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WO1997011972A1 - Proteine chimere de polypeptide et d'acide nucleique de liaison d'adn et d'adn methyltransferase et ses utilisations - Google Patents

Proteine chimere de polypeptide et d'acide nucleique de liaison d'adn et d'adn methyltransferase et ses utilisations Download PDF

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WO1997011972A1
WO1997011972A1 PCT/US1996/015576 US9615576W WO9711972A1 WO 1997011972 A1 WO1997011972 A1 WO 1997011972A1 US 9615576 W US9615576 W US 9615576W WO 9711972 A1 WO9711972 A1 WO 9711972A1
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dna
cell
protein
gene
methylation
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PCT/US1996/015576
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WO1997011972A9 (fr
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Timothy H. Bestor
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The Trustees Of Columbia University In The City Of New York
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Priority to AU73781/96A priority Critical patent/AU7378196A/en
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Publication of WO1997011972A9 publication Critical patent/WO1997011972A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

  • Methylation represses transcription directly by interference with binding of transcription factors (Joel et al. , 1993, and references therein) and methylated sequences are assembled into condensed chromatin that is inaccessible to transcription factors (reviewed by Bird, 1992) . It has been shown that methylation of CpG dinucleotides in the 5' long terminal repeat (LTR) of HIV-l suppresses viral transcription (reviewed by Bednarik, 1992) . Methylation can reduce transcription from the HIV-l 5' LTR to undetectable levels (Joel et al. , 1993) . It is also known that methylation patterns are transmitted by clonal inheritance
  • Retroviral DNA is especially prone to inactivation by de novo methylation upon germline transmission, and this has greatly reduced the usefulness of retroviral transducing vectors in the construction of transgenic animals (Jahner and Jaenisch, 1984; Jaenisch et al. , 1985) .
  • HIV-l infections in patients is unknown, but as HIV has only recently entered human populations, the host-parasite relationship may be far from equilibrium (Baltimore, 1995) and HIV proviral DNA may be inactivated with low efficiency under normal conditions (McCune, 1995) . It is clear, however, that HIV-l transcription is very sensitive to methylation of 5' LTR sequences (reviewed by Bednarik,
  • the present invention provides a chimeric protein which comprises a mutated DNA methyltransferase portion and a DNA binding protein portion that binds sufficiently close to a promoter sequence of a target gene, which promoter sequence contains a methylation site, to specifically methylate the site and inhibit activity of the promoter and thus inhibit expression of the target gene.
  • This invention also provides for a method for inhibiting the expression of a target gene which includes contacting a promoter of the target gene with the chimeric protein, so as to specifically methylate the promoter sequence of the target gene thus inhibiting expression of the target gene.
  • FIG. 1 Distribution of CpG sites and transcription factor binding sites in the 5' LTR of HIV-l (Sequence I.D. No. 1) . CpG sites that fall within the recognition sequence of a restriction methyltransferase or methylation-sensitive endonuclease are shown above downward-pointing arrows. Data are from Garcia and Gaynor, 1994.
  • Figure 2 Sequence of HIV-l 5' LTR. CpG dinucleotides are bold and underlined; CpG sites that lie within the recognition sequence of methylation-sensitive restriction endonuclease are bold and italicized. Major transcription start site is denoted by +1, and the underlined sequence following denotes the transcribed sequence; numbering is with respect to the transcription start site. Please refer to Figure 1 for location of protein binding sites. Strike through indicates the 9 bp binding site for the 3 finger protein developed by Wu et al. (1995) . Sequence and numbering from Garcia and Gaynor, 1994.
  • Figure 3 Gene silencing via targeted DNA methylation.
  • a chimera between a sequence-specific DNA binding protein and a DNA methyltransferase with attenuated DNA binding directs methylation only to sites in the vicinity of the recognition sequence of the DNA binding protein.
  • Figure 4 Domain organization of mammalian DNA methyl ⁇ transferase. This enzyme is comprised of a long N-terminal regulatory domain and a C-terminal catalytic domain that closely resembles bacterial restriction methyltransferase. See Bestor and Verdine (1994) for a recent review.
  • Protein-DNA contacts in the M. Haelll-DNA transition state intermediate.
  • the target cytosine is everted from the helix during methyl transfer, and is shown in the extra helical position.
  • Thin dotted lines indicate base-specific contacts in the major groove, bold lines indicate contacts with the sugar-phosphate backbone.
  • Mutation of residues involved in the sugar-phosphate contacts will reduce the affinity of DNA methyl-transferases for DNA, as will framework mutations that alter the disposition of the contact residues. Such mutations will make methyl transfer dependent on the DNA binding protein moiety of a DNA binding protein/DNA methyltransferase chimera, as described herein. Data from Reinisch et al .
  • FIG. 7 Cyclic in vitro/in vivo selection for DNA binding protein/DNA methyltransferase chimeras in which methylation is dependent on the DNA binding protein moiety. McrBC en ⁇ codes a nuclease that degrades heavily methylated DNA. Unmethylated DNA that has been linearized by a methylation- sensitive endonuclease does not transform. Use of these selective procedures on combinatorial libraries allows selection of chimeric DNA binding protein-DNA methyltransferase that target methylation only to a unique target site.
  • This selection scheme may be applied to LexA/DNA methyltransferase chimeras (Example 1) and to zinc finger/DNA methyltransferase chimeras (Examples 3-5) .
  • FIG. 9 Phage-display selection of a zinc finger proteins that bind to predetermined sequences in the HIV-l 5' LTR.
  • the strategy used to construct tridactyl zinc finger proteins that bind to specific sequences was independently developed by the laboratories of Berg (Desjarlais and Berg, 1993) , Klug (Choo and Klug, 1994) , Pabo (Rebar and Pabo, 1994),and Barbas (Wu et al. , 1994) .
  • the schemes are very similar; the one depicted here is that of Choo and Klug (1994) .
  • the method has been used to obtain proteins that bind to a number of predetermined sequences with high specificity and affinity.
  • the boxed sequence shown in the figure appears 20 base pairs 5' of the first Hpall (CCGG) site in the LTR sequence of Figure 2; it is shown for illustrative purposes only (Sequence I.D. No. 2) .
  • the actual target sequence will be determined as described in Example 2.
  • the viral genome of 3.2 kb is represented by a horizontal line. Positions of methylatable CpG sites are shown by vertical lines. Arrows indicate major viral transcripts. Notice that transcription of each major mRNA initiates in a cluster of CpG sites. CpG methylation has been shown to suppress viral transcription in transgenic animals (Miller and Robinson, 1993; Pourcel et al . , 1990) .
  • Figures IIA - 11B Targeted methylation of a preselected CpG site.
  • a Smal site is located adjacent ot a lexA binding site in the same plasmid.
  • LexA is predicted to position M.SssI over this site, and preferential methylation of theSmal site was expected (M.SssI has no intrinsic preference for Smal sites) .
  • the positions of other restriction sites are shown approximately to scale,* other features of the plasmid are omitted for the sake of clarity.
  • FIG. 13 Expression of LacI-M.SssI fusion in E. coli XLl- Blue [pLM9] .
  • the targeted methyltransferase is marked with an arrow.
  • the antibody used was anti-Lad.
  • An intact fusion protein was produced in good yield.
  • Lane marked pBS contained proteins extracted from cells that contained only the expression vector.
  • FIG 14. Targeted methylation demonstrated by bisulfite sequencing analysis.
  • the methylation target shown in Figure 12 was isolated from cells expressing Lad-M.SssI fusion proteins .
  • the bisulfite sequencing method was used to identify methylated sites.
  • pLM9-l and -2 are two sister bisulfite cones carrying BS-modified methylation target sequence derived from a pLM9 mutant that has attenuated activity. Methylation is limited to CpG sites in the immediate vicinity of the binding site of the sequence specific DNA binding protein Lad .
  • pBS is the precursor of pLM9 and has no lacI/M.SssI gene; no methylated sites are present .
  • pM is a pBS derivative that encodes fully active (non-targeted) M.SssI gene; all sites are methylated.
  • FIG. 17 Complete suppression of HIV-l transcription by LTR methylation.
  • Methods pLTR-CAT was treated with M.SssI to methylate all CpG sites. Methylated or unmethylated pLTR-CAT was cotransfected with unmethylated pLG-luc into HLtat cells and lysates were prepared at the times indicated. To control for the efficiency of transfection and recovery, volumes of lysate containing equivalent luciferase activity were assayed for CAT activity using the FASTCAT assay kit. Reaction products were quantitated using NIH Image. "CAT Activity" indicates conversion of 1- deoxychloramphenicol to 3-acetyl-l-deoxychloramphenicol, normalized to arbitrary units. Control cells were transfected with pLG-luc only and harvested 48 hours post- transfection.
  • the present invention provides for a chimeric protein which comprises a mutated DNA methyltransferase portion and a DNA binding protein portion that binds sufficiently close to a promoter sequence of a target gene, which promoter sequence contains a methylation site, to specifically methylate the site and inhibit activity of the promoter and thus inhibit expression of the target gene.
  • chimeric protein is a protein including at least two portions of two proteins positioned adjacent to each other so that the coding sequences of both portions are in frame and can be translated into one polypeptide.
  • the chimeric protein may include a portion of a DNA binding protein and a portion of a mutated or wild type DNA methyltransferase protein.
  • the two portions of the chimeric protein may be linked with intermediate sequences of 8,12,16 or 20 codons expressed as part of the chimeric protein.
  • adjacent includes two sequences that are positioned directly next to one another so that the codons of each sequence are in frame and contiguous. “Adjacent” also includes two sequences that are nearby one another linked to intermediate sequences and are in frame and can be translated into one polypeptide.
  • linked to encompasses a means of joining two separate portions of a protein, each of which may have a separate function.
  • the means of joining the functional portions of the protein may include a covalent or noncovalent association between functional polypeptides via an organic substance, an inorganic substance, a nucleotide, a polynucleotide, a peptide nucleic acid, a peptide, a triplex peptide, an electrostatic means, a triplex nucleic acid, or a self-assembling biomaterial.
  • DNA methyltransferase is a protein which is capable of methylating a particular DNA sequence, which particular DNA sequence may be -CpG- .
  • This protein may be a mutated DNA methyltransferase, a wild type DNA methyltransferase, a naturally occurring DNA methyltransferase, a variant of a naturally occurring DNA methyltransferase, a truncated DNA methyltransferase, or a segment of a DNA methyltransferase which is capable of methylating DNA.
  • the DNA methyltransferase may include mammalian DNA methyltransferase, bacterial DNA methyltransferase, M. SssI DNA methyltransferase and other proteins or polypeptides that have the capability of methylating DNA.
  • mutated DNA methyltransferase protein portion is a segment of a DNA methyltransferase protein or polypeptide containing an amino acid sequence which is different from the wild type DNA methyltransferase amino acid sequence.
  • a mutated DNA methyltransferase protein portion may include a segment of a naturally occurring variant of the wild type DNA methyltransferase protein, a segment of a DNA methyltransferase with a sequence altered from the wild type sequence, a truncated protein or portion of protein or polypeptide that is capable of methylating DNA.
  • This mutated DNA methyltransferase may be altered at one or more of its amino acids and retains part of the wild type methyltransferase activity, although the activity of the mutated DNA methyltransferase protein portion may be attenuated activity.
  • DNA binding protein portion is a segment of a DNA binding protein or polypeptide capable of specifically binding to a particular DNA sequence. The binding is specific to a particular DNA sequence site.
  • the DNA binding protein portion may include a truncated segment of a DNA binding protein or a fragment of a DNA binding protein.
  • a promoter sequence of a target gene is at least a portion of a non-coding DNA sequence which directs the expression of the target gene.
  • the portion of the non ⁇ coding DNA sequence may be in the 5' -prime direction or in the 3' -prime direction from the coding region of the target gene.
  • the portion of the non-coding DNA sequence may be located in an intron of the target gene.
  • the promoter sequence of the target gene may be a 5' long terminal repeat sequence of a human immunodeficiency virus-1 proviral DNA.
  • the target gene may be a retroviral gene, an adenoviral gene, a foamy viral gene, a parvo viral gene, a foreign gene expressed in a cell, an overexpressed gene, or a misexpressed gene.
  • methylation site in a DNA sequence, which methylation site may be -CpG-, wherein the methylation is restricted to particular methylation site(s) and the methylation is not random.
  • the chimeric protein may be a zinc three-finger DNA binding polypeptide linked to a CpG-specific DNA methyltransferase protein portion.
  • the chimeric protein may comprise a mutated Lex A DNA binding protein or polypeptide linked to a cytosine methyltransferase protein portion or polypeptide.
  • the chimeric protein may include at least a portion of a mutated M. SssI DNA methyltransferase protein or at least a portion of a mutated mammalian DNA methyltransferase.
  • an expression vector may code for the chimeric protein described hereinabove.
  • the expression vector may be a replicable vector and may include a pLS vector, a prokaryotic expression vector, a yeast expression vector, a baculovirus expression vector, a mammalian expression vector, or an episomal mammalian expression vector.
  • expression vector is a nucleic acid molecule which is capable of having a coding sequence of DNA or RNA ligated into it and then expressing that sequence when introduced into an appropriate host cell .
  • An expression vector may be a mammalian expression vector or a prokaryotic expression vector.
  • Another embodiment of this invention is a method for inhibiting the expression of a target gene which includes contacting a promoter sequence of the target gene with the chimeric protein described hereinabove, so as to specifically methylate the promoter sequence of the target gene thus inhibiting expression of the target gene.
  • the target gene may be an endogenous target gene which is native to a cell or a foreign target gene.
  • the foreign gene may be a retroviral target gene or a viral target gene.
  • the target gene in this embodiment may be associated with a cancer, a central nervous system disorder, a blood disorder, a metabolic disorder, a cardiovascular disorder, an autoimmune disorder, or an inflammatory disorder.
  • the cancer may be acute lymphocytic leukemia, acute myelogenous leukemia, B-cell lymphoma, lung cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, Hodgkin's disease, malignant melanoma, neuroblastoma, renal cell carcinoma or squamous cell carcinoma.
  • the central nervous system disorder may be Alzheimer's disease, Down's syndrome, Parkinson's disease, Huntington' s disease, schizophrenia, or multiple sclerosis.
  • the infectious disease may be cytomegalovirus, herpes simplex virus, human immunodeficiency virus, AIDS, papillomavirus, influenza, Candida albicans, mycobacteria, septic shock, or associated with a gram negative bacteria.
  • the blood disorder may be anemia, hemoglobinopathies, sickle cell anemia, or hemophilia.
  • the cardiovascular disorder may be familial hypercholesterolemia, atherosclerosis, or renin/angiotensin control disorder.
  • the metabolic disorder may be ADA, deficient SCID, diabetes, cystic fibrosis, Gaucher' s disease, galactosemia, growth hormone deficiency, inherited emphysema, Lesch-Nyhan disease, liver failure, muscular dystrophy, phenylketonuria, or Tay-Sachs disease.
  • the autoimmune disorder may be arthritis, psoriasis, HIV, or atopic dermatitis.
  • the inflammatory disorder may be acute pancreatitis, irritable bowel syndrome, Chrone's disease or an allergic disorder.
  • promoter sequences of the target genes listed below may be therapeutic target genes of the subject invention: dominant oncogenes, c-MYB, c-MYC, c-SIS, ERB A, ERB B, ERB B-l, ERB B-2, HER-2/NEU, c-SRC, c-YES, c-FPS, c-FES, c-FOS, c-JUN, c- ROS, c-ABL, c-FGR, c-MOS, c-RAS, c-RAF, c-MET, c-ETS, BCL-1, BCL-2, ETS, c-FMS, C-FES, C-BLK, TCL-1, TCL-2, TCL-3, TCL-5, ALL-1/HRX/MLL, PML promoter in PML/RAR- ⁇ fusion, RAR- ⁇ promoter in RAR- ⁇ /PML fusion, and NF-1.
  • Cancer related genes include: collagenase 92 Kd Type 4, collagenase 72 Kd Type 4, osteopontin, calcyclin, fibroblast growth factor, epidermal growth factor, matrilysin and stromolysin.
  • Viruses that establish chronic infections and which are involved in cancer or chronic diseases are also target genes of the subject invention.
  • Virus that have possible target genes include hepatitis C, hepatitis B, varicella, herpes simplex types I and II, Epstein-Barr virus, cytomegalovirus, JC virus and BK virus.
  • the promoter sequence of viral protein X and the promoter sequence of pre-S2/5 of the hepatitis B virus may be target genes
  • genes whose expression is involved in neurodegenerative disease may be target genes of the subject invention. These target genes may include beta amyloid precursor protein and the prion protein both of which are shown to be involved in Alzheimer's disease. In another embodiment of the subject invention, genes which are involved in the etiology of acromegaly may be target genes of the subject invention. These target genes may include somatostatin, growth-hormone releasing hormone and constitutive G-stimulatory protein mutants .
  • the target gene may be in a cell.
  • the cell may be a bacterial cell, an animal cell or a plant cell.
  • the cell may be a eukaryotic cell, a prokaryotic cell, a virus packaging cell, a somatic cell, a germ cell, a neuronal cell, a myocyte, a T lymphocyte, a CD4 + cell, a tumor cell, a CD4+ cell, or a stem cell.
  • the chimeric protein may be a mutated Lex A DNA binding protein portion or polypeptide linked to a cytosine DNA methyltransferase protein portion or polypeptide.
  • the DNA methyltransferase protein portion may be M. SssI DNA methyltransferase protein or the mammalian DNA methyltransferase protein.
  • the chimeric protein may include a tridactyl zinc finger protein or polypeptide capable of specifically binding the HIV-l 5'LTR nucleic acid sequence linked to a cytosine methyltransferase polypeptide.
  • the cytosine DNA methyltransferase polypeptide may be a M.
  • DNA methyltransferase protein or polypeptide or at least a portion of the mammalian DNA methyltransferase protein are particularly useful as a mammalian DNA methyltransferase protein.
  • (glyco) lipids (glyco) eptides, synthetic polymers, a triplex nucleic acid, naked nucleic acid transfer, particle- mediated nucleic acid transfer or cationic precipitation.
  • a further embodiment of this invention is a method for inhibiting expression of a target gene in a multicellular organism which includes contacting a promoter sequence of the target gene with the chimeric protein described hereinabove or a nucleic acid molecule encoding the chimeric protein, so as to specifically methylate the promoter sequence of the target gene and thus inhibit expression of the target gene in the multicellular organism.
  • the administration of the chimeric protein to the multicellular organism may comprise intralesional, intraperitoneal, intramuscular or intravenous injection; liposome-mediated delivery; viral infection; gene bombardment; topical, nasal, oral, anal, ocular or otic delivery.
  • the viral infection may be via a non-integrating, replication-defective virus.
  • the virus may comprise a replication-defective HIV-l provirus, a retroviral vector, an adeno-associated virus, a N2 retroviral vector, a SIM retroviral vector, a LNL6 vector, a LXSN vector or a MMuLV retroviral vector.
  • the process by which a plant or animal is rendered resistant to viral infection comprises introducing into the plant or animal a construct which on translation gives rise to the above-mentioned chimeric protein.
  • the introduction of the nucleic acid molecule is accomplished by genetic transformation of a part of the plant by a DNA sequence coding for the nucleic acid molecule, followed by the regeneration of a transgenic plant.
  • the transformation is carried out by the intermediary of Agrobacterium tumefaciens or Agrobacterium rhizogenes. (U.S. Patent Nos. 5,107,065 and 5,188,958) .
  • the present invention is further directed to a DNA construct for a plant, the construct comprising a genetic sequence and a promoter capable of directing expression of the genetic sequences wherein the genetic sequence on expression provides at least a portion of a sequence specific DNA binding protein linked to at least a portion of a mutated DNA methyltransferase protein.
  • the DNA construct may further be part of a DNA transfer vector suitable for transferring the DNA construct into a plant cell and insertion into a plant genome.
  • the DNA construct is carried by broad host range plasmid pGA470 which is capable of transformation into plant cells using Agrobacterium.
  • the present invention extends to other means of transfer such as genetic bullets (e.g. DNA-coated tungsten particles, high-velocity micro projectile bombardment) and electroporation amongst others (Maliga, 1993; Bryant, 1992; or Shimamoto, 1989) .
  • the gene encoding the chimeric protein having been introduced into the nonhuman mammal, or an ancestor of the nonhuman mammal at the single cell stage or an embryonic stage, is linked to a promoter and integrated into the genome of the nonhuman mammal.
  • One skilled in the art would be familiar with the experimental methods necessary to produce a transgenic mammal, as described in Leder et al. , U. S. Patent No. 4,736,866 and Krimpenfort and Berns, U. S. Patent No. 5,175,384 and Wagner and Chen, U. S. Patent No. 5,175,385.
  • a further embodiment of the subject invention is a method of treating a subject infected with a virus which comprises administering to the subject a therapeutic composition comprising a chimeric protein, or a nucleic acid molecule encoding the chimeric protein, capable of sequence specific methylation of a site(s) in a promoter sequence of a target viral gene under suitable conditions so as to specifically methylate the promoter sequence of the target gene thereby inhibiting expression of the target gene and thus treating the subject infected with the virus.
  • a therapeutic composition comprising a chimeric protein, or a nucleic acid molecule encoding the chimeric protein, capable of sequence specific methylation of a site(s) in a promoter sequence of a target viral gene under suitable conditions so as to specifically methylate the promoter sequence of the target gene thereby inhibiting expression of the target gene and thus treating the subject infected with the virus.
  • the therapeutic composition may comprise a nucleic acid encoding at least a portion of a mutated Lex A DNA binding protein or polypeptide linked to at least a portion of a mutated DNA methyltransferase protein or polypeptide.
  • the therapeutic composition may be a nucleic acid encoding a tridactyl zinc finger DNA binding protein or polypeptide capable of specifically binding the HIV-l 5' LTR nucleic acid sequence linked to at least a portion of a mutated DNA methyltransferase protein or polypeptide.
  • the therapeutic composition may comprise a nucleic acid encoding at least a portion of a sequence specific DNA binding protein or polypeptide linked to a mutated DNA methyltransferase protein or polypeptide.
  • the subject receiving the therapeutic composition of this invention may be a human or a non-human mammal.
  • the therapeutic composition may comprise a replicable expression vector chosen from the group consisting of a pLS vector, a prokaryotic expression vector, a yeast expression vector, a baculovirus expression vector, a mammalian expression vector, and an episomal mammalian expression vector.
  • the administration may comprise intralesional, intraperitoneal, intramuscular or intravenous injection; liposome-mediated delivery; viral infection; gene bombardment; topical, nasal, oral, anal, ocular or otic delivery.
  • the viral infection may be via a non-integrating, replication-defective virus.
  • nucleic acid molecule encoding a chimeric protein is incorporated into a liposome to allow for administration to the subject.
  • Methods of incorporation of nucleic acid molecules into liposomes are well known to those of ordinary skill in the art.
  • the nucleic acid encoding the chimeric protein may be delivered via transfection, injection, or viral infection.
  • RAC Recombinant DNA Advisory Committee
  • DNA to transfer genes, episomal vectors, retroviruses, adenoviruses, parvoviruses, and herpesviruses have been used
  • a separately preferred embodiment of the invention is a replicable expression vector encoding a chimeric protein capable of sequence specific DNA methylation.
  • the replicable expression vector may be chosen from the group consisting of a pLS vector, a prokaryotic expression vector, a yeast expression vector, a baculovirus expression vector, a mammalian expression vector, and an episomal mammalian expression vector.
  • the agent may comprise a mutated Lex A binding polypeptide linked to a methyltransferase polypeptide.
  • the agent may comprise a tridactyl zinc finger polypeptide that specifically binds to the HIV-l 5'LTR nucleic acid sequence linked to a methyltransferase polypeptide.
  • Another embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the replicable vector described hereinabove and a pharmaceutically acceptable carrier.
  • the carrier may comprise a diluent.
  • the pharmaceutically acceptable carrier may be an aerosol, intravenous, oral or topical carrier and is further described herein.
  • Another embodiment of the subject invention is a method of obtaining an expression vector including DNA encoding a desired chimeric protein that includes at least a portion of a mutated DNA methyltransferase protein and at least a portion of a DNA binding protein such chimeric protein being capable of inhibiting the expression of a target gene
  • a method of obtaining an expression vector including DNA encoding a desired chimeric protein that includes at least a portion of a mutated DNA methyltransferase protein and at least a portion of a DNA binding protein such chimeric protein being capable of inhibiting the expression of a target gene which comprises: (a) obtaining a population of expression vectors each of which expresses a chimeric protein including (1) a DNA corresponding to an endogenous promoter sequence of the target gene, which promoter contains at least one sequence specific methylation site; (2) a DNA encoding a mutated DNA methyltransferase; and (3) a DNA encoding a DNA binding protein, position adjacent the DNA of
  • “desired” includes optimal properties of a chimeric protein as described hereinabove.
  • the properties include specifically methylating a DNA sequence in a promoter sequence of a target gene and inhibiting the activity of the promoter and thus inhibiting expression of the target gene.
  • an "effective amount" is an amount which is effective to produce the sequence specific methyltransferase and inactivation of the target gene. Accordingly, the effective amount will vary with the subject being treated, as well as the condition to be treated.
  • the methods of administration are to include, but are not limited to, administration cutaneously, subcutaneously, intravenously, parenterally, orally, topically, or by aerosol.
  • suitable pharmaceutically acceptable carrier encompasses any of the standard pharmaceutically accepted carriers, such as phosphate buffered saline solution, water, emulsions such as an oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
  • a triglyceride emulsion useful in intravenous and intraperitoneal administration of the compounds is the triglyceride emulsion commercially known as Intralipid ® .
  • Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • Such carriers may also include flavor and color additives or other ingredients.
  • compositions comprising therapeutically effective amounts of polypeptide products of the invention together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • suitable diluents preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • a "therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
  • compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl., acetate, phosphate) , pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts) .
  • buffer content e.g., Tris-HCl., acetate, phosphate
  • additives such as albumin or gelatin to prevent absorption to surfaces
  • detergents e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts
  • solubilizing agents e.g., glycerol, polyethylene glycerol
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., Thimerosal, benzyl alcohol, parabens
  • bulking substances or tonicity modifiers e.g., lactose, mannitol
  • covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
  • the choice of compositions will depend on the physical and chemical properties of the protein. For example, a product derived from a membrane-bound form of a protein may require a formulation containing detergent.
  • Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils) .
  • particulate compositions coated with polymers e.g., poloxamers or poloxamines
  • particulate compositions coated with polymers e.g., poloxamers or poloxamines
  • compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.
  • Another embodiment of the subject invention is a transgenic non-human mammal whose somatic and germ cells contain and express a gene coding for a desired chimeric protein which is capable of inhibiting the expression of a target gene, the gene having been stably introduced into the non-human mammal at the single cell stage or an embryonic stage, and wherein the gene is linked to a promoter and integrated into the genome of the non-human mammal.
  • the desired chimeric protein may be the desired chimeric protein described hereinabove.
  • clonal inheritance of methylation patterns would render the inactivation essentially irreversible.
  • this irreversible inactivation is the basis of a therapeutic approach that has practical advantages over alternative molecular biological approaches (including antisense RNA, ribozymes, targeted nucleases, and intracellular anti-HIV antibodies; reviewed by Volberding, 1995) .
  • Provirus silencing by targeted cytosine methylation stimulates an existing host defense system and as an analogy, has the virtues of a vaccine: the clonal inheritance of methylation patterns is like the immunological memory, and the strong suppression of transcription is like the immune defense system in that both perturb the function of particular gene products.
  • the 5' LTR of HIV-l may be inactivated by targeted cytosine methylation.
  • the LTR that directs transcription of the provirus is rich in binding sites for transcription stimulatory proteins (Nabel, 1993; Garcia and Gaynor, 1994) and rich in CpG sites, the predominant site of methylation in vertebrates. It is also well-established that methylation of only a few of the CpG sites in the 5' LTR represses transcription to nearly undetectable levels (Bednarik et al. , 1990; Joel et al. , 1993, reviewed by Bednarik, 1992) , but the specific, critical sites that mediate this effect have not been identified.
  • Mammalian promoters are silenced by cytosine-5 methylation at 5'-CpG-3' dinucleotides.
  • the promoter within the 5' LTR of HIV-l may be silenced by directing enzymatic methylation to critical CpG sites. Because methylation patterns are subject to clonal inheritance, the silencing is essentially irreversible .
  • Example 1 developed in Example 1. Further rounds of cyclic in vivo/in vi tro selection (as developed in Example 1) may be used to obtain variants that methylate only CpG sites adjacent to the binding site of the zinc finger moiety.
  • Example 3 yields constructs that methylate critical sites in the HIV-l 5' LTR.
  • these constructs may be introduced into cultured human cells that express a reporter gene driven by the HIV-l 5' LTR as a means of testing the inhibitory effects of targeted de novo methylation.
  • constructs that prove effective in the studies of Example 4 may be introduced into HIV-l producing human T lymphocytes, and methylation of the target sequence and its effect on virus production can be measured directly.
  • the methylating construct may be delivered on a plasmid vector or as part of a recombinant adeno-associated virus (AAV) .
  • AAV adeno-associated virus
  • Zinc finger motifs have been shown to be remarkably versatile DNA binding structures (Choo and Klug, 1994) ; they are independently- folded protein modules that recognize the edges of 3 base pairs in the major groove. Over 1,300 zinc fingers have been identified, and the binding specificity of a large number has been determined (Choo et al. , 1995) .
  • a zinc finger protein designed to bind to a sequence adjacent to a CpG site in the HIV-l 5' LTR can be fused to an attenuated CpG- specific DNA methyltransferase, thereby achieving efficient, selective methylation of that CpG site.
  • a DNA methyltransferase with the desired binding properties has been cloned from a Spiroplasma species; this enzyme, known as M.SssI, has the same sequence specificity as the mammalian DNA methyltransferase (CpG) (as cloned by Bestor; Genbank Accession No. X14805) , but a higher turnover number and no requirement for hemimethylated substrates (Renbaum and Razin, 1992) .
  • One embodiment of this invention is the development and therapeutic application of zinc finger protein/DNA methyltransferase chimeras or agents whose general organization is similar to that of mammalian DNA methyltransferase. These agents selectively methylate and inactivate the 5' LTR of HIV-l. Such agents have considerable promise as therapeutic agents for retroviral diseases, and for other applications where selective inactivation of a promoter sequence is desired.
  • the zinc finger protein/DNA methyltransferase chimera of this invention is similar in design to mammalian DNA methyltransferase, which has been purified (Bestor and Ingram, 1983; Bestor and Ingram, 1985a) , characterized in terms of biochemical activities (Bestor and Ingram, 1985; Bestor, 1987) , the cDNA that encodes the enzyme cloned (Bestor, 1988; Genbank Accession No. X14805; Bestor et al . , 1988) , and the protein demonstrated to be essential for mouse development in a gene disruption study (Li et al. , 1992; Li et al . , 1993) .
  • mammalian DNA methyltransferase has an N-terminal regulatory domain and a C-terminal catalytic domain that closely resembles bacterial restriction methyltransferases.
  • mammalian DNA methyltransferase has an N-terminal regulatory domain and a C-terminal catalytic domain that closely resembles bacterial restriction methyltransferases.
  • embodiments of this invention differ from mammalian DNA methyltransferase in that the regulatory domain targets the catalytic domain to predetermined sequences .
  • the regulatory region of mammalian DNA methyltransferase suppresses the methylation of previously unmethylated sites (Bestor, 1992) , and directs the protein to sites of new DNA synthesis in S
  • the expression construct pCAL7 directs the expression of the DNA methyltransferase M.SssI (Renbaum and Razin, 1992) .
  • This enzyme in present in Spiroplasma species and has the same sequence specificity as mammalian DNA methyltransferase (CpG) , but a higher turnover number and no requirement for hemimethylated substrates (Renbaum and Razin, 1992) .
  • the C- terminal domain of mammalian DNA methyltransferase also has CpG-specific DNA methyltransferase activity and may also be used in practicing this invention, although k cat is much lower than that of M.SssI (Bestor, 1992; Renbaum and Razin, 1992) .
  • the plasmid construct described in Example 1 may be confirmed by restriction analysis and is described in full in the following section and depicted in Figure 5. This construct embodies (in a simple form) most of the principles of the targeted methylation approach described herein.
  • Table 1 shown below offers data which validates the selection against indiscriminate methylation via McrBC restriction of methylated DNA (Table 1) .
  • E. coli strain Input plasmid CFU ⁇ g DNA" 1 (Mer genotype) (methylation status)
  • pSE4 is an unmethylated control plasmid; pCAL7 directs synthesis of M.SssI (Renbaum et al. , 1992) under the control of the tac promoter. Plasmids were introduced into the indicated bacterial strains (Raleigh, 1992; Sutherland et al. , 1992) by electroporation, and transformation efficiencies determined by colony counts on selective media. The values shown are conservative, as IPTG induction was not used in this case, and the plasmid was only partially methylated as a result of basal expression from the p tac promoter; measurements of Hpall resistance indicated ⁇ 50% methylation. IPTG treatment increased the methylation level and increased restriction from a factor of ⁇ 10 3 to IO 4 .
  • a lambda repressor-integrase fusion protein has been shown to direct HIV-l integration to sites in the vicinity of lambda operators (Bushman, 1994) .
  • a chimeric DNA binding protein/DNA methyltransferase will cause preferential methylation of sites in the vicinity of the recognition sequence of the DNA binding protein through a very large increase in the local concentration of DNA methyltransferase. As shown in Figure 3, this may be used to selectively methylate and silence predetermined promoter sequences.
  • the design and selection of such chimeric proteins, and their development into potential therapeutic agents, is an embodiment of the subject invention.
  • the 5' LTR of HIV-l may be one target of inactivation, and a rational, step-wise approach to the this is described below in the Experimental Details.
  • methylating agents As mentioned above, the zinc finger/DNA methyltransferase chimeras discussed and described herein are analogous in structure to mammalian DNA methyltransferase, the enzyme that normally establishes and maintains methylation patterns in the genome. This enzyme has a zinc binding site that has been shown to participate in the discrimination of unmethylated and hemimethylated CpG sites (Bestor, 1992) . Replacement of this zinc-binding region with a tridactyl finger region of known binding specificity confers a new de novo specificity on the enzyme. Such an enzyme may be less immunogenic, and the efficiency of methylation may be increased because the coupling of DNA replication and methylation would be restored (Leonhardt et al. , 1992) . New generations of increasingly effective methylating agents may be developed using this approach.
  • Described herein is a new therapeutic agent. Many delivery agents are described and encompass embodiments of this invention which are technically sufficient to allow delivery to the target cell population.
  • the AAV transducing vector used in the experiments of Example 5 has many advantages, although other transducing vectors with superior properties are also encompassed in this invention. Delivery of the methylating agent to a target cell population has no addi ⁇ tional difficulties over those associated with other molecu ⁇ lar biological therapeutics.
  • the methylating agent need be present only transiently, since the new methylation pattern is subject to clonal inheritance, and gene silencing mediated by this agent is essentially irre ⁇ versible. This advantage is not enjoyed by alternative molecular biological therapeutics. In cases of recurrent infection or relapse due to silencing of an inadequate fraction of the proviral load, further treatments with the original agent or an agent that recognizes a different critical CpG site may be undertaken.
  • the highly versatile nature of the methylating agents makes such an approach more favorable than the extant molecular biological therapeutic agents.
  • Targeted methylation may also be applied to prophylaxis and the prevention of recurrent infections.
  • One promising route involves a zinc finger/DNA methyltransferase chimera that resides in the cytoplasm by virtue of the lack of nuclear localization signals, and which methylates incoming viral DNA during its transit through the cytoplasm.
  • retroviral DNA is accessible to nucleases (and therefore to methyltransferases of similar size) while in the cytoplasm (Bowerman et al. , 1989), and methylation prior to integration would yield a provirus that is transcriptionally silenced from the time of integration. Long-term, stable expression of such a construct could protect recurring infections with HIV-l or other susceptible viruses.
  • Successful development of constructs that can selectively silence specific viral promoters justifies the development of prophylactic derivatives for medical and veterinary applications and are also included as an embodiment of the instant invention.
  • methylating agents described here are very versatile, and can be targeted to other promoters by installation of zinc finger moieties of the appropriate sequence specificity.
  • the choice of HIV-l as a target in this proposal was motivated by the severe threat to public health posed by this virus rather than by any special vulnerability. Slight modification of the design and selection procedures can produce agents that methylate any predetermined sequence, and such agents may be useful in any case in medicine or experimental biology where it is desirable to silence a given promoter.
  • LexA was chosen for its proven ability to direct fused proteins to LexA binding sites (Brent and Ptashne, 1986)
  • M.SssI was chosen because of its high specific activity and the fact that it is the only bacterial methyltransferase that has the same specificity (5'-CpG-3',* Renbaum and Razin, 1992) as the mammalian enzyme (Bestor and Ingram, 1983) .
  • the product of the construct represented in Figure 6 yields an increased local concentration of DNA methyltransferase in the vicinity of LexA binding sites. As described above, this alone does not ensure targeted methylation, as the M.SssI moiety retains intrinsic activity towards all CpG sites and substantial methylation of collateral sites is to be expected; such indiscriminate methylation is lethal to mammalian cells. It is therefore necessary to make the DNA methyltransferase moiety dependent on LexA-mediated DNA binding; this may be accomplished by selection of mutant versions of M.SssI that have reduced intrinsic DNA binding activity. A novel cyclic in vivo/in vi tro selection protocol is used to select mutant proteins of the desired character.
  • Resistance to cleavage by a methylation- sensitive restriction endonuclease provides in vi tro selection for methylation of the target site; growth of cleavage-resistant pools of plasmid in McrBC * strains of E. coli selects against chimeras that methylate non-target sites.
  • McrBC modified cytosine restriction; the system causes the degradation of plasmid DNA that is methylated at many positions but has no effect on DNA that is methylated at single sites;Raleigh, 1992
  • the selection scheme is depicted in Figure 7.
  • the initial construct methylates both specific (that is, the Smal target site) and non-specific CpG sites elsewhere on the plasmid. It is therefore necessary to transfer DNA binding authority from the catalytic moiety to the DNA binding moiety; this is done by selecting for mutations that prevent the methylation of non ⁇ specific sites while allowing methylation of the specific site.
  • the M.SssI moiety is mutagenized by Mn ++ PCR (PCR amplification in the presence of Mn causes misincorporation of nucleotides and therefore transition and transversion mutations) from primers just external to the BamHl and Ascl sites (the primers are depicted as arrows in Figure 8) .
  • the PCR product is cleaved with BamHl and Ascl, the fragment purified by gel electrophoresis and ligated between the Ascl and BamHl sites of pLS ( Figure 6) .
  • An additional selection step may be added to the cyclic selection procedure if the previous two steps give insuffi ⁇ cient enrichment for the clones of interest .
  • Plasmid pools are extracted from McrBC_ bacteria and cleaved with Smal together with one of the 6 methylation-sensitive enzymes that have unique sites in pLS. If both sites are methylated, no cleavage occurs. If both sites are unmethylated, both will be cleaved. Only if one site is methylated and the other unmethylated will unit-length molecules with compatible ends be produced. These unit- length linear molecules are isolated by agarose gel electrophoresis, circularized by treatment with DNA ligase, and reintroduced into E. coli by electroporation. Note that this selection step, as in the case of the other two, can be completed in less than two days.
  • the number of iterations of the mutagenesis/selection protocol that is required to produce constructs with the required characteristics may be variable. However, the procedure is rapid, and multiple iterations are not demanding.
  • Constructs that have the desired binding and enzymatic specificity are sequenced in order to identify mutations that make DNA methyltransferase activity dependent on the binding energy of the DNA binding fusion partner.
  • the nature and position of the mutations are referred to the 3D structure of cytosine methyltransferases M.Hhal
  • the desired mutations may map to one of the many residues that make nonspecific contacts with the DNA backbone (reviewed by Bestor and Verdine, 1994) . These mutations are introduced into the starting constructs of Example 3 so as to reduce the amount of selection to obtain constructs of the desired character.
  • Example 2 Identification of those CpG sites in the HIV-l 5' LTR whose methylation produces maximal repression of transcription. As described previously, viral transcription is strongly repressed by methylation of the HIV-l 5' LTR (reviewed by Bednarik, 1992) . It is very likely that this effect is exerted through a subset of the 11 CpG sites present in the 5' LTR of HIV-l (see Figure 1) .
  • the critical CpG sites are identified by measurements of transcription rates of test constructs that bear single methylated CpG sites. The CpG site that gives the highest degree of inhibition when methylated serves as the target for Examples 3 and 4.
  • the primer is extended with a DNA polymerase; sequenase is the polymerase of choice for this invention because of its high processivity and low yield of partially extended products.
  • the nick at the 5' end of the primer is sealed by incubation with E. coli DNA ligase and the circular double stranded molecules are purified away from single stranded template and oligonucleotide primer by agarose gel electrophoresis.
  • the circular DNA is purified from gel slices, cleared of ethidium bromide by extraction with phenol, collected by ethanol precipitation, and mixed with equimolar amounts of an actin promoter-luciferase expression construct prior to transfection into HL2/3 cells (Ciminale et al. , 1990) .
  • This cell line expresses high levels of the HIV-l proteins Gag, Env, Tat, Rev, and Nef proteins and affords a good simulation of HIV-l transcription in infected T cells; it is especially important to identify CpG sites that suppress transcription when methylated and in the presence of the virus-encoded stimulatory factor Tat.
  • the hemimethylated sites introduced into the constructs are converted to symmetrically methylated sites immediately upon transfection (Busslinger et al. , 1983) .
  • the ratio of CAT activity to lu ⁇ ciferase activity are taken as the measure of inhibition by CpG methylation.
  • Control constructs are either completely unmethylated or completely methylated (by extension of the primer in the presence of dm 5 CTP) .
  • the location of the 11 CpG sites and the method of synthesis of the methylated expression constructs is shown in Figure 5.
  • the plasmid pUC-BENN-CAT and the cell line HL2/3 are distributed by the NIH AIDS Research and Reference Reagent Program, and have been obtained from that source. This experiment is straightforward, and the results are invaluable for the experiments described in Examples 3, 4, and 5.
  • the assays are done both in transiently transfected cells, and in cell clones that have stably integrated the construct; these clones are established by G418 selection after unlinked co- transformation with pSV2Neo and the pUC-BENN-CAT constructs.
  • Example 3 Design, selection, and affinity maturation of zinc finger-DNA methyltransferase chimeras that methylate critical CpG sites in the HIV 5' LTR. There are three important factors to be taken into account when selecting a site for targeted methylation in the HIV-l 5' LTR. First, the site must have the ability to repress transcription when methylated; this is established in the experiments of
  • the binding site for the zinc finger moiety must be adjacent to (rather than within) the recognition sequence of a regulatory factor.
  • the factors that bind to the 5' LTR are all host-encoded and are involved in the transcription of many cellular genes; methylation of such sites is expected to cause unacceptable toxicity through inactivation of vital cellular genes.
  • the recognition sequence is preferably from 6 to 25 base pairs in length and more preferably at least 9 base pairs in length, because such sequences occur by chance only every 4 9 (or 262,144) base pairs. This rarity ensures that target sites do not occur by chance in significant numbers of cellular promoters.
  • Zinc finger proteins that meet the above criteria are selected from combinatorial expression libraries by phage display, and fused to the CpG-specific DNA methyltransferase M.SssI as described in Example 1, and depicted in Figure 9. Rounds of cyclic in vivo/in vi tro selection (also described in Example 1) are used to obtain variants that methylate only CpG sites adjacent to the binding site of the zinc finger moiety. As before, more than one iteration of the mutagenesis/selection procedure may be required, but the process has no difficult or time-consuming steps. Only a few days are required for each iteration, and large numbers of clones (>10 9 per cycle) can be subjected to selection at once.
  • the method depicted in Figure 9 is used to select a tridactyl finger protein that binds to a 9 bp sequence adja- cent to a critical CpG site (identified in Example 2) .
  • the spacing between the recognition sequence and the target CpG site may be adjusted to conform to the sum of the estimated radii of the two globular proteins of masses 30,000 (M.SssI) and 20,000 (the tridactyl zinc finger protein) . This spacing is equivalent to roughly 25 base pairs.
  • the selected finger protein is fused to M.SssI methyltransferase via a random linker to create a second combinatorial expression library. From this point forward the protocol is very similar to that described in Example 1.
  • the construct differs from that shown in Figure 6 only in that LexA is replaced with the coding region for the zinc finger protein, and the LexA binding site is replaced with the HIV-l LTR sequence used for selection.
  • the c-MYC epitope tag is included to allow visualization of the protein on immunoblots and by immunofluorescence.
  • the sequence and spacing of the central CpG in the Smal site and the recognition sequence for the zinc finger protein is the same as that in the HIV-l LTR target sequence; a minimal change that converts the sequences flanking the CpG site to a Smal site is introduced. This change should not pose any problems, as the intrinsic sequence specificity of M.SssI is limited to the CpG dinucleotide (Renbaum and Razin, 1993) .
  • constructs in which the order of the zinc finger and M.SssI moieties has been reversed are constructed and subjected to the selection protocol described above.
  • the data indicate that it is possible to screen between IO 9 and 10 10 independent clones at once, and each iteration of the selection protocol requires only a few days.
  • Example 4 Inhibition of HIV-l 5' LTR-dependent transcription in cultured human cells that express zinc finger/DNA methyltransferase fusion proteins of novel and predetermined specificity.
  • Example 3 yields constructs that methylate critical sites in the HIV-l 5' LTR. These constructs are introduced into cultured human cells that express a reporter gene from the HIV-l 5' LTR as a means of testing the inhibitory effects of targeted de novo methy ⁇ lation.
  • the plasmid pUC-BENN-CAT (Gendelman et al. , 1986) is introduced into HL2/3 cells (Ciminale, 1990) together with unlinked pSV2Neo, and stable transfectants selected with G418.
  • the HL2/3 cell line expresses most of the HIV-l accessory proteins (including Tat) from a replication- defective HIV-l provirus (Ciminale et al. , 1990) .
  • the presence of HIV-l viral accessory proteins renders this assay a good simulation of HIV-l transcription in infected cells.
  • Transfectant clones that express the CAT gene from the HIV-l LTR are identified by RNA blot hybridization.
  • HL2/3-LTR-CAT Such cell clones are referred to as HL2/3-LTR-CAT.
  • HL2/3-LTR-CAT cells are transfected by the calcium phosphate technique with a mammalian expression vector that directs production of the zinc finger-M.SssI fusion protein (Example 3) from the cytomegalovirus (CMV) immediate early enhancer- promoter.
  • the starting vector is pEVRFO (Matthias et al . , 1990) , which contains the CMV promoter, a beta globin intron in the 3' untranslated region for enhanced transcript stability, and a polylinker region for convenient cloning. We have used this vector with good success in many constructs (Czank et al.
  • pCMV-ZMet This expression construct is termed pCMV-ZMet.
  • a pSV2Hyg resistance marker is co-transfected with pCMV-ZMet, and the cells subjected to double selection with G418 and Hygromycin.
  • Production of zinc finger/M.SssI fusion protein are assayed by immunoblot analysis; as with the LexA-M.SssI fusion protein of Figure 3, an N-terminal c-MYC epitope tag is present, and the protein is visualized with the 9E10 antibody.
  • Immunofluorescence microscopy is used to determine whether the methylating agent can gain access to nuclei. If it is found to be confined to the cytoplasm, the nuclear localization signal of SV40 large T antigen may be added to the N-terminus, as we have done for a class of fusion pro- teins to induce their translocation into nuclei (Leonhardt et al. , 1992) .
  • methylation may be assayed by cleavage of DNA with that enzyme followed by DNA blot hybridization. This is a simple and long-established technique for determination of the methylation status of individual CpG sites. If the critical site chosen for targeted methylation does not lie within such a restriction site, the genomic sequencing method of Grigg and Clark. (1994) may be used to identify methylated cytosines. The method of Collins and Myers
  • This method exploits the increased melting temperature of methylated DNA, which allows methylated and unmethylated fragments to be resolved by denaturing gradient gel electrophoresis (Collins and Myers, 1987) . Both methods are well-established and both will allow unambiguous localization of methylated sites in HIV-l LTR sequences.
  • the embodiments described herein of this invention indicate that expression of a finger protein/M.SssI chimera targeted to a critical CpG site in the HIV-l LTR methylates and inactivates expression of a reporter gene that is driven by the LTR.
  • Example 5 Inhibition of HIV-l replication in T lymphocytes productively infected with HIV-l.
  • Jurkat E6-1 cells are electroporated with infectious molecular clones of HIV-l strain NL4-3, and a productive infection established.
  • the HIV-producing cells are transfected with the finger protein/M.SssI construct described in the previous section, together with pSV2Neo as a selectable marker.
  • a vector con ⁇ trol and a finger protein-only control may be used. This latter control is necessary to distinguish inhibition via methylation from that resulting from direct binding of the finger protein to its target site, as has happened in one other case (Choo et al., 1995) .
  • the finger protein/M.SssI chimera is designed to cause de novo methylation and transcriptional repression of HIV-l proviral DNA. Methylation is assayed as described in the previous section, and viral transcription is assayed by RNA blot hy ⁇ bridization.
  • the plasmid-mediated system test described above is convenient and reliable but does not simulate conditions that may be encountered in a therapeutic application.
  • a vector that addresses the needs of therapeutic applications is therefore used and described below.
  • One of the most promising viral vectors is based on adeno-associated virus (AAV) , and the methylating agent may be delivered through use of recombinant AAV.
  • AAV adeno-associated virus
  • AAV has several advantages as a transducing vector: I. AAV is not associated with any disease, ii. The viral DNA is thought to integrate at a unique site on chromosome 19q, with little risk of insertional mutagenesis, iii. it has been reported that AAV can infect non-replicating cells
  • AAV can infect lymphoid cells (Mendelson et al. , 1992) , and transcription units borne by recombinant AAV strains are expressed in T lymphocytes (Chatterjee et al. , 1992) , and v. Virulent strains of AAV cannot arise by recombination within infected cells, as can occur with retroviral vectors.
  • the finger protein/DNA methyltransferase coding region under control of the CMV immediate early enhancer-promoter is amplified by PCR with primers that introduce Xbal sites at both ends of the construct. This expression cassette is ligated between the
  • clones of the desired specificity are termed pAV-ZMet.
  • Control clones that express only the zinc finger protein are constructed and termed pAV-Zif; clones that express only the mutant M.SssI are constructed and termed pAV-Met .
  • Infectious recombinant virus stocks are obtained by co-transfection of adenovirus-infected HeLa cells with pAV-ZMet, pAV-Zif, or pAV-Met, together with pTAAV, a plasmid that encodes proteins required for replication and packaging (Banerjee et al. , 1992) .
  • the titer of virus stocks obtained in this manner are commonly IO 7 mi 1
  • viruses are also capable of infecting CD4 * T lymphocytes such as Jurkat cell with high efficiency (Mendelson et al. , 1992) .
  • the recombinant viruses are termed AAV-ZMet, AAV-Zif, and AAV-Met, in keeping with the terminology of their plasmid precursors .
  • AAV-Met at an MOI that infects >90% of the cell population as established by immunofluorescence microscopy with antibodies against the c-MYC epitope encoded by the fusion proteins.
  • Jurkat cells are highly susceptible to infection by AAV (Mendelson et al. , 1992)
  • RNA blot hybridization is used to evaluate the effect of expression on transcript accumulation, as has been done in antisense inhibition experiments that utilized AAV expression vectors (Banerjee et al., 1992) .
  • the methylation status of integrated HIV-l proviral DNA may be evaluated as described in Example 3.
  • transcript levels, cell survival, and proviral methylation among cultures infected with vector control, AAV-ZMet, AAV-Zif, and AAV-Met can give a clear indication of the efficacy of targeted methylation in suppressing HIV-l expression under conditions that approximate those to be encountered in actual therapeutic applications.
  • Example 6 Design, selection, and affinity maturation of zinc finger-DNA methyltransferase chimeras that methylate critical CpG sites in a promoter of the hepatitis B virus.
  • One embodiment of the subject invention is to target the known promoters of the hepatitis B virus and inactivate the viral machinery via methylation.
  • the site for targeted methylation in the hepatitis B promoter is selected as follows. First , the site will have the ability to repress transcription when methylated; this is established in the experiments of Example 2 above. Second, the binding site for the zinc finger moiety will be adjacent to (rather than within) the recognition sequence of a regulatory factor. The factors that bind to the promoter of the viral X protein or the pre-S2/5 promoter are host-encoded and are involved in the transcription of many cellular genes; methylation of such sites is expected to cause unacceptable toxicity through inactivation of vital cellular genes. Third, the recognition sequence is optimally from 6 to 25 base pairs in length and more preferably at least 9 base pairs in length.
  • Zinc finger proteins that meet the above criteria are selected from combinatorial expression libraries by phage display, and fused to the CpG-specific DNA methyltransferase M.SssI as described in Example 1, and depicted in Figure 9. Rounds of cyclic in vivo/in vi tro selection (also described in Example 1) are used to obtain variants that methylate only CpG sites adjacent to the binding site of the zinc finger moiety. As before, more than one iteration of the mutagenesis/selection procedure may be required, but the process has no difficult or time-consuming steps. Only a few days are required for each iteration, and large numbers of clones (>10 9 per cycle) can be subjected to selection at once.
  • the method depicted in Figure 9 is used to select a tridactyl finger protein that binds to a 9 bp sequence adja ⁇ cent to a critical CpG site in either the promoter of viral protein X or the pre-S2/5 promoter of hepatitis B.
  • the spacing between the recognition sequence and the target CpG site may be adjusted to conform to the sum of the estimated radii of the two globular proteins of masses 30,000 (M.SssI) and 20,000 (the tridactyl zinc finger protein) . This spacing is equivalent to roughly 25 base pairs.
  • FIG 11 A and Figure 11B illustrate targeted methylation of a preselected CpG site in a pLS vector which encodes a lexA- ⁇ f.SssI fusion protein.
  • This data proves that a CpG- specific DNA methyltransferase can be directed to a target site that is adjacent to the binding site of a sequence- specific DNA binding protein. This targeted methylation was achieved in living bacterial cells. Mutagenesis and cyclic in vivo/in vi tro selection is being applied so as to obtain a chimeric protein that will methylate the target site but have no effect on collateral CpG sites.
  • the data shown above and specifically in Figures 11A-11B demonstrate that the principle of targeted methylation has been reduced to practice in the laboratory.
  • the Lac repressor was fused to the N-terminus of M.SssI by recombinant DNA technology and the chimeric protein was expressed in E. coli.
  • the construct used in these experiments is shown in detail in Figure 12.
  • the construct also contained a synthetic sequence that included the Lac repressor binding site and a cluster of CpG dinucleotides.
  • Figure 14 also shows that targeted methylation occurred 20- 30 nucleotides away from the 3' border of the Lac repressor binding site. This is taken to reflect steric interference between protein moieties. The result was expected and is not a limitation of the method.
  • Lac repressor-M.SssI fusion proteins confirmed the principle of targeted methylation.
  • Figure 15 depicts a new class of chimeric DNA methyltransferases that have been constructed and confirmed by DNA sequencing.
  • the sequence- specific DNA binding moiety is a three-finger zinc finger protein (Zif268) of known binding specificity. These agents are being subjected to the same characterization as the Lac repressor-M.SssI fusions. They have also been cloned into mammalian expression vectors and experiments in cultured primate cells are being performed.
  • chimeric DNA methyltransferase-DNA binding proteins have been shown to selectively methylated CpG sites in the vicinity of the recognition sequence of the DNA binding protein moiety.
  • the LTR of HIV-l has been cloned upstream of two reporter genes, chloramphenicol acetyltransferase (CAT) and firefly luciferase (Luc) . Both constructs drive high-level expression of the CAT of Luc reporter genes when transfected into tat-expressing HL2/3 or HLTAT human cervical carcinoma cells.
  • CAT chloramphenicol acetyltransferase
  • Luc firefly luciferase
  • Methylation is known to suppress transcription from most promoters but its effect on HIV-l LTR-driven transcription has not been documented in a system that resembles the infected cells.
  • Figure 17 shows that CpG methylation of 9 CpG sites in the HIV-l LTR reduces transcription to near background levels; the effect is at least 20 fold. The effect appears to be irreversible and does not require continued application or expression of any foreign agents. CpG methylation can therefore be regarded as the most powerful way to suppress the activity of HIV-l. Work is underway that will identify the CpG sites that mediate the biological effects of methylation.
  • M.SssI does not methylate single stranded DNA. This has led to the development of a new method for the region-specific methylation of a DNA fragment.
  • a single-stranded oligonucleotide or longer fragment synthesized by asymmetric PCR is annealed to a single-stranded circular DNA and treated with M.SssI.
  • CpG sites in the double stranded region of the plasmid are methylated while all single stranded regions are unmodified. This new method reduces the time required to identify critical CpG sites.
  • the regional methylation method is being used to evaluate the effects of CpG methylation on the HIV-l LTR versus the gag leader sequence; this latter sequence is rich and has strongly conserved CpG sites ( Figure 16) and methylation of the sites may suppress transcription of HIV-l as well or better than methylation of LTR sequences.
  • Ashburner, M. Drosophila A Labora tory Handbook . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1992. p303.
  • a targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71 , 865-874.
  • Prendergast, G. C, and E. B. Ziff (1991) Methylation- sensitive sequence-specific DNA binding by the c-Myc basic region. Science 251 , 186-189.
  • McrBC A multisubunit GTP-dependent restriction endonuclease. J. Mol . Biol . 225, 327-348. Raleigh, E. A. (1992) Organization and function of the mcrBC genes of Escherischia coli K-12. Mol . Micro . 6, 1079-1086.
  • Zinc finger phage affinity selection of fingers with new DNA-binding specificities. Science 263 , Gil -673.
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention porte sur une protéine chimère qui comprend une partie ADN méthyltransférase mutée, et une partie protéine de liaison d'ADN qui lie suffisamment étroitement une séquence promoteur d'un gène cible, ladite séquence promoteur contenant un site de méthylation, afin de méthyler spécifiquement le site et d'inhiber l'activité du promoteur et donc l'expression du gène cible. Cette invention porte également sur un procédé d'inhibition de l'expression d'un gène cible, qui consiste à mettre le gène cible en contact avec la protéine chimère, de façon à méthyler spécifiquement la séquence promoteur du gène cible, l'expression du gène cible étant ainsi inhibée.
PCT/US1996/015576 1995-09-28 1996-09-27 Proteine chimere de polypeptide et d'acide nucleique de liaison d'adn et d'adn methyltransferase et ses utilisations WO1997011972A1 (fr)

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* Cited by examiner, † Cited by third party
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US6200782B1 (en) * 1992-06-22 2001-03-13 City Of Hope Construction of nucleoprotein based assemblies comprising addressable components for nanoscale assembly and nanoprocessors
WO2001002553A3 (fr) * 1999-06-30 2001-05-03 Us Health Sequence et proteine de liaison a l'adn utilisees en tant qu'isolants dotes d'une activite de blocage d'amplificateurs specifiques pour la regulation d'une expression genique
WO2001032857A1 (fr) * 1999-10-29 2001-05-10 The Hospital For Sick Children Methylation de sequences instables
US6383784B1 (en) 1999-12-03 2002-05-07 City Of Hope Construction of nucleoprotein based assemblies comprising addressable components for nanoscale assembly and nanoprocessors
WO2001040478A3 (fr) * 1999-12-06 2002-05-10 Pasteur Institut Polynucleotides isoles a teneur reduite ou renforcee en motifs de regulation epigenetiques et leur utilisation
WO2001083793A3 (fr) * 2000-04-28 2002-06-20 Sangamo Biosciences Inc Modification ciblee de la structure de chromatine
GB2380195A (en) * 2001-07-24 2003-04-02 Gene Expression Technologies L Control of gene expression
WO2002026960A3 (fr) * 2000-09-29 2003-04-17 Sangamo Biosciences Inc Modulation de l'expression genique au moyen de domaines de localisation
WO2002006527A3 (fr) * 2000-07-13 2003-10-02 Transgenomic Inc Detection de proteines tronquees
US6919204B2 (en) 2000-09-29 2005-07-19 Sangamo Biosciences, Inc. Modulation of gene expression using localization domains
US7125857B2 (en) 1997-08-29 2006-10-24 The Regents Of The University Of California Modulators of DNA cytosine-5 methyltransferase and methods for use thereof
JP2011519280A (ja) * 2008-04-30 2011-07-07 サンバイオ，インコーポレイティド Ｄｎａメチル化における改変を伴う神経再生細胞連邦政府の援助に関する申告適用なし
EP3463484A4 (fr) * 2016-05-27 2019-10-30 The Regents of the University of California Procédés et compositions pour cibler des arn polymérases et la biogenèse de l'arn non codant sur des loci spécifiques
EP4262849A4 (fr) * 2020-12-15 2024-10-30 Flagship Pioneering Innovations V, Inc. Compositions et méthodes de modulation de l'expression de myc
USRE50635E1 (en) 2009-04-30 2025-10-14 Sanbio, Inc. Neural regenerating cells with alterations in DNA methylation

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ANN. REV. GENET., 1986, Vol. 20, PALMITER et al., "Germ-Line Transformation of Mice", pages 465-99. *
NUCLEIC ACIDS RESEARCH, 1992, Vol. 20, No. 22, ZHUKOVSKAYA et al., "Inactive 06-Methylguanine-DNA Methyltransferase in Human Cells", pages 6081-6090. *
PROC. NATL. ACAD. SCI. U.S.A., December 1995, Vol. 92, PRASAD et al., "Domains with Transcriptional Regulatory Activity within the ALL1 and AF4 Proteins Involved in Acute Leukemia", pages 12160-12164. *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6200782B1 (en) * 1992-06-22 2001-03-13 City Of Hope Construction of nucleoprotein based assemblies comprising addressable components for nanoscale assembly and nanoprocessors
US7138384B1 (en) 1997-08-29 2006-11-21 The Regents Of The University Of California Modulators of DNA cytosine-5 methyltransferase and methods for use thereof
US7125857B2 (en) 1997-08-29 2006-10-24 The Regents Of The University Of California Modulators of DNA cytosine-5 methyltransferase and methods for use thereof
WO2001002553A3 (fr) * 1999-06-30 2001-05-03 Us Health Sequence et proteine de liaison a l'adn utilisees en tant qu'isolants dotes d'une activite de blocage d'amplificateurs specifiques pour la regulation d'une expression genique
WO2001032857A1 (fr) * 1999-10-29 2001-05-10 The Hospital For Sick Children Methylation de sequences instables
US6383784B1 (en) 1999-12-03 2002-05-07 City Of Hope Construction of nucleoprotein based assemblies comprising addressable components for nanoscale assembly and nanoprocessors
WO2001040478A3 (fr) * 1999-12-06 2002-05-10 Pasteur Institut Polynucleotides isoles a teneur reduite ou renforcee en motifs de regulation epigenetiques et leur utilisation
WO2001083793A3 (fr) * 2000-04-28 2002-06-20 Sangamo Biosciences Inc Modification ciblee de la structure de chromatine
US7001768B2 (en) 2000-04-28 2006-02-21 Sangamo Biosciences, Inc. Targeted modification of chromatin structure
AU2001253914B2 (en) * 2000-04-28 2006-06-08 Sangamo Therapeutics, Inc. Targeted modification of chromatin structure
US8071370B2 (en) 2000-04-28 2011-12-06 Sangamo Biosciences, Inc. Targeted modification of chromatin structure
US7785792B2 (en) 2000-04-28 2010-08-31 Sangamo Biosciences, Inc. Targeted modification of chromatin structure
WO2002006527A3 (fr) * 2000-07-13 2003-10-02 Transgenomic Inc Detection de proteines tronquees
WO2002026960A3 (fr) * 2000-09-29 2003-04-17 Sangamo Biosciences Inc Modulation de l'expression genique au moyen de domaines de localisation
US6919204B2 (en) 2000-09-29 2005-07-19 Sangamo Biosciences, Inc. Modulation of gene expression using localization domains
GB2380195A (en) * 2001-07-24 2003-04-02 Gene Expression Technologies L Control of gene expression
JP2011519280A (ja) * 2008-04-30 2011-07-07 サンバイオ，インコーポレイティド Ｄｎａメチル化における改変を伴う神経再生細胞連邦政府の援助に関する申告適用なし
EP3118308A1 (fr) 2008-04-30 2017-01-18 SanBio, Inc. Cellules régénérantes neurales présentant des modifications dans la méthylation de l'adn
US10316288B2 (en) 2008-04-30 2019-06-11 Sanbio, Inc. Neural regenerating cells with alterations in DNA methylation
EP3851520A1 (fr) 2008-04-30 2021-07-21 SanBio, Inc. Cellules régénérantes neurales présentant des modifications dans la méthylation de l'adn
USRE50635E1 (en) 2009-04-30 2025-10-14 Sanbio, Inc. Neural regenerating cells with alterations in DNA methylation
EP3463484A4 (fr) * 2016-05-27 2019-10-30 The Regents of the University of California Procédés et compositions pour cibler des arn polymérases et la biogenèse de l'arn non codant sur des loci spécifiques
US11286493B2 (en) 2016-05-27 2022-03-29 The Regents Of The University Of California Methods and compositions for targeting RNA polymerases and non-coding RNA biogenesis to specific loci
US12043839B2 (en) 2016-05-27 2024-07-23 The Regents Of The University Of California Methods and compositions for targeting RNA polymerases and non-coding RNA biogenesis to specific loci
EP4262849A4 (fr) * 2020-12-15 2024-10-30 Flagship Pioneering Innovations V, Inc. Compositions et méthodes de modulation de l'expression de myc

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