WO2003035082A1

WO2003035082A1 - Drug for inhibiting expression of a target gene

Info

Publication number: WO2003035082A1
Application number: PCT/EP2002/011971
Authority: WO
Inventors: Stefan Limmer; Sylvia Limmer; Roland Kreutzer; Philipp Hadwiger
Original assignee: Ribopharma Ag
Priority date: 2001-10-26
Filing date: 2002-10-25
Publication date: 2003-05-01
Also published as: DE10230996A1

Abstract

The invention relates to a drug for inhibiting the expression of a target gene, said drug being present in at least one administration unit which contains a double strand ribonucleic acid suitable for inhibiting, through RNA interference, the expression of a target gene, in an amount allowing a dosage of less than 5 mg per kilogram of bodyweight per day.

Description

Medicament to inhibit the expression of a target gene

The invention relates to a medicament and a use for inhibiting the expression of a target gene.

From Jansen, B. et al. , The Lancet, Vol. 356, 2000, pp. 1728 to 1733, it is known to administer antisense oligonucleotides in doses of 0.6 to 6.5 mg / kg and day to patients. A dose of 0.6 mg / kg and day did not reduce the concentration of the protein encoded by the target gene. A permanent plasma concentration of over 1 mg / 1 is considered to be biologically relevant. This can be achieved by dosing the antisense oligonucleotide at about 2 mg / kg body weight and day. The therapy is only successful for some of the patients.

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. In particular, an effective medicament and a use for inhibiting the expression of a target gene are to be provided.

This object is solved by the features of claims 1 and 16. Advantageous configurations result from the features of claims 2 to 15 and 17 to 30.

BESTATIGUNGSKOPIE According to the invention, 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. In particular, individual non-complementary base pairs hardly or not at all impair the effectiveness. The maximum possible number of base pairs is the number of nucleotides in the shortest strand contained in the dsRNA. 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.

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.

Surprisingly, it has been shown that 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 μg, preferably at most 100 μg, particularly preferably at most 50 μg, in particular at most 25 μg, 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.

In one embodiment, 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. Although it is necessary in cell culture to introduce dsRNA into the cells by microinjection, lipofection, viruses, viroids, capsids, capsoids or other aids, this is surprisingly not necessary in vivo. The drug can also consist exclusively of the preparation mentioned. Such a drug is then suitable for intravenous applications, for example.

The solvent is preferably a physiological saline solution or a physiologically compatible buffer, in particular a phosphate-buffered saline solution.

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. In one embodiment, 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. In the simplest case, 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 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.

It has proven to be particularly advantageous if at least one end of the dsRNA has a single-stranded overhang formed from 1 to 4, in particular 2 or 3, nucleotides. Such 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. In one exemplary embodiment, 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 μg, preferably at most 50 μg, in particular at most 25 μg, per day and kg body weight allows.

Preferably, the dsRNA has a strand S2 in addition to the strand S1, i.e. it is made up of two single strands. The medicament is particularly effective when the strand S1 (anti-sense strand) has a length of 23 nucleotides, the strand S2 has a length of 21 nucleotides and the 3 'end of the strand S1 has a single-stranded overhang formed from two nucleotides , The end of the dsRNA located at the 5 'end of the strand S1 is smooth. The strand S1 can be complementary to the primary or processed RNA transcript of the target gene.

According to the invention, 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.

Because of the advantageous embodiment of the use according to the invention, reference is made to the preceding explanations.

The invention is explained below using the drawings as an example. As an abbreviation of the concentration "mol / 1" "M" is used. Show it:

1 GFP-specific immunoperoxidase staining on kidney paraffin sections of transgenic GFP mice, 2 GFP-specific immunoperoxidase staining on cardiac paraffin sections of transgenic GFP mice,

3 GFP-specific immunoperoxidase staining on pancreatic paraffin sections from transgenic GFP mice,

4 Western blot analysis of GFP expression in plasma,

5 Western blot analysis of GFP expression in the kidney,

6 Western blot analysis of GFP expression in the heart,

7 percentage GFP expression (FACS analysis) in the blood of GFP-transgenic mice after treatment with specific (GFP group) and non-specific (control group) dsRNA,

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,

9 Evaluation of a FACS analysis of the expressed

Surface marker proteins CDllb, CD3, CD4, CD8a and CD19 and

10 Western blot analysis of the GFP expression in the blood of the animals 4-7 treated with specific dsRNA (GFP-

Group, 4 lanes on the left) and animals 3-6 treated with non-specific dsRNA (control group, 4 lanes on the right). The double-stranded oligoribonucleotides used have the following sequences:

It was "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. The nonspecific dsRNA has neither homology to the GFP gene nor to a gene occurring in humans or mice. At the end of the experiment, the animals were sacrificed and GFP expression in tissue sections and in plasma was analyzed.

Test protocol:

Synthesis of the dsRNA:

Using an RNA synthesizer (type Expedite 8909, Applied Biosystems, Weiterstadt, Germany) and conventional chemical methods, the 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 Woertzgarten 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.

Experimental animal husbandry and experimentation: 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.

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, Lage, Germany was used as the litter. The animals received tap water and standard Altromin 1324 pelletized from Altromin ad libidum. First in vivo experiment:

For the experiment, 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 μl per 10 g body weight and the dose was 2.5 mg dsRNA or 50 μg per kg body weight. The division into the groups was as follows:

Group A: PBS (phosphate buffered saline) each 60 μl per 10 g body weight,

Group B: 2.5 mg per kg body weight of an unspecific control dsRNA (Kl control with blunt ends and a double-strand range of 22 nucleotide pairs),

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 μg Sl-dsRNA per kg body weight (i.e. 1/50 of the dose of group D).

After the last injection of a total of 10 injections, the animals were sacrificed after 14-20 h and organs and blood were removed as described.

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. A part was fixed for 24 h for immunohistochemical staining in Methyl Carnoys (MC, 60% methanol, 30% chloroform, 10% glacial acetic acid), a part for frozen sections and for protein isolation immediately shock-frozen in liquid nitrogen and stored at -80 ° C and a further, smaller part was frozen at -80 ° C in RNA-easy-Protect (QIAGEN GmbH, Max-Volmer-Strasse 4, 40724 Hilden) for RNA isolation. 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).

Processing the biopsies:

After the tissues had been fixed in MC for 24 h, the tissue pieces were dehydrated in an ascending alcohol series at RT (room temperature): 40% each 40% 70% methanol, 80% methanol, 2 x 96%

Methanol and 3 x 100% isopropanol. The tissues were then heated in 100% isopropanol to 60 ° C. in an incubator, subsequently incubated for 1 h in an isopropanol / paraffin mixture at 60 ° C. and 3 × for 2 h in paraffin and then embedded in paraffin. For immunoperoxidase stains, a Rotation microtome (Leica Microsystems Nussloch GmbH, Heidelberger Str. 17 - 19, D-69226 Nussloch, Germany) Tissue sections with a thickness of 3 μm were made on slides (Superfrost, Vogel GmbH & Co. KG, Medical Technology and Electronics, Marburger Strasse 81 , 35396 Gießen, Germany) and incubated for 30 min at 60 ° C in an incubator.

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. Incubation with the biotinylated secondary antibody (donkey anti-goat IgG; Santa Cruz Biotechnology; 1: 2000 dilution) was carried out for 30 min at RT, then for 30 min with avidin D peroxidase (1: 2000 dilution, Vector Laboratories, 30 Ingold Road, Burlingame, CA 94010, USA). After each antibody incubation, the sections were washed 3 × 3 min in PBS and buffer residues with cellulose were removed from the sections. All antibodies were diluted in 1% bovine serum albumin (BSA) / PBS. Staining with 3, 3 '-diaminobenzidine (DAB) was carried out using the DAB Substrate Kit (Vector Laboratories) according to the manufacturer's instructions. Hematoxylin III according to Gill

(Merck KgaA, Frankfurter Str. 250, D-64293 Darmstadt). After dehydration in an ascending alcohol series and 3 x 5 min xylene, the sections were covered with Entellan (Merck). The microscopic evaluation of the color was carried out using the 1X50 microscope from OLYMPUS Optical Co. (Europa) GmbH, Wendenstr. 14-18, D-20097 Hamburg, Germany equipped with a CCD camera (Hamamatsu Photonics KK, Systems Division, 812 Joko-cho Hamamtsu City, 431-3196 Japan).

Protein isolation from pieces of tissue:

800 μl of 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 β-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. KG, Walpersdorfer Strasse 12, D-91126 Schwabach), cooled in between. After 30 minutes of incubation on ice, the mixture was mixed and centrifuged for 20 minutes at 10000 × g, 4 ° C. (3K30, SIGMA Laborzentrifugen GmbH, An der Unteren Söse 50, D - 37507 Osterode am Harz). The supernatant was again incubated on ice for 10 minutes, mixed and 20

Minutes at 15000 x g, 4 ° C, centrifuged. A protein determination according to Bradford, 1976, modified according to Zor & Selinger, 1996, was carried out with the supernatant using the Roti-Nanoquant system from Carl Roth GmbH & Co., Schoe perlenstr. 1-5, 76185 Karls- ruhe, Germany according to the manufacturer's instructions. BSA (bovine serum albumin) was used in concentrations of 10 to 100 μg / ml for the protein calibration line.

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). For this purpose, 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 μl 10 % SDS, 3.3 ml double-distilled water, 250 μl ammonium persulfate (10%), 9 μl TE-MED (N, N, N ', N' -tetramethylenediamine) and covered with 0.1% SDS until polymerisation , The collecting gel was then poured: 0.83 μl acrylamide / bisacrylamide (30% / 0.9%), 630 μl 1 M Tris / HCl, pH 6.8, 3.4 ml double-distilled water, 50 μl 10% SDS, 50 ul 10% ammonium persulfate, 5 ul TEMED.

Before application to the gel, 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 μl plasma or 25 μg total protein). The electrophoresis was water-cooled at RT and constant 50 V. The protein gel marker Kaleidoscope Prestained Standard became the length standard

Bio-Rad Laboratories GmbH, Heidemannstrasse 164, 80939 Munich, Germany.

Western blot and immunodetection: The proteins were transferred from SDS-PAGE to a PVDF (polyvinyl difluoride) membrane (Hybond-P, Amersham Biosciences Europe GmbH, Munzinger Str. 9, D-79111 Freiburg, Germany) in semi-dry Method according to Kyhse-Anderson (J. Biochem. Biophys. Methods 10: 203-210, 1984) at RT and a constant current of 0.8 A / cm ² for 1.5 h. A Tris / glycine buffer was used as transfer buffer (39 M glycine, 46 mM Tris, 0.1% SDS and 20% methanol). To check the electrophoretic transfer, 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. To saturate non-specific bindings, 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. Incubation with the primary antibody (goat anti-GFP antibody, sc-5384, Santa Cruz Biotechnology) in a dilution of 1: 1000 was carried out for 1 h at RT. The mixture was then washed 3 × 5 min and incubated for 1 h at RT with a secondary antibody (donkey anti-goat IgG, labeled with horseradish peroxidase, Santa Cruz Biotechnology) in a dilution of 1: 10,000. The detection was carried out using the Amersham ECL system according to the manufacturer's instructions.

1 to 3 show the inhibition of GFP expression after intravenous injection of dsRNA specifically directed against GFP by means of immunoperoxidase staining of GFP on 3 μm paraffin sections. In the course of the experiment, dsRNA directed against GFP with a double-stranded region of 22 nucleotide (nt) pairs without overhangs at the 3 'ends (D) and the corresponding unspecific control dsRNA (B) as well as dsRNA specifically directed against GFP with a 19 nucleo - Double-strand region comprising tid pairs with 2nt overhangs at the 3 'ends (E) and the corresponding unspecific control dsRNA (C) applied in a 12-hour cycle over 5 days. (F) received 1/50 of the dose from group D. As a further control, animals without dsRNA administration (A) or WT animals were examined. 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, in FIG. 5 in the kidney and in FIG. 6 in the heart. 6 shows total protein isolates from different animals. The same total protein amounts per lane were found in each case applied. In 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. Animals that received GFP-directed dsRNA with 2nt overhangs at the 3 'ends of both strands and a double strand region comprising 19 nucleotide pairs showed significantly inhibited GFP expression in the examined tissues (heart, kidney, pancreas and blood) , compared to untreated animals (Fig. 1 to 6). In the animals of groups D and F, to which specific anti-GFP dsRNA with blunt ends and a 22 strand pairs comprising double strand region was applied, only those animals that received the dsRNA in a dose of 50 μg / kg body weight per day showed a specific Inhibition of GFP expression, which was, however, less pronounced than that of the animals in group E.

The summary evaluation of GFP inhibition in the tissue sections and in the Western blot shows that the inhibition of GFP expression is strongest in the blood and in the kidney (FIGS. 1, 4 and 5).

Second in vivo experiment:

In an additional study it was examined whether the dose of 5 mg / kg body weight (KG) and day applied in vivo, which had been shown to be effective in the first test, could be further reduced. For this, the 200-fold lower dose of 25 μg / kg body weight and day was chosen for the intravenous injection into the tail vein of the GFP transgenic mice. The GFP-specific dsRNA construct S7 / S11 derived from the GFP sequence was used, which had proven to be particularly effective in in vitro transcriptions. K4 was used as the non-specific control dsRNA. K4 has the same construction as S7 / S11 (dsRNA with 21 base pairings and a 2nt overhang at the 3 'end of the antisense strand S1). This- K4 sequence is derived from the 5 'end of the neomycin resistance gene.

In order to investigate the effectiveness of the GFP-specific 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 ,

During the experiment, the heterozygous

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 μl per 10 g body weight and the dose was 25 μg dsRNA (GFP-specific dsRNA) or 250 μg 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 μg / 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 μg / kg body weight. After the last injection on day 21, 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 μl 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. FACS analysis:

Before the 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 ™ (from Beckman Coulter GmbH) according to the manufacturer's protocol. For this purpose, 100 μl 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 ™ from Beckman Coulter GmbH.

In addition, a quantitative analysis of the B and T cells and the granulocytes / macrophages / monocytes was carried out by means of direct staining and subsequent FACS analysis. The following phycoerythrin-labeled monoclonal antibodies were used:

As a marker for B-lymphocyte rat anti-mouse CD19 (clone 1D3),

as a marker for granulocytes, macrophages, monocytes CDU (Clone Ml / 70),

as a marker for T-lymphocyte rats anti-mouse CD3 (Clone 17A2),

and for further differentiation of the T cells:

CD4 (Clone GK1.5) as a marker for natural killer T cells and

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 μl antibodies were placed in 5 ml FACS tubes and 100 μl 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).

SDS gel electrophoresis:

The electrophoretic separation was carried out in a multi-long electrophoresis chamber from Biometra with a denaturing, discontinuous 15% SDS-PAGE (polyacrylamide

Gel electrophoresis) according to Lämmli (Nature 277: 680-685, 1970). For this purpose, 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 μl 10 % SDS, 3.3 ml aqua bidist., 250 μl ammonium persulfate (10%), 9 μl TEMED (N, N, N ', N' -tetramethylendiamine) and covered with 0.1% SDS until polymerisation. The collecting gel was then poured: 0.83 μl acrylamide / bisacrylamide (30% / 0.9%), 630 μl 1 M Tris / HCl, pH 6.8, 3.4 ml double-distilled water, 50 μl 10% SDS, 50 ul 10% ammonium persulfate, 5 ul TEMED.

Before application to the gel, 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 μl 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.

Western blot: The transfer of the proteins from the SDS-PAGE to a PVDF (polyvinyl difluoride) membrane (Hybond-P, Amersham) was carried out in a semi-dry method according to Kyhse-Anderson (J. Bioche. Biophys. Methods 10: 203-210, 1984) at RT and a constant current of 0.8 mA / cm ² for 1.5 h. A Tris / glycine buffer was used as transfer buffer (39 mM glycine, 46 mM Tris, 0.1% SDS and 20% methanol). To check the electrophoretic transfer, 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). To saturate non-specific bindings, 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. Incubation with the primary antibody against GFP (polyclonal goat anti-GFP antibody, sc-5384, Santa Cruz Biotechnology) in a dilution of 1: 2000 was carried out for 1 h at RT together with the actin antibody (polyclonal goat anti-actin -Antibody, sc-1615, Santa Cruz Biotechnology) in a dilution of 1: 1000. The mixture was then washed for 5 minutes and incubated for 1 h at RT with a secondary antibody (donkey anti-goat IgG, labeled with horseradish peroxidase, Santa Cruz Biotechnology) in a dilution of 1: 10,000. The detection was carried out using the Amersham ECL system according to the manufacturer's instructions. FIGS. 7, 8 and 10 show the inhibition of GFP expression in the lymphocytes (FIGS. 7 and 8) analyzed in the FACS and in the whole blood by means of Western blot analysis (FIG. 10) after injection of specific dsRNA directed against GFP. The values shown in FIG. 7 correspond to the mean values of the values shown in FIG. 8. The application of 25 μg 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 μg 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. 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. In comparison, the total daily dose in the second in vivo test described here was 25 μg 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 μg / 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.

Claims

claims

1. Medicament for inhibiting the expression of a target gene, the medicament being present in at least one administration unit which contains a double-stranded ribonucleic acid (dsRNA) suitable for inhibiting the expression of the target gene by means of RNA interference in an amount which has a dosage of at most 5 mg per kg body weight and day.

2. Medicament according to claim 1, wherein the administration unit contains the dsRNA in an amount which has a dosage of at most 2.5 mg, in particular at most 200 μg, preferably at most 100 μg, particularly preferably at most 50 μg, in particular at most 25 μg, per kg Body weight and day allows.

3. Medicament according to one of the preceding claims, wherein the dsRNA is contained in the medicament in a preparation which, in particular exclusively, consists of a physiologically compatible solvent and the dsRNA dissolved therein.

4. Medicament according to one of the preceding claims, wherein the solvent is a physiological saline or a physiologically compatible buffer, in particular a phosphate-buffered saline.

5. Medicament according to one of the preceding claims, wherein the dsRNA is surrounded by a micellar structure, preferably a liposome, a capsid, a capsoid or a polymeric nano- or microcapsule, or is bound to a polymeric nano- or microcapsule.

6. Medicament according to one of the preceding claims, the medicament having a preparation which is suitable for inhalation, oral intake, infusion or injection, in particular which is suitable for intravenous, intraperitoneal or intratumoral infusion or injection.

7. Medicament according to one of the preceding claims, wherein a strand S1 of the dsRNA has a region complementary to the target gene, at least in sections, in particular consisting of fewer than 25 successive nucleotides.

8. Medicament according to claim 7, wherein the complementary region has 19 to 24, preferably 20 to 24, particularly preferably 21 to 23, in particular 22 or 23, nucleotides.

Less than 25, particularly preferably from 21 to 24, in particular 23, having ^'9. A medicament according to one of the preceding claims, wherein the strand Sl less than 30, preferably nucleotides.

10. Medicament according to one of the preceding claims, wherein at least one end of the dsRNA has a single-stranded overhang formed from 1 to 4, in particular 2 or 3, nucleotides.

11. Medicament according to claim 10, wherein the single-stranded overhang is located at the 3 'end of the strand S1.

12. Medicament according to one of the preceding claims, wherein the dsRNA has a single-stranded overhang only at one end, in particular at the 3 'end of the strand S1.

13. Medicament according to one of the preceding claims, wherein the dsRNA has a strand S2 in addition to the strand S1.

14. Medicament according to claim 13, wherein the strand Sl a

Length of 23 nucleotides, strand S2 a length of 21 nucleotides and the 3 'end of strand S1 has a single-stranded overhang formed from two nucleotides. points, while the end of the dsRNA located at the 5 'end of the strand S1 is smooth.

15. Medicament according to one of the preceding claims, wherein the strand S1 is complementary to the primary or processed KNA transcript of the target gene.

16. Use of a double-stranded ribonucleic acid

(dsRNA) in a dosage of at most 5 mg per kg body weight and day to inhibit the expression of a target gene by means of RNA interference in a mammal or human.

17. Use according to claim 16, wherein the dsRNA is used in a dosage of at most 2.5 mg, in particular at most 200 μg, preferably at most 100 μg, particularly preferably at most 50 μg, in particular at most 25 μg, per kg of body weight and day ,

18. Use according to claim 16 or 17, wherein the dsRNA is contained in a preparation which, in particular exclusively, consists of a physiologically compatible solvent and the dsRNA dissolved therein.

19. Use according to claim 18, wherein the solvent is a physiological saline or a physiologically acceptable buffer, in particular a phosphate-buffered saline.

20. Use according to any one of claims 16 to 19, wherein the dsRNA of a micellar structure, preferably one

Liposome, a capsid, a capsoid or a polymeric nano or microcapsule enclosed or bound to a polymeric nano or microcapsule.

21. Use according to one of claims 16 to 20, wherein the dsRNA is present in a preparation intended for inhalation, oral intake, infusion or injection, in particular for intravenous, intraperitoneal or intratumoral infusion or injection.

22. Use according to one of claims 16 to 21, wherein a strand S1 of the dsRNA has a region which is at least partially complementary to the target gene, in particular consists of fewer than 25 successive nucleotides.

23. Use according to claim 22, wherein the complementary region has 19 to 24, preferably 20 to 24, particularly preferably 21 to 23, in particular 22 or 23, nucleotides.

24. Use according to any one of claims 16 to 23, wherein the strand S1 has less than 30, preferably less than 25, particularly preferably 21 to 24, in particular 23, nucleotides.

25. Use according to one of claims 16 to 24, wherein at least one end of the dsRNA has a single-stranded overhang formed from 1 to 4, in particular 2 or 3, nucleotides.

26. Use according to claim 25, wherein the single-stranded overhang is located at the 3 'end of the strand S1.

27. Use according to any one of claims 16 to 26, wherein 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.

28. Use according to one of claims 16 to 27, wherein the dsRNA has a strand S2 in addition to the strand S1.

29. Use according to claim 28, wherein the strand Sl is a

30. Use according to any one of claims 16 to 29, wherein the strand S1 is complementary to the primary or processed RNA transcript of the target gene.