EP1438405A1

EP1438405A1 - Use of a double-stranded ribonucleic acid for specifically inhibiting the expression of a given target gene

Info

Publication number: EP1438405A1
Application number: EP02779511A
Authority: EP
Inventors: Stefan Limmer; Roland Kreutzer; Matthias John
Original assignee: Ribopharma AG
Current assignee: Alnylam Europe AG
Priority date: 2001-01-09
Filing date: 2002-10-25
Publication date: 2004-07-21
Also published as: EP2264173A2; EP2264173A3

Abstract

The invention relates to the use of a double-stranded ribonucleic acid (dsRNA) for specifically inhibiting the expression in a cell of a given target gene that has a point mutation as compared to an original gene. A strand S1 of said dsRNA has a region that is complementary to the target gene in which at least one nucleotide is not complementary to the target gene and at least one nucleotide more is not complementary to the original gene than is not complementary to the target gene.

Description

Use of a double-stranded ribonucleic acid for the targeted inhibition of the expression of a given target gene.

The invention relates to the use of a double-stranded ribonucleic acid (dsRNA) for the targeted inhibition of the expression in a cell of a given target gene that is point-mutated relative to an original gene. It further relates to the use of such a ribonucleic acid for the manufacture of a medicament, a medicament and a method for the targeted inhibition of the expression of said target gene in a cell.

DE 101 00 586 C1 discloses a method for inhibiting the expression of a target gene in a cell, in which 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.

From Elbashir, S.M. et al. , Nature 411 (2001), pages 494-498, it is known that a short dsRNA in which three nucleotides are not complementary to the target gene hardly inhibits the expression of a target gene. A completely complementary dsRNA, however, effectively inhibits the expression of the target gene.

From Holen, T. et al. , Nucleic Acids Research 30 (2002), pages 1757-1766, it is known that an inhibition of the expression of a gene by short dsRNA by means of RNA interference is also possible with dsRNAs whose one or two strands are not complementary to the target gene Has nucleotides.

Many diseases and degenerations of cells are based on the fact that a gene that is important for cells, often a proto-oncogene, is changed by one or a few point mutations. The problem in treating such an illness or such rather cells with the methods known hitherto is that the inhibition of the expression of the mutated gene often also leads to an inhibition of the non-mutated gene. This often has serious side effects.

The object of the present invention is to avoid the disadvantages of the prior art. In particular, the use of a dsRNA for the targeted inhibition of the expression of a target gene mutated with respect to an original gene in a cell is to be provided, in which the expression of the original gene remains largely unaffected. Furthermore, a medicament and a method for the targeted inhibition of the expression of a given target gene as well as a use for the production of the medicament are to be provided. This object is solved by the features of claims 1, 2, 18 and 33. Advantageous configurations result from the features of claims 3 to 17, 19 to 32 and 34 to 44.

According to the invention, the use of a double-stranded ribonucleic acid (dsRNA) for targeted inhibition of the expression in a cell of a given target gene which is point-mutated with respect to an original gene is provided, a strand S1 of the dsRNA having a region complementary to the target gene, in which at least one nucleotide is not complementary to the target gene and the number of nucleotides that are not complementary to the original gene is at least one higher than the number of nucleotides that are not complementary to the target gene. The invention further relates to the use of such a dsRNA for the production of a medicament for the targeted inhibition of the expression in a cell of a given target gene which is mutated point-wise relative to an original gene. For the purposes of this invention, a nucleotide is "complementary" to the target gene or original gene if it can form a specific Watson-Crick base pairing with a nucleotide corresponding to it in its sequence position. Under the “Complementary region” is understood to mean that the nucleotides contained therein are essentially complementary to the target gene. This means that not all nucleotides in the region are complementary to the target gene. The number of nucleotides that are not complementary to the target gene is one in the lowest case. The target gene is generally understood to mean the DNA strand - the double-stranded DNA present in the cell, which is complementary to a DNA strand which serves as a template during transcription, including all the areas transcribed. So it is in

Generally about the sense strand. The strand S1 can thus be complementary to an RNA transcript formed during expression or its processing product, such as e.g. an mRNA. For example, be sufficient if the strand S1 is complementary to part of the 3 'untranslated region of the mRNA. 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 original gene can be any gene which differs from the target gene to be inhibited by only one or a few point mutations. In general, it is a wild-type gene. A dsRNA is present when the ribonucleic acid consisting of one or two nucleic acid strands has a double-stranded structure. Not all nucleotides of the dsRNA must have canonical Watson-Crick base pairing within the dsRNA. The maximum possible number of base pairs is the number of nucleotides in the shortest strand contained in the dsRNA. The region complementary to the target gene can have fewer than 25 successive nucleotides, in particular 19 to 24, preferably 20 to 24, particularly preferably 21 to 23, in particular 22 or 23, nucleotides. The strand S1 can be less than 30, preferably less than 25, particularly preferably 21 to 24, in particular 23, Have nucleotides. It has been shown that short dsRNAs are particularly efficient in inhibiting the expression of a target gene. These dsRNAs are also known as siRNAs (short interfacing RNAs).

With targeted inhibition, the expression of the original gene is less inhibited than that of the target gene. Ideally, the expression of the original gene remains largely unaffected. A dsRNA that does not optimally inhibit the expression of the target gene is used for this purpose. In this way, a dsRNA can be provided which is so little complementary to the original gene that its expression remains largely unaffected. Side effects by inhibiting the original gene can be avoided or reduced.

Expression is inhibited by the dsRNA preferably according to the principle of RNA interference. The nucleotide which is not complementary to the target gene is preferably not located at the 3 'or 5' end of the region. Ideally, the non-complementary nucleotide is in the middle of the range. The

Target gene can have one or two point mutations compared to the original gene. Then the use according to the invention for the targeted inhibition of the expression of the target gene is particularly suitable for specifically inhibiting only this expression but not that of the original gene.

In one embodiment of the invention, the original gene is a proto-oncogene and the target gene is an oncogene derived therefrom. An oncogene often differs from the corresponding cellular proto-oncogene only by a single point mutation. The inhibition of the expression of the oncogene with conventional dsRNA therefore usually also causes an inhibition of the expression of the corresponding proto-oncogene. This is often associated with such serious side effects that the use of conventional dsRNA to inhibit the target gene in an organism is hardly possible.

The cell can be a tumor cell. In one embodiment of the invention, one nucleotide of the region is not complementary to the target gene and two nucleotides of the region are not complementary to the original gene. Even such a small difference in the number of complementary nucleotides can be sufficient to almost completely inhibit the expression of the target gene and to leave the expression of the original gene largely unaffected.

In one embodiment of the method, at least one base pair within the dsRNA is not complementary, i.e. the nucleotides of the base pair are not specifically paired according to Watson-Crick. The effectiveness of the dsRNA can be modulated by varying the number of non-complementary base pairs within the dsRNA. A reduced complementary action within the dsRNA reduces its stability in the cell.

The dsRNA preferably has at least at one end of the dsRNA a single-stranded overhang formed from 1 to 4, in particular 2 or 3, nucleotides. 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 then formed in each case by one end of strand S1 and one end of strand S2. Single-stranded overhangs reduce the stability of the dsRNA in blood, serum and cells and at the same time increase the expression-inhibiting effect of the dsRNA. It is particularly advantageous if the dsRNA has the overhang only at one end, in particular at its end which has the 3 'end of the strand S1. The other end is then formed smoothly, ie without overhangs, in the case of a dsRNA having two ends. Surprisingly, it has been shown that an overhang at one end of the dsRNA is sufficient to enhance the expression-inhibiting 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. The inhibition of expression is particularly effective if the overhang is at the 3 'end of the strand S1.

Preferably, the dsRNA has a strand S2 in addition to the strand S1, i.e. it is made up of two single strands. The dsRNA is particularly effective when the strand S1 (antisense 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. The dsRNA can be present in a preparation which is suitable for inhalation, oral ingestion, infusion and injection, in particular for intravenous, intraperitoneal or intratumoral infusion or injection. The preparation can, in particular exclusively, consist of a physiologically compatible solvent, preferably a physiological saline solution or a physiologically compatible buffer, and the dsRNA. The physiologically compatible buffer can be a phosphate-buffered saline solution. Surprisingly, it has been found that a dsRNA which is only dissolved and administered in such a solvent or buffer is taken up by the cell and the Expression of the target gene is inhibited without the dsRNA having to be packaged in a special vehicle.

The dsRNA is preferably located in a physiologically compatible solution, in particular a physiologically compatible buffer or a physiological saline solution, or enclosed by or on an icellar structure, preferably a liposome, a virus capsid, a capsoid or a polymeric nano or microcapsule polymeric nano or microcapsule bound. The physiologically compatible buffer can be a phosphate-buffered saline solution. An icular structure, a virus capsid, a capsoid or a polymeric nano- or microcapsule can facilitate the uptake of the dsRNA into infected cells. The polymeric nano or micro capsule consists of at least one biodegradable polymer, e.g. 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 dsRNA is preferably used in a dosage of at most 5 mg, in particular at most 2.5 mg, preferably at most 200 μg, particularly preferably at most 100 μg, 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 already has an excellent effectiveness in inhibiting the expression of the given target gene even at this dosage.

The invention further relates to a medicament for the targeted inhibition of the expression in a cell of a given target gene which is point-mutated with respect to an original gene, the medicament containing a double-stranded ribonucleic acid (dsRNA), a strand S1 of the dsRNA being one of the target genes. has a complementary region in which at least one nucleotide is not complementary to the target gene and the number of nucleotides which are not complementary to the original gene is at least one higher than the number of nucleotides which are not complementary to the target gene. The medicament is preferably present in at least one administration unit which contains the dsRNA in an amount which has a dosage of at most 5 mg, in particular at most 2.5 mg, preferably at most 200 μg, particularly preferably at most 100 μg, preferably at most 50 μg, in particular allows a maximum of 25 μg per kg body weight and day. 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 which enables the daily dose to be reached. The administration unit can also be designed for a single administration or ingestion 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.

According to the invention is also a method for the targeted inhibition of the expression of a given one

Source gene of point mutated target gene is provided in a cell, a double-stranded ribonucleic acid (dsRNA) being introduced into the cell and a strand S1 of the dsRNA having a region complementary to the target gene in which at least one nucleotide is not complementary to the target gene and the number of nucleotides which are not complementary to the original gene are at least one higher than the number of nucleotides which are not complementary to the target gene. Because of the further advantageous embodiments of the medicament according to the invention and the method according to the invention, reference is made to the preceding explanations.

The invention is explained below using the drawings as an example. Show it:

1 shows a graphic representation of the inhibition of the expression of an HCV-luciferase fusion protein by dsRNAs, which are to a different extent complementary to a sequence of a target gene and

2 shows a graphical representation of the inhibition of the expression of an HCV-luciferase fusion protein by dsRNAs, which are complementary to a different extent to a sequence of a target gene and are partially formed from RNA strands which are not completely complementary to one another.

To produce a reporter system, a 26 nucleotide long sequence of a cDNA sequence serving as the target gene and corresponding to the 3 'untranslated region of an HCV-RNA was fused with the open reading frame of the luciferase gene from the expression vector pGL3. The expression vector pGL3 comes from the Promega company and has been registered under the Gene Accession Number U47296 at the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Bethesda, MD 20894, USA. Nucleotides 280 to 1932 were used as the luciferase gene. The 26 nucleotide long sequence is a highly conserved sequence found in a large number of HCV genomes and their subtypes. In the HCV genome registered with NCBI under Gene Accession Number D89815, the 26 nucleotides correspond to nucleotides 9531 to 9556. They have the following sequence: 5'-gtcacggct agctgtgaaa ggtccgt-3 '(SEQ ID NO: 1).

The resulting fusion gene is a BamHI / NotI DNA fragment in the eukaryotic expression plasmid pcDNA 3.1 (+) from Invitrogen GmbH, Technologiepark Karlsruhe, Em y-Noether Strasse 10, D-76131 Karlsruhe, catalog No. V790-20 , has been cloned. The resulting plasmid is called p8.

The plasmid pCMVßGal from Clontech, Gene Accession Number U13186, NCBI, was used as a control for the transfection efficiency. This plasmid codes for the enzyme β-galactosidase and causes its expression.

The plasmid containing the fusion gene, the control plasmid and different dsRNAs are combined by transfection into cells of the HuH-7 liver cell line (JCRB0403, Japanese Collection of Research Bioresources Cell Bank, National Institute of Health Sciences, Kamiyoga 1-18-1 , Setagaya-ku, Tokyo 158, Japan). The inhibition of the expression of the luciferase gene brought about by the dsRNAs has been determined in relation to the expression of the β-galactosidase gene.

The dsRNAs used have the following sequences, designated SEQ ID NO: 2 to SEQ ID NO: 13 in the sequence listing:

HCV1 + 2, the strand S1 of which is completely complementary to the HCV sequence in the fused HCV luciferase gene:

S2: 5'- ACG GCU AGC UGU GAA AGG UCC GU-3 '(SEQ ID NO: 2) Sl: 3' -AG UGC CGA UCG ACA CUU UCC AGG -5 '(SEQ ID NO: 3) HCV3 + 4, which is neither complementary to the HCV nor to the luciferase sequence in the fused HCV-luciferase gene and serves as a negative control:

S2: 5'- AGA CAG UCG ACU UCA GCC U GG-3 '(SEQ ID NO: 12) Sl: 3'-GG UCU GUC AGC UGA AGU CGG A -5' (SEQ ID NO: 13)

HCV5 + 6, the strand S1 of which, apart from the nucleotide highlighted in bold, is complementary to the HCV sequence in the fused HCV luciferase gene:

S2: 5'- ACG GCU AGC UGU GAA UGG UCC GU-3 '(SEQ ID NO: 6) Sl: 3' -AG UGC CGA UCG ACA CUU ACC AGG -5 '(SEQ ID NO: 7)

HCV7 + 8, the strand S1 of which, apart from the two nucleotides highlighted in bold, is complementary to the HCV sequence in the fused HCV luciferase gene:

S2: 5'- ACG GCA AGC UGU GAA UGG UCC GU-3 '(SEQ ID NO: 8) Sl: 3' -AG UGC CGU UCG ACA CUU ACC AGG -5 '(SEQ ID NO: 9)

Lucl + 2, whose strand S1 is completely complementary to a luciferase sequence in the fused HCV luciferase gene and serves as a positive control:

S2: 5'- CGU UAU UUA UCG GAG UUG CAG UU-3 '(SEQ ID NO: 10)

Sl: 3'-GC GCA AUA AAU AGC CUC AAC GUC -5 '(SEQ ID NO: 11)

K3s + K3as, which is neither complementary to the HCV nor to a luciferase sequence in the fused HCV luciferase gene and serves as a negative control:

S2: 5 '- G AUG AGG AUC GUU UCG CAU GA-3' (SEQ ID NO: 4) Sl: 3'-UCC UAC UCC UAG CAA AGC GUA -5 '(SEQ ID NO: 5) HCV5 + 2, whose strand S1 is completely complementary to the HCV sequence and whose strand S2 is complementary to the HCV sequence in the fused HCV-luciferase gene apart from the nucleotide highlighted in bold:

S2: 5'- ACG GCU AGC UGU GAA UGG UCC GU-3 '(SEQ ID NO: 6) Sl: 3' -AG UGC CGA UCG ACA CUU UCC AGG -5 '(SEQ ID NO: 3)

HCV1 + 6, whose strand S2 is completely complementary to the HCV sequence and whose strand S1 is complementary to the HCV sequence in the fused HCV-luciferase gene, apart from the nucleotide highlighted in bold:

S2: 5'- ACG GCU AGC UGU GAA AGG UCC GU-3 '(SEQ ID NO: 2) Sl: 3' -AG UGC CGA UCG ACA CUU ACC AGG -5 '(SEQ ID NO: 7)

HuH-7 cells have been cultured in DMEM with 10% FCS. To prepare for a transfection, 2 x 10E4 cells were sown per well of a 96-well cell culture plate. The cells were transfected 24 hours after sowing using 110 μl of transfection medium per well of the 96-well cell culture plate and cultivated further in this total volume. Each transfection has been done in triplicate.

For this purpose, 3 μg of the plasmid pCMVßGal were first mixed with 1 μg of the plasmid p8. The transfection medium contained 0.25 μg of the mixture of the plasmids and 200, 100, 50, 25, 12.5 or 0 nmol / 1 of one of the dsRNAs mentioned per well.

"Gene Porter 2" from PEQLAB Biotechnologie GmbH, Carl-Thiersch-Str. 2 b, D-91052 Erlangen, catalog number 13-T202007 used according to the manufacturer's instructions. The cells were then incubated at 37 ° C. and 5% CO ₂ . One day after the transfection, 35 μl of fresh medium were added to each well and the cells were incubated for a further 24 h.

The effect of the dsRNAs used was determined by quantifying the expressed β-galactosidase using "Galacto-Star" from Tropix, 47 Wiggins Avenue, Bedford, MA 01730, USA catalog number BM100S, and the effect of the expressed luciferase using "Luciferase "from Tropix, catalog number BC100L, determined by chemiluminescence reaction. For this purpose, lysates of the cells were produced in accordance with the manufacturer's instructions and 2 μl of each for analysis for the detection of β-galactosidase and 5 μl for each analysis for detection of luciferase. The chemiluminescence was measured in a Sirius lunometer from Berthold Detection Systems GmbH, Bleichstrasse 56-68, D-75173 Pforzheim, Germany. The relative activity of the luciferase as a measure of the expression is determined by dividing the value of the chemiluminescence determined for luciferase by the value determined for β-galactosidase. An average is calculated for every 3 values determined in this way. The mean for cells transfected without dsRNA is arbitrarily defined as 1.0. The other mean values are related to this and are shown graphically in FIGS. 1 and 2.

Lucl + 2 (positive control) led to the greatest inhibition of luciferase activity (FIGS. 1 and 2). In the presence of HCV1 + 2, which was completely complementary to the target sequence for the reporter plasmid, a clear reduction in the luciferase activity can also be seen (FIGS. 1 and 2). As the concentration of HCV1 + 2 decreased, the luciferase activity increased. The oligonucleotide HCV5 + 6, which is not complementary to the target sequence in a nucleotide, is similarly efficient in the lucifera inhibition like HCVl + 2, especially at low concentrations (Fig. 1). For the specificity of this dsRNA, this means that it is not sufficient if the dsRNA is complementary to the target gene but is not complementary to the original gene with a nucleotide in order to specifically inhibit the expression of the target gene compared to the expression of the original gene.

HCV7 + 8 inhibits the expression of luciferase both at high and at low concentrations only to the extent of the negative controls HCV3 + 4 and K3S + K3AS (FIGS. 1 and 2). The low inhibition of luciferase activity is considered to be an unspecific effect. For the specificity of this dsRNA, this means that it is sufficient if the dsRNA is complementary to the target gene or not complementary to only one nucleotide, but is not complementary to the original gene in two nucleotides, in order for the expression of the target gene to be specific versus the expression of the original gene to inhibit.

In HCV5 + 2, a nucleotide in the sense strand S2 is not complementary to the antisense strand S1, the antisense strand S1 being completely complementary to the target gene. This dsRNA is as efficient as LUC1 + 2 and HCVl + 2 (Fig. 1 + 2). This is surprising since a complementarity reduced by one base pair within the dsRNA means that the stability of the dsRNA in the cell and therefore less efficiency could be expected.

In HCV6 + 1, a nucleotide in the antisense strand S1 is not complementary to the sense strand S2, and the antisense strand S1 is also not complementary to the target gene with a nucleotide. HCV6 + 1 inhibits expression less efficiently than HCV5 + 6, but more efficiently than HCV7 + 8 (Fig. 2). For the specificity and efficiency of the expression-inhibiting effect of the dsRNA, the sequence of the antisense strand S1 is more important than that of the sense strand S2. The HCV3 + 4 (FIG. 1) and K3S + K3AS (FIG. 2) serving as a negative control led to little or no inhibition of the luciferase activity. The low inhibition is unspecific, since it does not depend on the concentration of the dsRNAs used.

The data show that at least two nucleotides which are not complementary to an original gene are necessary in the antisense strand of a dsRNA in order to prevent inhibition of the expression of the original gene. Furthermore, the data show that the effectiveness of the inhibition of expression by a dsRNA can be modulated by reducing the degree of complementarity of the single strands forming the dsRNA.

Claims

claims

1. Use of a double-stranded ribonucleic acid

(dsRNA) for the targeted inhibition of the expression in a cell of a given target gene which is point-mutated with respect to an original gene, a strand S1 of the dsRNA having a region complementary to the target gene in which at least one nucleotide is not complementary to the target gene and the number of nucleotides which are not complementary to the original gene, is at least one higher than the number of nucleotides which are not complementary to the target gene.

2. Use of a double-stranded ribonucleic acid

(dsRNA) for the production of a medicament for the targeted inhibition of the expression in a cell of a given target gene which is mutated point-by-point to an original gene, a strand S1 of the dsRNA having a region complementary to the target gene, in which at least one nucleotide is not complementary to the target gene and the number of nucleotides that are not complementary to the original gene is at least one higher than the number of nucleotides that are not complementary to the target gene.

3. Use according to claim 1 or 2, wherein the nucleotide which is not complementary to the target gene is not located at the 3 'or 5' end of the region.

4. Use according to any one of the preceding claims, wherein the target gene has one or two point mutations compared to the original gene.

5. Use according to one of the preceding claims, wherein the source gene is a proto-oncogene and the target gene is an oncogene derived therefrom.

6. Use according to one of the preceding claims, wherein the cell is a tumor cell.

7. Use according to one of the preceding claims, wherein one nucleotide of the region is not complementary to the target gene and two nucleotides of the region are not complementary to the original gene.

8. Use according to one of the preceding claims, wherein at least one base pair within the dsRNA is not complementary.

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

10. Use according to claim 9, wherein the dsRNA has the overhang exclusively at one end, in particular at its 3 'end of the strand S1.

11. Use according to one of the preceding claims, wherein the dsRNA has a strand S2 in addition to the strand S1 and the strand S1 a length of 23 nucleotides, the strand S2 a length of 21 nucleotides and the 3 'end of the strand S1 one of two Nucleotides formed single-stranded overhang, while the end of the dsRNA located at the 5 'end of the strand S1 is smooth.

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

13. Use according to one of the preceding claims, wherein the dsRNA is present in 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.

14. Use according to claim 13, wherein the preparation, in particular exclusively, consists 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.

15. Use according to one of the preceding claims, wherein the dsRNA in a physiologically compatible solution, in particular a physiologically compatible buffer or a physiological saline solution, or of a micellar structure, preferably a liposome, a virus capsid, a capsoid or a polymeric nano- or enclosed in a microcapsule or bound to a polymeric nano- or microcapsule.

16. Use according to one of the preceding claims, wherein the dsRNA is administered orally, by inhalation, infusion or injection, in particular intravenous, intraperitoneal or intratumoral infusion or injection.

17. Use according to one of the preceding claims, wherein the dsRNA in a dosage of at most 5 mg, in particular at most 2.5 mg, preferably at most 200 μg, particularly preferably at most 100 μg, preferably at most 50 μg, in particular at most 25 μg, per kg of body weight and day is used.

18. Medicament for the targeted inhibition of the expression in a cell of a given target gene which is point-mutated with respect to an original gene, the medicament containing a double-stranded ribonucleic acid (dsRNA), one strand of which has a region complementary to the target gene, in which at least one nucleotide is not com- is complementary to the target gene and the number of nucleotides that are not complementary to the original gene is at least one higher than the number of nucleotides that are not complementary to the target gene.

19. Medicament according to claim 18, wherein the nucleotide which is not complementary to the target gene is not located at the 3 'or 5' end of the region.

20. Medicament according to claim 18 or 19, wherein the target gene has one or two point mutations compared to the original gene.

21. Medicament according to one of claims 18 to 20, wherein the original gene is a proto-oncogene and the target gene is an oncogene derived therefrom.

22. Medicament according to one of claims 18 to 21, wherein the cell is a tumor cell.

23. Medicament according to one of claims 18 to 22, wherein one nucleotide of the region is not complementary to the target gene and two nucleotides of the region are not complementary to the original gene.

24. Medicament according to one of claims 18 to 23, wherein at least one base pair within the dsRNA is not complementary

25. Medicament according to one of claims 18 to 24, wherein the dsRNA has at least at one end of the dsRNA a single-stranded overhang formed from 1 to 4, in particular 2 or 3, nucleotides.

26. Medicament according to claim 25, wherein the dsRNA has the overhang exclusively at one end, in particular at its end having the 3 'end of the strand S1.

27. Medicament according to claim 26, wherein the dsRNA has a strand S2 in addition to the strand S1 and the strand S1 has a length of 23 nucleotides, the strand S2 has a length of 21 nucleotides and the 3 ′ end of the strand S1 is formed from two nucleotides has single-stranded overhang, while the end of the dsRNA located at the 5 'end of the strand S1 is smooth.

28. Medicament according to one of claims 18 to 27, wherein the strand S1 is complementary to the primary or processed RNA transcript of the target gene.

29. Medicament according to one of claims 18 to 28, wherein the medicament has a preparation which is suitable for inhalation, oral intake, infusion or injection, in particular for intravenous, intraperitoneal or intratumoral infusion or injection.

30. Medicament according to claim 29, wherein the preparation, in particular exclusively, consists 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.

31. Medicament according to one of claims 18 to 30, wherein the dsRNA in the medicament in a solution, in particular a physiologically compatible buffer or a physiological saline solution, or from a micellar

Structure, preferably a liposome, a virus capsid, a capsoid or a polymeric nano- or microcapsule or is bound to a polymeric nano- or microcapsule.

32. Medicament according to one of claims 18 to 31, wherein the medicament is present in at least one administration unit which contains the dsRNA in an amount which comprises a Dosage of at most 5 mg, in particular at most 2.5 mg, preferably at most 200 μg, particularly preferably at most 100 μg, preferably at most 50 μg, in particular at most 25 μg, per kg of body weight and day.

33. A method for the targeted inhibition of the expression of a given target gene that is point-mutated with respect to an original gene in a cell, wherein a double-stranded ribonucleic acid (dsRNA) is introduced into the cell and a strand S1 of the dsRNA has a region complementary to the target gene, in which at least a nucleotide is not complementary to the target gene and the number of nucleotides which are not complementary to the original gene is at least one higher than the number of nucleotides which are not complementary to the target gene.

34. The method according to claim 33, wherein the nucleotide which is not complementary to the target gene is not located at the 3 'or 5' end of the region.

35. The method of claim 33 or 34, wherein the target gene has one or two point mutations compared to the original gene.

36. The method according to any one of claims 33 to 35, wherein the original gene is a proto-oncogene and the target gene is an oncogene derived therefrom.

37. The method according to any one of claims 33 to 36, wherein the cell is a tumor cell.

38. The method according to any one of claims 33 to 37, wherein one nucleotide of the region is not complementary to the target gene and two nucleotides of the region are not complementary to the original gene.

39. The method according to any one of claims 33 to 38, wherein at least one base pair within the dsRNA is not complementary.

40. The method according to any one of claims 33 to 39, wherein the dsRNA at least at one end of the dsRNA one of 1 to

4, in particular 2 or 3, nucleotides formed single-stranded overhang.

41. The method according to claim 40, wherein the dsRNA has the overhang exclusively at one end, in particular at its end having the 3 'end of the strand S1.

42. The method according to claim 41, wherein the dsRNA has a strand S2 in addition to the strand S1 and the strand S1 has a length of 23 nucleotides, the strand S2 a length of 21 nucleotides and the 3 ′ end of the strand S1 is formed from two nucleotides has single-stranded overhang, while the end of the dsRNA located at the 5 'end of the strand S1 is smooth.

43. The method according to any one of claims 33 to 42, wherein the strand S1 is complementary to the primary or processed RNA transcript of the target gene.

44. The method according to any one of claims 33 to 43, wherein the dsRNA in a solution, in particular a physiologically acceptable buffer or a physiological saline solution, or of a micellar structure, preferably a liposome, a virus capsid, a capsoid or a polymeric nanoscale or enclosed in a microcapsule or bound to a polymeric nano- or microcapsule.