WO2001002540A2 - Vecteurs adenoviraux de traitement de maladies - Google Patents

Vecteurs adenoviraux de traitement de maladies Download PDF

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WO2001002540A2
WO2001002540A2 PCT/US2000/017856 US0017856W WO0102540A2 WO 2001002540 A2 WO2001002540 A2 WO 2001002540A2 US 0017856 W US0017856 W US 0017856W WO 0102540 A2 WO0102540 A2 WO 0102540A2
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region
gene
composition
matter
cells
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PCT/US2000/017856
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WO2001002540A3 (fr
WO2001002540A9 (fr
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Terry Hermiston
Lynda K. Hawkins
Leisa Johnson
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Onyx Pharmaceuticals, Inc.
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Priority to EP00946890A priority Critical patent/EP1218527A2/fr
Priority to CA002373352A priority patent/CA2373352A1/fr
Priority to AU60578/00A priority patent/AU781775B2/en
Priority to JP2001508313A priority patent/JP2003504316A/ja
Publication of WO2001002540A2 publication Critical patent/WO2001002540A2/fr
Publication of WO2001002540A3 publication Critical patent/WO2001002540A3/fr
Publication of WO2001002540A9 publication Critical patent/WO2001002540A9/fr
Priority to AU2005209653A priority patent/AU2005209653B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention described herein relates generally to the field of gene therapy, and more specifically to adenoviral vectors that have prophylactic or therapeutic applications.
  • Adenovirus is a vector of choice for performing gene therapy. See. Jolly, D.. Cancer Gene Therapy, vol. 1. no. 1 , 1994: pp51 -64.
  • the well-characterized molecular genetics of adenovirus render it an advantageous vector in this regard.
  • Adenoviruses are nonenveloped icosohedral double-stranded DNA viruses with a linear genome of approximately 36 kilobase pairs. Each end of the viral genome has a short sequence known as the inverted terminal repeat (or ITR), which is required for viral replication. Portions of the viral genome can be readily substituted with DNA of foreign origin, and furthermore, recombinant adenoviruses are structurally stable.
  • the adenovirus replication cycle has two phases: and early phase, during which 4 transcription units El, E2, E3. and E4 are expressed, and a late phase which occurs after the onset of viral DNA synthesis when late transcripts are expressed primarily from the major late promoter (MLP).
  • MLP major late promoter
  • the late messages encode most of the virus ' s structural proteins.
  • the gene products of El , E2 and E4 are responsible for transcriptional activation, cell transformation, viral DNA replication, as well as other viral functions, and are necessary for viral growth.
  • most adenoviral vectors are based on viruses mutated in E l . E3 or a site upstream of E4 which provide for sites for the insertion of foreign DNA.
  • the majority of vectors are based on adenovirus mutants which lack the El region of the genome. By deleting this region, the virus is rendered replication incompetent while simultaneously allowing for the insertion of foreign genes.
  • adenovirus for gene therapy.
  • Smith, et al.. Nature Genetics. Vol. 5. pgs. 397-402 (1993) discloses the administration to mice of an adenoviral vector including a human Factor IX gene. Such administration resulted in efficient liver transduction and plasma levels of human Factor IX that would be therapeutic for hemophilia B patients. Human Factor IX levels, however, slowly declined to baseline by nine weeks after injection, and were not reestablished by a second vector injection. Smith, et al., also found that neutralizing antibodies to adenovirus block successful repeat administration of the adenovirus.
  • Kozarsky. et al.. J. Biol. Chem.. Vol. 269, No. 18, pgs. 13695- 13702 discloses the infusion of an adenoviral vector including DNA encoding the LDL receptor to rabbits. Stable expression of the LDL receptor gene was found in the rabbits for 7 to 10 days, and diminished to undetectable levels within 3 weeks. The development of neutralizing antibodies to the adenovirus resulted in a second dose being completely ineffective. Kass-Eisler, et al . Gene Therapy.
  • adenoviruses can be efficient in gene transfer into cells in vivo, and thus may be employed as delivery vehicles for introducing desired genes into eukaryotic cells
  • host responses include non-specific responses and specific immune responses
  • the nonspecific responses include inflammatory and non-inflammatory changes
  • An example of the latter is a change in host cell gene expression
  • Specific immune responses include various cellular responses and humoral antibody responses
  • Cellular responses include those mediated by T-helper lymphocytes, T- suppressor lymphocytes, cytotoxic T lymphocytes (CTL), and natural killer cells
  • the E3 region also contains open reading frames for two proteins,
  • E3 region While the E3 region is not essential for viral replication, it does play a key role in modulating the host immune or inflammatory responses to the virus
  • the immune response it is known that gpl 9K binds to MHC class 1 molecules in the endoplasmic reticulum, thus inhibiting its glycoslation and transport to the surface of the virallv infected cells Consequently, the infected cells are not recognized as foreign by cytotoxic lymphocvtes See, Burgert.
  • compositions and methods for constructing adenovirus having insertions or deletions in both the El and E3 regions See also, Ginsberg, H.S. et al., Proc. Natl. Acad. Sci. USA 1989 , vol. 86, pp. 3823-7.
  • a first object of the invention is to describe recombinant adenoviral vectors that have restriction sites in the E3 region that facilitate partial or total deletion of this region, or select genes contained therein, and if desired, substitute a heterologous gene, which gene will exhibit an expression pattern, both in terms of timing and degree of expression, substantially similar to the endogenous adenoviral gene that it replaces.
  • a second object of the invention is to describe recombinant adenoviral vectors that have restriction sites in the early region genes of the E3 region that encode the 6.7K and gpl9K proteins.
  • a third object of the invention is to describe recombinant adenoviral vectors that have restriction sites in the E3 region that encode the 10.4K, 1 1.6K, 14.5K, and 14.7K proteins.
  • a fourth object of the invention is to describe recombinant adenoviral vectors that have restriction sites in the E3 region that facilitate partial or total deletion of this region, or select genes contained therein, and compositions and methods for substituting foreign DNA therein.
  • a fifth object of the invention is to describe methods for making recombinant adenoviral vectors that have restriction sites in the E3 region that facilitate partial or total deletion of the E3 region, or select gene(s) contained therein.
  • a sixth object of the invention is to describe host cells containing recombinant adenoviral vectors that have a partial or total deletion of the E3 region, or select gene(s) contained therein.
  • a seventh object of the invention is a description of recombinant adenoviral mutants that have restriction sites in the E3 region that facilitate partial or total deletion of the E3 region, or select gene(s) contained therein.
  • An eighth object of the invention is a description of recombinant adenoviral mutants that have restriction sites in the E3 region that facilitate partial or total deletion of the E3 region, or select genes contained therein, including virions; E3SV, E3SV +V. E3SV + B, and E3SV +V + B.
  • a ninth object of the invention is a description of recombinant adenoviral mutants that have restriction sites in the E3 region that facilitate partial or total deletion of the E3 region wherein such mutants also have mutations elsewhere in the adenoviral genome, preferably in the E1A, E 1 B.
  • a tenth object of the invention is a description of methods and compositions for diagnosing or treating disease, preferably diseases involving unwanted cell growth, including neoplasia.
  • Preferred substituted genes include heterologous genes including negative selection genes, preferably cytosme deaminase and thymidine kinase
  • Figure 1 shows a map of the E3 region transc ⁇ ptional unit of adenovirus type 5
  • the split arrows indicate the spliced structures of the mRNAs (open rectangles or solid lines represent Exons, dashed lines, Introns), the thickness of the arrow indicates the relative abundance
  • the shaded bars above the arrows indicate the E3 proteins, which are named on the basis of their molecular masses
  • Figure 2 shows the production of recombinant virus using pNB and Ad5 TP-DNA (m u stands for map units)
  • Figure 3 shows the production of recombinant virus using pSN with the desired mutation and Ad5 TP-DNA The same strategy can also be used for the pG-based plasmids containing mutations
  • Figure 4 shows the restriction map of the E3 region of the adenovirus E3SV
  • Figure 5 shows the restriction map of the E3 region of the adenovirus E3SV +V
  • Figure 6 shows the restriction map of the E3 region of the adenovirus E3SV +B
  • Figure 7 shows the restriction map of the E3 region of the adenovirus E3SV + V +B
  • Figure 8 shows A549 cells mock infected or infected with Ad5
  • Figure 9 shows A549 cells infected with viruses Onyx 301. Onyx 302. Onyx 303. and Onvx
  • Figure 10 shows western blot analysis of gp l 9k from cell lysates prepared from cells infected with viruses Onyx 301. Onyx 302, Onyx 303, and Onvx 304 at different times post infection
  • Figure 1 1 shows a CD assay on cell lysates (0 5ug of protein) prepared from cells infected with ⁇ iruses Onyx 301 , Onyx 302. Onyx 303. Onyx 304. and Onvx 305 at different times post infection
  • Figure 12 shows a CD assay on cell 1 ⁇ sates prepared from cells infected with viruses Onyx 301 Onvx 302 Onyx 303, Onyx 304 and Onyx 305 at different times post infection using 0 6 ug protein/reaction
  • Figure 13 shows the cytopathic effect of viruses Onyx 305 and Onyx 320 at different times post infection
  • Figure 14 shows the cytopathic effect of virus Onyx 320 on cells that have, or have not had medium changes at certain times post infection
  • Figure 15 shows the expression of CD at different times by cells infected with virus Onyx 305
  • Figure 16 shows Western blots of the E3 proteins from A549 cells infected with Ad5 The numbers above each lane refer to times post-infection, p I
  • Figure 17 shows Western blots of E3 proteins from cells infected with Ad5 both in the absence and presence of araC, an inhibitor of DNA replication
  • the numbers above each lane refer to times post- mfection, p I
  • Figure 18 shows a Western blot of the pVIII protein from the L4 region from cells infected with either Ad5 or Onyx 320, or mock-infected cells (M)
  • the numbers above each lane refer to times post- lnfection, or p I
  • Figure 19 shows Western blots of the E3 proteins 1 1 6K, 14 5K and 14 7K from cells infected with 2 CD containing viruses, Onyx 303 and Onyx 304 These levels can be compared to the levels of E3 proteins produced by Ad5 virus shown in figure 16
  • Figure 20 shows a Western blot of 1 1 6K protein from cells infected with Ad5 or Onyx 305
  • Figure 21 shows a Western blot of the 26 kD intracellular form of mu ⁇ ne TNF (mTNF) synthesized by Onyx 320 infected cells As controls mock infected and Ad5 infected cells were also analyzed for the presence of mTNF
  • Figure 22 shows a Western blot of 17 kD mTNF secreted into the culture medium from Onyx 320 infected cells The medium was changed one hour before each indicated time point and then aliquots were taken at the indicated times post infection 25 ⁇ l of culture medium was run on a gel and blotted
  • Figure 23 shows the results of an ELISA assay which measured the amounts of secreted mTNF present in the culture supernatant as described in figure 22
  • Figure 24 shows Western blots of the E3 proteins gpl 9K 14 5K, and 14 7K from A549 cells infected with Onyx-320 As a control two time points from an Ad5 infection are included
  • the numbers above each lane refer to times post-infection p i
  • Figure 25 shows a Western blot of intracellular mTNF from cells infected with Onvx 320 and incubated in the presence or absence of araC
  • Figure 26 shows Western blots of the L4 protein, pVIII, from cells infected with Ad5 or Onyx 304, 305, and 320 and incubated in the presence or absence of araC.
  • Figure 27 shows Western blots of the E3 protein gpl 9K from cells infected with either Ad5 or Onyx 320 and incubated in the presence or absence of araC.
  • Figure 28 shows pictures of A549 cells which have been infected with either Ad5 or Onyx 321 and photographed at the indicated times post-infection.
  • Figure 29 shows Western blots of mTNF, and the E3 proteins 14.5K and 14.7K from A549 cells infected with either Ad5 or Onyx 321. The results of mock (M) infected cells at 24 hours are also shown. The numbers above each lane refer to times post-infection.
  • Figure 30 shows Western blots of the E3 proteins gpl9K and 1 1.6K from cells infected with
  • the numbers above each lane refer to times post- infection.
  • Figure 31 shows a Western blot of mTNF from cells infected with Onyx 321 incubated in the absence or presence of araC.
  • Figure 32 shows Western blots of the E3 proteins gpl 9K and 1 1.6K from cells infected with Onyx 321 either in the absence or presence of araC.
  • the numbers above each lane refer to times post- infection.
  • Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd, edition ( 1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, organic synthetic chemistry, and pharmaceutical formulation described below are those well known and commonly employed in the art.
  • the amino- and carboxy-terminal groups although often not specifically shown, will be understood to be in the form they would assume at physiological pH values, unless otherwise specified
  • the N-terminal H2 + and C-terminal-O " at physiological pH are understood to be present though not necessarily specified and shown, either in specific examples or in generic formulas
  • the left hand end of the molecule is the ammo terminal end and the right hand end is the carboxy-terminal end, in accordance with standard usage and convention
  • the basic and acid addition salts including those which are formed at non-physiological ph values are also included in the compounds of the invention
  • the ammo acid residues described herein are preferably in the "L" lsome ⁇ c form Stereoisomers (e g , D-amino acids) of the twenty conventional am o acids, unnatural ammo acids such as a,a-d ⁇ st ⁇ ubbed amino acids, N-alkyl am
  • isolated protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated protein” ( 1 ) is not associated w ith proteins found in nature, (2) is free of other proteins from the same source, e g free of human proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature
  • naturally-occurring refers to the fact that an object can be found in nature
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring
  • adenovirus indicates over 40 adenoviral subtypes isolated from humans, and as many from other mammals and birds See, Strauss, "Adenovirus infections in humans " in The Adenoviruses, Ginsberg, ed , Plenum Press. New York. NY, pp 451-596 (1984) The term preferably applies to two human serotypes, Ad2 and Ad5
  • polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ⁇ bonucleotides or deoxynucleotides or a modified form of either type of nucleotide
  • the term includes single and double stranded forms of DNA
  • ohgonucleotide referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring ohgonucleotide linkages
  • Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length Preferably oligonucleotides are 10 to 60 bases in length
  • Oligonucleotides are usually single stranded, e g for probes, although oligonucleotides may be double stranded, e g for use in the construction of a gene mutant
  • Oligonucleotides of the invention can be either sense or antisense
  • label refers to incorporation of a detectable marker, e g , by incorporation of a radiolabeled ammo acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidm (e g , streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colo ⁇ met ⁇ c methods)
  • marked avidm e g , streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colo ⁇ met ⁇ c methods
  • Various methods of labeling polypeptides and glycoprotems are known in the art and may be used Examples of labels for polypeptides include, but are not limited to, the following radioisotopes (e g , ⁇ H, ⁇ C, -> s 125j 5 1 * 1), fluorescent labels (e g , FITC, rhodamine, lanthamde phosphors), enzymatic labels (e g , horseradish
  • Two ammo acid sequences are homologous if there is a partial or complete identity between their sequences
  • 85% homology means that 85% of the am o acids are identical when the two sequences are aligned for maximum matching Gaps (in either of the two sequences being matched) are allowed in maximizing matching, gap lengths of 5 or less are preferred with 2 or less being more preferred
  • two protein sequences or polypeptide sequences derived from them of at least 30 ammo acids in length
  • are homologous, as this term is used herein, if they have an alignment score of more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater See Dayhoff, M O , in Atlas of Protein Sequence and Structure, 1972, volume 5, National Biomedical Research Foundation, pp 101- 1 10, and Supplement
  • reference sequence is a defined sequence used as a basis for a sequence comparison, a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing may comprise a complete cDNA or gene sequence Generally, a reference sequence is at least 20 nucleotides in length frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length Since two polynucleotides may each ( 1 ) comprise a sequence d e .
  • substantially pure means an object species is the predominant species present
  • a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present Generally a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%. 90%. 95% and 99% Most preferably the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species
  • substantially identical means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity
  • residue positions which are not identical differ by conservative amino acid substitutions
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains For example, a group of amino acids having aliphatic side chains is glycine. alanine, valine.
  • leucme, and isoleucine a group of amino acids having ahphatic-hydroxyl side chains is se ⁇ ne and threonine, a group of amino acids having amide-containing side chains is asparag e and glutamine. a group of amino acids having aromatic side chains is phenvlalanme, tyrosine. and tryptophan.
  • polypeptide fragment or "peptide fragment” as used herein refers to a polypeptide that has an ammo-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence Fragments typically 8- 10 amino acids long, preferably at least 10-20 amino acids long, and even more preferably 20-70 amino acids long Other chemistry terms herein are used according to conventional usage in the art,
  • DNA which encodes proteins that may be inserted into the adenoviral constructs of the instant invention in the E3 region can be obtained, in view of the instant disclosure by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate ceils or by combinations of these procedures, as illustrated below Screening of mRNA or genomic DNA may be carried out with ohgonucleotide probes generated from known gene sequence information Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemilummescent group in accordance with known procedures and used in conventional hybridization assays, as described in greater detail in the Examples below In the alternative, a gene sequence may be recovered by use of the polymerase chain reaction
  • a vector is a replicable DNA construct.
  • Preferred embodiment vectors described herein to realize the adenovirus E3 mutants are based on the pGEM vector series of Promega Corporation Vectors are used either to amplify DNA encoding a desired protein and/or to express DNA which encodes the protein
  • An expression vector is a replicable DNA construct in which a DNA sequence encoding a protein of interest is operably linked to suitable control sequences capable of affecting the expression of the protein in a suitable host The need for such control sequences will vary depending upon the host selected and the transformation method chosen Generally, control sequences include a transc ⁇ ptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation Amplification vectors do not require expression control domains All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.
  • Vectors useful for practicing the present invention include plasmids, viruses (including phage), and integratable DNA fragments (l e . fragments tegratable into the host genome by homologous recombination)
  • the vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself Suitable vectors will contain rephcon and control sequences which are derived from species compatible with the intended expression host
  • Transformed host cells are cells which have been transformed or transfected with the vectors constructed using recombinant DNA techniques
  • DNA regions are operably linked when they are functionally related to each other
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence
  • a ⁇ bosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation
  • operably linked means contiguous and, in the case of leader sequences. contiguous and in reading frame
  • a preferred embodiment promoter of the instant invention in those instances where certain E3 region DNA is deleted and DNA substituted therein is a tissue specific promoter which is operably linked to a negative selection gene
  • Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic cells
  • Prokaryotes include gram negative or gram positive organisms, for example Escherichia coh (E coli) or Bacilli
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below Exemplary host cells are DH5a , E. coli W31 10 (ATCC 27,325), E coli B, E. coli XI 776 (ATCC 31 ,537) and E. coli 294 (ATCC 31 ,446).
  • microbial vectors are available, and may have applications in constructing the instant adenoviral vectors.
  • a microbial vector will contain an origin of replication recognized by the intended host, a promoter which will function in the host and a phenotypic selection gene such as a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement. Similar constructs will be manufactured for other hosts.
  • E. coli is typically transformed using pBR322. See Bolivar et al, Gene 2, 95 ( 1977).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host.
  • Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature 275, 615 ( 1978); and Goeddel et al.. Nucleic Acids Res. 8, 4057 (1980) and EPO Application Publication Number 36,776) and the toe promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983)). While these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many promoters have been published, enabling a skilled worker to operably ligate them to DNA in plasmid or viral vectors (Siebenlist et al, Cell 20, 269, 1980)).
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribosome binding site, RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
  • the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources, including adenovirus.
  • viral sources including adenovirus.
  • a variety of viral and mammalian constitutive promoter elements can be used. See, Mittal et al., (1993) Virus Research, vol. 28, pp. 67-90.
  • commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Patent Number 4,599,308.
  • the early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication See Fiers e/ ⁇ / Nature 273. 1 13 ( 1978) Construction of Adenovirus E3 Mutants
  • adenovirus 5 genome is registered as Genbank accession #M73260, and the virus is available from the
  • adenovirus vector construction involves an initial deletion or modification of a desired region of the adenoviral genome, preferably the Ad5 genome, in a plasmid cassette using standard techniques
  • the adenoviral DNA, or a fragment thereof, present in pNB and which corresponds to the E3 region of the virus is subsequently cloned into another plasmid which may also be pGEM5zf+
  • another plasmid which may also be pGEM5zf+
  • the Spe l -Ndel fragment corresponding to bases 27082-31089, of the Ad5 genome can be excised from pNB and cloned into the Spe l and Nde l sites in the multiple cloning site (MCS) of pGEM5zf+
  • MCS multiple cloning site
  • This vector is termed pSN, and the adeno DNA present therein, bases 27082-31089, may be used to engineer the desired restriction sites into the E3 region to yield the appropriate E3 vectors, plasmids and viruses, discussed more below
  • the mutations in the E3 region described herein may be incorporated into adenoviral mutants that have mutations outside the E3 region Preferably such mutations would be in the E1B and/or El A and/or the E4orf6 regions of the adenoviral genome
  • the preferred mutations confer on adenovirus the to preferentially replicate in neoplastic cells compared to normal cells, wherein the neoplastic cells are functionallv defective in the tumor suppressor, p53
  • Such mutations typically occur in the E 1 B region that encodes the 55kD protein Defective p53 can arise in numerous ways, including a defect in those proteins that interact with p53.
  • the preferred mutations confer on adenovirus the ability to preferentially replicate in neoplastic cells compared to normal cells, wherein the neoplastic cells are functionally defective in the retinoblastoma tumor suppressor gene product, or p i 05 Rb
  • Such inactivating mutations in Ad5 typically occur in amino acids 30-85 in the E1 A CR1 domain, or nucleotide positions 697-790.
  • the CR3 domain of the adenoviral genome (spanning amino acids 150- 188) remains and is expressed as a truncated p289R polypeptide and is functional in transactivation of adenoviral early genes
  • Defective pRb can arise in numerous ways, including a defect in those proteins that interact with pRb, that is, a defect in the pRb pathway that renders pRb functionally inactive See, U S Patent No. 5, 677, 1 * 78
  • the E3 mutations described herein could be combined with the E l A deletion m the adenovirus Ad5 NT dl 1010
  • heterologous genes into the E1 B.
  • El A, or E4orf6 regions of an E3 mutant virus described herein would contain heterologous genes in E3, and optionally in E1B, El A or E4orf6
  • heterologous genes, or fragments thereof that encode biologically active peptides include those that encode immunomodulatory proteins, and prodrug activators (l e. cytosme deaminase, thymidme kinase, U S Patent Nos. 5. 358, 866, and 5, 677, 178)
  • Examples of the former would include interleukin 2, U S Patent Nos 4,738, 927 or 5.
  • Additional immunomodulatory proteins further include macrophage inflammatory proteins, including MIP-3, (See, Well, T N.
  • a preferred embodiment of a heterologous gene is a chime ⁇ c gene consisting of a gene that encodes a protein that traverses cell membranes, for example, VP22 or TAT, fused to a gene that encodes a protein that is preferably toxic to cancer but not normal cells
  • the initial step in the construction of recombinant adenoviral vectors having novel restriction sites in the E3 region that facilitate partial or total deletion of the E3 region genes, or select genes contained therein, is to make mutations in the adenoviral genome in a plasmid cassette using well established techniques of molecular biology, or modifications these techniques, referred to herein
  • the following restrictions sites were engineered into the E3 region of adenovirus 5 Pack Clal Pmel Swal BamHI BstBI, Sspl, Nhel.
  • the adenoviral vectors of the instant invention can also incorporate a tissue specific promoter in a part of the E3 region that has been deleted that will dnve the expression of another gene, preferablv a negative selection gene
  • a tissue specific promoter includes prostate specific antigen promoter See, PCT US95/14461
  • certain negative selection genes include cytosme deaminase, and thymidine kinase Regarding cytosme deaminase, see, U S Patent Nos 5 358, 866, and 5, 677, 178
  • a HSV tk gene cassette may be operably linked downstream of an E3 promoter Frequently, it is desirable to delete a nonessential portion (i e , for viral replication and packaging) of the adenoviral genome to accommodate the negative selection cassette, thus a substantial portion of the E3 gene region may be deleted and replaced with a negative selection cassette such as an HSV tk gene operably linked to either an E3 promoter, or a tissue specific promoter (and enhancer) or other suitable promoter/enhancer
  • a negative selection gene may be operably linked to an adenovirus late region promoter to afford efficient expression of the negative selection gene product in cells expressing a replication phenotype characterized by transcription from late gene promoters Expression of the HSV tk gene in a cell is not directly toxic to the cell unless the cell is exposed to a negative selection agent such as gancyclovir or FIAU Infected cells expressing a replication phenotype wherein a negative selection gene is substantially expressed may produce essentially no additional
  • gancyclovir is administered in an effective selective dosage, at which time the infected cells expressing the tk gene will be selectiveK ablated, thus negative selection can be used for enhanced cytopathic killing and/or to damp out further viral replication by killing ceils exhibiting a rephcative phenotype
  • a preferred embodiment is an HSV tk gene cassette (Zjilstra et al ( 1989) Nature 342 43 ⁇ Mansour et al ( 1988) Nature 336 348 Johnson et al ( 1989) Science 245 1234 Adair et al ( 1989 ) Proc Natl Acad Sci (U S A ) 86 4574, Capecchi M ( 1989) Science 244 1288 incorporated herein b ⁇ reference) operabK linked to an appropriate promoter and/or enhancer with a polvadenvlation site to form a tk expression cassette.
  • the tk expression cassette (or other negative selection expression cassette) is inserted into the adenoviral genome, for example, as a replacement for a substantial deletion of the E3 region.
  • the adenoviral vectors of the instant invention that encode a desired protein can be used for transformation of a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus and transducing a host cell with the virus or by transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216. 4,912,040, 4,740.461 , and 4,959.455. The transformation procedure used depends upon the host to be transformed.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Therapeutic Methods include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • neoplastic disease may be afforded by administering to a patient a composition comprising adenoviruses of the invention, and further comprising a negative selection gene.
  • a composition comprising adenoviruses of the invention, and further comprising a negative selection gene.
  • examples of the latter would include cytosine deaminase and thymidine kinase.
  • adenoviral constructs particularly in those instances where the E3 region of the virus encodes a protein useful for gene therapy of disease.
  • An example would be a cytokine, preferably an interleukin.
  • pancreatic carcinoma, bladder carcinoma, colon carcinoma, breast carcinoma, cervical carcinoma, ovarian carcinoma, or lymphocytic leukemias may be treated by administering an effective antineoplastic dosage of an appropriate adenovirus.
  • Suspensions of infectious adenovirus particles may be applied to neoplastic tissue by various routes, including intravenous, intraperitoneal, intramuscular, subdermal, and topical.
  • An adenovirus suspension containing about 10 ⁇ to 1012 or more virion particles per ml may be inhaled as a mist (e.g., for pulmonary delivery to treat bronchogenic carcinoma, small- cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma.
  • a tumor site for treating a tumor e.g., bronchogenic carcinoma, nasopharyngeal carcinoma, laryngeal carcinoma, cervical carcinoma
  • a tumor site for treating a tumor e.g., bronchogenic carcinoma, nasopharyngeal carcinoma, laryngeal carcinoma, cervical carcinoma
  • infusion e.g., into the peritoneal cavity for treating ovarian cancer, into the portal vein for treating hepatocarcinoma or liver metastases from other non-hepatic primary tumors
  • other suitable route including direct injection into a tumor mass (e.g.. a breast tumor), enema (e.g., colon cancer), or catheter (e.g., bladder cancer).
  • adenovirus mutants may be further evaluated by their capacity to reduce tumo ⁇ genesis or neoplastic cell burden in nu/nu mice harboring a transplant of neoplastic cells, as compared to untreated mice harboring an equivalent transplant of the neoplastic cells
  • E3 viruses may be combined with other antineoplastic protocols such as conventional chemotherapy Also in the event that the instant E3 adenoviral vectors, or viruses elicit an immune response that dampens their effect in a host animal, they can be administered with an appropriate immunosuppressive drug Propagation of Mutant Adenovirus
  • Adenoviral mutants of the invention typically are propagated as viral stocks in a cell line (e g , the 293 cell line ATCC # CRL 1573, American Type Culture Collection, Rockville, MD, Graham et al ( 1977) J Gen Virol 36 59, or A549 cells) that can provide certain desired viral functions, if needed, in trans to support replication and formation of infectious mutant vi ⁇ ons Formulations
  • Adenovirus E3 mutants may be formulated for therapeutic and diagnostic administration to a patient
  • a sterile composition containing a pharmacologically effective dosage of one or more species of adenovirus mutant is administered to a human patient or veterinary non-human patient for treatment, for example, of a neoplastic condition
  • the composition will comprise about 10 ⁇ to 1015 or more adenovirus particles in an aqueous suspension
  • a pharmaceutically acceptable carrier or excipient is often employed in such sterile compositions
  • a variety of aqueous solutions can be used, e_g_, water, buffered water, 0 4% saline, 0 3% glycine and the like
  • These solutions are sterile and generally free of paniculate matter other than the desired adenoviral vi ⁇ ons
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium
  • Adenoviruses of the invention, or the DNA contained therein, may be delivered to neoplastic cells by posome or immunoliposome delivery, such delivery may be selectively targeted to neoplastic cells on the basis of a cell surface property present on the neoplastic cell population (e g .
  • an aqueous suspension containing the vinons are encapsulated in liposomes or immunoliposomes
  • a suspension of adenovirus vinons can be encapsulated in micelles to form immunoliposomes bv conventional methods (U S Patent 5 043 164 U S Patent 4,957 735 U S Patent 4 925.661 , Connor and Huang ( 1985) J Cell Biol 101 582 Lasic DD ( 1992) Nature 355 279 Nov el Drug Delivery (eds Prescott LF and Nimmo WS Wiley New York 1989), Reddy et al ( 1992) J Immunol 148 page 1585) Immunoliposomes comprising an antibody that binds specifically to a cancer cell antigen (e g CALLA, CEA) present on the cancer cells of the individual may be used to target virions, or virion DNA to those cells.
  • a cancer cell antigen e g CALLA, CEA
  • compositions containing the present adenoviruses or cocktails thereof can be administered for prophylactic and/or therapeutic treatments of neoplastic disease.
  • compositions are administered to a patient already affected by the particular neoplastic disease, in an amount sufficient to cure or at least partially arrest the condition and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose” or “efficacious dose.” Amounts effective for this use will depend upon the severity of the condition, the general state of the patient, and the route of administration.
  • compositions containing the invention adenoviruses, or cocktails thereof are administered to a patient not presently in a neoplastic disease state to enhance the patient's resistance to recurrence of a neoplasm or to prolong remission time.
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amount ' s again depend upon the patient's state of health and general level of immunity.
  • Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of the antineoplastic adenoviruses of this invention sufficient to effectively treat the patient.
  • Antineoplastic adenoviral therapy of the present invention may be combined with other antineoplastic protocols, such as conventional chemotherapy.
  • adenoviral vectors/viruses described herein have multiple uses including applications in gene therapy.
  • a gene that encodes a medically useful protein may be cloned into the E3 region of the instant invention virions, and the virions used directly in gene therapy protocols to treat disease.
  • E3 mutant virions may also have deletions/mutations in other regions of the adenoviral genome, including in the E1B region and have substituted therefore a gene with desirable properties.
  • such genes might encode cytokines, including the interleukins, cell cycle regulatory proteins, including pi 6, ras, or proteins that induce cellular suicide or apoptosis, prodrug activators, including cytosine deaminase or thymidine kinase.
  • prodrug activators including cytosine deaminase or thymidine kinase.
  • tumor necrosis factor alpha, interferon gamma, and mip-3 may be utilized.
  • genes that encode anti-angiogenic factors could be used.
  • the instant adenoviral vectors may also be used to express proteins that are useful immunogens, or as a vaccine, and to transform cells which do not ordinarily express a particular protein to thereafter express this protein. Cells expressing these molecules are useful as intermediates for making cell membrane preparations useful for binding assays, which are in turn useful for drug screening.
  • Example 1 is illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
  • Example 1 is illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
  • Example 1 is illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
  • Adenoviral Vectors Vectors based on pGEM (Promega Corp.) were modified and used to clone, subclone, and mutagenize the appropriate E3 region of Ad5. This took advantage of the Ad5 existing restriction sites in and outside the E3 region, and these are shown in Table 1.
  • the vector designated pSN was subcloned by inserting the fragment from Ad5 Spel (27082) to Ndel (3 1089) into the Spel and Ndel sites in the multiple cloning site (MCS) in pGEM5Zf
  • the vector pGEM5 was further modified because it possesses 3 Sspl sites (2199, 2384, 2408); Sspl is one of the engineered sites in the E3 shuttle vector By deleting the Sspl sites in the vector, inserting genes into the E3 Sspl site would be facilitated since it would not involve partial restriction digests To delete the vector sites, the plasmid pGEM5 was cut with Sspl and EcoRV (present in the MCS at base 51 ) and rehgated. Unfortunately, the Sspl site at 2199 was not deleted and found in the resultant altered vectors. So the vector contains the deletion from Sspl at 2384 to EcoRV at 51.
  • the sites Pa and Clal were generated simultaneously by using mutant ohgonucleotide PacC plus PacNC and ClaC plus ClaNC, respectively, using the Transformer Site-Directed Mutagenesis kit (Clontech #K 1600- 1) as directed by the manufacturer
  • the sites Pmel and Swal were constructed separately using PmeC plus PmeNC and SwaC plus SwaNC. respectively, using the QuickChange Site- Directed Mutagenesis kit (Stratagene #200518) exactly as described by the manufacturer.
  • the Pmel site was cloned into the PacI/CIal-containing plasmid by inserting the Pmel-contai ng Kpnl-Xhol (natural ⁇ .d5 sites) fragment into this plasmid To this construct, the Swal site was inserted using the Hpal to Ndel fragment This resulting construct was called pG-PPCS and used for the next round of mutagenesis BamHI
  • the BamHI site was generated with the oligonucleotides BamC and SwaNC in the first PCR and this product with PmeC in the second PCR. using the above-described plasmid pG-PPCS as the template. This fragment (the product of the second PCR) was digested with Pmel and Swal to insert into pG-PPCS and this was termed pG-PPCS +B. It should be noted that this mutation also changes the 14.7K start codon to prevent premature initiation for any inserted gene at this site and that only two of the final four versions of the E3 shuttle vectors have the BamHI site. Vectors with this BamHI site are used when expression of 14.7K. is not desired, or when 14.7 is replaced with a foreign gene. BstBI. Nhel, and Stul
  • the BstBI site was created using BstBC and SwaNC in the first PCR and PmeC plus the first product in the second PCR.
  • the template used was pG-PPCS.
  • the second PCR product was digested with Muni and Swal and inserted into pG-PPCS, resulting in pG-PPBCS.
  • the fragment from Clal to Hpal with this site was inserted to make pG-PPBCS+B.
  • the Nhel site was made with NheC and PmeNC in the first PCR and SunC and the first product in the second PCR, using pG-PPBCS+/- B as the templates in separate reactions.
  • This fragment was digested with Pad and Pmel and inserted into the two versions of the above described constructs, pG-
  • PPBCS+/-B This construct was called pG-PNPBCS+/-B.
  • the Stul site was added by using StuC and
  • PNPBCS Stul-containing plasmid
  • the last two mutations were made using both the above-described plasmids as templates, pG- PNPBSCS and pG-PNPBSCS+B.
  • the Sspl site was created with SspC and HpaNC primers in the first PCR and NheC and the product of the first PCR in the second PCR. This fragment was digested with Xhol and Hpal to insert into the parental plasmid. with or without the BamHI site.
  • the EcoRV site was made using the EcoRVC and HpaNC primers in the first PCR and NheC and the first product in the second PCR. This fragment was cut with Xhol and Hpal to insert into the plasmids described in the above paragraph.
  • the Sspl and EcoRV sites were added together by cutting both BamHI-containing or BamHI-absent plasmids with Pmel and Clal and inserting the EcoRV-containing fragment into the parental plasmids. Note the EcoRV site also changes the start codon for 10.4K to prevent premature initiation of genes inserted into the Clal site at their 5' end.
  • the Clal site will be used, instead of the EcoRV site, for insertion of genes since the region with the EcoRV site is also involved in splicing. Deletion of this region might disrupt this event.
  • Figs. 4 through 7 These final versions of the E3 shuttle vectors are depicted in Figs. 4 through 7. These are called pGE3SV, pGE3SV+V, pGE3SV+B, and pGE3SV+V+B, the differences being the presence or absence of the EcoRV and BamHI sites. These plasmids are on deposit with the American Type Culture Collection, accession numbers XXX, XXX, XXX, and XXX, respectively.
  • shuttle vectors are used for construction of all subsequent plasmids, be they insertion of foreign genes or deletion of the Ad5 E3 genes.
  • the oligonucleotides that were used to mutagenize the desired E3 region are shown in Table 3.
  • each of the E3 genes was deleted using the engineered sites
  • the shuttle plasmids were cut with the following pairs of enzymes, filled in using T4 DNA polymerase, and rehgated Pad and Pmel, Sunl and Muni, Nhel and Pmel, BstBI and Stul (all in pG-E3SV), Clal and Swal (in pG-E3SV+V), Clal and Sspl ( pG-E3SV+V), and BamHI and Swal (in pG-E3SV+B)
  • Figure 2 shows another plasmid that could also be used, termed pNB
  • the pNB has two Spel sites one in the Ad5 insert and one in the pGEM5 MCS
  • the fragment from pNB which contained a portion of the MCS and Ndel 19549 to Spel 27082 was inserted into the Spel-cut pG-PPCS plasmid
  • the orientation was confirmed to be correct, and the resulting plasmid termed pNB-PPCS
  • All of the final versions of the E3 shuttle vectors were cloned into this plasmid by inserting the Pad to Swal region of the pG plasmids into the larger pNB-PPCS
  • the resulting plasmids were designated p B-E3SV, pNB-E3SV+V, pNB-E3SV+B and pNB-E3SV+V+B. and could be used to produce viruses similar to those produced using the
  • CD prodrug capabilities
  • ATCC ATCC (#40999, plasmid pCD2)
  • CD gene was amplified from this plasmid as follows
  • Ndel restriction site because we intended to use this particular enzyme to cut Ndel sites m the shuttle vectors, it was necessary to remove the Ndel site in the CD gene by PCR mutagenesis
  • the other CD-containing vectors were created by designing the appropriate primers which possess the desired restriction site at the 5 " or 3 ' end of the gene, the 5 primer always containing the ATG start codon
  • the CD gene was inserted into E3 regions using the following restrictions sites BstBI to Stul, Nhel to Muni, Nhel to Pmel. Pad to Pmel. Sunl to Muni, Clal to Swal. and BamHI to Swal
  • the plasmids were named pG-CDBstStu. pG-CDNheMun pG-CDNhePme, pG-CDPacPme, pG- CDSunMun.
  • CDBstStu may result in a greatly attenuated infection.
  • deletion of the 1 1.6K protein does not allow the infected cells to lyse at the proper time, compared to wild type infection. In this case the cell continues to metabolize, the virus production per cell is higher, and the cell basically becomes a factory for producing the foreign gene.
  • ADP is synthesized in large quantities using the major late promoter during the late phase of infection, a foreign gene inserted into this region would be expected to have the same expression characteristics. The results obtained with these viruses will be discussed below.
  • TNF Plasmids The plasmid containing the murine tumor necrosis factor (mTNF) gene was obtained from ATCC (#63169). This sequence contains the entire mTNF gene including the coding region for the prosequence. The mTNF gene was amplified from this plasmid by PCR (Table 4) and gel purified. The vector pGE3SV was cut with BstBI and Stul and gel-purified. At the same time, another vector, pG- E3SV+V was cut with Clal and Swal and gel purified. The purified PCR product was readily inserted into each of these vectors due to compatible ends.
  • mTNF murine tumor necrosis factor
  • constructs were called pG-mTNFBstStu and pG-mTNFClaSwa, respectively.
  • ATCC plasmid as a template once again.
  • mTNF gene was amplified by PCR.
  • the plasmid pG-E3SV+B and the PCR product were cut with BamHI and Swal, gel purified, and ligated together.
  • This construct was called pG-mTNFBamSwa. All constructs were sequenced extensively to check for unwanted mutations.
  • Example 6 Construction of CD and TNF Viruses
  • Ad5 TP-DNA was used for the mTNF containing viruses.
  • BstLink TP-DNA was used since it offers certain advantages.
  • the plasmid construction for this BstLink is described in Example 1 1. Note that all transfections were performed on 6 cm dishes and in duplicate; the quantities described here are per each 6 cm dish.
  • viruses E3-CD-PacPme (Onyx 301), E3-CD-NhePme (Onyx 302), E3-CD- SunMun (Onyx 303), E3-CD-NheMun (Onyx 304), E3-CD-BstStu (Onyx 305), and E3-mTNF-BstStu (Onyx 320) were made as follows: first, 0.5 micrograms of TP-DNA (Ad5 for CD viruses and BstLink for mTNF viruses) and ten micrograms of plasmid were cut with EcoRJ (20 units) at 37 degrees for 5 hours.
  • TP-DNA BstLink was cut with BstBI for the transfection.
  • the CD-containing plasmids were cut with Spel and Ndel and the mTNF-containing plasmids were cut with Pad and Ndel.
  • the fragments were gel-purified and eluted in water. For transfections, the cut TP-DNA and the isolated fragments were mixed together and used without any further manipulations.
  • the correct viruses were expanded by infecting a T150 of A549 cells with 500 ul of the viral suspension obtained from the 3 5 cm dish This was allowed to proceed to full CPE (when over 75% of the cells are no longer attached to the flask surface), which occurred in approximately 3 days Then 7 5 ml of this cell and medium mixture was used to infect a 3-liter spinner of KB cells and the virus produced was purified by CsCl-banding Plaque assays were performed to determine the infectious particles per unit volume
  • viruses were named at this point in such a way to make it obvious to tell which insert had been added and where the insert was placed Numbers for ease of reference were also assigned to each virus, and these appear in parenthesis Their names are E3-CD-PacPme (Onyx 301 ) E3-CD-NhePme
  • Onyx 302 E3-CD-SunMun (Onyx 303), E3-CD-NheMun (Onyx 304), E3-CD-BstStu (Onvx 305), E3- mTNF-BstStu (Onyx 320), and E3-mTNF-ClaSwa (Onyx 321 )
  • CD assay To assay for cytosine deaminase (CD) activity, the reaction was performed similar to that as described in Rogulski et al 1997 Briefly, A549 cells were seeded into 10 cm plates so that they were about 70 to 80%o confluent on the day of infection (about 2 to 4 million cells per plate) The cells were infected at an MOI (multiplicity of infection) of 10 pfu (plaque forming units) per cell for each of the E3-CD viruses Ad5 and mock infected cells were included as controls For the infection the proper volume of virus was suspended in 2 ml of DME per 10 cm plate and then added to the cell monolayer After one hour 8 ml of DME/2% FBS medium was added to each plate At various times post-infection (4, 8, 12 24 36 48 60 72 84 96 120 hours) the cells were rinsed 1 l ot cold PBS was added, cells were scraped (using disposable cell scrapers) and pelleted into 1 5 ml eppendort
  • Murine TNF alpha assa The A549 cells were plated onto 6 cm plates so that they would be approximately 80% confluent for the infection The infection was performed as described above at an M O I of 10 At various time points, the medium was removed and replaced with 3 ml of fresh DME/2% FBS. This was incubated at 37 ⁇ C for one hour.
  • the cells pellet was stored at -80 ⁇ C
  • Three hundred ul of ly sis buffer was added to each sample, freeze/thawed 3 times, and passed through a 22-gauge needle
  • a Bradford assay was performed to determine the quantity of protein
  • Ten micrograms of total protein was loaded onto a 4-20% SDS/PAGE gel and electrophoresed
  • the proteins were transferred onto a PVDF membrane, blocked with 3% dry milk in PBS.
  • Oligonucleotides used to amplify the CD and mTNF genes ( sequences are 5' to 3').
  • CD-containing viruses E3-CD-PacPme (Onyx 301 ), E3-CD-NhePme (Onyx 302), E3-CD-SunMun (Onyx 303), E3-CD-NheMun (Onyx 304).
  • the cell line A549 was infected with each of the viruses Onyx 301.
  • Onyx 302, Onyx 303, and Onyx 304 at a M.O.I.(multipIicity of infection) of ten.
  • samples were harvested as described in methods section for assay of CD activity. Also at each time point, a picture was taken of the cells to show phenotypic differences.
  • Figure 8 shows control mock infected and Ad5-infected cells
  • figure 9 shows cells infected with viruses with CD inserted into the gp l 9K region, that is, viruses , Onyx 301 , Onyx 302, Onyx 303, and Onyx 304.
  • the wild type infection proceeds normally and shows almost total CPE by 48 hr p.i.
  • the Onyx 304 virus shows near wild type levels of CPE at 48 hr p.i., while Onyx 303 shows a slightly attenuated infection The other two viruses.
  • Onyx 301 and Onyx 302 show an intermediate phenotype Interestingly, Onyx 303 is the CD substitution using the Sunl site and the infection is slower than wild ty pe likely due to the deletion of part of the y-leader, abrogating its addition to late messages and probably lowering their efficiency of translation
  • the viruses Onyx 301 and Onyx 302 lag behind Ad5 onK slightly It is noteworthy that 22 base pairs of the y-leader is deleted in the case of Onyx 301 , whereas Onyx 302 contains the entire y-leader
  • Onyx 303 produces wild type levels of the E3 proteins, namely 1 1 6K, 14 5K, and 14 7K Interestingly, Onyx 303 produces almost no 1 1 6K which could account for its attenuated phenotype
  • Onyx 303 produces almost no 1 1 6K which could account for its attenuated phenotype
  • ADP is predominately a late protein whose expression is thought to be driven bv the major late promoter and expressed at high levels during the late phase, that is.
  • a CD assay was performed on Onyx 305, and analysis for mTNF was performed on Onyx 320 As shown in figure 1 1, Onyx 305 does not show substantial CD activity until 12 hr p I , a time when the virus has entered into the late phase Also contrasted in figure 1 1 is the comparison with CD in the gpl9K region, an early region Clearly, there is a difference in the timing that the protein is synthesized To compare the amount of CD activity a CD assay was performed using 0 6 ug of total protein in each reaction (figure 15) Upon visual inspection and comparison to figure 12 (experiments were done at the same time) it appears that Onyx 305 does not synthesize quite as much as Onyx 301 -304 But it should be noted that the Onyx
  • CD inserted in place of ADP exhibits a similar time to expression as ADP
  • Example 8 Expression of mTNF in Onyx 320 Mirrors Endogenous Late Gene Expression Additional experiments were conducted to quantify the expression of mTNF from Onyx 320, and to confirm that mTNF exhibits a late expression pattern similar to 1 1.6K.
  • mTNF Quantification A549 cells were infected with Onyx 320 at an m.o.i. of 10 and the cells were harvested at the indicated times post-infection. To measure secretion of mTNF, the medium was removed one hour prior to the indicated time point and replaced with 5 ml of fresh medium. Aliquots of the newly added medium were then removed at the end of that hour to obtain data indicating the amount of mTNF produced per hour. As a control, Ad5 and mock infections were also conducted.
  • Figure 20 shows Western blot analysis of the timing and relative levels of expression of the endogenous E3 gene, 1 1.6K. Based on the detection limits of this assay, and previous published data, 1 1.6K is predominately synthesized after the start of the late phase of infection.
  • Figure 21 shows Western blot analysis of intracellular mTNF. This is the 27 kD uncleaved form of the molecule. See, Kriegler et al., Cell, 1988 Apr 8;53(l ):45-53. As is true of the 1 1.6K protein, mTNF expressed from this region is predominately synthesized and detected during the late phase of infection.
  • Figure 22 shows Western blot analysis of mTNF secreted into the medium during the one hour preceding the indicated time points. Twenty-five ul of culture medium was run on the gel to obtain this result. This shows that again detectable levels of the mTNF are produced at late times post-infection.
  • the mTNF produced is properly cleaved as it runs at the predicted molecular weight of 17kD.
  • Figure 23 shows the results of 4 different experiments, and the results are expressed as the amount of mTNF produced per hour per one million cells. As shown in the figure the infected cells express high levels of mTNF: indeed, about 43 to 68 nanograms were measured during the highest production period.
  • gp l 9K is synthesized in the presence of araC by both Ad5 (See also figure 17), and Onyx-320 Lastly, another control was run to further confirm that Onvx 320 infection truly reaches late phase in the absence of araC Western blot analysis was performed against another known late protein, pVIII Figure 26 shows that pVIII is not expressed in the presence of araC, but is in its absence This further supports that mTNF is expressed as a true late protein Note that figure 26 also shows the expression pattern for pVIII in Onyx 304 and Onyx 305, and the results with these viruses are similar to those observed for Onyx 320
  • Example 9 Expression of mTNF from the E3B Region of Onyx 321 As described above. Onyx 321 has the entire E3B region substituted with mTNF using the engineered sites Clal and Swal Experiments were conducted to determine the expression properties of mTNF from this region of the virus A549 cells were infected at an MOI of 10 and photographed at the indicated times post-infection ( Figure 28) At the 48 hour time point, Onyx-321 showed greater CPE than wild type virus at the same time post-infection Next.
  • the genes for the chemokines hMCP3-alpha and hMIP3 alpha were inserted into the 6 7Kygp l 9K region and viruses were made Specifically, the oligonucleotides used to PCR amplify these genes are listed in Table 4. each of these genes were amplified with the Nhel site on the 5 end and the Muni site on the 3 ' end
  • the MIP3-alpha gene was obtained in the form of an EST from ATCC (EST# 1 13153) and the EST for MCP3-alpha was obtained from Genome Systems (Image #485989).
  • the plasmid for insertion was pG-E3SV, both the plasmid and the PCR products were cut with Nhel and Muni All fragments were gel purified Then each of the chemokines was inserted into the vector individually Viruses were created using these constructs just as described above The viruses were plaque purified and confirmed by methods described above
  • Example 11 Construction of BstLink Virus/TP-DNA Insertion of a gene of choice into the adenovirus genome is an extensive process It involves cloning into the smaller plasmids first and then adding this into the larger, pNB-based vectors Ideally, the smaller plasmids could be used directly However, their use for co-transfections for virus construction is difficult because it allows a limited amount of overlapping sequences necessary for homologous recombination For example, there is only a 240 base pair overlap in sequence between the plasmid pSN and genome viral DNA at the 5' end when cutting TP-DNA with EcoRJ, the standard method Thus, to increase the region of overlap, a virus called BstLink was created as follows The plasmid pG-Bst— Stu was used because it deletes the 1 1 6K death gene, which, if used to generate viruses, results in much smaller plaques Thus, this plasmid was digested with Muni, filled in with T4 DNA polymerase, and

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Abstract

L'invention concerne des vecteurs adénoviraux, notamment des adénovirus mutants, présentant des sites de restriction dans la région E3, facilitant sa délétion partielle ou totale, ou sélectionnant des gènes contenus dans celle-ci, ainsi que des compositions et des méthodes de substitution de gène(s) hétérologue(s), si désiré, lequel/lesquels gènes présentent un type d'expression, à la fois en termes de durée et de degré d'expression, semblable à celui du gène adénoviral endogène qu'il remplace, et comportant également facultativement des mutations dans d'autres parties du génome adénoviral, notamment certaines régions E1B ou E1A, et ayant des applications en diagnostic ou en traitement de maladies, de préférence des maladies à croissance cellulaire indésirable, notamment le cancer.
PCT/US2000/017856 1999-07-02 2000-06-28 Vecteurs adenoviraux de traitement de maladies WO2001002540A2 (fr)

Priority Applications (5)

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EP00946890A EP1218527A2 (fr) 1999-07-02 2000-06-28 Vecteurs adenoviraux de traitement de maladies
CA002373352A CA2373352A1 (fr) 1999-07-02 2000-06-28 Vecteurs adenoviraux de traitement de maladies
AU60578/00A AU781775B2 (en) 1999-07-02 2000-06-28 Adenoviral vectors for treating disease
JP2001508313A JP2003504316A (ja) 1999-07-02 2000-06-30 疾患を処置するためのアデノウイルスベクター
AU2005209653A AU2005209653B2 (en) 1999-07-02 2005-09-09 Adenoviral vectors for treating disease

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US09/347,604 1999-07-02

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Cited By (4)

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US7109029B2 (en) 2001-02-23 2006-09-19 Cell Genesys, Inc. Vector constructs
CN104946602A (zh) * 2015-06-11 2015-09-30 华中科技大学同济医学院附属同济医院 具有肿瘤组织靶向性和抑癌基因修复性的重组溶瘤腺病毒Ad5-P16及其应用
CN111630060A (zh) * 2017-09-01 2020-09-04 盈珀治疗有限公司 用于在疾病的预防和/或治疗中使用的疫苗
WO2021092427A1 (fr) * 2019-11-06 2021-05-14 Memgen, Inc. Adénovirus oncolytiques à réplication améliorée

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CN112029737A (zh) * 2005-04-07 2020-12-04 宾夕法尼亚大学托管会 增强腺相关病毒载体功能的方法
CN101979606A (zh) * 2010-11-01 2011-02-23 陕西师范大学 一种以腺病毒表达系统产生的人逆转录泡沫病毒载体
CN105274142B (zh) * 2014-06-25 2018-12-04 中国科学院广州生物医药与健康研究院 复制型重组人55型腺病毒载体及其制备方法和应用
GB201804473D0 (en) * 2018-03-21 2018-05-02 Valo Therapeutics Oy Modified oncolytic adenoviruses

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WO1999055831A2 (fr) * 1998-04-24 1999-11-04 Onyx Pharmaceuticals, Inc. Vecteurs adenoviraux destines au traitement de maladies
WO2000030687A1 (fr) * 1998-11-19 2000-06-02 The Board Of Trustees Of The Leland Stanford Junior University Nouveau vecteur adenoviral et ses methodes de production et d'utilisation

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WO1999055831A2 (fr) * 1998-04-24 1999-11-04 Onyx Pharmaceuticals, Inc. Vecteurs adenoviraux destines au traitement de maladies
WO2000030687A1 (fr) * 1998-11-19 2000-06-02 The Board Of Trustees Of The Leland Stanford Junior University Nouveau vecteur adenoviral et ses methodes de production et d'utilisation

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7109029B2 (en) 2001-02-23 2006-09-19 Cell Genesys, Inc. Vector constructs
CN104946602A (zh) * 2015-06-11 2015-09-30 华中科技大学同济医学院附属同济医院 具有肿瘤组织靶向性和抑癌基因修复性的重组溶瘤腺病毒Ad5-P16及其应用
CN111630060A (zh) * 2017-09-01 2020-09-04 盈珀治疗有限公司 用于在疾病的预防和/或治疗中使用的疫苗
CN111630060B (zh) * 2017-09-01 2024-01-23 盈珀治疗有限公司 用于在疾病的预防和/或治疗中使用的疫苗
WO2021092427A1 (fr) * 2019-11-06 2021-05-14 Memgen, Inc. Adénovirus oncolytiques à réplication améliorée

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AU781775B2 (en) 2005-06-09
CN1357050A (zh) 2002-07-03
EP1218527A2 (fr) 2002-07-03
JP2003504316A (ja) 2003-02-04
WO2001002540A9 (fr) 2001-09-20
AU6057800A (en) 2001-01-22
CA2373352A1 (fr) 2001-01-11

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