KR20070031854A - Modulating viral transcrption - Google Patents

Abstract

A protein comprising a DNA binding domain comprising a plurality of zinc finger domains can bind to a target sequence in a viral nucleic acid, thereby regulating the transcription of the viral gene. In addition, comprising providing a subject cell with an agent capable of modulating viral gene transcription, wherein the agent comprises (1) a protein having a DNA binding domain and a protein delivery domain or (2) a DNA comprising a plurality of zinc finger proteins Methods for treating or preventing retroviral diseases are described, including nucleic acids encoding proteins comprising a binding domain.

Description

Transcriptional regulation of viruses {MODULATING VIRAL TRANSCRPTION}

AIDS is an important disease worldwide. In 2002, only 60 million people were reported infected with the Human immunodeficiency virus (HIV), one third of which died (Anthony S. Fauci, Nature Med. 9: 839-843 ( 2003)). Therefore, the new treatment will be very useful for AIDS patients.

summary

In one aspect, the invention features a protein (eg, isolated or recombinant protein) having a DNA binding domain comprising a plurality of zinc finger domains. The protein may bind to a target sequence in the nucleic acid of the virus, such as a promoter of the virus, such as a retrovirus such as HIV, such as HIV-1 such as HIV-1 subtypes B, A and / or E. Can bind to LTR. The protein can reduce transcription (eg, activity of the LTR promoter) in the absence of a transcriptional repression domain such as, for example, KRAB. For example, the protein may alter transcription (eg, activity of the LTR promoter, in particular transcription activated by TAT) in the absence of any transcription inhibitory domain in reporter assays 4, 5, 7, 8, 10, 12, 15, Or 16 times or more. In addition, when the protein is used in fusion with a transcription repression domain, such as a KRAB domain, the protein may be capable of 10, 15, 20, 25, 30, 40, or It can be reduced by 50 times or more.

In one embodiment, the DNA binding domain comprises three zinc finger domains, said zinc finger domains of the DNA contact residues of the motif group shown in Table 2 from the N-terminus to the C-terminus, or of the DNA binding domains described herein. At least 8, 9, 10 or 11 residues out of the 12 DNA contact residues of the three zinc finger domain sets.

In many embodiments, the DNA binding domains are non-natural domains or comprise two or three or more zinc finger domains derived from proteins of two or more different mammals (eg, humans).

In one embodiment, the DNA binding domain binds to a target sequence within the LTR of HIV such as subtype B, A or E. The DNA binding domain binds to a sequence in the LTR of either subtype compared to other subtypes, for example, can bind at least 2, 5, or 10 times more friendly. In other cases, the protein is effective to modulate (eg, inhibit) the transcriptional activity of LTR against a plurality of viral subtypes. For example, at least two or four times as effective for at least two subtypes. In one embodiment, the DNA binding domain binds to a target sequence that includes or overlaps one of the target sequences shown in Table 2. In one embodiment, the DNA binding domain reduces the affinity of a virus or host cell transcription factor for a binding site in a viral gene by binding to a target sequence within the viral gene, while the same for binding to a binding site in a cellular gene. Does not affect the degree. For example, the inhibitory effect on the viral gene is 2, 5, or 10 times or more.

In one embodiment, the target sequence comprises one or more of the following nucleotides: (i) G at position +42, G at position -50, (iii) T at position -49 of the LTR sequence shown in FIG. , (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.

The protein may comprise a protein transduction domain (PTD), such as a TAT derived PTD, or another PTD, such as another PTD described herein.

The protein may comprise a nuclear localization signal (hereinafter referred to as NLS) such as an NLS derived from a human protein such as the NLS described herein. Exemplary NLS sequences include: MyoD-NLS: CKRKTTNADRRKA (SEQ ID NO: 1); ppoI-NLS: KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 2); rrg1-NLS: DRNKKKKE (SEQ ID NO: 3), or fragments of these sequences that function to transport the polypeptide to the nucleus.

In one embodiment, the zinc finger protein comprises one or more sequences that stabilize proteins, sequences that increase protein expression, and / or sequences that facilitate protein purification. Such sequences may be on the N- or C-terminal side of the DNA binding domain, such as the N- or C-terminal end of the protein, between PTD and NLS, or between PTD and DNA binding domains. In one embodiment, the zinc finger protein does not comprise any foreign sequence.

A protein may 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 protein without other proteins of the same origin.

In one embodiment, the invention features a nucleic acid comprising a coding sequence encoding a protein described herein. The nucleic acid may further comprise a regulatory sequence, such as a promoter linked to the coding sequence to be operable. The coding sequence may be interrupted by one or more introns. The invention also features a method of making a protein comprising expressing the nucleic acid in a host cell and a cell such as a prokaryotic or eukaryotic cell, including the nucleic acid (eg as an integrated form or vector). The invention also provides for transgenic animals comprising one or more cells expressing a protein described herein or one or more cells expressing a protein described herein, such as transplanted or implanted cells. It is characterized by modified non-human animals, such as including animals. Methods of making transgenic animals, implants and media are known.

In another aspect, the invention provides a pharmaceutical composition comprising a medicament described herein (eg, a protein comprising PTD, or an expressible nucleic acid encoding a protein described herein) and a pharmaceutically acceptable carrier. It features.

In another aspect, the invention features a method of treating or preventing a retroviral disease, such as a disease mediated by HIV. The method includes an agent capable of controlling transcription of viral genes by treating a cell of a subject with a therapeutically or prophylactically effective amount. In one embodiment, the medicament comprises a protein having (i) a DNA binding domain that binds to a target sequence in the LTR of HIV, and (ii) a PTD. The protein may comprise one or more features described herein.

In other embodiments, the medicament comprises a nucleic acid encoding a protein comprising a DNA binding domain comprising a plurality of zinc finger domains. The DNA binding domain binds to a target sequence in the LTR of HIV. In some cases, the protein may reduce the transcriptional activity of LTR in the absence of a transcriptional repression domain. The protein may comprise one or more features described herein.

Treatment may include evaluating the subject, eg, determining whether the subject has been infected with a retrovirus, such as a particular subtype or designated retrovirus. The method may also include selecting a medicament based on the measurement results, eg, to provide a medicament that targets the appropriate subtype or other designation of the infectious virus.

In another aspect, the invention features a protein, eg, an isolated or recombinant protein, comprising, for example, a human NLS that is physically linked to an amino acid sequence that is not normally linked to a human NLS. For example, human NLS can be fused with other proteins or fragments thereof, such as other human or artificial proteins such as nucleic acid binding proteins comprising one or more human domains. For example, human NLS is fused with a chimeric zinc finger protein such as a chimeric protein comprising one or more human zinc finger proteins. Exemplary human NLS sequences include the following sequences: MyoD-NLS (SEQ ID NO: 1), ppoI-NLS (SEQ ID NO: 2), rrg1-NLS (SEQ ID NO: 3), or polypeptides into the nucleus A fragment of said sequences that carries. For example, such NLS sequences may be fused to a human MyoD protein or a protein that is not part of it, a poly (ADP-ribose) polymerase or a protein that is not part of it, or a protein that is not part of the human retinoic acid receptor or part thereof. Can be. The invention also includes a cell (eg, recombinant host cells) comprising a nucleic acid encoding such a protein and a vector comprising said nucleic acid.

1 shows the nucleic acid sequence (SEQ ID NO: 4) of the LTR promoter sequence found in HIV-1 subtype B used in the reporter construct. Also described are target sites of chimeric zinc finger proteins that recognize the binding sites of the transcription factors SP1 and NF-kB and the LTR region. The sign of +1 indicates the transcription start site.

FIG. 2 shows the LTR promoter nucleic acid sequences of HIV-1 subtype A (SEQ ID NO: 5) and subtype E (SEQ ID NO: 6) of the LTR promoter nucleic acids of HIV-1 subtype B (SEQ ID NOs: 1 to 208). In comparison with).

3 is a structural diagram of a plasmid encoding a PTD _TAT -ZFPKOX fusion protein.

Figure 4 shows the amino acid sequence of (a) PTD _TAT -LTR-65KOX (SEQ ID NO: 7), and (b) PTD _TAT -LTR-62KOX (SEQ ID NO: 8). Such proteins include the hexa-histidine sequence (box portion) for purification, and the sequences of Tat-peptide (box, bold portion), HA-NLS-ZFP (bold) and KOX domain (underlined) (SEQ ID NO: 23 ). Other configurations may be used, such as removing foreign sequences or including other linker sequences.

5A shows inhibition of LTR promoter activity by Tat-LTR-65-KOX protein, measured 24 hours after transfection. The activity of Firefly luciferase was normalized to the internal control Renilla luciferase (left). LTR-activity was expressed as inhibition-fold compared to the control (right).

Figure 5B shows inhibition of LTR promoter activity by Tat-LTR-65-KOX protein measured 48 hours after transformation.

5C shows inhibition of LTR promoter activity by Tat-LTR-62-KOX protein measured 24 hours after transfection.

5D shows inhibition of LTR promoter activity by Tat-LTR-62-KOX protein measured 48 hours after transformation.

Replication of retroviruses is governed by transcription from long terminal repeats (LTRs). Exemplary LTR sequences of HIV-1 are set forth in SEQ ID NO: 1. The HIV-1 LTR includes a binding site of viral transcription factor TAT and host transcription factors NF-kB and Sp1, and is also where several nucleotide sequence mutations have been reported between HIV-1 subtypes.

The transcriptional activity of the LTR can be modulated using artificial transcription factors such as zinc finger proteins comprising one or more zinc finger domains such as three zinc finger domains. Zinc finger proteins may bind to target sequences in the LTR, such as sequences having a length between 8 and 15 base pairs. For example, the target sequence includes some or all of one of six sequences in Table 2. The target sequence includes some or all of the binding site for viral transcription factors or host cell transcription factors such as Sp1 or NF-kB. In one embodiment, the target sequence comprises two adjacent transcription factor binding site regions, eg, two Sp1 target sites, such as Sp1-I and Sp1-II sites or Sp1-II and Sp1-III sites. In one embodiment, the zinc finger protein inhibits Sp1 from binding to one or more sites in the LTR of the virus, but binds to a site within an endogenous gene, such as to bind a cognate site within the p21 (WAF1 / CIP1) gene. It is not suppressed to the same extent. For example, an exemplary zinc finger protein inhibits Sp1 binding to LTR at least 2, 5, 10 or 50 times more than binding to cognate sites in the p21 (WAF1 / CIP1) gene.

Exemplary zinc finger domains include human zinc finger domains, and mutated domains derived from an artificial zinc finger domain such as a native zinc finger domain. In some embodiments, the zinc finger protein comprises a human framework, at least one, two, or three human domains or all human domains. Proteins with human inherent properties can minimize the possibility of immune rejection responses to the proteins. In some cases, the proteins are produced intracellularly, such that they are non-immunized, such as by modifying T cell epitopes to be minimal or absent.

Zinc Finger Domains

Zinc fingers are about 30 amino acid residues, four of which are cysteine or histidine, and are small polypeptides that are appropriately arranged to coordinate with zinc ions (Wolfe et al . , Annu. Rev. Biophys. Biomol.Struct . 3: 183-212 (1999)). Zinc finger domains, depending on the type of residue and zinc ion coordination bonding, for example, be categorized as Cys ₂ -His ₂ stream, Cys ₂ -Cys ₂ stream, Cys ₂ -CysHis and the like. The residues that coordinate with zinc in the Cys ₂ -His ₂ zinc finger are usually arranged as follows:

X _a -XCX _{2 -5} -CX ₃ -X _a -X ₅ -Ψ-X ₂ -HX _{3 -5} -H (SEQ ID NO: 9)

Wherein “Ψ (psi)” is a hydrophobic moiety. "X" represents any amino acid and "X _a " typically, but not always, typically represents phenylalanine or tyrosine, the subscript indicates the number of amino acids, and the two subscripted hyphens indicate a typical range of intervening amino acids. Point to. Although anti parallel β-sheets may be short, non-ideal and non-existent, typically intervening amino acids are folded to form antiparallel β-wrinkles that are charged against the α-helix. Due to the folding, the side chain coordinating with zinc is arranged to have a tetrahedral structure suitable for coordinating with zinc ions. Base contact residues are in the N-terminal and leading loop region of the finger.

For convenience, the major DNA contact residues of the zinc finger domain were numbered -1, 2, 3 and 6, as shown in the following example.

As noted in the above examples, the DNA contact residues are arginine (R), aspartic acid (D), glutamine (E) and arginine (R). The motif may be abbreviated as RDER. Such abbreviated notation as used herein refers to a specific poly from two preceding residues of the first cysteine (the beginning residue of SEQ ID NO: 10) to the last metal-chelating histidine (SEQ ID NO: 10). It is a compressed form that refers to a peptide sequence. In these and other motifs, X _a is usually, but not always, phenylalanine or tyrosine, and X _b is often hydrophobic. The lowercase letter “m” before the motif can be used to clarify that the abbreviation refers to a motif. For example, mRDE refers to a motif in which R appears at the -1 position, D at the 2 position, E at the 3 position, and R at the 6 position.

Zinc finger proteins typically comprise a DNA binding domain comprising a tandem arrangement 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 total zinc finger protein is less than 120, 110, 100, 95, 90, or 85 amino acids.

In an exemplary DNA binding domain, the zinc finger domains in which the motif is described continuously are not interspersed with other folded domains, but linkers such as the elastic linker described herein (SEQ ID NO: 22) between domains. It may include. For embodiments comprising a particular zinc finger protein described herein or an array thereof, the present invention provides a corresponding array having zinc fingers having the same DNA contact residues as the particular zinc finger protein or array thereof. Related embodiments include zinc finger proteins or arrays thereof. The corresponding zinc finger protein may differ by at least 1, 2, 3, 4 or 5 amino acids from the particular zinc finger protein described, eg, the sites of amino acids that are not DNA contact residues may differ. Other related embodiments include corresponding proteins with at least one, two or three zinc fingers, wherein the zinc fingers have the same DNA contact residues, eg, in the same order.

A number of exemplary human zinc finger domains are described in WO 01/60970, WO 03/016571, and US 2003-0165997. Zinc finger proteins can be designed to contain transcriptional regulatory domains. Exemplary transcriptional repression domains include the repression domains of KRAB, Kid, UME6, ORANGE, groucho, and WRPW. See, eg, Dawson et al . , Mol. Cell Biol. 15: 6923-31 (1995) and US 2004-0209277.

Exemplary zinc finger proteins have a dissociation constant of less than 10 ⁻⁷ , 10 ⁻⁸ , 10 ⁻⁹ , or 10 ⁻¹⁰ M to a target sequence in a retroviral LTR such as an HIV-1 or HIV-2 LTR. Can be combined. Dissociation constants are 20 mM Tris pH 7.7, 120 mM NaCl, 5 mM MgCl ₂ , 20 μM ZnSO ₄ , 10% Glycerol, 0.1% Nonidet P-40, 5 mM DTT, and 0.10 mg / mL BSA (bovine serum albumin), and gel shift analysis using purified proteins that are allowed to bind at room temperature. Further details are provided in the Examples below and in Rebar and Pabo, Science 263: 671-673 (1994).

Identification of Zinc Finger Proteins

Artificial zinc finger proteins can be identified by a variety of methods. For example, artificial DNA-binding proteins can be constructed such that target sequences are recognized by zinc finger domains characterized by a mixing and matching method. See, eg, WO 01/60970. Zinc finger domains can be separated and analyzed by various methods. US 2002-0061512 describes a method for evaluating domains using a yeast analysis system. Another method of making artificial DNA-binding proteins involves using phage display to select zinc finger domains with modified DNA-binding specificities (US Pat. No. 6,410,248).

 Domains that interact with the target sequence can be selected and used to prepare DNA binding proteins that bind to the target sequence. WO 01/60970 and WO 03/016571 also describe methods of designing DNA-binding proteins. The assembly structures of the zinc finger domains facilitate the rearrangement of the domains to prepare new DNA-binding proteins. Zinc finger domains in the native Zif268 protein are located in series along the DNA double helix. Each domain independently recognizes 3 to 4 base pairs of DNA portions. By linking three or more zinc finger domains, one can construct a DNA binding protein that specifically recognizes a DNA sequence of 9-base pairs or longer.

Bae et al. (Bae KH et al . , Nat Biotechnol. 21 (3): 275-80 (2003)) describe a novel method for assessing the specificity of DNA-binding domains in cells and using information derived from such intracellular assays. A method of making a DNA-binding protein is described.

To identify polypeptides comprising functional DNA binding domains that produce a desired phenotypic effect, nucleic acid libraries encoding different combinations of zinc finger domains may be screened. US 2003-0194727 describes an exemplary method for identifying useful zinc finger proteins by screening or screening. Generally, libraries of nucleic acids encoding polypeptides comprising different combinations of zinc finger domains and effector domains are prepared and introduced into cells. After expressing the library members, cells that exhibit a modified phenotype are isolated as compared to control cells (eg, untransformed cells or cells transformed with vector nucleic acids). Library nucleic acid in the cell is recovered and analyzed to identify the appropriate zinc finger protein. The method can be used to identify zinc finger proteins that directly or indirectly modulate the transcriptional activity of LTR.

Exemplary Zinc Finger Protein

The amino acid sequence of the DNA binding domain of an exemplary zinc finger protein that binds to HIV LTR is shown in Table 1 below.

The proteins may comprise additional sequences. For example, the following sequence, including a hemagglutinin tag and a nuclear localization signal, may be located at the N-terminus: YPYDVPDYAELPPKKKRKVGIRIPGEK (SEQ ID NO: 20).

In one embodiment, the proteins additionally comprise a transcriptional repression domain such as the KOX transcriptional repression domain, the "KRAB" domain (human finger protein 10; NCBI protein database AAH24182; GI: 18848329) of the human Kox1 protein, ie 2 to 97 of Kox1. amino acid:

DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 23).

Other useful proteins include proteins comprising: (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) DNA binding domains differing from at least one, but less than 10, 8, 6, 5, 4, 3, or 2 amino acids provided in one of the DNA binding domains provided in Table 1 (eg, the differences include insertion, deletion, and Substitutions such as conservative substitutions); And (iii) a DNA binding domain encoded by a nucleic acid hybridized (eg under very stringent conditions) to a nucleic acid encoding one of the DNA binding domains provided in Table 1.

The identity percent between two amino acid or nucleotide sequences is determined by Meyer and Miller's algorithm introduced in the alignment program (ALIGN version 2.0) (E. Meyers and W. Miller, CABIOS , 4: 11-17 (1989)). ] And PAM120 key residue table, gap length penalty 12 and gap penalty 4.

Conservative amino acid substitution refers to the possibility of interchange of residues with similar side chains. For example, amino acid groups having aliphatic side chains include glycine, alanine, valine, leucine, and isoleucine; Amino acid groups having aliphatic-hydroxyl side chains include serine and threonine; Amino acid groups having amide-containing side chains include asparagine and glutamine; Amino acid groups having aromatic side chains include phenylalanine, tyrosine and tryptophan; Amino acid groups having basic side chains include lysine, arginine and histidine; Amino acid groups having acidic side chains include aspartic acid and glutamic acid; And the amino acid groups having sulfur-containing side chains are cysteine and methionine.

As used herein, the term “hybridization under stringent conditions” refers to conditions that hybridize in 6 × sodium chloride / sodium citrate (SSC) at 45 ° C. and then wash twice with 0.2 × SSC, 0.1% SDS at 65 ° C. .

The zinc finger protein is also characterized by comprising one or more zinc finger domains having a set of residues identical to the DNA contact residues of one or more zinc fingers of the protein of Table 1. Exemplary proteins include the following motifs from the N terminus to the C terminus: 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 domain (PTD)

A "protein transfer domain" or "PTD" is an amino acid sequence that can cross a biological membrane, in particular a cell membrane. When PTD is attached to a heterologous polypeptide, it can enhance the migration of the heterologous polypeptide through the biological membrane. PTD is typically covalently linked (eg, by peptide bonds) to a heterologous DNA binding domain, such as a DNA binding domain capable of specifically binding to the LTR of a virus. For example, the PTD and the heterologous DNA binding domain can be encoded by a single nucleic acid, eg, in a common open reading frame or in one or more exons of a common gene. Exemplary PTDs may comprise between 10 and 30 amino acids and may form an amphipathic helix. Many PTDs have basic properties. For example, a basic PTD has 4, 5, 6, or 8 or more basic residues (eg arginine or lysine). PTDs can facilitate migration of polypeptides into eukaryotic cells, eg, mammalian cells such as vertebrate cells, such as humans, apes, mice, cattle, horses, cats.

PTDs can be linked to artificial transcription factors using, for example, a flexible linker. The flexible linker may comprise one or more glycine residues to confer free rotation. For example, the PTD can be spaced at least 10, 20, or 50 amino acids from the DNA binding domain of the transcription factor. PTD may be located at the N- or C-terminus relative to the DNA binding domain. Positioning N- or C-terminus for a particular domain does not require adjacent to that particular domain. For example, the PTD at the N-terminus relative to the DNA binding domain can be separated from the DNA binding domain by spacers and / or other types of domains.

The artificial transcription factor may also comprise a plurality of PTDs, eg, a plurality of different PTDs or two copies of the same PTD.

Exemplary PTDs include fragments derived from antennapedia protein, herpes simplex virus VP22 protein and HIV TAT protein. See, eg, WO2004-108883. The minimum Tat PTD contains between 47 and 57 residues of the human immunodeficiency virus Tat protein: YGRKKRRQRRR (SEQ ID NO: 21). This peptide sequence is referred to herein as "TAT-PTD". This peptide can mediate the introduction of at least 100 kDa of heterologous peptides and proteins into mammalian cells in vitro and in vivo (Ho et al., Cancer Res 61 (2): 474-7 (2001)). Schwarze et al., Including the brain where the fusion protein has been considered difficult due to blood-brain-barrier when 120 kDa β-galactosidase protein fused with TAT is injected intraperitoneally into mice. It has been found in all kinds of cells and tissues (Schwarze et al., Science 285 (5433): 1466-7 (1999)). The region of Tat used includes at least a PTD region but usually does not contain enough sequence to produce a Tat protein capable of activating LTR. For example, only residues in TAT-PTD, ie SEQ ID NO: 21 (above), are used.

In one embodiment, the PTD is obtained from a protein of human or other mammal. Exemplary mammalian PTDs are described in WO 03/059940 (human SIM-2) and WO 03/059941 (Mph).

Cell specific PTD. Some PTDs have specificity for specific cell types or conditions. US Publication Nos. 2002-0102265 and 6,451,527 describe exemplary methods for separating cell specific PTDs, such as using phage display. Exemplary cellular uptake signals include amino acid sequences specifically recognized by cell receptors or other surface proteins such as CD4 or CD8. Interactions between cell uptake signals and cells can also lead to internalization of transcription factors, including cell uptake signals.

Assay for Protein Delivery. Multiple assays can be used to determine which amino acid sequences can function as PTDs. For example, the amino acid sequence can be fused with a reporter protein such as β-galactosidase to form a fusion protein. This fusion protein is contacted with cultured cells. After washing the cells, assay for reporter activity. Another analysis detects the presence of a fusion protein comprising the amino acid sequence in question and other detectable sequences such as epitope labels. This fusion protein is contacted with cultured cells. After washing the cells, they are analyzed using western blot or immunofluorescence to detect the presence of detectable sequences in the cells. For example, WO 2004-108883 further describes protein delivery analysis and use.

Specific proteins described herein can include PTDs to form deliverable transcription factors, such as for treating or preventing HIV that can be used in therapy. In addition, one or more of the following motifs (eg, an array of zinc finger domains according to the series of motifs below) to form a transferable transcription factor, such as for treating or preventing HIV, which may be used in therapy. A protein comprising can be physically bound to PTD: 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.

Deliverable zinc finger proteins can facilitate treatment, such as the treatment of HIV-1 replication from infected cells, since the primary target cells for HIV are lymphocytes that circulate in the blood. Therapeutic proteins administered to the circulatory system (eg, blood) can be an effective and fast way to deliver the protein to the target.

cure

Agents capable of modulating the transcription of viral genes may be provided to a cell of a subject, eg, in a therapeutically effective amount or prophylactically effective amount, for the treatment or prevention of retroviral diseases, in particular various diseases such as AIDS and AIDS related complexes. Can be. The agent may be targeted against T lymphocytes (eg, CD4 + and / or CD8 + cells) or virus infected T lymphocytes. Exemplary agents include protein-based and nucleic acid-based drugs. One example of such a protein-based drug is a deliverable protein, such as a deliverable zinc finger protein. One example of such a nucleic acid-based drug is a nucleic acid encoding a zinc finger protein that modulates the transcriptional activity of LTR (eg, a nucleic acid wrapped in a carrier for delivery into a cell).

The amount of medicament, or “therapeutically effective amount,” effective for treating a disease is determined by a single or repeated administration to a subject to reduce one or more activities that contribute to the disease, such as to reduce the production of the virus. The reduction may include the activity of cells or tissues (eg, metastatic tissues) or the numerical reduction of viral particles produced, such as statistically significant reductions. The amount of an effective agent, or “prophylactically effective amount” of protein, refers to the amount of agent that is effective through a single or repeated administration to a subject to prevent or delay the onset or recurrence of a disease, such as a retroviral disease. .

The subject may be a human or other mammal, such as a non-human primate. Subjects infected with retroviruses (eg AIDS) or at risk of infection can be identified by standard methods, including immunoassay and PCR-based testing. The subject may be any one of several stages of HIV infection progression, for example, the stage may be acute primary infection syndrome; it may show no symptoms or may cause high fever, malaise, diarrhea and headache. Can be associated with influenza-like diseases with neurological symptoms such as), asymptomatic infection (long-term incubation with a gradual decrease in circulating CD4 + T cell numbers), and AIDS (which are more severe AIDS-defined diseases and / or Circulating CD4 cell number is defined as being reduced below the level required for effective immune function). In addition, treating or preventing HIV infection may include suspicions of past exposure to HIV, such as contact with HIV-contaminated blood, transfusion, exchange of fluids, unsafe sex with the infected person, accidental needle injury, tattoos with contaminated instruments, or Treatment of suspected HIV infection following the procedure of acupuncture, or viral transmission from mother to fetus during pregnancy, childbirth, or immediately after.

Treatment of HIV infection includes the prevention or treatment of AIDS-related signs of such a person, along with the treatment of an HIV family member of any retrovirus or person diagnosed with an active AIDS infection. HIV holders can be determined by any method known in the art. For example, a person may be identified as an HIV bearer based on an anti-HIV antibody positive or HIV positive or symptoms of AIDS.

In one embodiment, the present invention relates to retroviral diseases such as immunodeficiency viruses such as HIV, such as HIV I, HIV II, HIV III (also known as HTLV-II, LAV-1, LAV-2) and analogs. It provides a way to treat or prevent the disease. The term "HIV" includes all strains, forms, subtypes, clades and variants of the HIV family.

The methods of the present invention can include administering to a subject an agent that provides a zinc finger protein that modulates the transcriptional activity of the LTR promoter in an amount sufficient to treat or prevent retroviral disease. The agent can be a zinc finger protein itself, such as a protein delivery domain, or a nucleic acid encoding a zinc finger protein. For example, the disease may be due to virally infected cells such as virally infected T-cells. The methods of the present invention can be used to treat or prevent AIDS. The methods of the invention can also be carried out ex vivo, for example, by contacting the cells in an amount sufficient to transport the agent to one or more virally infected cells.

The agent may be administered by any suitable route (locally or systemically). For example, intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (eg, injection or infusion) is possible.

The medicament may, for example, be formulated into a pharmaceutical composition. Many methods for the preparation of such formulations are patented or generally known. See, eg, Sustained and Controlled Release Drug Delivery Systems , JR Robinson, ed., Marcel Dekker, Inc., New York, (1978).

For example, the medicament may be associated with at least one syndrome or HIV related to AIDS, or a syndrome or symptom defined by HIV infection, such as AIDS-related complex (ARC), progressive systemic lymphadenopathy ( progressive generalized lymphadenopathy (PGL), anti-HIV antibody positive symptoms, and HIV-positive symptoms, AIDS-related neurological symptoms (such as dementia or tropical paraparesis), Kaposi's sarcoma, thrombocytopenia (thrombocytopenia purpurea) and pneumocystis carinii pneumonia, Mycobacterial tuberculosis, esophageal candidiasis, toxoplasmosis of the brain, cytomegalovirus retinitis (CMV retinitis) Effective in improving related opportunistic infections such as brain diseases, HIV-related wasting syndrome, etc. To be administered.

In some embodiments, an effective amount of an agent that modulates the activity of LTR comprises at least about 10%, at least about 10%, transcriptional activity of the LTR promoter in infected cells (eg, in cells infected by an immunodeficiency virus) relative to the non-administration of the agent. An amount effective to reduce 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.

In some embodiments, the effective amount of the agent that modulates the transcriptional activity of the LTR promoter is such that the viral load in the individual is at least about 10%, at least about 20%, at least about 30%, at least about 40% relative to the non-administration of the agent. , At least about 50%, at least about 60%, at least about 70%, at least about 80%, or more.

In some embodiments, the therapeutically effective amount of a medicament that modulates the transcriptional activity of the LTR promoter may comprise an amount of detection of RNA molecules of the virus in a plasma sample taken from a subject of about 6000 RNA molecules / ml or less, about 4500 RNA molecules / ml or less, To about 3500 RNA molecules / ml or less, about 2500 RNA molecules / mL or less, or about 1500 RNA molecules / mL or less.

Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, it can be administered in a single tablet, divided into several doses, or reduced or increased at a constant rate depending on the urgency of the therapeutic situation. Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and consistency of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages to the subjects to be treated; Each unit contains a quantity of active compound and the necessary pharmaceutical carrier, calculated to produce the desired therapeutic effect. The dosage unit form design of the present invention is directly dependent on the inherent limitations of the art of incorporating such active ingredients for the treatment of (a) the inherent properties of the active compound and the particular therapeutic effect to be achieved, and (b) the individual sensitivity. have.

Exemplary non-limiting ranges of therapeutically or prophylactically effective protein or nucleic acid agents are from 0.1 to 20 mg / kg, preferably from 1 to 10 mg / kg or from 0.1 to 1 mg / kg. It should be noted that the value of the dosage may vary depending on the type and severity of the disease to be alleviated.

Formulations such as formulation as pharmaceutical compositions and deliverable zinc finger protein administration are described, for example, in WO 2004-108883. Appropriate 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 the formulation of a medicament described herein include: any or all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and physiologically compatible Analogs that can be. Preferably, the carrier is suitable for administration (eg, injection or infusion) intravenously, intramuscularly, subcutaneously, parenteral, spinal, or epithelium. Depending on the route of administration, the medicament may be coated with a material to protect it from the action of acids or other natural environments that may inactivate the medicament. "Pharmaceutically acceptable salts" refer to salts that do not have any undesirable toxic effects while maintaining the desired biological activity of the agent (Berge, SM, et al . , J. Pharm. Sci. 66: 1-19 ( 1977). Examples of such salts include acid addition salts and base addition salts. Acid addition salts are aliphatic mono- and dicarboxylic acids, phenyl substituted alkanoic acid, hydroxy alkanoic acid, aromatic acids as well as non-toxic inorganic acids derived from hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, trivalent phosphoric acid, and the like. Salts derived from non-toxic organic acids such as aliphatic and aromatic sulfonic acids and the like. Base addition salts are derived from alkaline earth metals such as sodium, potassium, magnesium and calcium, as well as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine and ethylenedia And those derived from non-toxic organic amines such as min, procaine and the like.

Agents that modulate the transcriptional activity of the LTR promoter (eg, a zinc finger protein or a nucleic acid encoding a deliverable zinc finger protein) that modulate the transcriptional activity of the LTR promoter may be administered alone or may be retroviral infections such as AIDS or ARC. It can be administered in combination with one or more existing ways to treat. For example, the medicament may be administered in combination with other anti-viral medicaments or agents that treat related diseases. As used herein, the term combination means that different agents are given at substantially the same time, either simultaneously or sequentially. If given sequentially above, the first agent at the start of the second agent administration will preferably be detected at a still effective concentration at the treatment site.

Examples of other agents that can be used to treat viral and / or related diseases include: acyclovir, amantadine, rimantadine, recombinant soluble CD4; rsCD4 , Fusion inhibitors (eg, T20 peptides, T-1249 peptides; Trimeris); Anti-CD4 antibodies; Anti-CCR5 antibodies (eg, Pro 140); CXCR4 blockers (eg, AMD 3100); HIV input inhibitors (eg, Pro-542; Progenics); CCR5 blockers (eg, SCH-C, SCH-D; Schering Plow); Anti-receptor antibodies (eg, antibodies against rhinoviruses), nevirapine (Viramune ^® ), emiravine (Coactinon ^® ), cidofovir (Vistide ^TM ), trisodium Trisodium phosphonoformate (Foscarnet ^™ ), famcyclovir, pencyclovir, valcyclovir, nucleic acid / cloning inhibitor, interferon, zidovudine (AZT, Retrovir ^™ ), Didanosine (dideoxyinosine, ddI, Videx ^TM ), stavudine (d4T, Zerit ^TM ), zalcitabine (dideoxycytosine, ddC, Hivid ^TM ), nevirapine (nerampine ^TM ) ), Lamivudine (Epivir ^TM , 3TC), protease inhibitors, saquinavir (Invirase ^TM , Fortovase ^TM ), ritonavir (Norvir ^TM ), nelfinavir (Viracept ^TM), efavirenz (efavirenz) (Sustiva ^TM), Abba cover (abacavir) to (Ziage n ^TM ), amprenavir (Agenerase ^TM ) indinavir (Crixivan ^TM ), ganciclovir, AzDU, delavirdine (Rescriptor ^TM ), kaletra, trigeber (trizivir), rifampin, clarithromycin, erythropoietin, colony stimulating factors (G-CSF and GM-CSF), non-nucleoside inverting transcriptase Inhibitors, nucleoside reverse transcriptase inhibitors, HIV protease inhibitors, adriamycin, beta-lactam antibiotics, tetratracyclines, chloramphenicol, neomycin, gramici Gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone, beta metazone ( betamethason e), dexamethasone, dexamethasone, fluocortolone, prednisolone, triamcinolone, indomethacin, sulindac, uorouracil, methotrexate ), Asparaginase and combinations thereof.

Examples of nucleoside reverse transcriptase inhibitors (RTI's) include AZT, ddI, 3TC, ddC, d4T, and abacavir. Examples of protease inhibitors include indinavir, saquinavir , ritonavir, nelfinavir, amprevanir, and lopinavir. Examples of non-nucleoside reverse transcriptase inhibitors include nevirapine, delavirdine, emiravine, and efavirenz. Exemplary anti-viral agents other than these include fusion inhibitors (eg, T20, T-1249); And / or CCR5 blockers (eg, SCH-C, SCH-D).

The zinc finger protein used in the combination therapy may comprise one or more zinc finger domains having DNA contact 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; mVSTR-mQSSR-mQSNK; mRSNR-mQSHV-mDSCR; 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.

Nucleic Acid Transport

DNA molecules encoding zinc finger proteins can be inserted into various DNA constructs and vectors for gene therapy purposes. As described herein, "vector" is a nucleic acid molecule capable of transporting another nucleic acid to a covalently linked site. Vectors include plasmids, cosmids, artificial chromosomes, viral elements, and RNA vectors (eg, based on the RNA viral genome). The vector can be replicated in the host cell or integrated into the host's DNA. Viral vectors include, for example, replication defective retroviruses, adenoviruses and adeno-associated viruses, and the like.

Gene therapy vectors are vectors designed for administration to a subject, such as a mammal, such that a cell of the subject can express a therapeutic gene contained in the vector. Gene therapy vectors can include regulatory elements such as 5 'regulatory elements, enhancers, promoters, 5' untranslated regions, signal sequences, 3 'untranslated regions, polyadenylation sites, and 3' regulatory regions. For example, a 5 'regulatory factor, enhancer or promoter can modulate the transcription of the DNA encoding the zinc finger protein. The regulation may be tissue specificity. For example, the regulation may involve transcription of a gene of interest in brain cells, such as cortical neurons or glia cells; Hematopoietic cells, such as T lymphocytes (eg, CD4 + and / or CD8 + T cells); Or endothelial cells. In addition, regulatory factors may include a response to foreign drugs such as steroids, tetracyclines, and the like. Therefore, the extent and timing of expression of therapeutic zinc finger proteins (eg, polypeptides that regulate the transcriptional activity of the LTR promoter) can be controlled.

Gene therapy vectors can be prepared for delivery, as nucleic acids themselves, as components of active or inactive viruses, or as components of liposomes or other delivery carriers. See, for example, US Pat. Nos. 2003-0143266 and 2002-0150626. In one embodiment, the nucleic acid is formulated in a lipid-protein-sugar matrix to form microparticles such as particles having a diameter between 50 nm and 10 μm. The particles can be prepared using any known lipid (eg, dipalmitoylphosphatidylcholine (DPPC), protein (eg, albumin), or sugar (eg, lactose)).

The gene therapy vectors can be delivered using a viral system. Vectors of exemplary viruses include vectors from retroviruses such as Moloney retrovirus, adenovirus, adeno-associated virus, and lentiviruses such as Herpes simplex viruses (HSV). Include them. For example, the HSV is potentially useful for infecting nervous system cells. See, for example, US Pat. Nos. 2003-0147854, 2002-0090716, 2003-0039636, 2002-0068362, and 2003-0104626. Gene delivery agents, such as viral vectors, can be prepared from recombinant cells that produce gene delivery systems.

Such gene therapy vectors can be used, for example, by intravenous injection, topical administration (see US Pat. No. 5,328,470) or by stereotactic injection, such as, eg, Chen et al . , Proc. Natl. Acad. Sci. USA 91: 3054-3057 ( 1994). Gene therapy agents can be further formulated to delay or delay the release of the agent, for example, using a sustained release substrate.

Other gene delivery methods are also available. Methods of formulating nucleic acid agents encoding zinc finger proteins are also known and can be performed, eg, as described in USSN 10 / 732,620. For example, such agents may be formulated as pharmaceutical compositions with a pharmaceutically acceptable carrier.

Some aspects of useful zinc finger proteins that bind to HIV LTR are further described by the following specific, non-limiting examples.

Example  One: HIV -1 of subtype B LTR  To recognize a promoter sequence Zinc Finger  Preparation of Protein

A chimeric zinc finger protein was constructed that recognizes the LTR promoter DNA sequence of HIV-1 subtype B, found mainly in North America and Europe. The names of the zinc finger proteins are shown in column 1 of Table 2. The DNA binding domains of this zinc finger protein comprise three zinc fingers. DNA contact residues of the finger are indicated by motifs in rows 2, 3 and 4, respectively. The names and sequences of target sites recognized by each zinc finger protein are shown in rows 5 and 6.

Figure 1 shows a fragment of the LTR promoter of HIV-1 subtype B, the binding sites of Sp1 and NF-kB, representative transcription factors of host cells recognizing the LTR promoter sequence of HIV-1 subtype B (Jeeninga, RE Et al. , J Virol. 74: 3740-3751 (2000)). Table 2 shows the chimeric zinc finger proteins that specifically recognize the target sequence in the promoter.

Target sequences bound by some proteins in Table 1 overlap the binding sites for specific transcriptional activators. For example, binding sites for the chimeric ZFP LTR -69, -66, -65, -62, -59, -56, -53 are binding sites of the transcriptional activator Sp1, and ZFP LTR-25 is an essential transcription factor. The binding site for TBP (TATA box binding protein) and LTR + 45R overlap the binding site for TAT, a transcriptional activator encoded by the HIV virus itself.

The amino acid sequence of the DNA binding domain of the zinc finger protein is provided in Table 1. Many component zinc finger domains are described in Bae et al. (Bae, KH et al. , Nature Biotechnol. , 21: 275-280 (2003)). The document also includes a general method for preparing zinc finger proteins.

Construction of a reporter gene comprising LTR promoter sequences of HIV-1 subtypes B, A, and E. A reporter plasmid comprising a 3'-region of the LTR promoter sequence (GenBank ^™ Accession No .; K03455 DNA sequence 9372-9621) operably linked to the reporter gene was prepared. The DNA fragment comprising the sequence of the LTR promoter region was obtained by cleaving pU3R-III CAT (Sodroski et al., Science , 227: 171-173, (1985)) with XhoI / HindIII, and similarly pGL3 with XhoI / HindIII Cloned into base plasmid (Promega). The resulting reporter plasmid was named LTR-B.

To prepare HIV-1 LTR subtypes A and E, synthetic oligonucleotides containing the sequences of each LTR subtype were annealed. Briefly, 100 pmole of oligonucleotides were mixed in 100 μl anneal buffer containing 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ and 1 mM dTT. The mixture of oligonucleotides was heated to 95 ° C for 10 minutes and then slowly cooled to 37 ° C. After the annealing process, the DNA double strand was cut with restriction enzymes KpnI and XhoI. The resulting insert fragment was cloned into pGL3-based plasmid (Promega) linearized with KpnI and XhoI. The reporter plasmids containing the sequences of the resulting HIV-1 LTR subtypes A and -E were named LTR-A and LTR-E, respectively.

The sequence of the oligonucleotides used to prepare LTR-A is as follows;

Af1 5'- CCGGGTACC gaagttgctgactgggactttccgctggggactttccaggggaggcgtgg tttgggcggagttggggagtggctaaccctcagatgctgc-3 '(SEQ ID NO .: 24), and

Af2 5'- CCCCTCGAG agagagctcccaggctcagatctggtctacctagagagacccagtacagg cgaaaagcagctgcttatatgcagcatctgagggttagcc-3 '(SEQ ID NO: 25).

The sequence of the oligonucleotides used to prepare LTR-E is as follows;

Ef1 5'- CCGGGTACC gaagtttctaacataggacttccgctggggactttccaggggaggtgtgg ccggggcggagttggggagtggctaaccctcagaagctgc-3 '(SEQ ID NO .: 26), and

Ef2 5'- CCCCTCGAG agggagctcccgggctcgacctggtctaacaagagagacccagtacaagcgaa aagcggctgcttttatgcagcttctgagggttagcc-3 '(SEQ ID NO: 27). Sequences for restriction enzymes KpnI and XhoI are shown in underlined capital letters and promoter sequences are shown in lowercase letters.

Reporter Assays. To perform cell-based assays, HeLa cells were expressed in an expression plasmid comprising an LTR-luminase reporter (LTR-B) plasmid and a chimeric ZFP (p3-ZFP) or a control plasmid that did not express chimeric ZFP (p3). Transformed into.

Two days after transformation, the cells were examined for reporter gene expression. Reporter activity in cells expressing ZFP was compared with reporter activity in cells not expressing ZFP. Transcription from LTR was activated by transforming together the plasmid (pTAT) encoding TAT, a potent viral transcriptional activator (Reynolds L. et al . , Proc. Natl. Acad. Sci. USA , 100: 1615-1620 ( 2003)).

HeLa cells were maintained in a 37 ° C. incubator with 5% CO ₂ in DMEM medium supplemented with 10% FBS and incubated. For transformation, 1 × 10 ⁴ cells were plated in 96-well plates and incubated overnight. DNA was delivered intracellularly using lipofectamine plus (LIPOFECTAMINE PLUS ^™ , Invitrogen). The DNA mixture contained 20 ng of ZFP-expressing plasmid (p3 ZFP), 20 ng of reporter plasmid (either LTR-B, LTR-A, or LTR-E), 20 ng of TAT expression plasmid (pTAT).

In order to calibrate different transfection efficiencies for each experimental group, 2 ng of Renilla luminase encoding plasmid (pRL-TK, Promega) was added to the DNA mixture. 1 μl of Plus reagent (Plus, Invitrogen) was added to each DNA mixture, incubated for 15 minutes at 25 ° C., and then incubated with 0.5 μl of lipofectamine (LIPOFECTAMINE ^™ , Invitrogen) for 15 minutes. Cells were treated with DNA-LIPOFECTAMIN ^™ complex and incubated at 37 ° C. for 2 days and then washed with PBS. 30 ul of passive lysis buffer (Promega) was added to the washed cells and allowed to react for 15 minutes. 10 μl of the cell lysate thus treated was taken and its luminase activity was measured using a dual luciferase assay system (Promgea). 50 μl of luminase assay substrate and 50 μl of Stop & Glo sample were added, and the degree of color development was measured using a luminescence analyzer according to the instructions of a person skilled in the art. Reporter LTR-B results were obtained from the average of two separate experiments using two wells, and reporter LTR-A and LTR-E results were obtained from the average of five individual wells.

result. Transcriptional inhibitory activity of chimeric ZFP on LTR promoter activity of HIV-1 subtype B.

When ZFP binds to the binding site of transcription factors, it is possible to inhibit transcription by blocking the interaction between endogenous transcription factors and promoter sequences (Kim, J.-S. et al., Proc, Natl., Acad., Sci., USA 94: 3616-3620 (1997). Table 2 shows the results of the reporter assay to investigate whether ZFP designed in the present invention exhibits such inhibitory activity.

Nine zinc finger proteins were tested. The effect of ZFP on the transcriptional activity of the LTR promoter in the absence of a transcriptional repression domain is shown in column 7 of Table 2. The target sites bound by LTR-62, LTR-65, LTR-59 partially overlap with the binding site for Sp1 in the LTR, namely Sp1-I and Sp1-II sites. Some of these zinc fingers in this assay inhibited the transcriptional activity of the LTR promoter by at least 16.6, 8.8 and 6.7 fold. This inhibitory effect is that obtained in the absence of any transcriptional repression domain, in contrast to some zinc finger proteins whose inhibitory activity is determined by fusion of the KRAB domain. One of the zinc finger proteins (ZFP-LTR-62) caused at least 16 fold inhibition in the absence of any inhibitory domain.

In addition, the zinc finger proteins of Table 2 inhibited the transcriptional activity of the LTR promoter when fused with an inhibitory domain. The ZFP shown in Table 2 was used in the Krppel-associated box (KRAB; Witzgall, R. et al . , Proc. Natl ) of rat KID-1 protein (NCBI accession no. AAB07673; amino acids, 12-74) . acad.Sci . USA , 91: 4514-4518, (1994)):

vsvtfedvavlftrdewkkldlsqrslyrevmlenysnlasmagflftkpkvisllqqgedpw (SEQ ID NO: 28).

Methods for cloning ZFP-KRAB are described in Bae et al. (Bae, K.-H et al. , Nature Biotechnol. 21: 275-280 (2003)), PCR amplification and restriction / ligation. Use a combination of methods. Column 8 of Table 2 shows the degree of inhibition of LTR promoter activity of HIV-1 subtype B mediated by expression of ZFP-KRAB fusion protein. These ZFPs (ZFP-KRAB) fused with the transcription repression domain KRAB showed about 1.4 to 9.8 fold greater inhibitory activity than ZFP without KRAB. ZFP-LTR-59, one of the ZFP-KRAB proteins, mediated at least a 58-fold inhibition of LTR activity.

We investigated whether the zinc finger protein that inhibits the LTR of HIV-1 subtype B has the same effect on the inhibition of the LTR promoters of A and E, the other two subtypes of HIV-1. Subtype-specific reporter plasmids were prepared. 2 is a schematic comparing the LTR promoter regions of subtypes A and E of HIV-1 with subtype B (Jeeninga, RE et al. , J. Virol. 74: 3740-3751, (2000)). Amplified the corresponding LTR regions derived from subtypes A and E of HIV-1 and cloned into pGL3-based plasmids (Promega) linearized by KpnI and XhoI, resulting in reporter plasmids LTR-A and LTR- It was named E.

To investigate the transcriptional inhibitory activity of chimeric zinc finger proteins, zinc finger proteins that were originally designed for LTR-B were analyzed along with other subtype-specific reporters. Of the exemplary ZFP-FRAB fusion proteins tested, ZFP-LTR-53-KRAB that binds to 8 of the 10 bases of the Sp1-I site showed pronounced inhibition with LTR-A and LTR-E. The degree of inhibition was about twice (Table 3). ZFP-LTR-53 without the KRAB domain also mediated about 2-fold inhibition with LTR-A and LTR-E (Table 4).

The Sp1-II site of subtype B, which had a high inhibitory effect when blocked by ZFP, mutated three bases in subtypes A and E, resulting in that the corresponding ZFP did not bind properly to the binding sites of subtypes A and E. ZFP effects are lost.

Such results according to the different types of HIV-1 result in the preparation of a zinc finger protein with high specificity that recognizes only a specific subtype of LTR and a wide range of subtypes despite the specificity for the target sequence within the LTR. It is indicated that it is possible to prepare zinc finger proteins that are effective for.

In some cases, changes in the LTR promoter sequence between subtypes can cause loss of inhibitory activity. Agents specific for a particular subtype may be used in a method that includes examining the patient to determine whether the patient is infected with a particular subtype of virus. Once the subtype is detected in the patient, the subtype-specific drug is administered to the patient.

On the other hand, the high specificity of the agent for a particular subtype may be beneficial as it allows the zinc finger protein to selectively target viral transcription without adversely affecting the transcription of host cell genes. Many endogenous transcription factors, such as SP1, that contribute to LTR activity are fatal for both HIV and host cell genes. Zinc finger proteins designed to recognize the border regions of Sp1 binding sites or sequences between Sp1 sites can selectively inhibit only LTR transcription of HIV-1, not genes in the host genome.

Example 2: LTR Inhibitory Activity of HIV-1 Subtype B of Deliverable ZFP

We investigated whether ZFP expressed from E. coli can be effectively transported into cells to inhibit HIV-1 LTR activity. The 3 ZFP construct designed in the previous experiment was used to reduce transcription from the LTR of HIV-1 subtype B.

Nucleic acids encoding ZFPs were inserted into the pTDTaT plasmid (see, eg, Dowdy et al., Science 285: 1569-1572 (1999)) using a combination of PCR and restriction cleavage and conjugation. After insertion into the pTAT-ZFP plasmid, a sequence encoding a polypeptide (from N terminus to C terminus) was prepared: ATG (initiation codon), hexa-histidine tag, HIV Tat PTD Sequence, nuclear position signal (NLS), array of zinc finger domains, and transcriptional regulatory domain (KOX).

The KOX is the "KRAB" domain of human Kox1 protein (Zink Finger Protein 10; NCBI Protein Database AAH24182; GI: 18848329) such as 2 to 97 amino acids of Kox1.

Expression of PTD _TAT- ZFPKOX Fusion Proteins. Expression vectors for deliverable chimeric ZFP were transformed into E. coli strain BL21 (DE3) pLysS and plated in LB-agarose containing ampicillin. Single colonies of cells transformed with PTD _TAT- ZFPKOX were picked and inoculated into selection medium. Initial cultures were inoculated in 300 ml LB selection medium and incubated until reaching 0.8 OD. Thereafter, 1 mM IPTG was added to induce protein expression and incubated at 37 ° C. for 2 hours. The amino acid sequences of the expressed proteins are shown in FIGS. 4A and 4B.

Purification of PTD _TAT- ZFPKOX Protein. Purified TAT-ZFP protein was prepared using Ni-NTA agarose (Qiagen) as directed by those skilled in the art. Briefly, BL21 cells expressing TAT-ZFP protein were spin down to lyse the cell pellet with lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-Cl, 8M Urea, Protease Inhibition Cocktail, pH 8.0). Ultrasonic crushing was carried out using ice 5 cycles for 10 seconds, which was allowed to stand for 30 seconds after crushing for 10 seconds (Fischer Scientific 550). Cell lysates were incubated with Ni-NTA agarose (Qiagen) for 45 minutes and washed _{twice with} wash solution (100 mM NaH ₂ PO ₄ , 10 mM Tris-Cl, 8M urea, pH 6.3). Protein was eluted in elution buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris-Cl, 8M urea, pH 4.5) and dialyzed with 4 ° C. refolding solution (20 mM Tris, 1 mM DTT, 100 mM ZnCl, pH 8.0). dialyze). Proteins were concentrated using CENTRICON ^™ filtration, quantified by Bradford assay, and examined by Coomassie blue staining after SDS-PAGE. Purified proteins were stored as 10% glycerol at −70 ° C.

Protein Delivery and Reporter Analysis. HeLa cells were maintained and cultured in 5% CO ₂ , 37 ° C. incubator with RPMI medium containing 10% FBS. Reporter assays are as described for the plasmid encoding the chimeric ZFP, except for the transformation process. A mixture of 20 ng of reporter plasmid (LTR-B), 20 ng of pTAT (transcriptional activator of HIV-1), and 2 ng of internal control Renilla Luminase (pRL-TK), lipofectamine 2000 ^TM (LIPOFECTAMIN 2000 ^TM Invitrogen) were transformed into 1 × 10 ⁴ HeLa cells cultured in 96-well plates. Simply lipofectamine 2000 ^TM Reagents Mixed with Opti-MEM medium and incubated at room temperature for 5 minutes. Then, the mixture was added and incubated for 20 minutes. DNA lipofectamine 2000 ^™ complex was added to the cells and incubated at 37 ° C. for one hour. Purified protein PTD _TAT- ZFPKOX was added directly to the cell culture at the desired concentration and incubated for two hours. The medium was exchanged with fresh growth medium and incubated in 5% CO ₂ , 37 ° C. incubator for 24 or 48 hours. After incubation, cells were washed twice with PBS. Thereafter, 30 µl of passive lysis buffer (Promega) was added and incubated for 15 minutes. 50 μl of luminase assay substrate and 50 μl of Stop & Glo sample were added to 10 μl of the resulting cell lysate, and luciferase activity was measured according to the instructions of the skilled person. The activity of the luminescent enzyme was measured using a luminescence analyzer.

result. LTR activity was strongly inhibited by the addition of deliverable chimeric ZFP, to a degree similar to that seen in previous experiments using plasmid transformation to provide nucleic acids encoding LTR-regulated ZFP.

Since reporter activity was very low in the absence of TAT, TAT was co-transformed with the reporter gene to promote the inhibitory effect of PTD _TAT- ZFPKOX protein. Subsequently, 200 μg of purified PTD _TAT- LTR-65KOX was added to the culture medium for 2 hours. This protein reduced the activity of luminase from the LTR-B reporter by about 16.4 fold (94% reduction) compared to the PBS treated control, both measured 24 hours after introduction (FIG. 5A). This inhibition was maintained up to 48 hours, showing a 20.2-fold inhibition (95% reduction) (FIG. 5B) compared to the control, comparable to measurements after 24 hours. The experiment also involved introducing another control protein, a deliverable zinc finger protein that does not interact with the LTR sequence (“free” ZFP called PTD _TAT- F435KOX). See Figures 5A and 5B. Within the test range of protein concentration, LTR activity was not modulated by the "irrelevant" PTD _TAT- F435KOX protein. This control shows that the decrease in LTR activity is due to the specific activity of the PTD _TAT -LTR-65KOX protein and other LTR binding proteins to the LTR promoter rather than to the addition of a foreign protein.

The inhibitory activity of PTD _TAT -ZFPKOX is protein concentration dependent. For example, treatment of 200 μg, 100 μg, 50 μg, and 25 μg of the protein for two hours for PTD _TAT- LTR-65KOX inhibited 16.4-fold, 11.1-fold, 1.9-fold, and 1.2-fold LTR activity, respectively. Induced (FIG. 5A). Treatment of the same concentration of “irrelevant” ZFP (PTD _TAT- F435KOX) did not cause inhibition. The measurements for these “unrelated” controls were 0.99, 1.03, 1.0 and 1.0 times, respectively. At all tested concentrations, cells survived intact (FIG. 5A).

In another test, chimeric ZFP (PTD _TAT -LTR-62KOX) also showed a concentration dependent inhibitory activity on the transcriptional activity of the LTR promoter (FIG. 5C and FIG. 5D).

All patents, patent applications, and references mentioned herein are incorporated by reference in their entirety. Other embodiments are also within the scope of the following claims.

                                  SEQUENCE LISTING <110> TOOLGEN, INC. <120> Modulating viral transcription <130> FPL / 200607-0129 / I <150> KR 10-2004-0002718 <151> 2004-01-14 <160> 28 <170> KopatentIn 1.71 <210> 1 <211> 13 <212> PRT <213> Homo sapiens <220> <221> SIGNAL (222) (1) .. (13) <223> MyoD-NLS <400> 1 Cys Lys Arg Lys Thr Thr Asn Ala Asp Arg Arg Lys Ala   1 5 10 <210> 2 <211> 20 <212> PRT <213> Homo sapiens <220> <221> SIGNAL (222) (1) .. (20) <223> ppoI-NLS <400> 2 Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys   1 5 10 15 Lys Ser Lys Lys              20 <210> 3 <211> 8 <212> PRT <213> Homo sapiens <220> <221> SIGNAL (222) (1) .. (8) <223> rrg1-NLS <400> 3 Asp Arg Asn Lys Lys Lys Lys Glu   1 5 <210> 4 <211> 250 <212> DNA <213> Human immunodeficiency virus type 1 <220> <221> promoter (222) (1) .. (250) HIV-1 subtype B LTR promoter <400> 4 tcacgtggcc cgagagctgc atccggagta cttcaagaac tgctgacatc gagcttgcta 60 caagggactt tccgctgggg actttccagg gaggcgtggc ctgggcggga ctggggagtg 120 gcgagccctc agatgctgca tataagcagc tgctttttgc ctgtactggg tctctctggt 180 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 240 aataaagctt 250 <210> 5 <211> 160 <212> DNA <213> Human immunodeficiency virus type 1 <220> <221> promoter <222> (1) .. (160) HIV-1 subtype A LTR promoter <400> 5 gaagttgctg actgggactt tccgctgggg actttccagg ggaggcgtgg tttgggcgga 60 gttggggagt ggctaaccct cagatgctgc atataagcag ctgcttttcg cctgtactgg 120 gtctctctag gtagaccaga tctgagcctg ggagctctct 160 <210> 6 <211> 159 <212> DNA <213> Human immunodeficiency virus type 1 <220> <221> promoter (222) (1) .. (159) HIV-1 subtype E LTR promoter <400> 6 gaagtttcta acataggact tccgctgggg actttccagg ggaggtgtgg ccggggcgga 60 gttggggagt ggctaaccct cagaagctgc ataaaagcag ccgcttttcg cttgtactgg 120 gtctctcttg ttagaccagg tcgagcccgg gagctccct 159 <210> 7 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> PTDTAT-LTR-65KOX <220> <221> SITE (222) (5) .. (10) <223> His6 tag <220> <221> DOMAIN (222) (40) .. (50) <223> Tat domain <220> <221> SITE (222) (73) .. (99) <223> HA-NLS sequence <220> <221> ZN_FING <222> (100) .. (186) <223> ZFP LTR-65 <220> <221> DOMAIN <222> (187) .. (282) <223> KOX domain <400> 7 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr   1 5 10 15 Gly Gly Asn Asn Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp              20 25 30 Arg Trp Gly Ser Lys Leu Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg          35 40 45 Arg Arg Gly Gly Ser Thr Met Ser Gly Tyr Pro Tyr Asp Val Pro Asn      50 55 60 Tyr Ala Gly Ser Met Ala Gly Thr Tyr Pro Tyr Asp Val Pro Asp Tyr  65 70 75 80 Ala Glu Leu Pro Pro Lys Lys Lys Arg Lys Val Gly Ile Arg Ile Pro                  85 90 95 Gly Glu Lys Pro Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe Gly Cys             100 105 110 Pro Ser Asn Leu Arg Arg His Gly Arg Thr His Thr Gly Glu Lys Pro         115 120 125 Phe Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu     130 135 140 Lys Thr His Thr Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Met 145 150 155 160 Glu Cys Gly Lys Ala Phe Asn Arg Arg Ser His Leu Thr Arg His Gln                 165 170 175 Arg Ile His Thr Gly Glu Lys Ala Ala Ala Asp Ala Lys Ser Leu Thr             180 185 190 Ala Trp Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe         195 200 205 Thr Arg Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr     210 215 220 Arg Asn Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr 225 230 235 240 Gln Leu Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu                 245 250 255 Pro Trp Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser             260 265 270 Glu Thr Ala Phe Glu Ile Lys Ser Ser Val         275 280 <210> 8 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> PTDTAT-LTR-62KOX <220> <221> SITE (222) (5) .. (10) <223> His6 tag <220> <221> DOMAIN (222) (40) .. (50) <223> Tat domain <220> <221> SITE (222) (73) .. (99) <223> HA-NLS sequence <220> <221> ZN_FING <222> (100) .. (186) <223> ZFP LTR-62 <220> <221> DOMAIN <222> (187) .. (282) <223> KOX domain <400> 8 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr   1 5 10 15 Gly Gly Asn Asn Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp              20 25 30 Arg Trp Gly Ser Lys Leu Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg          35 40 45 Arg Arg Gly Gly Ser Thr Met Ser Gly Tyr Pro Tyr Asp Val Pro Asn      50 55 60 Tyr Ala Gly Ser Met Ala Gly Thr Tyr Pro Tyr Asp Val Pro Asp Tyr  65 70 75 80 Ala Glu Leu Pro Pro Lys Lys Lys Arg Lys Val Gly Ile Arg Ile Pro                  85 90 95 Gly Glu Lys Pro Phe Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg             100 105 110 Ser Asp His Leu Lys Thr His Thr Arg Thr His Thr Gly Glu Lys Pro         115 120 125 Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe Gly Cys Pro Ser Asn Leu     130 135 140 Arg Arg His Gly Arg Thr His Thr Gly Glu Lys Pro Phe Gln Cys Lys 145 150 155 160 Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr                 165 170 175 Arg Thr His Thr Gly Glu Lys Ala Ala Ala Asp Ala Lys Ser Leu Thr             180 185 190 Ala Trp Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe         195 200 205 Thr Arg Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr     210 215 220 Arg Asn Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr 225 230 235 240 Gln Leu Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu                 245 250 255 Pro Trp Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser             260 265 270 Glu Thr Ala Phe Glu Ile Lys Ser Ser Val         275 280 <210> 9 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> coordinating residue <221> VARIANT <222> 1, 13 Xaa = Phe or Tyr <221> VARIANT 2, 4-8, 10-12, 14-18, 20-21, 23-27 X223 = any amino acid <221> VARIANT <222> 19 X223 = hydrophobic residue <400> 9 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa   1 5 10 15 Xaa Xaa Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Xaa His              20 25 <210> 10 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> purified polypeptide <221> VARIANT <222> 10 Xaa = Phe or Tyr <221> VARIANT <222> 2-5, 7-9, 11, 13, 17, 20-22 X223 = any amino acid <221> VARIANT <222> 16 X223 = hydrophobic residue <400> 10 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Glu Xaa   1 5 10 15 Xaa Arg His Xaa Xaa Xaa His              20 <210> 11 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> ZFP-LTR-69 <400> 11 Pro Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser   1 5 10 15 Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr Gly Glu Lys Pro Phe              20 25 30 Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys          35 40 45 Thr His Thr Arg Thr His Thr Gly Glu Lys Pro Tyr Thr Cys Ser Asp      50 55 60 Cys Gly Lys Ala Phe Arg Asp Lys Ser Cys Leu Asn Arg His Arg Arg  65 70 75 80 Thro his         <210> 12 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZPF-LTR-66 <400> 12 Pro Tyr Lys Cys Gly Gln Cys Gly Lys Phe Tyr Ser Gln Val Ser His   1 5 10 15 Leu Thr Arg His Gln Lys Ile His Thr Gly Glu Lys Pro Tyr His Cys              20 25 30 Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp Glu Leu Thr          35 40 45 Arg His Tyr Arg Lys His Thr Gly Glu Lys Pro Phe Gln Cys Lys Thr      50 55 60 Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr Arg  65 70 75 80 Thr     <210> 13 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZPF-LTR-65 <400> 13 Pro Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe Gly Cys Pro Ser Asn   1 5 10 15 Leu Arg Arg His Gly Arg Thr His Thr Gly Glu Lys Pro Phe Gln Cys              20 25 30 Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His          35 40 45 Thr Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Met Glu Cys Gly      50 55 60 Lys Ala Phe Asn Arg Arg Ser His Leu Thr Arg His Gln Arg Ile His  65 70 75 80 Thr     <210> 14 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZPF-LTR-62 <400> 14 Pro Phe Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His   1 5 10 15 Leu Lys Thr His Thr Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys              20 25 30 Lys Gln Cys Gly Lys Ala Phe Gly Cys Pro Ser Asn Leu Arg Arg His          35 40 45 Gly Arg Thr His Thr Gly Glu Lys Pro Phe Gln Cys Lys Thr Cys Gln      50 55 60 Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr Arg Thr His  65 70 75 80 Thr     <210> 15 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZFP-LTR-59 <400> 15 Pro Tyr Lys Cys Gly Gln Cys Gly Lys Phe Tyr Ser Gln Val Ser His   1 5 10 15 Leu Thr Arg His Gln Lys Ile His Thr Gly Glu Lys Pro Phe Gln Cys              20 25 30 Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His          35 40 45 Thr Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Gln Cys Gly      50 55 60 Lys Ala Phe Gly Cys Pro Ser Asn Leu Arg Arg His Gly Arg Thr His  65 70 75 80 Thr     <210> 16 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZFP-LTR-56 <400> 16 Pro Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser   1 5 10 15 Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr Gly Glu Lys Pro Tyr              20 25 30 Lys Cys Gly Gln Cys Gly Lys Phe Tyr Ser Gln Val Ser His Leu Thr          35 40 45 Arg His Gln Lys Ile His Thr Gly Glu Lys Pro Phe Gln Cys Lys Thr      50 55 60 Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr Arg  65 70 75 80 Thr     <210> 17 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZFP-LTR-53 <400> 17 Pro Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser   1 5 10 15 Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr Gly Glu Lys Pro Tyr              20 25 30 His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp Glu          35 40 45 Leu Thr Arg His Tyr Arg Lys His Thr Gly Glu Lys Pro Tyr Lys Cys      50 55 60 Gly Gln Cys Gly Lys Phe Tyr Ser Gln Val Ser His Leu Thr Arg His  65 70 75 80 Gln     <210> 18 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZFP-LTR-25 <400> 18 Pro Tyr Glu Cys Asn Tyr Cys Gly Lys Thr Phe Ser Val Ser Ser Thr   1 5 10 15 Leu Ile Arg His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys              20 25 30 Pro Asp Cys Gly Lys Ser Phe Ser Gln Ser Ser Ser Leu Ile Arg His          35 40 45 Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly      50 55 60 Lys Ala Phe Thr Gln Ser Ser Asn Leu Thr Lys His Lys Lys Ile His  65 70 75 80 Thr     <210> 19 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> ZFP-LTR + 45R <400> 19 Pro Tyr Ile Cys Arg Lys Cys Gly Arg Gly Phe Ser Arg Lys Ser Asn   1 5 10 15 Leu Ile Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys              20 25 30 Asp His Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu Asn Val His          35 40 45 Lys Arg Thr His Thr Gly Glu Lys Pro Tyr Thr Cys Ser Asp Cys Gly      50 55 60 Lys Ala Phe Arg Asp Lys Ser Cys Leu Asn Arg His Arg Arg Thr His  65 70 75 80 Thr     <210> 20 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> HA-NLS sequence <220> <221> SITE (222) (1) .. (9) <223> HA tag <220> <221> SIGNAL (222) (12) .. (19) <223> SV40-NLS <400> 20 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Glu Leu Pro Pro Lys Lys Lys   1 5 10 15 Arg Lys Val Gly Ile Arg Ile Pro Gly Glu Lys              20 25 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Minimal Tat PTD including a.a. residues 47-57 of the HIV Tat          protein <400> 21 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <220> <221> VARIANT <222> (1) <223> Xaa = Pro or Thr <220> <221> VARIANT <222> (5) <223> Xaa = Pro or Ala <400> 22 Xaa Gly Glu Lys Xaa   1 5 <210> 23 <211> 96 <212> PRT <213> Homo sapiens <220> <221> DOMAIN (222) (1) .. (96) <223> KRAB domain from the human Kox1 protein <400> 23 Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu Val Thr Phe Lys   1 5 10 15 Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys Leu Leu Asp Thr              20 25 30 Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu Asn Tyr Lys Asn          35 40 45 Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp Val Ile Leu Arg      50 55 60 Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg Glu Ile His Gln  65 70 75 80 Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile Lys Ser Ser Val                  85 90 95 <210> 24 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide for LTR-A construction <400> 24 ccgggtaccg aagttgctga ctgggacttt ccgctgggga ctttccaggg gaggcgtggt 60 ttgggcggag ttggggagtg gctaaccctc agatgctgc 99 <210> 25 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide for LTR-A construction <400> 25 cccctcgaga gagagctccc aggctcagat ctggtctacc tagagagacc cagtacaggc 60 gaaaagcagc tgcttatatg cagcatctga gggttagcc 99 <210> 26 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide for LTR-E construction <400> 26 ccgggtaccg aagtttctaa cataggactt ccgctgggga ctttccaggg gaggtgtggc 60 cggggcggag ttggggagtg gctaaccctc agaagctgc 99 <210> 27 <211> 98 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide for LTR-E construction <400> 27 cccctcgaga gggagctccc gggctcgacc tggtctaaca agagagaccc agtacaagcg 60 aaaagcggct gcttttatgc agcttctgag ggttagcc 98 <210> 28 <211> 63 <212> PRT <213> Rattus norvegicus <220> <221> DOMAIN (222) (1) .. (63) <223> Kruppel-associated box of KID-1 protein <400> 28 Val Ser Val Thr Phe Glu Asp Val Ala Val Leu Phe Thr Arg Asp Glu   1 5 10 15 Trp Lys Lys Leu Asp Leu Ser Gln Arg Ser Leu Tyr Arg Glu Val Met              20 25 30 Leu Glu Asn Tyr Ser Asn Leu Ala Ser Met Ala Gly Phe Leu Phe Thr          35 40 45 Lys Pro Lys Val Ile Ser Leu Leu Gln Gln Gly Glu Asp Pro Trp      50 55 60

Claims (39)

An isolated or recombinant protein comprising a DNA binding domain comprising a plurality of zinc finger domains, wherein (1) the DNA binding domain is a target sequence in the long-terminal repeat (LTR) of a human immunodeficiency virus And (2) the protein can reduce transcription of viral genes. The method of claim 1, A protein characterized in that in the absence of a transcription repression domain, transcription of viral genes can be reduced by more than twofold. The method of claim 1, And said target sequence overlaps the binding site of the transcription factor of the host cell but does not encompass said site. The method of claim 1, Wherein said DNA binding domain binds to the target sequence with a dissociation constant (Kd) of less than 10 ⁻⁸ M. 18. The method of claim 1, And said DNA binding domain has less than 90 amino acids. The method of claim 1, And said plurality of zinc finger domains comprise at least two human zinc finger domains from different human proteins. The method of claim 1, A protein characterized by further comprising a protein transduction domain. The method of claim 7, wherein And said protein delivery domain comprises a sequence derived from a human immunodeficiency virus tat protein, herpes simplex virus (HSV) VP22 protein, or Antennapedia homeodomia. The method of claim 1, The protein further comprises a transcription repression domain. The method of claim 9, The transcription inhibitory domain comprises a Kid or KOX inhibitory domain. The method of claim 1, A protein characterized by not including a transcription repression domain. The method of claim 1, A protein comprising a sequence of at least 95% identical to the sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, or 19. The method of claim 12, A protein comprising the sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, or 19. The method of claim 1, A protein characterized by inhibiting viral gene transcription four or more times. The method of claim 1, A protein characterized by inhibiting the transcription of viral genes at least fourfold in the absence of the KRAB domain. The method of claim 1, A protein characterized by inhibiting transcription of a viral gene four or more times in the absence of any transcription inhibition domain. The method of claim 1, Wherein said DNA binding domain comprises three zinc finger domains comprising the DNA contact residues of the motif group shown in Table 2 from N to C terminus. The method of claim 1, A protein characterized in that at least one zinc finger domain is a human zinc finger domain. The method of claim 1, A protein characterized in that in the absence of a transcription repression domain can reduce transcription from the viral promoter. The method of claim 1, A protein characterized by inhibiting the transcriptional activity of the LTR promoter at least 6-fold, having less than six zinc finger domains, no transcription inhibitory domain, and / or having less than 120 amino acids. The method of claim 1, A protein characterized in that said target sequence comprises the following nucleotides: Of the LTR sequence shown in Fig. 1, (i) G at position +42, (ii) G at position -50, (iii) T at position -49, (iv) G at -48 position, (v) G at position -47, (vi) C at position -46, (vii) G at position -45, (viii) T at position -25, (ix) A at -24 position, and / or (x) A at position -23. A protein comprising a protein binding domain and a DNA binding domain comprising a plurality of zinc finger domains, wherein (1) the DNA binding domain binds to a target sequence in the nucleic acid of the virus, and (2) the protein is responsible for transcription of the viral promoter. Protein, which can be reduced. The method of claim 22, Wherein said viral promoter is the LTR of a human immunodeficiency virus, and the protein is capable of reducing transcriptional activity of the LTR promoter. The method of claim 22, And said plurality of zinc finger domains comprise at least two human zinc finger domains from different human proteins. A pharmaceutical composition for treating or preventing retroviral diseases mediated by human immunodeficiency virus, comprising the protein according to any one of claims 1 to 24 and a pharmaceutically acceptable carrier. 26. A retroviral disease mediated by human immunodeficiency virus, which encodes a protein according to any one of claims 1 to 24 and comprises a nucleic acid capable of being expressed in a host cell and a pharmaceutically acceptable carrier. Pharmaceutical compositions for the treatment or prophylaxis. Providing a medicament capable of regulating viral gene transcription to a cell of a subject in a therapeutically or prophylactically effective amount, wherein the medicament A protein having (1) a DNA binding domain that binds to a target sequence in a promoter of a human immunodeficiency virus and (ii) a protein delivery domain; or (2) a nucleic acid encoding a protein comprising a DNA binding domain comprising 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 A method for treating or preventing a retroviral disease mediated by a human immunodeficiency virus, comprising a nucleic acid characterized by being capable of reducing this promoter transcription. The method of claim 27, The drug is a protein according to (1), wherein the DNA binding domain of the protein comprises a plurality of zinc finger domains. The method of claim 27, The protein delivery domain is derived from a human immunodeficiency virus Tat protein. The method of claim 27, Wherein said DNA binding domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, or 19. The method of claim 27, In the absence of a transcription repression domain, it is possible to reduce promoter transcription. The method of claim 27, The drug is a nucleic acid according to the above (2). The method of claim 32, Wherein said protein encoded by said nucleic acid inhibits promoter transcription more than fourfold. The method of claim 33, wherein And in the absence of any transcription repression domain, the protein encoded by the nucleic acid inhibits promoter transcription four or more times. The method of claim 34, wherein And in the absence of any transcription repression domain, the protein encoded by the nucleic acid inhibits promoter transcription at least 7-fold. The method of claim 27, Wherein said target sequence comprises the following nucleotides: Of the LTR sequence shown in Fig. 1, (i) G at position +42, (ii) G at position -50, (iii) T at position -49, (iv) G at -48 position, (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. The method of claim 27, The subject is a human infected with a human immunodeficiency virus. An isolated nucleic acid comprising a sequence encoding a protein according to any one of claims 1 to 24. A vector comprising the nucleic acid of claim 38.

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