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WO2004070041A1 - Vecteurs pour l'expression de toxines - Google Patents

Vecteurs pour l'expression de toxines Download PDF

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Publication number
WO2004070041A1
WO2004070041A1 PCT/GB2004/000441 GB2004000441W WO2004070041A1 WO 2004070041 A1 WO2004070041 A1 WO 2004070041A1 GB 2004000441 W GB2004000441 W GB 2004000441W WO 2004070041 A1 WO2004070041 A1 WO 2004070041A1
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WIPO (PCT)
Prior art keywords
vector
tcf
expression
copies
binding
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Ceased
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PCT/GB2004/000441
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English (en)
Inventor
Kai Stefan Lipinski
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Innovata Ltd
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ML Laboratories PLC
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Filing date
Publication date
Priority claimed from GB0302727A external-priority patent/GB0302727D0/en
Priority claimed from GB0312432A external-priority patent/GB0312432D0/en
Application filed by ML Laboratories PLC filed Critical ML Laboratories PLC
Publication of WO2004070041A1 publication Critical patent/WO2004070041A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • control elements include promoters, which are c/s-acting elements controlling expression of open reading frames to which they are operably linked.
  • promoters may be constitutive and non-tissue-specific, or they may be tissue-specific, allowing expression of the product encoded by the open reading frame only in cells or tissues of the appropriate type.
  • tissue-specific promoters are known, few are highly stringent (or 'tight') in their control of expression.
  • Some recombinant proteins are highly toxic in the cells usually used for manufacture of expression vectors. So, where vectors encoding highly toxic molecules are required, for example, for the ablation of cancer cells by gene therapy, it is currently very difficult to grow cells containing and synthesising such vectors because of the lethal effects of even low-level expression of the toxins in the cells. In the case of toxins such as the diphtheria toxin, expression of a few molecules, possibly even one, is fatal to the cell (Yamaizumi et a), 1978, Cell 15: 245-250). Although, in principle, use of a tissue-specific promoter that is not permissive for expression in a suitable host cell could be used for production of such vectors, in practice none is known with sufficient stringency to allow this approach.
  • Another approach is to attempt to control leaky transcription in producer cells by post-transcriptional blocking with specific antisense oligonucleotides (Raykov et al, 2002, Gene Therapy 9: 358-362). This approach requires efficient penetration of producer cells with the oligonucleotides and involves an extra and expensive intervention.
  • TCFs are a family of transcription factors within the High Mobility Group (HMG) of DNA-binding proteins (Love et al., Nature, 376, 791-795, 1995).
  • the family includes TCF-1 , TCF-3 and TCF-4 which are described in van der Wetering et. al, (EMBO J., 10, 123-132, 1991), EP-A-0 939 122 and Korinek et al. (Science, 275, 1784-1787, 1997).
  • TCF-4 has been shown to be involved in tumorigenesis related to Wnt Wingless signalling.
  • TCF and LEF-1 (lymphoid enhancer factor-1) are considered to mediate a nuclear response to Wnt signals by interacting with ⁇ - catenin.
  • Wnt signalling and other cellular events that increase the stability of ⁇ - catenin are considered to result in transcriptional activation of genes by LEF-1 and TCF proteins in association with ⁇ -catenin.
  • LEF- 1/TCF proteins repress transcription in association with Groucho and CBP (CREB binding protein).
  • ⁇ -catenin In the absence of Wnt signalling, ⁇ -catenin is found in two distinct multiprotein complexes.
  • One complex located at the plasma membrane, couples cadherins (calcium dependent adhesion molecules) with the actin cytoskeleton whereas the other complex (containing the proteins adenomatous polyposis coli protein (APC), axin and glycogen synthase kinase 3 ⁇ (GSK3 ⁇ )) targets ⁇ -catenin for degradation.
  • Wnt signalling antagonises the APC-axin-GSK3 ⁇ complex, resulting in an increase in the pool of free cytoplasmic ⁇ -catenin.
  • the free cytoplasmic ⁇ -catenin can translocate to the nucleus where it binds LEF-1/TCF factors and activates Wnt target genes.
  • LEF-1 /TCF transcription factors The regulation of LEF-1 /TCF transcription factors by Wnt and other signals is discussed in Eastman et al, (Current Opin. Cell Biology, U_, 233-240, 1999).
  • the APC gene is a tumour supressor gene that is inactivated in most colorectal cancers. Mutations of APC are considered to cause the accumulation of free ⁇ - catenin, which then binds TCF causing increased transcriptional activation of genes including genes important for cell proliferation (e.g. cyclin D1 (Tetsu er a/., Nature 398, 422-426, 1999 and Shtutman et al., PNAS USA, 96, 5522-5527, 1999) and c- myc (He et al., Science, 28_1, 1509-1512, 1998)). The involvement of APC in tumour development is discussed in He et al, (supra).
  • TCFs are known to recognise and bind TCF binding elements which have the nucleotide sequence CTTTGNN, wherein N indicates A or T (van der Wetering et al, supra).
  • TCF reporter genes have been constructed and are described in Korinek et al,
  • the TCF reporter gene is said to comprise three
  • TCF binding elements upstream of either a minimal c-Fos promoter driving luciferase expression or a minimal herpes virus thymidine kinase promoter driving chloramphenicol acetyl-transferase expression.
  • He et al discloses TCF reporter gene constructs comprising four TCF binding elements inserted into pBV-
  • Promoters containing TCF-binding elements are therefore useful for obtaining selective expression of operably-linked genes in cells in which there is an accumulation of nuclear ⁇ -catenin, which co-operates with TCF and activates expression in combination with other transcription factors.
  • alternative known therapeutic approaches include tumour-specific, replication-competent adenoviral vectors (Iggo et al, WO 00/56909).
  • tumour-specific, replication-competent adenoviral vectors Iggo et al, WO 00/56909.
  • tumours of the liver and prostate e.g. tumours of the liver and prostate, glioma, melanoma, various adenocarcinomas, osteosarcoma, and others.
  • a targeted strategy to treat these tumours with viral vectors would be highly valuable.
  • CTP1 comprises 5 copies of the TCF-binding element CCTTTGATC (SEQ ID NO:1 ) separated by gaps of 10, 12, 10 and 10 nucleotides, respectively.
  • the proximal binding element is separated from the TATA box of the SV40 promoter by 140 nucleotides.
  • CTP2 comprises the same arrangement of TCF-binding elements, but the proximal element is situated 46 nucleotides upstream of the TATA box of the adenovirus E1 A promoter.
  • CTP3 comprises 5 copies of the TCF-binding element separated by 4, 3, 4 and 3 nucleotides, respectively with the proximal element 25 nucleotides upstream of the E1A TATA box.
  • CTP4 comprises 10 copies of the TCF-binding element, arranged as 2 sets of the CTP3 arrangement separated by 40 nucleotides (that is spaced by 4, 3, 4, 3, 40, 4, 3, 4, 3) again situated 25 nucleotides upstream of the E1A TATA box.
  • This improved promoter construct was designed for selective expression of a variety of highly toxic proteins and peptides, in particular diphtheria toxin A (DTA) and the gibbon ape leukaemia virus fusogenic membrane glycoprotein [GaLV FMG] (Fielding et al, 2000, Hum Gene Ther 11: 817-826; Johnson et al, 2003, Gene Ther 10: 725- 732).
  • DTA diphtheria toxin A
  • GaLV FMG gibbon ape leukaemia virus fusogenic membrane glycoprotein
  • the invention provides a method of manufacturing an expression vector encoding one or more gene products that are toxic to the host cell.
  • the host cell is a eukaryotic cell. More preferably the method comprises introducing into a cell having no nuclear accumulation of ⁇ -catenin a vector encoding said toxic product, wherein expression of said toxic product is controlled by a control element comprising one or more TCF-binding elements.
  • a toxic product is one that is intrinsically toxic to cells, requiring no further activation or extrinsically added substrate or prodrug.
  • said cell is a producer cell suitable for the production of viral vectors, still more preferably, adenoviral vectors. Most preferably it is a cell line that complements the growth of replication-deficient adenoviral vectors, such as PerC6.
  • the vector comprises at least one copy of a TCF- binding element.
  • this element comprises the nudeotide sequence 5'- CTTTGNN-3' (SEQ ID NO:2), wherein N indicates A or T. More preferably it is 5'- CCTTTGATC-3' (SEQ ID NO:1)
  • the vector comprises up to 10 copies of a TCF-binding element.
  • the vector comprises 5 to 10 copies.
  • the copies are spaced by 10 to 12 nucleotides. Alternatively, they are spaced by 3 or 4 nucleotides.
  • the vector comprises 10 copies of a TCF-binding element arranged with spaces of 4, 3, 4, 3, 40, 4, 3, 4, and 3 intervening nucleotides respectively.
  • the spacing between the proximal TCF- binding element and the promoter TATA box is between 10 and 100 nucleotides. More preferably it is between 10 and 50 nucleotides. Further preferably it is between 20 and 40 nucleotides and, most preferably, it is 24 nucleotides.
  • Highly preferred embodiments comprise constructs CTP1 , CTP2, CTP3 and CTP4 as disclosed above and in Figure 1.
  • the vectors encode toxic molecules.
  • the toxic molecules may be proteins such as enzymes, enzyme inhibitors, fusogenic peptides, antibodies or functional fragments thereof, or may be ribonucleotides such as antisense RNA or ribozymes.
  • proteins selected from the list consisting of ricin, abrin, diphtheria toxin, botulinum toxin, gibbon ape leukaemia virus (GaLV) fusogenic membrane protein or cytotoxic peptides derived therefrom.
  • GaLV gibbon ape leukaemia virus
  • the invention provides a vector comprising up to 10 copies of a TCF-binding element, operably linked to an expressible gene encoding a toxic protein molecule.
  • the vector comprises 5 to 10 copies of a TCF-binding element.
  • the copies are spaced by 10 to 12 nucleotides. Alternatively, they are spaced by 3 or 4 nucleotides.
  • the vector comprises 10 copies of a TCF-binding element arranged with spaces of 4, 3, 4, 3, 40, 4, 3, 4, and 3 intervening nucleotides respectively.
  • the toxic molecules may be proteins such as enzymes, enzyme inhibitors, fusogenic peptides, antibodies or functional fragments thereof, or ribonucleotides such as antisense RNA or ribozymes.
  • ribonucleotides such as antisense RNA or ribozymes.
  • such toxic molecules are selected from the list consisting of ricin, abrin, diphtheria toxin, botulinum toxin, gibbon ape leukaemia virus (GaLV) fusogenic membrane protein or cytotoxic peptides derived therefrom.
  • An especially preferred embodiment comprises an expressible gene encoding diphtheria toxin A (DTA).
  • 'gene' means a nucleic acid sequence comprising an expressible open reading frame, with or without introns or other non-coding or regulatory sequences, encoding a desirable protein, peptide or RNA.
  • the vector is preferably a viral vector, more preferably an adenoviral vector.
  • nucleic acid constructs including plasmids, encoding all or part of such a viral vector, such as those used to manipulate the viral genome prior to packaging.
  • the vector comprises the control element CTP4, as shown in Figure 2.
  • host cells containing any such nucleic acid construct, plasmid, vector, or adenovirus.
  • the cytotoxic effect is derived from another mechanism such as conditional replication of a virus, preferably an adenovirus.
  • the present invention also provides the nucleic acid construct, vector or host cell of the present invention for use in therapy.
  • the nucleic acid construct, vector or host cell is used in the treatment of cancer.
  • the present invention also provides the use of the nucleic acid construct, vector or host cell of the present invention in the manufacture of a composition for use in the treatment of cancer.
  • the present invention also provides a method of treatment, comprising administering to a patient in need of such treatment an effective dose of the nucleic acid construct, vector or host cell of the present invention.
  • the patient is suffering from cancer.
  • the cancer is any cancer associated a nuclear accumulation of ⁇ -catenin, as occurs when the Wnt signalling pathway is deregulated, such as colorectal cancer, melanomas, breast, prostate and hepatocellular carcinomas, glioma, various adenocarcinomas or osteosarcoma.
  • the present invention also provides a pharmaceutical composition comprising the nucleic acid construct, vector or host cell of the present invention in combination with a pharmaceutically acceptable recipient.
  • the pharmaceutical compositions of the present invention may comprise the nucleic acid construct, vector or host cell of the present invention, if desired, in admixture with a pharmaceutically-acceptable excipient, buffer, carrier or diluent, for therapy to treat a disease.
  • the nucleic acid construct, vector or host cell of the invention or the pharmaceutical composition may be administered via a route which includes systemic, intramuscular, subcutaneous, intradermal, intravenous, aerosol, oral (solid or liquid form), topical, ocular, as a suppository, intraperitoneal and/or intrathecal and local direct injection.
  • the exact dosage regime will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact nature of the protein expressed by the therapeutic gene and the type of tissue that is being targeted for treatment.
  • the dosage also will depend upon the disease indication and the route of administration.
  • the amount of nucleic acid construct or vector delivered for effective treatment according to the invention will preferably be in the range of between about 50 ng - 1000 ⁇ g of vector DNA kg body weight; and more preferably in the range of between about 1-100 ⁇ g vector DNA/kg.
  • an ex vivo approach may be utilised whereby cells are removed from an animal, transduced with the nucleic acid construct or vector, and then re-implanted into the animal.
  • the liver for example, can be accessed by an ex vivo approach by removing hepatocytes from an animal, transducing the hepatocytes in vitro and re-implanting the transduced hepatocytes into the animal (e.g., as described for rabbits by Chowdhury et al., Science 254:1802-1805, 1991 , or in humans by Wilson, Hum. Gene Ther.
  • Such methods also may be effective for delivery to various populations of cells in the circulatory or lymphatic systems, such as erythrocytes, T cells, B cells and haematopoietic stem cells.
  • the present invention also provides a composition for delivering the nucleic acid construct or vector of the present, to a cell.
  • toxin expressed by means of the method described above, or using the nucleic acid constructs or expression vectors herein provided.
  • Figure 1 depicts four ⁇ -catenin-dependent promoter constructs used to express toxic products such as diphtheria toxin-A in cells permissible for vector propagation but not for ⁇ -catenin -dependent gene expression.
  • the numbers below each diagram refer to the number of nucleotides (10, 12, 4 or 3) between the CCTTTGATC repeats.
  • the gap between the proximal repeat and the E1A TATA box '140 to...', '46 to...' and '25 to...' is inclusive of the first nudeotide of the TATA box sequence (as made clear in Figure 2), and so the number of intervening nucleotides is 139, 45 and 24, respectively.
  • FIG. 2 shows the nudeotide sequence of construct CTP4.
  • the TCF-binding elements are underlined and the TATA box from the adenoviral serotype 5 E1A gene is in bold.
  • FIG 3. Shown is the DTA sequence as sequenced from the Ad.CTP4-DTA preparations LSP1 and LSP3 (see also Figure 4).
  • "Query” is the sequence obtained by sequencing adenoviral DNA which was BLASTed using the NCBI homepage showing the result "Sbjct”. The results indicate wild-type DTA DNA-sequence.
  • Ad.CTP4-DTA kills permissive, ⁇ -catenin-deregulated tumour cell lines.
  • SW480 mutant APC
  • HepG2 mutant ⁇ -catenin
  • Ad.CTP4-DTA black
  • MTS substrate assay Promega
  • Ad.CTP4-DTA shows no killing of non-permissive cells.
  • HeLa, HNX14C and HUVEC Human Umbilical Vein Endothelial Cells
  • Ad.CTP4- DTA Hatched
  • Ad.CTP4-LacZ control virus black
  • cell viability was analysed using the MTS substrate assay (Promega) and reading the OD450. Infections were done in triplicates and shown is the mean OD450 value. One out of at least two to three representative experiments is shown.
  • SW480, HeLa, HNX14C and HepG2 were obtained from ATCC (Manassas, VA, USA).
  • HUVEC were obtained from Promocell (Heidelberg, Germany) and cultured as recommended by the supplier.
  • PER.C6 were obtained from Crucell (Leiden, The Netherlands).
  • the adenoviral vector producer cell line 911 was used for plaque titering adenoviruses and was kindly provided by Prof. L. S. Young (Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK). 911 were cultured in DMEM containing 10% FCS, 10 mM MgCI 2 and antibiotics. All purchased cell lines were cultured as recommended by the supplier.
  • a cDNA coding for diphtheria toxin A was amplified by PCR (oligol : 5'-CATGCCATGGGCGCTGATGATGTTGTTG (SEQ IS NO:3); oligo2: ⁇ '-GCTCTA GACTATCGCCTGACACGATTTCCT) (SEQ ID NO:4) using the plasmid pDT201 (ATCC, Manassas, VA, USA) as template.
  • the resulting PCR product comprises amino acids 26-193 of wild-type DT protein with an additional codon for methionine in front of amino acid 26.
  • the PCR product was digested with A/col and Xba ⁇ and then cloned into Nco ⁇ IXba ⁇ digested CTP4-Luc thereby exchanging luciferase against DTA.
  • the correctness of the DTA open reading frame was confirmed by DNA sequencing the plasmid DNA (Seqlab GmbH, Goettingen, Germany).
  • the CTP4-DTA-p(A) expression cassette (1.1 kb) was cut out by Nhe ⁇ /BamH ⁇ , T4 blunted and then cloned into blunted pPS1128 (lit).
  • the virus Ad.CTP4-DTA were constructed by homologous recombination in PER.C6 cells using pPS1128/CTP4-DTA-p(A) and the overlapping adenoviral backbone vector pPS1 160 (lit), scaled-up and CsCI-purified and titred as described elsewhere using standard methods (lit).
  • Viral DNA was prepared from about 2-4x10 11 CsCI-banded virus particles by incubation with 100 ⁇ g/ml proteinase K in 20 mM Tris/HCI pH7.5, 5 mM EDTA pH8.0, 0.1% SDS for 3-4 hours at 37°C.
  • the crude DNA preparation was then extracted two times with an equal volume of Phenol:Chloroform:lsoamylalcohol (25:24:1), once with chloroform only and then precipitated with 1/10 Vol. 3.0 M NaAcetate and 2 Vol. 100% Ethanol. After centrifugation, (10 min at 13K at RT) the resultant DNA pellet was washed with 70% Ethanol, air-dried and then resuspended in water.
  • the CTP4 promoter and the coding sequence for DTA of LSP1 and LSP3 of Ad.CTP4-DTA were sequenced by Seqlab GmbH, Germany.
  • Ad.CTP4- DTA kills permissive SW480 cells in vitro but does not affect the viability of non-permissive HeLa cells.
  • Ad.CTP4-DTA is genetically stable and can be propagated to normal high titres.
  • Permissive tumour cell lines SW480 and HepG2 were infected with increasing MOIs of Ad.CTP4-DTA and control virus Ad.CTP4-LacZ. After four days cell killing was evaluated with the MTS solution assay (Promega). Both cell lines could be killed with relatively low MOIs (25-50; See Figure 5). In contrast, infection of the non-permissive tumour cell lines HeLa and HNX14C did not show more toxicity than the infection with the control virus Ads.CTP4-LacZ ( Figure 6) confirming again the high level of specificity of the CTP4 promoter.

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Abstract

L'invention concerne une méthode pour produire des vecteurs codant des protéines recombinantes ou des glycoprotéines recombinantes, toxiques pour les cellules dans lesquelles elles s'expriment. L'expression de ces protéines doit donc être strictement contrôlée pour permettre, par exemple, la production de vecteurs viraux pour la thérapie génique dans des cellules qui, sinon, seraient tuées par la toxine codée par les vecteurs. La présente invention porte également sur des vecteurs comportant des promoteurs synthétiques hautement spécifiques aux tissus et permettant l'expression de la toxine seulement dans les cellules dotées d'une accumulation nucléaire de beta-caténine.
PCT/GB2004/000441 2003-02-06 2004-02-05 Vecteurs pour l'expression de toxines Ceased WO2004070041A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0302727.3 2003-02-06
GB0302727A GB0302727D0 (en) 2003-02-06 2003-02-06 Vectors for expression of toxins
GB0312432A GB0312432D0 (en) 2003-05-30 2003-05-30 Vectors for expression of toxins
GB0312432.8 2003-05-30

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WO2004070041A1 true WO2004070041A1 (fr) 2004-08-19

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064739A1 (fr) * 2000-03-02 2001-09-07 Ml Laboratories Plc Element sensible au facteur de transcription de lymphocytes t (tcf)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064739A1 (fr) * 2000-03-02 2001-09-07 Ml Laboratories Plc Element sensible au facteur de transcription de lymphocytes t (tcf)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIPINSKI K S ET AL: "High-level, beta-catenin/TCF-dependent transgene expression in secondary colorectal cancer tissue.", MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY. UNITED STATES OCT 2001, vol. 4, no. 4, October 2001 (2001-10-01), pages 365 - 371, XP002285103, ISSN: 1525-0016 *

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