WO1999031118A1 - Inhibition de la proliferation de cellules tumorales a l'aide de ribozymes - Google Patents

Inhibition de la proliferation de cellules tumorales a l'aide de ribozymes Download PDF

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WO1999031118A1
WO1999031118A1 PCT/US1998/017292 US9817292W WO9931118A1 WO 1999031118 A1 WO1999031118 A1 WO 1999031118A1 US 9817292 W US9817292 W US 9817292W WO 9931118 A1 WO9931118 A1 WO 9931118A1
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neu
cells
ribozymes
tumor
expression
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PCT/US1998/017292
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Frank Czubayko
Anton Wellstein
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Georgetown University
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • This invention relates to use of ribozymes to inhibit growth and metastatic spread of cancer.
  • Tumor growth and, ultimately, metastasis is a complex 5 . precess regulated in part by factors controlling cellular proliferation and death as well as tumor angiogenesis.
  • HER-2/neu a member of the epithelial growth factor (EGF) tyrosine kinase receptor family has been implicated in 0 mediating growth factor signals as well as in modulating hormone responsiveness of breast cancer cells.
  • EGF epithelial growth factor
  • HER-2/neu is frequently over-expressed in a variety of adenocarcinomas, including those of lung, breast and ovarian cancer. In these tumors, HER-2/neu overexpression serves as a marker of faster 5 tumor progression and poor prognosis.
  • HER-2/neu expression is being evaluated as a potential therapeutic approach in breast cancer patients.
  • HER-2/neu expression 0 is mostly a marker or a rate-limiting factor in cancer cell growth.
  • Tumor-angiogenesis a process whereby factors stimulating the ingrowth of blood vessels into the tumor are secreted into the local tumor milieu by cancer and stroma cells, also plays 5 a critical role by regulating the balance between cell proliferation and cell death and by providing a route for distant spread.
  • 3 Both clinical and laboratory evidence suggests that spread of malignant cells from a localized tumor is directly related to the number of microvessels in the primary 0 tumor.
  • factors secreted by tumor and stroma cells which are potentially angiogenic, two have been confirmed as angiogenic factors which are rate-limiting in in vivo tumor models. The importance of one of these, vascular endothelial growth factor / vascular permeability factor 5 .
  • VEGF/VPF vascular endothelial growth factor
  • PTN pleiotrophin
  • Adenovirus constructs containing the appropriate vectors can be administered parenterally for 5 systemic effect or be administered directly into the tumor and/or into the specific arteries which supply the tumor for purposes of inhibiting tumor growth.
  • the invention provides a method for inhibiting growth of a malignant tumor by administration of recombinant adenovirus which expresses 0 ribozymes that target PTN mRNA or Her-2/neu in pharmaceutically acceptable carriers.
  • Figure 1 shows the overall genomic organization of the recombinant adenovirus ribozyme expression vectors. 5 Description of the Invention:
  • This invention involves constructed adenoviruses expressing hammerhead-ribozymes targeted to two factors: (1) the tyrosine kinase receptor HER-2/neu or (2) the growth factor pleiotrophin (PTN) .
  • Adenovirus- ediated transduction of either 0 HER-2/neu or PTN - targeted ribozymes depleted the respective RNAs and inhibited protein expression significantly in three different human cancer cell lines. This resulted in almost complete abrogation of HER-2/neu or PTN dependent cancer-cell proliferation.
  • ribozyme gene transfer technologies to biologically down-regulate expression of specific genes coupled with efficient gene delivery vehicles provides anti- cancer strategies to block expression of potentially rate- limiting tumor promoting factors such as HER-2/neu or PTN.
  • Hammerhead-ribozymes can be targeted to destroy specific mRNA transcripts by binding to and cleaving specific ribonucleotide sequences.
  • Such ribozymes targeted to specific oncogenes have been used successfully to down-regulate oncogene expression in both in vitro and in vivo models.
  • adenovirus vectors are well suited as an alternative to plasmid-based gene transfer technologies.
  • Adenovirus vectors provide highly efficient means for gene transfer and expression in vitro and in vivo, can be easily produced in high titers and have a broad host range. Further, adenovirus vectors have been used for the efficient delivery and expression of ribozymes into tumor cells in culture and mouse liver cells in vivo.
  • adenoviruses expressing hammerhead ribozymes for either HER-2/neu or PTN were constructed and evaluated in four separate human cancer cell lines. Each vector led to high level gene transfer and hammerhead ribozyme expression and to significant depletion of the respective gene products. Importantly, inhibition of either HER-2/neu or PTN gene expression in tumor cells abrogated the proliferative effects of these proteins, thus demonstrating the feasibility of these therapeutic approaches.
  • Ribozyme expressing vectors were derived from Ad-dl327 by homologous recombination as described in the Materials and methods section.
  • Ribozymes are expressed under the control of a viral CMV promoter and contain a SV40 polyadenylation signal to enable processing and increase transcript stability in eukaryotic cells.
  • the HER-2/neu ribozyme targets the HER-2/neu mRNA 1991 nucleotides (nt) downstream of the translation initiation site.
  • HER-2/neu specific antisense flanking regions of 9 nt and 8 nt on the 5' and 3' ends of the ribozyme were positioned around a minimized catalytic hammerhead ribozyme core of 22 nt, a sequence which has been used successfully in previous studies.
  • the specificity of this HER-2/neu ribozyme construct was demonstrated in in vitro cleavage assays as well as in transient co-transfection studies in NIH/3T3 cells using 5 . the pRc/CMV plasmid as a ribozyme expression vector.
  • a catalytically inactive mutant ribozyme and an unspecific ribozyme were used as controls in those experiments and had no effects.
  • the PTN ribozyme cleaves the PTN mRNA 66 nt downstream of the translation initiation site and contains PTN 0 specific antisense flanking regions of 12 nt and 11 nt at the 5' and 3' ends, respectively.
  • the specificity and efficacy of the RzPTN construct was previously demonstrated in in vitro cleavage assays, in transient co-transfection studies in SW-13 cells, in two human melanoma cell lines stably expressing the 5 ribozyme in cell culture, and finally, in in vivo experiments in nude mice.
  • two recombinant adenoviruses expressing either ⁇ -Galactosidase (AvlLacZ4) or Luciferase (AvlLuc) were used as controls in some of the experiments.
  • Recombinant adenoviruses expressing ribozymes were 0 generated by a homologous recombination method. (Berkner, K.L. , BioTechnique 1988; 6: 616-624.) They were plaque- purified, amplified and titrated in 293 cells as described under Materials and Methods section of this specification. Correct transgene integration and genomic organization of the 5 recombinant adenoviruses was confirmed by Southern analysis. A PCR assay for Ela with a sensitivity to detect contaminations of one copy of wild-type adenovirus in 10 7 copies of recombinant adenovirus was used to assure the absence of replication competent wild-type adenovirus.
  • ribozyme expression peaked 3 days after adenoviral infection, decreased rapidly after day 5 and was not detectable by Northern analysis after day 10.
  • Human adrenal carcinoma (SW-13) , glioblastoma (U87) , ovarian cancer (SK-OV-3) and embryonic kidney epithelial (293) cells were obtained from American Type Culture Collection (ATCC) and were grown as adherent cells in IMEM (Life Technologies, Gaithersburg, MD) with 10 % fetal bovine serum (FBS; Life Technologies) ; human melanoma cells (1205LU; gift from M. Herlyn, Wistar Institute, Philadelphia, PA) were maintained in KSFM/L15 media (Life Technologies) mixed at a ratio of 3:1 and supplemented with 5% FBS.
  • 32D mouse hematopoietic stem cells which were stably co-transfected with HER-2/neu and HER-3 5 .
  • Rz-coding sense and 0 antisense oligonucleotides sense: 5 ' -agcttcaagaccacctgatgagtccgttaggacgaaaccagcaga-3 ' (Seq. #1) ; and antisense: 5 ' -agcttctgctggtttcgtcctaacggactcatcaggtggtcttga-3 ' (Seq. #2) 5 were annealed together and ligated into the Hindlll site of the pRc/CMV plasmid (Invitrogen, San Diego, CA) .
  • This ribozyme contains HER-2/neu specific antisense flanking regions of 9 nt and 8 nt on the 5' and 3' ends of the 22nt catalytic Rz-core, that target the Rz to its cleavage site 1991 nt downstream of the translation initiation site in the HER-2/neu mRNA (GenBank accession no. M11730) .
  • the RzHER minigene expression cassette was excised as a NruI-EcoRV fragment (778 nt) and ligated into 5 . the EcoRV site of PAVS6A. The correct insertion of the PTN and HER-2/neu expression cassettes into PAVS6A was verified by DNA sequencing.
  • Recombinant replication-deficient adenoviral-vectors were constructed by a homologous recombination method (Berkner, 0 K.L., Development of Adenovirus Vectors for the Expression of Heterologous genes", BioTechnigue 1988; 6: 616-624.) using PAVS6A-RZ shuttle plasmids and the 35 Kb Clal fragment of Ad- d!327.
  • the 293 cells were co-transfected with the respective PAVS6A-plasmid and virus DNA and individual plaques containing 5 recombinant virus were purified and further amplified in 293 cells as described (Metereder, supra) .
  • Recombinant adenoviral- DNA was analyzed by restriction enzyme digestions with EcoRI, Ndel and Xbal followed by Southern analysis using radio-labeled oligonucleotides specific for RzPTN or RzHER as probes.
  • Adenoviruses with the correct restriction pattern were produced in large amounts and purified in a two-step CsCl ultracentrifu- gation procedure.
  • Viral titers were obtained by plaque forming assays in 293 cells and high titer virus stocks were aliquoted and stored at -80 °C.
  • RNA STAT-60 Total cellular RNA was isolated with the RNA STAT-60 method (Tel-Test, Friedenswood, Tx) , separated and blotted as described y Fang, W.J., et al., (J Biol Chem 1992; 267: 25889- 25897.)
  • a HER-2/neu cDNA probe (1.5 Kb EcoRI fragment) was hybridized, washed and exposed to film for 16 hours.
  • blots were stripped and reprobed with a Glyceraldehyde-3 phosphate dehydrogenase (G3PDH, Clontech, Palo Alto, CA) cDNA probe. Relative band intensities were measured by densitometry.
  • G3PDH Glyceraldehyde-3 phosphate dehydrogenase
  • Hybridization and washing conditions were modified as follows: the hybridization buffer contained only 25% formamide and the final washing step was done at 50 °C, 0.5 x SSC/0.1% SDS for 30 minutes. Fluorescence activated cell sorting (FACS)
  • HER-2/neu protein levels were trypsinized, washed once with serum containing growth medium, twice with PBS (SIGMA) and resuspended in PBS at 5xl0 5 cells/100 ⁇ l . The cells were incubated for 30 minutes at 4°C with 1: 100 dilution of a primary anti-human HER-2/neu mouse monoclonal antibody (Clone 9G6.10; Neomarkers, Fremont, CA) . Cells were washed twice with PBS and incubated for 30 minutes at 4°C in the dark with a 1:200 diluted FITC-labeled goat anti-mouse secondary antibody (Boehringer, Mannheim) .
  • SIGMA primary anti-human HER-2/neu mouse monoclonal antibody
  • SW-13/PTN cells (Fang, supra) were infected with Av-RzPTN or Av-RzHER at MOI' s of 100 for two hours. Infected cells were then trypsinized and 2xl0 4 cells were plated in 35 mm soft agar dishes in triplicates. Soft agar colony formation was evaluated after a 10 day incubation period as described previously. The 32D cell proliferation/survival assay was performed. (Goldstein, D.J.
  • Infected or non-infected cells were then grown for 48 hours in the absence or presence of Interleukin-3 , or in the presence of recombinant heregulin (Neomarkers) at 10 "9 M without addition of IL3. Proliferation was then assessed by counting surviving cells manually using a hemocytometer.
  • Adenovirus-mediated transduction of HER-2/neu ribozymes down- regulates HER-2/neu expression and inhibits HER-2/neu-mediated proliferation
  • SL-OV-3 cells were infected with Av-RzHER or Av-RzPTN as control virus at MOI ' s of 100. total RNA was harvested one and three days after the infection and HER-2/neu mRNA levels were quantified by Northern analysis. The Av-RzHER-specific ribozyme infection led to a specific and significant depletion of HER-2/neu mRNA by approximately 75% one day after the infection when compared to the Av-RzPTN control infection.
  • human MDA-MD-361 breast cancer cells were infected with Av-RzHER and Av-lLacZ4 as control at MOI ' s of 100.
  • HER-2/neu protein levels were determine by FACS analysis over a period of 7 days after adenoviral infection and the mean fluorescence intensities were noted. Infection with Av-RzHER led to a significant reduction of HER-2/neu protein expression compared to the control virus that had no effect on HER-2/neu levels throughout the entire study.
  • HER-2/neu levels were reduced three days after infection and remained low until day five, at which time the levels started to increase again. This time course correlated with the ribozyme expression levels described above.
  • SW- 5 13/PTN human adrenal carcinoma cells were used. These were stably transfected with a PTN expression plasmid. In contrast to parental SW-13 cells, which do not express PTN endogenously and are not colonogenic in soft agar, SW-13/PTN cells form colonies spontaneously at a high rate. Consequently, depletion of PTN expression by adenovirus-mediated ribozymes targeting should reverse colony formation in these cells.
  • the SW-13/PTN 5 . cells were infected with Av-RzPTN or AvlLacZ4 at MOI's from 1 to 100 plated in soft agar. Colony formation was measured after a 10 day incubation period.
  • the recombinant adenovirus containing the ribozymes which target PTN mRNA or HER-2/neu may be administered by means which will result in contact with the target tumor. If administered systemically , doses of about 10 3 to about 10 10 plaque-forming units (pfu) may be administered. When injected locally into 5 an artery that carries blood to the tumor, slightly higher doses on the order of up to about 10 11 pfu may be appropriate.
  • tumor cells When injected into a tumor, one would try to estimate the number of tumor cells (1 gram of tumor tissue contains approximately 10° cells) and would adjust the viral dose in 0 order to achieve at least a multiplicity of infection of 1 pfu/tumor cell into the tumor.
  • Appropriate carrier include the usual carriers used for administration such as saline, buffered saline, glucose in saline, Ringer's lactate, etc. 5
  • carriers used for administration such as saline, buffered saline, glucose in saline, Ringer's lactate, etc. 5
  • the constructs of the invention have been deposited in the

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Abstract

La transduction transitoire à médiation par adénovirus de ribozymes HER-2/neu inhibe l'expression de HER-2/neu dans des cellules cancéreuses. Des structures d'adénovirus contenant les vecteurs appropriés peuvent être administrées par voie parentérale pour produire un effet général ou peuvent être administrées directement dans la tumeur et/ou dans les artères spécifiques qui alimentent la tumeur afin d'empêcher la croissance de la tumeur. Cette invention concerne par conséquent un procédé permettant d'inhiber la croissance d'une tumeur maligne qui consiste à administrer un adénovirus de recombinaison exprimant des ribozymes qui ciblent l'ARNm PTN ou Her-2/neu dans des supports pharmaceutiquement acceptables.
PCT/US1998/017292 1997-08-22 1998-08-21 Inhibition de la proliferation de cellules tumorales a l'aide de ribozymes WO1999031118A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097114A2 (fr) * 2001-05-29 2002-12-05 Sirna Therapeutics, Inc. Traitement a l'acide nucleique de maladies ou d'affections associees aux taux de ras, her2 et hiv
US6613567B1 (en) 2000-09-15 2003-09-02 Isis Pharmaceuticals, Inc. Antisense inhibition of Her-2 expression

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599704A (en) * 1992-08-26 1997-02-04 Ribozyme Pharmaceuticals, Inc. ErbB2/neu targeted ribozymes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599704A (en) * 1992-08-26 1997-02-04 Ribozyme Pharmaceuticals, Inc. ErbB2/neu targeted ribozymes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CZUBAYKO F., ET AL.: "ADENOVIRUS-MEDIATED TRANSDUCTION OF RIBOZYMES ABROGATES HER-2/NEU AND PLEIOTROPHIN EXPRESSION AND INHIBITS TUMOR CELL PROLIFERATION.", GENE THERAPY, NATURE PUBLISHING GROUP, GB, vol. 04., no. 09., 1 September 1997 (1997-09-01), GB, pages 943 - 949., XP002920747, ISSN: 0969-7128, DOI: 10.1038/sj.gt.3300483 *
CZUBAYKO F., RIEGEL A. T., WELLSTEIN A.: "RIBOZYME-TARGETING ELUCIDATES A DIRECT ROLE OF PLEIOTROPHIN IN TUMOR GROWTH.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 269., no. 33., 19 August 1994 (1994-08-19), US, pages 21358 - 21363., XP002915486, ISSN: 0021-9258 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6613567B1 (en) 2000-09-15 2003-09-02 Isis Pharmaceuticals, Inc. Antisense inhibition of Her-2 expression
WO2002097114A2 (fr) * 2001-05-29 2002-12-05 Sirna Therapeutics, Inc. Traitement a l'acide nucleique de maladies ou d'affections associees aux taux de ras, her2 et hiv
WO2002097114A3 (fr) * 2001-05-29 2003-05-08 Ribozyme Pharm Inc Traitement a l'acide nucleique de maladies ou d'affections associees aux taux de ras, her2 et hiv

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