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WO2003035082A1 - Medicament destine a inhiber l'expression d'un gene cible - Google Patents

Medicament destine a inhiber l'expression d'un gene cible Download PDF

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Publication number
WO2003035082A1
WO2003035082A1 PCT/EP2002/011971 EP0211971W WO03035082A1 WO 2003035082 A1 WO2003035082 A1 WO 2003035082A1 EP 0211971 W EP0211971 W EP 0211971W WO 03035082 A1 WO03035082 A1 WO 03035082A1
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WIPO (PCT)
Prior art keywords
dsrna
strand
nucleotides
medicament
use according
Prior art date
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PCT/EP2002/011971
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German (de)
English (en)
Inventor
Stefan Limmer
Sylvia Limmer
Roland Kreutzer
Philipp Hadwiger
Original Assignee
Ribopharma Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10160151A external-priority patent/DE10160151A1/de
Priority claimed from PCT/EP2002/000151 external-priority patent/WO2002055692A2/fr
Priority to DE10230997A priority Critical patent/DE10230997A1/de
Priority to DE10230996A priority patent/DE10230996A1/de
Application filed by Ribopharma Ag filed Critical Ribopharma Ag
Priority to PCT/EP2002/011971 priority patent/WO2003035082A1/fr
Priority to JP2003538370A priority patent/JP2005506385A/ja
Priority claimed from PCT/EP2002/011968 external-priority patent/WO2003035868A1/fr
Publication of WO2003035082A1 publication Critical patent/WO2003035082A1/fr
Priority to US10/382,634 priority patent/US20040038921A1/en
Priority to US10/666,458 priority patent/US20040126791A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 nucleic acids [NA]
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • 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
    • C12N2320/50—Methods for regulating/modulating their activity

Definitions

  • the invention relates to a medicament and a use for inhibiting the expression of a target gene.
  • a method for inhibiting the expression of a target gene in a cell by means of RNA interference is known from DE 101 00 586 C1.
  • An oligoribonucleotide with a double-stranded structure is introduced into the cell.
  • One strand of the double-stranded structure is complementary to the target gene. It is not known how such an oligoribonucleotide can be used in vivo to inhibit a target gene.
  • the object of the present invention is to eliminate the disadvantages of the prior art.
  • an effective medicament and a use for inhibiting the expression of a target gene are to be provided.
  • a medicament for inhibiting the expression of a target gene is provided, the medicament being present in at least one administration unit which contains a double-stranded ribonucleic acid (dsRNA) which is suitable for inhibiting the expression of the target gene by means of RNA interference.
  • the dsRNA can be contained in the administration unit in an amount that enables a dosage of at most 5 mg per kg body weight and day.
  • a dsRNA is present when the ribonucleic acid consisting of one or two ribonucleic acid strands has a double-stranded structure. Not all nucleotides of the dsRNA need to have canonical Watson-Crick base pairings.
  • the target gene can be an oncogene, cytokine gene, idiotype protein gene (id protein gene), prion gene, gene for the expression of angiogenesis-inducing molecules, of adhesion molecules and cell surface receptors, of proteins which are involved in metastatic and / or invasive processes are involved, gene from proteinases and apoptosis and cell cycle regulating molecules, gene for expressing the EGF receptor, the multidrug resistance 1 gene (MDRI gene), preferably in pathogenic organisms Plasmodia, expressed gene or a component of a, in particular human pathogenic, virus.
  • id protein gene idiotype protein gene
  • prion gene gene for the expression of angiogenesis-inducing molecules, of adhesion molecules and cell surface receptors, of proteins which are involved in metastatic and / or invasive processes are involved, gene from proteinases and apoptosis and cell cycle regulating molecules, gene for expressing the EGF receptor, the multidrug resistance 1 gene (MDRI gene), preferably in pathogenic organisms
  • the administration unit can be designed for a single administration or intake per day. Then the entire daily dose is contained in one administration unit. If the administration unit is designed for repeated administration or ingestion per day, the dsRNA is contained therein in a correspondingly smaller amount that enables the daily dose to be reached.
  • the administration unit can also be used for a single administration or intake be designed for several days, e.g. B. by releasing the dsRNA over several days. The administration unit then contains a corresponding multiple of the daily dose.
  • the medicament contains the dsRNA in an amount sufficient to inhibit the expression of the target gene in an organism to be treated with it.
  • the drug can also be designed so that several units of the drug together contain the sufficient amount in total. The sufficient amount also depends on the pharmaceutical formulation of the drug.
  • the dsRNA is highly effective in vivo in a mammal or human in the low dosage of at most 5 mg per kg of body weight and day. This is particularly surprising because there are mechanisms in mammals and humans that recognize and break down double-stranded nucleic acids as foreign to the body.
  • the administration unit preferably contains the dsRNA in an amount which enables a dosage of at most 2.5 mg, in particular at most 200 ⁇ g, preferably at most 100 ⁇ g, particularly preferably at most 50 ⁇ g, in particular at most 25 ⁇ g, per kg of body weight and day. It has been shown that the dsRNA has an excellent effectiveness in inhibiting the expression of the target gene even in this even lower dosage.
  • the dsRNA is contained in the medicament in a preparation which, in particular, consists exclusively of a physiologically compatible solvent and the dsRNA dissolved therein.
  • the solvent is generally a buffer. It may contain additives, e.g. B. those that make the buffer physiologically compatible or durable. "Exclusively" means here that there are no substances which cause or mediate the uptake of the dsRNA into the target gene-expressing cells. Such substances are e.g. B. micellar structures, especially liposomes, or capsids. Surprisingly, it has been shown that a dsRNA which is only dissolved and administered in a physiologically compatible solvent is taken up by the cells containing the target gene and inhibits the expression of the target gene.
  • the dsRNA can be enclosed in the medicament by a micellar structure, preferably a liposome, a capsid, a capsoid or a polymeric nano- or microcapsule, or bound to a polymeric nano- or microcapsule.
  • a micellar structure, a capsid, a capsoid or a polymeric nano- or microcapsule can facilitate the uptake of the dsRNA in cells.
  • the dsRNA can be enclosed by a viral natural capsule or an artificial capsid produced by chemical or enzymatic means or a structure derived therefrom.
  • the polymeric nano- or microcapsule consists of at least one biodegradable polymer, for example polybutyl cyanoacrylate.
  • the polymeric nano- or microcapsule can transport and release dsRNA contained in or bound to it in the body.
  • the dsRNA can be administered orally, by inhalation, infusion or injection, in particular intravenous, intraperitoneal or intratumoral infusion or injection.
  • the medicament therefore has a preparation which is suitable for inhalation, oral intake, infusion or injection, in particular for intravenous, intraperitoneal or intratumoral infusion or injection. How such a preparation can be produced is known from pharmacy.
  • a preparation suitable for inhalation, infusion or injection can consist of a physiologically compatible solvent, preferably a physiological saline solution or a physiologically compatible buffer, in particular a phosphate-buffered salt solution, and the dsRNA.
  • a physiologically compatible solvent preferably a physiological saline solution or a physiologically compatible buffer, in particular a phosphate-buffered salt solution, and the dsRNA.
  • a strand S1 of the dsRNA preferably has a region which is at least partially complementary to the target gene and in particular comprises fewer than 25 successive nucleotides.
  • the “target gene” is generally understood to mean the DNA strand of the double-stranded DNA coding for a protein, which is complementary to a DNA strand which serves as a template during transcription, including all the areas transcribed. The target gene is therefore generally the sense strand.
  • the strand S1 can thus be complementary to an RNA transcript formed during the expression of the target gene or its processing product, such as e.g. an mKNA.
  • the target gene can also be part of a viral genome.
  • the viral genome can also be the genome of a (+) strand RNA virus, in particular a hepatitis C virus.
  • the complementary region of the dsRNA can have 19 to 24, preferably 20 to 24, particularly preferably 21 to 23, in particular 22 or 23, nucleotides.
  • a dsRNA with this structure is particularly efficient in inhibiting the target gene.
  • the Strand S1 of the dsRNA can have less than 30, preferably less than 25, particularly preferably 21 to 24, in particular 23, nucleotides. The number of these nucleotides is also the number of the maximum possible base pairs in the dsRNA. Such a dsRNA is particularly stable intracellularly.
  • dsRNA has a single-stranded overhang formed from 1 to 4, in particular 2 or 3, nucleotides.
  • a dsRNA has a better effectiveness in inhibiting the expression of the target gene at least at one end than a dsRNA without single-stranded overhangs.
  • One end is a region of the dsRNA in which there is a 5 'and a 3' strand end.
  • a dsRNA consisting only of strand S1 accordingly has a loop structure and only one end.
  • a dsRNA formed from the strand S1 and a strand S2 has two ends. One end is formed in each case by one end of strand S1 and one end of strand S2.
  • the single-stranded overhang is preferably located at the 3 'end of the strand S1. This localization of the single-stranded overhang leads to a further increase in the efficiency of the drug.
  • the dsRNA has a single-stranded overhang only at one end, in particular at the end located at the 3 'end of the strand S1.
  • the other end of a double-ended dsRNA is smooth, ie without overhangs. Surprisingly, it has been shown that an overhang at one end of the dsRNA is sufficient to increase the interference effect of the dsRNA, without lowering the stability to the same extent as by two overhangs.
  • a dsRNA with only one overhang has proven to be sufficiently stable and particularly effective both in various cell culture media and in blood, serum and cells. Inhibition of ex pression is particularly effective if the overhang is at the 3 'end of the strand S1. It is therefore sufficient, in particular in the case of a dsRNA with one or two smooth ends, if the administration unit contains the dsRNA in a quantity which comprises a dosage of at most 100 ⁇ g, preferably at most 50 ⁇ g, in particular at most 25 ⁇ g, per day and kg body weight allows.
  • the use of a double-stranded ribonucleic acid in a dosage of at most 5 mg per kg of body weight and day is also provided for inhibiting the expression of a target gene by means of RNA interference in a mammal or human.
  • FIG. 8 shows the GFP expression (FACS analysis) in the blood of the individual animals after treatment with specific (GFP group) and non-specific (control group) dsRNA,
  • the double-stranded oligoribonucleotides used have the following sequences:
  • GFP laboratory mice that express the green fluorescent protein (GFP) in all protein biosynthetic cells, double-stranded RNA (dsRNA) derived from the GFP sequence, or non-specific dsRNA intravenously injected into the tail vein.
  • dsRNA double-stranded RNA
  • non-specific dsRNA intravenously injected into the tail vein.
  • the nonspecific dsRNA has neither homology to the GFP gene nor to a gene occurring in humans or mice.
  • the animals were sacrificed and GFP expression in tissue sections and in plasma was analyzed.
  • RNA synthesizer type Expedite 8909, Applied Biosystems, Rothstadt, Germany
  • RNA single strands evident from the sequence listing and the RNA single strands complementary to them were synthesized. Then the Purification of the raw synthesis products with the help of HPLC. NucleoPac PA-100, 9x250 mm from Dionex GmbH, Am Woertzbaum 10, 65510 Idstein, Germany, were used as columns; Low salt buffer used: 20 mM Tris, 10 mM NaC10, pH 6.8, 10% acetonitrile; used high salt buffer 20 mM Tris, 400 mM NaC10, pH 6.8, 10% acetonitrile.
  • the flow was 3 ml / minute.
  • the hybridization of the single strands to the double strand was carried out by heating the stoichiometric mixture of the single strands in 10 mM sodium phosphate buffer, pH 6.8, 100 mM NaCl, to 80-90 ° C. and then slowly cooling to room temperature over 6 hours.
  • mice The transgenic laboratory mouse strain TgN (GFPU) 5Nagy (The Jackson Laboratory, Bar Harbor, ME, USA) was used, the GFP (with a beta-actin promoter and a CMV intermediate early enhancer) in all cells examined so far expressed (Hadjantonakis AK et al., 1993, Mech. Dev. 76: 79-90; Hadjantonakis AK et al., 1998 Nature Genetics 19: 220-222). GFP-transgenic mice can be clearly differentiated from the corresponding wild types (WT) based on the fluorescence (with a UV hand lamp). GFP-heterozygous animals were used for the experiments. These were bred by mating a corresponding WT animal with a heterozygous GFP-type animal.
  • WT wild types
  • the test was carried out in accordance with the German animal welfare regulations.
  • the animals were kept under controlled environmental conditions in groups of 3-5 animals in type III macro-lon cages from Ehret, Emmendingen, Germany at a constant temperature of 22 ° C. and a light-dark rhythm of 12 hours , Soft wood granulate 8/15 from Altromin, Germany was used as the litter.
  • the animals received tap water and standard Altromin 1324 pelletized from Altromin ad libidum.
  • the heterozygous GFP animals were kept in groups of 3 animals in cages as described above.
  • the injections of the dsRNA solution made intravenously (iv) into the tail vein in 12-hour cycle (between 5 and 30 7 00 17 30 as well as between 19 and 00 hours) for 5 days.
  • the injection volume was 60 ⁇ l per 10 g body weight and the dose was 2.5 mg dsRNA or 50 ⁇ g per kg body weight.
  • the division into the groups was as follows:
  • Group A PBS (phosphate buffered saline) each 60 ⁇ l per 10 g body weight
  • Group C 2.5 mg per kg of body weight of a further non-specific control dsRNA (K3 control with 2-nucleotide (nt) overhangs at both 3 'ends and a double-stranded region of 19 nucleotide pairs),
  • Group D 2.5 mg per kg of body weight dsRNA (specifically directed against GFP, hereinafter referred to as S1, with smooth ends and a double-stranded region of 22 nucleotide pairs),
  • Group E 2.5 mg dsRNA per kg body weight (specifically directed against GFP, hereinafter referred to as S7, with 2nt overhangs at the 3 'ends of both strands and a double strand region of 19 nucleotide pairs)
  • Group F 50 ⁇ g Sl-dsRNA per kg body weight (i.e. 1/50 of the dose of group D).
  • Organ removal Immediately after the animals were killed by CO inhalation, blood and various organs were removed (thymus, lungs, heart, spleen, stomach, intestine, pancreas, brain, kidney and liver). The organs were rinsed briefly in cold, sterile PBS and divided with a sterile scalpel.
  • the blood was kept on ice for 30 min immediately after removal, mixed, centrifuged for 5 min at 2000 revolutions per minute (Mini spin, Eppendorf AG, Barkhausenweg 1, 22331 Hamburg, Germany), the supernatant was removed and at -80 ° C stored (referred to here as plasma).
  • tissue pieces were dehydrated in an ascending alcohol series at RT (room temperature): 40% each 40% 70% methanol, 80% methanol, 2 x 96%
  • Immunoperoxidase staining against GFP The sections were deparaffinized 3 x 5 min in xylene, rehydrated in a descending alcohol series (3 x 3 min 100% ethanol, 2 x 2 min 95% ethanol) and then 20 min in 3% H 2 0 2 / Incubated methanol to block endogenous peroxidases. All of the incubation steps were subsequently carried out in a moist chamber. After washing 3 ⁇ 3 min with PBS, the first antibody (goat anti-GFP antibody, sc-5384, Santa Cruz Biotechnology, Inc., Bergheimer Str. 89-2, 69115 Heidelberg, Germany) was 1: 500 in 1% Incubated BSA / PBS overnight at 4 ° C.
  • isolation buffer 50 M HEPES, pH 7.5; 150 mM NaCl; 1 mM EDTA; 2.5 mM EGTA; 10% glycerol; 0.1% Tween; 1 mM DTT; 10 mM ⁇ -glycerol phosphate; 1 mM NaF; 0.1 M Na 3 V0 4 with a protease inhibitor tablet "Co plete” from Röche Diagnostics GmbH, Röche Applied Science, Sandhofer Str. 116, 68305 Mannheim) and homogenized 2 x 30 seconds with an Ultraturrax (DIAX 900, dispersing tool 6G, HEIDOLPH Instruments GmbH & Co.
  • DIAX 900 dispersing tool 6G, HEIDOLPH Instruments GmbH & Co.
  • SDS gel electrophoresis The proteins were electrophoretically separated in a multigel-long electrophoresis chamber from Whatman Biometra GmbH, Rudolf-Wissell-Str. 30, 37079 Göttingen, Germany using a denaturing, discontinuous 15% SDS-PAGE (polyacrylamide gel electrophoresis) according to Lä mli (Na ture 277: 680-685, 1970).
  • a separating gel with a thickness of 1.5 mm was first poured: 7.5 ml of acrylamide / bisacrylamide (30%, 0.9%), 3.8 ml of 1.5 M Tris / HCl, pH 8.4, 150 ⁇ l 10 % SDS, 3.3 ml double-distilled water, 250 ⁇ l ammonium persulfate (10%), 9 ⁇ l TE-MED (N, N, N ', N' -tetramethylenediamine) and covered with 0.1% SDS until polymerisation , The collecting gel was then poured: 0.83 ⁇ l acrylamide / bisacrylamide (30% / 0.9%), 630 ⁇ l 1 M Tris / HCl, pH 6.8, 3.4 ml double-distilled water, 50 ⁇ l 10% SDS, 50 ul 10% ammonium persulfate, 5 ul TEMED.
  • the proteins were mixed with an appropriate amount of 4-fold sample buffer (200 mM Tris, pH 6.8, 4% SDS, 100 mM DTT (dithiotreithol), 0.02% bromophenol blue, 20% glycerol) , denatured for 5 min in the heating block at 100 ° C, centrifuged briefly after cooling on ice and applied to the gel.
  • the same amounts of plasma or protein were used per lane (3 ⁇ l plasma or 25 ⁇ g total protein).
  • the electrophoresis was water-cooled at RT and constant 50 V.
  • the protein gel marker Kaleidoscope Prestained Standard became the length standard
  • both the gels after blotting and the blot branches after immunodetection were analyzed with Coomassie (0.1% Coomassie G250, 45% methanol, 10% ice cream). sig) colored.
  • Coomassie 0.1% Coomassie G250, 45% methanol, 10% ice cream.
  • sig colored.
  • the blot membrane was incubated for 1 h at RT after the transfer in 1% skim milk powder / PBS. The mixture was then washed three times for 3 min with 0.1% Tween-20 / PBS. All subsequent antibody incubations and washing steps were carried out in 0.1% Tween-20 / PBS.
  • FIG. 1 shows the inhibition of GFP expression in kidney sections
  • FIG. 2 in cardiac tissue and FIG. 3 in pancreatic tissue.
  • 4 to 6 Western blot analyzes of GFP expression in plasma and tissues are shown.
  • 4 shows the inhibition of GFP expression in plasma
  • FIG. 5 in the kidney
  • 6 shows total protein isolates from different animals. The same total protein amounts per lane were found in each case applied.
  • animals to which unspecific control dsRNA was administered (animals in groups B and C)
  • GFP expression was not reduced compared to animals which did not receive any dsRNA.
  • the percentage of GFP-positive lymphocytes in the blood was determined by means of FACS analysis (fluorescence activated cell sorting) after the end of the test, and the GFP expression in the whole blood was examined by Western blot analysis ,
  • GFP experimental animals with 2 to 3 animals each were kept in groups in cages as described above.
  • the injections were made in vaccination cages without anesthesia once a day, in the morning, intravenously (IV) into the tail vein over a period of 21 days.
  • the injection volume was 60 ⁇ l per 10 g body weight and the dose was 25 ⁇ g dsRNA (GFP-specific dsRNA) or 250 ⁇ g dsRNA (non-specific control dsRNA K4) per kg body weight.
  • the experimental animals were divided into two groups:
  • the GFP group consisted of 7 animals that received 25 ⁇ g / kg body weight of the GFP-specific dsRNA S7 / S11.
  • the control group consisting of 6 animals, received the non-specific control dsRNA K4 in a concentration of 250 ⁇ g / kg body weight.
  • the animals were sacrificed exactly 24 hours later, on day 22, with C0 2 , the brook space was opened and blood was immediately withdrawn by means of a heart puncture with a cannula. Approximately 100 ⁇ l whole blood was snap frozen in liquid nitrogen without further treatment for Western blot analysis. Most of the blood was mixed 1: 1 with 100 mM sodium citrate to inhibit blood coagulation, mixed gently, and kept in the dark at RT until FACS analysis.
  • an erythrolysis was carried out, which was automated using the Immunoprep Reagent Kit from Beckman Coulter GmbH - Diagnostics, Siemenstrasse 1, D-85716, Unterschleissheim, Germany on the Coulter® Q-Prep TM (from Beckman Coulter GmbH) according to the manufacturer's protocol.
  • 100 ⁇ l of the blood mixed with sodium citrate which were pipetted into a 5 ml FACS tube with a round bottom, were used.
  • the GFP-expressing cells were determined on the flow cytometer Coulter® EPICS XL TM from Beckman Coulter GmbH.
  • CD4 (Clone GK1.5) as a marker for natural killer T cells
  • CD8a (Clone 53-6.7) as a marker for cytotoxic T cells. All antibodies were obtained from BD Biosciences, Tullastrasse 8-12, 69126 Heidelberg, Germany. The staining with the corresponding antibodies was carried out before the erythrolysis described above. For this purpose, 10 ⁇ l antibodies were placed in 5 ml FACS tubes and 100 ⁇ l blood pipetted in, incubated darkened at RT for 30 min, and a 2-color fluorescence measurement was carried out after erythrolysis (excitation wavelength: 488 nm). As a control, the blood of two completely untreated GFP animals was also analyzed. The values of the percentage GFP expression for each individual animal thus result from the mean value of 6 individual measurements (a 1-color fluorescence measurement without staining and 5 2-color fluorescence measurements with antibody staining).
  • the electrophoretic separation was carried out in a multi-long electrophoresis chamber from Biometra with a denaturing, discontinuous 15% SDS-PAGE (polyacrylamide
  • the collecting gel was then poured: 0.83 ⁇ l acrylamide / bisacrylamide (30% / 0.9%), 630 ⁇ l 1 M Tris / HCl, pH 6.8, 3.4 ml double-distilled water, 50 ⁇ l 10% SDS, 50 ul 10% ammonium persulfate, 5 ul TEMED.
  • the whole blood was disrupted using ultrasound, with an appropriate amount of 4-fold sample buffer (200 M Tris, pH 6.8, 4% SDS, 100 mM DTT (dithiotreithol), 0.02% bromophenol blue, 20 % Glycerin) sets, denatured for 5 min in a heating block at 100 ° C, centrifuged briefly after cooling on ice and applied to the gel. 2 ⁇ l whole blood were used per lane. The run was water-cooled at RT and a constant electrical voltage of 50 V. The protein gel marker Kaleidoscope Prestained Standard from Bio-Rad was used as the length standard.
  • 4-fold sample buffer 200 M Tris, pH 6.8, 4% SDS, 100 mM DTT (dithiotreithol), 0.02% bromophenol blue, 20 % Glycerin
  • both the gels after blotting and the blot membranes after immunodetection were stained with Coomassie (0.1% Coomassie G250, 45% methanol, 10% glacial acetic acid).
  • Coomassie 0.1% Coomassie G250, 45% methanol, 10% glacial acetic acid.
  • the blot membrane was incubated for 1 h at RT after transfer in 1% lean milk powder / PBS. Then it was washed three times each for 3 min with 0.1% Tween-20 / PBS. All subsequent antibody incubations and washing steps were carried out in 0.1% Tween-20 / PBS.
  • FIG. 7 corresponds to the mean values of the values shown in FIG. 8.
  • the application of 25 ⁇ g GFP-specific dsRNA per kg body weight and day in the GFP group thus resulted in a significant and specific compared to the control group, which received 250 ⁇ g of an unspecific control dsRNA per kg body weight and day during the experiment Reduction of GFP expression.
  • the dsRNA concentrations were significantly lower than those used in the first in vivo experiment.
  • the injections were carried out over a period of 10 hours over a period of 10 days, resulting in a total daily dose of 5 mg dsRNA per kg body weight.
  • the total daily dose in the second in vivo test described here was 25 ⁇ g dsRNA per kg body weight (the injections were made once a day for 21 days). This total daily dose is reduced 200-fold compared to the first in vivo experiment.
  • the total dose of dsRNA per kg body weight over the entire test period was 50 mg per kg body weight (2.5 mg / kg body weight x 20 injections) in the first in vivo experiment and 0.525 mg per kg body weight in the second in vivo experiment (25 ⁇ g / kg body weight x 21 injections). This corresponds to an approximately 95-fold lower amount of dsRNA. However, the reduction in GFP expression in the blood is comparable in both studies.
  • FIG. 9 shows that the application of the dsRNAs mentioned leads to no change in the blood composition over a period of 21 days.
  • the reduction in GFP expression in the group treated with GFP-specific dsRNA is therefore not due to a reduction in GFP-expressing blood cells.

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Abstract

Médicament destiné à inhiber l'expression d'un gène cible, ce médicament se présentant sous forme d'au moins une unité d'administration. Cette unité contient un acide ribonucléique à double brin adapté pour inhiber, par interférence d'ARN, l'expression du gène cible, dans une quantité qui permet une posologie de 5 mg au plus par kg de poids corporel et par jour.
PCT/EP2002/011971 2001-10-26 2002-10-25 Medicament destine a inhiber l'expression d'un gene cible WO2003035082A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
DE10230997A DE10230997A1 (de) 2001-10-26 2002-07-09 Medikament zur Erhöhung der Wirksamkeit eines Rezeptor-vermittelt Apoptose in Tumorzellen auslösenden Arzneimittels
DE10230996A DE10230996A1 (de) 2001-10-26 2002-07-09 Medikament zur Behandlung eines Pankreaskarzinoms
PCT/EP2002/011971 WO2003035082A1 (fr) 2001-10-26 2002-10-25 Medicament destine a inhiber l'expression d'un gene cible
JP2003538370A JP2005506385A (ja) 2001-10-26 2002-10-25 膵臓癌を処置するための医薬
US10/382,634 US20040038921A1 (en) 2001-10-26 2003-08-11 Composition and method for inhibiting expression of a target gene
US10/666,458 US20040126791A1 (en) 2001-10-26 2003-09-19 Compositions and methods for treating trail-resistant cancer cells

Applications Claiming Priority (14)

Application Number Priority Date Filing Date Title
DE10155280 2001-10-26
DE10155280.7 2001-10-26
DE10158411.3 2001-11-29
DE10158411 2001-11-29
DE10160151.4 2001-12-07
DE10160151A DE10160151A1 (de) 2001-01-09 2001-12-07 Verfahren zur Hemmung der Expression eines vorgegebenen Zielgens
PCT/EP2002/000152 WO2002055693A2 (fr) 2001-01-09 2002-01-09 Procede pour inhiber l'expression d'un gene cible
EPPCT/EP02/00151 2002-01-09
PCT/EP2002/000151 WO2002055692A2 (fr) 2001-01-09 2002-01-09 Procede d'inhibition de l'expression d'un gene cible et medicament destine a la therapie d'une maladie tumorale
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DE10230996A DE10230996A1 (de) 2001-10-26 2002-07-09 Medikament zur Behandlung eines Pankreaskarzinoms
PCT/EP2002/011971 WO2003035082A1 (fr) 2001-10-26 2002-10-25 Medicament destine a inhiber l'expression d'un gene cible
PCT/EP2002/011968 WO2003035868A1 (fr) 2001-10-26 2002-10-25 Medicament qui augmente l'efficacite d'un remede declenchant l'apoptose mediee par recepteur dans des cellules tumorales

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US7473525B2 (en) 2001-01-09 2009-01-06 Alnylam Europe Ag Compositions and methods for inhibiting expression of anti-apoptotic genes
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EP2292739A1 (fr) 2006-03-24 2011-03-09 Institut National De La Recherche Agronomique Procédé de préparation de cellules aviaires differenciées et gènes impliqués dans le maintien de la pluripotence
US8101742B2 (en) 1999-01-30 2012-01-24 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
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US8114851B2 (en) 1999-01-30 2012-02-14 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
US9133454B2 (en) 1999-01-30 2015-09-15 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
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US9902955B2 (en) 1999-01-30 2018-02-27 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
US8168776B2 (en) 1999-01-30 2012-05-01 Alnylam Pharmaceuticals, Inc. Method for making a 21 nucleotide double stranded RNA chemically linked at one end
US8119608B2 (en) 1999-01-30 2012-02-21 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
US8202980B2 (en) 1999-01-30 2012-06-19 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
US8101584B2 (en) 1999-01-30 2012-01-24 Alnylam Pharmaceuticals, Inc. Method and medicament for inhibiting the expression of a given gene
US7829693B2 (en) 1999-11-24 2010-11-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US7473525B2 (en) 2001-01-09 2009-01-06 Alnylam Europe Ag Compositions and methods for inhibiting expression of anti-apoptotic genes
US7767802B2 (en) 2001-01-09 2010-08-03 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
US9074213B2 (en) 2001-01-09 2015-07-07 Alnylam Pharmacuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US9587240B2 (en) 2001-01-09 2017-03-07 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
US7745418B2 (en) 2001-10-12 2010-06-29 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting viral replication
US7763590B2 (en) 2001-10-12 2010-07-27 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a mutant gene
US7348314B2 (en) 2001-10-12 2008-03-25 Alnylam Europe Ag Compositions and methods for inhibiting viral replication
US7846907B2 (en) 2002-01-22 2010-12-07 Alnylam Pharmaceuticals, Inc. Double-stranded RNA (dsRNA) and method of use for inhibiting expression of a fusion gene
US7196184B2 (en) 2002-01-22 2007-03-27 Alnylam Europe Ag Double-stranded RNA (DSRNA) and method of use for inhibiting expression of the AML-1/MTG8 fusion gene
WO2005002594A1 (fr) * 2003-07-08 2005-01-13 Institute Of Hematology, Chinese Academy Of Medical Sciences Medicament antitumoral d'interference de l'arn utilise dans la resistance pleiotrope
DE10350256A1 (de) * 2003-10-01 2005-06-02 Grünenthal GmbH PIM-1-spezifische siRNA-Verbindungen
EP2292739A1 (fr) 2006-03-24 2011-03-09 Institut National De La Recherche Agronomique Procédé de préparation de cellules aviaires differenciées et gènes impliqués dans le maintien de la pluripotence

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