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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • 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
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/14011—Baculoviridae
  • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
  • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the invention relates to a coat protein-modified baculovirus vector for gene therapy; Areas of application are medicine, biotechnology and genetic engineering.
• baculovirus vector that can transfer therapeutic genes highly specifically and effectively into liver cells.
• Baculoviruses belong to a family of large DNA viruses, the host spectrum of which is naturally restricted to arthropods. Your genome (80kbp-200kpb) is packaged in flexible nucleocapsids that allow the insertion of large amounts of foreign DNA.
• the decisive prerequisite for bacuioviral gene transfer in mammalian cells is the insertion of an expression cassette that is functional in mammalian cells. This created an important prerequisite for the therapy of genetic diseases of the liver.
• the aim of this invention is the construction of a Baculoviru ⁇ vector which, by modifying the virus envelope, escapes inactivation by serum components and transfers therapeutic genes highly specifically and effectively in liver cells in vivo.
• the therapeutic DNA sequence for the vector according to the invention is the cDNA of a gene which is defective in the disease to be treated, i.e. missing or changed by mutation. It is also possible to use part of a genomic sequence which spans a mutation in the target gene and can homologously recombine with it.
• the first line serves as strong viral promoters, preferably the very early promoter of the Cyto arcadeieviru ⁇ (CMV). Cell-specific protorotors are also suitable.
• the establishment sequence has the task of stabilizing the vector in the cell without integration into the genome. It is used particularly in cases where long-term expression is required.
• Preferred establishment sequences according to the invention are viral core establishment sequences, such as the Ep ⁇ tein-Barr virus, or autonomous replication sequences from the mammalian genome.
• the new vectors are produced in the following essential steps:
• a novel vector for gene transfer is created which offers considerable advantages over the virus vectors developed so far (retroviruses, adenoviruses and unmodified baculoviruses). These include the stability in blood and serum, the liver specificity or free variation of the cell targeting, the almost unlimited possibility of incorporating foreign DNA, the infection of cells that are unable to divide, the lack of cytotoxicity and the simple generation of high-titre recombinant viruses.
• the envelope protein-modified Baculoviru ⁇ vectors enable a desired gene to be introduced into the affected organ of a patient and to optimally design his way to the functional site.
• the application of the vector can be local or systematic. A essential prerequisite for successful therapy of genetic and malignant diseases of humans is thereby created. The invention will be explained in more detail below by means of exemplary embodiments.
• Insect cells manufactured. 10 ul of the viruses were with 90 ul
• a sequence for an N-terminally modified baculovirus envelope protein (gp64) is cloned in a known manner in a recombination vector under the control of a baculoviral promoter.
• the modification is achieved by inserting the DNA sequence for amino acids 1-320 of the complement protection protein "decay accelerating factor (DAF)" between the signal sequence and the sequence of the baculovirus protein gp64 at the DNA level.
• DAF decay accelerating factor
• This construct is either cloned into the recombination vector, which contains the therapeutic DNA sequences together with the promoter, or is stably integrated into the virus packaging cell.
• the modified coat protein is inserted into the envelope of the baculovirus vector and thereby mediates:

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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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• 1997
  • 1997-08-15 DE DE1997135593 patent/DE19735593C2/de not_active Expired - Fee Related
• 1998
  • 1998-08-05 EP EP98947386A patent/EP1003896A1/de not_active Withdrawn
  • 1998-08-05 ES ES98947386T patent/ES2150894T1/es active Pending
  • 1998-08-05 CA CA002300362A patent/CA2300362A1/en not_active Abandoned
  • 1998-08-05 JP JP2000509856A patent/JP2003530064A/ja active Pending
  • 1998-08-05 WO PCT/DE1998/002255 patent/WO1999009193A1/de not_active Application Discontinuation

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