Doublestranded RNA (dsRNA) for inhibition the expression of a defined gene

0001The invention relates to methods for inhibiting the expression of a given target gene in a cell. It also relates to a medicament and the use of double-stranded oligoribonucleotides.

0002Such a method is known from the subsequently published WO 99/32619. The known method aims at inhibiting the expression of genes in cells of invertebrates. For this it is necessary that the double-stranded oligoribonucleotide has a sequence identical to the target gene with a length of at least 50 bases. To achieve efficient inhibition, the length of the identical sequence is 300 to 1000 base pairs. The production cost of such an oligoribonucleotide is high.

0003DE 196 31 919 C2 describes an anti-sense RNA with special secondary structures, the anti-sense RNA being in the form of a vector encoding it. The anti-sense RNA is an RNA molecule that is complementary to regions of the mRNA. Binding to these areas results in an inhibition of gene expression. This inhibition can be used in particular for the diagnosis and / or therapy of diseases, for example tumor diseases or viral infections. - The anti-sense RNA must disadvantageously be introduced into the cell in an amount which is at least as large as the amount of the mRNA. The effectiveness of the known anti-sense methods is not particularly high.

0004No. 5,712,257 discloses a medicament which contains mismatched double-stranded RNA (dsRNA) and biologically active mismatched fragments of dsRNA in the form of a ternary complex with a surface-active agent. The dsRNA used consists of synthetically produced single nucleic acid strands without a defined base sequence. The single strands form non-regular, so-called "non-Watson-Crick" base pairings with one another, so that mismatched double strands are formed. The known dsRNA is used to inhibit the multiplication of retroviruses, such as HIV. The multiplication of the virus can be inhibited if non-sequence-specific dsRNA is introduced into the cells. This leads to induction of interferon, which is intended to inhibit virus multiplication. The inhibitory effect or the effectiveness of this method is low.

0005From Fire, A. et.al, NATURE, Vol. 391, pp. It is known in 806 that dsRNA, one strand of which is complementary in sections to a gene of a roundworm to be inhibited, inhibits the expression of this gene with a high effectiveness. It is believed that the particular effectiveness of the dsRNA used in roundworm cells is not based on the anti-sense principle, but is possibly due to the catalytic properties of the dsRNA or the enzymes induced by it. - Nothing is said in this article about the effectiveness of specific dsRNA in inhibiting gene expression, particularly in mammalian and human cells.

0006The object of the present invention is to eliminate the disadvantages of the prior art. In particular, the most efficient method, medicament or as efficient an use as possible for producing a medicament is to be specified, with which a particularly effective inhibition of the expression of a given target gene can be brought about.

0007The object is achieved by the features of claim 1. Advantageous embodiments result from claims 2 to 32.

0008According to the invention, in order to inhibit the expression of a given target gene in a cell, an oligoribonucleotide having a double-stranded structure (dsRNA) and having 15 to 49 base pairs is introduced into the cell, wherein a strand of the dsRNA has a region I which has at least in sections complementary to the target gene at most 49 consecutive nucleotide pairs and a region II which is complementary within the double-stranded structure is formed from two separate RNA single strands. The oligoribonucleotide has a defined nucleotide sequence at least in sections. The defined section can be limited to the complementary area I. However, it can also be the case that the double-stranded oligoribonucleotide as a whole has a defined nucleotide sequence. The dsRNA can be longer than region I which is complementary to the target gene. The complementary region I can be arranged at the end or can be inserted into the dsRNA. Such a dsRNA can be produced synthetically or enzymatically using conventional methods.

0009It has surprisingly been found that an effective inhibition of the expression of the target gene can be achieved even with a length of the complementary region I of at most 49 base pairs. Corresponding oligoribonucleotides can be provided with less manufacturing effort.

0010In particular, dsRNA with a length of more than 50 nucleotide pairs induces certain cellular mechanisms in mammalian cells and human cells, for example the dsRNA-dependent protein kinase or the 2-5A system. This leads to the disappearance of the interference effect mediated by the dsRNA having a defined sequence. This blocks protein biosynthesis in the cell. This disadvantage in particular is eliminated by the present invention.

0011Furthermore, the uptake of dsRNA with a short chain length into the cell or into the cell nucleus is significantly easier compared to longer-chain dsRNAs.

0012It has proven advantageous for the dsRNA to be packaged in micellar structures, preferably in liposomes. The dsRNA can likewise be enclosed in viral natural capsids or in artificial capsids produced by chemical or enzymatic means or structures derived therefrom. - The aforementioned features enable the dsRNA to be introduced into predetermined target cells.

0013The gene to be inhibited is expediently expressed in eukaryotic cells. The target gene can be selected from the following group: oncogene, cytokine gene, Id protein gene, development gene, prion gene. It can also be expressed in pathogenic organisms, preferably in plasmodia. It can be part of a, preferably human pathogenic, virus or viroid. - The proposed method enables the production of agents for the therapy of genetically controlled diseases, for example Cancer, viral diseases or Alzheimer's disease.

0014The virus or viroid can also be an animal or phytopathogenic virus or viroid. In this case, the method according to the invention also allows the provision of agents for the treatment of animal or plant diseases.

0015According to a further design feature, the dsRNA is double-stranded in sections. The ends of the dsRNA can be modified to counteract degradation in the cell or dissociation into the single strands. Dissociation occurs especially when using low concentrations or short chain lengths. For particularly effective inhibition of dissociation, the cohesion of the complementary region II brought about by the nucleotide pairs can be increased by at least one, preferabl y two, further chemical linkages. A dsRNA according to the invention, the dissociation of which is reduced, has a higher stability against enzymatic and chemical degradation in the cell or in the organism.

0016The chemical linkage is expediently formed by a covalent or ionic bond, a hydrogen bond, hydrophobic interactions, preferably van der Waals or stacking interactions, or by metal-ion coordination. According to a particularly advantageous design feature, it can be produced at at least one, preferably at both, ends of the complementary region II.

0017It has also proven to be advantageous that the chemical linkage is formed by means of one or more connecting groups, the connecting groups preferably being poly (oxyphosphinicooxy-1,3-propanediol) and / or polyethylene glycol chains. The chemical linkage can also be formed by purine analogs used in the complementary areas II instead of purines. It is also advantageous that the chemical linkage is formed by azabenzene units introduced in the complementary regions II. It can also be formed by branched nucleotide analogs used in the complementary regions II instead of nucleotides.

0018It has proven expedient that at least one of the following groups is used to produce the chemical linkage: methylene blue; bifunctional groups, preferably bis (2-chloroethyl) amine; N-acetyl-N '- (p-glyoxylbenzoyl) cystamine; 4-thiouracil; Psoralen. Furthermore, the chemical linkage can be formed by thiophosphoryl groups attached to the ends of the double-stranded region. The chemical linkage is preferably produced at the ends of the double-stranded region by triple helix bonds.

0019The chemical linkage can expediently be induced by ultraviolet light.

0020The nucleotides of the dsRNA can be modified. This counteracts an activation in the cell of a protein kinase, PKR, which is dependent on double-stranded RNA. At least one 2'-hydroxyl group of the nucleotides of the dsRNA in the complementary region II is advantageously replaced by a chemical group, preferably a 2'-amino or a 2'-methyl group. At least one nucleotide in at least one strand of the complementary region II can also be a so-called "locked nucleotide" with a sugar ring chemically modified, preferably by a 2'-O, 4'-C-methylene bridge. Advantageously, several nucleotides are "locked nucleotides".

0021According to a further particularly advantageous embodiment, it is provided that the dsRNA is bound to, associated with or surrounded by at least one viral coat protein derived from a virus, derived therefrom or synthetically produced. The coat protein can be derived from the polyomavirus. The coat protein can contain the virus protein 1 (VP1) and / or the virus protein 2 (VP2) of the polyomavirus. The use of such coat proteins is, for example known from DE 196 18 797 A1, the disclosure content of which is hereby incorporated. - The aforementioned features significantly facilitate the introduction of the dsRNA into the cell.

0022When a capsid or capsid-like structure is formed from the coat protein, the one side preferably faces the interior of the capsid or capsid-like structure. The construct formed is particularly stable.

0023The dsRNA can be complementary to the primary or processed RNA transcript of the target gene. - The cell can be a vertebrate cell or a human cell.

0024At least two different dsRNAs can be introduced into the cell, a strand of each dsRNA being complementary, at least in sections, to one of at least two different target genes. This makes it possible to simultaneously inhibit the expression of at least two different target genes. In order to suppress the expression in the cell of a protein kinase, PKR, which is dependent on double-stranded RNA, one of the target genes is advantageously the PKR gene. This can effectively suppress PKR activity in the cell.

0025According to the invention, a medicament is also provided with at least one 15 to 49 base pair oligoribonucleotide with double-stranded structure (dsRNA) for inhibiting the expression of a given target gene in mammalian cells Area I, and a region II which is complementary within the double-stranded structure is formed from two separate RNA single strands. It has surprisingly been found that such a dsRNA is suitable as a medicament for inhibiting the expression of a given gene in mammalian cells. In comparison to the use of single-stranded oligoribonucleotides, the inhibition is brought about at concentrations which are at least one order of magnitude lower. The medicament according to the invention is highly effective. Less side effects are expected. It has surprisingly been found that an efficient inhibition of the expression of the target gene can be achieved even with a length of the complementary region I of at most 49 base pairs. Corresponding oligoribonucleotides can be provided with less manufacturing effort.

0026According to a further aspect of the invention, the use of a 15 to 49 base pair oligoribonucleotide with a double-stranded structure (dsRNA) is provided for the manufacture of a medicament for inhibiting the expression of a given target gene in mammalian cells, wherein a strand of the dsRNA has a region I which has at least in sections complementary to the target gene at most 49 consecutive nucleotide pairs and a region II which is complementary within the double-stranded structure is formed from two separate RNA single strands. - Surprisingly, such a dsRNA is suitable for the manufacture of a medicament for inhibiting the expression of a given gene. When using dsRNA, the inhibition compared to the use of single-stranded oligoribonucleotides is brought about at concentrations which are lower by an order of magnitude. The use according to the invention thus enables the production of particularly effective drugs.

0027With regard to advantageous configurations of the medicament and the use, reference is made to the description of the preceding features.

0028Exemplary embodiments of the invention are explained in more detail below with reference to the figures. Show it:<dl id="dl0001"><dt>Fig. 1</dt><dd>the schematic representation of a plasmid for the <i>in vitro</i>-Transcription with T7 and SP6 polymerase,</dd><dt>Fig. 2</dt><dd>RNA after electrophoresis on an 8% polyacrylamide gel and ethidium bromide staining,</dd><dt>Fig. 3</dt><dd>a representation of radioactive RNA transcripts after electrophoresis on an 8% polyacrylamide gel with 7 M urea using an "instant imager" and</dd><dt>Fig. 4 a - e</dt><dd>Texas red and YFP fluorescence in murine fibroblasts.</dd></dl>

<u>Example 1:</u>

0029The inhibition of transcription was determined by sequence homologous dsRNA in one <i>in vitro</i>-Transcription system with a core extract from human HeLa cells detected. The DNA template for this experiment was the medium<i>Bam</i>HI linearized plasmid pCMV1200.

Production of the matrix plasmids:

0030The plasmid shown in Fig. 1 was constructed for use in the enzymatic synthesis of the dsRNA. For this purpose, a polymerase chain reaction (PCR) with the "positive control DNA" of the HeLaScribe® Nuclear Extract was started<i>in vitro</i> Transcription kits from Promega, Madison, USA performed as a DNA template. One of the primers used contained the sequence one<i>Eco</i>RI cleavage site and the T7 RNA polymerase promoter according to sequence listing No. 1. The other primer contained the sequence one <i>Bam</i>HI cleavage site and the SP6 RNA polymerase promoter according to Sequence Listing No. 2. In addition, both primers had identical or complementary regions to the DNA template at their 3 'ends. The PCR was carried out using the "<i>Taq PCR core kits</i>"of the company Qiagen, Hilden, Germany according to the manufacturer's instructions. In a volume of 100 μl, 1.5 mM MgCl₂, each 200 µM dNTP, each 0.5 µM primer, 2.5 U <i>Taq</i>-DNA polymerase and about 100 ng "positive control DNA" used as a template in PCR buffer. After the initial denaturation of the template DNA by heating to 94 ° C. for 5 minutes, the amplification was carried out in 30 cycles of 60 seconds denaturation at 94 ° C., 60 seconds annealing at 5 ° C. below the calculated melting temperature of the primers and 1.5 - 2 minutes of polymerization at 72 ° C. After a final polymerization of 5 minutes at 72 ° C., 5 μl of the reaction mixture were analyzed by agarose gel electrophoresis. The length of the DNA fragment amplified in this way was 400 base pairs, 340 base pairs corresponding to the "positive control DNA". The PCR product was purified using<i>Eco</i>RI and <i>Bam</i>HI hydrolyzed and after re-purification for ligation with a likewise <i>Eco</i>RI and <i>Bam</i>HI hydrolyzed pUC18 vector used. There was a transformation from<i>E. coli</i> XL1-blue. The plasmid obtained (pCMV5) carries a DNA fragment which is flanked at the 5 'end by the T7 and at the 3' end by the SP6 promoter. By linearizing the plasmid with<i>Bam</i>HI can <i>in vitro</i> with the T7 RNA polymerase <i>run off</i>-Transcription of a 340 nucleotide long single-stranded RNA shown in Sequence Listing No. 3 can be used. If the plasmid with<i>Eco</i>RI linearized, it can be used for <i>run off</i>-Transcription with the SP6 RNA polymerase are used, whereby the complementary strand is formed. A 23 nucleotide longer RNA was also synthesized in accordance with the method described above. For this purpose, a DNA shown in sequence listing no<i>Eco</i>RI and <i>Bam</i>HI interfaces ligated to the pUC18 vector.

0031The plasmid pCMV1200 was constructed as a DNA template for in vitro transcription with HeLa core extract. For this purpose, an 1191 bp EcoRI / BamHI fragment from the HeLaScribe® Nuclear Extract<i>in vitro</i> Positive control DNA containing transcription kit amplified by PCR. The amplified fragment comprises the 828 bp "immediate early" CMV promoter and a 363 bp transcribable DNA fragment. The PCR product was ligated to the vector pGEM-T via "T-overhang" ligation. At the 5 'end of the fragment is a BamHI site. The plasmid was linearized by hydrolysis with BamHI and used as a template for run-off transcription.

<i>in vitro</i>-Transcription of the complementary single strands:

0032pCMV5 plasmid DNA was analyzed with <i>Eco</i>RI or <i>Bam</i>HI linearized. It was used as a DNA template for a<i>in vitro</i>-Transcription of the complementary RNA single strands with SP6 or T7 RNA polymerase used. The "Riboprobe<i>in vitro</i> Transcription "system from Promega, Madison, USA. According to the manufacturer, 2 µg linearized plasmid DNA in 100 µl transcription buffer and 40 U T7 or SP6 RNA polymerase were incubated for 5-6 hours at 37 ° C. The DNA was then incubated Matrix was degraded by adding 2.5 µl RNase-free DNase RQ1 and incubating for 30 minutes at 37 ° C. The transcription batch was made up with H₂O made up to 300 µl and purified by phenol extraction. The RNA was precipitated by adding 150 μl of 7 M ammonium acetate and 1125 μl of ethanol and stored at -65 ° C. until hybridization.

Production of the RNA double strands:

0033For the hybridization, 500 μl of the single-stranded RNA stored and precipitated in ethanol were centrifuged off. The resulting pellet was dried and taken up in 30 ul PIPES buffer, pH 6.4 in the presence of 80% formamide, 400 mM NaCl and 1 mM EDTA. 15 µl of the complementary single strands were combined and heated to 85 ° C for 10 minutes. The batches were then incubated at 50 ° C. overnight and cooled to room temperature.

0034Only approximately equimolar amounts of the two single strands were used in the hybridization. As a result, the dsRNA preparations contained single-stranded RNA (ssRNA) as contamination. In order to remove these ssRNA contaminations, the approaches after hybridization with the single-strand-specific ribonucleases RNase A from bovine pancreas and RNase T1 were identified<i>Aspergillus oryzae</i> treated. RNase A is an endoribonuclease specific for pyrimidines. RNase T1 is an endoribonuclease that preferentially cuts on the 3 'side of guanosines. dsRNA is not a substrate for these ribonucleases. For the RNase treatment, 1.2 µl RNaseA in a concentration of 10 mg / ml and 2 µl RNaseT1 in a concentration of 290 µg / were added to the batches in 300 µl Tris, pH 7.4, 300 mM NaCl and 5 mM EDTA. ml added. The batches were incubated for 1.5 hours at 30 ° C. The RNases were then denatured by adding 5 μl of proteinase K in a concentration of 20 mg / ml and 10 μl of 20% SDS and incubating at 37 ° C. for 30 minutes. The dsRNA was purified by phenol extraction and ethanol precipitated. In order to check the completeness of the RNase digestion, two control batches were treated with ssRNA analogously to the hybridization batches.

0035The dried pellet was taken up in 15 μl of TE buffer, pH 6.5 and subjected to a native polyacrylamide gel electrophoresis on an 8% gel. The acrylamide gel was then stained in an ethidium bromide solution and rinsed in a water bath. 2 shows the RNA visualized on a UV transilluminator. The one on track 1<i>sense</i>- and the one plotted on track 2 <i>antisense</i>Under the selected conditions, RNA showed a different running behavior than the dsRNA of the hybridization approach applied to lane 3. The RNase treated on lanes 4 and 5, respectively<i>sense</i>- or <i>antisense</i>RNA did not produce a visible band. This shows that the single-stranded RNAs have been completely broken down. The RNase-treated dsRNA of the hybridization approach applied on lane 6 is resistant to the RNase treatment. The band which migrates faster in the native gel compared to the dsRNA applied on lane 3 results from dsRNA which is free of ssRNA. In addition to the dominant main band, weaker, more rapidly migrating bands appear after RNase treatment.

<i>in vitro</i>-Transcription test with human cell nucleus extract:

0036Using the HeLaScribe® Nuclear Extract <i>in vitro</i> Transcription kits from Promega, Madison, USA, were used to determine the transcription efficiency of the above-mentioned DNA fragment contained in the plasmid pCMV1200 and homologous to the “positive control DNA” in the presence of the sequence homologous dsRNA (dsRNA-CMV5). In addition, the influence of the non-sequence homologous dsRNA (dsRNA-YFP) corresponding to the "yellow fluorescent protein" (YFP) gene was investigated. This dsRNA was produced analogously to the sequence homologous dsRNA. The sequence of a strand of this dsRNA can be found in sequence listing No. 5. As a matrix for the<i>run off</i>The plasmid pCMV1200 was used for transcription. It carries the "immediately early" promoter of the cytomegalovirus, which is recognized by the eukaryotic RNA polymerase II, and a transcribable DNA fragment. The transcription was carried out using the HeLa core extract, which contains all the necessary proteins for a transcription. By adding [α-³²P] rGTP for the transcription approach was obtained radioactively labeled transcript. The used [α-³²P] rGTP had a specific activity of 400 Ci / mmol, 10 mCi / ml. 3 mM MgCl₂, each 400 µM rATP, rCTP, rUTP, 16 µM rGTP, 0.4 µM [α-³²P] rGTP and, depending on the experiment, 1 fmol of linearized plasmid DNA and various amounts of dsRNA in transcription buffer. Each approach was made with H₂O to a volume of 8.5 ul. The approaches were mixed carefully. To start the transcription, 4 U HeLa core extract was added in a volume of 4 μl and incubated at 30 ° C. for 60 minutes. The reaction was terminated by adding 87.5 μl stop mix heated to 30 ° C. To remove the proteins, 100 μl of phenol / chloroform / isoamyl alcohol (25: 24: 1, v / v / v), saturated with TE buffer, pH 5.0, were added and the mixture was mixed vigorously for 1 minute. For phase separation, centrifugation was carried out at 12,000 rpm for about 1 minute and the upper phase was transferred to a new reaction vessel. 250 μl of ethanol were added to each batch. The batches were mixed well and incubated on dry ice / methanol for at least 15 minutes. To precipitate the RNA, the batches were centrifuged for 20 minutes at 12000 rpm and 4 ° C. The supernatant was discarded. The pellet was dried in vacuo for 15 minutes and in 10 ul H₂O resuspended. 10 μl of denaturing sample buffer were added to each batch. The free GTP was separated from the resulting transcript by means of denaturing polyacrylamide gel electrophoresis on an 8% gel with 7 M urea. The RNA transcripts formed in the denaturing sample buffer during the transcription with HeLa core extract were heated to 90 ° C. for 10 minutes and 10 μl of this was immediately applied to the freshly rinsed sample pockets. The electrophoresis was carried out at 40 mA. The amount of radioactive ssRNA formed in the transcription was determined after electrophoresis using a<i>Instant imager</i> analyzed.

0037Fig. 3 shows the means of <i>Instant imagers</i> Radioactive RNA shown from a representative test. Samples obtained from the following transcription batches were applied:<ul id="ul0001" list-style="none" compact="compact"><li>Lane 1: without template DNA, without dsRNA;</li><li>Lane 2: 50ng of template DNA, without dsRNA;</li><li>Lane 3: 50 ng template DNA, 0.5 µg dsRNA YFP;</li><li>Lane 4: 50 ng template DNA, 1.5 µg dsRNA YFP;</li><li>Lane 5: 50 ng template DNA, 3 µg dsRNA YFP;</li><li>Lane 6: 50 ng template DNA, 5 µg dsRNA YFP;</li><li>Lane 7: no template DNA, 1.5 dsRNA YFP;</li><li>Lane 8: 50ng of template DNA, without dsRNA;</li><li>Lane 9: 50 ng template DNA, 0.5 µg dsRNA-CMV5;</li><li>Lane 10: 50 ng template DNA, 1.5 µg dsRNA-CMV5;</li><li>Lane 11: 50 ng template DNA, 3 µg dsRNA-CMV5;</li><li>Lane 12: 50 ng template DNA, 5 µg dsRNA-CMV5;</li></ul>

0038There was a significant reduction in the amount of transcript in the presence of sequence-homologous dsRNA compared to the control batch without dsRNA and also to the batches with non-sequence-homologous dsRNA-YFP. The positive control in lane 2 shows that the<i>in vitro</i>- Transcription with HeLa core extract radioactive transcript was formed. The approach is used to compare with the transcription approaches that had been incubated in the presence of dsRNA. Lanes 3 to 6 show that the addition of non-sequence-specific dsRNA-YFP has no influence on the amount of the transcript formed. Lanes 9 to 12 show that the addition of a quantity of sequence-specific dsRNA-CMV5 lying between 1.5 and 3 μg leads to a decrease in the amount of transcript formed. In order to rule out that the observed effects are not based on the dsRNA, but rather on a contamination that may be unintentionally carried during the production of the dsRNA, a further control was carried out. Single-stranded RNA was transcribed as described above and then subjected to the RNase treatment. Using native polyacrylamide gel electrophoresis it could be shown that the ssRNA had been completely broken down. This approach, like the hybridization approaches, was subjected to phenol extraction and ethanol precipitation and then taken up in TE buffer. In this way a sample was obtained which did not contain any RNA but had been treated with the same enzymes and buffers as the dsRNA. Lane 8 shows that the addition of this sample had no effect on transcription. The decrease in the transcript when sequence-specific dsRNA is added can therefore be clearly attributed to the dsRNA itself. The reduction in the amount of transcript of a gene in the presence of dsRNA in a human transcription system indicates an inhibition of the expression of the corresponding gene. This effect is due to a novel mechanism caused by the dsRNA.

<u>Example 2:</u>

0039As a test system for this <i>in vivo</i>Experiments were carried out on the murine fibroblast cell line NIH3T3, ATCC CRL-1658. The YFP gene was introduced into the cell nuclei using microinjection. The expression of the YFP was examined under the influence of sequence homologous dsRNA which was simultaneously transfected. This dsRNA YFP is homologous to the 5 'region of the YFP gene over a length of 315 bp. The nucleotide sequence of a strand of the dsRNA-YFP is shown in Sequence Listing No. 5. The evaluation under the fluorescence microscope was carried out 3 hours after the injection using the green-yellow fluorescence of the YFP formed.

Construction of the template plasmid and production of the dsRNA:

0040As a template for the production of YFP dsRNA using T7 and SP6<i>in vitro</i>Transcription was a plasmid constructed according to the same principle as described in embodiment 1. The desired gene fragment was generated using the primer<i>Eco</i>_T7_YFP according to Sequence Listing No. 6 and <i>Bam</i>_SP6_YFP according to Sequence Listing No. 7 amplified by PCR and used analogously to the above description for the production of the dsRNA. The dsRNA-YFP obtained is identical to the dsRNA used as a non-sequence-specific control in exemplary embodiment 1.

0041A dsRNA (L-dsRNA) which was chemically linked to the 5 'end of the complementary RNA via a C18 linker group was prepared at the 3' end of the RNA according to sequence listing No. 8. Synthones modified with disulfide bridges were used for this. The 3'-terminal synthon is linked to the solid support via the 3'-carbon with an aliphatic linker group via a disulfide bridge. In the 5'-terminal synthon of the complementary oligoribonucleotide which is complementary to the 3'-terminal synthon of the one oligoribonucleotide, the 5'-trityl protective group is bonded via a further aliphatic linker and a disulfide bridge. After synthesis of the two single strands, removal of the protective groups and hybridization of the complementary oligoribonucleotides, the resulting thiol groups come into spatial proximity to one another. The single strands are linked by oxidation via their aliphatic linkers and a disulfide bridge. This is followed by cleaning using HPLC.

Preparation of cell cultures:

0042The cells were in DMEM with 4.5 g / l glucose, 10% fetal bovine serum under 7.5% CO₂- Incubated in culture dishes at 37 ° C and passaged before reaching confluence. The cells were detached using trypsin / EDTA. To prepare for microinjection, the cells were transferred to petri dishes and incubated further until microcolonies were formed.

Microinjection:

0043The culture dishes were removed from the incubator for about 10 minutes for microinjection. It was injected individually into approximately 50 cell nuclei per batch within a marked area using the AIS microinjection system from Carl Zeiss, Göttingen, Germany. The cells were then incubated for a further three hours. Borosilicate glass capillaries from Hilgenberg GmbH, Malsfeld, Germany with a tip diameter of less than 0.5 µm were prepared for microinjection. The microinjection was carried out using a micromanipulator from Narishige Scientific Instrument Lab., Tokyo, Japan. The injection duration was 0.8 seconds, the pressure was approximately 100 hPa. The plasmid pCDNA-YFP, which was approximately 800 bp in size, was used for the transfection<i>Bam</i>HI/<i>Eco</i>Contains RI fragment with the gene of the YFP in the vector pcDNA3. The samples injected into the cell nuclei contained 0.01 µg / µl pCDNA-YFP and Texas Red coupled to dextran-70000 in 14 mM NaCl, 3 mM KCl, 10 mM KPO₄, pH 7.5. In addition, about 100 μl of RNA with a concentration of 1 μM, or 375 μM in the case of the L-dsRNA, were added.

0044The cells were examined by means of a fluorescence microscope when excited with light of the excitation wavelength of Texas red, 568 nm or of YFP, 488 nm. Individual cells were documented using a digital camera. Figures 4 a - e show the result for NIH3T3 cells. In the cells shown in Fig. 4 a<i>sense</i>-YFP-ssRNA, in Fig. 4 b <i>antisense</i>-YFP-ssRNA, in FIG. 4 c dsRNA-YFP, in FIG. 4 d no RNA and in FIG. 4 e L-dsRNA were injected.

0045The left field shows the fluorescence of cells that were excited with 568 nm. The fluorescence of the same cells when excited with 488 nm can be seen on the right. The Texas red fluorescence of all the cells shown shows that the solution for injection was successfully applied to the cell nuclei and that the cells hit were still alive after three hours. Dead cells no longer showed Texas red fluorescence.

0046The fields on the right in FIGS. 4 a and 4 b show that the expression of the YFP was not visibly inhibited when the single-stranded RNA was injected into the cell nuclei. The right field of FIG. 4 c shows cells whose YFP fluorescence was no longer detectable after injection of dsRNA-YFP. 4 d shows as a control cells into which no RNA had been injected. The in Fig. 4th As a result of the injection of the L-dsRNA, which has regions homologous to the YFP gene, the cell shown shows a YFP fluorescence which is no longer detectable. This result proves that shorter dsRNAs can also be used for the specific inhibition of gene expression in mammals if the double strands are stabilized by chemical linking of the single strands.

Literature:

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