WO2005113770A1 - Siarn anti-rhoa et -rhoc et compositions therapeutiques renfermant ceux-ci - Google Patents

Siarn anti-rhoa et -rhoc et compositions therapeutiques renfermant ceux-ci Download PDF

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WO2005113770A1
WO2005113770A1 PCT/EP2005/006043 EP2005006043W WO2005113770A1 WO 2005113770 A1 WO2005113770 A1 WO 2005113770A1 EP 2005006043 W EP2005006043 W EP 2005006043W WO 2005113770 A1 WO2005113770 A1 WO 2005113770A1
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rhoa
sirna
cells
rhoc
isolated polynucleotide
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Claudine Soria
Hong Li
Jean-Yves Pille
Christophe Denoyelle
Claude Malvy
Jean-Rémi BERTRAND
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Institut Gustave Roussy
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention pertains to the field of anti-cancer therapy. More particularly, the invention is based on the demonstration, by the inventors, that the inhibition of rhoA and/or rhoC in cancer and/or angiogenic endothelial cells could inhibit tumor growth and tumoral angiogenesis .
  • the present invention hence relates to anti- cancercompositions, comprising means to inhibit the expression of RhoA and/or RhoC.
  • RhoA and RhoC Low molecular weight GTP/GDP binding GTPases of Ras superfamily, RhoA and RhoC, have been shown to promote cell proliferation and cell invasion (Aznar and Lacal, 2001; Schmitz et al., 2000). Accumulating studies indicate that Rho protein-dependant cell signalling might be important for malignant transformation (Denoyelle et al., 2003; Frame and Brunton, 2002; Price and Collard, 2001).
  • RhoA Once activated, protein RhoA triggers a complex set of signal transduction pathways, which include the activation of ROCK, responsible for actin polymerization required for cell locomotion, and the phosphatidyl inositol 3 phosphokinase/AKT pathway thought to be critical for cell survival and expression of genes involved in cell proliferation (Denoyelle et al., 2003). In several human cancers, RhoA is over-expressed during tumor, and this correlates with poor prognosis (Fritz et al., 2002; Kamai et al., 2003; Kamai et al., 2004; Sahai and Marshall, 2002).
  • RhoC another low molecular weight GTPase, was also shown to be involved in cancer invasion in melanoma (Fritz et al., 1999), inflammatory breast cancer (Clark et al., 2000; Kleer et al., 2002), and ovarian cancer (Horiuchi et al., 2003).
  • RhoA Several reports also demonstrate the implication of RhoA in angiogenesis, which participates to cancer growth and metastasis (Abecassis et al., 2003; Hoang et al., 2004; Liu and Senger, 2004; Vincent et al . , 2001; Vincent et al., 2002).
  • the low molecular weight GTPases require prenylation for their biological activity (Seabra, 1998) .
  • the prenylated derivatives, the farnesyl pyro-phosphate (FPP) and the geranyl-geranyl-pyro-phosphate (GGPP) which is a precursor of cholesterol, are respectively required for Ras- and Rho-regulated cell signalling (Malaney and Daly, 2001; Sahai and Marshall, 2002) .
  • the prenylation of Ras and Rho is controlled by enzymes farnesyl transferase and geranyl-geranyl-transferase .
  • FTI farnesylation inhibitors
  • GGTI geranyl- geranyl transferase inhibitors
  • RhoB-GG RhoB-GG
  • statins such as cerivastatin
  • HMGCoA reductase hydroxy-methyl-glutaryl coenzyme A reductase
  • cerivastatin induces in vi tro a strong inhibition of aggressive breast cells proliferation and cell invasion (Denoyelle et al., 2001).
  • statin has also been reported in other types of cancer cells (Kusama et al., 2002). Cerivastatin was also shown to inhibit angiogenesis (Vincent et al., 2001; Vincent et al., 2002).
  • the first effect was the inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) which was involved in cells spreading and cells motility and was activated during tumor invasion (Imamura et al., 2000; Sahai and Marshall, 2002; Sahai and Marshall, 2003; Vincent et al., 2003; Vincent et al., 2002) .
  • the second effect was the inhibition of the cell signal pathway RhoA/Fak/PI3 Kinase/AKT.
  • the inventors have investigated the anticancer action of specific RhoA and RhoC inhibitors and compared their effects with that of cerivastatin, FTI and GGTI in MDA-MB 231 cells and endothelial cells.
  • siRNA short interfering RNA
  • the siRNA triggers the degradation of the endogenous mRNA to which the siRNA hybridizes (Elbashir et al., 2001; Tuschl, 2002).
  • siRNA is able to penetrate into tumors, for example using cytofectin as a delivery system (Bertrand et al., 2002).
  • intravenous administration is more convenient for clinical use than intra-tumoral injection, and the IV route is likely to enable the siRNA to reach both primary tumor and metastasis. Therefore, the efficacy and the toxicity of IV administration of encapsulated anti-RhoA siRNA have also been investigated, using chitosan-coated nanoparticles as carrier.
  • Chitosan is deacylated derivative of chitin, which is one of the most abundant mucopolysaccharide in crustaceans and insects.
  • Nanoparticles entirely made of chitosan have already been described (Borchard, 2001) .
  • the electrostatic interactions between the negatively charged siRNA and cationic polymer result in the formation of a complex (speckled particle) that is protected by exposed steric polymer strands.
  • Chitosan is not toxic, easily biodegradable and was shown to protect nucleotides from enzymatic digestion both in vivo and in vi tro (Mao et al., 2001; Nsereko and A iji, 2002; Quong and Neufeld, 1998); moreover, the presence of serum does not interfere with the transfection ability (Mao et al., 2001).
  • chitosan nanoparticles containing reporter genes are being used in the literature for the transfection of mammalian cells both in vi tro and in vivo (Mansouri et al., 2004; Mao et al., 2001).
  • Chitosan-coated polyisohexylcyanoacrylate (PIHCA) nanoparticles represent a novel device for oligonucleotides transfer.
  • the chitosan used is 5000 Da.
  • the inventors have tested the efficacy of chitosan-coated nanoparticles loaded with anti-RhoA siRNA administered via the IV route, as antitumor therapy of xenografted aggressive breast tumor. The duration of its effect was also investigated.
  • chitosan-nucleotides nanoparticles may enable intracellular sustained release of nucleotides (Dastan and Turan, 2004); this might be beneficial for a more prolonged and controlled expression of siRNA.
  • the duration of drugs release depends on the drug tested (it can be complete in some hours while it can continue for several months with DNA) .
  • the in vivo efficacy of these nanoparticles as antitumor drug was determined by measuring growth of xenografted aggressive breast cancer (MDA-MB 231) in mice.
  • MDA-MB 231 xenografted aggressive breast cancer
  • the invention hence pertains to an isolated polynucleotide, which is able to inhibit RhoA or RhoC expression by transcriptional interference.
  • transcriptional interference is meant the RNAi mechanism described, for example, by Hutvagner and Zamore (Hutvagner and Zamore, 2002) .
  • the invention concerns an isolated double-stranded RNA molecule, such as a small interfering RNA (siRNA) , which is able to mediate interference of the human RhoA or RhoC mRNA, in particular to inactivate the RhoA or RhoC gene by transcriptional silencing .
  • siRNA small interfering RNA
  • an isolated polynucleotide according to the invention is able to down- regulate RhoA or RhoC mRNA and protein levels by at least 30%, preferably at least 50%, and more preferably at least 80% in cultured MDA-MB 231 cells or in cultured HMEC-1 cells, when said cells are transfected twice at 24 hours interval with 8.5 nM of said double-stranded RNA molecule.
  • Materials and methods appropriate for testing the down- regulation of RhoA and RhoC expression are described in the examples below.
  • Polynucleotides according to the invention also preferably have some functional characteristics that can be tested in vi tro .
  • the isolated polynucleotide according to the invention is an anti-RhoA (or anti-RhoC) siRNA
  • it is preferably in the form of a double-stranded RNA molecule, wherein each RNA strand has a length from 19 to 25 nucleotides, more preferably 19 to 23 nucleotides, and even more preferably 20 to 22 nucleotides.
  • one or both strand (s) has (have) a 3' overhang from 1 to 5 nucleotides, preferably from 1 to 3 nucleotides, and for example, 2 nucleotides.
  • each 3' overhang is stabilized against degradation.
  • an isolated double- stranded RNA molecule as described above is a chemically synthesized RNA, or an analogue of a naturally occurring RNA.
  • the isolated polynucleotide according to the invention can also be a DNA that is transcribed into anti-RhoA (or anti-RhoC) siRNA in mammalian cells.
  • it can be a plasmid comprising a palindromic sequence that will be transcribed in the cell into a single-strand RNA molecule designed so that it forms a short hairpin RNA (shRNA) .
  • shRNA short hairpin RNA
  • RNA molecules used in the experiments disclosed in the examples below, i . e . :
  • Anti-RhoA siRNA sense 5' GACAUGCUUGCUCAUAGUCTT 3' (SEQ ID No: 1) antisense 3' TTCUGUACGAACGAGUAUCAG 5' (SEQ ID No: 2), and
  • Anti-RhoC siRNA sense 5' GACCUGCCUCCUCAUCGUCTT 3' (SEQ ID No: 3) antisense 3' TTCUGGACGGAGGAGUAGCAG 5' (SEQ ID No: 4).
  • these sequences are not limitative, and other siRNA sequences can be used to perform the present invention, as well as DNA sequences that can be transcribed into shRNAs which will subsequently be processed into said siRNAs.
  • the isolated polynucleotides according to the invention can be used for the preparation of a pharmaceutical composition.
  • another object of the present invention is a pharmaceutical composition comprising an isolated polynucleotide as described above, and a pharmaceutically acceptable carrier.
  • a particular pharmaceutical composition according to the invention comprises a first isolated polynucleotide which is able to inhibit RhoA expression, a second isolated polynucleotide which is able to inhibit RhoC expression, and a pharmaceutically acceptable carrier.
  • the polynucleotides specific for each gene can be formulated in two separate pharmaceutical compositions, which will be administered to a patient in need thereof either simultaneously or sequentially, through the same route or not, depending on the physician's choice.
  • the invention hence also pertains to a kit of parts comprising at least one pharmaceutical composition comprising an isolated polynucleotide as described above, specific for RhoA, and another pharmaceutical composition comprising an isolated polynucleotide as described above, specific for RhoC.
  • the polynucleotide is in the form of an injectable solution.
  • this solution is preferably appropriate for intravenous and/or for local administration - examples of local administrations are intratumoral administration and administration to the eye, in order to decrease retinal vascularization.
  • the RhoA- (or RhoC-) down-regulating polynucleotide is comprised in a vector for introducing it into a mammalian cell.
  • a vector for introducing it into a mammalian cell The skilled artisan will choose, amongst the wide variety of vectors described in the scientific literature, an appropriate vector, depending on the target cells, the mode of administration that is contemplated (ex vivo or in vivo, intravenous or intratumoral, ...) , the expected duration of expression of the polynucleotide, etc.
  • Plasmids and viruses, such as lentiviruses can be cited as non-limiting examples (Abbas-Terki et al., 2002; Hannon and Conklin, 2004).
  • Such vectors can be used advantageously to deliver DNA that will subsequently be transcribed into shRNA, in the cells.
  • Integrative vectors carrying a sequence that can be transcribed into an anti-RhoA or anti-RhoC shRNA can advantageously be used to obtain a long-term expression, for example by local injection in tumors.
  • Other carriers that can be used in the pharmaceutical composition, in complement or as an alternative to the above-mentioned vectors comprise nanospheres or nanoparticles.
  • chitosan nanoparticles such as, for example, the chitosan-coated PIHCA nanoparticles described in WO 2004/00287, can be used to perform this aspect of the invention.
  • the molecular weight of chitosan in the nanoparticles which can be used as vehicles in the compositions according to the invention preferably ranges 2000 to 8000 Da.
  • chitosan of 5000 Da can be used in the pharmaceutical compositions according to the invention.
  • a pharmaceutical composition according to the invention is preferably formulated so as to enable the administration to a patient in need thereof, of between 100 to 2000 ⁇ g of the anti-RhoA or anti-RhoC isolated polynucleotide (s) , per kg body weight, in one day.
  • Greater amounts of polynucleotides according to the invention can also be administered to a patient, for example and the physician will be able to adjust the above quantities, depending on the patient, the type of cancer, and the administration route.
  • a pharmaceutical composition extratemporaneously .
  • the present invention hence also concerns a kit of parts comprising at least one isolated polynucleotide as described above, and a pharmaceutical vector which enables the transfection of cells by said polynucleotide, when combined with it.
  • the pharmaceutical compositions and kits of parts according to the invention can also further comprise an anticancer chemotherapeutic agent.
  • an isolated polynucleotide according to the invention which is specific for RhoA, will of course be advantageously used for the preparation of a pharmaceutical composition against a disease or condition associated with an over-expression of RhoA, and an isolated polynucleotide specific for RhoC will be advantageously used for the preparation of a pharmaceutical composition against a disease or condition associated with an over-expression of RhoC.
  • the polynucleotides and compositions of the invention are particularly useful for the treatment or prophylaxis of an hyperproliferative condition, such as a solid tumor cancer, in particular aggressive breast cancer.
  • polynucleotides according to the present invention can also be advantageously used for the preparation of a pharmaceutical composition targeting metastasis in cancer, especially pharmaceutical compositions formulated for systemic administration.
  • the present invention will be further illustrated by the additional description which follows, which refers to examples of obtention and use of siRNAs and compositions of the invention. It should be understood however that these examples are given only by way of illustration of the invention and do not constitute in any way a limitation thereof.
  • FIGURES Figure 1 Inhibition of RhoA and RhoC expression by anti-RhoA and anti-RhoC siRNA at both transcriptional and protein levels.
  • MDA-MB 231 cells were either untreated (lanes 1) or transfected with 8.5 nM anti-RhoA or anti-RhoC siRNA once (lanes 2) or twice at 24 hours interval (lanes 3). Assays were performed 24 h after the end of treatment.
  • For mRNA analyses cells were treated with (A) anti-RhoA siRNA or (B) anti-RhoC siRNA, and mRNA expression was followed by RT-PCR; ⁇ -actin served as an internal control.
  • Western Blots were performed to assay RhoA protein expression after treatment with anti-
  • RhoA siRNA (C) RhoC protein expression after treatment with anti-RhoC siRNA (D) . Note that a double transfection almost completely inhibited RhoA and RhoC expression .
  • Figure 2 Antiproliferative and anti-invasive effects of anti-RhoA or anti-RhoC siRNA on MDA-MB 231 breast cancer cells in comparison with action of GGTI and FTI Panel A : a ction on MDA-MB 231 prolifera tion 1.5.10 5 MDA-MB 231 cells were seeded and cells were counted in a particle counter after 24 hours or 48 hours of culture with a minimal concentration of SVF (2%) to assure viability of the cells. Control was done in the absence of agent.
  • MDA-MB 231 cells were treated with anti-RhoA or anti-RhoC siRNA (8.5 nM) or with FTI-277 (10 ⁇ M) , GGTI- 298 (10 ⁇ M) for 24 hours before adding to Transwell. Controls were performed in the absence of agent for FTI and GGTI and in the presence of cytofectin diluted 1/100 for siRNA. Then, the cells were detached, resuspended in the presence of agents to be tested at same concentrations as used during the incubation time and seeded into the transwell coated with matrigel.
  • HMEC-1 control incubated with the same concentration of cytofectin as used for siRNA transfection
  • cytofectin cytofectin as used for siRNA transfection
  • the formation of capillary tubes was photographed under an inverted light microscope at X 40 magnification. Note the cell rounding in HMEC-1 transfected with anti-RhoA siRNA, while no shape change was noted in anti-RhoC transfected cells.
  • Figure 4 Effect of anti-RhoA and anti-RhoC siRNA on nuclear ⁇ -catenin expression in MDA-MB 231 cells. MDA-MB 231 cells untreated (control) or incubated once or twice at 24 hours interval with 8.5 nM siRNA anti RhoA or anti RhoC were analysed.
  • FIG. 5 Effect of intratumoral injection of anti-RhoA or anti-RhoC siRNA on the growth and vascularisation of MDA-MB 231 tumors xenografted in the mice. 4.10 6 MDA-MB 231 cells were subcutaneously grafted in the dorsa of athymic mice. After 16 days, mice received an intratumoral injection of anti-RhoA or anti-RhoC siRNA (lOO ⁇ l at 85 nM) or cytofectin alone for control.
  • Figure 6 Effects of intravenous injections of anti-RhoA siRNA on the growth of MDA-MB-231 cells xenografted into Nude mice. 4 x 10 6 MDA-MB-231 cells were injected s . c. into the upper hind limb of athymic nude mice and allowed to grow until the tumor reached 20 mm 3 .
  • PIHCA Poly-isohexyl-cyanoacrylate
  • FIG. 7 intravenous injections of anti-RhoA siRNA inhibits MDA-MB tumor growth.
  • Tumors representative of control and anti-RhoA siRNA-treated groups are shown 10 and 30 days after the beginning of therapy, and at day 50, i.e., 20 days after the end of the therapy. Note the necrotic area in the center of tumour in one tumor in a mouse-treated withl500 ⁇ g anti RhoA siRNA-coated nanoparticles.
  • FIG 8 Toxicology studies of RhoA siRNA in the mice by examining the body weight gains.
  • Control group was constituted of 6 mice which were anesthezied every 3 days in order to be matched with treated group.
  • Body weight was measured in control mice and in both treated groups (150 and 1500 ⁇ g/Kg) in animals with or without xenografted tumour.
  • Figure 9 Liver examination after 30 days treatment with saline (control) or with siRNA antiRhoA- coated nanoparticles at 150 and 1500 ⁇ g/Kg
  • EXAMPLE 1 Anti-RhoA and Anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 Breast Cancer cells in vitro and in vivo Material and methods
  • MDA-MB 231 an aggressive human breast carcinoma cell line, was grown in RPMI-1640 medium (Eurobio, Les Ulis, France) supplemented with 10% heat- inactivated foetal calf serum (FCS, Costar, Brumath, France) , 2 mM L-glutamine (Gibco BRL Life Technologies, Grand Island, NY) , and 100 IU/ml penicillin/streptomycin (Sarbach, Suresnes, France/Diamant , Puteaux, France). Cells were cultured at 37°C in a humidified 5% C02 atmosphere.
  • the human microvascular endothelial cell-1 (HMEC-1) line was kindly provided by Dr Ades (Center for Disease Control and Prevention, Atlanta, GA.
  • HMEC-1 vascular endothelial growth factor
  • HMEC-1 cells were cultured in a complete MCDB131 medium (Sigma, Paris, France) supplemented with 15% foetal calf serum (FCS), 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, 10 ng/ml epidermal growth factor (EGF) (Euromedex, Souffelweyersheim, France) and 1 ⁇ g/ml hydrocortisone (Pharmacia-Upjohn, St Quentin en Yvelines, France). HMEC-1 cells were used before the 15th passage, because after 25 passages, changes in the morphology and functions of endothelial cells would make them unsuitable for angiogenesis assessment.
  • SiRNA treatment Two siRNA against human RhoA or Rho C were designed.
  • RhoA and RhoC were scanned to identify AA(N19)TT sequences.
  • Candidate sequences were compared with the cDNA sequences of RhoA and RhoC and also searched against the non-redundant human DNA database using a BLAST algorithm (accession by international NCBI) .
  • a control siRNA was also tested. It was selected because it exhibited no cellular toxicity.
  • sequences selected for the sense and antisense strands are: For the anti-RhoA siRNA sense 5' GACAUGCUUGCUCAUAGUCTT 3' (SEQ ID No: 1) antisense 3' TTCUGUACGAACGAGUAUCAG 5' (SEQ ID No: 2) For the anti-RhoC siRNA sense 5' GACCUGCCUCCUCAUCGUCTT 3' (SEQ ID No: 3) antisense 3' TTCUGGACGGAGGAGUAGCAG 5'.
  • siRNA sense 5' CAGUCAGGAGGAUCCAAAGTG 3' (SEQ ID No: 5) antisense 3' TTGUCAGUCCUCCUAGGUUUC 5' (SEQ ID No: 6) They were synthesized as synthetic oligonucleotides by Eurogentech (Belgium) and annealed to form a short double-stranded RNA with a 3 ' -dithymidine overhang. Hybridization was performed in a buffer containing 2 mM sodium acetate, 100 mM potassium acetate and 30 mM Hepes buffer, pH 7.4.
  • siRNAs were introduced into MDA-MB 231 or HMEC-1 cells by cytofectin-mediated transfection (Ozyme, Paris, France) using the method and siRNA concentration described by Bertrand et al. (Bertrand et al., 2002). Cells were cultured in 6-well plates in 200 ⁇ l serum- enriched medium. When confluence reached 50 %, 20 ⁇ l of 85 nM siRNA (final concentration) in 1/100 diluted cytofectin were added dropwise to the cell cultures and incubated with the cells for indicated time.
  • RNA Extraction and Reverse Transcriptase-Polymerase Chain Reaction Assay (RT-PCR) . Briefly, after siRNA transfection, cells were detached with the non enzymatic cell dissociation solution (CDS, Sigma) and washed twice in PBS. Total RNA extraction was performed using the "SV total RNA isolation system" (Promega, Charbonnieres-les-Bains, France) according to the manufacturer's instructions. Primers were chosen using a biomolecular sequences databases (Genbank) and oligonucleotides used as primers were synthesized by Genset (Paris, France); the sequences were as follows: For RhoA :
  • Reverse primer 5' -CTTCGGAATGATGAGCACAC-3' (from position of 361-380 bp) (SEQ ID No: 8).
  • RhoC 5' -CTTCGGAATGATGAGCACAC-3' (from position of 361-380 bp) (SEQ ID No: 8).
  • Reverse primer 5' -TACCCGGACACTGATGTCATC-3' (from position of 220-240 bp) (SEQ ID No: 10)
  • ⁇ -actin 5' -TACCCGGACACTGATGTCATC-3' (from position of 220-240 bp) (SEQ ID No: 10)
  • PCR products were respectively 287 bp for RhoA, 147 bp for RhoC and 838 bp for actin Reverse transcription was performed at 48 °C for 45 min. The reaction products were then subjected to 30 cycles of PCR for RhoA and RhoC and 35 for actin. An amplification cycle consisted of 30 sec at 94 °C for denaturation, 30 sec at 60°C and 30 sec at 68°C. Finally, an extension step at 68 °C (7 min) improved the quality of the final product by extending truncated product to full length. RT-PCR was done on control cells and after one or two treatment with siRNA.
  • PVDF polyvinylidene difluoride
  • Binding of primary antibody was detected by enhanced chemiluminescence visualization system (ECL, Amersham) using the horseradish peroxidase (HRP) -coupled anti-mouse or anti-goat antibody (1:10,000; Dako, Glostrup, Denmark) for 40 min at room temperature. The autoradiography was carried-out for 1 min to 10 min. Western blot was performed 24 hours after one or two treatments with siRNA.
  • ECL enhanced chemiluminescence visualization system
  • HRP horseradish peroxidase
  • mda-mb-231 cells were washed in cold PBS, lysed in ice-cold lysis buffer containing phosphatase and protease inhibitors, and centrifuged at 100,000g for 30 min. at 4°C; the supernatant collected at this step is referred to as the cytosolic fraction.
  • Pellets were rehomogenized in the same lysis buffer containing 2% Triton X-114 (Sigma) and centrifuged at 800g for 10 min. at 4°C; the supernatant collected at this step is the nuclear fraction. Protein concentrations were determined as above (Bradford, 1976) .
  • ⁇ -catenin 20 ⁇ g of protein extract were subjected to Western blotting using mouse ⁇ -catenin monoclonal antibody (Santa Cruz) diluted 1/500.
  • Cell proliferation assay To ensure cell viability, minimal concentration of FCS were added to the medium: 2% for MDA- MB 231, and 7.5% for HMEC-1 cells. Briefly, after trypsination, the cells were seeded at a concentration of 1.5.10 5 cells per well in a 24-well-plate (Costar, Cambridge, MA) . The agent to be tested was added at the appropriate concentration. For siRNA treatment, two transfections were carried out, at T 0 and after 24 hours, as described above.
  • the cell number was measured after 48 hours by a particle counter (Coulter Zl, Coultronics, France) after detachment with a non-enzymatic cell dissociation solution (CDS, Sigma) .
  • CDS non-enzymatic cell dissociation solution
  • Cell invasiveness through matrigel This was performed as previously described (Denoyelle et al . , 2001).
  • An 8 ⁇ m-diameter pore Transwell Dutscher, Brumath, France
  • MDA-MB 231 cells were used untreated (incubated in medium alone), or treated for 24 hours FTI- 277 (10 ⁇ M), GGTI-298 (10 ⁇ M) or transfected by the siRNA for indicated time, or treated with cytofectin alone.
  • Afetre 24h of incubation the cells were detached by the non enzymatic cell dissociation solution, washed twice with PBS, and resuspended in RPMI 1640 with 0.2 mg/ml bovine serum albumin (BSA, Sigma) in the presence or absence of agents to be tested (at the same concentration as that used for the previous incubation) ; in the case of the siRNAs, this corresponds to a second transfection 24h after the first. In each case, 2.
  • Results were calculated with reference to control values observed after incubation in medium alone (untreated control, for FTI-277 and GGTI-298 treatments), or cytofectin-containing medium (control for all siRNA treatments), arbitrarily set at 100%. Similar experiment was done with HMEC-1 cells, except that detached cells re-suspended in MCDB-131 medium with 0.2 mg/ml bovine serum albumin (BSA, Sigma). Capillary tube formation on matrigel matrix Endothelial cells controls or transfected at indicated conditions using siRNA anti RhoA or anti RhoC were used. Matrigel matrix (Becton Dickinson, France) was kept on ice during 24 hours. Then, 200 ⁇ l per well of matrigel was added to a 24-well culture plate.
  • BSA bovine serum albumin
  • mice L'doubtedlysle, France), 6 week old, were kept in a temperature-controlled room where humidity and light were carefully monitored.
  • MDA-MB 231 cells (4.10 6 cells in a volume of 200 ⁇ l) were injected subcutaneously (s.c.) on the right side of the dorsal area behind the last limb.
  • s.c. subcutaneously
  • Intratumoral injections were repeated every 3 days for a total period of 20 days. Tumor sizes were routinely measured every day (perpendiculars diameters) and tumor volumes were systematically calculated. On day 20, animals were euthanized, tumors were removed and then subjected to immunohistochemistry analysis to assess the extent of intratumoral vascularization within the different experimental groups. Immunohistochemical staining was performed as previously described (Li et al., 2001). The tumors were fixed overnight in absolute ethyl alcohol and embedded in paraffin. Five-micrometer sections were then prepared.
  • tumor sections were deparaffinated in toluene, rehydrated, permeabilized by microwave oven treatment in citrate buffer (pH 6.0), quenched by 3% H 2 0 2 for 5 min to remove endogenous peroxidase activity, washed in PBS and then .incubated for 1 hour with a rat antibody against mouse PECAM-1 (platelet-endothelial cell adhesion molecule-1; Pharmingen, France) and then with a biotinylated goat anti-rat IgG antibody for 15 min. After washing, sections were incubated with streptavidin-peroxydase and the vessels were revealed by peroxydase substrate diaminobenzidin .
  • citrate buffer pH 6.0
  • RhoA and RhoC mRNA and protein levels by anti RhoA and anti RhoC siRNA transfection to MDA-MB 231 cells and HMEC-1 Efficacy of the inhibition of RhoA and RhoC synthesis by siRNA in MDA-MB 231 and HMEC-1 was analysed by both RT-PCR and Western blot for expression of RhoA and RhoC. As shown in figure 1, both mRNA and protein expressions of RhoA and RhoC were down regulated when MDA- MB 231 cells were incubated with siRNA. 90 % inhibition was obtained when treatment with siRNA was repeated two times at 24 hours interval.
  • RhoA and RhoC proteins expression increased, because siRNA in mammalian cells was transient (not shown) . Therefore, a double transfection was selected for testing the effect of RhoA or RhoC gene expression inhibition. Similar inhibition was observed with HMEC-1 cells (not shown) . This down regulation is specific as siRNA antigen
  • RhoA did not modify RhoC mRNA expression expression and siRNA anti RhoC did not modify the expression of RhoA mRNA (fig 1) .
  • ⁇ -actin mRNA was not modified by the treatment with siRNA.
  • a 9-hour bFGF stimulation of endothelial cells triggers capillary tube formation, and sprouting of new capillaries is also visible: closed polygons are easily visible and complex mesh like structures with several shunts and branches are markedly formed.
  • siRNA were transfected in HMEC-1, the formation of capillary tubes was inhibited and endothelial cells remain well separated. A cell rounding was observed when cells were transfected with siRNA anti RhoA but not with siRNA anti RhoC. Inhibitory action by anti RhoA or anti RhoC siRNA on ⁇ - catenin nuclear localization As observed in figure 4, the transfection of MDA-MB 231 cells with anti RhoA or anti RhoC siRNA inhibited ⁇ -catenin nuclear fraction.
  • PECAM-1 immunostaining was dramatically reduced in tumors excised from anti RhoA siRNA-injected mice.
  • the angiogenesis index were 30.5 ⁇ 4.12 in the control group and 8.75 ⁇ 3.30 in the anti RhoA siRNA- treated group and 22.5 ⁇ 3.32 in the anti RhoC siRNA- treated group.
  • RhoA is overexpressed in cancer and Rho-protein- dependant cell signalling might be important for malignant transformation (Sahai and Marshall, 2002) . Therefore the inhibition of Rho proteins might be a good approach to inhibit cancer cell aggressivity.
  • This strategy was already tested by using inhibitors of Rho prenylation, a step required for Rho-cell signaling.
  • cerivastatin a potent inhibitor of HMG-CoA reductase, efficiently reduced cancer cell proliferation and invasion (Denoyelle et al., 2003) and also angiogenesis (Vincent et al., 2001; Vincent et al . , 2002).
  • cerivastatin Despite the potency of cerivastatin on cancer cell aggressivity and on angiogenesis in vi tro, it cannot be considered as a potential anticancer agent due to its rapid clearance by the liver.
  • the action of cerivastatin on MDA-MB 231 can also be mimicked by GGTI (Denoyelle et al., 2003).
  • GGTI Denoyelle et al., 2003
  • several other proteins could also be geranyl-geranylated and their role in Rho- dependant cell signalling remained to be confirmed.
  • siRNA chemically synthesized short interfering RNA (siRNA) was used. siRNA can specifically and effectively direct homology-dependent post-transcriptional gene silencing.
  • anti-RhoA and anti-RhoC siRNAs can suppress the proliferation and the migration of aggressive breast human cancer cells (MDA-MB231 cells) , characterized by an oncogenic mutation of Ras and an over-expression of RhoA and RhoC.
  • MDA-MB231 cells aggressive breast human cancer cells
  • RhoA and RhoC over-expression of RhoA and RhoC.
  • the effect of siRNAs was compared with that of FTI, GGTI or cerivastatin used at optimal concentration (25 ng/ml) which totally inhibit prenylation of Rho to cell membrane (Denoyelle et al., 2003).
  • Anti-RhoA or anti- RhoC siRNA was used in conditions which have been determined to suppress more than 90% of mRNA and protein synthesis (2 successive transfections at 8.5 nM in cytofectin at 24 hours interval) . Indeed, an unique transfection of RhoA or RhoC siRNA did not achieved a total inhibition of mRNA and protein expression, and consequently induced a weak inhibition in MDA-MB231 cells proliferation. The specificity of the siRNA was confirmed by the absence of action on actin mRNA expression. It was also observed that anti-RhoA siRNA did not modify RhoC mRNA expression and vice-versa (siRNA anti RhoC inhibits only RhoC expression) .
  • anti-RhoA or anti-RhoC siRNA was more effective than Rho cell signalling inhibitors, GGTI and cerivastatin, for inhibiting the proliferation and invasion of MDA-MB 231 cells and cytokine-stimulated proliferation from endothelial cells. These siRNA also inhibited the formation of capillary-like tubes by endothelial cells.
  • Anti-RhoA and -RhoC siRNA also induced a potent inhibition of Rho-cell signalling, as demonstrated by the inhibition of ⁇ -catenin translocation into the nucleus of MDA-MB 231 cells.
  • RhoB-GG was shown to inhibit anchorage-dependent and -independent growth, induce apoptosis, inhibit constitutive activation of Erk and insulin-like growth factor-1 stimulation of Akt and to suppress tumor growth in nude mice (Chen et al., 2000).
  • MDA-MB 231 tumor cells were xenografted subcutaneously in nude mice. As indicated by in vi tro results, the efficiency of siRNA is transient and to be efficient the tranfection of siRNA into tumor or endothelial cells has to be repeated. Therefore, the siRNA was introduced (lOO ⁇ l per injection with 85 nMol siRNA) directly in the tumor every three days during 20 days.
  • anti-RhoA siRNA could inhibit both the proliferation of tumor cells from human origin and angiogenesis derived from murine endothelial cells, while anti-RhoC siRNA could inhibit only the proliferation of tumor cells.
  • the angiogenic index was greatly inhibited in anti-RhoA siRNA -treated group (8.75 ⁇ 3.30 versus 30.5 ⁇ 4.12 for control group) and less in anti-RhoC siRNA-treated group (22.5 ⁇ 3.32).
  • siRNA anti-RhoA and anti-RhoC siRNA represent a powerful tool to reduce aggressive breast cancer growth by inhibiting both cancer cells invasion and proliferation and angiogenesis under both in vi tro and in vivo conditions. Therefore, siRNA anti-RhoA and -RhoC may have potential therapeutic utility in the treatment of aggressive breast cancers.
  • EXAMPLE 2 Intravenous delivery in mice of anti-RhoA siRNA loaded in nanoparticles of chitosan: safety and efficacy in xenografted aggressive breast cancer
  • Example 1 Anti-RhoA siRNA The double-stranded anti-RhoA siRNA described in Example 1 was used. Chitosan nanoparticles Type 2 Chitosan-PIHCA nanoparticles from
  • siRNA nanoparticles 1 ml of anti RhoA siRNA at 30 ⁇ M was added to 300 ⁇ l of the suspension of chitosan nanoparticles. After an incubation of 15 minutes at room temperature, the mixture was vortexed for 5 minutes. A diluted preparation was also tested: the previous suspension was diluted 1/10 in 10 mM hepes and 100 mM NaCI buffer .
  • mice Female athymic nude ⁇ nu/nu mice (Iffa Credo,
  • mice were anesthesthetized. Anesthesy was done by inhalation using isoflurane (aerrane, BAXTER, Paris France). In control groups, the same anesthesia was done every 3 days because it influences the body weight . Tumors were measured (perpendicular diameters) every day and their volumes calculated. After siRNA treatment was stopped (day 30) in 6 mice of each group, blood was collected for biochemical analysis. Mice were then euthanazied for examination of different organs after laparatomy. In the 6 other mice of treated groups, the mice were kept for another 30 days period for examination of the tumor growth as previously described.
  • Toxicological assays For toxicological assays, a similar procedure of siRNA administration was done in mice with or without xenografted tumor. Toxicology studies of RhoA siRNA were conducted in the mice by examining the following parameters: Body weight gains and final mean body weights of mice Biochemical analysis: blood samples were collected in Eppendorf tubes and then centrifuged at 1000 g for 10 minutes at room temperature and the serum separated and stored at - 20 °C until examination. Serum of mice from each group was mixed to obtain a sufficient amount of serum for examination. Biochemical analysis were done in auto analysesr (Advia 1650 , Bayer, France) .
  • alkaline phosphatase alkaline phosphatase
  • alanine aminotransferase alanine aminotransferase
  • g-glutamyltransferase for examination of liver
  • lipase for pancreatic examination.
  • Proteins determination was done to determine nutritional status of mice.
  • Urea and creatinin were also measured to examine the renal function.
  • calcium, phosphore and alakaline phosphatase were evaluated for the study of bone metabolism. organs were also examined after mice necropsy at day 30 and day 60.
  • Intravenous injection of anti-RhoA siRNA inhibits tumor growth
  • the effects of intravenous administration of anti-RhoA siRNA on tumor growth were investigated. To that effect, intravenous injection was done after anaesthesia in order to reach 150 and 1500 ⁇ g anti-RhoA siRNA per kg body weight.
  • the 150 ⁇ g anti-RhoA siRNA injected group achieved an important decrease in tumor growth (> 90% decrease in mean tumor volume on day 30) .
  • a complete inhibition of tumor growth was obtained in all mice of this group; moreover, in 5 mice out of 12 that had 6
  • PIHCA chitosan-coated nanoparticles-mediated intravenous delivery of siRNA in the growth of tumors was hence tested.
  • the effect of this therapy was tested on xenografted tumor (MDA-MB 231, characterised by an overexpression of RhoA and a constitutive activation of RhoA) growth in mice.
  • the amount of siRNA used was that deducted from the results disclosed in Example 1, showing that the antitumoral action of RhoA siRNA was obtained by injecting 0,12 ⁇ g in tumour mass every 3 days. Taking into account the dilution in body mass, 150 ⁇ g/kg body weight was administered intravenously every 3 days for 30 days and thereafter treatment was stopped.
  • a dose 10 times higher was also administered by the same way to assess both efficacy and toxicity (1500 ⁇ g/kg) .
  • the 150 ⁇ g/kg treatment regimen resulted in an important decrease in mean tumour volume (decrease more than 90 % in comparison to untreated mice) in anti RhoA- siRNA-treated mice in comparison to untreated mice.
  • Tumour growth was totally stopped using 1500 ⁇ g/kg, and an important zone of central necrosis in tumor was observed in 5/12 animals. This is probably in relation with the anti-RhoA-induced inhibition of angiogenesis.
  • the efficacy of this therapy disappeared when the treatment was stopped and the delay for the relapse depended on the dose of siRNA administered (2 days for the lower dose and 8 days for the higher one) .
  • siRNA This absence of denutrition is in good agreement with the absence of a toxic action of siRNA, as measured by the serum proteins level (Table 1) .
  • the cytotoxicity of siRNA was also evaluated using the serum activities of enzymes markers after 30 days treatment. It was shown that siRNA treatment did not induce any cytolytic action on the liver as aminotransferase level was similar in control groups and the two treated groups.
  • RhoA siRNAs represent a powerful tool for inhibiting cancer aggressivity in vivo, with potential therapeutic utility for the treatment of aggressive breast cancers, or cancers of diverse origins in which RhoA is overexpressed .
  • RhoA induces MMP-9 expression at CD44 lamellipodial focal complexes and promotes HMEC-1 cell invasion.
  • Denoyelle, C Albanese, P., Uzan, G., Hong, L., Vannier, J.P., Soria, J. and Soria, C. (2003) Molecular mechanism of the anti-cancer activity of cerivastatin, an inhibitor of HMG-CoA reductase, on aggressive human breast cancer cells. Cell Si gna l, 15, 327-338. Denoyelle, C, Vasse, M., Korner, M., Mishal, Z., Ganne, F. , Vannier, J.P., Soria, J. and Soria, C.
  • Rho GTPases in human breast tumours expression and mutation analyses and correlation with clinical parameters.
  • RNAi nature abhors a double-strand. Curr Opin Genet Dev, 12, 225-232. Imamura, F. , Mukai, M., Ayaki, M. and Akedo, H. (2000) Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Jpn J Cancer Res, 91, 811-816. Kamai, T., Tsujii, T., Arai, K. , Takagi, K. ,
  • Rho-family GTPases Regulation of the cytoskeleton by Rho-family GTPases: implications for tumour cell invasion. Semin Cancer Biol , 11, 167-173. Quong, D. and Neufeld, R.J. (1998) DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads. Biotechnol Bioeng, 60, 124-134. Sahai, E. and Marshall, C.J. (2002) RHO- GTPases and cancer. Na t Rev Cancer, 2 , 133-142. Sahai, E. and Marshall, C.J. (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.
  • Cerivastatin an inhibitor of 3-hydroxy-3- methylglutaryl coenzyme a reductase, inhibits endothelial cell proliferation induced by angiogenic factors in vitro and angiogenesis in in vivo models.
  • Arterioscler Thromb Vase Biol 22, 623-629. Weidner, N., Semple, J.P., Welch, W.R. and

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Abstract

L'invention concerne l'utilisation de siARN anti-RhoA et -RhoC afin d'inhiber de manière spécifique la synthèse de RhoA ou RhoC. L'invention concerne, en outre, des molécules d'ARN double brin isolées et capables de médier une interférence des ARNm des RhoA et RhoC humains, notamment aux fins d'inactivation de ces gènes par silençage transcriptionnel. L'invention concerne enfin des compositions thérapeutiques comprenant de tels siARN anti-RhoA et/ou anti-RhoC, ainsi que l'utilisation de celles-ci dans la fabrication d'une composition thérapeutique, notamment contre le cancer du sein agressif.
PCT/EP2005/006043 2004-05-13 2005-05-12 Siarn anti-rhoa et -rhoc et compositions therapeutiques renfermant ceux-ci WO2005113770A1 (fr)

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WO2008003329A2 (fr) 2006-07-07 2008-01-10 Aarhus Universitet Nanoparticules pour l'administration d'acides nucléiques
WO2010065961A2 (fr) 2008-12-05 2010-06-10 Whitehead Institute For Biomedical Research Compositions et procédés concernant le mir-31
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WO2011163436A1 (fr) * 2010-06-24 2011-12-29 Quark Pharmaceuticals, Inc. Composés à base d'arn double brin pour le gène rhoa et leur utilisation
EP2592147A1 (fr) 2007-10-12 2013-05-15 F. Hoffmann-La Roche AG Expression protéinique pour plusieurs acides nucléiques
EP3042955A4 (fr) * 2013-09-06 2017-07-26 The University of Tokyo Utilisation de rhoa dans le diagnostic du cancer et criblage d'inhibiteur
WO2018162517A1 (fr) 2017-03-10 2018-09-13 F. Hoffmann-La Roche Ag Procédé de production d'anticorps multispécifiques
CN114929257A (zh) * 2020-11-13 2022-08-19 Elmed株式会社 包含rhoa肽抑制剂作为有效成分的抗癌剂

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Cited By (23)

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WO2008003329A3 (fr) * 2006-07-07 2008-02-21 Univ Aarhus Nanoparticules pour l'administration d'acides nucléiques
EP2295045A1 (fr) * 2006-07-07 2011-03-16 Aarhus Universitet Nanoparticules pour l'administration d'acides nucléiques
WO2008003329A2 (fr) 2006-07-07 2008-01-10 Aarhus Universitet Nanoparticules pour l'administration d'acides nucléiques
EP2397123A1 (fr) * 2006-07-07 2011-12-21 Aarhus Universitet Nanoparticles for nucleic acid delivery
US7875449B2 (en) 2006-09-15 2011-01-25 Fmc Biopolymer As Oligonucleotide non-viral delivery systems
EP2592147A1 (fr) 2007-10-12 2013-05-15 F. Hoffmann-La Roche AG Expression protéinique pour plusieurs acides nucléiques
US8771988B2 (en) 2007-10-12 2014-07-08 Hoffmann-La Roche Inc. Protein expression from multiple nucleic acids
EP2592148A1 (fr) 2007-10-12 2013-05-15 F. Hoffmann-La Roche AG Expression protéinique pour plusieurs acides nucléiques
EP2370091A2 (fr) * 2008-12-05 2011-10-05 Whitehead Institute For Biomedical Research Compositions et procédés concernant le mir-31
EP2370091A4 (fr) * 2008-12-05 2013-03-06 Whitehead Biomedical Inst Compositions et procédés concernant le mir-31
JP2012511031A (ja) * 2008-12-05 2012-05-17 ホワイトヘッド・インスティテュート・フォー・バイオメディカル・リサーチ ｍｉＲ−３１に関連する組成物および方法
AU2009322137B2 (en) * 2008-12-05 2015-09-10 Whitehead Institute For Biomedical Research Compositions and methods relating to miR-31
WO2010065961A2 (fr) 2008-12-05 2010-06-10 Whitehead Institute For Biomedical Research Compositions et procédés concernant le mir-31
JP2014221834A (ja) * 2008-12-05 2014-11-27 ホワイトヘッド・インスティテュート・フォー・バイオメディカル・リサーチ ｍｉＲ−３１に関連する組成物および方法
US9045755B2 (en) 2010-06-24 2015-06-02 Quark Pharmaceuticals, Inc. Double stranded RNA compounds to RhoA and use thereof
AU2011270896B2 (en) * 2010-06-24 2015-04-09 Quark Pharmaceuticals, Inc. Double stranded RNA compounds to RhoA and use thereof
WO2011163436A1 (fr) * 2010-06-24 2011-12-29 Quark Pharmaceuticals, Inc. Composés à base d'arn double brin pour le gène rhoa et leur utilisation
EP3042955A4 (fr) * 2013-09-06 2017-07-26 The University of Tokyo Utilisation de rhoa dans le diagnostic du cancer et criblage d'inhibiteur
US10563265B2 (en) 2013-09-06 2020-02-18 Chugai Seiyaku Kabushiki Kaisha Use of RHOA in cancer diagnosis and inhibitor screening
EP3591050A3 (fr) * 2013-09-06 2020-03-25 The University of Tokyo Utilisation de rhoa dans le diagnostic du cancer et le dépistage d'inhibiteurs
EP4335920A3 (fr) * 2013-09-06 2024-05-22 The University of Tokyo Utilisation de rhoa dans le diagnostic du cancer et le criblage d'inhibiteurs
WO2018162517A1 (fr) 2017-03-10 2018-09-13 F. Hoffmann-La Roche Ag Procédé de production d'anticorps multispécifiques
CN114929257A (zh) * 2020-11-13 2022-08-19 Elmed株式会社 包含rhoa肽抑制剂作为有效成分的抗癌剂

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