WO1998037207A1 - Constructions genetiques antitumorales - Google Patents

Constructions genetiques antitumorales Download PDF

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Publication number
WO1998037207A1
WO1998037207A1 PCT/GB1998/000551 GB9800551W WO9837207A1 WO 1998037207 A1 WO1998037207 A1 WO 1998037207A1 GB 9800551 W GB9800551 W GB 9800551W WO 9837207 A1 WO9837207 A1 WO 9837207A1
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gene
cells
genetic construct
cell
promoter
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PCT/GB1998/000551
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Ian David Hickson
Susan Nicola Edwards
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Imperial Cancer Research Technology Limited
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Priority to AU63007/98A priority Critical patent/AU6300798A/en
Publication of WO1998037207A1 publication Critical patent/WO1998037207A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • 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
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)

Definitions

  • the present invention relates to cancer therapy, and in particular to the treatment of cancer using gene therapy.
  • Cancer is a major killer. Although much progress has been made in understanding the molecular basis of cancer there remains the need for new and improved methods of cancer treatment.
  • tumour suppressor genes include p53, Rb, FAP, DCC, NF1, NF2, WT1, BRCA1, BRCA2 and DPC4.
  • the tumour suppressor gene products such as p53 and Rb are known to modulate transcription of various genes.
  • Ginsberg et al (1991) Proc. Natl. Acad. Sci. USA 88, 9979-9983 shows that wild-type p53 can down-modulate the activity of various promoters.
  • WO 97/12970 describes anti-tumour vector constructs and methods.
  • An object of the present invention is to provide new and improved cancer therapies.
  • a first aspect of the invention provides a genetic construct comprising a promoter element responsive to a tumour suppressor gene product and a cytotoxic gene wherein the promoter element is substantially inactive in a cell which contains said tumour suppressor gene product encoded by a wild type tumour suppressor gene and is active in a cell which contains a variant said tumour suppressor gene product encoded by a mutant tumour suppressor gene, or does not contain said tumour suppressor gene product.
  • tumour suppressor gene product By a “genetic element responsive to a tumour suppressor gene product” we include those genetic elements (either DNA or RNA, but usually DNA) which either directly or indirectly respond to a wild-type tumour suppressor gene product to cause suppression of the expression of a gene to which said element is operatively linked.
  • tumour suppressor gene product and the genetic element interact indirectly via one or more additional factors.
  • the effect is that, when present in a cell which contains a tumour suppressor gene product encoded by a wild type tumour suppressor gene the genetic construct is substantially unable to express the said cytotoxic gene whereas when present in a cell which contains a variant tumour suppressor gene product encoded by a mutant tumour suppressor gene, or when present in a cell which lacks said tumour suppressor gene product, the genetic construct is able to express said cytotoxic gene.
  • tumour suppressor gene will lead to the production of a tumour suppressor gene product which is substantially incapable of suppressing the activity of the said promoter element. It is these mutations which lead to the variant tumour suppressor gene products as defined in the first aspect of the invention.
  • the genetic construct when present in a normal cell which contains wild-type copies of the given tumour suppressor gene the genetic construct expresses substantially no cytotoxic gene product, or at least a very low level compared to the level of expression of said cytotoxic gene product in a tumour cell wherein both copies of the tumour suppressor gene are mutated or absent (and therefore inactive).
  • the level of expression of the cytotoxic gene product when the genetic construct is present in non-tumour cells is such that there is substantially no toxicity to said non-tumour cells due to the cytotoxic gene product.
  • the level of expression of the cytotoxic gene in the tumour cell is at least three times the level in the normal cell; more preferably the level in tumour cell is at least ten times the level in the normal cell; still more preferably it is at least fifty times, and most preferably it is at least one hundred times.
  • cytotoxic gene we mean a gene which encodes a molecule (either polypeptide or RNA) having a directly or indirectly cytotoxic function.
  • cytotoxic we mean that the molecule encoded by the gene may itself be toxic (for example ricin; interferon-gamma (IFN- ⁇ ); ribonuclease; deoxyribonuclease; Pseudomonas exotoxin A) or it may be metabolised to form a toxic product, or it may act on something else to form a toxic product.
  • ricin interferon-gamma
  • ribonuclease deoxyribonuclease
  • Pseudomonas exotoxin A cytotoxic
  • the gene may encode an enzyme, the enzyme being one that converts a relatively non-toxic prodrug to a toxic drug.
  • the enzyme cytosine deaminase converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU) (Mullen et al (1922) PNAS 89, 33); the herpes simplex enzyme thymidine kinase sensitises cells to treatment with the antiviral agent ganciclovir (GCV) or aciclovir (Moolten (1986) Cancer Res. 46, 5276; Ezzedine et al (1991) New Biol 3, 608).
  • the cytosine deaminase of any organism for example E. coli or Saccharomyces cerevisiae, may be used.
  • the gene encodes a cytosine deaminase which is able to convert 5-fluorocytosine (5FC) to 5- fluorouracil.
  • the gene encodes thymidine phosphorylase (TP).
  • TP is identical to platelet- derived endothelial cell growth factor (PD-ECGF).
  • PD-ECGF platelet- derived endothelial cell growth factor
  • TP enhances cell killing by the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) as disclosed in Patterson et al (1995) Br. J. Cancer 72, 669-675.
  • the gene encodes thymidine kinase which is able to active ganciclovir or acyclovir.
  • pro-drug/enzyme combinations include those disclosed by BagshaWe et al (WO 88/07378), namely various alkylating agents and the Pseudomonas spp. CPG2 enzyme, and those disclosed by Epenetos & Rowlinson-Busza (WO 91/11201), namely cyanogenic pro-drugs (for example amygdalin) and plant-derived -glucosidases.
  • the nitroreductase/CB1954 system described by Bridgewater et al (1995) Eur. J. Cancer 31A, 2362-2370 is another example of an enzyme/prodrug combination suitable for use in the invention.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumour cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form (see, for example, D.E.V. Wilman "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions 14, 375-382 (615th Meeting, Harbor 1986) and V.J. Stella et al "Prodrugs: A Chemical Approach to Targeted Drug Delivery” Directed Drug Delivery R. Borchardt et al (ed.) pages 247-267 (Humana Press 1985)).
  • Enzymes that are useful in this embodiment of the invention include, but are not limited to, alkaline phosphatase useful for converting phosphate- containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5- fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D- alanylcarboxypeptidases, useful for converting prodrugs that contain D- amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ - galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; / 3-
  • antibodies with enzymatic activity can be used to convert the prodrugs of the invention into free active drugs [see, eg R J Massey, Nature, 328, pp. 457-458 (1987)].
  • abzymes antibodies with enzymatic activity
  • the genes and cDNAs encoding at least some of these enzyme and abzyme are known in the art.
  • the prodrugs of this invention include, but are not limited to, the above-listed prodrugs, eg phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide- containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, 3-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5- fluorouridine prodrugs which can be converted by the enzyme of the conjugate into the more active, cytotoxic free drug.
  • the prodrugs of this invention include, but are not limited to, the above-listed prodrugs, eg phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide- containing prodrugs, D-amino acid-modified prodrugs, glycos
  • the gene delivered to the target cell encodes a ribozyme capable of cleaving targeted RNA or DNA.
  • the targeted RNA or DNA to be cleaved may be RNA or DNA which is essential to the function of the cell and cleavage thereof results in cell death.
  • Ribozymes which may be encoded in the genomes of the viruses or virus- like particles herein disclosed are described in Cech and Herschlag "Site- specific cleavage of single stranded DNA” US 5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P" US 5,168,053, Cantin et al "Ribozyme cleavage of HIV- 1 RNA” US 5,149,796; Cech et al “RNA ribozyme restriction endoribonucleases and methods", US 5,116,742; Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods, US 5,093,246; and Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterification” , US 4,987,071, all in
  • the genetic construct can be DNA or RNA it is preferred if it is DNA.
  • the genetic construct is adapted for delivery to a cell, preferably a human cell. More preferably, the genetic construct is adapted for delivery to a cell in an animal body, more preferably a mammalian body; most preferably it is adapted for delivery to a cell in a human body.
  • the constructs of the invention may be introduced into the tumour cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the tumour cell.
  • retroviruses provide a potential means of selectively infecting cancer cells because they can only integrate into the genome of dividing cells; most normal cells surrounding cancers are in a quiescent, non-receptive stage of cell growth or, at least, are dividing much less rapidly than the tumour cells.
  • Retroviral DNA constructs which contain a promoter segment which comprise a promoter element responsive to a tumour suppressor gene product and a cytotoxic gene as defined may be made using methods well known in the art.
  • To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo R gene).
  • tumour cells which produce retroviruses are injected into the tumour.
  • the retrovirus-producing cells so introduced are engineered to actively produce retroviral vector particles so that continuous productions of the vector occurred within the tumour mass in situ.
  • proliferating tumour cells can be successfully transduced in vivo if mixed with retroviral vector- producing cells.
  • Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into preexisting viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).
  • Immunoliposomes are especially useful in targeting to cancer cell types which over-express a cell surface protein for which antibodies are available (see Table for examples).
  • MPB-PE N-[4-(p- maleimidophenyl)butyryl]-phosphatidylethanolamine
  • MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface.
  • the liposome is conveniently loaded with the DNA or other genetic construct of the invention for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 ⁇ m and 0.2 ⁇ m pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB-PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the tumour.
  • monoclonal antibodies or other molecules that bind to tumour cell surface antigens are useful in targeting the DNA construct of the invention, for example as part of an immunoliposome.
  • Monoclonal antibodies which will bind to many of these antigens are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens.
  • the antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]).
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982).
  • non-human antibodies can be “humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • Such "humanized” antibodies, or fragments thereof, are preferred as they may give rise to a lower anti- antibody reaction than rodent antibodies.
  • variable heavy (V H ) and variable light (V L ) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855). That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V H and V L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341, 544).
  • dAbs single domain antibodies
  • ScFv molecules we mean molecules wherein the V H and V L partner domains are linked via a flexible oligopeptide.
  • antibody fragments rather than whole antibodies
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
  • Effector functions of whole antibodies, such as complement binding, are removed.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.
  • Other molecules immunologically reactive with the target cell surface molecule include, for example minimal recognition units (MRU) and complementarity dete ⁇ nining regions.
  • adenoviruses carrying external DNA via an antibody-polylysine bridge see Curiel Prog. Med. Virol. 40, 1-18
  • transferrin-poly cation conjugates as carriers
  • a polycation-antibody complex is formed with the DNA construct or other genetic construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody.
  • the polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone.
  • the adenovirus because it contains unaltered fibre and pentos proteins, is internalized into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is polylysine.
  • the DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.
  • a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids.
  • Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell.
  • the transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs).
  • complexes of transferrin-polycation and the DNA constructs or other genetic constructs of the invention are supplied to the tumour cells, a high level of expression from the construct in the cells is expected.
  • High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used.
  • This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.
  • This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types.
  • tumours may be desirable to locally perfuse a tumour with the suitable delivery vehicle comprising the genetic construct for a period of time; additionally or alternatively the delivery vehicle or genetic construct can be injected directly into accessible tumours.
  • delivery vehicle or genetic construct can be injected directly into accessible tumours.
  • naked DNA and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the patient to be treated.
  • Non- viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1144.
  • a further aspect of the invention provides a composition comprising genetic construct as defined in the invention and means for introducing said genetic construct into a cell, preferably the cell of an animal body.
  • adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle.
  • Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein.
  • Mutant adenoviruses which replicate selectively in p53-def ⁇ cient human tumour cells such as those described in Bischoff et al (1996) Science 214, 373- 376 are also useful for delivering the genetic construct of the invention to a cell.
  • a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention.
  • suitable viruses or virus-like particles include HSV, AAV, vaccinia and parvovirus.
  • tumour suppressor gene is p53.
  • the p53 gene is known to be mutated or deleted in over 50% of human cancers.
  • the seven most common human tumour-derived p53 mutants are Hisl75,
  • the distribution of p53 mutations identified in various tumour types is described in Holstein et al (1991) Science 253, 49-53 and de Fromental & Soussi (1992) Genes, Chromosomes & Cancer 4, 1-15.
  • the human tumour to be treated has a mutant p53 wherein the p53 protein has a mutation in the DNA binding domain (residues 102- 292). Missense point mutations in this domain are associated with the development of human cancers. Human cancer cells which have p53 Val 138 or p53 His273 mutations are particularly suited to treatment with the genetic constructs of the present invention.
  • the promoters useful in the genetic construct of the first aspect of the invention are those promoters which are substantially repressed by wild-type p53 gene products but are active in the presence of a variant p53 gene product encoded by a mutant p53 gene or in the absence of any p53 gene product.
  • the product of the p53 gene may be absent due to a deletion in both copies of the p53 gene or by mutations in the p53 gene which prevent expression of its product or a combination of deletion and mutation.
  • the promoters described in Santhanam et al (1991) Proc. Natl. Acad. Sci. USA 88, 7605-7609 and in Ginsberg et al (1991) Proc. Natl. Acad. Sci. USA 88, 9979-9983 may also be suitable for use.
  • the promoter may be any one of the promoters from c-fos, -actin, p53, hsc70, c-jun and IL-6 or a suitable fragment or portion or variant thereof which retains the appropriate activity.
  • the promoter element is one which is active in proliferating but is substantially inactive in non-proliferating cells.
  • the level of /37207 is one which is active in proliferating but is substantially inactive in non-proliferating cells.
  • 19 expression of the cytotoxic gene in a proliferating is at least three times the level in a non-proliferating cell; more preferably, the level is at least ten times, still more preferably, it is at least fifty times.
  • a proliferating cell is one which is actively going through the cell cycle, ie is not in G 0 .
  • the promoter distinguishes between proliferating cells and quiescent cells.
  • Quiescent cells are out of cycle or are G 0 cells.
  • the promoter element of the genetic construct is the human topoisomerase He. gene promoter or a portion thereof.
  • proliferative cell-specific or selective expression can be obtained using promoters which drive expression of cell-cycle genes such as cyclin and PCNA. These may be used in conjunction with the promoter element of the genetic construct of the invention to confer proliferative cell selectivity.
  • the promoter element comprises the polynucleotide described in Figure 8. (This is the same as Figure 1 in Isaacs et al (1996) J. Biol. Chem. 271, 16741-16747).
  • the promoter element comprises the polynucleotide defined by the sequence -500 to +93 in Figure 8; more preferably the promoter element comprises the polynucleotide defined by the sequence -400 to +.93 in Figure 8; and still more preferably the promoter element comprises the polynucleotide defined by the sequence -200 to +93 in Figure 8.
  • the promoter element comprises at least the polynucleotide defined by the sequence -144 to +93 in Figure 8.
  • the transcription start site in Figure 8 is nucleotide + 1.
  • promoter particularly attractive for use in the invention are (a) its very small size; and (b) that two different regulatory processes act independently on such a short fragment of DNA, that is to say that the topoisomerase He. promoter is active in proliferating cells but not in non-proliferating cells, and the promoter is substantially inactive in the presence of wild-type p53 but is active in the absence of wild-type p53.
  • the topoisomerase lice promoter is expressed in proliferating cells (including tumour cells) but since proliferating cells in the body (other than tumour cells) have wild-type p53, the topollce promoter in the genetic construct of the invention is under negative regulation in these cells.
  • Topoisomerase lie is expressed in all normal proliferating cells; however, specificity arises from the higher proliferation index of the tumour cells coupled with a higher level of topoisomerase lice promoter activity in the absence of wild-type p53.
  • topoisomerase lice promoter in which one or more base pair changes or deletions have been made will also work in the invention, and that others will not.
  • inverted CCA AT box number 2 (ICB2 in Figure 8) from ATTGG to ATTCC abolishes the down regulation of promoter activity that normally occurs when cells stop proliferating.
  • a promoter carrying this mutation would express in both proliferating tumour cells and non-proliferating normal cells.
  • the usefulness of a particular mutant promoter can be determined by linking the mutant promoter to a reporter gene, such as the chloramphenicol acetyl transferase (CAT) gene, or, indeed the topoisomerase lice gene itself and studying its expression in particular tumour and normal cells at different stages of the cell cycle (see Isaacs et al (1996) J. Biol. Chem. 271, 16741-16747; Hochhauser et al (1992) J. Biol. Chem. 267, 18961- 18965; and Sandri et al (1996) Nucl. Acids Res. 24, 4464-4470).
  • CAT chloramphenicol acetyl transferase
  • the invention includes the use of a human DNA topoisomerase lice promoter element to differentially express therapeutic genes in tumour cells which lack wild-type p53.
  • Example 1 The results presented in Example 1 define a 101 bp region of the topoisomerase lice (topo lice) promoter which is negatively regulated by wild-type p53, but not by mutant p53.
  • the p53 gene is mutated or deleted in over 50% of human malignancies.
  • expression from the topo lice promoter element will not be repressed in the cells of many human tumours (this may be a reason for the differential sensitivity of tumour cells to topoisomerase II-targeting drugs).
  • expression from the topo lice promoter is elevated in cells which are rapidly dividing. This provides another level of selectivity of expression since tumour cells are highly proliferative.
  • the topo lice promoter element drives the expression of a therapeutic gene such that it was expressed at higher levels in tumour cells (lacking wild-type p53) than in normal tissues (containing wild-type p53).
  • the therapeutic gene could encode, for example, an enzyme capable of activating an inert form of a drug to a form which will kill the cell. Thus cell killing would selectively occur in the tumour cells where the enzyme was present at higher levels than in normal cells.
  • additional elements conferring tissue specificity and/or enhanced expression levels could be incorporated into the vehicle used to deliver the therapeutic gene.
  • the topo lice promoter element could thus be generally useful in cancer gene therapy.
  • the tumour suppressor gene is the Rb (retinoblastoma) gene.
  • the retinoblastoma susceptibility gene product, pRb acts as a transcriptional inhibitor and mutation or deletion of pRb (which occurs in at least some tumour cells) derepresses gene expression.
  • the mechanism appears to be the release from sequestration (by pRb) of a transcription factor that then activates gene expression.
  • Rb loss of function is particularly associated with retinoblastomas, osteosarcomas, SCLCs, prostate and breast cancers.
  • Promoters repressed by Rb and therefore useful in the practice of the invention include those from the genes for c-myc, cdc2 and IL-6 (see, for example, Salcedo et al (1995) Arch. Med. Res. 26, 5157-5162; Yu et al (1992) J. Biol. Chem. 267, 10203-10206; Hamel et al (1992) Mol. Cell. Biol. 12, 3431-3438; Dalton (1992) EMBO J. 11, 1797-1804; and Santhanam et al (1991) Proc. Natl. Acad. Sci. USA 88, 7605-7609. ⁇ ⁇ « Struktur ⁇ ⁇ -, PCT/GB98/00551 O 98/37207
  • the genetic construct of the invention (whether it contains a topoisomerase lice-derived promoter element or some other suitable promoter element as defined) further comprises a genetic element which generally enhance the expression of the cytotoxic gene, at least once its expression has been activated in the tumour cell.
  • Suitable enhancers include viral enhancers such as those from SV40, CMV or retroviral LTRs which would generally enhance expression of the cytotoxic gene.
  • viral enhancers such as those from SV40, CMV or retroviral LTRs which would generally enhance expression of the cytotoxic gene.
  • the genetic construct of the invention (whether it contains a topoisomerase lice-derived promoter element or some other suitable promoter element as defined) further comprises a genetic element which confers tissue-selective or cell-type-selective expression, or further comprises a genetic element which confers further tumour-selectivity.
  • the tyrosinase and TRP-1 genes both encode proteins which play key roles in the synthesis of the pigment melanin, a specific product of melanocytic cells.
  • the 5' ends of the tyrosinase and tyrosinase-related protein (TRP-1) genes confer tissue specificity of expression on genes cloned downstream of these promoter elements.
  • Prostate-specific antigen is one of the major protein constituents of the human prostate secretion. It has become a useful marker for the detection and monitoring of prostate cancer.
  • the gene encoding PSA and its promoter region which directs the prostate-specific expression of PSA have been described (Lundwall (1989) Biochem. Biophys. Res. Comm. 161, 1151-1159; Riegman et al (1989) Biochem. Biophys. Res. Comm. 159, 95-102; Brawer (1991) Ada Oncol. 30, 161-168).
  • Carcinoembryonic antigen is a widely used tumour marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues.
  • the complete gene encoding CEA has been cloned and its promoter region analysed.
  • a CEA gene promoter construct containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the adenocarcinoma cell line SW303, compared with the HeLa cell line. This indicates that s-acting sequences which convey cell type specific expression are contained within this region (Schrewe et al (1990) Mol. Cell. Biol. 10, 2738-2748).
  • the mucin gene, MUC1 contains 5' flanking sequences which are able to direct expression selectively in breast and pancreatic cell lines, but not in non-epithelial cell lines as taught in WO 91/09867.
  • the alpha-fetoprotein (AFP) enhancer may be useful to drive pancreatic tumour-selective expression (Su et al (1996) Hum. Gene Ther. 1, 463- 470).
  • the genetic constructs of the invention can be prepared using methods well known in the art.
  • a variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.
  • the combination of these activities therefore generates blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying polymeric linker sequences at their ends.
  • These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.
  • a desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
  • the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • the present invention also relates to a host cell transformed with a genetic (preferably DNA construct) construct of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).
  • Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney- derived COS-1 cells available from the ATCC as CRL 1650.
  • Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.
  • reagents useful in transfecting such cells for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life
  • Electroporation is also useful for transforming cells and is well known in the art for transforming yeast cell, bacterial cells and vertebrate cells.
  • bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25 ⁇ FD.
  • Successfully transformed cells ie cells that contain a DNA construct of the present invention
  • cells resulting from the introduction of an expression construct of the present invention can be grown to produce the cytotoxic gene product as defined in the invention.
  • Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208.
  • the presence of the protein in the supernatant can be detected using antibodies as described below.
  • successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein.
  • cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • the genetic construct is a plasmid DNA construct it can be purified.
  • the DNA construct of the invention is purified from the host cell using well known methods.
  • plasmid vector DNA can be prepared on a large scale from cleaved lysates by banding in a CsCl gradient according to the methods of Clewell & Helinski (1970) Biochemistry 9, 4428-4440 and Clewell (1972) J. Bacteriol. 110, 667-676. Plasmid DNA extracted in this way can be freed from CsCl by dialyse against sterile, pyrogen-free buffer through Visking tubing or by size-exclusion chromatography.
  • plasmid DNA may be purified from cleared ly sates using ion-exchange chromatography, for example those supplied by Qiagen. Hydroxy apatite column chromatography may also be used.
  • composition comprising the genetic construct and means for introducing said genetic construct into a cell; and virus or virus-like particle comprising a genetic construct of the invention can all be used to treat a cancer patient.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a genetic construct as defined; or a composition comprising the said genetic construct and means for introducing said genetic construct into a cell; or said virus or virus-like particle and a pharmaceutically acceptable carrier.
  • the carrier or carriers must be "acceptable" in the sense of being compatible with the genetic construct or composition or virus or virus-like particle of the invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • a further aspect of the invention provides a method of treating a host with a cancer, or a host which host may develop cancer, the method comprising administering to said host a therapeutically effective amount of a genetic construct of the invention or a virus or virus-like particle of the invention or a composition comprising a genetic construct of the invention and means for introducing said genetic construct into a cell.
  • the host is a mammal with a cancer; more preferably the host is a human patient with a cancer.
  • the tumour suppressor gene status of the host is determined either prior to or during treatment to determine that the tumour suppressor gene has been mutated in a relevant place in the gene or deleted. This can be done by methods known in the art such as PCR/sequencing or immunohistochemistry.
  • tumour suppressor gene which encode a portion of the tumour suppressor gene product which is involved, directly or indirectly, in the control of transcription from the said promoter.
  • the appropriate genetic construct can be chosen for administration to a tumour type in which it will be maximally effective.
  • the tumour is shown to express mutant p53 or p53 is absent, the genetic construct comprises a promoter element responsive to p53 in the said manner.
  • tumours it will be appreciated that it is preferable to administer the said genetic construct or said virus or virus-like particle or said composition locally at the site of the tumour or into the vasculature of the tumour.
  • the vascularisation of many tumours, and the high permeability of tumour vasculature, allows efficient delivery to tumours via this route.
  • said genetic construct or said virus or virus-like particle or said composition may be administered systemically.
  • cytotoxic gene product When the cytotoxic gene product is directly cytotoxic it may be sufficient to administer said genetic construct or said virus or virus-like particle or said composition alone. However, as discussed below, it may be desirable to use the therapeutic method of the present invention with other forms of therapy.
  • the cytotoxic gene product is indirectly cytotoxic it is preferable that the said genetic construct or said virus or virus-like particle or said composition is administered to the host and that any additional component to make the method cytotoxic is also administered to the host.
  • the additional component such as a prodrug activated by an enzyme which is the indirectly cytotoxic gene product
  • the invention provides a method of treating a mammal harbouring a tumour.
  • the mammal is first prepared for tumour therapy by administering a said genetic construct or said virus or virus-like particle or said composition of the invention and allowing the genetic material therein to localise to and be expressed in the tumour.
  • the indirectly cytotoxic gene encodes an enzyme which is able to convert a relatively non-toxic prodrug into a cytotoxic drug.
  • the expression of said enzyme in the tumour can, if desirable, be measured in the tumour using methods well known in the art.
  • the method then further comprises administering to the mammal a component (preferably a prodrug) which is acted upon by the indirectly cytotoxic gene product (preferably an enzyme which converts said prodrug to a cytotoxic drug).
  • the said genetic constructs or said virus or virus-like particles or said compositions are administered in any suitable way, usually parenterally, for example intravenously, intraperitoneally or intra-vesically, in standard sterile, non- pyrogenic formulations of diluents and carriers, for example isotonic saline (when administered intravenously).
  • the invention also provides a therapeutic system comprising a said genetic construct or a said virus or virus-like particle or a said composition wherein said cytotoxic gene is indirectly cytotoxic and an additional component acted upon by said indirectly cytotoxic gene or its product and which becomes cytotoxic by said action.
  • the indirectly cytotoxic gene is cytosine deaminase or thymidine kinase and the said additional component is 5-fluorocytosine or ganciclovir, respectively.
  • the invention also provides a therapeutic system comprising a said genetic construct or a said virus or virus-like particle or a said composition and means for determining the tumour gene status of a tumour in a patient.
  • the said means include reagents for detecting mutations in tumour suppressor genes.
  • Such means and reagents are well known in the art and include PCR primers, restriction enzymes, oligonucleotide probes and the like. Mutants of p53 can also be detected using antibodies.
  • one embodiment of the invention provides for the introduction of a genetic construct of the invention into the germ line.
  • the genetic construct would lay "dormant" (ie without expressing substantially any cytotoxic gene product) in normal, non-cancer cells.
  • the cytotoxic gene would be expressed to a cytotoxic level in those cells where a mutation or deletion of the tumour suppressor gene occurred (as when the cell becomes cancerous or more virulently cancerous).
  • the system may be used as a monitor of, and destroyer of cancer cells, in mammals, especially humans, who are healthy individuals. It will be appreciated that when the cytotoxic gene is an indirectly cytotoxic gene that the additional component (such as a prodrug when appropriate) is administered periodically to "sweep out" (ie kill) the tumour cells.
  • the preferred method is to treat a host which host has a cancer.
  • the memods of the invention can be used to treat any cancer in which mutation of a tumour suppressor gene is implicated.
  • p53 is mutated in at least 50% of all human cancers.
  • the method is particularly suitable for:
  • Tumour types displaying a high frequency of p53 mutations (these are generally not as responsive to currently available therapies as tumours that rarely harbour p53 mutations).
  • types displaying a high frequency of p53 mutations include lung, colon, head and neck, lymphoma, pancreas and stomach.
  • Breast and prostate have a lower frequency (20-25 %) but are common cancers.
  • Tumour types expressing high levels of topoisomerase lice include Hodgkin's disease, high grade lymphomas, squamous non-small cell lung cancers, seminomas and drug resistant colon tumours. Tumour types with both these attributes would be particularly suitable.
  • the method is used to treat cancers in which the p53 tumour suppressor gene is mutated or deleted. It is particularly preferred to use a genetic construct wherein the element comprises the human topoisomerase lice promoter or a portion thereof.
  • the genetic construct comprising the human topoisomerase lice promoter or a portion thereof is particularly preferred for the treatment of such tumours.
  • an analysis of the growth fraction of a particular tumour is performed.
  • This can be done using methods well known in the art such as by immunohistology using antibodies to cell proliferation antigens such as Ki-67 or proliferating cell nuclear antigen (PCNA) (see, for example,
  • the p53 status of the tumour is carried out using methods well known in the art, for example by PCR/sequencing or by immunohistochemistry.
  • Preferred mutations are those that relieve repression of the topoisomerase lice promoter.
  • tumour types where p53 is mutated frequently are given above; thus, it may not be necessary to determine the p53 status in many cases.
  • the method of treatment may include determination of the p53 status, and in particular the presence of particular p53 mutations, before or during treatment.
  • Cytotoxic chemo- or radio-therapy is known to upregulate wild-type p53 in normal tissues thereby inhibiting expression of the cytotoxic gene product from the genetic construct.
  • Cancer chemotherapeutic agents include: alkylating agents including nitrogen mustards such as mechlorethamine (HN 2 ), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole- carboxamide); Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5- fluorouracil; 5-FU), floxuridine (fluorode
  • Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L- asparaginase; and biological response modifiers such as interferon alphenomes.
  • VLB vinblastine
  • epipodophyllotoxins such as etoposide and teniposide
  • antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C)
  • enzymes such as L- asparaginase
  • Miscellaneous agents including platinum coordination complexes such as cisplatin (cw-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N- methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,p'-OOO) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.
  • platinum coordination complexes such as cisplatin (cw-DDP) and carboplatin
  • anthracenedione such as mitoxantrone and anthracycline
  • substituted urea such as hydroxyurea
  • methyl hydrazine derivative such as procarbazine (N- methylhydrazine, MI
  • the genetic construct is one which comprises an element responsive to the p53 gene product it is desirable that the cytotoxic gene is indirectly cytotoxic and that its product activates a prodrug. Any cytotoxic drug that was released from the prodrug and which was available to normal cells which contain wild-type p53 will activate p53 expression and switch off expression of the cytotoxic gene from the genetic construct that may have been taken up by the normal cells. This should limit the overall cytotoxic effect in adjacent normal tissue.
  • the topoisomerase lice gene promoter may be used in a dual control unit.
  • a preferred embodiment of the invention includes a first genetic construct comprising a topoisomerase lice gene promoter and a cytotoxic gene and (either as part of the first genetic construct or as a separate genetic construct) a second genetic construct in which expression of a second gene for down-regulating the cytotoxic gene in non-tumour cells is controlled by a second promoter that is up-regulated in non-tumour cells.
  • This type of dual control unit system is described in WO 97/12970 incorporated herein by reference.
  • the dual construct strategy exploits differences in the transcriptional activation and stability functions of the p53 protein between normal and tumour cells. More particularly, it utilises the dual transcriptional regulatory function of wild-type p53 (wtp53) to eliminate or minimise expression of the antitumour gene in normal cells and to enhance its expression in tumour cells.
  • the strategy proposes a combination of two genetic units.
  • genetic unit 1 the topoisomerase lice promoter linked to a cytotoxic gene
  • the cytotoxic gene is controlled by a promoter whose function is suppressed by wtp53, but is not suppressed by mutant p53 (mp53), indeed it may even be up-regulated by mp53.
  • a gene for down-regulating the cytotoxic gene in normal cells is controlled by a promoter containing the p53 binding site, and which is therefore potently up-regulated by wtp53 but not by mp53.
  • the wtp53 in normal cells is used to up-regulate a gene which downregulates the expression of the cytotoxic agent in those normal cells.
  • the down- regulating gene may for example express antisense RNA to the cytotoxic gene, or a specific ribozyme for cytotoxic gene RNA, or a specific transcription suppressor for the cytotoxic gene.
  • the p53 tumour suppressor gene product is believed to control at least in part the expression of the cytotoxic gene; however, the dual construct strategy may be used in an analogous fashion with respect to any tumour suppressor gene product which is able to control at least in part the expression of the cytotoxic gene.
  • this embodiment of the invention is preferably based on the p53 gene, because of its central role in tumour suppression and the differences- in its function in normal and tumour cells.
  • the general concept of this aspect of the invention may be applicable using other genes to upregulate the cytotoxic agent in tumour cells and to down-regulate it in normal cells. Since the natural tumour suppressor pathways are complex and involve many genes, at least some of them could be used in a similar fashion.
  • Constructs for use in this embodiment of the invention comprise two separate genetic units.
  • the cytotoxic gene is driven by a topoisomerase lice promoter ("type I") promoter which is not suppressed by, and may even be stimulated by, mutant p53 but is suppressed by wild-type p53.
  • the second genetic unit is designed to suppress any leaky expression of the cytotoxic gene in normal cells.
  • the promoter for this unit typically contains the p53 consensus binding sequence, which is only activated by wild-type p53, within the context of a minimal promoter comprising a transcriptional start site and TATA Box.
  • the p53 binding sequence may be included upstream of the minimal promoter or downstream. It may be useful if the p53 binding sequence is downstream from the promoter in genetic unit 2.
  • tumour suppression pathway Apart from p53, other genes associated with the tumour suppression pathway may be utilised in the present invention.
  • one approach employs an antisense construct (eg for the cytotoxic gene) driven by the type II promoter (eg p53 responsive). No antisense transcript should therefore be produced in tumours harbouring only mutant p53, but this construct should be transcriptionally activated within normal cells and lead to down-regulation of any functional cytotoxic gene transcript driven by the type I promoter. It should be noted that even though normal cells express only very low levels of wild-type p53, the introduction of gene constructs by transfection has been shown to induce expression of detectable wild-type p53. Such induction may therefore help to improve discrimination between normal and tumour cells. Low dose radiation, which would be expected to induce wt p53 in normal but not tumour cells, may also improve discrimination.
  • an antisense construct eg for the cytotoxic gene driven by the type II promoter (eg p53 responsive). No antisense transcript should therefore be produced in tumours harbouring only mutant p53, but this construct should be transcriptionally activated within normal cells and lead to down-regulation of any functional cyto
  • a third approach uses a sequence-specific transcriptional suppressor.
  • transcriptional suppressors have been characterised. Experiments may include use of the binding domain of the yeast Gal-4 transcription factor and the suppression domain of the Drosophila even-skipped protein under the control of the type II promoter to suppress expression by means of a tandem repeat of the Gal-4 target sequence cloned into the type I promoter.
  • a combination of two or more such approaches may be utilised. However, the most appropriate approach may vary according to the cytotoxic gene being expressed.
  • the sequence expressed by the Type II unit must be tailored to the sequence expressed by the Type I unit, adding a level of complexity to experimentation.
  • the use of a transcriptional suppressor has the advantage of allowing the construction of a "universal cassette" into which different coding /37207
  • 40 sequences eg different cytotoxic genes, may be inserted under the control of the Type I promoter without there being a need for tailoring of the Type II unit.
  • the type I and type II promoter constructs may be tested individually, to assess the magnitude of the effects which can be generated. Co-transfections may then be carried out to assess the combined effects of both constructs in determining overall expression levels of a reporter gene such as luciferase in the presence of wild-type or mutant p53.
  • both genetic units may be incorporated into a single construct in either a head-to-head, tail-to-tail or head-to-tail arrangement, in the latter case having the type II promoter upstream of the type I promoter, or vice versa.
  • Figure 1 shows the concentration-dependent inhibition of human topoisomerase lice promoter activity by wild-type p53. Different concentrations of wild-type p53 expressing plasmid were co-transfected with 10 ⁇ g of topoisomerase IIce-CAT construct in SKOV3 cells. After 48 hours, cells were lysed and a CAT assay was performed on the extract.
  • Figure 2 shows the topoisomerase lice promoter activity in cells expressing no p53 (SKN), mutant p53 (SK23 at 37 °C) or wild-type p53 (SK23 at 32 °C).
  • the transfected cells were divided between two dishes, one of which was grown at 32°C and the other at 37°C. After 48 hours, cells were lysed and a CAT assay was performed on the extract. Three independent assays of CAT activity on extracts from SKN cells and two independent assays on extracts from SK23 cells are shown.
  • Figure 3 shows a Western blot analysis showing transcriptional activation of p21 upon shift down of Saos-2 ts p53 cells to 32 °C. Proteins were transferred to a nitrocellulose membrane and probed with antibodies to both p53 and p21. The positions of p53 and p21 are indicated on the right. Note the presence of p21 in the sample from cells grown at 32 °C, but not those grown at 37 °C.
  • Figure 4a is a diagrammatic representation of the promoter constructs employed. The 5' limit of each promoter fragment is indicated on the left. The position of the GC boxes (GC), inverted CCAAT boxes (ICB), ATF site (ATF) and growth hormone reporter gene (HGH) are indicated. The major transcription start site is marked with an arrow.
  • Figure 4b shows the percentage of growth hormone (HGH) expression 120 h after transfection of Saos-2 ts p53 cells with various different lengths of the topoisomerase lice promoter (indicated on the X axis).
  • HGH growth hormone
  • Figure 4c shows the percentage of growth hormone (HGH) expression as a function of cell number 120 h after transfection of Saos-2 ts p53 cells 5 with 144 bp and 101 bp topoisomerase lice promoter constructs.
  • the shaded bars represent those cells cultured at 37 °C (p53 mutant) and the black bars represent cells grown at 32 °C (p53 wt).
  • Figure 5 shows the percentage of growth hormone expression (HGH) 120 o h following transfection of Saos-2 cells with mutated forms of the topoisomerase lice promoter (ICB1 and GC1) alone (shaded bars), or following co-transfection with the wild-type p53-expressing construct pLSVhp53c62 (black bars).
  • HGH expression level in the absence of p53 was arbitrarily given a value of 100%.
  • Figure 6 shows the cell cycle distribution of Saos-2 ts p53 cells transfected with the topoisomerase lice 101 bp promoter construct and the control promoters PGKGH (fusion construct of human phosphoglycerate kinase 1 promoter and human growth hormone) and FGH (fusion construct of i mouse ferritin heavy subunit promoter and human growth hormone) .
  • the cells were cotransfected with CD2 cell surface marker and the cell cycle distribution of the CD2-positive transfectants was analysed 120 hours after transfection.
  • the upper traces represent cells cultured at 37 °C and the lower black traces cells cultured at 32°C.
  • Figure 7 shows the percentage of growth hormone expression (HGH) 120 h after transfection of Saos-2 ts p53 cells with the control promoters PGKGH and FGH.
  • the shaded bars represent expression of HGH when the cells were cultured at 37 °C (mutant p53) and the black bars cells i cultured at 32°C (wt p53).
  • the expression level of HGH at 37°C in each case was arbitrarily given a value of 100% .
  • Figure 8 shows a, sequence of the human topoisomerase lice promoter.
  • Bases are numbered (as indicated on the left) with respect to the major transcription start site, which is designated + 1 and marked with an arrow.
  • Putative cw-acting elements include GC boxes (Spl sites) at position -562 and -51, ICBs at positions -385, -259, -175, -108, and -68, and an ATF site at -226.
  • the ATG start codon is overlined.
  • b schematic representation of putative transcription factor binding motifs and the truncated promoter derivatives constructed. The 5' limit of each promoter fragment is indicated on the left.
  • the major transcription start site is marked with an arrow.
  • the positions of the GC boxes (GC), ICBs Q, ATF site (ATF), and growth hormone reporter gene (HGH) are indicated.
  • Figure 9 shows that the CRB and 3H10 antisera detect 170 kDa and 180 kDa proteins, respectively, in human cell nuclear extracts.
  • a 0.35 M NaCl nuclear protein extract prepared from NCI460 cells (lanes 2 and 4) or NCI460/pV8 cells (lanes 1 and 3) was electrophoresed alongside molecular weight standards on a 9% SDS-polyacrylamide gel, transferred to Hybond-N, and the membrane was exposed either to the CRB antiserum (Lanes 1 and 2) or to a mixture of the CRB and 3H10 antisera (Lanes 3 and 4).
  • Antibody detection was the ECL system.
  • Molecular weights (in kDa) are indicated on the left.
  • the positions of the 170 kDa topoisomerase lice and the 180 kDa topoisomerase II/3 protein are indicated on the right.
  • Figure 10 shows the staining of tissues for topoisomerase lice and ⁇ .
  • Left hand side pictures are stained for topoisomerase ce ( Figures A, C, E and G).
  • Right hand side pictures are stained for topoisomerase ⁇ (B, D, F, H).
  • a and B show tonsil sections at low power and C and D are tonsil sections at higher power.
  • Topoisomerase ce (A & C) is mainly restricted to the larger cells in the germinal centre (centroblasts), whereas topoisomerase ⁇ (B & D) is very widely distributed in all cell types, including the B and T cell areas.
  • E and F show sections of a squamous cell carcinoma of the lung and G and H show a case of Hodgkin's disease.
  • the distribution of topoisomerase lice staining is similar to that seen with anti-proliferation- associated antibodies (such as Ki-67), whereas topoisomerase ⁇ is found in the majority of cell types, including Reed-Sternberg cells in Hodgkin's disease (Figure H).
  • FIG 11 shows the expression of topoisomerase lice and ⁇ in cytospin preparations of the SUDHL-1 cell line using immunoperoxidase staining.
  • B and D localisation of topoisomerase lice using the CRB antibody. The antigen is present on the whole of the nucleus with stronger staining in nucleolar areas (cells in mitosis show antigen associated with condensed chromatin, not in the cytoplasm).
  • DNA topoisomerase lice is an essential enzyme for chromosome segregation during mitosis. Consistent with a cell division-specific role, the expression of the topoisomerase lice gene is strongly influenced by the proliferation status of cells.
  • the p53 protein is one of the most important regulators of cell cycle progression in mammals, with an apparent dual role in the induction of cell cycle arrest following cytotoxic insults and in the regulation of the apoptotic cell death pathway.
  • p53 plays a role in regulating expression of the human topoisomerase lice gene. We show that wild-type, but not mutant, p53 is able to decrease substantially the activity of the foil length topoisomerase lice gene promoter.
  • topoisomerase lice is independent of all characterised transcription factor binding sites and is directed at the minimal gene promoter.
  • expression of wild-type p53 induces down-regulation of the human topoisomerase lice promoter by acting on the basal transcription machinery.
  • Table 1 Percentage inhibition of CAT expression from the 2.5 kb fragment of topoisomerase Il ⁇ promoter by wild-type or mutant p53. The vector only control was arbitrarily given a value of 100%.
  • p53 was capable of down- regulating the topoisomerase lice gene promoter
  • an expression construct designated pLTRp53cGVal 135, encoding a murine p53 protein that assumes a wild-type conformation at 32 °C, but a mutant conformation at 37 °C (Michalovitz et al, 1990).
  • the construct containing the CAT reporter gene under the control of the 2.5 kb fragment of the topoisomerase lice promoter was transfected into a clone of SKOV3 cells (designated SK23a), into which the construct encoding the temperature sensitive p53 had been stably integrated.
  • p53 acts on the minimal topoisomerase lice gene promoter
  • the wild type p53 was shown to be functional since expression of the p53-regulated p21 protein (WAF1/CIP1) was seen in cells grown at 32 °C but not at 37 °C ( Figure 3).
  • WAF1/CIP1 p53-regulated p21 protein
  • Figure 3 For the analyses of Saos-2 ts p53 cells, we used a series of topoisomerase lice promoter constructs generated for analysis of the growth-state regulation of the promoter (Isaacs et al, 1996). Instead of the CAT gene, these constructs contain human growth hormone (HGH) as a reporter gene cloned downstream of various different fragments of the human topoisomerase lice promoter.
  • HGH human growth hormone
  • HGH from constructs containing 617, 210, 144 and 101 bp of the promoter was in each case substantially lower in Saos-2 ts p53 cells cultured at 32 °C, than in those cells cultured at 37 °C ( Figure 4b).
  • Figure 4c To exclude effects of incubation temperature on cell cycle transit times, we measured cell numbers at the end of each experiment. HGH expression o as a function of cell number still showed a substantially reduced level at 32°C as compared to 37°C ( Figure 4c). Indeed, temperature shift did not dramatically affect cell proliferation over the time-course of these experiments, since cell numbers generally differed by no more than 50% in me transfected cell populations incubated at 32°C and 37°C.
  • p53 acts specifically on the topoisomerase Il . gene promoter
  • wild-type p53 is a negative regulator of the activity of the human topoisomerase lice promoter and that this effect is mediated o through the minimal sequences required for topoisomerase lice promoter activity. This regulation is apparently independent of a perturbation in the cell cycle distribution of the transfected cells, and has been demonstrated in different cell lines using different constructs encoding either human or murine p53. Moreover, we have shown that p53 has some apparent
  • p53 interacts with DNA in a sequence-specific fashion binding to DNA o containing two contiguous monomers of the sequence 5'-PuPuPuC (T/A)
  • topoisomerase lice promoter does not contain a precise match for this consensus p53 binding element. However, a similar sequence
  • topoisomerase lice promoter could be responding to cell cycle perturbation induced by p53, and not to a direct effect of p53 on the transcription machinery. No evidence was obtained for accumulation of cells in any particular cell cycle phase, in those cells in which wild-type p53 was expressed, at least over the time course of these experiments.
  • the other genetic changes mat accompany the acquisition of a transformed state, such as alteration in the retinoblastoma susceptibility gene (which is not expressed in Saos-2 cells) or cyclin-dependent kinase inhibitors, could be influencing the efficiency with which wild-type p53 can mediate cell cycle arrest in the cell lines chosen for study here.
  • me topoisomerase lice promoter responds to growth arrest signals (Isaacs et al, 1996).
  • the regulatory elements in the promoter that respond to changes in growth state are distinct from those responding to p53, and are located upstream of the minimal topoisomerase lice gene promoter defined in this work.
  • the 101 bp minimal promoter which we have shown to be regulated by p53, lacks normal negative regulation brought about by inhibition of proliferation (Isaacs et al, 1996).
  • topoisomerase II is a cell cycle regulated gene, recent data indicate that the variation in mRNA expression during the cell cycle is largely due to changes in transcript stability, not promoter activity (Goswami et al, 1996).
  • At least one action of p53 as a negative regulator of gene transcription appears to be directed towards components of the basal transcription machinery.
  • An important feature of this effect may be the formation of complexes between p53 and TATA box-binding protein (TBP)-associated factors, possibly TBP itself.
  • TBP TATA box-binding protein
  • Liu and Berk (1995) have shown recently that p53 may act through direct or indirect interactions with both TFHB and TFIID, which act as basal transcription factor complexes.
  • Our data are consistent with the hypothesis that wild-type p53 acts via negatively regulating the basal transcription machinery required to effect expression of me topoisomerase lice gene.
  • p53 appears to disrupt the ability of a protein complex to bind to DNA that includes either the transcription factor Spl (GC box binding factor) (Perrem et al, 1995), or CBF (CCAAT box binding factor) (Agoff et al, 1993).
  • Spl transcription factor
  • CBF CCAAT box binding factor
  • the human ovarian cancer cell line SKOV3 which does not express p53 mRNA or protein (Yaginuma and Westphal, 1992), was grown in RPMI- 1640 medium supplemented with 10% foetal calf serum (FCS).
  • FCS foetal calf serum
  • the SK23a and SKN cell clones (Vikhanskaya et al, 1994) were bom derived from SKOV3 cells.
  • SKOV3 cells were co-transfected wim a plasmid encoding murine temperature sensitive mutant p53 (Michalovitz et al, 1990) and the pSV2neo vector containing the neomycin selectable marker genes.
  • the SKN cell line was derived by transfection of SKOV3 cells wim the neomycin expression vector alone, and served as a negative control.
  • the human osteosarcoma cell line Saos- 2 which expresses no p53 (Diller et al, 1990) and no functional pRb protein (Huang et al, 1988), as well as a derivative (designated Saos-2 ts p53) that stably expresses a temperature sensitive human p53 protein due to a valine to alanine substitution at amino acid 138, were maintained in RPMI-1640 supplemented with 10% FCS.
  • DMEM Dulbecco's modified Eagles 's Medium
  • the construct encoding the wild-type human p53 designated pLSVhp53c62 (Zakut-Houri et al, 1985) utilises me SV40 early promoter in the vector pLSV.
  • the construct for expression of mutant murine p53 (pLTRp53cGVal 135; kindly supplied by Dr M. Oren) has been described in detail elsewhere (Michalovitz et al, 1990), and encodes a temperature sensitive p53 that adopts a wild-type conformation at 32 °C, but a mutant conformation at 37 °C due to a mutation at position 135 (valine to alanine).
  • the vector for the expression of mutant human p53 contains the mutated cDNA under the control of me CMV promoter (kindly supplied by Dr P. Chumakov). Plasmids encoding the E. coli CAT gene under me control of different fragments of the human topoisomerase lice gene promoter, have been reported previously (Hochhauser et al, 1992).
  • Plasmids carrying the mouse ferritin (FGH) and me human phosphoglycerate kinase 1 (PGKGH) gene promoters linked to the human growth hormone (HGH) coding region were kindly provided by Dr J. Firth (Oxford, UK).
  • the pKV461/CD2 (kindly provided by Dr C.J. Norbury) contains a truncated rat CD2 cDNA and was generated by excising me CD2 cDNA from pERCD2-2 (He et al, 1988) and cloning it into the BgRl site of pKV461 (kindly supplied by Dr M. Sowden; (Sowden et al, 1989).
  • HGH human growth hormone
  • the plasmid used for all of the HGH constructs was PGEM7Zf+ (PROMEGA), containing me 1.8 kb HGH cDNA as a reporter gene.
  • This plasmid, designated pSVGH was a generous gift from Dr J. Firth, Oxford, UK.
  • the SV40 promoter in pSVGH was replaced by the topoisomerase lice promoter to generate pHGH, as described previously (Isaacs et al, 1996).
  • Deletion constructs containing various truncated forms of the topoisomerase lice promoter were generated by PCR from the full length 2.5 kb fragment of the promoter (Hochhauser et al, 1992).
  • PCR primers incorporated restriction sites to enable directional cloning via the Xbal and Hindlll, or the Bam ⁇ sites in pHGH. Sequences for the 5' primers (with the 5' limit indicated on me left) used in generating the promoter truncations were as follows. Numbering begins at + 1, the transcription start site.
  • the cells were then washed in phosphate buffered saline, trypsinized, and seeded at a ratio of 1 :2 in RPMI-1640 medium. Cultures were then maintained either at 32 °C or at 37 °C for up to 120 hours.
  • CAT activity was measured by the conversion of 14 C-labelled chloramphenicol to its acetylated forms using standard techniques. The % conversion was determined by excising radioactive spots from the min layer chromatography plates and measuring the level of radioactivity in each sample in a scintillation counter.
  • Promoter activity was determined by measuring the level of the HGH gene product in the media of transfected cells in culture.
  • the assay was conducted on aliquots of medium using an immuno-radiomimetric assay (IRMA) kit supplied by the Norm East Thames Radioimmune Assay (Netria) Co. This assay utilises a sheep polyclonal anti-HGH solid phase antibody and an 125 I-labelled murine monoclonal anti-HGH tracer antibody. All assays were kindly performed by Gillian Campling at Littlemore Hospital, Oxford, UK.
  • IRMA immuno-radiomimetric assay
  • Nucleotide sequencing was performed on double-stranded plasmid templates using the dideoxy chain termination method and Sequenase enzyme, as recommended by me suppliers (US Biochemical Corp.).
  • me CD2 cell surface antigen was achieved using a FITC- conjugated anti-rat CD2 monoclonal antibody (OX-34; SeroTec), as described by O'Connell et al (1994).
  • OX-34 FITC- conjugated anti-rat CD2 monoclonal antibody
  • Cells were fixed for 30 minutes in ice-cold 70% ethanol in PBS, collected by centrifugation and were treated wim RNase A (100 ⁇ g/ml final concentration) and propidium iodide (40 ⁇ g/ml) in PBS for 30 minutes at 37°C.
  • Cell cycle distribution was men determined using a Becton-Dickinson FACScan, and me data were analyzed using Lysys II software.
  • Example 2 The distribution and expression of the two isoforms of DNA topoisomerase II in normal and neoplastic human tissues
  • topoisomerase II-targeting drugs (breast, lung, lymphoma and seminoma) or of those that show de novo drug resistance (colon). Topoisomerase lice was expressed exclusively in the proliferating compartments of all normal tissues, and was detectable in both the cell nucleus and cytoplasm. In biologically aggressive or rapidly proliferating tumours (eg high grade
  • topoisomerase 11/3 was expressed ubiquitously in vivo and was localised in both me nucleoli and the ⁇ nucleoplasm. This isoform was present in quiescent cell populations, but was expressed at a generally higher level in all tumours and proliferating cells than in normal quiescent tissues.
  • topoisomerase lice is a strict proliferation marker in normal and neoplastic cells in vivo, but mat topoisomerase IIjS has a much more general cell and tissue ) distribution than has topoisomerase lice.
  • the apparent upregulation of topoisomerase ll ⁇ in neoplastic cells has implications for the response of patients to antitumour therapies that include topoisomerase Il-targeting drugs.
  • topoisomerase II The normal tissue distribution in humans of the topoisomerase lice and ⁇ proteins using isoform-specific monoclonal antibodies has been studied.
  • topoisomerase II/3 is expressed widely and in all tissue types, including within quiescent cell compartments.
  • mat topoisomerase 11/3 is detectable both in the nucleoplasm and in nucleoli, and is expressed at a generally higher level in neoplastic than normal tissues.
  • cytospin samples were prepared on glass slides using a Shandon cytocentrifuge. The cytospin samples were then air dried and fixed in PBS containing 3.7% formalin for 15 minutes prior to immunostaining.
  • the cell lines used were as follows:- SUDHL-1 (T- cell lymphoma), MCF-7 (breast carcinoma), SuSa (testicular teratoma), NCI460 (non small cell lung cancer) and CEM (erymro-leukaemia).
  • the CRB antibody was raised in rabbits to an extreme C-terminal peptide of me human topoisomerase lice protein (ARG-ALA-LYS-LYS-PRO-ILE-
  • luminescence detection was as recommended by the supplier (Amersham), with die blocking buffer comprising 20 mM Tris-HCl, pH 7.6, 0.9% NaCl, 0.05% Tween-20 and 1 % low fat milk powder.
  • a range of normal tissues (tonsil, spleen, lymph node, thymus, skin, pancreas, testis, colon, kidney, liver, brain and lung) and mmours (9 breast carcinomas, 10 colon carcinomas, 13 lung carcinomas, 10 cases of Hodgkin's disease, 13 large cell non-Hodgkin's lymphomas (NHL), 8 cases of lymphocytic lymphoma (CLL) and 8 seminomas of the testis; see Table 2) was obtained from the frozen tissue bank stored at -70 °C in the University Department of Cellular Science, John Radcliffe Hospital, Oxford, UK. Cryostat sections of 8 mm were obtained and were mounted on poly- 1 -lysine coated glass slides. After drying for between 30 minutes and 8 hours, the sections were fixed in PBS containing 3.7% formalin for 15 minutes and men immediately immunostained using an immunoperoxidase Duet kit (Dako, Denmark).
  • the mmours were classified according to the proportion of labelled cell nuclei as follows: 0-5%, 5-30%, 30-60% and greater man 60% . These were established initially by counting the number of unlabelled and labelled nuclei throughout the section. It was found wim experience that this system could be reproduced without formal counting by visual inspection of the section. This was validated by reviewing mmours in the series and comparing visual estimate with the previously established percentages. Tumours were consistently placed wimin the same proliferation category. Table 2: Staining of human tumours for topoisomerase Il ⁇ expression
  • Tumours are classified by type and % of nuclei staining positive for topoisomerase lice.
  • the anti-topoisomerase lice peptide antiserum, CRB produced nuclear staining in all of the normal tissues studied wim a distribution very similar to that seen with known proliferation-associated antigens, such as Ki-67 ( Figure 10).
  • proliferation-associated antigens such as Ki-67
  • FIG. 10 For example, in lymphoid tissue, topoisomerase Il-ce expressing cells were numerous in me germinal centres, but scarce in mantle zones. In epithelium and testicular tubules, positive staining for topoisomerase lice was present in the basal layers, but not in the more mature superficial cells. In colon and lung, positive staining was present in a minority of basal and alveolar epimelial cells, respectively.
  • the anti-topoisomerase U ⁇ peptide 3H10 antiserum produced positive staining in virtually all cell nuclei wimin all of the normal tissues studied ( Figure 10).
  • a punctate pattern of nuclear staining was evident, which was localised bom within nucleoli and dispersed diroughout the nucleoplasm.
  • the nucleoli appeared larger, and there was a greater dispersion of immunoreactive material into the surrounding nucleoplasm.
  • colon there were scattered nuclear dots in most of the cells.
  • the staining pattern for topoisomerase lice protein seen in the range of 20 tumours examined reflected mat of the normal tissues described above.
  • the CRB antibody gave a pattern of nuclear staining that strongly correlated with that seen with antibodies to the established proliferation marker, Ki-67 antigen.
  • Ki-67 antigen was the striking positivity of the abnormal mono- and multi-nucleate cells in cases of Hodgkin's disease :5 ( Figure 10). Cytoplasmic staining with the CRB antibody was noted and was generally more evident in me tumour biopsies than it was in the normal tissue samples.
  • tumour cells staining positive for topoisomerase lice o ranged from less than 5% to more man 60% , and mis was related to tumour type and grade ( Figure 10 and Table 2).
  • high grade lymphomas had a higher proportion of positively-staining cells than did low grade lymphomas and lymph nodes from patients with chronic lymphatic leukaemia.
  • the squamous tumours had a higher proportion of positively staining cells man did adenocarcinomas or carcinoid tumours.
  • Seminomas showed the highest percentage of cells staining positive for topoisomerase lice, while expression was generally low in breast cancers.
  • the intrinsically drug resistant colon tumours analysed showed a generally high percentage of cells staining positive for topoisomerase lice (Table 2).
  • topoisomerase II/3-specific antibody 3H10 produced granular nuclear staining in virtually all of the cell types in every tumour analysed. No direct association wim proliferative index (and therefore with topoisomerase lice expression) was evident, although there was a generally higher intensity of staining in tumour tissue than that seen in normal tissues ( Figure 10). In each tumour sample, a minimum of 50% of the cells stained positive for topoisomerase U ⁇ , although in most cases more than 90% of cells expressed topoisomerase 11/3. As with normal tissues, staining within both nucleoli and in the nucleoplasm was evident wim me
  • Staining with 3H10 on all of the lines showed a different pattern from that of CRB.
  • the pattern was nuclear, but showed a granular distribution (Fig 11).
  • me cells undergoing mitosis or cells that had just divided
  • Example 3 Genetic constructs expressing cytosine deaminase or thymidine phosphorylase from the topoisomerase Ho? promoter
  • the -144 to +93 promoter element from the human topoisomerase Hot gene is linked to me E. coli cytosine deaminase gene (or to thymidine phosphorylase gene/cDNA) in several different vector systems.
  • a simple plasmid vector is used and transfected into cell lines using standard techniques.
  • the expression of the pro-drug activating gene is assessed by quantifying ability to kill the transfected cells using 5-fluorocytosine (which is converted to 5-fluorouracil by cytosine deaminase) or 5'-deoxy- 5-fluorouridine (which is converted to 5-fluorouracil by thymidine phosphorylase).
  • the cell lines are Saos-2 (p53null) or Saos-2 expressing p53 wt or temperature-sensitive p53. Clonogenic survival curves are determined at 37°C for the first two cell lines and at bom 37°C and 32°C for me latter host cell line. Because transfection is a relatively inefficient process, the transfectants are extracted from the bulk of the untransfected population by use of me pHook system. In this, the cells are co- transfected with cDNA encoding a cell surface marker which permits transfectants to be recovered using magnetic beads coated in antibody to the surface marker. In all cases cell survival in cases where p53 is wild- type and where it is mutant are compared.
  • Retroviral vectors containing me genetic constructs are made and these vectors give near 100% infection frequencies, but only replicate in proliferating cells and as such are mainly useful for confirming me p53-mediated regulation of the promoter.
  • cells can be grown to confluence to analyse proliferation-specific regulation of gene expression.
  • Example 4 Transient co-transfection assays using the topoisomerase lice promoter and luciferase reporter gene
  • the topoisomerase lice promoter element (-144 to +93) is linked to a luciferase reporter gene.
  • This genetic construct is co-transfected with different dosages of a genetic construct expressing wild-type or mutant (143, 248 or 216) ⁇ 53 genes into a p53-minus human leukaemia cell line, K562.
  • Example 5 Inclusion of the topoisomerase lice promoter element in a "dual control unit"
  • a "dual control unit" analogous to tiiose described in WO 97/12970 is made.
  • Genetic unit I contains a topoisomerase lice promoter (-144 to +93) with lac operator driving a renilla luciferase (rl) reporter.
  • Genetic unit II contains a d GC3p53 promoter (contains a consensus ⁇ 53 binding site upstream of the minimal HSV-thymidine kinase promoter) driving a firefly luciferase reporter. Insulator sequences from chick /3-globin gene are used.
  • the genetic construct is co-transfected into K562 cells in the presence or absence of wild-type p53 and the activities of both luciferases are measured. Details of the luciferase reporter genes, tik:GC3p53 promoter and chick ⁇ - globin insulator sequences are described in WO 97/12970.

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Abstract

L'invention concerne une construction génétique comprenant un élément promoteur sensible à un produit génique suppresseur de tumeurs et un gène cytotoxique dont l'élément promoteur est sensiblement inactif dans une cellule contenant le produit génique suppresseur de tumeur, codé par un gène suppresseur de tumeur de type sauvage, cet élément promoteur étant actif dans une cellule contenant une variante du produit génique suppresseur de tumeur, codé par un gène mutant suppresseur de tumeur, ou ne contenant pas ce produit génique suppresseur de tumeur. Cette construction génétique est utile dans le traitement du cancer.
PCT/GB1998/000551 1997-02-21 1998-02-20 Constructions genetiques antitumorales WO1998037207A1 (fr)

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WO2000078949A1 (fr) * 1999-06-22 2000-12-28 Institut National De Sante Et De Recherche Medicale Polypeptide icbp90 et ses fragments et polynucleotides codant lesdits polypeptides et applications au diagnostic et au traitement du cancer
WO2004005546A1 (fr) * 2002-07-04 2004-01-15 Imperial College Innovations Limited Criblage de composes anti-ovulatoires
WO2004009112A1 (fr) * 2002-07-18 2004-01-29 Helix Biopharma Corp. Utilisation d'urease pour inhiber la croissance de cellules cancereuses
WO2005103251A2 (fr) * 2004-04-02 2005-11-03 Board Of Regents, The University Of Texas System Promoteurs specifiques du cancer
US7264800B2 (en) 2002-07-18 2007-09-04 Helix Biopharma Corporation Method and composition for inhibiting cancer cell growth
US20180044686A1 (en) * 2015-03-09 2018-02-15 Sinai Health System Tools and methods for using cell division loci to control proliferation of cells

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GB2305920A (en) * 1995-10-02 1997-04-23 Cancer Res Campaign Tech Antitumour Vector Constructs

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HUBER B E ET AL: "VDEPT: AN ENZYME/PRODRUG GENE THERAPY APPROACH FOR THE TREATMENT OFMETASTATIC COLORECTAL CANCER", ADVANCED DRUG DELIVERY REVIEWS, vol. 17, no. 3, 5 December 1995 (1995-12-05), pages 279 - 292, XP000654839 *
SANDRI MI ET AL: "P53 REGULATES THE MINIMAL PROMOTER OF THE HUMAN TOPOISOMERASE IIALPHA GENE", NUCLEIC ACIDS RESEARCH, vol. 24, no. 22, 15 November 1996 (1996-11-15), OXFORD GB, pages 4464 - 4470, XP002068824 *
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7199218B1 (en) 1999-06-22 2007-04-03 Institut National De La Sante Et De La Recherche Medicale :(Inserm) ICBP90 polypeptide and its fragments and polynucleotides coding for said polypeptides and applications for diagnosing and treating cancer
FR2795414A1 (fr) * 1999-06-22 2000-12-29 Ass Pour Le Dev De La Rech En Polypeptide icbp90 et ses fragments et polynucleotides codant lesdits polypeptides et applications au diagnostic et au traitement du cancer
WO2000078949A1 (fr) * 1999-06-22 2000-12-28 Institut National De Sante Et De Recherche Medicale Polypeptide icbp90 et ses fragments et polynucleotides codant lesdits polypeptides et applications au diagnostic et au traitement du cancer
WO2004005546A1 (fr) * 2002-07-04 2004-01-15 Imperial College Innovations Limited Criblage de composes anti-ovulatoires
WO2004009112A1 (fr) * 2002-07-18 2004-01-29 Helix Biopharma Corp. Utilisation d'urease pour inhiber la croissance de cellules cancereuses
US7211250B2 (en) 2002-07-18 2007-05-01 Helix Biopharma Corporation Method and composition for inhibiting cancer cell growth
US7264800B2 (en) 2002-07-18 2007-09-04 Helix Biopharma Corporation Method and composition for inhibiting cancer cell growth
WO2005103251A3 (fr) * 2004-04-02 2006-04-27 Univ Texas Promoteurs specifiques du cancer
WO2005103251A2 (fr) * 2004-04-02 2005-11-03 Board Of Regents, The University Of Texas System Promoteurs specifiques du cancer
US7723104B2 (en) 2004-04-02 2010-05-25 Board Of Regents, The University Of Texas System Cancer specific promoters
US7816131B2 (en) 2004-04-02 2010-10-19 Board Of Regents, The University Of Texas System Cancer specific promoters
US20180044686A1 (en) * 2015-03-09 2018-02-15 Sinai Health System Tools and methods for using cell division loci to control proliferation of cells
US20220325289A1 (en) * 2015-03-09 2022-10-13 Sinai Health System Tools and methods for using cell division loci to control proliferation of cells

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