WO2007024029A1 - Agent antiviral et inhibiteur de replication virale - Google Patents

Agent antiviral et inhibiteur de replication virale Download PDF

Info

Publication number
WO2007024029A1
WO2007024029A1 PCT/JP2006/317295 JP2006317295W WO2007024029A1 WO 2007024029 A1 WO2007024029 A1 WO 2007024029A1 JP 2006317295 W JP2006317295 W JP 2006317295W WO 2007024029 A1 WO2007024029 A1 WO 2007024029A1
Authority
WO
WIPO (PCT)
Prior art keywords
zfp
antiviral agent
replication
domain
protein
Prior art date
Application number
PCT/JP2006/317295
Other languages
English (en)
Inventor
Takashi Sera
Original Assignee
Kyoto University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyoto University filed Critical Kyoto University
Priority to JP2008527641A priority Critical patent/JP2009506025A/ja
Publication of WO2007024029A1 publication Critical patent/WO2007024029A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an antiviral agent and a viral replication inhibitor for animal DNA viruses.
  • One conventional method to inhibit binding of a viral replication protein to a replication origin is, for example, to use a "dominant-negative variant" of that protein so that the variant only has the DNA binding site of the viral replication protein (Orozco et ah (2000) J. Biol. Chem. 275-' 6114- 6122).
  • a dominant-negative variant When such a dominant-negative variant is bound to the replication origin of the virus, it inhibits binding of the protein that induces initiation of viral replication to that replication origin.
  • the binding ability of the dominant-negative variant is almost equal to that of the target viral replication protein, and accordingly, expression of a large amount of the dominant-negative variant is required to effectively inhibit binding to the replication origin by a competition between the variant and the wild-type viral replication protein.
  • a problem arises because expression of the variant protein in an amount sufficient to inhibit the binding to the replication origin in vivo is extremely difficult.
  • high level expression of the dominant-negative variant can be achieved, such levels may induce cellular toxicity via non-specific binding of the variant to sequences other than the desired target sequence. Accordingly, there is a need for high-affinity DNA binding proteins since these can be used at lower expression levels .
  • the ZFPs reported therein attempted to provide a means for preventing viral infection of plants using those rationally-designed ZFPs. For example, prevention of plant DNA virus infection using artificial ZFPs was reported by Sera (2005) J. Virology 79: 2614-2619.
  • An object of the present invention is to provide an antiviral agent and a viral replication inhibitor against viruses that are harmful to animals including human. To achieve this object, the present invention provides that viral replication is effectively preventable by inhibiting the binding of a replication protein of an animal virus to a replication origin of that virus. Based on such binding inhibition, excellent antiviral agents are obtainable.
  • the present invention thus provides the following antiviral agents and viral replication inhibitors which are substances that inhibit binding of a replication protein derived from an animal DNA virus to a replication origin of the virus as an active ingredient, wherein said the substance is a zinc finger protein.
  • an antiviral agent comprising an artificial zinc finger protein (ZFP) and/or a nucleic acid encoding said ZFP, wherein the ZFP is capable of inhibiting binding of a replication protein from an animal DNA virus to a replication origin of that virus.
  • the replication protein can have two or more binding sites in the replication origin of the DNA virus. In such cases, the ZFP can binds to one, two or more of such binding sites to prevent binding of the replication protein at the replication origin.
  • the antiviral agent is for a human papilloma virus (HPV), preferably HPV18, but it can also be for another high risk HPV such as HPV type 16, 31, 35, 39, 45, 51, 52, 58 or 59.
  • HPV human papilloma virus
  • the preferred target replication protein is the E2 protein and the artificial ZFP binds at, near or overlapping with the E2 binding site 3 (E2BS3) and/or the E2 binding site 4 (E2BS4).
  • the antiviral agent for HPV targets DNA represented as GAAAACGGTCGGGACCGAA or GGTCGGGACCGAAAACGGT.
  • the ZFP of the antiviral agent comprises 6 zinc finger domains with the domains covalently joined with from 0 to 10 amino acid residues.
  • the amino acids at positions -1, 2, 3 and 6, (which are sometimes referred to as the recognition amino acids) of the ⁇ -helix of the zinc finger domains are, in order, respectively,
  • amino acids at positions -1, 2, 3 and 6 of the domains are, in order, respectively,
  • recognition amino acids can be part of the framework provided by the formula
  • the ZFP comprises an amino acid sequence of SEQ ID NO: 2 or 3.
  • Antiviral agents of the invention also include ZFPs which comprise at least three zinc finger domains covalently joined to each other with from 0 to 10 amino acid residues, and amino acids at positions -1, 2, 3 and 6 of the ⁇ - helix of the zinc finger domains are selected as follows ⁇ the amino acid at position —1 is arginine, glutamine, threonine, methionine or glutamic acid; the amino acid at position 2 is serine, asparagine, threonine or aspartic acid; the amino acid at position 3 is histidine, asparagine, serine or aspartic acid; and the amino acid at position 6 is arginine, glutamine, threonine, tyrosine, leucine or glutamic acid.
  • antiviral agents of the invention include ZFPs comprising at least three zinc finger domains, and wherein each zinc finger domain is independently represented by the following formula :
  • X is, independently, any amino acid
  • X n represents the number of occurrences of X in the polypeptide chain!
  • Z' 1 is arginine, glutamine, threonine, methionine or glutamic acid
  • Z 2 is serine, asparagine, threonine or aspartic acid
  • Z 3 is histidine, asparagine, serine or aspartic acid.
  • Z 6 is arginine, glutamine, threonine, tyrosine, leucine or glutamic acid, and said domains are covalently joined to each other with from 0 to 10 amino acid residues.
  • the ZFP can further comprise a nuclear- localization signal (NLS) and/or a cell-penetrating peptide (CPP), which may be located to the N- or C-terminus of the ZFP open reading frame (ORF).
  • NLS nuclear- localization signal
  • CPP cell-penetrating peptide
  • ORF ZFP open reading frame
  • any of the ZFPs or nucleic acids encoding those ZFPs are a viral replication inhibitor which is the ZFP or nucleic acid of any one of claims 1-17.
  • a still further aspect of the invention is directed to the use of any of the ZFPs and/or a nucleic acid encoding those ZFP of the invention for the manufacture of an antiviral agent.
  • Another embodiment of the invention provides a method for prophylactic and/or therapeutic treatment of animal viral infection by administering a prophylactically- and/or therapeutically effective amount of an antiviral agent of the invention to an animal to thereby treat, reduce or ameliorate said viral infection.
  • a further embodiment of the invention relates to a method of inhibiting replication of an animal virus in a living body of an animal which comprises administering an effective amount of a ZFP of the invention and/or a nucleic acid encoding a ZFP of the invention to thereby inhibit replication of the virus.
  • the method is used to inhibit human DNA viruses in a human, including HPV, and preferably an HPV type associated with a high risk of cancer such as HPV type 16, 18, 31, 35, 39, 45, 51, 52, 58 or 59.
  • FIG. 1 is a schematic diagram showing the binding of one unit of a zinc finger domain to a four base pair DNA target site. The residues at positions - 1, 2, 3, and 6 each independently contact one base. Position -1 is the start of the ⁇ -helix in a zinc finger domain.
  • FIG. 2 shows known and possible base interactions with amino acids. Interactions similar to those shown between guanine (G) and histidine (His) can be made with other amino acids that donate hydrogen bonds (serine, lysine and the like). Interactions similar to those shown between thymidine and threonine can be made with other hydrophobic amino acids. Interactions similar to those shown between thymidine and threonine/serine can be made with other amino acids that donate hydrogen bonds.
  • FIG. 3 shows the recognition of 4th base in a four base pair DNA target sequence by amino acids at position 2 in a zinc finger domain.
  • FIG. 4 shows a ZFP (zinc finger domain) target sequence and particularly shows the organization of the HPV- 18 replication origin and amino acid sequences for ZFP inhibition of DNA replication.
  • the boxes indicate the E2"binding site (E2BS) E2BS-3 and E2BS-4, respectively, and indicate the 12-bp DNA sequence recognized by the E2 protein.
  • the numbers below the boxes indicate their locations (in nt) in the HPV- 18 genome.
  • the two 19-bp DNA targets chosen for ZFPHPVI and ZFPHPV2 are also indicated.
  • FIG. 5 shows the amino acid sequences of six zinc finger domains for each of ZFPHPVI and ZFPHPV'2.
  • the underlined amino acids in each finger domain indicate the recognition amino acids at positions -1, 2, 3, and 6 in the ⁇ -helix of the finger domain.
  • FIG. 6 shows the autoradiographs from DNA binding assays with ZFPHPVI and ZFPHPV"2 (two left panels) and with E2 (right panel).
  • ZFPHPVI and ZFP H pv2 a 32 P-labeled 56"bp probe including E2BS-3 and E2BS-4 was used.
  • a 32 P-labeled 4Obp probe containing a single E2BS was used for the E2 protein. Protein concentrations are indicated above the lanes. The locations of DNA-ZFP complex and free DNA are indicated.
  • FIG. 7 shows inhibition of HPV- 18 DNA replication by ZFP genes in a transient replication assay.
  • Transient replication assays were performed with pRL-El, pRL-E2, and pUOOril77 along with the ZFP expression plasmid indicated below each lane.
  • Hirt-extracted samples were digested with Dpnl before Southern blot hybridization with a probe specific for the ampicillin resistant gene of pUCOril77.
  • FIG. 8 shows inhibition of E2 binding by ZFPs.
  • the competitive binding experiment with E2 and ZFPHPVI or ZFPHPV'2 were performed with the 32p-labeled 56-bp probe containing E2BS-3 and E2BS-4 along with a constant level of E2 protein (100 nM) in the presence of increasing concentrations of the ZFP (nM), from lane 8 to lane 5 ZFPHPVI and from lane 12 to lane 9 for ZFPHPV2.
  • Lanes 1 to 4 indicate band positions of free DNA, DNA bound to E2, DNA bound to ZFPHPVI, and DNA bound to ZFPHPV"2, respectively, which are used as markers for lanes 5 to 12.
  • the concentrations of ZFP and E2 protein used are indicated below each lane.
  • FIG. 9 shows HPV- 18 DNA replication inhibition by a ZFP with a cell penetrating domain and a nuclear localization signal using the transient viral replication assay.
  • FIG. 10 shows in vitro specific binding of ZFPHPVI and ZFPHPV2 DNA binding to the replication origin using mutant DNA probes.
  • the top panel (a) provides the DNA sequences of the mutant probes. Mutations are underlined and the lane numbers correspond to those shown above panels a to g.
  • Panels b, d and f show the autoradiographs for 10, 100 and 1000 pM ZFPHPVI.
  • Panels c, e and g show the autoradiographs for 10, 100 and 1000 pM ZFPHPV2. The locations of DNA-ZFP complex and free DNA are indicated.
  • FIG. 11 shows specific binding of an artificial DNA binding protein to a replication origin in a cell as measured in a transient replication assay.
  • the mutant probes and mutant ori plasmids are shown in panel a.
  • the mutations were introduced into the 4-bp spacer DNA (i.e., 5_-CGGG-3_J located between E2BS-3 and E2BS-4 to minimize any negative effect on HPV- 18 DNA replication due to the mutations.
  • Panel b shows a gel shift assay for mutant probes
  • panel c shows transient replication of HPV- 18 mutant ori plasmids.
  • the transient replication assays used pRLrEl, pRLr E2, and a pUOOril77 derivative with pCMV- ZFP H pv"2or pcDNA 3.1 (as indicated below each lane.
  • FIG. 12 compares inhibition of the viral replication by E2C-R9 and ZFPHPV2-R9 in a transient replication assay.
  • FIG. 13 shows a Western blot analysis of the time course of the amount of the ZFP HPV 2"CPP (cell membrane-penetrating peptide) -fused proteins taken up by cells after addition of those proteins to the cellular medium.
  • ZFP HPV 2"CPP cell membrane-penetrating peptide
  • animal viruses include various animal DNA viruses that can infect mammals including human and cattle, birds including chicken, cultivable fish and shellfish and the like.
  • human viruses such as papilloma viruses, herpes viruses, EB viruses, rotaviruses, hepatitis viruses such as HBV, adenoviruses, and poxviruses
  • HPVs human papilloma viruses
  • HPVs human papilloma viruses
  • HPVs human papilloma viruses
  • HPVs human papilloma viruses
  • HPV- 16, HPV- 18 and the like are believed to cause uterine cervix cancer, and thus it is expected that a prophylactic or therapeutic effect for uterine cervix cancer can be obtained by inhibiting replication of papilloma virus. Therefore, human papilloma viruses are preferred targets of the antiviral agent of the present invention.
  • ZFPs having an antiviral action or viral replication inhibitory action are disclosed in the Sera application, its corresponding non-U.S. applications, the Sera 2002 reference and the like. Those ZFPs are generally for inhibition of plant viruses.
  • ZFPs that can be used as an active ingredient of the antiviral agent of the invention preferably recognize and specifically bind, per a single finger, to, for example, four (4) specific bases of double -stranded DNA at the replication origin of an animal DNA virus.
  • the zinc finger proteins may bind to a binding site of one replication protein, or may have two or more units that can bind to binding sites of two or more different replication proteins. Any number of binding sites at the origin of replication can be targeted for binding by a ZFP to produce an antiviral agent in accordance with the invention.
  • Animal DNA viruses have one or more unique replication proteins that can bind to a replication origin to initiate viral replication.
  • human papilloma viruses have replication proteins El and E2, and if binding of one or both of these replication proteins is inhibited, replication of human papilloma viruses is inhibited.
  • the E2 binding sites are more preferred as the target thereof.
  • the E2 binding site comprises 4 binding sites (E2BS-1, E2BS-2, E2BS-3 and E2BS-4) in the replication origin. Among them, E2BS-3 and E2BS-4 shown in FIG. 4, are more preferred as targets of the ZFP.
  • E2BS-3 and E2BS-4 correspond to positions 42 to 53 and 58 to 69, respectively, on the 7857 base pairs of the genome of HPV 18, and corresponding positions in papilloma viruses other than HPV- 18 can be easily identified by those of skill in the art by comparison of the appropriate DNA sequences.
  • the sequence of the ZFP so that the agent can bind to both of E2BS-3 and E2BS-4.
  • the ZFP has 6 domains and recognizes 19 contiguous base sequences (each domain recognizes four bases)
  • ZFPHPVI and ZFPHPV described in detail in the Examples, were designed for target sequences 44-62 and 51-69, respectively.
  • binding of the replication protein to the replication origin can be more specifically inhibited by designing a ZFP to bind across the two or more binding sites.
  • Zinc finger proteins that can be suitably used as the antiviral agent of the invention have at least one zinc finger domain using, for example, four base pair target sequence.
  • Multi-fingered (multi-domained) ZFPs, designed for longer target sequences, as discussed below, are useful as the antiviral agent of the invention.
  • ZFP zinc finger protein
  • a ZFP polypeptide having DNA binding domains that are stabilized by zinc.
  • Each of the DNA binding domains is typically referred to as “finger” or a “zinc finger domain,” such that a ZFP has at least one finger, more typically two fingers, more preferably three fingers, or even more preferably four or five fingers, to at least six or more fingers.
  • each finger binds three or four base pairs of DNA, and thereby ZFP specifically binds to.
  • a nucleic acid sequence called a target nucleic acid sequence.
  • Each finger usually comprises an approximately 30-amino acid, zinc-chelating, DNA-binding subdomain.
  • a representative motif of one class, the Cys2"His2 class is -CYS-(X)2-4"CYS-(X) 1 2"His-(X)3-5"His, where X is any amino acid, and a ZFP containing this motif consists of an alpha helix containing the two invariant histidine residues and the two cysteine residues of a single beta turn (see, e.g., Berg et al., Science 271:1081-1085 (1996)) that binds a zinc cation.
  • the zinc finger protein contains one or more, preferably 3 to 40, more preferably 3 to 15 of zinc finger domains of the following formula ⁇
  • the zinc finger protein used as the active ingredient of the antiviral agent of the present invention may be a multi-domained ZFP, in which each zinc finger domain is independently represented by the formula above.
  • the target nucleic acid sequence has a length of 3N+1 base pairs, wherein N is the number of overlapping four base pair segments in that target and is obtained by dividing the target nucleic acid sequence into overlapping four base-pair segments, wherein the fourth base of each segment, up to the N-I segment, is the first base of the immediately following segment.
  • zinc finger protein used as the active ingredient of the antiviral agent of the present invention comprises at least three zinc finger domains covalently joined to each other with from 0 to 10 amino acid residues, wherein the amino acids at positions -1, 2, 3 and 6 of the alpha-helix of the zinc finger are selected from the preferred embodiments mentioned above.
  • ZFPs of preferred embodiment may comprise at least three zinc finger domains, each independently represented by the following formula
  • X represents amino acids in a framework of a Cys2- His2 zinc finger domain and can be a known zinc finger framework, a consensus framework, a framework obtained by varying the sequence of any of these frameworks, or any artificial framework that maintains the overall structure of zinc finger domain.
  • known frameworks are used to determine the identities of each X.
  • Preferred frameworks are those from SpIC and Zif268.
  • a more preferred framework is domain 2 form SpIC.
  • the ZFPs used for the antiviral agent of the present invention comprise from 3 to 40 zinc finger domains, and preferably, 3 to 15 domains, 3 to 12 domains, 3 to 9 domains or 3 to 6 domains, as well as ZFPs with 3, 4, 5, 6, 7, 8 or 9 domains.
  • ZFPs are those wherein, independently or in any combination, Z' 1 is methionine in at least one of said zinc finger domains; Z' 1 is glutamic acid in at least one of said zinc finger domains; Z 2 is threonine in at least one of said zinc finger domains; Z 2 is serine in at least one of said zinc finger domains; Z 2 is asparagine in at least one of said zinc finger domains!
  • Z 6 is glutamic acid in at least one of said zinc finger domains; Z 6 is threonine in at least one of said zinc finger domains; Z 6 is tyrosine in at least one of said zinc finger domains; Z 6 is leucine in at least one of said zinc finger domains and/or Z 2 is aspartic acid in at least one of said zinc finger domains, but Z" 1 is not arginine in the same domain.
  • ZFPs used as the antiviral agents of the present invention may contain one or more nuclear localization signal (NLS) and/or one or more cell membrane -penetrating peptide (CPP). Addition of these CPP and NLS are particularly useful when the antiviral agent is a protein.
  • the active ingredient of the antiviral agent of the present invention preferably contains both of the nuclear localization signal and the cell membrane-penetrating peptide, which may be located on either the N- terminal or C-terminal side of the ZFP ORF. Specific examples thereof include the following structures ' -
  • the ZFPs, and the nucleic acids encoding those ZFPs include both a CPP and an NLS.
  • NLS is an amino acid sequence necessary to localize proteins in the nucleus of a eukaryotic cell through the nuclear membrane structure in the cell.
  • the type of NLS is not particularly imported provided that the NLS has an activity to localize proteins in the nucleus.
  • NLS derived from SV40 large-T antigen polynucleotide coding for the region of 126th to 132nd amino acids of the large-T antigen, Proc. Natl. Acad. Sci. (1989) 86: 9327-9331
  • NLS of HIF- l ⁇ and the like are preferred.
  • the CPP can be any sequence that promotes penetration of a ZFP into cells.
  • peptides containing many basic amino acids such as Tat peptide and polylysine are preferred.
  • cell membrane-penetrating peptide include, but not limited to, the followings. Those derived from natural proteins:
  • HIV TAT amino acids 47-57
  • Antp homeodomain (amino acids 43-58)
  • the method for adding a CPP and/or NLS to a ZFP is not particularly limited.
  • DNA encoding such domains can be directly ligated to DNA encoding ZFP with or without a cleavable peptide linker (which may have a protease cleavage site) in E. coli, insect cells, animal cells or whatever cells that are used as the expression system.
  • a CPP and/or NLS domain can be chemically bound to a ZFP via a bifunctional or polyfunctional linker including succinimide and/or maleimide.
  • such proteins can be prepared as an aggregate based on a non-covalent bond using protein interactions such as cJun-cFos.
  • a binding product obtained by binding CPP to cJun and a binding product obtained by binding cFos to ZFP may be used to prepare an aggregate thereof.
  • the active ingredient of the antiviral agent or the viral replication inhibitor of the present invention may be a polynucleotide that can express a zinc finger protein such as an expression vector containing a nucleic acid encoding the zinc finger protein.
  • the expression vector can be constructed by a known method using a DNA or RNA virus vector or plasmid vector that can express a protein in animal cells such as mammal cells (see, Molecular Cloning, Cold Spring harbor Laboratory, A laboratory manual, 1989 or any other recombinant DNA text).
  • coli such as the pET series, ⁇ CR4, pCR2.1, pBR322, pBR325, pUC12 and pUC13, plasmids derived from yeast (pSH19, pSHl ⁇ etc.), bacteriophage such as ⁇ phage, animal viruses such as retroviruses, vaccinia viruses and adenoviruses and the like may be used.
  • These expression vectors can be introduced into animal (especially human) cells by using liposomes (especially cationic liposomes), if necessary, or other techniques know to those of skill in the art.
  • an antiviral agent comprising a zinc finger protein as an active ingredient is administered to an animal, it is preferable to administer it as a fusion protein with at least a CPP.
  • nucleic acid- contacting residues in zinc finger domains are primarily responsible for determining specificity and affinity and occur in the same position relative to the first consensus histidine and second consensus cysteine.
  • the first residue is seven residues to the N-terminal side of the first consensus histidine and six residues to the C-terminal side of the second consensus cysteine. This residue is hereinafter referred to as the residue at " ⁇ 1 position.”
  • the other three amino acids are two, three and six residues removed from the C-terminus of the residue at position -1, and are referred hereafter to as the residues at "2 position", "3 position” and "6 position", respectively. These positions are interchangeably referred to as the Z' 1 , Z 2 , Z 3 and Z 6 positions.
  • Position - 1 is the start of the alpha helix in a zinc finger domain.
  • the location of amino acid positions -1, 2, 3 and 6 in a zinc finger domain, and the bases they contact in a four base pair DNA target sequence, are shown schematically in FIG. 1.
  • a zinc finger-nucleic acid recognition code is shown in Table 1 and is based on known and possible base-amino acid interactions (FIG. 2). Some interactions listed in FIG. 2 are also identified in different proteins such as H- T-H protein, cro and the lambda repressor. For recognition of the first and third DNA bases in a four base pair region, amino acids containing longer side chains can be chosen. For recognition of the second and fourth bases, amino acids containing shorter side chains can be chosen. For example, in the case of guanine base recognition, arginine can be chosen as an amino acid at positions "1 and 6, histidine can be chosen as an amino acid at position 3 and serine can be chosen as an amino acid at position 2.
  • amino acids shown in Table 1 there is stable interaction with specific DNA bases of the replication origin serving as a target (e.g., in the examples, E2 protein binding site of papilloma virus) by hydrogen bonding.
  • DNA bases of the replication origin serving as a target
  • amino acids having hydrophobic side chains are also chosen (i.e., leucine for first thymidine base and methionine for third thymidine base).
  • Other DNAbase-amino acid interactions are possible * ' however, amino acids with the highest affinity can be chosen.
  • arginine can be preferably chosen because of additional hydrogen bonding.
  • the recognition of the four bases in a four base pair DNA sequence (1st base of a neighboring 3' triplet DNA) by amino acids at position 2 is shown in FIG. 3. Asp, Thr, Asn and Ser at position 2 of a zinc finger domain preferentially bind to C, T, A and G, respectively.
  • the fourth base is in the anti-sense nucleic acid strand.
  • the bases are always provided in 5' to 3' order.
  • the fourth base listed in the table is always the complement of the fourth base provided in the target sequence.
  • the target sequence is written as ATCC, then it means a sense strand target sequence of 5'-ATCO3' and an antisense strand of 3'-TAGG"5 ⁇
  • the first base of A means there is glutamine at position 6
  • the second base of T means there is serine at position 3
  • the third base of C means there is glutamine at position -1.
  • the fourth base written as C it means that it is the complement of C, i.e., G, which is found in the table and used to identify the amino acid of position 2. In this case, the amino acid at position 2 is serine.
  • Z 6 is threonine if the first base is T and where Z' 1 is threonine if the third base is T.
  • a recognition code enlarged to generally provide additional conservative amino acids for those present in the recognition code of Table 1 is below provided in Table 2.
  • Table 2 the order of amino acids listed in each box represents, from left to right, the most preferred to least preferred amino acid at that position.
  • the framework determined by the identity of X can be a known zinc finger framework, a consensus framework or an alteration of any one of these frameworks provided that the altered framework maintains the overall structure of zinc finger domain.
  • Preferred frameworks are those from SpIC and Zif268.
  • a more preferred framework is domain 2 form SpIC.
  • the proteins containing the designed zinc finger domain can be prepared either synthetically or recombinantly, preferably recombinantly, using any of the multitude of techniques well-known in the art.
  • the codon usage can be optimized for high expression in the organism in which that ZFP is to be expressed.
  • organisms include bacteria, fungi, yeast, animals, and insects, but are not limited to these examples.
  • the antiviral agent is to be a ZFP protein, one could express large quantities of that protein in a protein expression from any of those organisms, purify it and then use for administration to an animal.
  • a multi-domained (i.e., a multi-fingered) ZFP the above method for designing a single domain ⁇ e.g., a method of using a preferred framework such as the domain 2 from SpIC) can be followed, especially if the domains are not contiguous.
  • a preferred framework such as the domain 2 from SpIC
  • ZFPs designed by dividing the target sequence into overlapping four base pair segments provides a context-independent zinc finger recognition code from which to produce ZFPs, and typically, ZFPs with high binding affinity, especially when there are more than 3 zinc finger domains in the ZFP.
  • the target sequence has a length of 3N+1 base pairs, wherein N is the number of overlapping four base pair segments in the target and is determined by dividing the target sequence into overlapping four base pair segments, where the fourth base of each segment, up to the N-I segment, is the first base of the immediately following segment.
  • the remainder of the design method for each four base pair segment follows that of a single domain with respect to determining the identities of each X, Z" 1 , Z 2 , Z 3 and Z 6 .
  • This method is useful for designing ZFPs having from 3 to 15 domains (i.e., N is any number from 3 to 15), and more preferably from 3 to 12 domains, from 3 to 9 domains or from 3 to 6 domains. Since ZFPs with more than 40 domains are known in the art, if desired, N can range to at least 40, if not more.
  • the zinc finger domains designed are either covalently joined directly one to another or can be separated by a linker region of from 1 to 10 amino acids or a peptide.
  • the linker amino acids can provide flexibility or some degree of structural rigidity.
  • the choice of linker can be, but is not necessarily, dictated by the desired affinity of the ZFP for its cognate target sequence. It is within the skill of the art to test and optimize various linker sequences to improve the binding affinity of the ZFP for its cognate target sequence. Methods of measuring binding affinity between ZFPs and their targets are well known. Typically gel shift assays are used but transient expression assays, for example as provided in the Examples hereof, can also be used.
  • the amino acid linker is preferably be flexible to allow each three finger domain to independently bind to its target sequence and avoid steric hindrance of each other's binding.
  • the ZFPs of antiviral agent of the invention include any ZFP having one or more combination of amino acids for positions -1, 2, 3 and 6 as provided by the recognition code in Table 1.
  • Antiviral agents or viral replication inhibitors comprising a ZFP of the invention (with or without CPP and NLS domains) or a nucleic acid encoding such ZFP as an active ingredient can be prepared by using additives for pharmaceutical preparations such as physiologically acceptable carriers, excipients or stabilizers, for example, in the form of a lyophilized formulation or an aqueous solution.
  • additives for pharmaceutical preparations include, but not limited to, buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides,
  • antiviral agents or viral replication inhibitors of the invention may be mixed with other pharmaceutical ingredients so far that the active ingredient provided by the present invention is not adversely effected.
  • Such pharmaceutical ingredients can be combined in amounts effective for the intended prophylactic and/or therapeutic purpose.
  • known antiviral agents and the like may be combined.
  • the active ingredients of the antiviral agents provided by the present invention may be entrapped in microcapsule prepared, for example, by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and polydnethyl methacrylate) microcapsule, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microcemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems e.g., liposomes, albumin microspheres, microcemulsions, nano-particles and nanocapsules
  • the antiviral agents or viral replication inhibitors of the invention may be prepared as sustained-release preparations.
  • sustained-release matrices for preparing the sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide variant, which matrices are provided in the form of shaped articles, e.g., films or microcapsule.
  • sustained-release matrices include polyester hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides (U.S. Patent No.
  • copolymers of L- glutamic acid and ethyl-L-glutamate non-degradable ethylene -vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • polyD-(-)-3-hydroxy butyric acid and the like.
  • polymers such as ethylene -vinyl acetate and lactic acid-glycolic acid enable release of molecules over 100 days, certain hydrogels release proteins for shorter time periods.
  • stabilization when encapsulated antibodies remain in the body for a long time, for example, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism of destabilization. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio- disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • the dosages of the antiviral agents or viral replication inhibitors of the invention are not particularly limited, and appropriate dosages can be selected by those of skill in the art based on the type of the active ingredient, type or body weight of an animal as a target of administration, type of the animal virus, type and symptoms of viral infection, and the like.
  • ZFPHPV'1 and ZFPHPV Two ZFPs were designed, ZFPHPV'1 and ZFPHPV"2, for the DNA sequences as indicated in FIG. 4 and constructed as described in Sera et al. (2002) Biochemistry 41-7074-7081.
  • ZFPW A control ZFP, referred to as "ZFPW, was constructed in the same system but with alanine substituted for all the recognition amino acids (i.e., amino acids at positions -1, 2, 3 and 6 in each zinc finger domain is alanine).
  • An K coli expression plasmid encoding HPV- 18 E2 was prepared by cloning the E2 open reading frame (ORF) (HPV-18 nucleotides [nt] 2817 to 3914) from pHPV-18 (American Type Culture Collection) into the Ec ⁇ RI/ HindLll sites of pET21a.
  • ORF E2 open reading frame
  • ZFPs with CPP and NLS domains were prepared for ZFP HPV "2 as follows.
  • the first ZFP is referred to as "PTD4-ZFPHPV-2 and has the PTD4 sequence (TyrAlaArgAlaAlaAlaArgGlnAlaArgAla), a three glycine linker, the NLS sequence (ProLysLysLysArgLysVal), an additional linker (GlyGlyGlyGlySer), the T7 Tag (MetAlaSerMetThrGlyGlyGlnGlnMetGly) preceding the N-terminus of the ZFPHPV2 ORF in pET-21a.
  • the T7 tag is used to detect proteins by Western blotting and to protect PTD4 from attack by exoproteases.
  • the second ZFP is referred to as ZFPHPV"2-R9 and has the ZFPHPV2 ORF followed at the Oterminus by a linker (GlyGlyGlyGlySer), the same NLS sequence, a three glycine linker, the R9 sequence (ArgArgArgArgArgArgArgArgArgArg) and a V5-tag
  • PTD4-NLS The structures of PTD4-NLS and NLS-R9 are shown below.
  • PTD4-NLS The structures of PTD4-NLS and NLS-R9 are shown below.
  • Both PTD4-ZFP and ZFP-R9 have T7-tag from pET-21a on the N-turminus
  • V5-tag is simultaneously inserted into the C-turminus for only ZFP-R9
  • pRL-El, pRL-E2 and pUCOril77 were constructed as follows.
  • the vectors p RLrEl and pRL ⁇ 2 were constructed by cloning the El gene (nt 904 to 2887 from the HPV- 18 gene) or the E2 gene (nt 2817 to 3914 from the HPV- 18 gene) into the Nhel/Xbal sites of a pRL-SV40 vector (Promega), respectively.
  • pUC-Oril77 a vector having the HPV- 18 replication origin, was constructed by cloning the 177-bp AluI/BamHI fragment (HPV- 18 nt 7800 to 7857 and 1 to 119), designated Oril77 and including the HPV 18 replication origin, into the HincII/BamHI sites of pUC-19.
  • Mammalian expression vectors PCMV-ZFPHPV'1, PCMVZFPH PV 2 and pCMV-ZFPAia, for expressing ZFPHFV-1, ZFPHPV"2 and ZFPAia, respectively in animal cells were constructed by cloning the respective ZFP ORF into a modified pcDNA3.1 (Invitrogen).
  • the modified plasmid contains an N- terminal T7 tag, a nuclear localization signal from the simian virus 40 large T antigen, and a multicloning site for insertion of the ZFP.
  • DNA binding assays were conducted with a double-stranded DNA probe of 56 base pairs: 5'-
  • TA 4 GGAGTAACCGAAAACGGTCGGGACCGAAAACGGTGTATAT
  • TA 4 -S' (SEQ ID NO: 4) probe having two E2 binding sites (E2 binding sites are underlined) or with a double-stranded DNA probe of 40 base pairs, 5'- (TA) 4 GGAGTAACCGAAAACGGTGTATAT(TA) 4 ⁇ ' (SEQ ID NO: 5) having one E2 binding site.
  • Each probe was labeled by using [cr 32 P]dATP or [cr 32 P]dTTP and the Klenow fragment.
  • FIG. 6 shows the results for ZFPHPVI and ZFPHPV2 binding to DNA (two left panels) and indicates that these ZFPs bound to the E2 binding sites of HPV at concentration of about 10 pM.
  • the apparent dissociation constant (Kd) of the E2 protein, where half-maximal binding was observed was approximately 40 nM.
  • Kd apparent dissociation constant
  • control protein ZFPAM
  • ZFPAM was unable to shift the target DNA even at 1 ⁇ M in the presence of an excess amount of cold poly(dA"dT)2 competitor DNA, showing that specific recognition amino acids in both ZFPHP V 1 and ZFPHPV'2 are required to recognize the 19-bp DNA targets.
  • E2 final concentration: 100 nM
  • ZFPHPVI or ZFPHPV 2 final concentration: 0.1, 1, 10 or 100 nM
  • FIG. 8 indicates that both ZFPHPVI and ZFPHPV-2 inhibited DNA replication of HPV and that ZFPHPV2 has potent replication inhibitory action, with lane 8 to lane 5 showing the results with ZFPHPVI and lane 12 to lane 9 showing the results for ZFPHPV"2.
  • Lanes 1 to 4 indicate band positions of free DNA, DNA bound to E2, DNA bound to ZFPHPVI, and DNA bound to ZFPHPV2, respectively.
  • a transient viral replication system (as described below) with a variant replication origin was prepared by leaving the binding sites of the replication protein E2 unchanged, and only mutating the binding site in the linker region of the ZFPs from CGGG (WT) to CGGT (MT2), CTGT (MTlO), ATAT (MTIl) or ATATATAT (MT12). These mutant probes are also shown in the top panel of FIG. 11.
  • the gel shift assay results for DNA probes cotransfected with pCMV" ZFP HPV "2 are provided in FIG. 11, panel b.
  • the gel shift assay results for mutant ori plasmids cotransfected with PCMV-ZFPHPV"2 are provided in FIG. 11, panel c. Inhibition of replication of the transient variant viral origins was not observed (FIG. 11, panel c), which suggests that ZFPHP V '2 does not bind to this variant replication origin.
  • a total of 8 x 10 5 cells per well of the human cell line 293H (Invitrogen) were plated onto a BioCoat polyD-lysine of a 12-well plate (Becton Dickinson) and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 0.1 mM nonessential amino acids and 10% fetal bovine serum (Invitrogen).
  • the cells were cotransfected with the three vectors needed for transient replication, pRL-El at 1.5 ⁇ g, pRLrE2 at 0.17 ⁇ g and pUC-Oril77 at 0.17 ⁇ g, and 0.17 ⁇ g of the appropriate ZFP vector for animal cell expression: pCMV-ZFP H pvl, pCMV-ZFP H Pv2, pCMV-ZFPAia or pcDNA3.1 (expression control for ZFP; Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
  • plasmids were isolated from the cells by the Hirt extraction, method [Hirt (1967) J. MoI. Biol. 26:365-369] and linearized by digestion with HincBIl.
  • each sample was treated with an excess of Dpnl to remove the unreplicated, methylated input DNA pUCOril77.
  • One percent of the remaining half of each linearized sample was used to confirm that equal amounts of the plasmids used for each transient replication assay were introduced into 293H cells.
  • the DNA samples were separated by electrophoresis (in 0.8% agarose gels with 0.5x Tris-borate- EDTA buffer), followed by Southern blot hybridization.
  • DNA band intensities on X-ray films were digitized and quantitated by UN-SCAN-IT (Silk Scientific, Inc.). Average DNA band intensities of replicated pUCOri derivatives were calculated from four independent experiments and normalized with DNA band intensities of input pUC-Ori derivatives.
  • ZFPAia was a negative control.
  • FIG. 7 show that ZFPHPV"1 and ZFPHPV2 inhibited DNA replication.
  • ZFPHPV'2 especially, reduced the replication level to about 12% that of the control pcDNA3.1.
  • the control protein, ZFPAia was unable to inhibit DNA replication (FIG. 7, lane 4), showing that the specific recognition amino acids (i.e., those at positions -1, 2, 3 and 6) in ZFPHPV 1 and ZFPHPV'2 are required for inhibition.
  • PTD4"ZFPHPV2 or ZFPHPV2-R9 were added to 8 x 10 5 of 293H cells (per well in 12-well plates) at the indicated final concentration.
  • the cells were cultured for one hour and transfected with pRL-El (1.5 ⁇ g), pRL-E2 (0.17 ⁇ g) and pUC"Oril77 (0.17 ⁇ g).
  • the replication of pUCOril77 was analyzed in the same manner as in the above (a).
  • ZFPHPV2-R9 can penetrate the cells, enter into the nuclei and inhibit viral replication when added to the medium of in the transient viral replication system. Accordingly, this protein is capable of acting as an effective antiviral agent or viral replication inhibitor.
  • the protein E2OR9 was prepared by recombinantly fusing the R9 domain (for cell penetrating ability) to the DNA binding domain located at the C terminus (amino acids 283 to 365) of the E2 protein (i.e., dominant-negative variant). Transient viral replication was examined for E2C-R9 inhibition and showed that ZFP H PV2-R9 had a greater replication inhibitory ability than that of the dominant-negative variant E2C-R9 (FIG. 12). Again, suggesting that a ZFP with high DNA binding ability can act as a highly effective as an antiviral agent.
  • the antiviral agent and viral replication inhibitor of the present invention are useful for prophylactic and/or therapeutic treatment of animal virus infection.
  • the antiviral agent or viral replication inhibitor comprising a zinc finger protein as an active ingredient, which is bound with a peptide having a cell penetrating ability and a nuclear localization signal, has an effect markedly higher than that of conventional antiviral agents and is extremely useful as a medicament.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne un agent antiviral qui comprend une protéine en doigt de zinc et/ou un acide nucléique codant la protéine en doigt de zinc afin que la liaison d'une protéine de réplication dérivée d'un virus ADN animal à une origine de réplication du virus soit inhibée.
PCT/JP2006/317295 2005-08-26 2006-08-25 Agent antiviral et inhibiteur de replication virale WO2007024029A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2008527641A JP2009506025A (ja) 2005-08-26 2006-08-25 抗ウイルス剤及びウイルス複製阻害剤

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2005-246876 2005-08-26
JP2005246876 2005-08-26
JP2006038575 2006-02-15
JP2006-038575 2006-02-15

Publications (1)

Publication Number Publication Date
WO2007024029A1 true WO2007024029A1 (fr) 2007-03-01

Family

ID=37771753

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/317295 WO2007024029A1 (fr) 2005-08-26 2006-08-25 Agent antiviral et inhibiteur de replication virale

Country Status (2)

Country Link
JP (1) JP2009506025A (fr)
WO (1) WO2007024029A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009018003A2 (fr) * 2007-07-27 2009-02-05 Ensemble Discovery Corporation Analyses de détection et utilisation de celles-ci
EP3733847B1 (fr) 2012-10-23 2022-06-01 Toolgen Incorporated Composition pour le clivage d'un adn cible comprenant un arn guide spécifique de l'adn cible et un acide nucléique codant pour la protéine cas ou la protéine cas, et leur utilisation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6183879B2 (ja) * 2012-08-31 2017-08-23 国立大学法人弘前大学 新規ペプチドおよびその医薬用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008286A2 (fr) * 2000-07-21 2002-01-31 Syngenta Participations Ag Code de reconnaissance pour domaines en doigt de zinc et ses utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008286A2 (fr) * 2000-07-21 2002-01-31 Syngenta Participations Ag Code de reconnaissance pour domaines en doigt de zinc et ses utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEE D. ET AL.: "Identification of sequence requirement for the origin of DNA replication in human papillomavirus type 18", VIRUS RESEARCH, vol. 52, 1997, pages 97 - 108, XP003008661 *
SVERDRUP F. ET AL.: "The E2 Binding Sites Alone Are Sufficient To Function as the Minimal Origin of Replication of Human Papillomavirus Type 18 DNA", JOURNAL OF VIROLOGY, vol. 69, no. 2, 1995, pages 1319 - 1323, XP003008660 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009018003A2 (fr) * 2007-07-27 2009-02-05 Ensemble Discovery Corporation Analyses de détection et utilisation de celles-ci
WO2009018003A3 (fr) * 2007-07-27 2009-07-16 Ensemble Discovery Corp Analyses de détection et utilisation de celles-ci
EP3733847B1 (fr) 2012-10-23 2022-06-01 Toolgen Incorporated Composition pour le clivage d'un adn cible comprenant un arn guide spécifique de l'adn cible et un acide nucléique codant pour la protéine cas ou la protéine cas, et leur utilisation

Also Published As

Publication number Publication date
JP2009506025A (ja) 2009-02-12

Similar Documents

Publication Publication Date Title
JP2869396B2 (ja) Ｔａｔ由来の輸送ポリペプチド
AU2018208708A1 (en) Delivery system for functional nucleases
KR101873773B1 (ko) 류마티즘 관절염 예방 또는 치료 조성물
WO2005084158A2 (fr) Transduction polypeptidique et peptides fusogenes
US11618780B2 (en) Composition and method for activating latent human immunodeficiency virus (HIV)
WO1989001519A1 (fr) Proteine humaine liant le mannose
WO2002019965A2 (fr) Réaction du type choc thermique et réplication virale
WO2009095500A1 (fr) Inhibiteurs de la réplication lentivirale
US20220265837A1 (en) Complex for intracellular delivery of molecules
WO2007024029A1 (fr) Agent antiviral et inhibiteur de replication virale
US20130237476A1 (en) Adipose tissue targeted peptides
US20230054711A1 (en) Multimerization delivery system for intracellular delivery of molecule
US11865181B2 (en) Peptidic materials that traffic efficiently to the cell cytosol and nucleus
JP4831410B2 (ja) 抗ウイルス性ペプチドおよび抗ウイルス剤
JP6994714B2 (ja) 抗ウイルス性ペプチドおよびその利用
Avram et al. Evaluation of the therapeutic properties of mastoparan-and sifuvirtide-derivative antimicrobial peptides using chemical structure-function relationship-in vivo and in silico approaches
JP4927546B2 (ja) ヘプタペプチドパターン及び細胞透過ドメインを含む相互作用ポリペプチド
US20140303093A1 (en) Micro-utrophin polypeptides and methods
RU2432359C2 (ru) ПРИМЕНЕНИЕ СЛИТОГО ПОЛИПЕПТИДА, СОДЕРЖАЩЕГО БЕЛОК MeCP2 И ДОМЕН ТРАНСДУКЦИИ БЕЛКА, ФАРМАЦЕВТИЧЕСКАЯ КОМПОЗИЦИЯ, СОДЕРЖАЩАЯ ТАКОЙ БЕЛОК, И СПОСОБ ЛЕЧЕНИЯ И/ИЛИ ПРОФИЛАКТИКИ СВЯЗАННОГО С РАЗВИТИЕМ НЕРВНОГО ЗАБОЛЕВАНИЯ
US20190367567A1 (en) Peptides and Uses for Managing Viral Infections
JP2020059662A (ja) 抗ウイルス性ペプチドおよびその利用
US20060160732A1 (en) Compositions and methods for inhibiting cell senescence and hyperproliferative disorders
KR20190087760A (ko) 세포투과성 MxA 재조합 단백질
KR20020009315A (ko) 세포침투성 티에이티-씨3 트랜스페라제 융합단백질, 이융합단백질의 발현벡터 및 티에이티-씨3 트랜스페라제를이용한 로 단백질의 생리적 기능 분석방법
AU2426288A (en) Human mannose binding protein

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2008527641

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06783155

Country of ref document: EP

Kind code of ref document: A1