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EP1364038A2 - Particules d'adenovirus avec proteines de fibres mutagenisees - Google Patents

Particules d'adenovirus avec proteines de fibres mutagenisees

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Publication number
EP1364038A2
EP1364038A2 EP01962700A EP01962700A EP1364038A2 EP 1364038 A2 EP1364038 A2 EP 1364038A2 EP 01962700 A EP01962700 A EP 01962700A EP 01962700 A EP01962700 A EP 01962700A EP 1364038 A2 EP1364038 A2 EP 1364038A2
Authority
EP
European Patent Office
Prior art keywords
adenoviral
fiber
protein
cell
particle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01962700A
Other languages
German (de)
English (en)
Inventor
John Leonard Jakubczak
Michele Lynette Rollence
David A. Stewart
Susan C. Stevenson
Paul L. Hallenbeck
Neeaja Idamkanti
Michael Kaleko
Theodore Smith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Pharma GmbH Austria
Novartis AG
Original Assignee
Novartis Pharma GmbH Austria
Novartis AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Pharma GmbH Austria, Novartis AG filed Critical Novartis Pharma GmbH Austria
Publication of EP1364038A2 publication Critical patent/EP1364038A2/fr
Withdrawn legal-status Critical Current

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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
    • C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
    • C12N2710/10222—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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
    • C12N2710/10241—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10243—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
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C12N2810/405—Vectors comprising RGD peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6009—Vectors comprising as targeting moiety peptide derived from defined protein from viruses dsDNA viruses
    • C12N2810/6018—Adenoviridae

Definitions

  • adenoviral vectors The wide tropism of adenoviral vectors is one of its advantages as a gene-delivery vehicle. However, there are a number of reasons why targeted vectors are desirable. Adenoviral vectors with increased transduction specificity should show reduced toxicity, since lower doses could be delivered to achieve the same desired therapeutic benefit. In addition, these lower doses should reduce potential immune responses to the viruses. This increased safety of targeted vectors would then allow for new routes of delivery, such as systemic administration, that would be applicable to a number of indications, like cancer and cardiovascular disease. Adenoviral particles with mutagenized fiber proteins are useful in the preparation of targeted adenoviruses.
  • Figure 1 shows the strategy used for the production of pseudotyped adenoviral vectors with transiently expressed fiber proteins using the transient transfection/infection system.
  • Figure 1 A shows a schematic diagram of the genomic structure of Ad5. ⁇ gal. ⁇ F.
  • Figure IB shows the transient transfection/infection system.
  • the fiber deleted adenoviral vector, Ad5. ⁇ gal. ⁇ F, as shown in panel A can be grown in packaging cell lines transiently or stably expressing different fiber proteins to generate Ad5. ⁇ gal. ⁇ F/F+ fiber containing adenoviral particles.
  • the vector is used to infect 293T cells that have been transfected with a fiber expression plasmid.
  • the resulting particles will have new receptor tropisms dependent on the fiber protein.
  • FIG 2 shows the differential fiber-dependent adenoviral transduction properties of HeLa cells using pseudotyped adenoviral vectors.
  • HeLa cells were transduced with 1000 total particles per cell with the indicated pseudotyped adenoviral vector. After 24 hours, the cells were analyzed for ⁇ -galactosidase activity using a chemiluminescence reporter assay.
  • the relative ⁇ -galactosidase activity of each pseudotyped adenoviral vector containing a mutated fiber protein was determined and normalized as a percentage of Ad5. ⁇ gal. ⁇ F/wt, which contains a wildtype fiber protein. All values are the mean percentage of Ad5. ⁇ gal. ⁇ F/wt, ⁇ standard deviation (sd) derived from 5 to 6 separate transductions.
  • Figure 3 is a plasmid map for p5FloxPRGD.
  • Figure 4 is a plasmid map for pAvlhlpr.
  • Figure 5 is a plasmid map for pSKO2, containing fiber mutations in combination with a cRGD targeting moiety.
  • Figure 6 shows the transduction efficiency of adenovirus with retargeting ligand and detargeting fiber mutations.
  • HDF Fig. 6A
  • HeLa Fig. 6B
  • CHO-K1 Fig. 6C
  • PC3 Fig. 6D
  • AvlnBg is the parental control with an unmodified fiber gene
  • AvlnBgHIRGD has been genetically altered to include cRGD in the HI loop
  • AvlnBgHIRGDKOl has been genetically altered to include cRGD in the HI loop and the S408E, P409A mutation in fiber knob
  • AvlnBgHIRGDKO2 has been genetically altered to include cRGD in the HI loop and the ⁇ V441, K442 mutation in fiber knob.
  • the cells were analyzed for ⁇ - galactosidase reporter gene activity using a chemiluminescence reporter assay.
  • Figure 7 shows that AvlnBgHIRGDKO2 can compete transduction of HDF cells with
  • AvlGFPHTRGD HDF cells were infected at 1000 particles per cell with AvlGFPHIRGD, an adenoviral vector expressing GFP and which has been genetically altered to include cRGD in the HI loop. The infections were competed with AvlnBg, AvlnBgHIRGD, AvlnBgHIRGDKOl, and AvlnBgfflRGDKO2 at doses ranging from 1000 to 128,000 particles per cell in four-fold dose increments. After 24 hours, the cells were analyzed for GFP expression by measuring the fraction of cells that were positive for GFP expression by FACS analysis. The data was normalized as a percentage of AvlGFPHIRGD without competitor.
  • Figure 8 is a plasmid map for pSKOl, containing fiber mutations in combination with a cRGD targeting moiety.
  • Figure 9 is a plasmid map of pFLAv3nBgKOl containing the full-length adenoviral genome with the KOI fiber AB loop mutation.
  • Figure 10 shows the transduction efficiency of Hela (Fig. 10 A) and HDF (Fig. 10B) cells using adenoviral vectors containing fiber AB loop mutations.
  • Figure 11 shows the transduction efficiency of Hep3B (Fig. 11 A), HepG2 (Fig. 1 IB) and mouse hepatocytes (Fig. 11C) using adenoviral vectors containing fiber AB loop mutations.
  • Figure 12 shows a competition viral transduction assay.
  • Figure 13 shows in vivo adeno viral-mediated expression of ⁇ -galactosidase by an analysis of ⁇ -galactosidase activity in mouse livers.
  • Figure 14 shows in vivo adenoviral-mediated transduction of mouse livers by hexon PCR analysis.
  • Figure 15 shows in vivo adenoviral-mediated expression expression of ⁇ - galactosidase by an analysis of ⁇ -galactosidase activity in C57BL/6, Balb/C, and CD-I mouse livers.
  • Figure 16 shows in vitro adenoviral-mediated transduction of isolated primary CD-I mouse hepatocytes.
  • This invention relates to mutated adenoviral fiber proteins and adenovirus particles containing such proteins. It further relates to polynucleotides encoding the proteins and vectors containing the polynucleotides. It also relates to methods for making and using the adenoviral particles. With the mutated fiber proteins, the adenovirus particles no longer bind to their natural cellular receptor. They can then be "retargeted" to a specific cell type through the addition of a ligand to the virus capsid, which causes the virus to bind to and infect such cell.
  • adenovirus or "adenoviral particle” is used to include any and all viruses that may be categorized as an adenovirus, including any adenovirus that infects a human or an animal, including all groups, subgroups, and serotypes.
  • adenoviruses are ones that infect human cells.
  • Such adenoviruses may be wild-type or may be modified in various ways known in the art or as disclosed herein. Such modifications include modifications to the adenovirus genome that is packaged in the particle in order to make an infectious virus.
  • modifications include deletions known in the art, such as deletions in one or more of the El, E2a, E2b, E3, or E4 coding regions.
  • Such modifications also include deletions of all of the coding regions of the adenoviral genome.
  • adenoviruses are known as "gutless" adenoviruses.
  • the terms also include replication- conditional adenoviruses; that is, viruses that replicate in certain types of cells or tissues but not in other types. These include the viruses disclosed in U.S. Patent No. 5,998,205, issued December 7, 1999 to Hallenbeck et al. and U.S. Patent No. 5,801,029, issued September 1, 1998 to McCormick, the disclosures of both of which are incorporated herein by reference in their entirety.
  • Such viruses are sometimes referred to as cytolytic or cytopathic viruses (or vectors), and, if they have such an effect on neoplastic cells, are referred to as oncolytic viruses (or vectors).
  • the mutated adenoviral fiber protein of the invention is a fiber protein where at least one amino acid in the CD loop of a wild-type fiber protein of an adenovirus from subgroup C, subgroup D, subgroup E, or selected viruses from subgroup F, (in particular those having the long fiber) have been mutated to reduce or substantially eliminate the ability of the fiber protein to bind to the cellular receptor known as the coxsackievirus-adenovirus receptor (CAR) to which the wild-type fiber of these subgroups, as well as subgroup A, bind.
  • CAR coxsackievirus-adenovirus receptor
  • Subgroup A includes adenovirus serotypes 12, 18, and 31.
  • Subgroup C includes adenovirus serotypes 1, 2, 5, and 6.
  • Subgroup D includes adenovirus serotype 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-49.
  • Subgroup E includes adenovirus serotype 4.
  • Subgroup F includes adenovirus serotypes 40 and 41. These latter two serotypes have both a long and a short fiber protein. Only the long fiber protein binds to CAR.
  • the preferred adenovirus serotype of the invention is adenovirus serotype 5.
  • the reduction or elimination of the ability of the mutated adenovirus fiber protein to bind CAR as compared to the corresponding wild-type fiber protein is measured by comparing the transduction efficiency (gene transfer and expression of a marker gene) of an adenovirus particle containing the mutated fiber protein compared to an adenovirus particle containing the wild-type fiber protein for cells having CAR.
  • the term "substantially eliminate” refers to a transduction efficiency less than about 11% of the efficiency of the wild-type fiber containing virus on Hela cells using the transient transfection/infection system described in Example 1.
  • the efficiency is less than about 9%.
  • the efficiency is less than about 8%.
  • the phrase “reduce” or “reduction” refers to a change in the efficiency of transduction by the adenovirus containing the mutated fiber as compared to the adenovirus containing the wild- type fiber to a level of about 75% or less of the wild-type on Hela cells using the transient transfection infection system described in Example 1.
  • the change in efficiency is to a level ofabout 65% or less than wild-type. Most preferably, it is about 55% or less.
  • the fiber proteins of the invention are modified by chemical and biological techniques known to those skilled in the art. Such techniques permit the mutation of at least one amino acid in the CD loop of the wild-type fiber protein to change the ability of the protein to bind to CAR.
  • mutate or “mutation” or similar terms refers to the deletion or change of at least one amino acid in this part of the protein.
  • the amino acid can be changed by substitution or by modification in a way that derivatizes the amino acid.
  • the preferred fiber protein of the invention is a mutated adenovirus serotype 5 fiber protein.
  • the amino acid sequence of the wild-type protein is shown in SEQ ID NO:2.
  • the CD loop in the wild-type adenovirus 5 protein extends from the amino acid at position 441 to the amino acid at position 453.
  • the amino acid at position 441 and/or the amino acid at position 442 of the wild-type fiber protein is mutated.
  • Such mutation may involve a deletion of the amino acid at either or both of positions 441 and 442 (SEQ ID NOS:6, 10, 12, 13). Alternatively, substitution at either or both of these positions may be made.
  • the amino acid at position 441 of the wild-type fiber protein is changed from valine to alanine.
  • the amino acid at position 442 of the wild-type fiber protein is changed from lysine to alanine.
  • the amino acid at position 441 of the wild- type fiber protein is changed from valine to alanine, and the amino acid at position 442 of the wild-type fiber protein is changed from lysine to alanine (SEQ ID NO: 14).
  • the present inventors have also discovered that certain mutations in other parts of the wild-type adenovirus 5 fiber protein reduce or substantially eliminate the ability of an adenoviral particle with the mutated fiber to bind to CAR.
  • the mutations are at one or more of amino acid positions 408, 409, 460, 509, 510, 538, and 539 of the wild-type protein.
  • the fiber protein is mutated at amino acid positions 408 and 409, preferably by substituting glutamic acid for serine at position 408 and substituting alanine for proline at position 409 (SEQ ID NO:4).
  • the fiber protein is mutated at amino acid position 460 of the wild- type fiber protein, most preferably by substituting glutamic acid for arginine (SEQ ID NO: 16). In another preferred embodiment, the fiber protein is mutated at at least one of amino acid positions 509 and 510 of the wild-type fiber protein, preferably by deleting the amino acids at both positions (SEQ ID NO: 18). In another preferred embodiment, the fiber protein is mutated at at least one of amino acid positions 538 and 539 of the wild-type fiber protein, preferably by deleting the amino acids at both positions (SEQ ID NO:20). Any or all of these mutations may be combined with mutations in the CD loop of adenovirus 5.
  • the mutated fiber protein of the invention comprises at least one mutation at amino acid positions 441 and 442 of the wild-type fiber protein plus a mutation at one or more of amino acid positions 408, 409, 460, 509, 510, 538, and 539 of the wild-type fiber protein.
  • SEQ ID NO:8 amino acid positions 408, 409, 460, 509, 510, 538, and 539 of the wild-type fiber protein.
  • the mutated adenoviral fiber protein of the invention is a fiber protein where at least one amino acid in the AB loop of a wild-type fiber protein of an adenovirus from subgroup C, subgroup D, subgroup E, or selected viruses from subgroup F (in particular those having a long fiber), have been mutated to reduce or substantially eliminate the ability of the fiber protein to bind to CAR.
  • the preferred fiber protein of the invention is a mutated adenovirus serotype 5 fiber protein.
  • the mutated adenovirus serotype 5 fiber protein contains mutations at amino acid positions 408 and/or 409 of the wild-type fiber protein.
  • the mutations are at both positions.
  • such mutations may be deletions, substitutions, or a modification in a way that derivitizes the amino acid.
  • the same type of mutation need not be made at each position.
  • glutamic acid is substituted for serine at position 408.
  • alanine is substituted for proline at position 409.
  • glutamic acid is substituted for serine at position 408, and alanine is substituted for proline at position 409 (SEQ ID NO:4).
  • the invention also comprises polynucleotides that encode the proteins of the invention.
  • polynucleotide means a nucleic acid molecule, such as DNA or RNA, that encodes a polynucleotide.
  • the molecule may include regulatory sequences.
  • the polynucleotide is DNA.
  • Such polynucleotides are prepared or obtained by techniques known by those skilled in the art in combination with the teachings contained therein. Examples of such polynucleotides are shown in SEQ TD NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19.
  • the polynucleotides of the invention also include polynucleotides that differ in certain bases but still encode the proteins of the invention due to the redundancy of the genetic code.
  • the invention further comprises vectors including the polynucleotides of the invention. Such vectors include partial or complete adenoviral genomes and plasmids. Such vectors are constructed by techniques known to those skilled in the art.
  • the packaging cells of the invention are cells that provide complementing functions to the functions provided by the genes in the adenovirus genome that are to be packaged into the adenovirus particle.
  • the production of such particles require that the genome be replicated and that those proteins necessary for assembling an infectious virus be produced.
  • the particles may also require certain proteins necessary for the maturation of the viral particle.
  • Such proteins may be provided by the vector or by the packaging cell.
  • the packaging cells of the invention may contain the polynucleotide encoding the mutated fiber protein.
  • Such polynucleotide may be transfected into the cell, preferably as part of a plasmid, or it may be infected into the cell with a viral vector. It may be stably incorporated into the genome of the cell, thus providing for a stable cell line. Alternatively, it may be unincorporated into the genome, in which case a transient complementing cell will be provided.
  • the adenovirus genome to be packaged is transferred into the complementing cell by techniques known to those skilled in the art. These techniques include transfection or infection with another virus.
  • the polynucleotide encoding the mutated fiber protein may be in this genome instead of in the packaging cell.
  • the packaging cell may also encode a fiber protein.
  • Such protein may assist in the maturation and packaging of an infectious particle.
  • Such protein may be a wild-type fiber protein or one modified so as to be unable to attach to the penton base protein.
  • the packaging cells are cultured under conditions that permit the production of the desired viral particle.
  • the viral particles are recovered by standard techniques.
  • a preferred way of making the adenoviral particles of the invention is as follows.
  • the polynucleotide encoding the mutated fiber protein is made using standard techniques in an adenoviral shuttle plasmid.
  • This plasmid contains the right end of the virus, in particular from the end of the E3 region through the right ITR. It also contains a recombinase site, such as a lox site.
  • This plasmid is co-transfected into a complementing cell line along with a helper plasmid, which contains the remaining portion of the adenovirus genome, except for the El region and sometimes also the E2a region.
  • a third plasmid which is an expression plasmid containing a gene encoding a recombinase such as Cre, is also transfected into the complementing cell.
  • the complementing cell is preferably a 293 cell, which contains the adenoviral El genes, or an AEl-2a cell ⁇ Gorziglia, Kadan, et al. 1996 ⁇ , which contains the adenoviral El and E2a genes. Most preferably, the complementing cell is a 633 cell ⁇ Von
  • Seggern, Huang, et al. 2000 ⁇ which stably expresses the adenovirus serotype 5 wild-type fiber protein, and was derived from the AEl-2a cell line.
  • the transfected complementing cells are maintained under standard cell culture conditions.
  • the adenoviral plasmids recombine to form the adenoviral genome that is packaged.
  • the particles are infectious, but replication deficient because their genome is missing at least the El genes.
  • the particles contain both wild-type and mutated fiber proteins. They are recovered from the crude viral lysate and are purified by standard techniques.
  • the recovered particles are preferably used to infect 293 or AEl-2a cells. This permits the recovery of particles whose capsids contain only the desired mutated fiber. This two-step procedure provides high titer batches of the adenoviral particles of the invention.
  • the adenoviral particles may be replication competent or replication incompetent.
  • the particles selectively replicate in certain predetermined target tissue but are replication incompetent in other cells and tissues.
  • the adenoviral particles replicate in abnormally proliferating tissue, such as solid tumors and other neoplasms.
  • Such replication conditional adenoviral particles and vectors may be produced by techniques known to those skilled in the art, such as those disclosed in the above-referenced U.S. Patent Nos. 5,998,205 and 5,801,029. These particles and vectors may be produced in adenoviral packaging cells as disclosed above.
  • the preferred packaging cells are those that have been designed to limit homologous recombination that could lead to wild-type adenoviral particles. Such cells are disclosed in U.S. Patent Nos. 5,994,128, issued November 30, 1999 to Fallaux, et al., and 6,033,908, issued March 7, 2000 to Bout, et al.
  • the modified fiber polynucleotide also includes sequences that encode a targeting ligand. Accordingly, such sequences are transfected into the complementing cell by the shuttle plasmid.
  • the targeting ligand sequences may be included in the penton or hexon proteins. In such cases, they would be in the helper plasmid.
  • the packaged adenoviral genome may also contain a heterologous polynucleotide.
  • This polynucleotide is usually included in the deleted El region of the helper plasmid.
  • the polynucleotide may be in the E3 region, in which case it is included in the shuttle plasmid.
  • the adenovirus particles of the invention include the mutated fiber proteins. Such particles may include different types of the mutated fibers of the invention. They may also include wild-type fibers along with the mutated fibers.
  • the adenoviral particles of the invention preferably further comprise a targeting ligand included in a capsid protein of the particle.
  • the ligand may be included in any of the capsid proteins, such as fiber, hexon, or penton.
  • the ligand is included in a fiber protein, which is preferably a mutated fiber protein of the invention.
  • the targeting ligand is included within the HI loop of the fiber protein. Any ligand that can fit in the HI loop and still provide a functional virus may be used. Such ligands may be as long as 80-90 amino acids.
  • Such ligands are added by techniques known in the art, such as those disclosed in PCT application PCT/US99/02549, published as WO99/39734 on August 12, 1999, and Example 12 of U.S. patent application number 09/482,682, filed January 14, 2000, the disclosures of both of which are hereby incorporated herein by reference.
  • a targeting ligand may be any chemical moiety that preferentially directs the adenoviral particle to a desired cell type.
  • the categories of such ligands include peptides, polypeptides, single chain antibodies, and multimeric proteins.
  • Specific ligands include the TNF superfamily of ligands which include tumor necrosis factors (or TNF's) such as, for example, TNF-alpha and TNF-beta, lymphotoxins (LT), such as LT- ⁇ and LT- ⁇ , Fas ligand which binds to Fas antigen; CD40 ligand, which binds to the CD40 receptor of B- lymphocytes; CD30 ligand, which binds to the CD30 receptor of neoplastic cells of Hodgkin's lymphoma; CD27 ligand, NGF ligand, and OX-40 ligand; transferrin, which binds to the transferrin receptor located on tumor cells, activated T-cells, and neural tissue cells; Apo
  • Cyclic RGD (cRGD) is preferred.
  • cyclic RGD (or cRGD) refers to any amino acid that binds to ⁇ integrins on the surface of cells and contains the sequence RGD (Arg- Gly-Asp). The sequence in SEQ ID NO:43 is particularly preferred.
  • the ligand also has a trimeric structure.
  • the ligand is selected from the TNF superfamily of ligands hereinabove described. Such ligands are trimeric and of similar size to the fiber head domain. Such ligands may be incorporated into the fiber protein using the techniques disclosed in U.S. Patent No. 5,756,086, issued May 26, 1998 to McClelland et al., the disclosure of which is incorporated herein by reference.
  • the adenovirus particles may further include at least one heterologous polynucleotide.
  • heterologous polynucleotide means a polynucleotide derived from a biological source other than an adenovirus, which encodes a polypeptide when the adenovirus infects a cell. Such polynucleotides are included in the adenoviral genome within the particle and are added to that genome by techniques known in the art. Any heterologous polynucleotide of interest may be added.
  • a preferred polynucleotide is one that encodes an immunostimulating protein, such as an interleukin, interferon, or colony stimulating factor.
  • Mammalian GM-CSF is preferred.
  • GM-CSF is a primate GM-CSF; most preferably, it is human GM-CSF.
  • An alternative preferred polynucleotide encodes herpes simplex virus thymidine kinase (HSV-TK), which is useful as a safety switch as described in U.S. Patent Application No. 08/974,391, filed November 19, 1997, which published as PCT Publication No. WO/9925860, the disclosure of which is incorporated herein by reference.
  • HSV-TK herpes simplex virus thymidine kinase
  • the adenoviral particles of the invention are used to genetically engineer a cell to express a protein that it otherwise does not express or does not express in sufficient quantities. This is accomplished by infecting the desired cell with an adenoviral particle of the invention whose genome includes a desired heterologous polynucleotide. This permits the expression of the heterologous polynucleotide in the cell.
  • the cell is a mammalian cell. More preferably, the mammalian cell is a primate cell. Most preferably, the primate cell is a human cell. The cell may be inside the body of the animal (in vivo) or outside the body (in vitro).
  • the adenoviral particle includes a targeting ligand as described above.
  • a targeting ligand as described above.
  • the adenoviruses of the invention can be used to study cell transduction and gene expression in vitro or in various animal models. The latter case includes ex vivo techniques, in which cells are transduced in vitro and then administered to the animal. They may also be used to conduct gene therapy on humans or other animals. Such gene therapy may be ex vivo or in vivo.
  • the adenoviral particles of the invention in a pharmaceutically-acceptable carrier are delivered to a human in a therapeutically effective amount in order to prevent, treat, or ameliorate a disease or other medical condition in the human through the introduction of a heterologous gene that encodes a therapeutic protein into cells in such human.
  • the adenoviruses are delivered at a dose ranging from approximately 1 particle per kilogram of body weight to approximately 10 14 particles per kilogram of body weight. Preferably, they are delivered at a dose of approximately 10 6 particles per kilogram of body weight to approximately 10 13 particles per kilogram of body weight. Most preferably, the dose ranges from approximately 10 9 particles per kilogram of body weight to approximately 10 particles per kilogram of body weight.
  • adenoviral particles of the invention with the above-identified modifications in the AB loop, particularly those with modifications at amino acid positions 408 and 409 of the wild-type adenovirus serotype 5 fiber protein, and most particularly those where glutamic acid is substituted for serine at position 408 and alanine is substituted for proline at position 409 (SEQ DD NO:4), have additional desirable utilities.
  • the inventors have unexpectedly discovered that such viral particles provide enhanced gene transfer to and expression in hepatocytes in the liver of an animal as compared to adenoviral particles with the wild-type fiber protein.
  • the invention includes a method of enhancing adenoviral-mediated gene transfer to and expression in cells in the liver of an animal by administering adenoviruses having such AB loop modification in at least one of their fiber proteins to an animal under conditions where cells in the liver are transduced.
  • the hepatocytes are the cells that are primarily transduced.
  • an adenovirus particle comprising a mutated adenovirus serotype 5 fiber protein, wherein glutamic acid is substituted for serine at amino acid position 408 and alanine is substituted for proline at amino acid position 409 (SEQ ID NO:4), is used to deliver the heterologous gene.
  • Such adenoviral particles would be particularly useful for gene therapy where it is desired to express a heterologous gene in a patient's liver. This could be used, for example, in the treatment of diabetes, hemophilia, and diseases related to increased cholesterol or triglyceride blood levels in a patient such as atherosclerosis. It would also include anti- angiogenesis treatment methods involving the delivery of one or more anti-angiogenic genes to the hepatocytes of a patient's liver.
  • the dose for these types of particles would be approximately 1 particle per kilogram of body weight to approximately 10 13 particles per kilogram of body weight. Preferably, the dose would be approximately 10 5 particles per
  • the dose ranges from approximately 10 8 particles per kilogram of body weight to approximately 10 n particles per kilogram of body weight.
  • Such particles are delivered by routes of administration known to those skilled in the art.
  • One such route is intravenous injection.
  • An alternative route is intraparenchymal injection.
  • the particles may also be delivered by injection into the hepatic artery, portal vein, or bile duct.
  • Example 1 Adenovirus Type 5 Viral Particles Pseudotyped With Mutagenized Fiber Proteins Show Diminished
  • Ad5 human adenovirus type 5
  • CAR coxsackie adenovirus receptor
  • Fiber is a homotrimeric protein present twelve times on the viral capsid. It has three domains: an N-terminal tail that interacts with the penton base in the viral capsid, a rod-like shaft containing 22 copies of a 15 amino-acid beta sheet structure, and a globular knob domain. It is the knob domain that mediates binding to CAR during cell attachment.
  • the fiber-deleted Ad vector is Ad5. ⁇ gal. ⁇ F, which is an El- E3- and fiber- gene deleted adenovirus that expresses cytoplasmic ⁇ -galactosidase under the control of the SV40 promoter ⁇ Von Seggern, Chiu, et al. 1999 ⁇ (Fig. 1A).
  • the modified fiber proteins for pseudotyping are produced from expression plasmid constructs designed for high levels of fiber protein expression ⁇ Von Seggern, Huang, et al. 2000 ⁇ .
  • the primary advantage of this system is that modified fiber proteins can be quickly incorporated into virions and functionally analyzed in their most relevant context for their effect on CAR interaction and subsequent gene transfer and expression.
  • pDV60 contains the CMV promoter, the first Ad5 tripartite leader exon (TPL), the natural first intron and the fused second and third TPL exons upstream of the Ad5 fiber gene. All amino acid changes were incorporated into the fiber cDNA using the pDV60 plasmid as the template. Individual amino acid residues in pDV60 were mutagenized using the QuickChange Site-Directed Mutagenesis system (Stratagene, La Jolla CA).
  • the oligonucleotide primers used for the incorporation of amino acid changes are listed in Table 1 for each single or double amino acid modification.
  • the thermal cycler protocol was 95°C for 30 sec, followed by 18 cycles of 95°C for 30 sec, 55°C for 1 min, and 68°C for 20 min.
  • the entire knob domain of the Ad5 fiber was deleted from amino acids 404 to 581.
  • a 31 amino acid peptide derived from the GCN4 leucine zipper ⁇ Harbury, Zhang, et al. 1993 ⁇ was fused immediately after the fiber TLWT sequence at the fiber shaft-head junction using PCR gene overlap extension ⁇ Horton, Cai, et al. 1990 ⁇ .
  • This reaction fused the Ad5 fiber tail and shaft regions (amino acids 1 to 403) to the GCN4 isoleucine 31 amino acid peptide to form the KOI 1 mutant and was cloned into pDV60 to create pDKOll.
  • the pDV55 control plasmid is similar to pDV60, except that it lacks the fiber gene ⁇ Von Seggern, Huang, et al. 2000 ⁇ .
  • Ad5. ⁇ gal.wt is a first generation E1-, E3-deleted adenovirus containing a lacZ reporter cassette in the El region ⁇ Von Seggern, Chiu, et al. 1999 ⁇ .
  • Ad5. ⁇ gal. ⁇ F is identical to Ad5. ⁇ gal.wt except that the fiber gene is deleted ⁇ Von Seggern, Chiu, et al. 1999 ⁇ .
  • Human 293T cells were obtained from ATCC (CRL 11268) and were cultured in the DMEM containing 10% FBS. The 293T cells stably express the SV40 large T antigen that allows for the amplification of plasmids from the SV40 origin of replication.
  • the 633 cells stably express the Ad5 fiber protein ⁇ Von Seggern, Huang, et al. 2000 ⁇ and are derived from AEl-2a, a cell line that complements El a- and E2a-deleted adenoviral vectors ⁇ Gorziglia, Kadan, et al. 1996 ⁇ .
  • 633 cells were grown in Richter's CM (Life Technologies #C-2671) and 10% FBS.
  • Hela cells (ATCC CCL-2) were grown in Dulbecco's modified Eagle's media supplemented with 10% FBS.
  • AEl-2a cells also known as S8 cells
  • 633 cells see Example 6F and 6G of U.S. Patent Application number 09/482,682, filed January 14, 2000, which disclosure is incorporated herein by reference.
  • the transfected 293T cells were then infected with Ad5. ⁇ Gal. ⁇ F/F + virus at a particle per cell ratio of 350.
  • the AdS. ⁇ GaL ⁇ F F* virus is an El, E3, fiber-deleted Ad5 vector ⁇ Von Seggern, Chiu, et al. 1999 ⁇ that was propagated in the fiber-complementing cell line, 633, such that the capsid contains wildtype Ad5 fiber protein ⁇ Von Seggern, Huang, et al. 2000 ⁇ .
  • the growth media was aspirated and 2.5ml of infection media (DMEM and 2% FBS) containing Ad5. ⁇ Gal. ⁇ F/F + was added and slowly rocked at 37°C, 5% CO 2 for 2 hours.
  • CPE cytopathic effect
  • the transfected/infected 293T cells were harvested after complete CPE by gently dislodging the cells, pelleting by centrifugation, and resuspending in 1ml phosphate buffered saline.
  • a crude viral lysate (CVL) was prepared by five freeze-thaw cycles to disrupt the cells and release the virus.
  • the virus was purified by CsCl gradient centrifugation using standard procedures.
  • the virus particle titer was determined spectrophotometrically as described ⁇ Mittereder, March, et al. 1996 ⁇ . Yields of Ad5. ⁇ Gal. ⁇ F virus pseudotyped with
  • 1 1 19 modified fiber protein typically ranged from 10 to 10 particles.
  • Western immunoblot analysis The expression and incorporation of each fiber protein onto adenoviral particles was verified by denaturing sodium dodecyl-sulfate (SDS) 4 to 12% polyacrylamide gel electrophoresis (PAGE) and Western immunoblot analysis. An aliquot of each adenoviral vector preparation corresponding to 5.0xl0 9 particles per lane was analyzed. The proteins were transferred to a nitrocellulose membrane with a minitransblot apparatus (Novex Inc.) for 90 minutes at 30V.
  • SDS sodium dodecyl-sulfate
  • PAGE polyacrylamide gel electrophoresis
  • the membrane was blocked for at least 1 hour at room temperature in lO M Tris, ⁇ H7.4 containing 150mM NaCl, 2mM EDTA, 0.04% Tween-20, and 5% dried milk.
  • the blocked membrane was incubated for 1 hour with a 1:1000 dilution of a primary rabbit anti-Ad5 fiber polyclonal antiserum.
  • the membrane was then developed with a 1:5000 dilution of the secondary donkey anti-rabbit IgG horseradish peroxidase- conjugated antibody (Amersham Lifesciences, Arlington Heights, IL) using an enhanced chemiluminescense system (Amersham Lifesciences).
  • the membrane was exposed to film for approximately 1 to 20 seconds.
  • the membrane was then used to reprobe for detection of the adenoviral penton protein to ensure equivalent loading of viral particles. Briefly, the membrane was incubated for 1 hour with a 1:5000 dilution of the primary rabbit anti-Ad5 penton polyclonal antiserum. The membrane was then re-developed with a 1:5000 dilution of the secondary goat anti-rabbit IgG horseradish peroxidase-conjugated antibody as described above. Production of anti-Ad5 fiber and anti-Ad5 penton-specific antiserum.
  • Both of the rabbit primary antibodies used in the anti-fiber and anti-penton Western immunoblot analysis were generated by immunizations of New Zealand White rabbits (Loftstrand Labs, Ltd., Gaithersburg, MD).
  • the Ad5 fiber and penton proteins were expressed using the baculoviral expression system.
  • the purified Ad5 fiber protein and partially purified penton base proteins were used for immunizations according to standard protocols.
  • the antiserum obtained was tested for immunoreactivity against the Ad5 fiber and penton proteins by Western immunoblot analysis.
  • Adenoviral transduction Hela cells were infected with the adenoviral vectors containing mutated fiber proteins to evaluate the effects of fiber amino acid mutations on CAR interaction and subsequent gene expression. Monolayers of HeLa cells in 12 well dishes were infected with 1000 particles per cell for 2 hours at 37°C in a total volume of 0.35 ml of the DMEM containing 2% FBS. The infection medium was then aspirated from the monolayers and 1ml of complete DMEM containing 10% FBS was added per well. The cells were incubated for an additional 24 hours to allow for ⁇ -galactosidase expression. ⁇ -galactosidase expression analysis.
  • ⁇ -galactosidase encoded by the adenoviral vectors in the infected cells was measured by a chemiluminescence reporter assay and by histochemical staining with a chromogenic substrate.
  • the relative levels of ⁇ - galactosidase activity were determined using the Galacto-Light chemiluminescence reporter assay system (Tropix, Bedford, MA). Briefly, the cell monolayers were washed with PBS and processed according to the manufacturer's protocol. The cell homogenate was transferred to a microfuge tube and centrifuged to remove cellular debris.
  • Total protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Pierce, Inc., Rockford, IL) with bovine serum albumin as the assay standard. An aliquot of each sample was then incubated with the Tropix ⁇ -galactosidase substrate for 45 minutes in a 96 well plate. A luminometer was used to determine the relative light units (RLU) emitted per sample and then normalized for the amount of total protein in each sample (RLU/ug total protein).
  • BCA bicinchoninic acid
  • RLU relative light units
  • the cell monolayers were fixed with 0.5% glutaraldehyde in PBS, and then were incubated with a mixture of 1 mg of 5-bromo-4-chloro- 3-indolyl- ⁇ -D-galactoside (X-gal) per ml, 5mM potassium ferrocyanide, 5mM potassium ferricyanide and 2 mM MgCl 2 in 0.5 ml of PBS.
  • the monolayers were washed with PBS and the blue cells were visualized by light microscopy with a Zeiss JD03 microscope.
  • Transient transfection/infection system To rapidly evaluate a panel of potential CAR- binding fiber mutants in the context of viral particles, we have developed a transient transfection/infection system.
  • This system which is based on pseudotyping a fiberless virus with the mutant fiber proteins, consists of two components. The first is an El, E3, fiber- deleted adenovirus, Ad5. ⁇ gal. ⁇ F ⁇ Von Seggern, Chiu, et al. 1999 ⁇ (Fig. 1A).
  • This virus when grown on a non fiber-complementing cell line such as 293T, yields viral particles lacking fiber protein.
  • these fiberless virions are designated Ad5. ⁇ gal. ⁇ F/F " .
  • Ad5. ⁇ gal. ⁇ F is produced on the fiber-complementing cell line 633 ⁇ Von Seggern, Huang, et al. 2000 ⁇
  • the virions contain a full complement of wildtype fiber protein on the surface and is referred to as Ad5. ⁇ gal. ⁇ F/F*.
  • the second component of the system is an expression plasmid that supplies fiber protein to the assembling virus in trans. This plasmid, pDV60, is designed to express high levels of fiber protein ⁇ Von Seggern, Huang, et al. 2000 ⁇ .
  • the transient transfection/infection system is shown schematically in Fig. IB.
  • Transfection of 293T cells by the pDV60-based fiber-expression plasmid results in high levels of fiber production in the cells.
  • the cells are infected with Ad5. ⁇ gal. ⁇ F F + that has been previously pseudotyped with wildtype fiber by growth in 633 cells.
  • the infected cells are collected and viral particles, now pseudotyped with the fiber protein supplied in trans by the fiber-expression plasmid, are purified. In this way, any plasmid-encoded fiber proteins that are capable of trimerization and incorporating into the viral particles will complement Ad5. ⁇ gal. ⁇ F.
  • Ad5. ⁇ gal. ⁇ F that is pseudotyped either by growth in 633 cells or by transient transfection with a fiber expression plasmid is designated AdS. ⁇ gal. ⁇ F/F 1" .
  • the function of these modified fiber proteins in the context of a viral particle can then be tested for their ability to mediate fiber-dependent Ad infection and gene transfer.
  • the level of pDV60-encoded fiber protein incorporated into the AdS. ⁇ gal. ⁇ F/F 1' pseudotyped virions using the transient transfection/infection system was equivalent to the level of fiber protein in the Ad5. ⁇ gal.wt particles.
  • the equivalent loading of viral particles was demonstrated by detection of the 68kDa penton monomer for each vector.
  • Fiber mutation analysis The transient transfection/infection system was then used to evaluate a series of mutations in the fiber knob for their effect on CAR-mediated gene transfer of Ad5 particles.
  • a panel of expression plasmids encoding fourteen mutant fiber proteins was constructed (Table 2).
  • the wildtype fiber (pDV60) and a null construct (pDV55) were used ⁇ Von Seggern, Huang, et al. 2000 ⁇ .
  • These plasmids were transfected into 293T cells, followed by infection with AdS. ⁇ gal. ⁇ F/F*.
  • the resulting virions obtained from this procedure were thus pseudotyped with the plasmid-encoded fibers.
  • each fiber protein into the adenoviral capsid was examined by Western immunoblot analysis of the CsCl-purified virus stocks.
  • the relative levels of fiber protein on the capsid were compared with the amount of penton protein to control for equal loading of viral particles in each lane.
  • the fiber proteins encoded by most mutants were sufficiently expressed, trimerized and incorporated into the Ad5. ⁇ gal. ⁇ F viral particles and the 62kDa fiber monomer was detected in this analysis.
  • Analysis of the KO11 mutant displayed the expected protein of approximately 48kDa although this truncated fiber protein was not incorporated to the same level as wild-type fiber.
  • mutants KO2 SEQ ID NO:6
  • KO1+2 SEQ ID NO:8
  • KO2a SEQ ID NO: 10
  • KO11 showed lower levels of incorporated fiber protein although KO11 may have a reduced immunoreactivity with an antiserum that was generated against the full-length wildtype Ad5 fiber protein.
  • Analysis of the relative expression level and trimerization ability of these mutants on non-denaturing polyacrylamide gels showed lower levels of fiber monomer and trimer, indicative of deficiencies in the steady-state levels of these mutant proteins.
  • the KO2 (SEQ ID NO:6) mutation resulted in an approximately 167-fold reduction with 0.6% wt activity.
  • the KO2a (SEQ ID NO: 10), KO2b (SEQ ID NO: 12), and KO2c (SEQ TD NO: 14) constructs were designed to identify the amino acid mutation responsible for the significant reduction in CAR interaction. In this comparison, it was revealed that the deletion of amino acid V441 reduced CAR interaction greatest as this single deletion in KO2a (SEQ ID NO: 10) resulted in the 167-fold reduction of wt activity and the deletion of K442 had no further effect.
  • the most potent mutation was found with combining the KOI (SEQ ID NO:4) and KO2 (SEQ ID NO:6) mutations in the KO1+2 (SEQ ID NO: 8) construct as this combination resulted in a 1000-fold reduction with only 0.1% wt levels found.
  • a dramatic reduction in transduction efficiency was also found for the KO11 mutant with a 125-fold reduction of ⁇ -galactosidase activity when the entire knob domain was deleted.
  • Significant decreases were also observed for KO3 (SEQ ID NO: 16), KO4 (SEQ ID NO: 18), and KO5 (SEQ ID NO:20) although not as dramatic.
  • mutants KOI SEQ ID NO:4
  • KO2a SEQ ID NO: 10
  • KO2 SEQ TD NO:6
  • KO2c SEQ ID NO: 14
  • KO4 showed an intermediate reduction in ⁇ -galactosidase activity and in the number of X- gal stained positive cells while KO10 had little effect on transduction efficiency by either measure (Fig. 2).
  • One approach to selective cell transduction is to manipulate the fiber protein to redirect the receptor specificity to a particular cell type.
  • All mutants that incorporated amino acid changes within this region displayed a reduction in fiber-mediated gene transfer including KO1+2 (SEQ TD NO:8), KO2 (SEQ ID NO:6), KO2a (SEQ ID NO: 10), KOI (SEQ ID NO:4), KO2b (SEQ ID NO: 12) and KO2c (SEQ ID NO: 14) that resulted in a 1000 to 12-fold reduction.
  • the KOI +2 mutation containing a two amino acid substitution in the A:B loop and a two amino acid deletion in the C:D loop demonstrated the most potent reduction in gene transfer which is greater than either mutation alone.
  • KO2 ( ⁇ V441,K442) (SEQ ID NO:6) and KO2a ( ⁇ V441) (SEQ ID NO:10) mutants showed a significant decrease in transduction efficiency, greater than 160-fold. A portion of this reduction is undoubtedly due to the lower levels of the mutant fiber protein on the viral capsid. However, a significant reduction in transduction efficiency has also been observed for a virus that has the identical KO2 mutation introduced genetically into the viral genome. This virus has a full complement of the mutant fiber protein on the capsid and still shows a dramatic reduction in transduction efficiency in all cell types tested.
  • the second requirement for an adenovirus that transduces in a cell-type specific manner is the introduction of a novel tropism.
  • the most efficient means is by genetic modification of the fiber gene.
  • Krasnykh et al. ⁇ Krasnykh, Dmitriev, et al. 1998 ⁇ have shown that the HI loop is an appropriate location in the fiber protein to insert peptides with novel receptor specificities.
  • the cRGD ligand (SEQ ID NO:43) ⁇ Pasqualini, Koivunen, et al. 1995 ⁇ inserted into the HI loop has been shown to expand the tropism of Ad both in vitro ⁇ Dmitriev, Krasnykh, et al.
  • Seq. ID 24 GATATTGGAGCCAAACTGCCGGCCAAAACTGAAACTGT-3' pDK02a Seq. ID 25 5 ACAGTTTCAGTTrTGGCTAAAGGCAGTTTGGCTCCA-3 , ⁇ V441
  • Seq. ID 26 5'-TGGAGCCAAACTGCCT ⁇ AGCCAAAACTGAAACTGT-3' pDK ⁇ 2b Seq. ID 27 S'-GTTTCAGTTTTGGCTGTTGGCAGTTTGGCTCCAATA-S' ⁇ K442
  • Seq. ID 30 5'-TGGAGCCAAACTGCCTGCAGCAGCCAAAACTGAAAC-3' pDK03 Seq. ID 31 5'-GCTCATCTTATTATAGAATTCGACGAAAATGGAGTG-3 ' R460E
  • Seq. ID 32 5'-CACTCCAR ⁇ TCGTCGAATTCTATAATAAGATGAGC-3* pDK04 Seq. ID 33 5'-GCTTATCCAAAATCTCACACTGCCAAAAGTAACATTGTC-3 • ⁇ G509, K510
  • Seq. ID 42 5'-TT ⁇ ACCGTGAGAT-T ⁇ GCATAAGCTGATAGGT-3 , tnumbering of amino acid residues as in Xia et al 1994.
  • Fiber Fiber mutant Representative avg Mean % wt ( ⁇ expression Dgal activity SD)** plasmid Designation Mutation* (RLU/Og protein) pDV60 Wildtype (wt) None 529882.0 100.0 ( ⁇ 0.9) pDV55 F Null 266.3 0.1 ( ⁇ 0.1)* pDKOl KOI S408E. P409A 7618.0 1.4 ( ⁇ 0.8)* pDKOl+2 KOl+2 S408E, P409A, ⁇ V441, K442 472.3 0.1 ( ⁇ 0.0)* pDK02 K02 ⁇ V441.
  • RLU relative light units. Value represents the average of three wells. numbering of fiber amino acid residues as in Xia et al. 1994.
  • **% wildtype represents the mean ( ⁇ SD) of the ⁇ gal activity of Ad5. ⁇ gal. ⁇ F pseudotyped with each corresponding fiber mutant in 5-6 separate transductions. All values were normalized to wildtype (pDV60) at 100%.
  • Plasmid Description The following three plasmids were used to rescue infectious adenoviruses containing modified fibers.
  • p5FloxPRGD is a shuttle plasmid used to incorporate modifed fibers into the Ad genome ( Figure 3). It consists of the final 6kb of DNA from the right end of an Avl genome. A lox site was inserted upstream of the fiber gene.
  • the Ad5 packaging signal has been inserted near the right inverted terminal repeat (PJTR).
  • pAvlhlpr is a helper plasmid that consists of an entire Avl genome with the exception of the right ITR ( Figure 4).
  • pCre is a plasmid that constitutively expresses the Cre recombinase. It consists of the Cre gene cloned into the expression plasmid pcDNAl.lzeo + (Invitrogen). None of these plasmids are capable of producing infectious Ad on their own.
  • the Cre protein mediates recombination between the lox sites in p5FloxPRGD and pAvlhlpr, reconstituting a full length Avl genome, which is then capable of producing infectious virus.
  • KO2 fiber mutations All amino acid changes were incorporated into the fiber gene using the p5FloxPRGD adenoviral shuttle plasmid as the template.
  • This shuttle plasmid encodes a fiber that contains a cRGD peptide sequence, HCDCRGDCFC (SEQ ID NO:43), inserted in the HI loop.
  • the cRGD peptide has been shown to bind to ovintegrins on the cell surface.
  • Amino acid residues V441 and K442 in the CD loop of the fiber gene were deleted using the QuickChange Site-Directed Mutagenesis system (Stratagene, La Jolla CA).
  • adenoviral vectors with CD loop mutations The mutagenized fiber gene was incorporated into the adenoviral DNA backbone by cre-lox recombination. To do this, the pSKO2 shuttle plasmid and the pAvlhlpr helper plasmid were cotransfected with pCre into 633 cells, a cell line that expressed wildtype fiber ⁇ Von Seggern, Huang, et al. 2000 ⁇ . Expression of the Cre recombinase from pCre mediates recombination between lox sites in pSKO2 and pAvlhlpr, resulting in full length Avl viral DNA, with nuclear ⁇ -Gal transgene in the El region.
  • this viral DNA is capable of being packaged into infectious viral particles containing a mixture of wildtype fiber and mutant fibers.
  • the virus was purified by standard CsCl centrifugation procedures.
  • This virus was designated AvlnBgHTRGDKO2(633).
  • this viral preparation was used to infect AEl-2a cells, which do not express fiber ⁇ Gorziglia, Kadan, et al. 1996 ⁇ . Viral particles were purified as above. This virus was designated AvlnBgHTRGDKO2.
  • Comparable fiber incorporation in AylnBgHIRGDKO2 To ensure that the levels of the mutant fiber on the AvlnBgHTRGDKO2 viral particles were normal relative to viruses with wildtype fiber levels, Western blot analysis was performed. Equivalent amounts of AvlnBg and AvlnBgHIRGDKO2 were subjected to SDS-PAGE. This gel was transferred to a membrane and incubated with rabbit anti-Ad5 fiber and rabbit anti-Ad5 penton polyclonal antisera. The fiber penton ratio on AvlnBgHIRGDKO2 viral particles is indistinguishable from that of AvlnBg, demonstrating that there was no effect of the fiber mutations on the level of fiber protein assembled on the viral capsid.
  • AylnBgHTRGDKO2 As shown previously in Example 1, adenoviruses pseudotyped with fiber proteins containing deletions of V441 and K442 in the CD loop are severely affected in their ability mediate gene transfer in the CAR-expressing cell line Hela. In order to test the idea that gene transfer by these mutant viruses can be mediated by alternative ligand/receptor interactions, we tested the ability of the AvlnBgHIRGDKO2 virus to transduce HDF, Hela, PC3 and CHO-K1 cell lines (Fig 6). All of these cells lines express ⁇ v integrins on the cell surface and, with the exception of Hela, show poor transduction by adenovirus due to a known or presumed deficiency in CAR levels.
  • AvlnBgHTRGDKO2 This reduction in the inability of AvlnBgHTRGDKO2 to transduce cells through ovintegrins is not due to a defect in the RGD targeting moiety.
  • AvlnBgHTRGDKO2 is purified from 633 cells, the resulting virus, AvlnBgHTRGDKO2 (633) contains a mixture of the wildtype fiber expressed from the 633 cells, and the mutant fiber expressed from the adenoviral genome.
  • This virus which has both types of fiber on the virion surface, is now able to mediate efficient transduction of HDF cells. This indicates that the RGD in the fiber is able to mediate transduction of HDF cells, even in the context of the V441, K442 deletion.
  • the RGD retargeting ligand sequence in a fiber containing the V441, K442 deletion is functional in its ability to mediate binding of ⁇ v - integrins (by its ability to inhibit transduction of AvlGFPHIRGD, see Fig. 4) and transduction of cells low in CAR (by its ability to transduce HDF cells when the virus is purified from 633 cells).
  • AvlnBgHIRGDKO2 was able to compete AvlGFPHIRGD transduction of HDF cells which are low in CAR and high in o integrins.
  • We conclude that the V441 K442 deletion has dramatically reduced ability to mediate transduction of CAR-expressing cells and that fibers containing these deletions and alternatives targeting ligands are functional.
  • Two recombinant adenoviral vectors were prepared that contain the KOI fiber mutation and are designated Av3nBgFKOl and AvlnBgKOlRGD. These vectors contain the KOI fiber mutation alone or in combination with a cRGD targeting moiety. The construction of these vectors is described below. Genetic incorporation of the KOI fiber mutation in combination with the cRGD targeting moiety to generate AylnBgKOlRGD. All amino acid changes were incorporated into the fiber gene using the p5FloxPRGD adenoviral shuttle plasmid as the template as previously described in Example 2.
  • This shuttle plasmid encodes a fiber that contains a cRGD peptide sequence, HCDCRGDCFC, inserted in the fiber HI loop.
  • the cRGD peptide has been shown to bind to ⁇ v-integrins on the cell surface.
  • Amino acid residues 408 and 409 in the AB loop of the fiber gene were changed using the Quickchange site-directed mutagenesis system (Stratagene, La Jolla CA). Substitution of these residues has been shown using the transient transfection/infection system to dramatically inhibit transduction of HeLa cells which express the adenoviral receptor, CAR.
  • the resulting shuttle plasmid was called pSKOl ( Figure 8).
  • the mutagenized fiber gene and the cRGD targeting moiety were incorporated into the adenoviral DNA backbone by cre-lox recombination.
  • the pSKOl shuttle plasmid ( Figure 8) and the pAvlhlpr plasmid ( Figure 4) were co-transfected with pCRE (described in Example 2) into 633 cells, a cell line that expresses the wildtype fiber ⁇ Von Seggern et al. 2000 ⁇ .
  • KOI fiber mutation Genetic incorporation of the KOI fiber mutation into the adenoviral genome.
  • the KOI mutation alone was incorporated genetically into the adenoviral genome to generate Av3nBgFKOl.
  • the KOI mutation was cloned into a plasmid containing the full-length Av3 adenoviral genome ⁇ Gorzigliz, Kadan, et al., 1996 ⁇ to generate ⁇ FLAv3nBgFKOl ( Figure 9).
  • Transfections were carried out in 633 cells, and in this fiber complementing cell line, the resulting viral DNA containing the KOI mutation is capable of being packaged into infectious viral particles containing a mixture of wildtype fiber and mutant fiber proteins.
  • the virus Upon observation of CPE, the virus was purified by standard CsCl centrifugation procedures. In order to obtain viral particles containing only the adenoviral encoded mutated KOI fiber protein with the S408E, P409A mutations, this viral preparation was used to infect AEl-2a cells, which do not express fiber. Viral particles were purified as described above.
  • Av3nBg An El, E2a, E3-deleted adenoviral vector encoding a nuclear localizing ⁇ -galactosidase
  • Av3nBgFKOl The same as Av3nBg but containing the KOI mutation in the fiber gene
  • Avl5FHTRGD An El, E3-deleted vector encoding a nuclear localizing ⁇ - galactosidase and containing a cRGD ligand in the HI loop of fiber
  • AvlnBgKOlRGD An El, E3-deleted vector encoding a nuclear localizing ⁇ - galactosidase and containing both the KOI fiber mutation and a cRGD ligand in the HI loop.
  • adenoviral vectors containing AB loop mutations Transduction efficiency of adenoviral vectors containing AB loop mutations.
  • adenoviruses pseudotyped with fiber proteins containing the S408E, P409A substitutions in the AB loop are severely affected in their ability to mediate gene transfer in HeLa cells, a CAR-expressing cell line.
  • the four vectors listed in Table 3 were compared for transduction efficiency on HeLa cells, human diploid fibroblasts (HDFs), two different human hepatocellular carcinoma cell lines, Hep3Bs and HepG2s, and a mouse hepatocyte cell line, FL83b.
  • the cells were seeded into the wells of a 24- well dish at 1-2 x 10 5 cells per well. The next day, the exact number of cells per well was determined for each cell line by counting a representative well for each cell type.
  • the cells were transduced with various numbers of particles per cell (PPC), in triplicate, using each of the four vectors.
  • PPC particles per cell
  • HDFs which express little or no CAR on their surface, were poorly transduced by both Av3nBg and Av3nBgFKOl ( Figure 10B).
  • efficient transduction of HDFs was observed using the two vectors containing a cRGD ligand in the HI loop, Avl5FHIRGD and AvlnBgKOlRGD ( Figure 10B).
  • adenoviral vectors containing the KOI fiber mutation In vivo analysis of adenoviral vectors containing the KOI fiber mutation. Efficient in vivo targeting of adenoviral vectors requires both ablation of the normal tropism and the addition of a new tropism. We generated adenoviral vectors which were designed to achieve both of those requirements. (See Table 3.) To ablate the normal tropism, the adenovirus fiber protein was genetically modified to knockout its interaction with the coxsackie-adenovirus receptor (CAR). To retarget the vector to a different receptor, a cRGD targeting moiety was inserted into the HI loop of the fiber knob.
  • CAR coxsackie-adenovirus receptor
  • KOI fiber knockout mutant
  • mice In vivo study design. The study included 5 cohorts of 5 mice each. Adenoviral vectors encoding nuclear targeted ⁇ -galactosidase (nBg) were administered by tail vein injection. The dose was 1 x 10 13 particles per kilogram. Mice were sacrificed 3 days after vector administration. Tissues, including liver, lung, heart, kidney, and spleen, were collected. Several assays were utilized to assess the efficiency of liver transduction and the vector biodistribution and included hexon PCR analysis, ⁇ -gal immunohistochemistry, and the ⁇ -gal Tropix assay. One group of mice received Hanks Balanced Salt Solution (HBSS) instead of adenoviral vector and served as a negative control.
  • HBSS Hanks Balanced Salt Solution
  • a second cohort received Av3nBg, which contains a "wild-type" fiber protein and served as a positive control.
  • a third group received Av3nBgFKOl, a fourth group received AvlnBgKOlHIcRGD, and a fifth group received AvlnBgHIcRGD.
  • mice in- groups 2 through 6 were injected with a volume of 10 ml kg to achieve a vector dose of 1 x 10 13 particles per kg.
  • the HBSS control group received an equivalent dose volume.
  • the remaining tissue from each organ was placed into a 1 ml cryovial and frozen in dry ice to preserve it for hexon PCR analysis to determine vector content.
  • pieces of each lobe were frozen in dry ice to preserve it for hexon PCR analysis and other pieces of each lobe were placed in a 'Tropix" vial, and frozen on dry ice.
  • the results of the immunohistochemical staining for ⁇ -galactosidase expression showed that Av3nBg, Av3nBgFKOl, Avl5FHIRGD, and AvlnBgKOlRGD all yielded efficient transduction of hepatocytes.
  • Av3nBgFKOl yielded a higher percentage of ⁇ - galactosidase expressing cells and a more intense staining than Av3nBg. This result was completely unexpected since Av3nBgFKOl transduction of various cells in culture was dramatically reduced. Evaluation of ⁇ -galactosidase expression in mouse livers by a chemiluminescent assay ( Figure 13) confirmed the results of the immunohistochemical staining. Mice that received Av3nBgFKOl demonstrated higher levels of expression than those that received Av3nBg. A measurement of the vector content in hepatocytes was determined by a semi-quantitative hexon PCR assay ( Figure 14).
  • the results were consistent with both the immunohistochemical staining and the chemiluminescent assay.
  • the vector content in hepatocytes was approximately 35% higher in the mice that received Av3nBgFKOl than in those that received Av3nBg.
  • the fiber AB loop mutation contained in Av3nBgFKOl ablates interaction with human and mouse CAR in vitro.
  • this fiber AB loop mutation behaves quite unexpectantly as it was found to dramatically enhance adenoviral- mediated gene transfer to liver and other organs and results in increasing vector potency.
  • This fiber modification will be useful for in vivo gene therapy strategies and will allow for lower doses of adenoviral vectors to be used systemically.
  • adenoviral vectors containing the KOI fiber mutation C57BL/6, Balbc, and CD1 male mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). When the mice were 5 weeks of age they received either HBSS (vehicle control), Av3nBg, or Av3nBgFKOl via tail vein injection at a dose of 1 x 10 13 particles per kg which is approximately 2xl0 ⁇ particles per mouse. Cohorts of five mice received each treatment. The vector was diluted to 1 x 10 12 particles per ml using Hanks Balanced Salt Solution (HBSS) immediately prior to injection. Three days after vector delivery, the animals were sacrificed and tissues including liver, lung, heart, kidney, and spleen were collected.
  • HBSS Hanks Balanced Salt Solution
  • the transduction efficiencies of Av3nBg and Av3nBgFKOl were evaluated on primary mouse hepatocytes.
  • Hepatocyte Wash medium enriched William's E; Life Technologies
  • Hepatocye Attachment Medium Modified William's E, supplemented with 1% pen-strep and 5% FBS; Life Technologies.
  • Viability was assessed by trypan blue exclusion.
  • Cells were plated at approximately 1 x 10 5 viable cells per well on collagen type I-coated 24-well plates and allowed to attach for 2 hr at 37°C in 5% CO2. After 2 hr, unattached cells and media were removed, cells were washed and cultured in HepatoZYME- SFM (Life Technologies). Immunohistochemical staining for albumin confirmed the identity of these cells as hepatocytes.
  • Av3nBgFKOl Transduction efficiency of Av3nBgFKOl on primary murine hepatocytes.
  • the cells were transduced with the adenoviral vectors Av3nBg and Av3nBgFKOl at various numbers of particles per cell, ranging from 0 to 12,500.
  • the cells were incubated with adenoviral vector for 1 hour at 37° C in a total volume of 0.2 ml of culture medium.
  • the cell monolayers were washed once with PBS, then 1 ml of the appropriate culture medium was added to each well.
  • the cells were incubated for 24 hours to allow for ⁇ -galactosidase expression.
  • the cell monolayers were then fixed and stained with X-Gal for 24 hours.
  • An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 72:9706-9713.
  • Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines Improved Transduction of Epstein-Barr Virus-Transformed B Cells. J Virol 74:354-362.

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Abstract

Cette invention se rapporte à des protéines de fibres adénovirales mutées et à des particules d'adénovirus contenant ces protéines, à des polynucléotides codant ces protéines et à des vecteurs contenant ces polynucléotides, ainsi qu'à des procédés de production et d'utilisation de ces particules adénovirales. Grâce à ces protéines de fibres mutées, les particules d'adénovirus ne se fixent plus à leur récepteur cellulaire naturel. Elles peuvent être alors reciblées sur un type de cellule spécifique grâce à l'addition d'un ligand au capside du virus, qui amène le virus à se fixer à une telle cellule et à l'infecter. On a établi la liste des mutations de fibres spécifiques qui suppriment la fixation au récepteur naturel. On a découvert que des particules d'adénovirus ayant certaines mutations de fibres augmentent le transfert génique et l'expression dans le foie par rapport à des particules virales ayant une fibre de type sauvage.
EP01962700A 2000-06-02 2001-06-01 Particules d'adenovirus avec proteines de fibres mutagenisees Withdrawn EP1364038A2 (fr)

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US7232899B2 (en) 1996-09-25 2007-06-19 The Scripps Research Institute Adenovirus vectors, packaging cell lines, compositions, and methods for preparation and use
WO2004101799A2 (fr) 2003-05-14 2004-11-25 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Elargissement du tropisme de l'adenovirus
FR2856928A1 (fr) * 2003-07-04 2005-01-07 Inst Nat Sante Rech Med Nouveau compose adjuvant de l'immunite, compositions le contenant et procedes mettant en oeuvre ledit compose adjuvant
JP4478775B2 (ja) * 2003-07-31 2010-06-09 財団法人名古屋産業科学研究所 増殖制御型組換えアデノウイルスベクターの効率的な作製方法及びその作製用キット
US7473418B2 (en) 2004-03-25 2009-01-06 Cell Genesys, Inc. Pan cancer oncolytic vectors and methods of use thereof
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US5846782A (en) * 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
FR2761689B1 (fr) * 1997-04-02 1999-06-25 Transgene Sa Fibre adenovirale modifiee et adenovirus cibles
JP2002513290A (ja) * 1997-05-08 2002-05-08 ジェネティック・セラピー・インコーポレテッド 修飾されたファイバー蛋白質を有するアデノウイルスによる遺伝子移入
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IL153143A0 (en) 2003-06-24
WO2001092299A9 (fr) 2002-09-19

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