US20080031855A1 - Anticancer Agent Containing Minus-Strand Rna Virus - Google Patents

Anticancer Agent Containing Minus-Strand Rna Virus Download PDF

Info

Publication number
US20080031855A1
US20080031855A1 US11/630,522 US63052205A US2008031855A1 US 20080031855 A1 US20080031855 A1 US 20080031855A1 US 63052205 A US63052205 A US 63052205A US 2008031855 A1 US2008031855 A1 US 2008031855A1
Authority
US
United States
Prior art keywords
virus
minus
gene
strand rna
cells
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US11/630,522
Other languages
English (en)
Inventor
Shinji Okano
Yoshikazu Yonemitsu
Katsuo Sueishi
Satoko Shibata
Mamoru Hasegawa
Haruhiko Kondo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dnavec Research Inc
Original Assignee
Dnavec Research Inc
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
Priority claimed from PCT/JP2004/016089 external-priority patent/WO2005042737A1/ja
Application filed by Dnavec Research Inc filed Critical Dnavec Research Inc
Assigned to DNAVEC RESEARCH INC. reassignment DNAVEC RESEARCH INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SHIBATA, SATOKO, KONDO, HARUHIKO, OKANO, SHINJI, SUEISHI, KATSUO, YONEMITSU, YOSHIKAZU, HASEGAWA, MAMORU
Publication of US20080031855A1 publication Critical patent/US20080031855A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464408—Platelet-derived growth factor receptors [PDGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • A61K39/464456—Tyrosinase or tyrosinase related proteinases [TRP-1 or TRP-2]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18832—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18871—Demonstrated in vivo effect

Definitions

  • the present invention relates to the field of cancer therapy.
  • Virotherapy is a therapy that infects tumor cells with replicative viruses, such as HSV-1 or adenovirus, and uses the viral cytocidal effect that accompanies viral propagation for treating tumor.
  • replicative viruses such as HSV-1 or adenovirus
  • the replicative viruses for tumor therapy are mutant viruses whose viral genome has been genetically engineered, such that while their ability to replicate in tumors is maintained, their pathogenicity in normal human tissue is suppressed to a minimum.
  • Therapeutic replicative viruses infect tumor cells and replicate inside the cells, and the infected cells die out in this process.
  • Anticancer virotherapy has a wide range of applications and provides excellent practicality: for example, it can be used in combination with conventional therapy such as surgery, radiation therapy, and chemotherapy; it can be applied to solid cancers in general; it can be administered repeatedly; and it can be made to directly incorporate into the viral genome therapeutic genes such as those of cytokines to enhance antitumor effect. Development of a more effective virotherapy is expected to contribute greatly to cancer therapy.
  • An objective of the present invention is to provide a more effective anticancer virotherapy and anticancer agents. More specifically, the present invention provides methods for producing anticancer agents comprising a minus-strand RNA virus, and cancer therapeutic methods using a minus-strand RNA virus.
  • RNA virus To verify the therapeutic effect of in vivo administration of RNA virus on tumor, the present inventors performed experiments using mice subjected to ventral subcutaneous inoculation with melanoma cell lines. As a result, the present inventors found that tumor size is significantly reduced when minus-strand RNA virus that does not carry any therapeutic gene is injected into the tumor of the animals. Therefore, this revealed that in vivo administration of a minus-strand RNA virus exhibits antitumor effects even when the virus does not carry any therapeutic gene.
  • Minus-strand RNA viruses are considered to be effective anticancer agents themselves. Moreover, direct injection of such viruses into tumor sites (cancer tissues) is expected to yield effective antitumor effects, and such viruses are useful for treating cancer.
  • the present invention relates to antitumor agents comprising a minus-strand RNA virus, methods for producing the antitumor agents, and methods for suppressing cancer using the RNA virus, and more specifically, it relates to inventions described in each claim.
  • An invention or a combination of inventions set forth in a claim(s) citing the same claim is already included in inventions described in the claims. More specifically, the present invention relates to:
  • FIG. 1 depicts the therapeutic effects of in vivo administration of a GFP-expressing SeV, a soluble FGF receptor-expressing SeV, or a soluble PDGFR ⁇ -expressing SeV on melanoma.
  • FIG. 2 depicts the growth curve of B16 melanoma cells that were inoculated subcutaneously.
  • FIG. 3 depicts the results of 51 Cr release assay for YAC-1 target cells.
  • FIG. 4 depicts the results of 51 Cr release assay for TRP2 peptide +EL-4.
  • the present invention provides anticancer agents (carcinostatic agents) comprising a minus-strand RNA virus as an active component.
  • an RNA virus refers to a virus carrying an RNA genome.
  • the RNA virus that works as an active component of the anticancer agents of the present invention is preferably a minus-strand RNA virus.
  • a minus-strand RNA virus refers to viruses that include a minus strand (an antisense strand corresponding to a sense strand encoding viral proteins) RNA as the genome.
  • the minus-strand RNA is also referred to as negative strand RNA.
  • the minus-strand RNA virus used in the present invention particularly includes single-stranded minus-strand RNA viruses (also referred to as non-segmented minus-strand RNA viruses).
  • the “single-strand negative strand RNA virus” refers to viruses having a single-stranded negative strand [i.e., a minus strand] RNA as the genome.
  • viruses include viruses belonging to Paramyxoviridae (including the genera Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus ), Rhabdoviridae (including the genera Vesiculovirus, Lyssavirus, and Ephemerovirus ), Filoviridae, Orthomyxoviridae, (including Influenza viruses A, B, and C, and Thogoto-like viruses), Bunyaviridae (including the genera Bunyavirus, Hantavirus, Nairovirus, and Phlebovirus ), Arenaviridae, and the like.
  • Paramyxoviridae including the genera Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus
  • Rhabdoviridae including the genera Vesiculovirus, Lys
  • a minus-strand RNA virus preferably used in the context of the preset invention includes, for example, Sendai virus belonging to Paramyxoviridae.
  • Sendai virus belonging to Paramyxoviridae include Newcastle disease virus, mumps virus, measles virus, respiratory syncytial virus (RS virus), rinderpest virus, distemper virus, simian parainfluenza virus (SV5), and human parainfluenza viruses 1, 2, and 3; influenza virus belonging to Orthomyxoviridae; and vesicular stomatitis virus and rabies virus belonging to Rhabdoviridae.
  • Sendai virus SeV
  • HIV-1 human parainfluenza virus-1
  • HPIV-3 human parainfluenza virus-3
  • PDV canine distemper virus
  • a more preferred example is a virus selected from the group consisting of Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV), rinderpest virus (RPV), Hendra virus (Hendra), and Nipah virus (Nipah).
  • Sendai virus SeV
  • HIV-1 human parainfluenza virus-1
  • HPIV-3 human parainfluenza virus-3
  • PDV phocine distemper virus
  • CDV canine distemper virus
  • DMV dolphin molbillivirus
  • PDPR measles virus
  • RPV rinderpest virus
  • Hendra virus Hendra
  • Nipah virus Nipah virus
  • viruses of the present invention are preferably those belonging to Paramyxoviridae (including Respirovirus, Rubulavirus, and Morbillivirus ) or derivatives thereof, and more preferably those belonging to the genus Respirovirus (also referred to as Paramyxovirus ) or derivatives thereof.
  • the derivatives include viruses that are genetically-modified or chemically-modified in a manner not to impair their gene-transferring ability.
  • viruses of the genus Respirovirus are human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), bovine parainfluenza virus-3 (HPIV-3), Sendai virus (also referred to as murine parainfluenza virus-1), and simian parainfluenza virus-10 (HPIV-10).
  • the minus-strand RNA viruses of the present invention are most preferably Sendai viruses.
  • the minus-strand RNA viruses of the present invention may be derived from natural stains, wild-type strains, mutant strains, laboratory-passaged strains, artificially constructed strains, or such. More specifically, the minus-strand RNA viruses may be minus-strand RNA viruses isolated from nature, or minus-strand RNA viruses artificially generated by genetic recombination. Furthermore, as long as the ability to replicate genomic RNA in infected cells is maintained, any of the genes carried by wild-type minus-strand RNA viruses can have mutations or deletions. For example, minus-strand RNA viruses comprising a mutation or deletion in at least an envelope protein or coat protein-encoding gene of the minus-strand RNA viruses can be preferably used.
  • minus strand RNA viruses can replicate the RNA genome in infected cells but cannot form infectious virions. Since there is no need to be concerned about spread of infection to the surrounding, the viruses are very safe.
  • a minus-strand RNA virus that does not comprise at least one or a combination of the genes encoding spike proteins or envelope proteins such as F, H, HN, or G can be used (WO00/70055 and WO00/70070; Li, H. —O. et al., J. Virol. 74(14) 6564-6569 (2000)).
  • a genome can be replicated in the infected cells if proteins that are necessary for genome replication (for example N, P, and L proteins) are encoded supplying to virus-producing cells, products of the deficient genes or proteins which can complement the deficient genes (WO00/70055 and WO00/70070; Li, H. —O. et al., J. Virol. 74(14) 6564-6569 (2000)).
  • viruses carrying the M protein gene release non-infectious virions (VLP) even if they do not carry genes encoding spike proteins such as F protein or HN protein; therefore, VLP can be produced without complementing the viral proteins (WO00/70070).
  • VLP non-infectious virions
  • RNP comprising genomic RNA and the N, L, and P proteins can be amplified in cells even if they do not have any envelope protein genes; therefore, RNP can be collected from cell lysates by centrifugation and such.
  • the anticancer agents of the present invention can be produced using mutant RNA viruses.
  • mutant RNA viruses For example, many temperature-sensitive mutations of envelope proteins and coat proteins are known. RNA viruses comprising these temperature-sensitive mutant protein genes can be preferably used in the present invention.
  • Temperature-sensitive mutations refer to mutations that significantly decrease protein activity at normal temperatures (for example, 37° C. to 38° C.) of the virus' host, as compared with the activity at low temperatures (for example, 30° C. to 32° C.). Such proteins harboring temperature-sensitive mutations are useful because they enable virus production at permissible temperatures (low temperatures).
  • temperature-sensitive mutations of the M gene of minis-strand RNA viruses include amino acid substitutions at a site arbitrarily selected from the group consisting of G69, T116, and A183 in the Sendai virus M protein (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246).
  • the amino acids of the homologous sites in the M protein of other minus-strand RNA viruses can be identified easily, and specifically, examples of homologous sites in M proteins that correspond to G69 of the SeV M protein include (the letter and number denote the amino acid and its position; abbreviation of the names is shown inside the parentheses): G69 in human parainfluenza virus-1 (HPIV-1); G73 in human parainfluenza virus-3 (HPIV-3); G70 in phocine distemper virus (PDV) and canine distemper virus (CDV); G71 in dolphin molbillivirus (DMV); G70 in peste-des-petits-ruminants virus (PDPR), measles virus (MV), and rinderpest virus (RPV); G81 in Hendra virus (Hendra) and Nipah virus (Nipah); G70 in human parainfluenza virus-2 (HPIV-2); E47 in human parainfluenza virus-4a (HPIV-4a) and human parainfluenza virus
  • M protein homologous sites that correspond to T116 in the SeV M protein include: T116 in human parainfluenza virus-1 (HPIV-1); T120 in human parainfluenza virus-3 (HPIV-3); T104 in phocine distemper virus (PDV) and canine distemper virus (CDV); T105 in dolphin molbillivirus (DMV); T104 in peste-des-petits-ruminants virus (PDPR), measles virus(MV), and rinderpest virus (RPV); T120 in Hendra virus (Hendra) and Nipah virus (Nipah); T117 in human parainfluenza virus-2 (HPIV-2) and simian parainfluenza virus 5 (SV5); T121 in human parainfluenza virus-4a (HPIV-4a) and human parainfluenza virus-4b (HPIV-4b); T119 in mumps virus (Mumps); and S120 in Newcastle disease virus (NDV).
  • HPIV-1 human parainfluenza virus-1
  • M protein homologous sites that correspond to A183 in the SeV M protein include: A183 in human parainfluenza virus-1 (HPIV-1); F187 in human parainfluenza virus-3 (HPIV-3); Y171 in phocine distemper virus (PDV) and canine distemper virus (CDV); Y172 in dolphin molbillivirus (DMV); Y171 in peste-des-petits-ruminants virus (PDPR), measles virus (MV), and rinderpest virus (RPV); Y187 in Hendra virus (Hendra) and Nipah virus (Nipah); Y184 in human parainfluenza virus-2 (HPIV-2); F184 in simian parainfluenza virus 5 (SV5); F188 in human parainfluenza virus-4a (HPIV-4a) and human parainfluenza virus-4b (HPIV-4b); F186 in mumps virus (Mumps); and Y187 in Newcastle disease virus (NDV).
  • HPIV-1 human
  • viruses comprising a genome encoding, as their M protein, a mutant M protein in which the amino acids of any one of the three sites, preferably a combination of any two of these sites. More preferably, all three sites mentioned above are substituted with other amino acids are used in the present invention.
  • a preferred amino acid mutation is substitution to another amino acid whose side chains have different chemical properties.
  • substitution is carried out with an amino acid whose BLOSUM62 matrix value (Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) is 3 or less, preferably 2 or less, more preferably 1 or less, and even more preferably 0 or less.
  • BLOSUM62 matrix value Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919
  • G69, T116, and A183 of the Sendai virus M protein or homologous sites of other viral M proteins can be substituted wit Glu (E), Ala (A), and Ser (S), respectively.
  • Mutations can be introduced, for example, by using oligonucleotides and such according to well known methods for introducing mutations.
  • Examples of temperature-sensitive mutations of the HN gene include amino acid substitutions at any of the sites selected from the group consisting of A262, G264, and K461 in the Sendai virus HN protein (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246).
  • A262, G264, and K461 in the Sendai virus HN protein or homologous sites of other viral HN proteins are substituted with Thr (T), Arg (R), and Gly (G), respectively.
  • the amino acids at positions 464 and 468 in the HN protein can be mutated by referring to the temperature-sensitive mumps virus vaccine strain, Urabe AM9 (Wright, K. E. et al., Virus Res. 2000: 67; 49-57).
  • Minus-strand RNA viruses may comprise mutations in the P gene or L gene. Specific examples of such mutations include mutation of Glu at position 86 (E86) of the SeV P protein, substitution of Leu at position 511 (L511) of the SeV P protein to another amino acid, or substitution of homologous sites in the P protein of a different minus-strand RNA virus. Specific examples include substitution of the amino acid at position 86 to Lys, and substitution of the amino acid at position 511 to Phe.
  • examples include substitution of Asn at position 1197 (N1197) and/or Lys at position 1795 (K1795) in the SeV L protein to other amino acids, or substitution of homologous sites in the L protein of another minus-strand RNA virus, and specific examples include substitution of the amino acid at position 1197 to Ser, and substitution of the amino acid at 1795 to Glu.
  • Mutations of the P gene and L gene can significantly increase the effects of persistent infectivity, suppression of the release of secondary virions, and suppression of cytotoxicity. Furthermore, by combining mutation and/or deletion of the envelope protein gene, these effects can be increased dramatically.
  • viruses that comprise in their envelope, proteins that are different from the envelope proteins originally carried by the viruses.
  • viruses comprising such protein can be produced.
  • Such protein is not particularly limited, and desired proteins that confer the ability to infect mammalian cells are used.
  • a specific example is the vesicular stomatitis virus (VSV) G protein (VSV-G).
  • VSV-G protein may be derived from any VSV strain, and for example, VSV-G protein derived from the Indiana serotype strain (J. Virology 39: 519-528 (1981)) can be used, but it is not limited thereto.
  • the minus-strand RNA viruses used in the present invention can comprise an arbitrary combination of envelope proteins derived from other viruses.
  • a characteristic of the minus-strand RNA viruses of the present invention is that they themselves have anticancer effects. Specifically, the minus-strand RNA viruses do not need to comprise foreign proteins or nucleic acids encoding the proteins. Therefore, the present invention relates to anticancer agents comprising minus-strand RNA viruses that do not encode foreign proteins.
  • the minus-strand RNA viruses of the present invention may or may not encode a foreign protein or a foreign gene in their genomic RNA. Since minus-strand RNA viruses that do not encode any foreign proteins also exhibit anticancer (carcinostatic) effects, foreign genes are not necessarily required.
  • RNA viruses used in the present invention may be RNA viruses that do not encode proteins with anticancer therapeutic effects.
  • Such viruses include RNA viruses encoding desired foreign proteins that do not have any anticancer therapeutic effects.
  • RNA viruses encoding marker proteins such as green fluorescent protein (GFP), luciferase, or various peptide tags can be used to detect the introduction of RNA viruses.
  • GFP green fluorescent protein
  • luciferase or various peptide tags can be used to detect the introduction of RNA viruses.
  • the carcinostatic (anticancer) effects can also be further enhanced.
  • Recombinant minus-strand RNA viruses that carry foreign genes can be reconstituted using well-known methods. Specific procedures for such production typically include the steps of (a) transcribing a cDNA encoding the genomic RNA of a minus-strand RNA virus in a cell expressing viral proteins necessary for virion formation, and (b) collecting the culture supernatant comprising the produced virus.
  • the viral proteins can be expressed from the transcribed viral genomic RNA, or they may be provided in trans from sources other than the genomic RNA. When viral genes that are required for virion formation are deficient from the genomic RNA, these viral genes are separately expressed in the virus-producing cells to complement virion formation.
  • a vector in which a DNA encoding the protein or the genomic RNA is linked downstream of a suitable promoter that functions in the host cells, is introduced into the host cells. Transcribed genomic RNAs are replicated in the presence of viral proteins, and infectious virions are formed. When producing deficient viruses that lack envelope protein genes and such, the deficient proteins, viral proteins that can complement their functions, or such are expressed in the virus-producing cells.
  • the minus-strand RNA viruses of the present invention can be produced using the following known methods (WO97/16539; WO97/16538; WO00/70055; WO00/70070; WO01/18223; Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997; Kato, A. et al., 1997, EMBO J. 16: 578-587; Yu, D. et al., 1997, Genes Cells 2: 457-466; Durbin, A. P. et al., 1997, Virology 235: 323-332; Whelan, S. P. et al., 1995, Proc. Natl. Acad. Sci.
  • minus-strand RNA viruses comprising parainfluenza virus, vesicular stomatitis virus, rabies virus, measles virus, rinderpest virus, and Sendai virus can be reconstituted from DNA.
  • minus-strand RNA viruses especially single-stranded minus-strand RNA viruses, more preferably viruses of the Paramyxoviridae family, and even more preferably viruses of the genus Respirovirus.
  • proteins include hormones, cytokines, growth factors, receptors, intracellular signaling molecules, enzymes, antibodies (including full-length antibodies, antibody fragments such as Fab, single-chain antibodies, etc), and peptides.
  • the proteins may be secretory proteins, membrane proteins, cytoplasmic proteins, nuclear proteins, and such.
  • Artificial proteins include, for example, fusion proteins of chimeric toxins and such, dominant negative proteins (including soluble receptor molecules or membrane-bound dominant negative receptors), cell surface molecules and truncated cell adhesion molecules.
  • the proteins may also be proteins to which a secretory signal, membrane-localization signal, nuclear translocation signal, or such has been attached.
  • the function of a particular gene can be suppressed by expressing antisense RNA molecules, RNA-cleaving ribozymes, or the like as transgenes.
  • Carcinostatic effects may be enhanced by preparing viruses using a therapeutic gene showing carcinostatic effects as a foreign gene.
  • a gene that inhibits vascularization or angiogenesis can further enhance antitumor effects.
  • genes that are known to promote vascularization or angiogenesis include fibroblast growth factor 2 (FGF2) (Baffour, R. et al., J. Vasc. Surg. 16(2):181-91, 1992), endothelial cell growth factor (FCGF) (Pu, L. Q. et al., J. Surg. Res. 54(6):575-83, 1993), vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) (Takeshita, S. et al., Circulation 90 (5 Pt 2):II228-34, 1994; Takeshita, S.
  • FGF2 fibroblast growth factor 2
  • FCGF endothelial cell growth factor
  • VEGF vascular endothelial growth factor
  • VPF vascular permeability factor
  • HGF/SF hepatocyte growth factor/scatter factor
  • minus-strand RNA viruses that encode soluble FGF-R can be preferably used in the present invention.
  • soluble FGF-R naturally occurring soluble FGF-R may be used, or fragments comprising an extracellular domain of membrane-bound FGF-R (FGF-R1 and such) may be used (A. Hanneken and A. Baird, Investigative Ophthalmology & Visual Science, Vol 36, 1192-1196, 1995; Takaishi, S. et al., Biochem. Biophys. Res. Commun., 267(2):658-62, 2000; Seno M, et al., Cytokine, 10(4):290-4, 1998; and Hanneken, A., FEBS Lett. 489:176, 2001).
  • minus-strand RNA viruses into which cytokine-encoding genes have been introduced are also useful.
  • a minus-strand RNA virus carrying a gene encoding an immunostimulatory cytokine will serve as an effective anticancer agent having the activity to induce tumor immunity.
  • immunostimulatory cytokines comprise interleukins (for example, IL-1alpha, IL-1beta IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-18, IL-19, IL-20, IL-21, IL-23, and IL-27), interferons (for example, IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factor (TNF), transforming growth factor (TGF)-beta, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), insulin-like growth factor (IGF)-I, IGF-2, Flt-3 ligand, Fas ligand, c-kit ligand, and other immunomodulatory proteins (such as chemokines and costimulatory molecules).
  • interleukins
  • IL-4 for example, Arai et al. (1989), J. Immunol. 142(1) 274-282; for IL-6, for example, Yasukawa et al. (1987), EMBO J., 6(10): 2939-2945; for IL-12, for example, Wolf et al. (1991), J. Immunol. 146(9): 3074-3081; for IFN-alpha, for example, Gren et al. (1984) J. Interferon Res. 4(4): 609-617, and Weismann et al. (1982) Princess Takamatsu Symp. 12: 1-22.
  • IFN-beta sequences include the sequence from position 139 to 636 of Accession number NM — 002176 (corresponding to position 22 to 187 of the amino acid sequence of NP — 002167) (Derynck, R. et al., Nature 285, 542-547 (1980); Higashi, Y. et al., J. Bio. Chem. 258, 9522-9529 (1983); Kuga, T. et al., Nucleic Acid Res. 17, 3291(1989). Moreover, one may refer to: for TNF, for example, Pennica et al. (1984) Nature 312: 724-729; for G-CSF, for example, Hirano et al.
  • nucleic acid sequence encoding GM-CSF includes sequences containing the sequences from position 84 to 461 of Accession number NM — 000758 (corresponding to position 18 to 144 of the amino acid sequence of NP — 000749).
  • the nucleic acid sequence encoding IL-4 includes sequences contra the sequences from position 443 to 829 of Accession number NM — 000589 (corresponding to position 25 to 153 of the amino acid sequence of NP — 000580).
  • Signal peptides can be appropriately substituted with signal peptide sequences of other proteins. Using natural genes encoding these cytokines or using the degeneracy of the genetic code, mutant genes encoding functional cytokines can be constructed and used.
  • genes may be modified to express modified forms of the cytokines.
  • a cytokine that has two forms, the precursor form and mature form for example, cytokines producing active fragments by cleavage of their signal peptides or by limited proteolysis
  • Other modified forms for example, fusion proteins formed between an active fragment of a cytokine and a heterologous sequence (for example, a heterologous signal peptide) cam also be used.
  • recombinant virus refers to a virus produced via a recombinant polynucleotide or to an amplification product of a virus.
  • Recombinant polynucleotide refers to a polynucleotide in which one or both ends are not linked as in the natural condition.
  • a recombinant polynucleotide is a polynucleotide in which the linkage of the polynucleotide chain has been artificially modified (cleaved and/or liked).
  • Recombinant polynucleotides can be produced using well known gene recombination methods by combining polynucleotide synthesis, nuclease treatment, ligase treatment, and such.
  • Recombinant viruses can be produced by expressing a genetically engineered polynucleotide encoding a viral genome, and then reconstituting the virus. For example, methods for reconstructing a virus from cDNA that encodes the viral genome are known (Y. Nagai, A. Kato, Microbiol. Immunol., 43, 613-624 (1999)).
  • “gene” refers to a genetic substance, a nucleic acid having a sequence to be transcribed in a sense or antisense strand. Genes may be RNAs or DNAs. In this invention, a nucleic acid encoding a protein is referred to as a gene of that protein. Further, a gene may not encode a protein. For example, a gene may encode a functional RNA, such as a ribozyme or antisense RNA. A gene may be a naturally-occurring or artificially designed sequence. Furthermore, in the present invention, “DNA” includes both single-stranded and double-stranded DNAs. Moreover, “encoding a protein” means that a polynucleotide includes an ORF that encodes an amino acid sequence of the protein in a sense or antisense strand, so that the protein can be expressed under appropriate conditions.
  • Minus-strand RNA viruses may encode an antisense strand for a foreign gene in the genomic RNA as required.
  • Genomic RNA refers to RNA that has the function to form a ribonucleoprotein (RNP) with the viral proteins of a minus-strand RNA virus. Genes contained in the genome are expressed by the RNP, genomic RNA is replicated, and daughter RNPs are formed.
  • RNP ribonucleoprotein
  • the genome of a minus-strand RNA virus is constituted so that the viral genes are situated in an antisense orientation between the 3′-leader region and 5′-trailer region.
  • E sequence transcription ending sequence
  • I sequence intervening sequence
  • S sequence transcription starting sequence
  • NP, P, M, F, HN, and L genes refer to genes encoding nucleocapside-, phospho-, matrix-, fusion-, hemagglutinin-neuraminidase-, and large-proteins respectively. Genes in each virus belonging to Paramyxovirinae are commonly listed as follows. In general, NP gene is also listed as “N gene.” Respirovirus NP P/C/V M F HN — L Rubulavirus NP P/V M F HN (SH) L Morbillivirus NP P/C/V M F H — L
  • the database accession numbers for the nucleotide sequences of each of the Sendai virus genes are: M29343, M30202, M30203, M30204, M51331, M55565, M69046, and X17218 for NP gene; M30202, M30203, M30204, M55565, M69046, X00583, X17007, and X17008 for P gene; D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, and X53056 for M gene; D00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152, and X02131 for F gene; D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, and X56131 for HN gene; and D00053, M30202, M30203, M30204, M509
  • viral genes encoded by other viruses are: CDV, AF014953; DMV, X75961; HPIV-1, D01070, HPIV-2, M55320; HPIV-3, D10025; Mapuera, X85128; Mumps, D86172; MV, K01711; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV, X68311; SeV, X00087; SV5, M81442; and Tupaia, AF079780 for N gene; CDV, X51869; DMV, Z47758; HPIV-1, M74081; HPIV-3, X04721; HPIV-4a, M55975; HPIV-4b, M55976; Mumps, D86173; MV, M89920; NDV, M20302; PDV, X75960; RPV, X68311; SeV, M30202; SV5, AF
  • the ORFs encoding these viral proteins and ORFs of the foreign genes are arranged in the antisense direction in the genomic RNAs, via the above-described E-I-S sequence.
  • the ORF closest to the 3′-end of the genomic RNAs requires only an S sequence between the 3′-leader region and the ORF, and does not require an E or I sequence.
  • the ORF closest to the 5′end of the genomic RNA requires only an E sequence between the 5′-trailer region and the ORF, and does not require an I or S sequence.
  • two ORFs can be transcribed as a single cistron, for example, by using an internal ribosome entry site (IRES) sequence.
  • IRS internal ribosome entry site
  • RNA genome in wild type paramyxoviruses, includes a 3′-leader region, six ORFs encoding the N, P, M, F, HN, and L protein in the antisense direction and in this order, and a 5′-trailer region on the other end.
  • the viral gene orientation in the genomic RNAs of the present invention is not restricted, but similarly to the wild type viruses, it is preferable that ORFs encoding the N, P, M, F, HN, and L proteins are arranged in this order, after the 3′-leader region, and before the 5′-tailer region.
  • viruses maintaining the N, P, and L genes can autonomously express genes from the RNA genome in cells, replicating the genomic RNA. Furthermore, by the action of genes such as the F and HN genes, which encode envelope proteins, and the M gene, infectious virions are formed and released to the outside of cells. Thus, such viruses become viruses with propagation ability. “With propagation ability” indicates that when a virus infects a host cell, the virus is amplified in the cell and infections virions are produced. In the present invention, a foreign gene may be inserted into a protein-noncoding region in this genome, as required.
  • a minus-strand RNA virus of this invention may be deficient in any of the wild type virus genes.
  • a virus that excludes the M, F, or HN gene, or any combination thereof can be preferably used in this invention.
  • Such viruses can be reconstituted, for example, by externally supplying the products of the deficient genes. Similar to wild type viruses, the viruses thus prepared adhere to host cells and cause cell fusion, but they cannot firm daughter virions that retain the same infectivity as the original virus, because the virus genome introduced into cells is deficient in viral genes. Therefore, such viruses are useful as safe viruses that can only introduce genes once (for example, foreign genes). Examples of genes in which the genome may be deficient are the F gene and/or HN gene.
  • viruses can be reconstituted by transfecting host cells with a plasmid expressing a recombinant minus-strand RNA viral genome deficient in the F gene, along with an F protein expression vector and expression vectors for the NP, P, and L proteins (WO00/70055 and WO00/70070; Li, H. —O. et al., J. Virol. 74(14) 6564-6569 (2000)).
  • Viruses can also be produced, for example, using host cells that have incorporated the F gene into their chromosomes.
  • amino acid sequences do not need to be the same as the viral sequences, and a mutant or homologous gene from another virus may be used as a substitute, so long as the activity in nucleic acid introduction is the same as, or greater than, that of the natural type.
  • viruses that include an envelope protein other than that of the virus from which the viral genome was derived may be prepared as viruses used in this invention.
  • a virus including a desired envelope protein can be generated by expressing an envelope protein other than the envelope protein encoded by the basic viral genome.
  • Such proteins are not particularly limited.
  • a desired protein that confers an ability to infect cells may be used.
  • examples of such proteins include the envelope proteins of other viruses, for example, the G protein of vesicular stomatitis virus (VSV-G).
  • the VSV-G protein may be derived from an arbitrary VSV strain. For example, VSV-G proteins derived from Indiana serotype strains (J.
  • the virus of the present invention may include any arbitrary combination of envelope proteins derived from other viruses.
  • Preferred examples of such proteins are envelope protein derived from viruses that infect human cells.
  • Such proteins are not particularly limited, and include retroviral amphotropic envelope proteins and the like.
  • the envelope proteins derived from mouse leukemia virus (MuLV) 4070A strain can be used as the retroviral amphotropic envelope proteins.
  • envelope proteins derived from MuMLV 10A1 strain may also be used (for example, pCL-10A1 (Imgenex) (Naviaux, R. K. et al., J. Virol. 70:5701-5705 (1996)).
  • the proteins of Herpesviridae include, for example, gB, gD, gH, and gp85 proteins of herpes simplex viruses, and gp350 and gp220 proteins of EB virus.
  • the proteins of Hepadnaviridae include the S protein of hepatitis B virus. These proteins may be used as fusion proteins in which the extracellular domain is linked to the intracellular domain of the F or HN protein.
  • the viruses used in this invention include pseudotype viruses that include envelope proteins, nick as VSV-G, derived from viruses other than the virus from which the genome was derived. If the viruses are designed such that these envelope proteins are not encoded in RNA genomes, the proteins will never be expressed after virion infection of the cells.
  • viruses used in this invention may be, for example, viruses that include on the envelope spice thereof, proteins such as adhesion factors capable of adhering to specific cells, ligands, receptors, antibodies or fragments, or viruses that include a chimeric protein with these proteins in the extracellular domain and polypeptides derived from the virus envelope in the intracellular domain.
  • proteins such as adhesion factors capable of adhering to specific cells, ligands, receptors, antibodies or fragments
  • proteins such as adhesion factors capable of adhering to specific cells, ligands, receptors, antibodies or fragments
  • These protein may be encoded in the viral genome, or supplied through the expression of genes not in the viral genome (for example, genes carried by other expression vectors, or genes in
  • any viral gene contained in the virus may be modified from the wild type gene in order to reduce the immunogenicity caused by viral proteins, or to enhance RNA transcriptional or replicational efficiency, for example.
  • modifying at least one of the replication factors N, P, and L genes is considered to enhance transcriptional or replicational function.
  • the HN protein which is an envelope protein, has both hemagglutinin activity and neuraminidase activity, it is possible, for example, to improve viral stability in blood if the former activity is attenuated, and infectivity can be controlled if the latter activity is modified.
  • the epitopes of the F protein and/or HN protein which can be cell surface antigenic molecules, can be analyzed, and using this, viruses with reduced antigenicity to these proteins can be prepared.
  • the minus-strand RNA virus may be deficient in one or more accessory genes.
  • the pathogenicity of SeV toward hosts such as mice is remarkably reduced, without hindering gene expression and replication in cultured cells (Kato, A. et al., 1997, J. Virol. 71: 7266-7272; Kato, A. et al., 1997, EMBO J. 16: 578-587; Curran, J. et al., WO01/04272, EP1067179).
  • Minus-strand RNA viruses can be made, for example, to play a role as a vector to introduce foreign genes that are expected to have a synergistic effect with their own anticancer effect. These vectors do not have a DNA phase and carry out transcription and replication only in the host cytoplasm, and consequently, chromosomal integration does not occur (Lamb, R. A. and Kolakofsky, D., Paramyxoviridae: The viruses and their replication. In: Fields B N, Knipe D M, Howley P M, (eds). Fields of Virology. Vol. 2. Lippincott-Raven Publishers: Philadelphia, 1996, pp. 1177-1204).
  • minus-strand RNA viruses contributes greatly to safety when it is used as a vector. For example, results on foreign gene expression show that even after multiple continuous passages of SeV, almost no nucletide mutation is observed. This suggests that the viral genome is highly stable and the inserted foreign genes are stably expressed over long periods of time (Yu, D. et al., Genes Cells 2, 457-466 (1997)). Further, there are qualitative advantages associated with SeV not having a capsid structural protein, such as packaging flexibility and insert gene size. Thus, minus-strand RNA viruses can be made to play an additional role as a highly efficient vector for human gene therapy. For example, SeV with propagation ability are capable of introducing foreign genes of up to at least 4 kb in size, and can simultaneously express two or more kinds of genes by adding the transcriptional units.
  • SeV is known to be pathogenic in rodents causing pneumonia, but is not pathogenic for human. This is also supported by a previous report hat nasal administration of wild type SeV does not have severely harmful effects on non-human primates (Hurwitz, J. L. et al., Vaccine 15: 533-540, 1997). These SeV characteristics suggest that SeV can be applied therapeutically on humans, supporting the proposition that SeV are a promising choice of gene therapy for cancer.
  • minus-strand RNA viruses of this invention may encode foreign genes in their genomic RNA.
  • a recombinant virus harboring a foreign gene is obtained by inserting a foreign gene into an above-described viral genome.
  • the foreign gene can be any desired gene expected to have a synergistic effect and such with the anticancer effect of the RNA virus of this invention, and may be a gene that encodes a naturally-occurring protein, or protein modified from a naturally-occurring protein by deletion, substitution, or insertion of amino acid residues.
  • the foreign gene can be insured at any desired position in a protein-noncoding region of the virus genome, for example.
  • nucleic acid can be inserted, for example, between the 3′-leader region and the viral protein ORB closest to the 3′-end; between each of the viral protein ORFs; and/or between the viral protein ORF closest to the 5′-end and the 5′-trailer region.
  • nucleic acids encoding the foreign genes can be inserted into those deficient regions.
  • An E-I-S sequence should be arranged between the inserted foreign gene and the viral ORF. Two or more genes can be inserted in tandem via E-I-S sequences.
  • Expression levels of a foreign gene carried in a minus-strand RNA virus can be controlled using the type of transcriptional initiation sequence added upstream (to the 3′-side of the negative strand) of the gene (WO01/18223).
  • the expression levels can also be controlled by the position at which the foreign gene is inserted in the genome: the nearer to the 3′-end of the negative strand the insertion position is, the higher the expression level; while the nearer to the 5′-end the insertion position is, the lower the expression level.
  • the insertion position of a foreign gene can be appropriately controlled such that the combination with genes encoding the viral proteins before and after the foreign gene is most suitable.
  • a foreign gene is inserted between the 3′-leader region and the viral protein ORF closest to the 3′-end.
  • a foreign gene may be inserted between the ORFs of the viral gene closest to the 3′-end and the second closest viral gene.
  • the viral protein gene closest to the 3′-end of the genome is the N gene
  • the second closest gene is the P gene.
  • the gene expression level from the virus can be suppressed to obtain an appropriate effect, for example, by inserting the foreign gene at a site in the virus as close as possible to the 5′-side of the negative strand, or by selecting an inefficient transcriptional initiation sequence.
  • RNA virus To prepare a minus-strand RNA virus, a cDNA encoding a genomic RNA of a virus is transcribed in mammalian cells, in the presence of viral proteins (i.e., N, P, and L proteins) essential for reconstitution of an RNP, which is a component of a virus.
  • Viral RNP can be reconstituted by producing either the negative strand genome (that is, the same antisense strand as the viral genome) or the positive strand (the sense stand encoding the viral proteins). Production of the positive strand is preferable for increased efficiency of minus-strand RNA virus reconstitution.
  • the RNA terminals preferably reflect the terminals of the 3′-leader sequence and 5′-tailer sequence as accurately as possible, as in the natural viral genome.
  • the RNA polymerase may be expressed within a cell using the recognition sequence of T7 RNA polymerase as a transcription initiation site.
  • a self-cleaving ribozyme can be encoded at the 3′-end of the transcript, allowing accurate cleavage of the 3′-end with this ribozyme (Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997; Kato, A. et al., 1997, EMBO J. 16: 578-587; and Yu, D. et al., 1997, Genes Cells 2: 457-466).
  • a recombinant Sendai virus carrying a foreign gene can be constructed as follows, according to descriptions in: Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997; Kato, A. et al., 1997, EMBO J. 16: 578-587; Yu, D. et al., 1997, Genes Cells 2: 457-466; or the like.
  • a DNA sample including a cDNA sequence of an objective foreign gene is prepared.
  • the DNA sample is preferably one that can be confirmed to be a single plasmid by electrophoresis at a concentration of 25 ng/ ⁇ l or more.
  • the following explains the case of using a NotI site to insert a foreign gene into a DNA encoding a viral genomic RNA, with reference to examples.
  • the nucleotide sequence is altered using site-directed mutagenesis or the like, such that the encoded amino acid sequence does not change, and the NotI site is preferably excised in advance.
  • the objective gene fragment is amplified from this sample by PCR, and then recovered.
  • both ends of the amplified fragments become NotI sites E-I-S sequences are designed to be included in primers such that, after a foreign gene is inserted into the viral genome, one E-I-S sequence each is placed between the ORF of the foreign gene, and either side of the ORFs of the viral genes.
  • the forward side synthetic DNA sequence has a form in which any desired sequence of not less than two nucleotides (preferably four nucleotides not including a sequence derived from the NotI recognition site, such as GCG and GCC, and more preferably ACTT) is selected at the 5′-side, and a NotI recognition site ‘gcggccgc’ is added to its 3′-side. To that 3′-side, nine arbitrary nucleotides, or nine plus a multiple of six nucleotides are further added as a spacer sequence.
  • any desired sequence of not less than two nucleotides preferably four nucleotides not including a sequence derived from the NotI recognition site, such as GCG and GCC, and more preferably ACTT
  • a NotI recognition site ‘gcggccgc’ is added to its 3′-side.
  • nine arbitrary nucleotides, or nine plus a multiple of six nucleotides are further added as a spacer sequence.
  • the 3′-nd of the forward side synthetic oligo DNA is preferably about 25 nucleotides, selected from the desired cDNA such that the final nucleotide becomes a G or C.
  • no less am two arbitrary nucleotides are selected from the 5′-side, a NotI recognition site ‘gcggccgc’ is added to its 3′-side, and to that 3′ is further added an oligo DNA insert fragment for adjusting the length.
  • the length of this oligo DNA is designed such that the chain length of the NotI fragment of the final PCR-amplified product will become a multiple of six nucleotides (the so-called “rule of six”); Kolakofski, D., et al., J. Virol.
  • the complementary strand sequence of the Sendai virus S, I, and E sequences preferably 5′-CTTTCACCCT-3′ (SEQ ID NO: 1), 5′-AAG-3′, and 5′-TTTTTCTTACTACGG-3′ (SEQ ID NO: 2), respectively; and further to this 3′-side is added a complementary strand sequence corresponding to about 25 nucleotides, counted backwards from the termination codon of a desired cDNA sequence, whose length has been selected such that the final nucleotide of the chain becomes a G or C, to make the 3′-end of the reverse side synthetic DNA.
  • PCR can be performed according to conventional methods, using Taq polymerase or other DNA polymerases. Objective amplified fragments may be digested wit NotI, and then inserted into the NotI site of plasmid vectors such as pBluescript. The nucleotide sequences of PCR products thus obtained are confirmed with a sequencer, and plasmids that include the correct sequence are selected. The inserted fragment is excised from these plasmids using NotI, and cloned into the NotI site of a plasmid composed of genomic cDNA. A recombinant Sendai virus cDNA can also be obtained by inserting the fragment directly into the NotI site of a genomic cDNA, without using a plasmid vector.
  • a recombinant Sendai virus genomic cDNA can be constructed according to methods described in the literature (Yu, D. et al., Genes Cells 2: 457-466, 1997; Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997).
  • an 18 bp spacer sequence (5′-(G)-CGGCCGCAGATCTTCACG-3′) (SEQ ID NO: 3), including a NotI restriction site, is inserted between the leader sequence and the ORF of N protein of the cloned Sendai virus genomic cDNA (pSeV(+)), obtaining plasmid pSeV18 + b(+), which includes an auto-cleavage ribozyme site derived from the antigenomic strand of delta hepatits virus (Hasan, M. K. et al., 1997, J. General Virology 78: 2813-2820).
  • a recombinant Sendai virus cDNA including a desired foreign gene can be obtained by inserting a foreign gene fragment into the NotI site of pSeV18 + b(+).
  • a virus can be reconstituted by transcribing a DNA encoding a genomic RNA of a recombinant virus thus prepared, in cells in the presence of the above-described viral proteins (L, P, and N).
  • the recombinant viruses can also be reconstituted by methods known in the art (WO97/16539; WO97/16538; Durbin, A. P. et al., 1997, Virology 235: 323-332; Whelan, S. P. et al., 1995, Proc. Natl. Acad. Sci. USA 92: 8388-8392; Schnell. M. J. et al., 1994, EMBO J. 13: 4195-4203; Radecke, F. et al., 1995, EMBO J. 14: 5773-5784; Lawson, N. D. et al., Proc. Natl. Acad. Sci.
  • minus-strand RNA viruses including parainfluenza virus, vesicular stomatitis virus, rabies virus, measles virus, rinderpest virus, and Sendai virus can be reconstituted from DNA.
  • viruses of this invention can be reconstituted according to these methods.
  • a viral DNA is made F gene-, HN gene-, and/or M gene-deficient, such DNAs do not form infectious virions as is.
  • infectious virions can be formed by separately introducing host cells with these deficient genes, and/or genes encoding the envelope proteins of other viruses, and then expressing these genes therein.
  • the viruses can be prepared by the steps of: (a) transcribing cDNAs encoding genomic RNAs of minus-strand RNA viruses (negative strand RNAs), or complementary strands thereof (positive strands), in cells expressing N, P, and L proteins; and (b) harvesting culture supernatants thereof including the produced minus-strand RNA viruses.
  • a DNA encoding a genomic RNA is linked downstream of an appropriate promoter.
  • the genomic RNA thus transcribed is replicated in the presence of N, L, and P proteins to form an RNP complex.
  • M, HN, and F proteins virions enclosed in an envelope are formed.
  • a DNA encoding a genomic RNA can be linked downstream of a T7 promoter, and transcribed to RNA by T7 RNA polymerase.
  • Any desired promoter can be used as a promoter, in addition to those including a T7 polymerase recognition sequence.
  • RNA transcribed in vitro may be transfected into cells.
  • Enzymes essential for the initial transcription of genomic RNA from DNA can be supplied by introducing the plasmid or viral vectors that express them, or, for example, by incorporating the RNA polymerase gene into a chromosome of the cell so as to enable induction of its expression, and then inducing expression at the time of viral reconstitution.
  • genomic RNA and viral proteins essential for virus reconstitution are supplied, for example, by introducing the plasmids that express them.
  • helper viruses such as the wild type or certain types of mutant minus-strand RNA viruses can be used.
  • Methods for introducing DNAs expressing the genomic RNAs into cells include, for example, (i) methods for making DNA precipitates which target cells can internalize; (ii) methods for making complexes including DNAs that are suitable for internalization by target cells, and have a low-cytotoxic positive charge; and (iii) methods for using electric pulses to instantaneously create holes in the target cell membrane, which are of sufficient size for DNA molecules to pass through.
  • transfection reagents can be used.
  • DOTMA Roche
  • Superfect QIACEN #301305
  • DOTAP DOTAP
  • DOPE DOSPER
  • transfection methods using calcium phosphate can be cited, and although DNAs transferred into cells by this method are internalized by phagosomes, a sufficient amount of DNA is known to enter the nucleus (Graham, F. L. and Van Der Eb, J., 1973, Virology 52: 456; Wigler, M. and Silverstein, S., 1977, Cell 11: 223).
  • chloroquine may also be added to enhance the effect (Calos, M. P., 1983, Proc. Natl. Acad. Sci. USA 80: 3015).
  • the methods of (iii) are referred to as electroporation methods, and are used more in general than methods (i) or (ii) because they are not cell-selective. The efficiency of these methods is presumed to be good under optimal conditions for: the duration of pulse electric current, shape of the pulse, potency of electric field (gap between electrodes, voltage), conductivity of buffer, DNA concentration, and cell density.
  • the methods of (ii) are simple to operate and facilitate examination of many samples using a large amount of cells, making transfection reagents suitable for the transduction into cells of DNA for virus reconstitution.
  • the Superfect Transfection Reagent QIAGEN, Cat No. 301305
  • the DOSPER Liposomal Transfection Reagent (Roche, Cat No. 1811169) is used; however, the transfection reagents are not limited to these.
  • virus reconstitution from cDNA can be carried out, for example, as follows:
  • simian kidney-derived LLC-MK2 cells (ATCC CCL-7) are cultured up to about 100% confluency, using minimum essential medium (MEM) including 10% fetal calf serum (FCS) and antibiotics (100 units/ml penicillin G and 100 ⁇ g/ml streptomycin).
  • MEM minimum essential medium
  • FCS fetal calf serum
  • antibiotics 100 units/ml penicillin G and 100 ⁇ g/ml streptomycin
  • One hour after infection 2 to 60 ⁇ g, and more preferably 3 to 20 ⁇ g. of DNA encoding the genomic RNA of a recombinant Sendai virus is transfected along with the plasmids expressing trans-acting viral proteins essential for viral RNP production (0.5 to 24 ⁇ g of pGEM-N, 0.25 to 12 ⁇ g of pGEM-P, and 0.5 to 24 ⁇ g of pGEM-L) (Kato, A. et al., Genes Cells 1: 569-579, 1996), using the lipofection method or the like with Superfect (QIAGEN).
  • the ratio of the amounts of expression vectors encoding the N, P, and L proteins is preferably 2:1:2, and the plasmid amounts are appropriately adjusted in the range of 1 to 4 ⁇ g of pGEM-N, 0.5 to 2 ⁇ g of pGEM-P, and 1 to 4 ⁇ g of pGEM-L.
  • the transfected cells are cultured, as desired, in serum-free MEM composed of 100 ⁇ g/ml of rifampicin (Sigma) and cytosine arabinoside (AraC), more preferably only 40 ⁇ g/ml of cytosine arabinoside (AraC) (Sigma).
  • Optimal drug concentrations are set so as to minimize cytotoxicity due to the vaccinia virus, and to maximize virus recovery rate (Kato, A. et al., 1996, Genes Cells 1: 569-579). After culturing for about 48 to 72 hours after transfection, cells are harvested, and then disintegrated by repeating freeze-thawing three times.
  • LLC-MK2 cells are re-transfected with the disintegrated materials including RNP, and cultured. Alternatively, the culture supernatant is recovered, added to a culture solution of LLC-MK2 cells to infect them, and the cells are then cultured. Transfection can be conducted by, for example, forming a complex with lipofectamine, polycationic liposome, or the like, and introducing the complex into cells. Specifically, various transfection reagents can be used. For example, DOTMA (Roche), Superfect (QIAGEN #301305), DOTAP, DOPE, and DOSPER (Roche #1811169) may be cited. In order to prevent decomposition in the endosome, chloroquine may also be added (Calos, M.
  • Viruses thus obtained can be stored at ⁇ 80° C.
  • LLC-MK2 cells express the envelope protein may be used for transfection, or a plasmid expressing the envelope protein may be cotransfected.
  • a defective type virus can be amplified by culturing the transfected cells overlaid with LLK-MK2 cells expressing the envelope protein (see WO00/70055 and WO00/70070).
  • Titers of viruses thus recovered can be determined, for example, by measuring CIU (Cell-Infected Unit) or hemagglutination activity (HA) (WO00/70070; Kato, A. et al., 1996, Genes Cells 1: 569-579; Yonemitsu, Y. & Kaneda, Y., Hemaggulutinating virus of Japan-liposonme-mediated gene delivery to vascular cells. Ed. by Baker A H. Molecular Biology of Vascular Diseases. Method in Molecular Medicine: Humana Press: pp. 295-306, 1999).
  • CIU Cell-Infected Unit
  • HA hemagglutination activity
  • Titers of viruses carrying GFP (green fluorescent protein) marker genes and the like can be quantified by directly counting infected cells, using the marker as an indicator (for example, as GFP-CIU). Titers thus measured can be treated in the same way as CIU (WO00/70070).
  • GFP green fluorescent protein
  • the host cells used in the reconstitution are not particularly limited.
  • cultured cells such as LLC-MK2 cells and CV-1 cells derived from monkey kidney, BHK cells derived from hamster kidney, and cells derived from humans can be used.
  • suitable envelope proteins By expressing suitable envelope proteins in these cells, infectious virions including the proteins in the envelope can also be obtained.
  • a virus obtained from an above-described host can be infected to embrionated hen eggs, to propagate the virus. Methods for manufacturing viruses using hen eggs have already been developed (Nakanishi, et al., ed.
  • a fertilized egg is placed in an incubator, and cultured for nine to twelve days at 37 to 38° C. to grow an embryo. After the virus is inoculated into the allantoic cavity, the egg is cultured for several days (for example, three days) to proliferate the virus. Conditions such as the period of culture may vary depending upon the recombinant Sendai virus being used. Then, allantoic fluids including the virus are recovered.
  • Sendai viruses defective in F gate can be performed as described below (see WO00/70055 and WO00/70070).
  • a PCR product formed in PCR by using the pair of primers [forward: 5′-gttgagtactgcaagagc/SEQ ID NO: 5, reverse: 5′-tttgccggcatgcatgtttcccaaggggagagtttgcaacc/SEQ ID NO: 6] is connected upstream of F, and a PCR product formed using the pair of primers [forward: 5′-atgcatgccggcagatga/SEQ ID NO: 7, reverse: 5′-tgggtgaatgagagaatcagc/SEQ ID NO: 8] is connected downstream of the F gene with EcoT22I.
  • the plasmid thus obtained is digested with SacI and SalI to recover a 4931 bp fragment of the region including the F gene-defective site, which is cloned into pUC1 to form pUC18/dFSS.
  • This pUC18/dFSS is digested with DraIII, the fragment is recovered, replaced with the DraIII fragment of the region including the F gene of pSeV18 + , and ligated to obtain the plasmid pSeV18 + / ⁇ F.
  • a foreign gene is inserted, for example, into the NsiI and NgoMIV restriction enzyme sites in the F gene-defective site of pUC18/dFSS.
  • a foreign gene fragment may be, for example, amplified using an NsiI-tailed primer and an NgoMIV-tailed primer.
  • the SeV-F gene is amplified by PCR, and inserted to the unique SwaI site of the plasmid pCALNdlw (Arai, T. et al., J. Virology 72, 1998, p 1115-1121), which is designed to enable the inducible expression of a gene product by Cre DNA recombinase, thus constructing the plasmid pCALNdLw/F.
  • a helper cell line expressing SeV-F protein is established.
  • the monkey kidney-derived LLC-MK2 cell line which is commonly used for SeV propagation, can be used as the cells, for example. LLC-MK2 cells are cultured in MEM supplemented with 10% heat-inactivated fetal bovine set (FBS), penicillin G sodium (50 units/ml), and streptomycin (50 ⁇ g/ml) at 37° C. in 5% CO 2 .
  • FBS heat-inactivated fetal bovine set
  • penicillin G sodium 50 units/ml
  • streptomycin 50 ⁇ g/ml
  • the SeV-F gene product is cytotoxic
  • the above-described plasmid pCALNdLw/F which was designed to enable inducible expression of the F gene product with Cre DNA recombinase, is transfected to LLC-MK2 cells by the calcium phosphate method (using a mammalian transfection kit (Stratagene)), according to protocols well known in the art.
  • the plasmid pCALNdLw/F (10 ⁇ g) is introduced into LLC-MK2 cells grown to 40% confluency using a 10cm plate, and the cells are then cultured in MEM (10 ml) including 10% FBS, in a 5% CO 2 incubator at 37° C. for 24 hours. After 24 hours, the cells are detached and suspended in the medium (10 ml). The suspension is then seeded into five 10-cm dishes, 5 ml into one dish, 2 ml each into two dishes, and 0.2 ml each into two dies, and cultured n MEM (10 ml) including G418 (GIBCO-BRL) (1200 ⁇ g/ml) and 10% FBS.
  • MEM MEM
  • G418 G418
  • the cells were cultured for 14 days, exchanging the medium every two days, to select cell lines into which the gene is stably introduced.
  • the cells grown from the above medium that show G418 resistance are recovered using a cloning ring. Culture of each clone thus recovered is continued in 10-cm plates until confluent.
  • LLC-MK2 cells are seeded on 100-mm dishes at 5 ⁇ 10 6 cells/dish.
  • T7 RNA polymerase To transcribe the genomic RNA using T7 RNA polymerase, the cells are cultured for 24 hours, and then recombinant vaccinia virus, which expresses T7 RNA polymerase (PLWUV-VacT7: Fuerst, T. R. et al., Proc. Natl. Acad. Sci.
  • UV Stratalinker 2400 with five 15-watt bulbs (Catalog No. 400676 (100V); Stratagene, La Jolla, Calif., USA).
  • plasmid expressing the genomic RNA and expression plasmids each expressing N, P, L, F, or HN protein of the minus-strand RNA virus are transected into the cells using an appropriate lipofection reagent.
  • the plasmid ratio is preferably, but is not limited to, 6:2:1:2:2:2 in this order.
  • the expression plasmid for the genomic RNA, and the expression plasmids each of which expresses N, P, or L protein, or F and HN proteins pGEM/NP, pGEM/P, pGEM/L, and pGEM/F-HN; WO00/70070, Kato, A.
  • the suspension is subjected to three freeze-thaw cycles, and mixed with lipofection reagent DOSPER (Boehringer Mannheim) (10 6 cells/25 ⁇ l DOSPER). After the mixture is allowed to stand at room temperature for 15 minutes, it is transfected to F-expressing helper cells (10 6 cells/well in 12-well-plate) cloned as described above. The cells are cultured in serum-free MEM (containing 40 ⁇ g/ml AraC and 7.5 ⁇ g/ml trypsin), and the supernatant is collected. Viruses deficient in genes other than F, for example, HN and M genes, can be prepared by a similar method as described above.
  • a naturally derived vim may also be used as a minus-strand RNA virus of the present invention, and one can refer to “Uirusu Jikken-gaku kakuron (Virology Experiments in detail)”, second revised edition (edited by Researcher's Associates at the National Institute of Health, Maruzen, 1982) for methods of purifying, multiplying, and obtaining isolated stains of each of the RNA viruses.
  • each type of parainfluenza virus such as Sendai virus belonging to the Paramyxoviridae family
  • Vero cells are most widely used, it can be propagated using CV1, FL, KB, HeLa, HEp2, and such (p 351).
  • Viruses belonging to the Rhabdoviridae family are propagated by tissue culture methods using BHK, CE, Vero cells, and the like. Purification methods involve the steps of adjusting the pH of a culture solution on the third to fourth day of infection to 7.4 or more, and concentrating the solution after removing cell debris by low-speed centrifugation (p 376).
  • Viruses belonging to the Arenaviridae family propagate well in most cultured cells that are subcultured in vitro, and it can be propagated by infection into HK-21/13S cells, followed by culturing as a suspension in agar (Sedwik W. D., J. Virol. 1, 1224 (1967)) (p 240).
  • Viruses belonging to the Orthomyxoviridae family such as the influenza virus, can be propagated in developing hen eggs and MDCK cells (p 295). Purification can be carried out by methods such as centrifugation, and adsorption to and release from red blood cells (Layer W. C., Fundamental Techniques in Virology, 82 (1969)) (p 317).
  • proteins include, for example, hormones, cytokines, growth factors, receptors, intracellular signaling molecules, enzymes, and peptides.
  • the proteins may be secretory proteins, membrane proteins, cytoplasmic proteins, nuclear proteins, and the like.
  • Artificial proteins include, for example, fusion proteins of chimeric toxins and such, dominant negative proteins (including soluble receptor molecules or membrane-bound dominant negative receptors), cell surface molecules and truncated cell adhesion molecules.
  • the proteins may also be proteins to which a secretory signal, membrane-localization signal, nuclear translocation signal, or the like has been attached.
  • RNA molecules RNA-cleaving ribozymes, or the like as the introduced gene.
  • gene therapy can be performed by administering this virus.
  • the virus of the present invention can be released into extracellular fluid of virus producing cells at a titer of, for example, 1 ⁇ 10 5 CIU/ml or higher, preferably 1 ⁇ 10 6 CIU/ml or higher, more preferably 5 ⁇ 10 6 CIU/ml or higher, more preferably 1 ⁇ 10 7 CIU/ml or higher, more preferably 5 ⁇ 10 7 CIU/ml or higher, more preferably 1 ⁇ 10 8 CIU/ml or higher, and more preferably 5 ⁇ 10 8 CIU/ml or higher.
  • the titer of virus can be determined according to methods described herein or elsewhere (Kiyotani, K. et al., Virology 177(1), 65-74 (1990); and WO00/70070).
  • the recovered viruses can be purified to be substantial pure. Tee purification can be achieved using known purification/separation methods, including filtration, centrifugation, adsorption, and column purification, or any combinations thereof.
  • substantially pure means that the virus component constitutes a major proportion of a solution comprising the virus.
  • a viral composition can be confirmed to be substantially pure by the fact that the proportion of protein contained as the viral component to the total protein (excluding proteins added as carriers and stabilizers) in the solution is 10% (w/w) or greater, preferably 20% or greater, more preferably 50% or greater, preferably 70% or greater, more preferably 80% or greater, and even more preferably 90% or greater.
  • Specific purification methods for, for example, the paramyxovirus include methods using cellulose sulfate ester or cross-linked polysaccharide sulfate ester (Japanese Patent Application Kokoku Publication No. (JP-B) S62-30752 (examined, approved Japanese patent application published for opposition), JP-B S62-33879, and JP-B S62-30753) and methods including adsorbing to fucose sulfate-containing polysaccharide and/or degradation products thereof (WO97/32010), but are not limited thereto.
  • the minus-strand RNA viruses of the present invention may be, for example, infectious virions or non-infectious virions.
  • the RNA viruses of the present invention may also be genomic RNA-protein complexes (ribonucleoprotein complexes; RNPs).
  • RNPs genomic RNA-protein complexes
  • these virions or complexes are preferably mixed with a lipofection reagent and administered in vivo.
  • virions and complexes can be mixed with lipofectamine or a polycationic liposome and this mixture can be administered in vivo (WO00/70055). In this method, various transfection reagents can be used.
  • DOTMA Boehringer
  • Superfect QIAGEN #301305
  • DOPE DOPE
  • DOSPER Boehringer #1811169
  • Chloroquine can be added to prevent degradation in endosomes (Calos, M. P., 1983, Proc. Natl. Acad. Sci. USA 80: 3015).
  • the minus-stan RNA virus can be mixed (combined as necessary with a desired pharmaceutically acceptable carrier or medium.
  • the present invention provides methods for producing an anticancer agent, which comprises the step of mixing a minus-strand RNA virus with a pharmaceutically acceptable carrier or medium.
  • the “pharmaceutically acceptable carrier or medium” refers to materials that can be administered together with a minus-strand RNA virus and which do not significantly inhibit infection by the minus-strand RNA virus.
  • Such carrier or medium includes, for example, deionized water, sterilized water, sodium chloride solution, dextrose solution, culture medium, serum, phosphate buffered saline (PBS), and Ringer's solution containing dextrose, sodium chloride, and lactic acid, and they may be appropriately combined with a minus-strand RNA virus in formulations. Further, they may be concentrated by centrifugation when necessary, and resuspended in a physiological solution such as a culture solution or physiological saline solution.
  • a composition of the present invention may take the form of an aqueous solution, capsule, suspension, syrup, or such.
  • a virus composition of the present invention may be a composition in the form of a solution, freeze-dried product, or aerosol. In the case of a freeze-dried product, it may include sorbitol, sucrose, amino acids, various proteins, and such as stabilizers.
  • the minus-strand RNA viruses of the present invention have anticancer activity, and they can exhibit anticancer (carcinostatic) effects when administered to tumors (cancer tissue). Therefore, the present invention relates to a method for suppressing cancer (method for suppressing cancer cell growth, or method for treating cancer), which comprises the step of administering a minus-strand RNA virus to cancer tissues (in vivo administration).
  • cancer therapy can be performed on cancer patients.
  • This method comprises the step of administering a minus-strand RNA virus (an anticancer agent of the present invention). Specifically, the method comprises the step of administering a therapeutically effective amount of a minus-strand RNA virus to a patient.
  • Use of the present method is expected to suppress the growth of cancer cells compared to when a minus-strand RNA virus of the present invention is not administered.
  • the minus-strand RNA virus may not carry a foreign gene, or it may carry a gene (foreign gene) encoding one or more cancer antigens, immunostimulatory cytokines, proteins that inhibit angiogenesis, or such.
  • the present invention can be applied to desired solid cancers, and such examples include tongue cancer, gum cancer, malignant lymphoma, malignant melanoma, upper jaw cancer, nose cancer, nasal cavity cancer, larynx cancer, pharyngeal cancer, glioma, meningioma, glioma, lung cancer, breast cancer, pancreatic cancer, gastrointestinal cancer (esophageal cancer, stomach cancer, duodenal cancer, colon cancer), squamous cancer, adenocarcinoma, alveolar cell cancer, testis tumor, prostate cancer, thyroid cancer, liver cancer, ovarian cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, and chondrosarcoma.
  • the target cancer is preferably epithelial cancer, and is more preferably skin cancer including skin squamous cancer, skin basal cell cancer, Bowen's disease, Paget's disease, and skin malignant melanoma.
  • the in vivo dose of the minus-strand RNA virus of this invention varies depending on the disease, patient's weight, age, sex, and symptom, purpose of administration, form of administered composition, administration method, introduced gene, and the like, but can be appropriately determined by those skilled in the art.
  • the route of administration can be appropriately selected, and includes, for example, percutaneous, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, intraarticular, and subcutaneous administration.
  • the administration may be local or systemic.
  • the virus at a dose within the range of preferably about 10 5 to about 10 11 CIU/ml, more preferably about 10 7 to about 10 9 CIU/ml, and most preferably about 1 ⁇ 10 5 to about 5 ⁇ 10 8 CIU/ml, in a pharmaceutically acceptable carrier.
  • the amount per dose for human is preferably 2 ⁇ 10 5 to 2 ⁇ 10 11 CIU, which is administered once or more within a range where the side effects are clinically acceptable. The same applies to the number of doses per day.
  • a dose converted from the above-described dose based on the body weight ratio between the subject animal and human or the volume ratio (e.g., mean value) of the target site for administration.
  • the propagation of the transmissible minus-strand RNA virus when it becomes necessary to suppress the propagation of the transmissible minus-strand RNA virus after administration to subjects or cells due to the completion of treatment or the like, through the administration of an RNA-dependent RNA polymerase inhibitor, the propagation of the virus can be specifically suppressed without damaging the host.
  • the minus-strand RNA viruses of the present invention are preferably administered to the cancerous lesion of a patient.
  • “Cancerous lesion” refers to a region of cancer tissue or its surrounding area (for example, within 5 mm of the cancer, or preferably within 3 mm of the cancer).
  • the dose may be appropriately adjusted depending on the type and stage of the cancer, the presence or absence of an introduced gene, and such.
  • antitumor effects are expected of minus-strand RNA viruses even if they do not carry foreign genes, higher synergistic effects can be expected by loading RNA viruses with, for example, an IFN-beta gene or soluble FGF receptor gene.
  • the minus-strand RNA virus can be administered one or more times as long as the side effects are clinically acceptable, and the same applies to the number of doses administered per day.
  • Subjects receiving the administration are not particularly limited, and examples include birds and mammals (human and non-human mammals) such as chicken, quail, mouse, rat, dog, pig, cat, bovine, rabbit, sheep, goat monkey, and human.
  • mammals human and non-human mammals
  • an amount calculated from the above-mentioned dose according to the body weight ratio between human and the animal of interest can be administered.
  • the present invention also relates to the use of a minus-strand RNA virus in cancer therapy. Furthermore, the present invention relates to the use of a minus-strand RNA virus in the production of anticancer agents (or carcinostatic agents, agents for suppressing cancer growth, or such).
  • the present invention relates to a package comprising a minus-strand RNA virus, which includes descriptions on the use of the minus-strand RNA virus for cancer suppression (as an anticancer agent).
  • the minus-strand RNA virus may be suspended in a solution such as culture solution or physiological saline solution.
  • “Use in cancer suppression” means that for example, a minus-strand RNA virus, or a composition comprising such RNA virus, is used in tumor growth suppression, cancer degeneration, cancel therapy, treatment and therapy of cancer patients, prolongation of the life of cancer patients, or as an anticancer agent.
  • the descriptions can be printed directly on the package; alternatively, a piece of paper or a sticker containing the descriptions may be comprised in the package.
  • the package may be a container comprising a minus-strand RNA virus, and in such case, the container may be, for example, a bottle, tube, plastic bag, vial syringe, or such. Furthermore, the package of the present invention may comprise a bag, outer box, or such that stores the container.
  • the package may comprise instructions describing the method for administering a minus-strand RNA virus, and may further comprise a syringe, catheter, and/or injection needle for administering the minus-strand RNA virus.
  • kits for treating cancer or for producing an anticancer agent which comprises at least a minus-strand RNA virus as a component.
  • a kit of the present invention may appropriately comprise, for example, carries, physiological saline solution, buffer, bottle, tube, plastic bag, vial, and syringe. Instructions for use can also be packaged into the kit.
  • Five days (day 5) and 12 days (day 12) after inoculation 1 ⁇ 10 8 PFU of Sendai virus that does not comprise any specific therapeutic gene (GFP-expressing SeV; SeV-GFP).
  • Sendai virus expressing a human soluble FGF receptor (SeV-sFGFR), or Sendai virus expressing human soluble PDGFR ⁇ (SeV-hsPDGFR ⁇ ) was injected into the tumor. Then, the tumor size was measured over time.
  • the tumor size significantly decreased compared to the tumor size of the nonadministered group ( FIG. 1 ). Therefore, tn vivo administration of SeV was able to exhibit antitumor effects, even when the SeV did not carry any therapeutic gene.
  • SeV expressing a soluble FGF receptor was administered, a stronger effect of tumor growth suppression was confirmed as compared to that in the SeV-GFP-administered group.
  • the antitumor effect was most significant when the soluble PDGFR ⁇ -expressing SeV was administered, since the tumor size hardly increased.
  • SeV exhibits a sufficient antitumor effect even if it does not carry any foreign gene such as a therapeutic gene.
  • This Example shows an example of a method for beating a tumor by in vivo and ex vivo administration of an RNA virus
  • a B16 melanoma-transplanted model that expresses MHC class I at only a very low level and exhibits poor immunogenicity was used as a tumor model.
  • C57BL/6 mice (6- to 8-week-old; female) (CHARLES RIVER JAPAN, INC.) were used as the tumor model mice, and dendritic cells were collected from C57BL/6 mice (8week-old; female) (CHARLES RIVER JAPAN, INC.).
  • the dendritic cells were obtained by collecting bone marrow from thigh bones of C57BL/6 mice; removing T cells using SpinSepTM, murine hematopoietic progenitor enrichment cocktail (anti-CD5 antibody, anti-CD45R antibody, anti-CD11b antibody, anti-Gr-1 antibody, anti-TER119 antibody, anti-7/4 antibody; Stem Cell technology); then culturing the cells for one week with the addition of IL-4 and GM-CSF. On day 0, 1 ⁇ 10 5 /100 ⁇ L of B16 melanoma cells were subcutaneously (s.c.) inoculated into the abdominal area of the mice.
  • s.c. subcutaneously
  • dendritic cells without stimulation for activation dendritic cells activated with LPS (LPS DC), or dendritic cells activated by introducing SeV-GFP or SeV-IFN ⁇ expressing mouse interferon ⁇ (SeV GFP DC and SeV IFN ⁇ DC, respectively) were administered in the area surrounding the tumor.
  • LPS DC LPS-driven dendritic cells activated by introducing SeV-GFP or SeV-IFN ⁇ expressing mouse interferon ⁇
  • tumor antigens tumor antigens
  • SeV was introduced into dendritic cells by infecting dendritic cells cultured for one week as described above with SeV-IFN ⁇ at a MOI of 40, and culturing the cells for 8 hours.
  • SeV-IFN ⁇ suppressed tumor growth, both in the case of direct intratumoral injection and in the case of ex vivo administration through dendritic cells
  • a significantly strong tumor suppressing effect was observed in mice treated with DC/SeV-IFN ⁇ .
  • NK natural killer
  • spleens were excised from mice of each of the therapeutic groups described above after 7 days from the end of three rounds of DC therapy to prepare effector cells.
  • 51 Cr release assay was performed using Yac-1 as the target.
  • the spleen cells remaining from the NK cell activity assay described above were cultured for 5 days with TRP-2 peptide, a B16 tumor antigen, to use them as effector cells.
  • the effector cells were co-cultured with EL-4 target cells pulsed with mTRP-2 peptide, and then 51 Cr release assay was performed.
  • the rate of specific 51 Cr release was calculated as follows: [(sample (cpm) ⁇ spontaneous emission (cpm))/(maximum emission (cpm) ⁇ spontaneous emission (cpm))] ⁇ 100 where the maximum emission was determined using target cells incubated with 1% triton X, while spontaneous emission was determined using target cells incubated with culture medium alone.
  • NK natal killer cells
  • CTLs cytotoxic T lymphocytes
  • the present invention provides anticancer agents that contain a minus-strand RNA virus as an active ingredient.
  • the RNA viruses of the present invention exhibit effective anticancer therapeutic effects without carrying any foreign gene that has therapeutic effects. Therefore, they may contribute greatly to the reduction of the cost for introducing foreign genes and to the reduction of the operational time for virus preparation.
  • the present invention enables new virotherapies that use minus-strand RNA viruses.
US11/630,522 2004-06-24 2005-04-28 Anticancer Agent Containing Minus-Strand Rna Virus Abandoned US20080031855A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
JP2004-187028 2004-06-24
JP2004187028 2004-06-24
PCT/JP2004/016089 WO2005042737A1 (ja) 2003-11-04 2004-10-29 遺伝子導入された樹状細胞の製造方法
JPPCT/JP2004/016089 2004-10-29
PCT/JP2005/008124 WO2006001120A1 (ja) 2004-06-24 2005-04-28 マイナス鎖rnaウイルスを含む抗癌剤

Publications (1)

Publication Number Publication Date
US20080031855A1 true US20080031855A1 (en) 2008-02-07

Family

ID=37866022

Family Applications (2)

Application Number Title Priority Date Filing Date
US11/630,522 Abandoned US20080031855A1 (en) 2004-06-24 2005-04-28 Anticancer Agent Containing Minus-Strand Rna Virus
US11/630,532 Expired - Fee Related US8889118B2 (en) 2004-06-24 2005-04-28 Anticancer agent containing dendritic cell having RNA virus transferred thereinto

Family Applications After (1)

Application Number Title Priority Date Filing Date
US11/630,532 Expired - Fee Related US8889118B2 (en) 2004-06-24 2005-04-28 Anticancer agent containing dendritic cell having RNA virus transferred thereinto

Country Status (9)

Country Link
US (2) US20080031855A1 (zh)
EP (2) EP1775343A4 (zh)
JP (3) JPWO2006001120A1 (zh)
KR (2) KR20070028573A (zh)
CN (2) CN101006171A (zh)
AU (2) AU2005257105A1 (zh)
CA (2) CA2571844A1 (zh)
HK (1) HK1106794A1 (zh)
WO (2) WO2006001122A1 (zh)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020002143A1 (en) * 2000-03-30 2002-01-03 Munehide Kano AIDS virus vaccines using sendai virus vector
US20050266566A1 (en) * 1995-11-01 2005-12-01 Dnavec Research Inc. Recombinant sendai virus
US20060104950A1 (en) * 2002-10-24 2006-05-18 Shinji Okano Methods of Tranducing genes into T cells
US20070009949A1 (en) * 1999-05-18 2007-01-11 Kaio Kitazato Paramyxovirus-derived RNP
US20070161110A1 (en) * 2004-01-22 2007-07-12 Akihiro Iida Methods for producing minus-strand rna viral vectors using hybrid promoter comprising cytomegalovirus enhancer and chicken beta-actin promoter
US20070248627A1 (en) * 2004-01-13 2007-10-25 Dnavec Research Inc. Gene Therapy for Tumors Using Minus-Strand Rna Viral Vectors Encoding Immunostimulatory Cytokines
US20070269414A1 (en) * 2003-11-04 2007-11-22 Shinji Okano Method for Producing Gene Transferred Denritic Cells
US20080014183A1 (en) * 2004-06-24 2008-01-17 Dnavec Research Inc. Anticancer Agent Containing Dendritic Cell Having Rna Virus Transferred Thereinto
US20080038234A1 (en) * 2004-03-23 2008-02-14 Shuji Hayashi Bone Marrow-Related Cells Associated With Tissue Maintenance And/Or Repair
US20090053790A1 (en) * 2006-01-31 2009-02-26 Ishihara Sangyo Kaisha, Ltd. Polypeptide Having Affinity for Envelope Virus Constituent and Use Thereof in Transferring Substance Into Cell
US20100167341A1 (en) * 2006-01-17 2010-07-01 Dnavec Corporation Novel protein expression system
US20110162093A1 (en) * 2005-06-14 2011-06-30 Yasuji Ueda Methods for producing antibodies
US20110223148A1 (en) * 2008-09-16 2011-09-15 Genomidea Inc. Therapeutic/prophylactic agent for prostate cancer
US20120107829A1 (en) * 2009-03-31 2012-05-03 Leukocare Ag Stabilizing compositions for immobilized biomolecules
US20170070889A1 (en) * 2008-05-13 2017-03-09 At&T Mobility Ii Llc Administration of access lists for femtocell service
US20200140887A1 (en) * 2016-06-22 2020-05-07 Icahn School Of Medicine At Mount Sinai Viral Delivery of RNA Utilizing Self-Cleaving Ribozymes and CRISPR-Based Applications Thereof

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2377925A1 (en) 2006-04-14 2011-10-19 Advanced Cell Technology, Inc. Hemangio-colony forming cells
JPWO2008065752A1 (ja) * 2006-11-30 2010-03-04 国立大学法人北海道大学 ｄｉＲＮＡを有効成分とする免疫治療用薬剤
KR101647843B1 (ko) 2007-03-12 2016-08-11 온콜리틱스 바이오테크 인코포레이티드 변형된 서열을 갖는 레오바이러스
WO2008136438A1 (ja) * 2007-04-27 2008-11-13 Kyushu University, National University Corporation 遺伝子治療用ウイルスベクター
US8283163B2 (en) * 2007-05-17 2012-10-09 Dnavec Corporation Method for production of dendritic cell
WO2010055900A1 (ja) 2008-11-14 2010-05-20 ディナベック株式会社 樹状細胞の製造方法
US20100266642A1 (en) * 2009-02-20 2010-10-21 Bind Biosciences, Inc. Modified cells for targeted cell trafficking and uses thereof
CN102822332A (zh) * 2009-12-04 2012-12-12 干细胞及再生医学国际股份有限公司 从源于人胚胎干细胞的成血管细胞产生自然杀伤细胞和树突细胞的方法
DE102010018961B4 (de) * 2010-04-23 2012-09-20 Eberhard-Karls-Universität Tübingen Universitätsklinikum Genetisch modifiziertes Paramyxovirus zur Behandlung von Tumorerkrankungen
KR101365208B1 (ko) * 2011-12-06 2014-02-20 가톨릭대학교 산학협력단 바이러스를 이용하여 제조된 심근염 치료용 면역관용 수지상 세포 및 이의 제조 방법
CA2896053A1 (en) 2012-12-21 2014-06-26 Ocata Therapeutics, Inc. Methods for production of platelets from pluripotent stem cells and compositions thereof
DE202016008300U1 (de) 2015-07-02 2017-07-14 PrimeVax Immuno-Oncology, Inc. Zusammensetzungen zur Kombinationstherapie mit Denguevirus und dendritischen Zellen
MX2018003757A (es) 2015-09-26 2018-09-21 Primevax Immuno Oncology Inc Composiciones y metodos para producir celulas dendriticas.
CN110139671A (zh) 2016-11-16 2019-08-16 普莱瓦克斯免疫肿瘤学公司 用于治疗癌症的组合免疫疗法

Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124148A (en) * 1988-04-27 1992-06-23 United Cancer Research Institute Method for treating viral diseases with attenuated virus
US6077519A (en) * 1993-01-29 2000-06-20 University Of Pittsburgh Methods for isolation and use of T cell epitopes eluted from viable cells in vaccines for treating cancer patients
US6300090B1 (en) * 1994-07-29 2001-10-09 The Rockefeller University Methods of use of viral vectors to deliver antigen to dendritic cells
US20020002143A1 (en) * 2000-03-30 2002-01-03 Munehide Kano AIDS virus vaccines using sendai virus vector
US20020098576A1 (en) * 1995-11-01 2002-07-25 Yoshiyuki Nagai Recombinant sendai virus
US20020123479A1 (en) * 1999-12-08 2002-09-05 Song Elizabeth S. Immunostimulation mediated by gene-modified dendritic cells
US20020169306A1 (en) * 1999-05-18 2002-11-14 Kaio Kitazato Envelope gene-deficient paramyxovirus vector
US20030022376A1 (en) * 1999-05-18 2003-01-30 Kaio Kitazato Paramyxovirus-derived RNP
US20030166252A1 (en) * 1999-05-18 2003-09-04 Kaio Kitazato Paramyxovirus-derived RNP
US20030170266A1 (en) * 1999-05-18 2003-09-11 Kaio Kitazato Envelope gene-deficient paramyxovirus vector
US6645760B2 (en) * 1995-10-31 2003-11-11 Dnavec Research Inc. Negative strand RNA viral vector having autonomous replication capability
US6746860B1 (en) * 2000-05-18 2004-06-08 Dnavec Research, Inc. Paramyxovirus vectors used for transfer of foreign genes
US20040265272A1 (en) * 2001-09-28 2004-12-30 Aikichi Iwamoto Mammalian cell-infecting virus vector encoding epitope-bound beta2m and utilization thereof
US20050130123A1 (en) * 2001-09-18 2005-06-16 Makoto Inoue Methods of examining (-) strand rna virus vectors having lowered ability to form grains and method of constructing the same
US20050191617A1 (en) * 2002-06-03 2005-09-01 Makoto Inoue Pramyxovirusl vectors encoding antibody and utilization thereof
US20060104950A1 (en) * 2002-10-24 2006-05-18 Shinji Okano Methods of Tranducing genes into T cells
US7056689B1 (en) * 1993-04-30 2006-06-06 Wellstat Biologics Corporation Methods of treating and detecting cancer using viruses
US20070141705A1 (en) * 2004-01-22 2007-06-21 Makoto Inoue Method for producing viral vectors
US20070161110A1 (en) * 2004-01-22 2007-07-12 Akihiro Iida Methods for producing minus-strand rna viral vectors using hybrid promoter comprising cytomegalovirus enhancer and chicken beta-actin promoter
US20070248627A1 (en) * 2004-01-13 2007-10-25 Dnavec Research Inc. Gene Therapy for Tumors Using Minus-Strand Rna Viral Vectors Encoding Immunostimulatory Cytokines
US20070269414A1 (en) * 2003-11-04 2007-11-22 Shinji Okano Method for Producing Gene Transferred Denritic Cells
US20080014183A1 (en) * 2004-06-24 2008-01-17 Dnavec Research Inc. Anticancer Agent Containing Dendritic Cell Having Rna Virus Transferred Thereinto
US20080038234A1 (en) * 2004-03-23 2008-02-14 Shuji Hayashi Bone Marrow-Related Cells Associated With Tissue Maintenance And/Or Repair
US20080199438A1 (en) * 2004-03-16 2008-08-21 Dnavec Research Inc. Methods For Suppressing Tumor Proliferation

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6734014B1 (en) 1996-02-08 2004-05-11 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for transforming dendritic cells and activating T cells
WO1998006437A2 (en) 1996-08-13 1998-02-19 Chiron Corporation Compositions and methods for polynucleotide delivery
WO1998053048A1 (en) 1997-05-21 1998-11-26 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods and compositions for making dendritic cells from expanded populations of monocytes and for activating t cells
JP2000253876A (ja) 1999-03-08 2000-09-19 Dnavec Research Inc センダイウイルスベクターを用いたワクチンおよびワクチンタンパク質
DK1186667T3 (da) * 1999-05-18 2007-10-22 Dnavec Research Inc Kappegendeficient virusvektor af Paramyxoviridae
EP1179594B1 (en) 1999-05-18 2008-06-25 Dnavec Research Inc. Ribonucleoprotein complex in paramyxovirus
US6472208B1 (en) 1999-12-13 2002-10-29 Héma-Québec Method of producing human IFN-α using sendai virus-infected hematopoietic stem cells
JP4791651B2 (ja) 2000-05-18 2011-10-12 株式会社ディナベック研究所 外来遺伝子導入用パラミクソウイルスベクター
US20050013810A1 (en) 2001-05-08 2005-01-20 Waller Edmund K Regulating immune response using dendritic cells
EP1509595A4 (en) 2001-07-05 2006-02-22 Phylogix Inc METHODS FOR ISOLATING DENDRITIC CELLS
EP1441591B1 (en) 2001-09-06 2016-06-29 NorthWest Biotherapeutics, Inc. Compositions and methods for priming monocytic dendritic cells and t cells for th-1 response
WO2003029274A2 (en) * 2001-09-28 2003-04-10 University Of North Carolina At Chapel Hill Paramyxoviruses as gene transfer vectors to lung cells
FR2838123B1 (fr) 2002-04-04 2005-06-10 Sanofi Synthelabo Nouveaux derives d'indolozine-1,2,3 substituee, inhibiteurs selectifs du b-fgf
AU2002951082A0 (en) 2002-08-30 2002-09-12 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Therapeutic cellular agents
US7785583B2 (en) 2002-12-10 2010-08-31 Argos Therapeutics, Inc. In situ maturation of dendritic cells
AU2006237903A1 (en) 2005-04-20 2006-10-26 Dna Vec Corporation Highly safe intranasally administrable gene vaccines for treating Alzheimer's disease
KR20090027693A (ko) 2006-05-31 2009-03-17 디나벡크 가부시키가이샤 알츠하이머병 치료제
US8283163B2 (en) 2007-05-17 2012-10-09 Dnavec Corporation Method for production of dendritic cell

Patent Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5124148A (en) * 1988-04-27 1992-06-23 United Cancer Research Institute Method for treating viral diseases with attenuated virus
US6077519A (en) * 1993-01-29 2000-06-20 University Of Pittsburgh Methods for isolation and use of T cell epitopes eluted from viable cells in vaccines for treating cancer patients
US7056689B1 (en) * 1993-04-30 2006-06-06 Wellstat Biologics Corporation Methods of treating and detecting cancer using viruses
US6300090B1 (en) * 1994-07-29 2001-10-09 The Rockefeller University Methods of use of viral vectors to deliver antigen to dendritic cells
US6645760B2 (en) * 1995-10-31 2003-11-11 Dnavec Research Inc. Negative strand RNA viral vector having autonomous replication capability
US6723532B2 (en) * 1995-10-31 2004-04-20 Dnavec Research Inc. Negative strand RNA viral vector having autonomous replication capability
US20020098576A1 (en) * 1995-11-01 2002-07-25 Yoshiyuki Nagai Recombinant sendai virus
US20050266566A1 (en) * 1995-11-01 2005-12-01 Dnavec Research Inc. Recombinant sendai virus
US20020169306A1 (en) * 1999-05-18 2002-11-14 Kaio Kitazato Envelope gene-deficient paramyxovirus vector
US20070009949A1 (en) * 1999-05-18 2007-01-11 Kaio Kitazato Paramyxovirus-derived RNP
US20030166252A1 (en) * 1999-05-18 2003-09-04 Kaio Kitazato Paramyxovirus-derived RNP
US20030022376A1 (en) * 1999-05-18 2003-01-30 Kaio Kitazato Paramyxovirus-derived RNP
US20030170266A1 (en) * 1999-05-18 2003-09-11 Kaio Kitazato Envelope gene-deficient paramyxovirus vector
US20020123479A1 (en) * 1999-12-08 2002-09-05 Song Elizabeth S. Immunostimulation mediated by gene-modified dendritic cells
US20020002143A1 (en) * 2000-03-30 2002-01-03 Munehide Kano AIDS virus vaccines using sendai virus vector
US6746860B1 (en) * 2000-05-18 2004-06-08 Dnavec Research, Inc. Paramyxovirus vectors used for transfer of foreign genes
US20050130123A1 (en) * 2001-09-18 2005-06-16 Makoto Inoue Methods of examining (-) strand rna virus vectors having lowered ability to form grains and method of constructing the same
US20040265272A1 (en) * 2001-09-28 2004-12-30 Aikichi Iwamoto Mammalian cell-infecting virus vector encoding epitope-bound beta2m and utilization thereof
US20050191617A1 (en) * 2002-06-03 2005-09-01 Makoto Inoue Pramyxovirusl vectors encoding antibody and utilization thereof
US20060104950A1 (en) * 2002-10-24 2006-05-18 Shinji Okano Methods of Tranducing genes into T cells
US20070269414A1 (en) * 2003-11-04 2007-11-22 Shinji Okano Method for Producing Gene Transferred Denritic Cells
US20070248627A1 (en) * 2004-01-13 2007-10-25 Dnavec Research Inc. Gene Therapy for Tumors Using Minus-Strand Rna Viral Vectors Encoding Immunostimulatory Cytokines
US20070141705A1 (en) * 2004-01-22 2007-06-21 Makoto Inoue Method for producing viral vectors
US20070161110A1 (en) * 2004-01-22 2007-07-12 Akihiro Iida Methods for producing minus-strand rna viral vectors using hybrid promoter comprising cytomegalovirus enhancer and chicken beta-actin promoter
US20080199438A1 (en) * 2004-03-16 2008-08-21 Dnavec Research Inc. Methods For Suppressing Tumor Proliferation
US20080038234A1 (en) * 2004-03-23 2008-02-14 Shuji Hayashi Bone Marrow-Related Cells Associated With Tissue Maintenance And/Or Repair
US20080014183A1 (en) * 2004-06-24 2008-01-17 Dnavec Research Inc. Anticancer Agent Containing Dendritic Cell Having Rna Virus Transferred Thereinto

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7442544B2 (en) 1995-11-01 2008-10-28 Dnavec Research Inc. Recombinant sendai virus
US20050266566A1 (en) * 1995-11-01 2005-12-01 Dnavec Research Inc. Recombinant sendai virus
US20070009949A1 (en) * 1999-05-18 2007-01-11 Kaio Kitazato Paramyxovirus-derived RNP
US20020002143A1 (en) * 2000-03-30 2002-01-03 Munehide Kano AIDS virus vaccines using sendai virus vector
US8217019B2 (en) 2000-03-30 2012-07-10 Dnavec Research Inc. AIDS virus vaccines using Sendai virus vector
US20060104950A1 (en) * 2002-10-24 2006-05-18 Shinji Okano Methods of Tranducing genes into T cells
US20070269414A1 (en) * 2003-11-04 2007-11-22 Shinji Okano Method for Producing Gene Transferred Denritic Cells
US20070248627A1 (en) * 2004-01-13 2007-10-25 Dnavec Research Inc. Gene Therapy for Tumors Using Minus-Strand Rna Viral Vectors Encoding Immunostimulatory Cytokines
US7521043B2 (en) 2004-01-13 2009-04-21 Dnavec Research Inc. Gene therapy for tumors using minus-strand RNA viral vectors encoding immunostimulatory cytokines
US8741650B2 (en) 2004-01-22 2014-06-03 Dnavec Research Inc. Methods for producing minus-strand RNA viral vectors using hybrid promoter comprising cytomegalovirus enhancer and chicken β-actin promoter
US20070161110A1 (en) * 2004-01-22 2007-07-12 Akihiro Iida Methods for producing minus-strand rna viral vectors using hybrid promoter comprising cytomegalovirus enhancer and chicken beta-actin promoter
US20080038234A1 (en) * 2004-03-23 2008-02-14 Shuji Hayashi Bone Marrow-Related Cells Associated With Tissue Maintenance And/Or Repair
US8889118B2 (en) 2004-06-24 2014-11-18 Dna Vec Research Inc. Anticancer agent containing dendritic cell having RNA virus transferred thereinto
US20080014183A1 (en) * 2004-06-24 2008-01-17 Dnavec Research Inc. Anticancer Agent Containing Dendritic Cell Having Rna Virus Transferred Thereinto
US20110162093A1 (en) * 2005-06-14 2011-06-30 Yasuji Ueda Methods for producing antibodies
US20100167341A1 (en) * 2006-01-17 2010-07-01 Dnavec Corporation Novel protein expression system
US20090053790A1 (en) * 2006-01-31 2009-02-26 Ishihara Sangyo Kaisha, Ltd. Polypeptide Having Affinity for Envelope Virus Constituent and Use Thereof in Transferring Substance Into Cell
US20170070889A1 (en) * 2008-05-13 2017-03-09 At&T Mobility Ii Llc Administration of access lists for femtocell service
US20110223148A1 (en) * 2008-09-16 2011-09-15 Genomidea Inc. Therapeutic/prophylactic agent for prostate cancer
US8691212B2 (en) * 2008-09-16 2014-04-08 Genomldea Inc. Therapeutic/prophylactic agent for prostate cancer
US20120107829A1 (en) * 2009-03-31 2012-05-03 Leukocare Ag Stabilizing compositions for immobilized biomolecules
US9797895B2 (en) * 2009-03-31 2017-10-24 Leukocare Ag Stabilizing compositions for immobilized biomolecules
US20200140887A1 (en) * 2016-06-22 2020-05-07 Icahn School Of Medicine At Mount Sinai Viral Delivery of RNA Utilizing Self-Cleaving Ribozymes and CRISPR-Based Applications Thereof
US10988779B2 (en) * 2016-06-22 2021-04-27 Icahn School Of Medicine At Mount Sinai Viral delivery of RNA utilizing self-cleaving ribozymes and CRISPR-based applications thereof

Also Published As

Publication number Publication date
EP1775343A4 (en) 2007-11-14
JP5296983B2 (ja) 2013-09-25
KR101270839B1 (ko) 2013-06-05
EP1775342A8 (en) 2007-06-20
EP1775343A1 (en) 2007-04-18
AU2005257105A1 (en) 2006-01-05
CN101006172B (zh) 2012-09-05
KR20070028573A (ko) 2007-03-12
HK1106794A1 (en) 2008-03-20
JPWO2006001120A1 (ja) 2008-04-17
US20080014183A1 (en) 2008-01-17
CA2571849A1 (en) 2006-01-05
EP1775343A8 (en) 2007-09-26
US8889118B2 (en) 2014-11-18
EP1775342A1 (en) 2007-04-18
WO2006001122A1 (ja) 2006-01-05
JPWO2006001122A1 (ja) 2008-04-17
WO2006001120A1 (ja) 2006-01-05
CN101006172A (zh) 2007-07-25
EP1775342A4 (en) 2007-11-07
CA2571844A1 (en) 2006-01-05
CN101006171A (zh) 2007-07-25
KR20070032022A (ko) 2007-03-20
JP2013151523A (ja) 2013-08-08
AU2005257107A1 (en) 2006-01-05

Similar Documents

Publication Publication Date Title
US20080031855A1 (en) Anticancer Agent Containing Minus-Strand Rna Virus
EP1179594B1 (en) Ribonucleoprotein complex in paramyxovirus
US7521043B2 (en) Gene therapy for tumors using minus-strand RNA viral vectors encoding immunostimulatory cytokines
KR100852078B1 (ko) 심혈관계로의 유전자 전달을 위한 파라믹소바이러스 벡터
EP2048232A1 (en) Non-replicating paramyxoviridae virus vector
EP1561819A1 (en) Method of transferring gene into t cells
EP1891215B1 (en) Highly safe intranasally administrable gene vaccines for treating alzheimer's disease
US20100167341A1 (en) Novel protein expression system
EP1690937A1 (en) Method of constructing transgenic dendritic cell
US20050191617A1 (en) Pramyxovirusl vectors encoding antibody and utilization thereof
WO2006137517A1 (ja) 幼少個体への遺伝子導入用ベクター
US20100203027A1 (en) Viral vector for gene therapy
JP4903159B2 (ja) アルツハイマー病の治療のための安全性に優れた鼻腔内投与可能遺伝子ワクチン
EP1642966B1 (en) Minus strand rna viral vectors carrying a gene with altered hypermutable regions
EP1598424A1 (en) Paramyxovirus vector encoding ribozyme and utilization thereof

Legal Events

Date Code Title Description
AS Assignment

Owner name: DNAVEC RESEARCH INC., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:OKANO, SHINJI;YONEMITSU, YOSHIKAZU;SUEISHI, KATSUO;AND OTHERS;REEL/FRAME:019396/0288;SIGNING DATES FROM 20070412 TO 20070424

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION