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WO1998048842A1 - Utilisation d'un baculovirus modifie contenant un acide nucleique exogene pour preparer un medicament servant a administrer ledit acide nucleique aux hepatocytes - Google Patents

Utilisation d'un baculovirus modifie contenant un acide nucleique exogene pour preparer un medicament servant a administrer ledit acide nucleique aux hepatocytes Download PDF

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Publication number
WO1998048842A1
WO1998048842A1 PCT/GB1998/001249 GB9801249W WO9848842A1 WO 1998048842 A1 WO1998048842 A1 WO 1998048842A1 GB 9801249 W GB9801249 W GB 9801249W WO 9848842 A1 WO9848842 A1 WO 9848842A1
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Prior art keywords
nucleic acid
baculovirus
hbv
gene
cells
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Ceased
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PCT/GB1998/001249
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English (en)
Inventor
Michael Joseph Mcgarvey
Howard Christopher Thomas
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Ip2ipo Innovations Ltd
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Imperial College Innovations Ltd
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Application filed by Imperial College Innovations Ltd filed Critical Imperial College Innovations Ltd
Priority to AU72217/98A priority Critical patent/AU7221798A/en
Priority to EP98919340A priority patent/EP0979106A1/fr
Publication of WO1998048842A1 publication Critical patent/WO1998048842A1/fr
Anticipated expiration legal-status Critical
Priority to US09/729,856 priority patent/US20010000228A1/en
Ceased legal-status Critical Current

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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
    • 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
    • C12N2710/14011—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 present invention relates to materials for use in gene therapy and novel methods of gene therapy.
  • it relates to novel methods of 5 delivering selected nucleic acid to hepatocytes in order to treat viral infections of such cells, for instance DNA encoding an effector enzyme capable of converting a prodrug to its active form.
  • Hepatitis viruses e.g. hepatitis B (HBV) and hepatitis C (HCV)
  • HBV hepatitis B
  • HCV hepatitis C
  • gene therapy represents a potential method of targeting specific cells, e.g. liver cells.
  • a working system could provide various options for interfering with the replication cycle of viruses, particularly those which can cause chronic infections such as HBV and HCV.
  • the system could be used to deliver nucleic acid encoding therapeutic agents which when expressed within liver cells will protect them against the viruses.
  • adenoviruses can deliver nucleic acid encoding the therapeutic agent to liver cells, they can also infect other cells and deliver the nucleic acid to them. In addition, they can elicit a strong cell mediated immune response and the virus itself can have cytopathic effects. Retroviruses usually have low virus titres making it difficult to deliver a large enough dose of the effector gene(s).
  • the integration of the retrovirus proviral DNA into the genome of the target cell could alter the pattern of gene expression within the cell and this could have deleterious effects on the cell.
  • liposomes are relatively non-specific in their uptake by liver cells and this could lead to the delivery of the nucleic acid encoding the therapeutic agent to non-target cells.
  • the asialoglycoprotein receptor is liver specific, and can consequently direct the uptake of effector genes coupled to the asialoglycoprotein ligand (ASGPL), this system for gene delivery involves relatively complex chemical processes to couple the effector genes to the ASGPL. This involves the use of spacer molecules e.g. peptides.
  • Baculoviruses are insect viruses which have shown hepatocyte specificity (Hofman et al, PNAS 92:10099-10103 (1995); Boyce and Bucher, PNAS, 93:2348-2352 (1996)). Thus, although these viruses can "infect" liver cells, they do not replicate and so should represent less risk as compared to retroviruses for instance.
  • Another key feature of Baculoviruses is that they have a large potential genetic "payload", around 15kB; i.e. up to 15kB of the baculovirus genome can be replaced by nucleic acid which encodes therapeutic agents.
  • the present invention provides the use of a Baculovirus containing therapeutic and/or prophylactic nucleic acid to deliver said nucleic acid to one or more hepatocytes.
  • therapeutic nucleic acid includes DNA or RNA whose expression is capable of directly or indirectly treating an infection in the hepatocytes or providing a prophylactic effect.
  • the therapeutic nucleic acid is directed against viral infections, particularly HBV and HCV infections.
  • the nucleic acid will be provided in the form of a nucleic acid construct which will also comprise regulatory nucleic acid, e.g. a suitable promoter sequence, to provide for expression of the therapeutic/prophylactic nucleic acid in the target cell(s).
  • regulatory nucleic acid e.g. a suitable promoter sequence
  • a mammalian promoter can be used, examples of which include the CMV and SV40 promoters.
  • the construct will be provided in the form of a vector, e.g. a plasmid construct, which in turn will be "packaged" by the baculovirus.
  • the packaging of the nucleic acid which encodes a therapeutic/prophylactic agent into baculoviruses is carried out by recombination between the polyhedrin gene in baculovirus DNA and baculovirus polyhedrin sequences that form part of the recombinant plasmid or "shuttle" vector in which the nucleic acid which encodes a therapeutic/prophylactic agent has been cloned.
  • the result of this recombination event is the production of a recombinant baculovirus which contains the nucleic acid which encodes a therapeutic/prophylactic agent as an integral part of the baculovirus genomic DNA.
  • the baculovirus system can be used to deliver a gene encoding an enzyme for instance which can convert a prodrug compound to its active form.
  • An example of such an approach would be the delivery of a gene encoding a (TK) enzyme, eg. the Herpes Simplex Virus (HSV) thymidine kinase, which is capable of converting ganciclovir or araM to their active forms.
  • HSV Herpes Simplex Virus
  • araM Herpes Simplex Virus
  • liver cells of a subject can be "enabled” with respect to this conversion which should result in therapeutically effective levels of active drug being formed in the liver upon administration of ganciclovir or araM.
  • This embodiment of the invention can be further refined by the inclusion in the construct of one or more regulatory sequences which is/are only activated in the presence of the infective agent against which the therapy/prophylaxis is directed.
  • regulatory sequences which is/are only activated in the presence of the infective agent against which the therapy/prophylaxis is directed.
  • examples of such an approach would include the use of viral promoters in the construct which would lead to expression only in the presence of the target virus (where such a promoter is only recognised by a viral nucleic acid polymerase encoded by the virus itself).
  • Another example would be to utilise a specific activator, such as the HBV X protein. This is a transactivator protein capable of "switching on" certain control sequences, e.g. the NFKB sequence (Lucito and Schnieder, J Virol.
  • fusion proteins of Herpes simplex virus (HSV) protein VP22 with CAT and HSV-TK, encoded by such a baculovirus should allow these proteins to spread from baculovirus infected to uninfected cells since it has been shown that the Herpes simplex virus (HSV) protein, VP22, can spread from cell to cell very efficiently when the gene for this protein is transfected into cells (Elliot G and O ⁇ are P (1997) Cell 88: 223-233).
  • HSV Herpes simplex virus
  • an alternative approach is based on the use of "antisense” nucleic acid.
  • This is nucleic acid which when transcribed results in a nucleic acid molecule which is complementary to at least part of the genome of an infective agent, such as a virus.
  • This complementary nucleic acid will thus bind to the nucleic acid of the infective agent, e.g. a hepatitis virus, and will prevent its transcription and ultimate translation.
  • the construct of the invention could encode antisense nucleic acid directed against the HBV replicase gene. Prevention of transcription of this gene will therefore prevent the formation of new viral particles.
  • the antisense nucleic acid could be directed against the HBV capsid protein or envelope protein genes.
  • Ribozymes which contain antisense RNA coupled to RNA domains capable of catalytically cleaving target RNAs and which are specifically designed to degrade HBV and HCV viral genomic and/or messenger RNAs (Lieber et al., J Virol. 70 8782-8791 (1996)) can also be encoded in these recombinant baculoviruses.
  • the present invention can also be used to deliver therapeutic agents directly.
  • One example of such an approach would be a construct encoding a particular interferon- ⁇ subtype for use in the treatment or prophylaxis of a hepatitis virus infection. It is known for instance that certain interferon- ⁇ subtypes are particularly effective against viral infections in certain cell types. Thus, in WO-A-9524212 it is disclosed that IFN ⁇ ⁇ is particularly effective against viral infection of liver cells.
  • a construct of the invention encoding IFN ⁇ , and under the control of a selective expression system as described above, would provide a defence against HBV for instance which would be activated by the presence of the virus.
  • IL-12 interleukin-12
  • IL-12 interleukin-12
  • IL-12 is particularly effective against viral infections in certain cell types IL-12 has been shown to shift the Th2 mediated immune response to HBV e-antigen to a Th1 response. There is evidence that this Th2 to Th1 shift may favour viral clearance of in chronic HBV infection (Milich et al., Proc. Nat'l. Acad. Sci. USA 92 6847-6851 (1995)).
  • the delivery of genes which can convert a prodrug to its active form capable of killing liver tumour cells can be achieved with the baculovirus gene delivery system described.
  • Baculoviruses have been shown to be able to deliver genes to the liver tumour cell lines HepG2 and Huh7 and that when regulated by mammalian promoters these genes can be expressed in these tumour cell lines (Hofmann C, et al. Proc Natl Acad Sci USA 92: 10099-10103. (1995); Boyce FM, Bucher NLR Proc Natl Acad Sci USA 93: 2348-2352 (1995)).
  • the expression of the HSV TK gene can convert ganciclovir to its active form which is capable of killing tumour cells.
  • the HSV TK gene By regulating the expression of the HSV TK gene in a baculovirus vector sequencing such as promoter and enhancer elements that can be activated in liver tumour cells but not in adult liver cells (a good example are the promoter and enhancer elements from the alpha-foetoprotein gene) it should be possible to restrict the expression of the HSV TK gene and the activation of ganciclovir to liver tumour cells and not normal hepatocytes, thus increasing the specificity of the anti-tumour therapy.
  • a baculovirus vector sequencing such as promoter and enhancer elements that can be activated in liver tumour cells but not in adult liver cells (a good example are the promoter and enhancer elements from the alpha-foetoprotein gene) it should be possible to restrict the expression of the HSV TK gene and the activation of ganciclovir to liver tumour cells and not normal hepatocytes, thus increasing the specificity of the anti-tumour therapy.
  • the present invention provides a baculovirus containing therapeutic and/or prophylactic nucleic acid .
  • a baculovirus can have any one or more of the features as discussed herein.
  • the baculovirus can be used in the manufacture of a medicament for use in the treatment or prophylaxis of an infection, e.g. a viral infection such as HBV or HCV, or for use in the prophylaxis or treatment of liver cancer and form further aspects of the invention.
  • the present invention provides a pharmaceutical formulation comprising a baculovirus as defined in claim 21 or claim 22 optionally together with one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • This pharmaceutical composition will generally be provided in a sterile unit dosage form in a sealed container.
  • compositions within the scope of the present invention may include one or more of the following: preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colorants, odourants, salts, buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the modified baculovirus of the present invention.
  • compositions within the scope of the present invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
  • oral including buccal or sublingual
  • rectal nasal
  • topical including buccal, sublingual or transdermal
  • vaginal or parenteral including subcutaneous, intramuscular, intravenous or intradermal
  • parenteral including subcutaneous, intramuscular, intravenous or intradermal
  • compositions adapted for oral administration may be provided as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or whips; or as emulsions.
  • Tablets or hard gelatine capsules may comprise lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
  • Soft gelatine capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
  • Solutions and syrups may comprise water, polyols and sugars.
  • suspension oils e.g. vegetable oils
  • oil- in-water or water-in-oil suspensions may be used to provide oil- in-water or water-in-oil suspensions.
  • compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis (Iontophoresis is described in Pharmaceutical Research, 3(6):318 (1986)).
  • compositions adapted for topical administration may be provided as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • compositions adapted for topical administration in the mouth include lozenges, pastilles and mouthwashes.
  • compositions adapted for rectal administration may be provided as suppositories or enemas.
  • compositions adapted for nasal administration which use solid carriers include a coarse powder (e.g. having a particle size in the range of 20 to 500 microns). This can be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nose from a container of powder held close to the nose.
  • coarse powder e.g. having a particle size in the range of 20 to 500 microns. This can be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nose from a container of powder held close to the nose.
  • compositions adopted for nasal administration which use liquid carriers include nasal sprays or nasal drops. These may comprise aqueous or oil solutions of the active ingredient.
  • compositions adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of apparatus, e.g. pressurised aerosols, nebulisers or insufflators.
  • apparatus e.g. pressurised aerosols, nebulisers or insufflators.
  • Such apparatus can be constructed so as to provide predetermined dosages of the active ingredient.
  • compositions adapted for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions. These may contain antioxidants, buffers, bacteriostats and solutes which render the compositions substantially isotonic with the blood of an intended recipient. Other components which may be present in such compositions include water, alcohols, polyols, glycerine and vegetable oils, for example.
  • Compositions adapted for parenteral administration may be presented in unit-dose or multi- dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of a sterile liquid carrier, e.g. sterile water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Dosages
  • Dosages will be readily deteiminable by routine trials, and will be under the control of the physician or clinician.
  • the guiding principle for determining a suitable dose will be delivery of a suitably efficacious but non-toxic, or acceptably toxic, amount of material.
  • the pharmaceutical formulations of the invention can be used in the treatment or prophylaxis of infections, such as viral infections as well as in methods for the prophylaxis treatment of liver cancer. Such methods form yet further aspects of the invention. In preferred embodiment the methods are for the treatment or prophylaxis of HBV or HCV.
  • baculoviruses which contain the chloramphenicol acetyl transferase (CAT) reporter gene, under the control of a baculovirus polyhedrin promoter (CAT), the CAT gene under the control of a CMV promoter, CCAT the CAT gene under the control of a NFKB promoter, NCAT and the thymidine kinase (TK) gene under the control of a NFKB promoter, NTK, using the shuttle vector pCRBac (Invitrogen Inc).
  • the CAT coding sequence (CAT) was obtained from the plasmid pCDNA3.1 (InVitrogen Inc) by the polymerase chain reaction (PCR).
  • CAT DNA sequence was then ligated into the baculoviral shuttle vector pCRBac according to the procedure described in the Baculovirus TA Cloning Kit, to produce the plasmid pCAT.
  • the DNA sequence that contained a CMV promoter at the 5" end of the molecule and the CAT coding sequence at the 3' end was obtained from the plasmid pCDNA3.1 by using the polymerase chain reaction (PCR).
  • CMV-CAT DNA sequence To amplify the CMV-CAT DNA sequence, forward and reverse primers of 20 nucleotides that corresponded to the 5' end of the CMV promoter and 3' end of the CAT coding sequence were used. The CMV-CAT DNA was then ligated into pCRBac (as described above) to produce pCCAT.
  • a shuttle vector that contained the CAT coding sequence under the control of the NFKB promoter
  • the same PCR procedure (as described above) was used but with a forward primer which was 50 nucleotides in length that contained both a 30 nucleotide sequence that corresponded to the NFKB promoter and a 20 nucleotide sequence that corresponded to the sequence 50-70 nucleotides upstream of the CAT coding sequence in pCAT and a reverse primer that corresponds to the 3' end of the CAT coding sequence.
  • the NF ⁇ B-CAT DNA was then ligated into pCRBac (as described above) to produce pNCAT.
  • the TK coding sequence was amplified from herpes simplex 1 (HSV1) DNA by the same PCR procedure (as described above) using a forward primer, which was 50 nucleotides in length and which was comprised of a 30 nucleotide sequence that corresponded to the NFKB promoter and a 20 nucleotide sequence that corresponded to the 5' end of the TK coding sequence and a reverse 20 nucleotide primer that corresponded to the 3' end of the TK coding sequence.
  • HSV1 DNA was extracted from 10 8 HSV1 particles according to a procedure previously described (Garson et al.
  • the NF ⁇ B-TK DNA was then ligated into pCRBac (as described above) to produce pNTK.
  • a plasmid was also made that expresses the HBV X protein coding sequence under the control of a CMV promoter by ligating the HBV X coding sequence into pcDNA3.1 to produce the plasmid pcDX.
  • the HBV X coding sequence was obtained by PCR amplification using forward and reverse primers that corresponded to the 20 nucleotides at the 5' and 3' ends of the HBV X coding sequence from plasmid pHBV130.4 (Gough and Murray J. Mol. Biol. 162 43-67 (1982)).
  • Recombinant baculoviruses were made with the shuttle plasmids described above with the Bac-N-Blue transfection kit (InVitrogen). All the procedures use to make these recombinant baculoviruses were as described in the Bac-N-Blue transfection kit. This resulted in baculoviruses that expressed, CAT under the control of the CMV promoter (CCAT), CAT under the control of the NFKB promoter (NCAT), and TK under the control of the NFkB promoter (NTK). These baculoviruses were used to infect tissue culture, hepatocyte cell lines Huh-7 and HepG2, HepG2 (2.2.15) cells (Acs G et al.
  • FIGs 2 and 3 show the infection of HepG2 cells and Huh7 cells with either CCAT, NCAT, NCAT in cells which express the HBV-X protein (NCAT + pcDX) or CAT (no mammalian promoter) in cells which express the HBV-X protein (CAT + pcDX) and uninfected cells which express the HBV-X protein (pcDX).
  • Figure 4 shows a comparison of the stimulation of NCAT in HepG2.2.15 cells (which express the HBV-X protein constitutively) and HepG2 cells. In all three experiments the expression of the HBV-X protein shows a stimulatory effect on NCAT expression. Expression of the HBV X protein in these cells was achieved by transfection of pcDX into the cells by using Transfectin (Life Technologies) and the transfection procedure used was the one supplied with Transfectin.
  • Baculovirus CCAT was used to infect human primary hepatocytes obtained from liver biopsies. These human primary hepatocytes were prepared by mincing human liver samples followed by digestion with collagenase. Briefly, the minced liver was washed five times with Phosphate buffered saline (PBS) and then a pre-warmed (to 37°C) solution of 0.05% collagenase (Life Technologies) in PBS was added. The collagenase solution/ liver mixture was stirred at 37°C for five minutes.
  • PBS Phosphate buffered saline
  • TK activity of baculovirus NTK was measured by the use of a genetic complementation assay (Metzger C, et al (1994). J.Vi ol.69: 8423 - 8427). Briefly, monolayers of HepG2 cells, which were also transfected with pcDX to express the HBV-X protein, were infected with baculovirus NTK. Cells were either grown in cell culture medium (RPMI plus 10% bovine foetal serum) or the same cell culture medium supplemented with ganciclovir to a final concentration of 1mM.
  • TK thymidine kinase deficient
  • NTK was shown to be active in vivo in woodchucks chronically infected with woodchuck hepatitis virus (WHV)- Approximately 0.4ml aliquots of NTK, containing about 10 9 plaque forming units per ml were injected into the livers of WHV chronically infected woodchucks. Needle biopsies of the same regions of the woodchuck livers were stained with mouse monoclonal antibody RFHBe2 (to hepatitis B virus e antigen and has previously been shown to react with WHV infected liver cells). In control liver approximately 10% of the cell stained for WHV e-antigen whereas in the liver infected with NTK the number of cells staining for WHV e-antigen was reduced to 4%.
  • RFHBe2 mouse monoclonal antibody

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Abstract

Particules de baculovirus modifié contenant un acide nucléique prophylactique et/ou thérapeutique, ainsi que leur utilisation pour le traitement d'états hépatiques, tels que des infections virales.
PCT/GB1998/001249 1997-04-29 1998-04-29 Utilisation d'un baculovirus modifie contenant un acide nucleique exogene pour preparer un medicament servant a administrer ledit acide nucleique aux hepatocytes Ceased WO1998048842A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU72217/98A AU7221798A (en) 1997-04-29 1998-04-29 Use of a modified baculovirus containing exogenous nucleic acid for the manufacture of a medicament for delivering said nucleic acid to the hepatocytes
EP98919340A EP0979106A1 (fr) 1997-04-29 1998-04-29 Utilisation d'un baculovirus modifie contenant un acide nucleique exogene pour preparer un medicament servant a administrer ledit acide nucleique aux hepatocytes
US09/729,856 US20010000228A1 (en) 1997-04-29 2000-12-06 Use of a modified baculovirus containing exogenous nucleic acid for the manufacture of a medicament for delivering said nucleic acid to the hepatocytes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9708698.7A GB9708698D0 (en) 1997-04-29 1997-04-29 Materials for therapy
GB9708698.7 1997-04-29

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WO1998048842A1 true WO1998048842A1 (fr) 1998-11-05

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AU (1) AU7221798A (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077233A3 (fr) * 1999-06-10 2001-12-06 Gen Hospital Corp Virus d'adn non mammalien resistant au complement et utilisations de ces virus

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009074A1 (fr) * 1994-09-23 1996-03-28 The General Hospital Corporation Utilisation d'un virus a adn non mammalien en vue de l'expression d'un gene exogene dans une cellule mammalienne
WO1998011243A2 (fr) * 1996-09-11 1998-03-19 The General Hospital Corporation Virus a adn non mammalien qui possede une proteine de coque modifiee

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009074A1 (fr) * 1994-09-23 1996-03-28 The General Hospital Corporation Utilisation d'un virus a adn non mammalien en vue de l'expression d'un gene exogene dans une cellule mammalienne
WO1998011243A2 (fr) * 1996-09-11 1998-03-19 The General Hospital Corporation Virus a adn non mammalien qui possede une proteine de coque modifiee

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077233A3 (fr) * 1999-06-10 2001-12-06 Gen Hospital Corp Virus d'adn non mammalien resistant au complement et utilisations de ces virus

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GB9708698D0 (en) 1997-06-18
EP0979106A1 (fr) 2000-02-16

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