- 1 is described in SEQ ID NO : 1. The HIV- 1 LTR includes binding sites for the viral transcription factor TAT and for host transcription factors NF-kB and Spl. Several mutations in these sequences are reported among HIV-l subtypes. Transcriptional activity of LTR can be modulated using artificial transcription factors, e.g., zinc finger proteins that include one or more zinc finger domains, e.g., three zinc finger domains. The zinc finger protein can bind to a target sequence within the LTR, e.g., a sequence having a length between 8-15 basepairs. For example, the target sequence includes or overlaps with one of the sequences in column 6 of Table 2. The target sequence may overlap with or include a binding site for a viral transcription factor or a host cell transcription factor, e.g., Spl or NF-κB. h one embodiment, the target sequence includes regions of two adjacent binding sites for transcription factors, e.g., two Spl binding sites, e.g., the Spl-I and Spl -LI sites or the Spl -II and Spl-LTI sites, hi one embodiment, the zinc finger protein inhibits the binding of Spl to one or more sites in a viral LTR but does not inhibit Spl from binding to a site in an endogenous gene, e.g., the binding of Spl to its cognate sites in the p21(WAFl/CLPl) gene, to the same extent. For example, an exemplary zinc finger protein inhibits Spl binding to the LTR at least 2, 5, 10, or 50 fold more than it inhibits Spl binding to its cognate sites in the p21(WAFl/CIPl) gene. Exemplary zinc finger domains include human zinc finger domains and artificial zinc finger domains, e.g., mutated domains derived from naturally occurring zinc finger domains. In some embodiments, the zinc finger protein includes a human framework, at least one, two, or three human domains, or all human domains. A protein with human characteristics can minimize the potential for an adverse immunological response against the protein. In some cases, because the protein is produced intracellularly, it is deimmunized, e.g., modified to have a minimum or no T cell epitopes.

Zinc Fingers Domains Zinc fingers are small polypeptide domains of approximately 30 amino acid residues in which there are four amino acids, either cysteine or histidine, appropriately spaced such that they can coordinate a zinc ion (see, e.g., Wolfe et al., (1999) Annu. Rev. Biophys. Biomol. Struct. 3:183-212). 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 Cys -His zinc fingers are typically spaced as follows: X_a-X-C-X_2-5-C-X₃-X_a-X₅-ρsi-X₂-H-X_3-5-H (SEQ ID NO: 9), where psi is a hydrophobic residue. "X" represents any amino acid, wherein X_a is typically, but not always, phenylalanine or tyrosine, the subscript indicates the number of amino acids, and the subscript with two hyphenated numbers indicates a typical range of intervening amino acids. 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 in a tetrahedral conformation appropriate for coordinating the zinc ion. The base contacting residues are at the N-terminus of the finger and in the preceding loop region. For convenience, the primary DNA contacting residues of a zinc finger domain are numbered: -1, 2, 3, and 6, as illustrated by the following example: - 1 1 2 3 4 5 6 C-X₂_₅-C-X₃-X_a-X-R-X-D-E-X_b-X-R-H-X₃-₅-H (SEQ ID NO: 10), As noted in the example above, the DNA contacting residues are Arg (R), Asp (D), Glu (E), and Arg (R). The above motif can be abbreviated RDER. As used herein, such abbreviation is a shorthand that refers to a particular polypeptide sequence from the second residue preceding the first cysteine (above, initial residue of SEQ ID NO: 10) to the ultimate metal-chelating histidine (ultimate residue of SEQ ID NO: 10). In the above motif and others, X_a is typically, but not always, phenylalanine or tyrosine, and X is frequently hydrophobic. The small letter "m" before a motif can be used to make explicit that the abbreviation is referring to a motif. For example, mRDER refers to a motif in which R appears at positions -1, D at position 2, E at position 3, and R at position 6. A zinc finger protein typically includes a DNA binding domain which may include a tandem array of two, three or more zinc finger domains. The DNA binding domain may be less than 100, 95, 90, or 85 amino acids. In some cases, the entire zinc finger protein is less than 120, 110, 100, 95, 90, or 85 amino acids. In an exemplary DNA binding domain, zinc finger domains, whose motifs are listed consecutively herein, are not interspersed with other folded domains, but may include a linker, e.g., a flexible linker described herein (SEQ ID NO: 22) between domains. For an implementation that includes a specific zinc finger protein or an array thereof described herein, the invention also features a related implementation that includes a corresponding zinc finger protein or an array thereof having an array with zinc fingers that have the same DNA contacting residues as the specific zinc finger protein or array thereof. The corresponding zinc finger protein may differ by at least one, two, three, four, or five amino acids from the disclosed specific zinc finger protein, e.g., at an amino acid position that is not a DNA contacting residue. Other related implementations include a corresponding protein that has at least one, two, or three zinc fingers that have the same DNA contacting residues, e.g., in the same order. Many exemplary human zinc finger domains are described in WO 01/60970,

WO 03/016571, and US 2003-0165997. Zinc finger proteins can be engineered to include a transcriptional regulatory domain. Exemplary transcriptional repression domains include repression domains from KRAB, Kid, UME6, ORANGE, groucho, and WRPW. See, e.g., Dawson et al., (1995) Mol. Cell Biol. 15:6923-31 and US 2004-0209277. Exemplary zinc finger proteins can bind to a target sequence in a retroviral LTR, e.g., a

HIV-l or fflV-2 LTR, with a dissociation constant of less than 10^"7, 10^"8, 10^"9, or 10^"10 M. The dissociation constant can be determined by gel shift analysis using a purified protein that is allowed to bind in 20 mM Tris pH 7.7, 120 mM NaCl, 5 mM MgCl₂, 20 μM ZnSO₄, 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 Rebar and Pabo (1994) Science 263 :671- 673.

Identifying Zinc Finger Proteins Artificial zinc finger proteins can be identified by a variety of methods. For example, an artificial DNA-binding protein can be constructed to recognize a target sequence by mixing and matching characterized zinc finger domains. See, e.g., WO 01/60970. Zinc finger domains can be isolated and characterized using a variety of methods. US 2002-0061512 describes a method for evaluating domains using a yeast assay system. Another method for constructing an artificial DNA-binding protein includes using phage display to select for zinc finger domains with altered DNA-binding specificity (US 6,410,248). Domains that interact with a target site can be selected and used to generate a DNA binding protein that binds to the target sequence. WO 01/60970 and WO 03/016571 also describe methods for designing DNA-binding proteins. The modular structure of zinc finger domains facilitates their rearrangement to construct new DNA-binding proteins. Zinc finger domains in the naturally-occurring Zif268 protein are positioned in a tandem array that can straddle the DNA double helix. Each domain independently recognizes a different 3-4 basepair DNA segment. By linking three or more zinc finger domains, a DNA binding protein that specifically recognizes a 9-bp or longer DNA sequence can be engineered. Bae KH et al. (2003) Nat Biotechnol. 21(3):275-80 describes a method for evaluating the specificity of DΝA-binding domains in cells and a method of constructing new DΝA- binding proteins using information from such cellular assays. It is also possible to screen libraries of nucleic acids encoding different combinations of zinc finger domains to identify a polypeptide that includes a functional DΝA binding domain that produces a desired phenotypic effect. US 2003-0194727 describes exemplary methods of identifying useful zinc finger proteins by screening or selection. Generally, a library of nucleic acid that encodes polypeptides that include different combination of zinc finger domains and an effector domain is prepared and introduced into cells. After expressing the library members, cells that exhibit an altered phenotype relative to a reference cell (e.g., an untransformed cell or a cell transformed with a vector nucleic acid) are isolated. The library nucleic acid in the cell is recovered and characterized to identify the relevant zinc finger protein. This method can be used to identify zinc finger proteins that modulate transcriptional activity of LTR directly or indirectly.

Exemplary zinc finger proteins The amino acid sequences of the DΝA binding domains of exemplary zinc finger proteins that bind to the HIN LTR are show in Table 1. Table 1

ZFP Name Amino acid sequence of DNA binding domain

ZFP-LTR-69 PYHCD DGCGWKFARSDELTRHYRKHTGEKPFQCKTCQRKFSRSDHLKT HTRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 11) ZFP-LTR-66 PYKCGQCGKFYSQVSHLTRHQKIHTGEKPYHCDWDGCG KFARSDELTR HYRKHTGEKPFQCKTCQRKFSRSDHLKTHTRT (SEQ ID NO: 12) ZFP-LTR-65 PYKCKQCGKAFGCPSNLRRHGRTHTGEKPFQCKTCQRKFSRSDHLKTHT RTHTGEKPYKCMECGKAFNRRSHLTRHQRIHT (SEQ ID NO: 13) ZFP-LTR-62 PFQCKTCQRKFSRSDHLKTHTRTHTGEKPYKCKQCGKAFGCPSNLRRHG RTHTGEKPFQCKTCQRKFSRSDHLKTHTRTHT (SEQ ID NO: 14) ZFP-LTR-59 PYKCGQCGKFYSQVSHLTRHQKIHTGEKPFQCKTCQRKFSRSDH KTHT RTHTGEKPYKCKQCGKAFGCPSNLRRHGRTHT (SEQ ID NO: 15) ZFP-LTR-56 PYHCD DGCGWKFARSDELTRHYRKHTGEKPYKCGQCGKFYSQVSHLTR HQKIHTGEKPFQCKTCQRKFSRSDHLKTHTRT (SEQ ID NO: 16 ZFP-LTR-53 PYHCD DGCG KFARSDELTRHYRKHTGEKPYHCD DGCGWKFARSDEL TRHYRKHTGEKPYKCGQCGKFYSQVSHLTRHQ (SEQ ID NO: 17) ZFP-LTR-25 PYECNYCGKTFSVSSTLIRHQRIHTGEKPYKCPDCGKSFSQSSSLIRHQ RTHTGEKPYKCEECGKAFTQSSNLTKHKKIHT (SEQ ID NO: 18) ZFP- PYICRKCGRGFSRKSNLIRHQRTHTGEKPYECDHCGKSFSQSSHLNVHK LTR+45R RTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHT (SEQ ID NO: 19)

These proteins can include additional sequences. For example, the following sequence which includes a hemagglutinin tag and a nuclear localization signal can be positioned at the N-terminus: YPYDVPDYAELPPKKKRKVGIRtPGEK (SEQ ID NO: 20). In one embodiment, the proteins can further include a transcriptional repression domain, e.g., the KOX transcriptional repression domain, a "KRAB" domain from the human Koxl protein (Zinc finger protein 10; NCBI protein database AAH24182; GL18848329), i.e., amino acids 2-97 of Koxl : DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQINYRNVMLENYKNLVSL

GYQLTKPDVΓLRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ TD NO: 23) Other useful proteins include proteins that include: (i) a DNA binding domain that is at least 85, 90, 92, 94, 95, 96, 97, 98, or 99% identical to one of the DNA binding domains provided in Table 1; (ii) a DNA binding domain that differs by at least one, but fewer than ten, eight, six, five, four, three or two amino acids from one of the DNA binding domains provided in Table 1 (e.g., differences include insertions, deletions and substitutions, e.g., conservative substitutions); and (iii) a DNA binding domain that is encoded by a nucleic acid that hybridizes (e.g., under high stringency conditions) to a nucleic acid that encodes one of the DNA binding domains provided in Table 1. The percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, amino acids having aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; amino acids having aliphatic-hydroxyl side chains are serine and threonine; amino acids having amide-containing side chains are asparagine and glutamine; amino acids having aromatic side chains are phenylalanine, tyrosine, and tr ptophan; amino acids having basic side chains are lysine, arginine, and histidine; amino acids having acidic side chains are aspartic acid and glutamic acid; and amino acids having sulfur-containing side chains are cysteine and methionine. As used herein, the term "hybridizes under high stringency conditions" refers to conditions for hybridization in 6 x sodium chloride/sodium citrate (SSC) at 45°C, followed by two washes in 0.2 x SSC, 0.1% SDS at 65°C. Also featured are zinc finger proteins that include one or more zinc finger domains that have a set of DNA contacting residues that are identical to those of one or more zinc fingers of a protein of Table 1. Exemplary proteins include the following motifs in N to C-terminal order: mRDER-mRDHT-mDSCR; mQSHR-mRDER-mRDHT; mCSNR-mRDHT-mRSHR; mRDHT-mCSNR-mRDHT; mQSHR-mRDHT-mCSNR; mRDER-mQSHR-mRDHT; mRDER-mRDER-mQSHR; mVSTR-mQSSR-mQSNK; and mRSNR-mQSHV-mDSCR.

Protein Transduction Domains 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, e.g., a DNA binding domain that can specifically bind to a viral LTR. 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. For example, a basic PTD can 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 eukaryotic cell, e.g., a vertebrate cell, e.g., a mammalian cell, such as a human, simian, murine, bovine, equine, or feline cell. A PTD can be linked to an artificial transcription factor, for example, using a flexible linker. Flexible linkers may include one or more glycine residues to allow 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. Being located N- or C-terminal to a particular domain does not require being adjacent to that particular domain. For example, a PTD N-terminal to a DNA binding domain can be separated from the DNA binding domain by a spacer and/or other types of domains. An artificial transcription factor 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 segments from the antennapedia protein, the herpes simplex virus VP22 protein and HIV TAT protein. See, e.g., WO2004-108883. The minimal Tat PTD includes residues 47-57 of the human immunodeficiency virus Tat protein: YGRKKRRQRRR (SEQ ID NO: 21). This peptide sequence is referred to as "TAT-PTD" herein. This peptide can mediate the introduction of heterologous peptides and proteins in excess of 100 kDa into mammalian cells in vitro and in vivo (Ho et al. (2001) Cancer Res 61(2):474-7). Schwarze et al. showed that when the 120 kDa β-galactosidase protein fused with TAT-PTD was injected into a mouse intraperitoneally, the fusion proteins were found in all types of cells and tissues even including brain, which has been thought to be difficult because of the blood-brain-barrier (Schwarze et al. (1999) Science 285(5433): 1466-7). The region of Tat that is used includes at least the PTD region, but typically does not include sufficient sequences to produce a Tat protein capable of activating LTR. For example, only residues in the TAT-PTD, i.e., SEQ ID NO: 21(above), are used. In one embodiment, the PTD is obtained from a human or other mammalian protein. Exemplary mammalian PTD's are described in WO 03/059940 (human SLM-2) and WO 03/059941 (Mph). Cell-specific PTD's. Some PTD's are specific for particular cell types or states. US 2002-0102265 and US 6,451,527describe exemplary method for isolating cell specific PTD's, e.g., using phage display. Exemplary cellular uptake signals include amino acid sequences that are specifically recognized by cellular receptors or other surface proteins, e.g., CD4 or CD8. Interaction between the cellular uptake signal and the cell can also cause internalization of the transcription factor that includes the cellular uptake signal. Assays for protein transduction. A number of assays are available to determine if an amino acid sequence can function as a PTD. For example, the amino acid sequence can be fused to a reporter protein such as β-galactosidase to form a fusion protein. This fusion protein is contacted with culture cells. The cells are washed and then assayed for reporter activity. Another assay detects the presence of a fusion protein that includes the amino acid sequence in question and another detectable sequence, e.g., an epitope tag. This fusion protein is contacted with culture cells. The cells are washed and then analyzed by Western or immunofluorescence to detect the presence of the detectable sequence in cells. See, e.g., WO 2004-108883 for further description of protein transduction assays, uses, and assays. A specific protein described herein can include a PTD to form a transducible transcription factor that can be used in a therapeutic method, e.g., to treat or prevent HIV. hi addition, a protein that includes one or more of the following motifs (e.g. an array of zinc finger domains according to a series of motifs below) can be physically associated with a PTD (e.g., as a fusion protein) and a transcriptional repression domain to form a transducible transcription factor that can be used in a therapeutic method, e.g., to treat or prevent HIV: mRDER-mRDNT-mRDHT; mRDVR-mRDHT-mDSVR; mDAHR-mRDHT-mDANK; mAADR-mNSDR-mTSNK; mHSDR-mQSDK-mQATR; mDSSK-mQAHT-mDSSK; mADDQ-mRSDR-mQAHK; and mRDAQ-mDANT-mASTK. Transducible zinc finger proteins can facilitate the treatment, e.g., of HIV-l replication from infected cells, because major target cells for HIV are lymphocytes circulating in blood. A therapeutic protein administered into circulation (e.g., the bloodstream) maybe an efficient and rapid mode for delivering the protein to a target.

Treatments Agents that can regulate viral gene transcription can be provided to cells of a subject, e.g., in therapeutically effective or prophylactically effective amounts, to treat or prevent a variety of disorders, such as retroviral disorders, particularly ALDS and AIDS-related complex. The agents can be targeted to T lymphocytes (e.g., CD4+ and/or CD8+ cells) or to virus infected T lymphocytes. Exemplary agents include protein-based and nucleic acid-based agents. An example of a protein-based agent is a transducible protein, e.g., a transducible zinc finger protein. An example of a nucleic acid-based agent is a nucleic acid that encodes a zinc finger protein that regulates transcriptional activity of LTR (e.g., a nucleic acid packaged in a vehicle for delivery into a cell). An amount of an agent effective to treat a disorder, or a "therapeutically effective amount" refers to the amount of the agent that is effective, upon single or multiple dose administration to a subject, to reduce at least one activity that contributes to disease, e.g., to reduce virus production. Such reduction can include a reduction, e.g., a statistically significant reduction, in the activity of a cell or tissue (e.g., metastatic tissue) or the number of viral particles produced. An amount of an agent effective to prevent a disorder, or a "a prophylactically effective amount" of the protein refers to the amount of the protein, which is effective, upon single- or multiple-dose administration to the subject, in preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g., a retroviral disorder. The subject can be a human or other mammal, e.g., a non-human primate. Subjects who have a retroviral infection (e.g., ADDS), or are at the risk of retroviral infection, can be identified by standard methods, including immunoassays and PCR-based assays. The subject can be at any one of the several stages of HIN infection progression, which, for example, include acute primary infection syndrome (which can be asymptomatic or associated with an influenza-like illness with fevers, malaise, diarrhea and neurologic symptoms such as headache), asymptomatic infection (which is the long latent period with a gradual decline in the number of circulating CD4+ T cells), and ADDS (which is defined by more serious ATDS- defining illnesses and/or a decline in the circulating CD4 cell count to below a level that is compatible with effective immune function). In addition, treating or preventing HIV infection includes treating suspected infection by HIV after suspected past exposure to HTV by, e.g., contact with HlV-contaminated blood, blood transfusion, exchange of body fluids, unsafe sex with an infected person, accidental needle stick, receiving a tattoo or acupuncture with contaminated instruments, or transmission of the virus from a mother to a baby during pregnancy, delivery or shortly thereafter. Methods of treating HIN infection include treatment of a carrier of any of the HIV family of retro viruses or a person who is diagnosed to have active ADDS, as well as the treatment or prophylaxis of the AIDS-related conditions in such persons. A carrier of HIV may be identified by any methods known in the art. For example, a person can be identified as an HIV carrier on the basis that the person is anti-HIN antibody positive, or is HIV-positive, or has symptoms of AIDS. In one aspect, this disclosure provides a method of treating or preventing a retroviral disorder, e.g. a disorder mediated by an immunodeficiency virus, e.g., HTV, e.g., HIV I, HIV II, HTV HI (also lαiown as HTLV-fl, LAV-1, LAV-2), and the like. "HTV" includes all strains, forms, subtypes, clades and variations in the HTV family. The method can include administering to a subject an agent that provides a zinc finger protein that regulates transcriptional activity of LTR promoter, in an amount sufficient to treat or prevent the retroviral disorder. The agent can be the zinc finger protein itself, e.g., a protein that includes a protein transduction domain, or a nucleic acid that encodes the zinc finger protein. For example, the disorder can be caused by a virus infected cell, e.g., virus infected T-cell. The method can be used to treat or prevent AIDS. The method can also be performed in vitro, e.g. by contacting the agent to virus infected cells in an amount sufficient to deliver the agent into one or more of the cells. The agent can be administered by any appropriately route, (locally or systemically), e.g., by intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The agent can be formulated accordingly, e.g., as a pharmaceutical composition. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. For example, the agent can be administered in an amount effective to ameliorate at least one symptom associated with ADDS or symptoms or conditions associated with, or defined by, HIV infection such as AIDS-related complex (ARC), progressive generalized lymphadenopathy (PGL), anti-HIV antibody positive conditions, and HIV-positive conditions, ADDS-related neurological conditions (such as dementia or tropical paraparesis), Kaposi's sarcoma, thrombocytopenia purpurea and associated opportunistic infections such as Pneumocystis carinii pneumonia, Mycobacterial tuberculosis, esophageal candidiasis, toxoplasmosis of the brain, CMV retinitis, HlV-related encephalopathy, HIN-related wasting syndrome, and so forth. In some embodiments, an effective amount of an agent that modulates the activity of LTR is an amount that is effective to reduce transcriptional activity of LTR prompter in an infected cell (e.g., in a cell infected with the immunodeficiency virus) by at least about 10%>, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%), at least about 80%, or more, compared to the amount of transcriptional activity of LTR promoter in the absence of administration of the agent. In some embodiments, an effective amount of an agent that modulates transcriptional activity of LTR promoter is an amount that is effective to reduce viral load in an individual by at least about 10%o, at least about 20%, at least about 30%, at least about 40%, at least about 50%), at least about 60%, at least about 70%), at least about 80%, or more, compared to the viral load in the individual in the absence of treatment with the agent. In some embodiments, a therapeutically effective amount of an agent that modulates transcriptional activity of LTR promoter decreases the detectable level of viral RΝA molecules in a plasma sample from the subject to less than about 6000 RΝA molecules/ml, less than about 4500 RNA molecules/ml, less than about 3500 RNA molecules/ml, less than about 2500 RNA molecules/ml, or less than about 1500 RNA molecules/ml. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in a dosage unit form for ease of administration and uniformity of the dosage. The dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a protein agent or a nucleic acid agent is 0.1-20 mg/kg, more preferably 1-10 mg/kg or 0.1-1 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. Methods for formulating, e.g., as pharmaceutical compositions, and administering transducible zinc finger proteins are described, e.g., in WO 2004-108883. Suitable dosages of the molecules used may depend on the age and weight of the subject and the particular drug used. Exemplary pharmaceutically acceptable carriers for formulating agents described herein include: any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the agent may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the agent. A

"pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the agent and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. An agent that modulates the transcriptional activity of LTR promoter (e.g., a nucleic acid encoding a zinc finger protein that regulates transcriptional activity of LTR promoter or a transducible zinc finger protein) can be administered alone or in combination with one or more of the existing modalities for treating retroviral infections, e.g., AIDS or ARC. For example, the agent can be administered in combination with another anti- viral agent or an agent that treats an associated disorder. The term "in combination" in this context means that different agents are given substantially contemporaneously, either simultaneously or sequentially. If given sequentially, at the onset of administration of the second agent, the first of the two agents is preferably still detectable at effective concentrations at the site of treatment. Examples of other agents that can be used to treat a viral disorder and/or associated disorders include acyclovir, amantadine, rimantadine, recombinant soluble CD4 (rsCD4), a fusion inhibitor (e.g., a T20 peptide, a T-1249 peptide; Trimeris); an anti-CD4 antibody; an anti-CCR5 antibody (e.g., Pro 140); a CXCR4 blocker (e.g., AMD 3100); an HIV entry inhibitor (e.g., Pro-542; Progenies); a CCR5 blocker (e.g., SCH-C, SCH-D; Schering Plough); anti-receptor antibodies (e.g., for rhinoviruses), nevirapine (Viramune^®), emiravine (Coactinon^®), cidofovir (Vistide™), trisodium phosphonoformate (Foscarnet™), famcyclovir, pencyclovir, valacyclovir, nucleic acid/replication inhibitors, interferon, zidovudine (AZT, Retrovir™), didanosine (dideoxymosine, ddl, Videx™), stavudine (d4T, Zerit™), zalcitabine (dideoxycytosine, ddC, Hivid™), nevirapine (Viramune™), lamivudine (Epivir™, 3TC), protease inhibitors, saquinavir (hivirase™, Fortovase™), ritonavir (Norvir™), nelfinavir (Viracept™), efavirenz (Sustiva™), abacavir (Ziagen™), amprenavir (Agenerase™) indinavir (Crixivan™), ganciclovir, AzDU, delavirdine (Rescriptor™), kaletra, trizivir, rifampin, clarithromycin, erythropoietin, colony stimulating factors (G-CSF and GM-CSF), non- nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors, HIN protease inhibitors, adriamycin, beta-lactam antibiotics, tetracyclines, chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone, betamethasone, dexamethasone, fluocortolone, prednisolone, triamcinolone, indomethacin, sulindac, uorouracil, methotrexate, asparaginase and combinations thereof. Examples of nucleoside reverse transcriptase inhibitors (RTI's) include AZT, ddl, 3TC, ddC, d4T, and abacavir). Examples of protease inhibitors include indinavir, saquinavir, ritonavir, nelfmavir, amprevanir, and lopinavir). Examples of non-nucleoside reverse transcriptase inhibitors include nevirapine, delavirdine, emiravine, and efavirenz). Other exemplary anti-viral agents include a fusion inhibitor (e.g., T20, T-1249); and/or a CCR5 blocker (e.g., SCH-C, SCH-D). The zinc fmger protein that is used in combination therapy can include one or more zinc fmger domains that have DNA contacting residues corresponding to the following motifs: mRDER-mRDHT-mDSCR; mQSHR-mRDER-mRDHT ; mCSNR-mRDHT-mRSHR; mRDHT-mCSNR-mRDHT; mQSHR-mRDHT-mCSNR; mRDER-mQSHR-mRDHT; mRDER-mRDER-mQSHR; mNSTR-mQSSR-mQSNK; mRSNR-mQSHV-mDSCR; mRDER-mRDNT-mRDHT; mRDNR-mRDHT-mDSNR; mDAHR-mRDHT-mDAΝK; mAADR-mΝSDR-mTSΝK; mHSDR-mQSDK-mQATR; mDSSK-mQAHT-mDSSK; mADDQ-mRSDR-mQAHK; and niRDAQ-mDAΝT-mASTK.

Nucleic Acid Delivery DNA molecules that encode a zinc finger protein can be inserted into a variety of DNA constructs and vectors for the purposes of gene therapy. As used herein, a "vector" is a nucleic acid molecule competent to transport another nucleic acid molecule to which it has been covalently linked. Vectors include plasmids, cosmids, artificial chromosomes, viral elements, and RNA vectors (e.g., based on RNA virus genomes). The vector can be competent to replicate in a host cell or to integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses. A gene therapy vector is a vector designed for administration to a subject, e.g., a mammal, such that a cell of the subject is able to express a therapeutic gene contained in the vector. The gene therapy vector can contain regulatory elements, e.g., a 5' regulatory element, an enhancer, a promoter, a 5' untranslated region, a signal sequence, a 3' untranslated region, a polyadenylation site, and a 3' regulatory region. For example, the 5' regulatory element, enhancer or promoter can regulate the transcription of the DNA encoding the zinc fmger protein. The regulation can be tissue specific. For example, the regulation can restrict the transcription of the desired gene to brain cells, e.g., cortical neurons or glial cells; hematopoietic cells, e.g., T lymphocytes (e.g., CD4+ and/or CD8+ T cells); or endothelial cells. Alternatively, regulatory elements can be included that respond to an exogenous drug, e.g., a steroid, tetracycline, or the like. Thus, the level and timing of expression of the therapeutic zinc finger protein (e.g., a polypeptide that regulates the transcriptional activity of LTR promoter) can be controlled. Gene therapy vectors 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. See, e.g., US 2003-0143266 and 2002-0150626. In one embodiment, the nucleic acid is formulated in a lipid-protein-sugar matrix to form microparticles, e.g., having a diameter between 50 nm to 10 micrometers. The particles may be prepared using any known lipid (e.g., dipalmitoylphosphatidylcholine, DPPC), protein (e.g., albumin), or sugar (e.g., lactose). The gene therapy vectors can be delivered using a viral system. Exemplary viral vectors include vectors from retroviruses, e.g., Moloney retrovirus, adenoviruses, adeno- associated viruses, and lentiviruses, e.g., Herpes simplex viruses (HSV). HSV, for example, is potentially useful for infecting nervous system cells. See, e.g., US 2003-0147854, 2002-0090716, 2003-0039636, 2002-0068362, and 2003-0104626. The gene delivery agent, e.g., a viral vector, can be produced from recombinant cells that produce the gene delivery system. A gene therapy vector 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. Other gene delivery methods can also be used. Methods for formulating nucleic acid agents that encode a zinc fmger protein are also known and can be performed, e.g., as described in USSN 10/732,620. For example, such agents can be formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier. Some aspects of useful zinc fmger proteins that bind to the HIV LTR are further illustrated by the following specific and non-limiting examples. EXAMPLE

Production of zinc finger proteins that recognize LTR promoter sequences of HIN- 1 subtype B. We have generated chimeric zinc finger proteins that recognize DΝA sequences of

LTR promoter of HIN-1 subtype B that is commonly found in North America and Europe. The names of these zinc fmger proteins are provided in column 1 of Table 2. The DNA binding domains of these zinc fmger proteins include three zinc fingers. The DNA contacting residues in these fingers are indicated by the motifs in columns 2, 3, and 4 respectively. The name and the sequence of the target site that is recognized by each zinc finger protein is provided in columns 5 and 6.

Table 2

Col. 1 Col. 2 Col. 3 Col. 4 Col. 5 Col. 6 Col. 7 Col. 8 Fold fmger 1 fmger2 fmger 3 Target Site Fold Rep. w/

ZFP Name motif motif motif Name Target Site sequence Rep. KRAB

ZFP-LTR-69 RDER RDHT DSCR LTR-69 5' GCC TGG GCG 3' 4±0.4 18.4±0.4

ZFP-LTR-66 QSHR RDER RDHT LTR-66 5' TGG GCG GGA 3' 1.9±0.6 5.4±0.1

ZFP-LTR-65 CSNR RDHT RSHR LTR-65 5' GGG CGG GAC 3' 8.8±0.4 43.7±0

ZFP-LTR-62 RDHT CSNR RDHT LTR-62 5' CGG GAC TGG 3' 16.6±0.1 42.2±0.7

ZFP-LTR-59 QSHR RDHT CSNR LTR-59 5' GAC TGG GGA 3' 6.7±0.7 58.4±0.1

ZFP-LTR-56 RDER QSHR RDHT LTR-56 5' TGG GGA GTG 3' 5.5±0.2 14.5±0.8

ZFP-LTR-53 RDER RDER QSHR LTR-53 5' GGA GTG GCG 3' 3±0.3 29.5±0.3

ZFP-LTR-25 VSTR QSSR QSNK LTR-25 5' TAA GCA GCT 3' 7.3±0.4 lO.l±O

ZFP-

LTR+45R RSNR QSHV DSCR LTR+45R 5' GCC AGA GAG 3' 7±0.2 21±0.2

Control + Tat 1.0 1.0

Fig. 1 describes a segment of LTR promoter of HIN-1 subtype B, including binding sites for Spl and ΝF-κB, major host transcription factors recognizing LTR promoter sequences of HIN subtype B (Jeeninga, R. E. et al., 2000, J Nirol. 74:3740-3751). Table 2 lists chimeric zinc finger proteins that specifically recognize target sequences in the promoter. The target sequences bound by some of the proteins in Table 1 overlap with binding sites for specific transcriptional activators. For example, the binding sites for chimeric ZFP LTR-69, -66, -65, -62, -59, -56, -53 overlap with binding site for transcriptional activator Spl, the ZFP LTR-25 overlaps with binding site for essential transcription factor TBP (TATA box binding protein), and the LTR+45R overlaps with the binding site for TAT, a transcription activator encoded by the HIN virus itself. The amino acid sequences of the DΝA binding domains of these zinc fmger proteins are provided in Table 1. Many of the component zinc fmger domains have been described in Bae, K.-H. et al, Nature Biotechnol. 2003, 21:275-280. This reference also includes general methods for constructing zinc finger proteins. Construction of reporter gene containing LTR promoter sequences of HIV-l subtype B, subtype A and subtype E. We constructed a reporter plasmid that included 3'- region of LTR promoter sequences (GenBank™ accession number K03455; DNA sequence 9372-9621) operably linked to a reporter gene. The DNA fragment containing the sequences of this LTR promoter region was obtained from digestion of pU3R-III CAT (Sodroski et al, Science, 1985, 227:171-173) with XhoLHindTfl, and ligated with pGL3-basic plasmid (Promega) after digestion with XhoI/HindHT. The resulting reporter plasmid was designated LTR-B. To construct HIV-l LTR subtype A and E, we annealed synthetic oligonucleotides that contained sequences of each LTR subtypes. Briefly, 100 pmole of oligonucleotides were mixed in lOOul annealing buffer containing 50mM NaCl, lOmM Tris-HCl, lOmM MgCl₂ and ImM dTT. Mixture of oligonucleotides were heated to 95°C for 10 minutes then gradually cooled to 37°C. The double strand DNA after annealing procedure was then digested with Kpnl and Xhol. The insert fragments thus prepared were ligated into pGL3 -basic plasmid (Promega) linearized with Kpnl and Xhol. The resulting reporter plasmids that contain sequence of HIV-l LTR subtype A and -E were designated LTR- A and LTR-E. The sequences of oligonucleotides used for construction of LTR- A were; Afl 5 ' -CCGGGTACCgaagttgctgactgggactttccgctggggactttccaggggaggcgtggtttgggcgga gttggggagtggctaaccctcagatgctgc-3' (SEQ ID NO: 24), and Af2

5'-CCCCTCGAGagagagctcccaggctcagatctggtctacctagagagacccagtacaggcgaaaagcagctgcttatatg cagcatctgagggttagcc-3' (SEQ ID NO: 25). The sequences of oligonucleotides used for construction of LTR-E were; Efl 5 ' -CCGGGTACCgaagtttctaacataggacttccgctgggg actttccaggggaggtgtggccggggcggagttggggagtggctaaccctcagaagctgc-3' (SEQ ID NO: 26), and Ef2

5'-CCCCTCGAGagggagctcccgggctcgacctggtctaacaagagagacccagtacaagcgaaaagcggctgcttttatgc agcttctgagggttagcc-3' (SEQ ID NO: 27). The sequences for restriction enzymes Kpnl and Xhol are indicated as underlined capital letters, and the promoter sequences are written in lower case lettering.

Reporter Assays. To perform cell-based assays, HeLa cells were transfected with LTR-luciferase reporter (LTR-B) plasmid and with expression plasmids containing chimeric ZFP (p3-ZFP) or a control plasmid (p3) that do not express chimeric ZFP. The cells were examined for the reporter gene expression two days post-transfection. Reporter activity from cells that expressed ZFPs was compared to that of cells that do not express ZFP. Transcription from LTR was activated by cotransfection of plasmid (pTAT) that encodes the strong viral transcriptional activator TAT (Reynolds L. et al., Proc. Natl. Acad. Sci. USA, 2003, 100:1615-1620). HeLa cells were maintained and cultivated in DMEM media supplemented with 10% FBS in a 37°C incubator supplied with 5% CO₂. For the transfection, lxlO⁴ cells were plated in 96-well culture plate and incubated overnight. We used LIPOFECTAMINE PLUS™ (Invitrogen) for delivery of DNAs into cells. The DNA mixture included 20 ng of ZFP- expressing plasmid (p3-ZFP), 20 ng of reporter plasmid (either LTR-B or LTR-A or LTR-E), 20ng of expression plasmid for TAT (pTAT). To correct for differential transfection efficiencies, 2 ng of a plasmid that encodes renilla luciferase (pRL-TK; Promega) was added to the DNA mixtures. Each DNA mixture was added to 1 μl of Plus reagent (Invitrogen) and incubated for 15 min at 25°C, then combined with 0.5 μl of LIPOFECTAMIN™ (Invitrogen) and incubated for 15 min. The cells were treated with DNA-LIPOFECTAMIN™ complex, and incubated for two days at 37°C. After the two-day incubation, cells were washed with PBS, then treated with 30 μl of passive lysis buffer (Promega) for 15 minutes. 10 μl of each cell lysate was evaluated for luciferase activity using a dual luciferase assay system (Promega). The analysis was performed with a luminometer measuring luminescences after adding 50 μl of luciferase assay substrate and 50 μl of Stop and Glo reagent as recommended by manufacturer's protocol. The results from reporter LTR-B was obtained from average of two independent experiments performed with duplicate setting, and those from reporter LTR-A, and -E were obtained from average of five independent wells. Results. Transcriptional inhibitory effect of chimeric ZFPs on LTR promoter activity of HIV-l subtype B. The ZFP can inhibit transcription by blocking interactions between endogenous transcription factors and promoter sequences when the ZFP bind to sites for transcription factors (Kim, J.-S. et al, 1997, Proc, Natl., Acad., Sci., USA, 94:3616-3620). Table 2 indicates the results of reporter assays to examine whether our designed ZFPs show such inhibitory activities. Nine zinc finger proteins were tested. Their effect on transcriptional activity of LTR promoter in the absence of a transcriptional repression domain is shown in Table 2, column 7. The target sites bound by LTR-62, LTR-65, LTR-59 partially overlap the binding sites for Spl in the LTR, namely the Spl-I and Spl -IT sites. Some of these zinc fingers inhibit transcriptional activity of LTR promoter in these assays at least 16.6, 8.8, and 6.7 fold. This inhibitory effect was achieved in the absence of any transcriptional repression domain, in contrast to some zinc fmger proteins whose inhibitory effect depended on fusion to a KRAB domain. One of the zinc finger proteins (ZFP-LTR-62) caused at least al 6-fold suppression in the absence of any repression domain. The zinc finger proteins in Table 2 also inhibited transcriptional activity of LTR promoter when fused to a repression domain. We fused the ZFPs shown in Table 2 with Kriippel-associated box (KRAB, Witzgall, R. et al., Proc. Natl. acad. Sci. USA, 1994, 91:4514-4518) of KLD-1 protein of rat (NCBI accession number AAB07673; amino acids, 12- 74): VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASMAGFLFTKPKV ISLLQQGEDPW (SEQ ID NO: 28). The method for cloning of ZFP-KRAB is described in Bae, K.-H. et al, Nature Biotechnol. 2003, 21 :275-280, and uses a combination of PCR amplification and restriction ligation. Column 8 of Table 2 shows the extent of inhibition of LTR promoter activities of HIN-1 subtype B mediated by expression of the ZFP-KRAB fusion proteins. These ZFP fused with transcriptional repression domain KRAB (ZFP-KRAB) caused inhibition about 1.4 to 9.8 fold greater than the corresponding ZFP without KRAB. One of the ZFP-KRAB proteins, ZFP-LTR-59 mediated at least a 58 fold-inhibition of LTR activity. We have investigated whether the zinc fmger proteins that inhibit the LTR of HIV-l subtype B are also effective at inhibiting LTR promoters of two other subtypes of HIV-l, subtypes A and E. Subtype-specific reporter plasmids were produced. Fig. 2 depicts schematics of the LTR promoter region of subtypes A and E of HIN-1 compared with subtype B (Jeeninga, R. E. et al., 2000, J. Virol. 74:3740-3751). Corresponding LTR regions derived from subtype A and E HIV-l were amplified and cloned into pGL3 -basic plasmid (Promega) linearized with Kpnl and Xhol, and the resulting reporter plasmids were designated LTR-A and LTR-E. To examine transcriptional inhibitory activity of the chimeric zinc finger proteins, zinc finger proteins originally designed for LTR-B were assayed with other subtype-specific reporters. Of the exemplary ZFP-KRAB fusion proteins that were tested, ZFP-LTR-53-KRAB that recognize 8 bases out of 10 of Spl-I site showed significant inhibition with LTR-A and LTR-E. The extent of inhibitions was about 2-fold inhibition (Table 3). ZFP-LTR-53, without a KRAB domain, also mediated about 2-fold inhibition with LTR-A and LTR-E (Table 4). The Spl -IT site of subtype B that was highly effective when blocked by ZFP was mutated in subtype A and E with 3 bases and the corresponding ZFP could not bind to the sites properly with subtype A and E, resulting loss of ZFP effect.

Table 3. Fold inhibition of LTR promoter activities of subtype A and subtype E mediated by ZFP-KRAB

Table 4. Fold inhibition of LTR promoter activities of subtype A and subtype E mediated by ZFP without KRAB domains.

These results with different subtypes of HIN-1 indicate that it is possible to produce zinc fmger proteins that are highly specific and recognize only LTRs of a particular subtype and to produce zinc fmger proteins that, while specific for a target sequence in the LTR, are effective for a broader range of subtypes. In some cases, changes to LTR promoter sequences between subtypes can be responsible for loss of inhibitory activity. Agents that are specific for a particular subtype can be used in a method that includes evaluating a patient to determine if they are infected with a virus of a particular subtype. Once the subtype is identified in the patient, then the subtype- specific agent is administered to the patient. On the other hand, high specificity of agents for particular subtypes may be advantageous because it enables the zinc finger protein to specifically target viral transcription without detrimentally affecting the transcription of host cell genes. Many endogenous transcription factors that contribute LTR activity, for example SP1, are critical for the transcription of genes of both HIV and the host cell. Zinc finger proteins designed to recognize boundaries regions of Spl binding sites or sequences between Spl sites may selectively inhibit transcription of the LTR of HIV-l but not genes in the host genome.

Transducible ZFPs can inhibit LTR activity of HIV-l subtype B We investigated whether the ZFPs expressed from E. coli can be efficiently delivered into cells and inhibit HIV-l LTR activity. We used 3 ZFP constructs previously designed to reduce transcription from LTR of HIV-l subtype B. Nucleic acids encoding the ZFPs were inserted into pTDTaT plasmid (see, e.g., Dowdy^' et al. (1999) Science 285: 1569-1572) using a combination of PCR and restriction digestion and ligation. After insertion into the pTAT-ZFP plasmid, a sequence is produced that encodes a polypeptide that includes (from N to C terminus): ATG (start codon), a hexa-histidine tag, the HIN Tat PTD sequence, a nuclear localization signal (ΝLS), an array of zinc finger domains, and a transcriptional regulatory domain (KOX). The KOX is a "KRAB" domain from the human Koxl protein (Zinc fmger protein 10; ΝCBI protein database AAH24182; G 18848329), e.g., amino acids 2-97 of Koxl. Expression of PTD_TAT-ZFPKOX fusion proteins. The expression vector for transducible chimeric ZFPs were transformed into E.coli strain BL21(DE3) pLysS, plated onto LB-agarose containing amphicilin. Single colonies of cells transformed with PTD_TAT- ZFPKOX were picked and inoculated into selection medium. The initial culture was inoculated into 300ml LB selection medium and cultivated until the culture reached to about 0.8 O.D. Then lmM IPTG was added to induce protein expression for about 2 hours at 37°C. The amino acid sequences of expressed proteins are indicated in Fig. 4A and Fig. 4B. Purification of PTD_TAT-ZFPKOX proteins. Purified TAT-ZFP proteins were prepared using Ni-NTA agarose (Qiagen) following manufacturer's instructions. Briefly, the BL21 cells expressing TAT-ZFP proteins were spun down and the cell pellets were lysed with lysis buffer (100 mM NaH₂PO , 10 mM Tris-Cl, 8 M Urea, protease inhibitor cocktail, pH 8.0), sonicated in ice using five cycles of 10 seconds in which sonication for 10 seconds was followed by a 30 second pause (Fischer Scientific 550). The lysates were incubated with Ni- NTA agarose (Qiagen) for 45 minutes and washed twice in wash solution (100 mM NaH₂PO , 10 mM Tris-Cl, 8 M urea, pH 6.3). Protein was eluted in elution buffer (100 mM NaH₂PO₄, 10 mM Tris-Cl, 8 M urea, pH 4.5) and dialyzed into refolding solution (20mM Tris, 1 mM DTT, 100 μM ZnCl, pH 8.0) at 4°C. The proteins were concentrated using CENTRICON™ filtration, quantified with Bradford assay and examined on SDS-PAGE with Commassie blue staining. The purified proteins were stored with 10% glycerol at -70°C. Protein transduction and reporter assay. HeLa cells were maintained and cultivated in RPMI medium containing 10% FBS in a 5% CO₂, 37°C incubator. Reporter assays were as described above for plasmids encoding chimeric ZFPs except for the transfection procedure. A mixture of 20 ng of reporter plasmid (LTR-B), 20 ng of pTAT (transcription activator of HIN-1), and 2 ng of internal control renilla luciferase (pRL-TK) was transfected into lxlO⁴ HeLa cells cultivated in 96-well plate using LIPOFECTAMIΝ 2000™ (Invitrogen). Briefly, Lff OFECTAMLΝE 2000™ reagent was mixed with Opti-MEM medium and incubated at room temperature for 5 minutes. Then the DΝA mixture was added then incubated for 20 minutes. The DΝA LIPOFECTAMIΝE 2000™ complex was added to cells and incubated for one hour at 37°C. The purified protein PTD_TA_T-ZFPKOX was added to desired concentration directly into cell culture and incubated for two hours. The medium was exchanged with fresh growth medium and incubated for 24 hours or 48 hours in a 5% CO₂, 37°C incubator. After incubation for 24 hours or 48 hours, cells were washed twice with PBS then added with 30 μl of passive lysis buffer (Promega) and incubated for 15 minutes. 10 μl of cell lysates thus prepared was used for measurement of luciferase activity, by addition of 50 μl of Luciferase assay substrate and 50 μl of Stop and Glo reagent as recommended by manufacturer. Luciferase activity was analyzed with luminometer. Results. LTR activity was strongly inhibited by addition of transducible chimeric ZFPs, to a similar extent as seen in previous experiments using plasmid transfection to provide nucleic acid encoding the LTR-regulating ZFPs. Since, in the absence of TAT, reporter activity was very low, we co-transfected TAT with reporter gene to facilitate inhibitory effect of PTD_TAT-ZFPKOX protein, hi one series, 200 μg of the purified PTDT_AT-LTR-65KOX was added to culture medium for 2 hours. This protein reduced luciferase activity from LTR-B reporter by about 16.4-fold (a 94% reduction) compared to control PBS treatment, both measured at 24 hours post-transduction (Fig. 5A). This inhibition was maintained to 48 hours at a similar extent by 20.2-fold (a 95% reduction) compared to control (Fig. 5B). The experiment also included a transduction with another control protein, a transducible zinc finger protein that does not interact with LTR sequences (a "non-relevant" ZFP called PTD_TAτ-F435KOX). See Fig. 5A and Fig. 5B. Within the tested range of protein concentrations, LTR activity was not modulated by the "non-relevant" PTD_TAT-ZFPKOX protein. This control demonstrates that the reduction of LTR activity was not due to addition of exogenous proteins, but rather to the specific activity of the PTD_TA_T- LTR-65KOX protein and other LTR binding proteins for the LTR promoter. The inhibitory effect of PTD_TAT-ZFPKOX was dependent on protein concentration. For example, in the case of PTD_TA_T-LTR-65KOX, treatment with 200 μg, 100 μg, 50 μg, and 25 μg of protein for two hours resulted in 16.4-fold, 11.1-fold, 1.9-fold, 1.2-fold inhibition of LTR activity respectively (Fig. 5A). Treatment with the "non-relevant" ZFP (PTD_TAτ- F435KOX), at the same concentrations did not cause inhibition. Detected values for this "non-relevant" control were 0.99-fold, 1.03-fold, 1.0-fold, 1.0-fold respectively. At all tested concentrations, the cells were intact and viable (Fig. 5A). In another example, a chimeric ZFP (PTD_TAT-LTR-62KOX) also showed concentration dependent inhibitory effect on transcriptional activity of LTR promoter (Fig. 5C and Fig. 5D).

All references, patents, and patent applications cited herein are hereby incorporated by reference in their entireties. Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated or recombinant protein comprising a DNA binding domain that includes a plurality of zinc finger domains, wherein (1) the DNA binding domain binds to a target sequence in the long-terminal repeat (LTR) of a human immunodeficiency virus, and (2) the protein can reduce transcription of a viral gene.

2. The protein of claim 1 which can reduce transcription of the viral gene at least 2 fold in the absence of a transcriptional repression domain.

3. The protein of claim 1 wherein the target sequence overlaps with but does not span the binding site of a host cell transcription factor.

4. The protein of claim 1 wherein the DNA binding domain binds to the target sequence with a Kd of less than 10^"8 M.

5. The protein of claim 1 wherein the DNA binding domain has fewer than 90 amino acids.

6. The protein of claim 1 wherein the plurality of zinc finger domains comprise at least two human zinc fmger domains from different human proteins.

7. The protein of claim 1 wherein the protein further comprises a protein transduction domain.

8. The protein of claim 7 wherein the protein transduction domain comprises a sequence from an HTV tat protein, HSV VP22 protein, or Antennapedia homeodomain.

9. The protein of claim 1 wherein the protein further comprises a transcriptional repression domain.

10. The protein of claim 9 wherein the transcription repression domain comprises a Kid or KOX repression domain.

11. The protein of claim 1 wherein the protein does not include a transcriptional repression domain.

12. The protein of claim 1 wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, or 19.

13. The protein of claim 12 wherein the protein comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, or 19.

14. The protein of claim 1 wherein the protein inhibits transcription of the viral gene at least 4 fold.

15. The protein of claim 1 wherein the protein inhibits transcription of the viral gene at least 4 fold in the absence of a KRAB domain.

16. The protein of claim 1 wherein the protein inhibits transcription of the viral gene at least 4 fold in the absence of any transcriptional repression domain.

17. The protein of claim 1 wherein the DNA binding domain comprises three zinc finger domains that comprise DNA contacting residues of sets of motifs shown in Table 2 from N- to C-terminus.

18. The protein of claim 1 wherein at least one of the zinc finger domains is a human zinc finger domain.

19. The protein of claim 1 which can reduce the transcription from a viral promoter in the absence of a transcriptional repression domain.

20. The protein of claim 1 which inhibits transcriptional activity of LTR promoter at least 6 fold, has fewer than six zinc finger domains, does not include a transcriptional repression domain, and/or has fewer than 120 amino acids.

21. The protein of claim 1 wherein the target sequence includes: (i) G at position +42, (ii) G at position -50, (iii) T at position -49, (iv) G at position -48, (v) G at position -47, (vi) C at position -46, (vii) G at position -45, (viii) T at position -25, (ix) A at position -24, and or (x) A at position -23 of the LTR sequence shown in FIG. 1.

22. A protein comprising a DNA binding domain that includes a plurality of zinc finger domains and a protein transduction domain, wherein (1) the DNA binding domain binds to a target sequence in a viral nucleic acid, and (2) the protein can reduce transcription of a viral promoter.

23. The protein of claim 22 wherein the viral promoter is the LTR of a human immunodeficiency virus and the protein can reduce transcriptional activity of LTR promoter.

24. The protein of claim 22 wherein the plurality of zinc fmger domains comprise at least two human zinc finger domains from different human proteins.

25. A pharmaceutical composition for treating or preventing a retroviral disorder mediated by a human immunodeficiency virus, comprising a protein according to any one of claims 1 to 24, and a pharmaceutically acceptable carrier.

26. A pharmaceutical composition for treating or preventing a retroviral disorder mediated by a human immunodeficiency virus, comprising a nucleic acid that can be expressed in a host cell and encodes a protein according to any one of claims 1 to 24, and a pharmaceutically acceptable carrier.

27. A method of treating or preventing a retroviral disorder mediated by a human immunodeficiency virus, comprising providing an agent that can regulate viral gene transcription to cells of a subject in therapeutically effective or prophylactically effective amount, wherein the agent comprises (1) a protein that has (i) a DNA binding domain that binds to a target sequence in a promoter of a human immunodeficiency virus and (ii) a protein transduction domain, or (2) a nucleic acid that encodes a protein comprising a DNA binding domain that includes a plurality of zinc finger domains, wherein (i) the DNA binding domain binds to a target sequence in a promoter of a human immunodeficiency virus, and (ii) the protein can reduce promoter transcription.

28. The method of claim 27 wherein the agent is the protein according to (1), and the

DNA binding domain of the protein includes a plurality of zinc finger domains.

29. The method of claim 28 wherein the protein transduction domain is derived from the HIV Tat protein.

30. The method of claim 27 wherein the DNA binding domain comprises a sequence that is at least 95% identical to SEQ DD NO: 11, 12, 13, 14, 15, 16, 17, 18, or 19.

31. The method of claim 27 wherein the protein can reduce promoter transcription in the absence of a transcriptional repression domain

32. The method of claim 27 wherein the agent is the nucleic acid according to (2).

33. The method of claim 32 wherein the protein encoded by the nucleic acid inhibits promoter transcription at least 4 fold

34. The method of claim 33 wherein the protein encoded by the nucleic acid inhibits promoter transcription at least 4 fold in the absence of any transcriptional repression domain.

35. The method of claim 34 wherein the protein encoded by the nucleic acid inhibits promoter transcription at least 7 fold in the absence of any transcriptional repression domain.

36. The method of claim 27 wherein the target sequence includes: (i) G at position +42, (ii) G at position -50, (iii) T at position -49, (iv) G at position -48, (v) G at position -47, (vi) C at position -46, (vii) G at position -45, (viii) T at position -25, (ix) A at position -24, and/or (x) A at position -23 of the LTR sequence shown in FIG. 1.

37. The method of claim 27 wherein the subject is a human who is infected with a human immunodeficiency virus.

38. An isolated nucleic acid that comprises a sequence encoding the protein according to any one of claims 1 to 24.

39. A vector comprising the nucleic acid of claim 38.