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WO1998002564A1 - Vecteur hybride de virus herpetique/virus de epstein-barr - Google Patents

Vecteur hybride de virus herpetique/virus de epstein-barr Download PDF

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WO1998002564A1
WO1998002564A1 PCT/CA1997/000487 CA9700487W WO9802564A1 WO 1998002564 A1 WO1998002564 A1 WO 1998002564A1 CA 9700487 W CA9700487 W CA 9700487W WO 9802564 A1 WO9802564 A1 WO 9802564A1
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vector
polynucieotide
virus
nucleic acid
heφes
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Craig A. Strathdee
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Robarts Research Institute
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Robarts Research Institute
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16241—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16243—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to viral vectors and more specifically to a polynucieotide cassette containing elements of the Epstein Ban * virus (EBV) and herpes simplex virus (HSV) for use in a viral vector.
  • EBV Epstein Ban * virus
  • HSV herpes simplex virus
  • the delivery of DNA to target tissues presents a variety of problems that differ with each potential therapeutic application.
  • the challenge for the therapeutic use of viral vectors is to achieve efficient and often extended expression of an exogenous gene while evading the host defenses.
  • the introduction of a particular foreign or native gene into a host ceil is typically facilitated by first introducing the gene sequence into a suitable nucleic acid vector.
  • a variety of methods have been developed which are capable of mediating the introduction of such a recombinant vector into a desired host cell for the purpose of altering phenotype or terminating existence of the cell.
  • the delivery of the appropriate gene to a patient with a recessive inherited disease should correct the genetic defect and potentially ameliorate the disease state.
  • delivery of a gene encoding a toxin is desirable to kill a cancer cell, whereas another gene may be desirable to an kill infectious organism.
  • HSV Herpes simplex virus
  • HSV is a commonly used virus for transduction.
  • HSV is a human neurotropic herpesvirus which causes lifelong infections through latent maintenance in the ganglia of sensory neurons. Two sets of cis elements are required for packaging the HSV genome.
  • the first, ori L or ori s is recognized by the viral DNA poiymerase which initiates rolling circle replication and produces concatamers during productive infection.
  • the second, the site is specifically cleaved during packaging to generate linear dsDNA molecules that are terminally redundant.
  • An advantage of the HSV as a potential vector system is that the virus is able to infect an extremely wide range of mammalian cell types. The initial stages of infection by HSV involve a fairly non-specific interaction with cell surface sulfated glycans such as heparin sulfate. and as such, there are no cell types to which the virus fails to attach. This suggests that the HSV particle may be a universally efficient gene delivery reagent.
  • the major problem involved in using the HSV as a vector system is that the viral genome is poorly suited to serve as a general purpose platform for long term gene expression.
  • Epstein-Barr virus is a human herpesvirus that causes lifelong latent infections in humans and is endemic to the global population, with over 95% of adults being seropositiv .
  • EBV is one of the most highly transforming viruses known in viiro. yet causes no detectable disease in the overwhelming majority of cases.
  • the EBV genome is maintained episomally at a relatively low copy number and replicates only once in the S phase of the cell cycle with a fidelity similar to that of chromosomally-encoded genes.
  • EBV genes Only two EBV genes are required for episomal replication and maintenance during latent infection: o P which functions in cis as the episomal origin of replication, and EBNA-1 which functions in trans to bind oriP to facilitate subsequent replication and segregation during mitosis.
  • Bacterial plasmids that contain the EBNA ⁇ and oriP genes can replicate in most human cell types and can provide stable long term expression. This suggests that the EBV may be a universally efficient gene expression reagent.
  • the major problem in using the EBV as a vector system is that it relies on a physical method of gene delivery. As described above, these are at present too inefficient for practical application.
  • An idealized vector system for therapeutic application of cloned genes would combine the high efficiencies of gene transfer, the ability to inco ⁇ orate larger amounts of non-viral DNA and retain the flexibility to take advantage of developments in physical transfer methods.
  • the present invention was developed to inco ⁇ orate, in a polynucieotide cassette, desirable elements of the he ⁇ es simplex virus (HSV) and the Epstein Barr virus (EBV).
  • HSV he ⁇ es simplex virus
  • EBV Epstein Barr virus
  • a vector containing such a cassette is efficiently packaged into infectious particles when the appropriate HSV helper virus is provided.
  • the vector has enhanced replicational stability, a trait which is retained when the vector is delivered to cells as a transducing particle.
  • vectors containing the EBV/HSV polynucieotide cassette are functional in both mitotic and postmitotic cells.
  • the invention provides a polynucieotide cassette comprising a HSV origin of replication, a HSV repetitive element, an EBV origin of replication and an EBV EBNA-l coding sequence.
  • the polynucieotide cassette includes the EBNA-l and oriP components of the EBV. and the or ⁇ F or ori L and a site components of the HSV.
  • the present invention describes methods for combining the aforementioned cassette on a single gene delivery/expression platform which may contain, in addition to the HSV EBV essential components, a cDNA expression cassette, a plasmid backbone to propagate the vector in bacteria, and a marker gene to facilitate selection in mammalian cells.
  • the invention provides a vector which includes the polynucieotide cassette of the invention.
  • the invention includes a method of producing a recombinant he ⁇ es virus including transfecting a suitable packaging host cell with the vector and recovering the recombinant virus. Further, the invention includes a method for introduction and expression of a heterologous nucleic acid sequence in a target cell including infecting the cell with the recombinant virus and expressing the heterologous nucleic acid sequence in the cell.
  • Figure 1 shows an illustration of the structure of the pHERO 200 vector.
  • EBV latent replicon of EBV
  • oriP oriP genes
  • a cDNA expression cassette inco ⁇ orating the RSV 3'-LTR and rabbit ⁇ -globin gene polyadenylation signal
  • the bacterial hph gene to provide for selection in mammaiian cells by resistance to hygromycin B
  • HSV lytic replicon which consists of the a sequence and the ori s
  • the colEl replicon and bacterial bla gene to provide for propagation in bacteria by resistance to ampicillin.
  • two reporter genes are indicated in the MCS of the cDNA expression cassette, the bacterial ⁇ -galactosidase reporter gene diiven by the RSV 3'-LTR and SV40 polyadenylation signals, as well as the firefly luciferase gene driven by the CMV promoter and rabbit ⁇ -globin polyadenylation signal.
  • Figure 2 shows an illustration of the structure of the pHERO 400 vector.
  • EBV which consists of the EBNA-l and oriP genes
  • a cDNA expression cassette inco ⁇ orating the RSV 3'-LTR and rabbit ⁇ -globin gene polyadenylation signal
  • the bacterial zeo gene to provide for selection in mammalian cells as well as bacteria by resistance to Zeocin
  • the HSV lytic replicon which consists of the a sequence and the ori s
  • the colEl replicon to provide for propagation in bacteria.
  • Unique restriction enzyme sites are indicated.
  • Figures 3a and 3b show an illustration of the structure of the pHERO 600 and pCIG 2 vectors, respectively.
  • pHERO There are 4 functional domains present in pHERO: (i) the latent replicon of EBV, which consists of the EBNA-l and oriP genes; (ii) the bacterial zeo gene to provide for selection in mammalian cells as well as bacteria by resistance to Zeocin; (iii) the HSV lytic replicon, which consists of the a sequence and the o t ; and (iv) the colEl and fl ori replicons and bacterial bla gene to provide for propagation in bacteria be resistance to ampicillin.
  • pCIG There are 2 functional domains in pCIG: i) the cDNA expression cassette, which consists of the CMV promoter, a multiple cloning site, and the bovine growth hormone polyadenylation signal; and ii) the colEl replicon and bacterial neo gene to provide for propagation in bacteria by resistance to kanamycin.
  • Figure 4 shows the repiicational stability of vectors of the invention using the ⁇ - galactosidase or luciferase reporter system.
  • the present invention provides a polynucieotide cassette for inco ⁇ oration into a suitable vector to provide a means for delivery and expression of a desirable gene and gene product to a cell.
  • the polynucieotide cassette encodes HSV/EBV components and can be inco ⁇ orated into any vector.
  • a "polynucieotide cassette”, as the term is used herein, is a nucleic acid (preferably DNA) encoding a he ⁇ es virus origin of replication, a he ⁇ es virus repetitive element, an Epstein-Barr virus origin of replication and an Epstein-Barr virus EBNA-l coding sequence.
  • a he ⁇ es virus origin of replication includes ori s or ori L .
  • a he ⁇ es virus repetitive element includes an a site.
  • An Epstein- Barr virus origin of replication includes oriP.
  • An example of this cassette contained in a vector is illustrated in the exemplary vector of the invention, the pHERO600 vector, in which the HSV EBV components are inco ⁇ orated such that they are flanked by unique restriction enzyme sites ( Figure 3). It is understood that, in a cassette format, the HSV/EBV components can be added as a final phase of vector construction, after initial experiments aimed at optimizing cDNA expression have been performed by standard techniques.
  • a principal advantage to the cassette system is that it can be used in conjunction with any existing expression system.
  • existing systems include transient expression vectors as well as vectors designed for use with other viral systems, for example murine retroviral vectors or adenoviral vector constructs.
  • a "viral vector”, as used herein, is a nucleic acid molecule in which a gene sequence to be transferred is operably linked to a subset of viral sequences.
  • operably linkage refers to functional linkage between a promoter sequence and the structural gene regulated by the promoter nucleic acid sequence.
  • the operablv linked promoter controls the expression of the polypeptide encoded by the structural gene, or in a preferred embodiment of the invention, the heterologous gene nucleic acid sequences.
  • the orientation or placement of the elements of the vector is not strict, e.g., an enhancer element can also be utilized, so long as the "operable linkage" requirement is fulfilled for control of and expression of the nucleic acid sequences.
  • the viral sequences and the total genome size is selected such that the vector is capable of being encapsulated in a viral particle and thus be capable of binding to. and introducing its gene sequences into a virus-sensitive host cell.
  • the infective properties of such a virion are, therefore, the same as those containing the wild type viral genome.
  • Modifications to the vector delivery system can be made to increase its versatility and range of host cell infectivity. For example, alterations to the sequences that flank the cassette can be made such that a new series cf unique restriction endonuclease sites could be inco ⁇ orated in order to facilitate the subcloning process. In another example, alterations to the helper virus used to package the vector could be used torn enhance its tropism for a particular tissue.
  • the promoter in operable linkage with the heterologous gene can be any viral or housekeeping gene promoter, including for example, LTR. cytomegalovirus (CMV) immediate early, DHFR, SV40 early or late, Moloney murine leukemia virus and Rous Sarcoma virus LTR promoters.
  • the promoter sequence may be homologous or heterologous to the nucleic acid sequence.
  • a wide range of promoters may be utilized, including viral or tissue specific promoter. Cell or tissue specific promoters can be utilized to target expression of gene sequences in specific cell populations. Suitable mammalian and viral promoters for the present invention are available in the art. However, the promoter operablv linked with the heterologous gene must be responsive to the regulatable transactivating element.
  • site-specific recombination rather than subcloning, can be used to introduce the cassette into a suitable expression vector.
  • a loxP recombination site could be introduced into the cassette to facilitate cre-mediated recombination with an existing ioxP site in the expression vector (see for example, U.S. Patent No. 4,959.317, inco ⁇ orated herein by reference).
  • An advantage of this arrangement is that no unique restriction endonuclease sites are required, and thus recombination with Bacterial Artificial Chromosome (BAC)-based vectors that contain large inserts of genomic DNA is possible. This would facilitate the use of very large gene-based expression systems which has the advantage of providing an expression system that utilizes all of the endogenous sequences required for authentic regulation of gene expression.
  • BAC Bacterial Artificial Chromosome
  • a reporter gene sequence is inco ⁇ orated in the vector to facilitate identification of a cell inco ⁇ orating a functional vector.
  • a "reporter gene sequence”, as used herein, is any gene sequence which, when expressed, results in the production of a protein whose presence or activity can be monitored.
  • a reporter or selectable marker gene sequence is said to be “heterologous” if it is not naturally present in a wild type he ⁇ es virus genome.
  • suitable reporter genes include the gene for chloramphenicol acetyltransferase, luciferase, ⁇ - galactosidase. ⁇ -lactamase. etc.
  • the reporter gene sequence may be any gene sequence whose expression produces a gene product which affects the target tissue physiology.
  • a constitutively active selectable marker is used to provide a mechanism to enrich for transduced cells.
  • a "selectable marker gene sequence” may be any gene sequence capable of expressing a protein whose presence permits one to selectively propagate a cell in which it is contained.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler,et al., 1977. Cell Y ⁇ _: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Na . Acad. Sci.
  • ODC omithine decarboxyiase
  • 2- (difluoromethyl)-DL-ornithine 2- (difluoromethyl)-DL-ornithine
  • DFMO McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.
  • tTA inducible system driving the zeo gene, in which the marker is active in a packaging cell line to permit ease of growth, but is not active when delivered to other cell types either in vitro or in vivo.
  • inducible promoters could be employed, such as the ecdysone-inducible promoters (Invitrogen Inc.. San Diego, CA.).
  • Other selectable markers and promoters are known to those of skill in the art.
  • a mammalian promoter driving the selectable marker gene is removed, such that the gene is expressed only in bacteria and no selection is possible in mammalian cells.
  • This vector is useful for applications where no selection is required, such as direct in vivo application gene delivery.
  • the bacterial gene product would contribute to an immune response directed against transduced cells, and would be detrimental to long term survival of the cells.
  • the recombinant vector can be packaged into infectious particles using any suitable HSV packaging system.
  • replication-defective helper virus is utilized to package the vector. This method is based on the fact that the vector is able to compete with the HSV genome for packaging into infectious particles during the HSV life cycle.
  • Any replication-defective strain of HSV can be used in this packaging protocol, provided that a suitable complementing cell line is available. Examples include, but are not limited to, the IE3 deletion mutants dl20 and D30EBA which can be grown on the E5 cell line, the 14H ⁇ 3 IE3 + VP16 deletion mutant that can be grown on the E5 cell line, and the 5dll .2 IE2 deletion mutant that can be grown on the 2-2 cell line.
  • Vero cells monkey kidney
  • Vero cells monkey kidney
  • G418 is typically included in the culture medium
  • N.A. DeLuca and P.A. Schaf ⁇ c ⁇ Nucleic Acids Res. 15:4491-451 1 ; T. Paterson and R.D. Everett (1990) J. Gen. Virol. 71 :1775-1783; P.A. Johnson et al. (1994) J. Virology 68:6347-6362; A.M. McCarthy et al. ( 1989) J. Virology 63 : 18-27; respectively)
  • a helper virus-free packaging system is available for HSV, and is compatible with the pHERO vector.
  • HSV functions are provided in the form of an overlapping set of 5 cosmid constructs that together comprise the - 155 kb HSV genome from which the a site sequences have been specifically deleted.
  • the cosmid set can recombine and replicate similar to the intact viral genome but cannot be packaged into infectious HSV particles (Fraefel et al., J. Virology 70:7190-7197, 1996). If the pHERO vector is included in the initial transfection. then all of the infectious particles produced will contain only the packaged vector.
  • Any cell line that can support the growth of HSV and that is easily transfectable can be used to transfect the vector.
  • An example described here is the BHK-21 hamster kidney cell line. This cell line is grown and transfected similarly to the 293 cell line, with the exception that they are typically split at a ratio of 1 : 10 to account for their greater growth rate.
  • the recombinant vector can be introduced into the cultured cells using conventional transfection technologies. Suitable methods include calcium phosphate co-precipitation, electroporation, and cationic liposomes, and conventional mechanical procedures such as microinjection or biollistic introduction. Other methods are known to those skilled in the art.
  • the cells were split by a factor of 20. and plated in medium supplemented with the appropriate antibiotic to select for transfectants. For example, hygromycin B was used for pHERO200, and Zeocin for pHERO400. After 10 to 14 days individual colonies can be isolated using standard methodologies.
  • the presence of the pHERO vector of the invention is verified by assaying for cDNA expression using conventional techniques known to those skilled in the art.
  • this incudes ⁇ -galactosidase and luciferase reporter genes.
  • episomal DNA was extracted from each clone using standard methodologies, shuttled back into bacteria through electroporation. and then mapped with restriction endonucleases. Usually approximately 80% of clones tested will maintain the pHERO vector as a stable episome, and these are further expanded by continuous growth in medium supplemented with the appropriate antibiotic.
  • the example above is merely illustrative and should be understood that other selectable markers, for example, or comparable parameters can be utilized to achieve the same pu ⁇ ose.
  • the invention provides a method for introduction and expression of a heterologous nucleic acid sequence in a target cell comprising infecting the cell with the recombinant virus and expressing the heterologous nucleic acid sequence in the cell.
  • the product encoded by the heterologous nucleic acid delivered by the method of the invention includes any composition which would have a desired biological effect at the site of transduction.
  • products would include antirestenotic and antiproliferative compositions.
  • Other products include platelet receptor and mediator inhibitors, smooth muscle cell proliferation inhibitors, growth factor inhibitors, Gp ⁇ b/ ⁇ ia antagonists, agents that inhibit cell adhesion and aggregation, tumor suppressor genes, protooncogenes, DNA repair genes, genes coding for cytokines, genes coding for bacterial enzymes involved in the conversion of nontoxic prodrugs to their active molecules, and multi-drug resistance genes.
  • heterologous genes include Factor VIII or Factor IX, TNF, tissue factor, VLA-4, growth-arrest homeobox gene, gax. L-arginine. retinoids.
  • the heterologous gene may encode a protein product important in normal neuronal cell function, e.g., tyrosine hydroxylase in the production of dopamine; choline acetyl transferase for the production of acetylcholine. It is envisioned that any heterologous gene can be cloned into the vector of the invention for genetic modification of a cell.
  • nucieic acid sequences that interfere with the gene's expression at the translational level can be delivered.
  • This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense polynucieotide or triplex agent, or by cleaving it with a ribozyme.
  • Antisense polynucleotides in the context of the present invention includes both short sequences of DNA known as oligonucleotides of usually 10-50 bases in length as well as longer sequences of DNA that may exceed the length of the target transcript itself.
  • Antisense polynucleotides useful for the present invention are complementary to specific regions of a corresponding target mRNA. Hybridization of antisense polynucleotides to their target transcripts can be highly specific as a result of complementary base pairing. The capability of antisense polynucleotides to hybridize is affected by such parameters as length, chemical modification and secondary structure of the transcript which can influence polynucieotide access to the target site. (Stein et al, Cancer Research 48:2659, 1988). An antisense polynucieotide can be introduced to a cell by introducing a DNA segment that codes for the polynucieotide into the cell such that the polynucieotide is made inside the cell.
  • An antisense polynucieotide can also be introduced to a cell by adding the polynucieotide to the environment of the cell such that the ceil can take up the polynucieotide directly.
  • the latter route is preferred for the shorter polynucleotides of up to about 20 bases in length.
  • the product encoded by the heterologous nucleic acid can be expressed by using inducible or constitutive regulatory elements for such expression.
  • inducible or constitutive regulatory elements for such expression.
  • Commonly used constitutive or inducible promoters, for example, are known in the art.
  • the subject is a human, however, it is envisioned that the method of sustained in vivo delivery of compositions via transduction as described herein can be performed on any animal.
  • the vector containing the polynucieotide cassette of the claimed invention can be administered in vivo by injection or by gradual perfusion over time.
  • the composition can be administered intravenously, intraperitoneally, intramuscularly, subcutaneous ly, intracavity, or transdermally, and preferably is administered intravascularly at or near the site of targeted for transduction with the recombinant virus.
  • Other methods of introducing infectious materials to an organism or to target tissues are known in the art.
  • the vector containing the polynucieotide cassette of the claimed invention can be administered ex vivo by transfer of genetic material to cells located outside the host. Following transfer of the genetic material, the transduced cells can be implanted back in the host.
  • the dosage ranges for the administration of the compositions in the method of the invention are those large enough to produce the desired effect in which the symptoms of the disease are ameliorated. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.
  • the method of the invention can also be used for treatment of a neuronal disorder wherein the heterologous nucleic acid may encode a gene which encodes a receptor, or a gene which encodes a ligand.
  • receptors include receptors which respond to dopamine, GABA. adrenaline, noradrenaiine, serotonin, glutamate, acetylcholine and other neuropeptides, as described above.
  • neuronal cells can be genetically altered in a subject having a disorder of the basal ganglia, such as Parkinson's disease, wherein the cells can be modified to contain an exogenous gene encoding L-DOPA, the precursor to dopamine. Parkinson's disease is characterized by a loss of dopamine neurons in the substantia nigra of the midbrain, which have the basal ganglia as their major target organ.
  • Alzheimer's disease is characterized by degeneration of the cholinergic neurons of the basal forebrain.
  • the neurotransmitter for these neurons is acetylcholine, which is necessary for their survival.
  • Following a stroke there is selective loss of cells in the CA1 of the hippocampus as well as cortical cell loss which may underlie cognitive function and memory loss in these patients. Once identified, molecules responsible for CA1 cell death can be inhibited by the methods of this invention.
  • antisense sequences or a gene encoding an antagonist can be transferred to a neuronal progenitor and implanted into the hippocampal region of the brain.
  • a neuronal progenitor or a gene encoding an antagonist can be transferred to a neuronal progenitor and implanted into the hippocampal region of the brain.
  • the replicative stability of the standard pHERO vector was established using a cultured human cell line designated as HEK293 (Human embryonic kidney). HEK293 cells are grown in standard DMEM culture medium supplemented with 10% FCS. When 100% confluent, monolayers are split by a factor 1 :5 and replaced in fresh medium, typically this is required every 3-5 days. The cell line can be serially passaged in this fashion for up to 4 months, at which point the cells are discarded and a new vial of cells retrieved from a previously banked stock in long term storage stored in liquid nitrogen. The replicative stability of pHERO200 was compared to that of a standard yet closely EBV-based vector designated pREP45.
  • pREP45 is derived from the well described pREP4 vector in that it contains the identical dual reporter gene cassette in pHERO200 BL ( Figure 1).
  • the pREP and pHERO200 vectors were transfected into 293 cells using the Lipofectamine cationic liposome reagent (Life Technologies. Inc.. Bethesda, Md.) And stable clones selected as described in the preferred embodiments. Two independent clones of each vector were expanded and these were then each split into duplicate cultures.
  • the pREP45 vector ha a half-life of - 10 weeks under these assay and culture conditions, whereas the pHERO200 vector was fully stable up to the conclusion of the experiment at 19 weeks. These values were confirmed in a separate measure of stability, in which the proportion of cells in the non-selected cu.tures at week 11 that contained each vector construct was directly assayed by plating out serial dilutions under selective and non-selective conditions.
  • the parental pREP45 vector was present in cells representative of 15-41 % of the culture, whereas the pHERO200 vector was present in 75- 100% of the cells, respectively.
  • the pHERO vector has greatly improved stability over that of other EBV-based vectors. Since replicational stability and long term gene expression in vivo are both controlled by EBNA-l in the EBV viral genome, it is expected that the ability of the pHERO vector to provide for long term gene expression in vivo will also be greatly enhanced.
  • a transient transfection protocol can be used (F. Lim et al, (1996) BioTechniques 20:460-469).
  • the appropriate complementing cell line is plated out and grown until the monolayers reach -80-90% confluence, at which time the medium is removed and the cells washed and then transfected with the pHERO vector DNA using an appropriate transfection protocol, as described for 293 cells previously. Following recovery of the cells overnight, the culture media is replaced with fresh media containing ImM N, N'-hexamethylene bis-acetamide and then the monolayers are infected with the corresponding HSV helper virus at a multiplicity of infection between 0.1 and 1.0.
  • the culture is then grown until the entire monolayer shows the distinctive cytopathic effect characteristic of productive HSV replication.
  • the infected cells are harvested and the HSV particles purified using standard methodologies.
  • the relative titers of the helper virus and vector in the resulting lysate are determined by direct plaque assay on the corresponding complementing cell line, or by assays for reporter gene expression on a suitable non- complementing cell line.
  • the results of a typical packaging reaction for pHERO400 ⁇ are presented in Table 1, in which the vector expressed the ⁇ -galactosidase reporter gene.
  • the advantage of this packaging system is that relatively high viral titers can be achieved, although the fraction of packaged vector in the overall lysate is very low.
  • the ⁇ -gal+ titer was obtained by X-Gal histochemical staining 2 days after infecting Vero cells, and represents the number of lacZ transducing units/ml of lysate.
  • the HSV titer was obtained by screening for plaque formation on 2-2 cells for the 5dl 1.2 helper virus and on ES cells for the dl20 and 14H ⁇ 3 helper virus, and represents the number of infectious helper virus units/ml of lysate.
  • An alternate method of packaging the pHERO vector, utilizing an HSV helper virus, makes use of the fact that the vector is stably maintained in the packaging cell line as an episome.
  • the STAR14 cell line for use with this protocol which is based on the 293 cell line and contains the HSV IE2 and IE3 genes maintained by growth selection in 1 ⁇ g/ml puromycin and 400 ug/ml G418.
  • To package the pHERO vector stable transfectants are selected and cloned out using standard methodologies described for 293 cells previously. Individual STAR 14 clones containing the vector are plated out and grown until the monolayers reach -80 to 90% confluence.
  • the culture medium is then replaced with fresh medium supplemented with ImM N.N'-hexamethylene-bis-acetamide and containing no antibiotic selection.
  • the appropriate helper virus is added to provide for a multiplicity of infection of between 0.1 and 1.0, and the culture is then grown until the entire monolayer shows the distinctive cytopathic effect characteristic of productive HSV replication.
  • the infected cells are then harvested and the HSV particles purified using standard methodologies.
  • the relative titers of the helper virus and vector in the resulting lysate are determined as for the transient packaging system described previously.
  • the results of a typical packaging reaction for pHERO400 ⁇ are presented in Table 2, in which the vector expressed the ⁇ -galactosidase reporter gene.
  • the advantage of this packaging system is that a much higher proportion of the vector is packaged into infectious particles when compared to the helper virus, although the actual viral titers is much lower.
  • the ⁇ -gal+ titer was obtained by X-Gal histochemical staining 2 days after infecting Vero cells, and represents the number of lacZ transducing units/ml of lysate.
  • the HSV titer was obtained by screening for plaque formation on 2-2 cells for the 5dl 1.2 helper virus and on ES cells for the dl20 and 14H ⁇ 3 helper virus, and represents the number of infectious helper virus units/ml of lysate.
  • a helper virus free packaging system can be used to package the pHERO vector.
  • the BHK-21 cell line is used. Cells are plated out and grown until the monolayers reach 80- 90% confluence, at which time they are washed and transfected with a 1 :4 mixture of the pHERO vecto ⁇ HSV cosmid DNA using Lipofectamine reagent as described for 293 cells. After recovery of the ceils overnight, fresh medium containing ImM N, N'-hexamethylene bis-acetamide is added and the culture is grown until the entire monolayer shows the distinctive cytopathic effect characteristic of productive HSV replication. The infected cells are then harvested and the HSV particles purified using standard methodologies described previously.
  • the relative titers of the helper virus and vector in the resulting lysate are determined as for the transient packaging system described previously.
  • the results of a typical packaging reaction for pHERO400 ⁇ are presented in Table 3.
  • the advantage of this packaging system is that there is no helper virus present in the lysate, although the absolute number of infectious particles produced is quite low.
  • the replicative stability of the packaged pHERO vector was assayed using the helper virus- free lysate to infect 293 cells, followed by selection for stably transduced cells by growth in Zeocin. Since it was possible to recover stable colonies at a high frequency (Table 3), this demonstrates that the packaged vector retains the stability of the parental pHERO vector, and can be used to deliver genes to mitotically active as well as post-mitotic cell types.
  • the ⁇ -gal+ titer was obtained by X-Gal histochemical staining 2 days after infecting Vero cells, and represents the number of lacZ transducing unitis/ml of lysate.
  • the Zeo R titer was obtained by selecting cultures for 14 days in Zeocin starting 1 day after infecting
  • the HSV titer was obtained by screening for plaque formation on Vero cells, and represents the number of replication-competent infectious viral units/ml of lysate. This is the background reversion rate of the packaging-defective HSV helper virus.
  • Figure 1 shows an illustration of the structure of the pHERO 200 vector. There are 5 functional domains present: (i) the latent replicon of EBV, which consists of the EBNA-l and oriP genes.
  • the EBNA-l gene is expressed from the HSV IE promoter to provide consistently high levels of gene product; (ii) a cDNA expression cassette, inco ⁇ orating the RSV 3'-LTR, a multiple cloning site and rabbit ⁇ globin gene polyadenylation signal; (iii) the bacterial hph gene to provide for selection in mammalian cells by resistance to hygromycin B.
  • the gene is expressed using the HSV thymidine kinase promoter and polyadenylation signals; (iv) the HSV lytic replicon, which consists of the a sequence and the ori s ; and (v) the colEl replicon and bacterial bla gene to provide for propagation in bacteria by resistance to ampicillin.
  • two reporter genes are indicated in the MCS of the cDNA expression cassette, the bacterial ⁇ - galactosidase reporter gene driven by the RSV 3'-LTR and SV40 polyadenylation signals, as well as the firefly luciferase gene driven by the CMV promoter and rabbit ⁇ -globin polyadenylation signal.
  • FIG. 2 shows the structure of the pHERO 400 vector.
  • the EBNA-l gene is expressed from the HSV IE promoter to provide consistently high levels of gene product;
  • a cDNA expression cassette inco ⁇ orating the RSV 3'-LTR and rabbit ⁇ -globin gene polyadenylation signal;
  • the bacterial zeo gene to provide for selection in mammalian cells as well as bacteria by resistance to Zeocin;
  • the HSV lytic replicon which consists of the a sequence and the ori s ; and
  • a multiple cloning site and also the colEl replicon to provide for propagation in bacteria. Useful unique restriction enzyme sites are indicated.
  • the bacterial ⁇ -galactosidase reporter gene was cloned into the multiple cloning site of the cDNA expression cassette
  • Figures 3a and 3b show the structure of the pHERO 600 and pCIG 2 vectors, respectively.
  • pHERO There are 4 functional domains present in pHERO; i) the latest replicon of EBV, which consists of the EBNA-l and oriP genes.
  • the EBNA-l gene is expressed from the HSV IE promoter to provide consistently high levels of gene product; (ii) the bacterial zeo gene to provide for selection in mammalian cells as well as bacteria by resistance to Zeocin; (iii) the HSV lytic replicon, which consists of the a site sequence and the or ⁇ S ; and (iv) the colEl and fl ori replicons and bacterial bla gene to provide for propagation in bacteria by resistance to ampicillin.
  • pCIG There are 2 functional domains in pCIG: i) the cDNA expression cassette, which consists of the CMV promoter, a multiple cloning site, and the bovine growth hormone polyadenylation signal; and ii) the colEl replicon and bacterial neo gene to provide for propagation in bacteria by resistance to kanamycin.
  • the cDNA to be expressed is first subcloned into the pCIG vector.
  • Expression of the cDNA can be studied using transient transfection assays, and if desired, optimized by switching promoter or enhance elements.
  • the corresponding cassette from pHERO600 is subcloned into either the BglH or Sail sites of pCIG.
  • the efficiency of the subcloning process is enhanced by providing a zeo resistance marker in the HSV/EBV cassette of pHERO600, such that only recombinants containing the cassette can be directly selected in bacteria.
  • the system is functional with any existing vector other than pCIG, provided that a compatible cloning site outside of the cDNA expression cassette of the vector is available.
  • Figure 4 shows the replicational stability of EBV vectors.
  • Duplicate cultures of stably transfected 293 cells were used to investigate the replicational stability of each construct over time.
  • maintenance of the constructs was directly selected by growth in hygromycin, while in the other set no selection was employed.
  • the level of reporter gene activity in the non- selected culture as compared to that of the selected culture provided an index of the proportion of the culture that retained the vector construct at any give time point.
  • the overall replicational stability was infeired from the progressive loss of reporter gene activity over time for each construct. The experiment was continued for 19 weeks, with the cultures being assayed at 2 week intervals beginning at week 3.

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Abstract

Cassette polynucléotide contenant des éléments du virus de l'herpès simplex et du virus Epstein-Barr et qui peut être incorporé dans une particule infectieuse appropriée capable d'infecter et de se propager dans des cellules mitotiques et non mitotiques. De tels vecteurs servent dans la production et l'expression des séquences d'acides nucléiques hétérologues dans des cellules cibles.
PCT/CA1997/000487 1996-07-12 1997-07-11 Vecteur hybride de virus herpetique/virus de epstein-barr Ceased WO1998002564A1 (fr)

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AU33320/97A AU3332097A (en) 1996-07-12 1997-07-11 Hybrid herpes virus/epstein-barr virus vector

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GBGB9614699.8A GB9614699D0 (en) 1996-07-12 1996-07-12 Hybrid herpes virus-based vector for gene delivery and expression
GB9614699.8 1996-07-12

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2793414A1 (fr) * 1999-05-10 2000-11-17 Centre Nat Rech Scient Conjugue acide nucleique-anticorps pour delivrer un acide nucleique etranger dans les cellules
WO2001042478A1 (fr) * 1999-11-25 2001-06-14 Gene Bio-Tech Corporation Limited Procede de lutte contre les infections neoplasiques associees au virus epstein barre (ebv) par vecteurs viraux recombinants, procede de construction et de proliferation et applications connexes
WO2012089338A1 (fr) 2010-12-29 2012-07-05 Curevac Gmbh Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4939088A (en) * 1987-02-18 1990-07-03 Meloy Laboratories Inc. Sustained production of recombinant gamma interferon using an Epstein-Barr virus replicon
EP0488528A1 (fr) * 1990-10-30 1992-06-03 Applied Immune Sciences, Inc. Vecteurs recombinants issus de virus associés à l'adénovirus (AAV)
WO1995032299A1 (fr) * 1994-05-20 1995-11-30 The Board Of Trustees Of The Leland Stanford Junior University Systeme de replication autonome pour cellules mammaliennes
WO1996029421A1 (fr) * 1995-03-23 1996-09-26 Cantab Pharmaceuticals Research Limited Vecteurs d'apport de genes
WO1997005263A1 (fr) * 1995-07-26 1997-02-13 Children's Medical Center Corporation Systeme d'encapsidation du vecteur de l'herpesvirus depourvu de virus auxiliaire
WO1997019182A1 (fr) * 1995-11-17 1997-05-29 Centre National De La Recherche Scientifique Production de vecteurs retroviraux par l'intermediaire de vecteurs herpetiques

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4939088A (en) * 1987-02-18 1990-07-03 Meloy Laboratories Inc. Sustained production of recombinant gamma interferon using an Epstein-Barr virus replicon
EP0488528A1 (fr) * 1990-10-30 1992-06-03 Applied Immune Sciences, Inc. Vecteurs recombinants issus de virus associés à l'adénovirus (AAV)
WO1995032299A1 (fr) * 1994-05-20 1995-11-30 The Board Of Trustees Of The Leland Stanford Junior University Systeme de replication autonome pour cellules mammaliennes
WO1996029421A1 (fr) * 1995-03-23 1996-09-26 Cantab Pharmaceuticals Research Limited Vecteurs d'apport de genes
WO1997005263A1 (fr) * 1995-07-26 1997-02-13 Children's Medical Center Corporation Systeme d'encapsidation du vecteur de l'herpesvirus depourvu de virus auxiliaire
WO1997019182A1 (fr) * 1995-11-17 1997-05-29 Centre National De La Recherche Scientifique Production de vecteurs retroviraux par l'intermediaire de vecteurs herpetiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FRENKEL N ET AL: "THE HERPES XIMPLEX VIRUS AMPLICON A VERSATILE DEFECTIVE VIRUS VECTOR", GENE THERAPY, vol. 1, no. SUPPL. 01, 16 August 1993 (1993-08-16), pages S40 - S46, XP000574909 *
SPAETE R R ET AL: "THE HERPES SIMPLEX VIRUS AMPLICON: A NEW EUCARYOTIC DEFECTIVE-VIRUS CLONING-AMPLIFYING VECTOR", CELL, vol. 30, no. 1, 1 August 1982 (1982-08-01), pages 295 - 304, XP000574822 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2793414A1 (fr) * 1999-05-10 2000-11-17 Centre Nat Rech Scient Conjugue acide nucleique-anticorps pour delivrer un acide nucleique etranger dans les cellules
WO2000067697A3 (fr) * 1999-05-10 2001-06-28 Centre Nat Rech Scient Conjugue acide nucleique-anticorps pour delivrer un acide nucleique etranger dans les cellules
WO2001042478A1 (fr) * 1999-11-25 2001-06-14 Gene Bio-Tech Corporation Limited Procede de lutte contre les infections neoplasiques associees au virus epstein barre (ebv) par vecteurs viraux recombinants, procede de construction et de proliferation et applications connexes
WO2012089338A1 (fr) 2010-12-29 2012-07-05 Curevac Gmbh Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh
WO2012089225A1 (fr) 2010-12-29 2012-07-05 Curevac Gmbh Combinaison de vaccination et d'inhibition de la présentation des antigènes restreinte par le cmh de classe i

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