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WO2002081716A1 - Vecteur adenoviral ovin ameliore - Google Patents

Vecteur adenoviral ovin ameliore Download PDF

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Publication number
WO2002081716A1
WO2002081716A1 PCT/AU2002/000443 AU0200443W WO02081716A1 WO 2002081716 A1 WO2002081716 A1 WO 2002081716A1 AU 0200443 W AU0200443 W AU 0200443W WO 02081716 A1 WO02081716 A1 WO 02081716A1
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WO
WIPO (PCT)
Prior art keywords
ovine adenovirus
transcription unit
nucleic acid
recombinant
genome
Prior art date
Application number
PCT/AU2002/000443
Other languages
English (en)
Inventor
Gerald Wayne Both
Original Assignee
Commonwealth Scientific And Industrial Research Organisation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Commonwealth Scientific And Industrial Research Organisation filed Critical Commonwealth Scientific And Industrial Research Organisation
Publication of WO2002081716A1 publication Critical patent/WO2002081716A1/fr

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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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to ovine adenovirus vectors and to the use of these vectors for the delivery and expression of nucleic acid sequences.
  • Gene therapy has the potential to be a treatment of the future for a variety of acute and chronic diseases. This promise is largely unrealised because of the high expectations placed upon the technology and the lack of appreciation of which vectors are best suited to certain problems. There are difficulties in delivering genes to enough cells in vivo to generate a therapeutic effect. Identifying new vectors with different properties, such as a different cell tropism, will result in a range of vectors from which to choose to find the best vector for a particular purpose. The ability to overcome immunity to the vector that may pre-exist will also enhance the efficiency of gene delivery. An ability to produce stable, recombinant vectors that replicate to high titre in a robust manner will also improve the prospects of therapeutic success.
  • Adenoviruses are of great interest as delivery vectors for gene therapy and vaccine applications.
  • the most commonly used vectors are derived from human adenovirus type 5 as this virus is of low pathogenicity and oncogenicity as well as one of the best studied at the molecular level.
  • prospective patients develop immunity to human adenoviruses as a result of natural infection. Therefore, vectors based on non-human adenoviruses are also being developed.
  • the ovine adenovirus isolate OAdV287 (OAdV) has been characterised and adapted as an alternative gene delivery vector (WO 96/03508 and WO 97/0682).
  • the genomic sequence of this virus has been deposited with GenBank under accession No. U40839.
  • the ovine adenovirus is not neutralised by human antisera that neutralise human adenovirus type 5 (Hofmann etal., 1999) and thus should provide an initial advantage in gene delivery.
  • the nucleotide sequence of the OAdV genome revealed that its structure was not typical of most human and animal adenoviruses which are classified as members of the mastadenoviruses (Vrati etal., 1996a).
  • OAdV has now been recognised as the prototype of a proposed new genus of adenoviruses, tentatively called the atadenoviruses (Harrach and Benko, 1999).
  • the OAdV genome was successfully cloned into a plasmid that could be propagated in E. coli and from which infectious DNA could be generated (Vrati etal., 1996b). Because the functional importance of some OAdV genes remains to be determined, the strategy to adapt the virus as a gene delivery vector was to identify probable non-essential intergenic sites and use these to insert foreign gene cassettes. Thus, site I between the pVIII and fiber genes was identified and exploited.
  • site III was identified in plasmid OAV600 (WO 96/03508). Unique cloning sites were inserted at a location that was thought to be immediately adjacent to the 3' end of the right hand transcription unit. Subsequently, an overview transcription map of the OAdV genome was elucidated (Khatri and Both, 1998). This revealed that site III was located between 3' end of the transcription unit for the Right Hand End and the promoters for the probable E4 region.
  • the present inventors have now surprisingly found that there are specific sites in the ovine adenovirus genome at which foreign genetic material may be inserted leading to recombinant ovine adenovirus vectors with enhanced stability.
  • the present inventors have found that an increase in stability is obtained when the foreign DNA is inserted between coding regions of transcription units which are adjacent in the ovine adenovirus genome.
  • the present invention provides a recombinant ovine adenovirus vector, the vector comprising an ovine adenovirus genome in which a heterologous nucleic acid molecule is inserted between coding regions of transcription units which are adjacent in ovine adenovirus genome.
  • heterologous nucleic acid sequence is inserted between the Right Hand End transcription unit and the E4 transcription unit or between the Left Hand End transcription unit and the IVa 2 transcription unit.
  • the present invention provides a plasmid comprising a recombinant ovine adenovirus vector of the first aspect.
  • the plasmid comprises an origin of replication and a selectable marker.
  • the present invention provides a method of delivering a heterologous nucleic acid molecule to a target cell, the method comprising transfecting the cell with a recombinant ovine adenovirus vector of the first aspect.
  • the present invention provides a method of delivering a heterologous nucleic acid molecule to an animal cell, the method comprising administering to the animal a recombinant ovine adenovirus vector of the first aspect such that the vector infects the cell.
  • the present invention provides a method of gene transfer to human cells, the method comprising administering to the cells a recombinant ovine adenovirus vector of the first aspect such that the vector infects at least one cell and the infected cell expresses the heterologous nucleic acid molecule.
  • the present inventors have now found that recombinant ovine adenovirus vectors which comprise foreign genes inserted at site III, ie. between the 3' end of the transcription unit for the Right Hand End and the promoters for the E4 region (see Figure 1) are surprisingly more stable than ovine adenovirus vectors which comprise foreign genes inserted at other sites.
  • the present invention provides a recombinant ovine adenovirus vector, the vector comprising an ovine adenovirus genome in which a heterologous nucleic acid molecule is inserted between coding regions of transcription units which are adjacent in ovine adenovirus genome.
  • heterologous nucleic acid sequence is inserted between the Right Hand End transcription unit and the E4 transcription unit or between the Left Hand End transcription unit and the -Va 2 transcription unit.
  • the heterologous nucleotide sequence inserted between the Right Hand End transcription unit and the E4 transcription unit is inserted between nucleotide 26,682 (the 3' end of the RHE transcription unit) and the 5' end of the E4 promoter (around nucleotide 26,555) of the ovine adenovirus genome. More preferably, the heterologous nucleotide sequence is inserted between nucleotides 26,682 and 26,622, more preferably between nucleotides 26,682 and 26,675, and most preferably between nucleotides 26,675 and 26,676.
  • the heterologous nucleotide sequence inserted between the Left Hand End transcription unit and the IVa 2 transcription unit is inserted between nucleotides 3063 and 3072, corresponding to the 3' end of the LHE transcription unit and the 3' end of the IVa 2 transcription unit of the ovine adenovirus genome, respectively.
  • the ovine adenovirus genome is the genome of ovine adenovirus OAV287 (as described in GenBank Accession No. U40839) or a functionally equivalent sequence.
  • the phrase "functionally equivalent sequence” is intended to cover minor variations in the OAV287 genome which, due to degeneracy in the genetic code, does not result in the genome encoding different viral polypeptides. Further, this term is intended to cover alterations in the genomic sequence which lead to changes in the encoded polypeptides, but in which such changes do not substantially affect the biological activities of these viral polypeptides.
  • a portion of the ovine adenovirus genome not essential for the maintenance or viability of the native adenovirus is deleted or altered.
  • Open reading frames (RH1-6) in the RHE transcription unit appear to be functionally redundant in that a substantial portion of this region can be deleted without affecting virus viability eg. virus OAdV603 (Xu et al, 1997).
  • virus OAdV603 Xu et al, 1997
  • the heterologous nucleic acid molecule may be any nucleic acid molecule of interest.
  • the nucleic acid of interest may comprise a therapeutic gene or may encode an antigenic peptide.
  • the therapeutic gene may be, for example, an oncogene or a tumour supressor gene.
  • the therapeutic gene may encode a product such as an enzyme, a blood derivative, a hormone, a lymphokine, an interleukin, an interferon, a TNF, a growth factor, neurotransmitter, a trophic factor, etc.
  • the therapeutic gene may also encode a macromolecule which complements a genetic defect in a somatic cell, or a macromolecule which catalyses one or more processes leading to cell death.
  • Cell death may occur directly as a result of gene expression or indirectly as a result of an immune response to an expressed foreign antigen.
  • the gene encodes an enzyme such as herpesvirus thymidine kinase or non-mammalian cytosine deaminase which metabolises a prodrug. More preferably the gene encodes prokaryotic purine nucleoside phosphorylase.
  • expression of the gene by the transfected cell preferably leads to metabolism of the prodrug giving rise to a toxic product which leads to cell death.
  • the heterologous nucleic acid comprises a cell-specific promoter linked to the therapeutic gene, such that the gene is only expressed in a desired target cell.
  • promoters are well known in the art.
  • the promoter may be selected from the probasin, prostate specific antigen (PSA) or prostate specific membrane antigen (PSMA) promoters.
  • PSA prostate specific antigen
  • PSMA prostate specific membrane antigen
  • the erbB-2 promoter may be used.
  • the carcinoembryonic antigen (CEA) promoter may be used.
  • Example 1 Construction and stability of OAdV expressing green fluorescent protein
  • the plasmid pOAdV217A, containing the HCMV/GFP cassette in site I was constructed as follows. The coding portion of the GFP gene was blunt- cloned into the Xhol/Smal sites of plasmid pCI (Promega Corp, Madison WI) to place it under the control of the HCMV promoter. The entire cassette was excised by Bglll/BamHI digestion and blunt-cloned into the Xbal site of pGemllzf (Promega Corp, Madison WI).
  • a clone with a 5' Apal and 3' Notl site was selected and the insert was cloned into these sites in pOAV200 (site I insertion) for virus rescue (Vrati et al., 1996b). Subsequently, the cassette was further subcloned and modified by Aflll digestion and blunt end ligation to remove the intron provided in pCI. The virus was rescued after transfection of CSL503 cells as described previously (Vrati et al., 1996b) except that cationic lipids were used (Cameron et al., 1999) in place of lipofectamine. The virus proved difficult to rescue and several attempts were made.
  • the virus was subsequently passaged to expand the stock.
  • Viral DNA was extracted from a portion of each passage, digested with BamHI and analysed by agarose gel electrophoresis.
  • Passage 1 virus had a similar amount of the 3.1kb band that was diagnostic for the cassette compared to the starting plasmid from which the virus was rescued. However, by passage three this band was significantly depleted relative to the band immediately above it and a smaller product of ⁇ 1.7kb had appeared, demonstrating the instability of this particular genome.
  • PCR amplification across the site of the inserted cassette and nucleotide sequencing revealed that a variety of deletion events had resulted in the loss of all or part of the HCMV promoter and the GFP coding and polyadenylation sequence.
  • Example 2 Construction and stability of OAdV carrying an HCMV alkaline phosphatase cassette
  • the corresponding viruses were rescued and passaged in CSL503 cells. From gel electrophoresis it was apparent that the 1.95 and 1.8kb bands representing the cassette in OAdV216 were lost rapidly after passage and by passage two had been replaced by a ⁇ 1.4kb band. For OAdV616 however, the genome is stable. The diagnostic 1.8kb band was retained, even after four passages.
  • Example 3 Construction and stability of an OAdV carrying cassettes for prostate cancer gene therapy
  • Plasmids pOAdV220 and 222 contained the promoter from Rous sarcoma virus and HCMV, respectively.
  • Plasmids pOAdV223 and 623 contained a prostate-specific promoter/ enhancer element derived from the prostate-specific membrane antigene gene linked to the promoter the rat probasin gene promoter. These cassettes were inserted in the left to right orientation into the Apal/Notl sites of pOAdV200 (site I) and pOAdV600 (site III), respectively.
  • site III is the preferred site for insertion of foreign gene cassettes. As site III is located between recently defined transcription units (Khatri and Both, 1998) the insertion of a discrete transcription cassette may not interfere with other viral functions. With this precedent, it is anticipated that expression cassettes could also be inserted into the OAdV genome between the Left Hand End and IVa2 transcription units (see Figure 1).
  • Vrati S., Brookes, D. E., Strike, P., Khatri, A., Boyle, D. B., and Both, G. W. (1996a).
  • Unique genome arrangement of an ovine adenovirus Identification of new proteins and proteinase cleavage sites. Virology 220(1), 186-199.
  • Vrati S., Macavoy, E. S., Xu, Z. Z., Smole, C, Boyle, D. B., and Both, G. W. (1996b). Construction and transfection of ovine adenovirus genomic clones to rescue modified viruses. Virology 220(1), 200-203.

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Abstract

La présente invention concerne un vecteur adénoviral ovin recombinant. Ce vecteur comprend un génome adénoviral ovin dans lequel une molécule d'acide nucléique hétérologue est insérée entre des régions de codage d'unités de transcription qui sont adjacentes dans le génome adénoviral ovin. La séquence d'acide nucléique hétérologue est insérée de préférence entre l'unité de transcription d'extrémité droite et l'unité de transcription E4 ou entre l'unité de transcription d'extrémité gauche et l'unité de transcription IVa2.
PCT/AU2002/000443 2001-04-06 2002-04-08 Vecteur adenoviral ovin ameliore WO2002081716A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPR4268 2001-04-06
AUPR4268A AUPR426801A0 (en) 2001-04-06 2001-04-06 Improved ovine adenovirus vector

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WO2002081716A1 true WO2002081716A1 (fr) 2002-10-17

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003508A1 (fr) * 1994-07-26 1996-02-08 Commonwealth Scientific And Industrial Research Organisation Adn codant un adenovirus ovin (oav287) et son utilisation comme vecteur viral

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003508A1 (fr) * 1994-07-26 1996-02-08 Commonwealth Scientific And Industrial Research Organisation Adn codant un adenovirus ovin (oav287) et son utilisation comme vecteur viral

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KHATRI A., BOTH G.W.: "Identification of transripts and promoter regions of ovine adenovirus OAV287", VIROLOGY, vol. 245, 1998, pages 128 - 141 *
VRATI S. ET AL.: "Construction and transfection of ovine adenovirus genomic clones to rescue modified viruses", VIROLOGY, vol. 220, 1996, pages 200 - 203 *
VRATI S. ET AL.: "Unique genome arrangement of an ovine adnovirus: identification of new proteins and proteinase cleavage sites", VIROLOGY, vol. 220, 1996, pages 186 - 199 *
XU Z.Z. ET AL.: "An ovine adenovirus vector lacks transforming ability in cells that are transformed by AD5 E1 E1 A/B sequences", VIROLOGY, vol. 270, 2000, pages 162 - 172 *
XU Z.Z. ET AL.: "Construction of ovine adenovirus recombinants by gene insertion or deletion of related terminal region sequences", VIROLOGY, vol. 230, 1997, pages 62 - 71 *

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