EP1198579A1 - Vecteur adenoviral non humain destine au transfert genique, son application et sa production - Google Patents

Vecteur adenoviral non humain destine au transfert genique, son application et sa production

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Publication number
EP1198579A1
EP1198579A1 EP00949457A EP00949457A EP1198579A1 EP 1198579 A1 EP1198579 A1 EP 1198579A1 EP 00949457 A EP00949457 A EP 00949457A EP 00949457 A EP00949457 A EP 00949457A EP 1198579 A1 EP1198579 A1 EP 1198579A1
Authority
EP
European Patent Office
Prior art keywords
use according
vector
cells
human
adenovirus
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.)
Withdrawn
Application number
EP00949457A
Other languages
German (de)
English (en)
Inventor
Peter LÖSER
Christian Hofmann
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.)
Develogen AG
Original Assignee
Develogen AG
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 DE10019006A external-priority patent/DE10019006A1/de
Application filed by Develogen AG filed Critical Develogen AG
Publication of EP1198579A1 publication Critical patent/EP1198579A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10111—Atadenovirus, e.g. ovine adenovirus D
    • C12N2710/10141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the production of a non-human adenoviral vector for gene transfer in mammalian cells.
  • This vector is particularly suitable for gene transfer into the muscle, in particular into the skeletal muscle, or into cell types that occur in the muscle or skeletal muscle. Areas of application are medicine, biotechnology and genetic engineering. When functional DNA sequences are inserted, the vector is suitable for the treatment of altered, also pathological phenomena in cells or cell complexes, for the production of biological material and for vaccination.
  • vectors derived from retroviruses, adeno-associated viruses (AAV) or human adenoviruses are used for gene transfer e.g. favored with the goal of gene therapy.
  • AAV adeno-associated viruses
  • human adenoviruses are used for gene transfer e.g. favored with the goal of gene therapy.
  • the adenoviral vectors of the so-called first generation have been intensively researched as gene transfer vectors over the past decade (review by: Bramson, JL et al. (1 995). Curr. Op. Biotech. 6, 590-595). They are derived from the human adenovirus of serotype 5 and are, in the essential E1 region, often also deleted in the non-essential E3 region, whereby up to 8 KBp of foreign DNA can be inserted into the virus genome. These vectors can complement E1 deficiency Cells with high titers can be stored well and mediate an effective gene transfer in vitro and in vivo. In vivo, however, there is a rapid loss of expression of the transgenes and of the vector genome.
  • adenoviral vector which can transfer DNA sequences into target cells, in particular mammalian cells, and where there results a preferably transient expression of the transduced gene in the cell or in the organism
  • the non-human adenoviral vector is suitable for efficient gene transfer in vivo, e.g. for transduction of mammalian cell types, which in
  • Muscle occur, or in the skeletal muscles.
  • the field of application is the transfer of genetic material into cells, for example with the goals: therapy of genetically determined and acquired
  • An object of the invention is thus the use of a non-human adenovirus vector for the production of an agent for the transfer of genetic material, comprising the envelope of a non-human adenovirus and genetic material packaged therein, which (a) DNA sequences from a non-human adenovirus and
  • (b) contains one or more DNA sequences which code for peptides or polypeptides which are heterologous to the non-human adenovirus, in operative linkage with expression control sequences.
  • the genetic material of the gene transfer vector according to the invention contains DNA sequences from a non-human adenovirus, i.e. an adenovirus naturally occurring in a non-human species selected, for example, from mammals and birds, as listed in Russell, W.C. and Benkö, M. (1 999), Adenoviruses (Adenoviridae): Animal viruses. In: Granoff, A. and Webster, R.G. (Eds): Encyclopedia of Virology.
  • a non-human adenovirus i.e. an adenovirus naturally occurring in a non-human species selected, for example, from mammals and birds, as listed in Russell, W.C. and Benkö, M. (1 999), Adenoviruses (Adenoviridae): Animal viruses. In: Granoff, A. and Webster, R.G. (Eds): Encyclopedia of Virology.
  • the virus is preferably a sheep or bovine adenovirus.
  • sheep adenoviruses are ovine mastadenoviruses or ovine atadenoviruses such as OAV isolate 287, whose nucleotide sequence is given under Genbank Acc. No. U40389.
  • Suitable bovine adenoviruses are bovine atadenoviruses or bovine mastadenoviruses, negative bovine and ovine atadenoviruses being particularly interesting in the complement fixation assay.
  • the gene transfer vector according to the invention contains one or more DNA sequences which code for peptides or polypeptides which are heterologous with respect to the non-human adenovirus, in operative linkage with
  • Expression control sequences ie an expression cassette for one or several transgenes.
  • the expression cassette for the transgene can, for example, be inserted into a cloning site.
  • the transgene expression cassette preferably contains expression control sequences which allow expression in mammalian cells, for example in human cells.
  • the expression control sequences can be constitutively active and / or regulatable in the desired target cell.
  • the expression control sequences can be of viral or cellular origin or comprise a combination of viral and cellular elements.
  • suitable promoters are viral promoters, e.g. RSV promoter, CMV immediate early promoter / enhancer, SV40 promoter, or tissue-specific, especially liver-specific promoters, e.g. the human albumin promoter (Ponder et al., Hum. Gene Ther.
  • the expression regulation sequences conveniently comprise a polyadenylation signal, for example that of bovine growth hormone gene (Goodwin & Rottman, J. Biol. Chem.
  • the viral gene transfer vector can be used to transfer heterologous nucleic acids into permissive cells, cell groups, organs and organisms, in particular for gene therapy or for vaccination.
  • the genomic sequence or the cDNA of a gene can be used, the product of which is missing in the patient to be treated, occurs in unphysiological amounts or / and is defective. You can also use part of a genomic sequence that a mutation in Target gene spanned and can be homologously recombined with this.
  • various genes can be used, which slow down the growth or kill the tumor cells, possibly in combination with pharmaceuticals or by immunostimulation.
  • One or more, possibly modified genes of a pathogenic organism against which immunization is to be achieved can be used for the purpose of vaccination.
  • the gene transfer vector according to the invention can contain genetic material from other viruses, for example expressed or regulatory sequences of hepatitis B and hepatitis C virus (HBV, HCV), and of bacteria and pathogenic single or multi-cells, for example Plasmodium faiciparum.
  • viruses for example expressed or regulatory sequences of hepatitis B and hepatitis C virus (HBV, HCV), and of bacteria and pathogenic single or multi-cells, for example Plasmodium faiciparum.
  • transgenes for the purpose of substitution gene therapy are genes for secreted serum factors (for example human blood coagulation factors IX (FIX) and VIII (FVIII), erythropoietin (Epo) ⁇ -1 - antitrypsin (AAT)), and genes for proteins which could be used in muscle diseases (e.g. dystrophin, utrophin), and the gene defective in Wilson's disease (ATP7B).
  • preferred specific transgenes are tumor suppressor genes such as p 1 6 or p53 (individually or in combination, e.g. P1 6 / p53), genes for various interleukins (individually or in combination, e.g. IL2 / IL7) and suicide genes, e.g. Herpes simplex virus type I thymidine kinase (HSV-TK).
  • the vector is suitable for gene transfer in cells or cell complexes which have changed, also pathological phenomena, for example for gene therapy, for example for the therapy of inherited or malignant diseases.
  • the vector is also suitable for vaccination, for example for vaccination against pathogens, in particular viruses, bacteria and eukaryotic single or multi-cells, or for vaccination against malignant or non-malignant cells or cell populations.
  • pathogens in particular viruses, bacteria and eukaryotic single or multi-cells
  • efficient expression of the transgene can be achieved even after multiple administration of the vector. This leads to considerable advantages, especially for applications in the field of gene therapy and vaccination.
  • Another advantage is the - compared to human adenovirus vectors - observed reduced expression of vector-inherent genes of the vector after the gene transfer into the target cell, preferably essentially no expression of vector-inherent genes after the gene transfer. This leads to a significantly increased safety for use in the organism, especially in humans.
  • the vector according to the invention can be used to transfer genetic material into a target cell and preferably to express this genetic material in the target cell and preferably to express this genetic material in the target cell.
  • the target cell is preferably a human cell.
  • non-human target cells in particular non-human mammalian cells, can also be used, for example, for veterinary or research applications.
  • the gene transfer can take place in vitro, ie in cultured cells, but also in vivo, ie in living organisms or specific tissues or organs of such organisms.
  • the vector according to the invention is particularly preferably used for gene transfer into muscle, in particular into the skeletal muscle. This finding is all the more surprising since previously used human adenoviral vectors only poorly infected the skeletal muscle.
  • the muscle cells are preferably selected from myocytes / myotubes / myofibers, fibroblasts, dendritic cells, endothelial cells and combinations thereof.
  • the preferred mode of administration of the vector depends on the intended application.
  • For muscle-directed gene transfer or transfer to a solid tumor for example, local application of the vector by intramuscular / intratumoral injection is preferable.
  • a systemic introduction for example by intra-arterial or intravenous injection, can take place for gene transfer to other target organs or tissues.
  • the directed transfer into special tissues or organs can take place either by a natural or modified tropism of the vector for certain cell types or by selecting vessels which supply the tissue to be found.
  • the dosage can only be decided after more detailed studies on the efficiency of gene transfer by the respective vector. Animal studies typically use 1 0 7 to 1 0 13 , eg 1 0 9 to 10 1 1 viral particles / kg body weight. However, the exact dosage can be modified depending on the type of vector, the type and severity of the disease and the type of administration.
  • a relatively low dose of the vector for example 1 0 7 to 1 0 9 viral particles per kilogram of body weight, is preferably used in the first doses in order to prevent an immune response from developing prevent the vector that could render the administration of subsequent doses ineffective.
  • non-human gene transfer adenovirus vectors can be carried out by inserting transgene expression cassettes and, if appropriate, further genetic elements into a base vector, e.g. B. a natural non-human adenovirus or a variant derived therefrom, e.g. B. a partially deleted variant and propagation of the resulting vector in suitable permissive cells.
  • a base vector e.g. B. a natural non-human adenovirus or a variant derived therefrom, e.g. B. a partially deleted variant and propagation of the resulting vector in suitable permissive cells.
  • Figure 1 Efficient expression of human oyAntitrypsin (hAAT) after injection of OAVhaat into the quadriceps muscle.
  • Blood was drawn three days after infection and the concentration of hAAT in the serum was determined by ELISA. Each pillar represents an individual animal.
  • FIG. 2 The skeletal muscle is the site of OAV infection and the site of OAV-mediated gene expression after intramuscular injection of OAVhaat in mice.
  • Balb / C mice were injected with 1x10 9 infectious particles OAVhaat (2 and 3) or with buffer (1) as described in Figure 1 and killed three days after application.
  • DNA and RNA were prepared from the injected skeletal muscle as well as from liver and spleen according to standard methods, a) Southern blotting of 20 ⁇ g DNA from skeletal muscle, liver and spleen digested with EcoRI. The hybridization was carried out against an OAV-specific probe, the position of the 2399 bp EcoRI fragment from the OAV vector is indicated.
  • OAV DNA digested with EcoRI equivalent to 5 copies per cell, was identified as Positive control used. Identical numbers denote identical animals, b) RNase protection assay with 20 ⁇ g total RNA from skeletal muscle. The hybridization was carried out against a 364 base long probe that spanned the EcoNI fragment of the human aat gene. 20 and 40 pg of a haat mRNA synthesized in vitro were used as a positive control; the position of the band specific to the haat transcript is indicated by the arrow.
  • FIG. 3 Dose-response relationship of OAV-dependent expression and the possibility of intramuscular readministration of OAV vectors in mice.
  • C57 / BI-6 mice received the specified amounts of infectious particles of OAVhaat (10 9 to 3x1 0 7 ) in an intramuscular injection.
  • the expression of the haat gene was determined by measuring the concentration of ⁇ antitrypsin in the serum of the mice three days after infection using an ELISA (dark columns). The second injection took place on day 35 after the first application of the vector, all animals received a dose of 5x10 8 infectious particles.
  • Serum hAAT was determined three days after the second vector administration (light gray columns).
  • HEK-293 cells human embryonic kidney cells ATCC CRL-1 573
  • permissive for E1-deleted human adenoviruses were cultivated in DMEM (Gibco BRL) supplemented with 2 mM glutamine and 10% fetal calf serum at 5% CO 2 .
  • CSL 503 cells fetal ovine lung cells, permissive for OAV, Pye et al, Austr. Vet. J. 66 (1 998), 231-232
  • OAVhAAT was used as the viral vector, which encodes the human ⁇ 1-antitrypsin (hAAT) cDNA under transcriptional control of the Rous Sarcoma virus 3'LTR contains (Hofmann et al., J. Virol 73 (1 999), 6930-6936).
  • Ad ⁇ haat an E1-deleted human Ad5 adenovirus vector, which contains the identical haat expression cassette as OAVhaat was used as a control.
  • the viruses were cultivated in permissive cell lines and purified as previously described (Sandig et al., Gene Therapy 3 (1 996), 1 002 - 1 009). Virus titers were determined by an endpoint dilution assay with permissive cell lines.
  • Typical titers for OAV and Ad5 vectors were 0.5 - 1 x 10 0 and 0.5 - 1 x 1 0 1 1 infectious particles per ml.
  • the ratio of particles to infectious particles for recombinant Ad5 and OAV Vectors was 40: 1.
  • mice Eight week old female Balb / C and C57 / BI-6 mice were purchased from Charles River, Germany. The mice were given intramuscular injections into the quadriceps muscle with a maximum volume of 35 ⁇ per injection. Blood was obtained from the outer tail vein to determine the serum ⁇ i-antitrypsin level. HAAT was determined by ELISA as described in Cichon and Strauss (Gene Therapy 5 (1 998) 85-90).
  • the titer of neutralizing antibodies against viral vectors was determined based on the ability of serum to inhibit infection of 293 cells by human Ad vectors and of CSL503 cells by OAV vectors as described in Hoffmann et al. (1 999), supra.
  • the antibody titer against hAAT in serum was determined by the method of Morral et al. (Hum Gene Ther 8 (1 997), 1 275 - 1 286).
  • RNA and total RNA from mouse tissues and cultured cells was isolated using standard methods. Southern blots for Detection of OAV-specific DNA in mouse organs was carried out as previously described (Hoffmann et al. (1 999), supra) using 20 ⁇ g genomic DNA. RNase protection tests were performed on 20 g of total skeletal muscle RNA using standard procedures. A radiolabeled 362 bp RNA fragment comprising the EcoN 1 fragment of human hAAT cDNA was used as a probe.
  • RT-PCR was performed with 2 ⁇ g total RNA from skeletal muscle using the Titan TM kit (Röche Diagnostics Mannheim, Germany) according to the manufacturer's instructions.
  • the efficient expression of the transgene and the presentation of the antigens encoded by the transgene are required.
  • the efficiency of the vaccination is reflected in the antibody titer against the transgene / antigen.
  • the potential of OAV as a vaccination vector was determined by a simple experiment. Balb / C mice received an intramuscular injection of 5 x 10 8 infectious particles OAVhAAT. On day 30 after infection, the antibody titers against the transgene product, human ⁇ vAntitrypsin, in the serum of the mice were determined as follows: 96-well plates were tested with 100 ⁇ l of an antibody directed against hAAT (DiaSorin, Stillwater, USA, cat. No.
  • the enzyme activity was determined using standard methods using OPD as the substrate, and the absorption was measured at 595 nm.
  • the antibody titer against hAAT was greater than 1 00000 after an injection of approx. 10 9 infectious particles.
  • the antibody titer, according to the administered dose of the vector OAVhaat was between 1 000 000 and 10 (see Table 1).
  • an infection with the human adenovirus Ad 5haat (1 0 10 infectious particles) and a corresponding transcript detection were carried out.
  • IMR 90 cells (Nichols et al., Science 1 96 (1 977), 60-63) were infected with OAVhaat at an MOI of 1. After 3 days, the expression of recombinant hAAT was quantified by an ELISA. The amount of the recombinant protein was determined with 1 mg / ml cell culture supernatant / 1 0 6 cells.

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  • Wood Science & Technology (AREA)
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  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

La présente invention concerne la production d'un vecteur adénoviral non humain, destiné au transfert génique dans des cellules de mammifères. Ce vecteur est particulièrement adapté au transfert génique dans le muscle, notamment dans le muscle du squelette, ou dans des types cellulaires présents dans le muscle ou dans le muscle du squelette. Les domaines d'application sont la médecine, la biotechnologie et le génie génétique. Si des séquences d'ADN fonctionnelles sont insérées, ledit vecteur est adapté au traitement de phénomènes modifiés, même pathologiques, dans des cellules ou dans des complexes cellulaires, ainsi qu'à la production de matériaux biologiques et à la vaccination.
EP00949457A 1999-08-06 2000-08-04 Vecteur adenoviral non humain destine au transfert genique, son application et sa production Withdrawn EP1198579A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19938332 1999-08-06
DE19938332 1999-08-06
DE10019006 2000-04-17
DE10019006A DE10019006A1 (de) 1999-08-06 2000-04-17 Neuartiger adenoviraler Vektor für den Gentransfer, seine Anwendung und seine Herstellung
PCT/EP2000/007601 WO2001011066A1 (fr) 1999-08-06 2000-08-04 Vecteur adenoviral non humain destine au transfert genique, son application et sa production

Publications (1)

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EP1198579A1 true EP1198579A1 (fr) 2002-04-24

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Application Number Title Priority Date Filing Date
EP00949457A Withdrawn EP1198579A1 (fr) 1999-08-06 2000-08-04 Vecteur adenoviral non humain destine au transfert genique, son application et sa production

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EP (1) EP1198579A1 (fr)
AU (1) AU6280600A (fr)
WO (1) WO2001011066A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1453537A1 (fr) 2001-12-12 2004-09-08 FH Faulding & Co. Limited Composition de preservation virale

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5820868A (en) * 1993-12-09 1998-10-13 Veterinary Infectious Disease Organization Recombinant protein production in bovine adenovirus expression vector system
AUPN477695A0 (en) * 1995-08-14 1995-09-07 Commonwealth Scientific And Industrial Research Organisation Gene therapy
FR2763959A1 (fr) * 1997-06-02 1998-12-04 Transgene Sa Nouveaux vecteurs adenoviraux recombinants comprenant une sequence d'epissage
EP1001030A1 (fr) * 1998-09-22 2000-05-17 Boehringer Ingelheim International GmbH CELO virus recombinant et l' ADN du virus CELO

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0111066A1 *

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WO2001011066A1 (fr) 2001-02-15
AU6280600A (en) 2001-03-05

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