DE10100588A1 - Inhibiting expression of target genes, useful e.g. for treating tumors, by introducing into cells two double-stranded RNAs that are complementary to the target - Google Patents
Inhibiting expression of target genes, useful e.g. for treating tumors, by introducing into cells two double-stranded RNAs that are complementary to the targetInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C12N2310/111—Antisense spanning the whole gene, or a large part of it
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12N2330/30—Production chemically synthesised
Abstract
Description
Die Erfindung betrifft ein Verfahren, eine Verwendung und ei nen Stoff zur Hemmung der Expression eines Zielgens.The invention relates to a method, a use and egg NEN substance for inhibiting the expression of a target gene.
Aus der WO 99/32619 und der WO 00/44895 sind Verfahren zur Hemmung der Expression von medizinisch oder biotechnologisch interessanten Genen mit Hilfe eines doppelsträngigen Oligori bonukleotids (dsRNA) bekannt. Die bekannten Verfahren sind nicht besonders effektiv.WO 99/32619 and WO 00/44895 describe processes for Inhibition of medical or biotechnological expression interesting genes with the help of a double-stranded oligori bonucleotides (dsRNA) known. The known methods are not particularly effective.
Aufgabe der vorliegenden Erfindung ist es, die Nachteile nach dem Stand der Technik zu beseitigen. Es soll insbesondere ein möglichst wirksames Verfahren, eine möglichst wirksame Ver wendung und ein Stoff angegeben werden, mit denen eine noch effizientere Hemmung der Expression eines Zielgens erreichbar ist.The object of the present invention is to overcome the disadvantages to eliminate the state of the art. It is supposed to be a the most effective method, the most effective ver application and a substance can be specified with which one more more efficient inhibition of the expression of a target gene can be achieved is.
Diese Aufgabe wird durch die Merkmale der Ansprüche 1, 36 und 72 gelöst. Vorteilhafte Ausgestaltungen ergeben sich aus den Merkmalen der Ansprüche 2 bis 35, 37 bis 71 und 73 bis 99.This object is achieved by the features of claims 1, 36 and 72 solved. Advantageous configurations result from the Features of claims 2 to 35, 37 to 71 and 73 to 99.
Mit den erfindungsgemäß beanspruchten Merkmalen wird überra schender Weise eine drastische Erhöhung der Effektivität der Hemmung der Expression eines Zielgens erreicht. Die genauen Umstände dieses Effekts sind noch nicht geklärt.With the features claimed according to the invention is surprising a drastic increase in the effectiveness of the Inhibition of expression of a target gene achieved. The exact Circumstances of this effect have not yet been clarified.
Die gleichzeitige Applikation mehrerer erfindungsgemäßer Oli goribonukleotide mit zu unterschiedlichen Bereichen bzw. Ab schnitten des Zielgens komplementären Sequenzen bewirkt eine stärkere Hemmung der Expression des Zielgens schon bei Ver wendung sehr niedriger Konzentrationen. The simultaneous application of several oils according to the invention Goribonucleotides with too different areas or Ab cutting the target gene complementary sequences causes a stronger inhibition of the expression of the target gene already in Ver very low concentrations.
Die Gesamtzahl der verwendeten unterschiedlichen erfindungs gemäßen Oligoribonukleotide kann bis zu 100 betragen. In ei nem besonderen Fall können die komplementären Bereiche der erfindungsgemäßen Oligoribonukleotide die gesamte Sequenz des Zielgens lückenlos überdecken. Dabei sind auch Überlappungen in den überdeckten Bereichen möglich.The total number of different fiction used according to oligoribonucleotides can be up to 100. In egg In a special case, the complementary areas of the Oligoribonucleotide invention the entire sequence of Cover target gene completely. There are also overlaps possible in the covered areas.
Nach einem Ausgestaltungsmerkmal kann zumindest ein Ende des ersten und/oder des zweiten Oligoribonukleotids zumindest ein nicht nach Watson & Crick gepaartes Nukleotid aufweisen. Es wird angenommen, dass durch die besondere Ausbildung des zu mindest eine Endes zumindest eines der Oligoribonukleotide die Stabilität desselben erhöht wird. Durch die Erhöhung der Stabilität. wird die wirksame Konzentration in der Zelle er höht. Die Effektivität ist gesteigert.According to a design feature, at least one end of the at least one of the first and / or the second oligoribonucleotide nucleotide not paired according to Watson & Crick. It it is believed that due to the special training of the at least one end of at least one of the oligoribonucleotides the stability of the same is increased. By increasing the Stability. the effective concentration in the cell becomes he increased. The effectiveness is increased.
Die Effektivität kann weiter gesteigert werden, wenn das Ende einen aus 1 bis 4 Nukleotiden gebildeten einsträngigen Ab schnitt und/oder ungepaarte Nukleotide aufweist. Eine beson dere Erhöhung der Stabilität des erfindungsgemäßen Oligoribo nukleotids ist beobachtet worden, wenn das Ende das 3'-Ende eines Strangs der doppelsträngigen Struktur ist.The effectiveness can be further increased when the end a single-stranded Ab formed from 1 to 4 nucleotides cut and / or unpaired nucleotides. A special their increase in the stability of the oligoribo according to the invention nucleotide has been observed when the end is the 3 'end one strand of the double-stranded structure.
Als besonders vorteilhaft hat es sich erwiesen, die Zelle vor dem Einführen der Oligoribonukleotide mit Interferon zu be handeln. Auf diese Weise können besonders effektiv Tumore be kämpft werden.It has proven to be particularly advantageous to place the cell in front the introduction of the oligoribonucleotides with interferon act. In this way, tumors can be particularly effective to be fought.
Es hat sich gezeigt, dass durch eine solche aufeinanderfol gende Applikation von Interferon und erfindungsgemäßen Oligo ribonukleotiden die Nachteile, wie sie bei der bekannten al leinigen Verwendung von langkettigen Oligoribonukleotiden auftreten, vermieden und die Vorteile der Verwendung von kur zen Oligoribonukleotiden mit weniger als 50 Nukleotidpaaren zur Hemmung der Genexpression besser ausgenutzt werden kön nen. Darüber hinaus wird der durch die Oligoribonukleotide vermittelte hemmende Effekt auf die Genexpression verstärkt.It has been shown that such a successive application of interferon and oligo according to the invention ribonucleotides the disadvantages, as in the known al line use of long chain oligoribonucleotides occur, avoided and the benefits of using cure zen oligoribonucleotides with less than 50 nucleotide pairs can be better used to inhibit gene expression NEN. In addition, the oligoribonucleotides mediated inhibitory effect on gene expression enhanced.
Nach einem weiteren Ausgestaltungsmerkmal wird die Effektivi tät des Verfahrens erhöht, wenn zumindest ein weiteres Oligo ribonukleotid in die Zelle eingeführt wird, welches eine dop pelsträngige aus mindestens 49 aufeinanderfolgenden Nukleo tidpaaren gebildete Struktur aufweist, wobei ein Strang oder zumindest ein Abschnitt des Strangs der doppelsträngigen Struktur des weiteren Oligoribonukleotids komplementär zu ei nem dritten Bereich des Zielgens ist. Die Hemmung der Expres sion des Zielgens ist in diesem Fall deutlich gesteigert.According to a further design feature, the effectiveness Activity of the procedure increased if at least one other oligo ribonucleotide is introduced into the cell, which has a dop pelstring from at least 49 consecutive nucleos Has tid pairs formed structure, wherein a strand or at least a portion of the strand of the double-stranded Structure of the further oligoribonucleotide complementary to egg is a third region of the target gene. Inhibition of express The target gene is significantly increased in this case.
Nach einem weiteren Ausgestaltungsmerkmal kann das erste und/oder das zweite Oligoribonukleotid eine doppelsträngige aus weniger als 25 aufeinanderfolgenden Nukleotidpaaren ge bildete Struktur aufweisen.According to a further design feature, the first and / or the second oligoribonucleotide is a double-stranded from less than 25 consecutive pairs of nucleotides have formed structure.
Der erste, zweite und dritte Bereich können abschnittsweise überlappen, aneinandergrenzen oder auch voneinder beabstandet sein.The first, second and third areas can be in sections overlap, adjoin or also spaced apart his.
Die erfindungsgemäßen Oligoribonukleotide können dann beson ders einfach in die Zelle eingeschleust werden, wenn sie in micellare Strukturen, vorteilhafterweise in Liposomen, einge schloßen werden. Es ist auch möglich das/die Oligoribonukleo tid/e in virale natürliche Kapside oder in auf chemischem oder enzymatischem Weg hergestellte künstliche Kapside oder davon abgeleitete Strukturen einzuschließen. The oligoribonucleotides according to the invention can then in particular which are simply introduced into the cell when they are in micellar structures, advantageously in liposomes be closed. It is also possible that the oligoribonucleo tid / e in viral natural capsids or in chemical or artificial capsids or enzymatically produced including structures derived therefrom.
Das Zielgen kann nach einem weiteren Ausgestaltungsmerkmal eine der in dem anhängenden Sequenzprotokoll wiedergegebenen Sequenzen SQ001 bis SQ140 aufweisen. Es kann auch aus der folgenden Gruppe ausgewählt sein: Onkogen, Cytokin-Gen, Id- Protein-Gen, Entwicklungsgen, Priongen.The target gene can have a further design feature one of the reproduced in the attached sequence listing Sequences SQ001 to SQ140 have. It can also come from the the following group can be selected: oncogene, cytokine gene, id Protein gene, development gene, prion gene.
Das Zielgen wird zweckmäßigerweise in pathogenen Organismen, vorzugsweise in Plasmodien, exprimiert. Es kann Bestandteil eines Virus oder Viroids, insbesondere eines humanpathogenen Virus oder Viruids, sein. Das Virus oder Viruid kann auch ein tier- oder pflanzenpathogenes Virus oder Viroid sein.The target gene is conveniently found in pathogenic organisms, preferably expressed in plasmodia. It can be part a virus or viroid, especially a human pathogen Virus or viruids. The virus or viruid can also be a animal or phytopathogenic virus or viroid.
Nach einem weiteren Ausgestaltungsmerkmal ist vorgesehen, dass die ungepaarten Nukleotide durch Nukleosidthiophosphate substituiert sind.According to a further design feature, that the unpaired nucleotides by nucleoside thiophosphates are substituted.
Die doppelsträngige Struktur der erfindungsgemäßen Oligoribo nukleotide kann weiter durch eine chemische Verknüpfung der der beiden Stränge stabilisiert werden. Die chemische Ver knüpfung kann durch eine kovalente oder ionische Bindung, ei ne Wasserstoffbrückenbindung, hydrophobe Wechselwirkungen, vorzugsweise von-der-Waals- oder Stapelungswechselwirkungen, oder durch Metall-Ionenkoordination gebildet werden. Es hat sich weiter als zweckmäßig und die Stabilität erhöhend erwie sen, wenn die chemische Verknüpfung in der Nähe des einen oder in der Nähe der beiden Enden des erfindungsgemäßen Oli goribonukleotids gebildet ist. Weitere vorteilhafte Ausge staltungen hinsichtlich der chemischen Verknüpfung können den Merkmalen der Ansprüche 23 bis 29 entnommen werden, ohne dass es dafür einer näheren Erläuterung bedarf.The double-stranded structure of the oligoribo according to the invention nucleotides can be further linked by chemically linking the of the two strands are stabilized. The chemical ver tying can be by a covalent or ionic bond, egg ne hydrogen bonding, hydrophobic interactions, preferably von der Waals or stacking interactions, or be formed by metal ion coordination. It has proved to be more expedient and increased stability when the chemical linkage is close to one or near the two ends of the oli according to the invention goribonucleotide is formed. Further advantageous Ausge chemical linkage events can Features of claims 23 to 29 can be taken without it requires a more detailed explanation.
Zum Transport der erfindungsgemäßen Oligoribonukleotide hat es sich ferner als vorteilhaft erwiesen, dass diese an minde stens ein von einem Virus stammendes, davon abgeleitetes oder ein synthetisch hergestelltes virales Hüllprotein gebunden, damit assoziiert oder davon umgeben werden. Das Hüllprotein kann vom Polyomavirus abgeleitet sein. Das Hüllprotein kann insbesondere das Virus-Protein 1 und/oder das Virus-Protein 2 des Polyomavirus enthalten. Nach einer weiteren Ausgestaltung ist vorgesehen, dass bei Bildung eines Kapsids oder kapsidar tigen Gebildes aus dem Hüllprotein die eine Seite zum Inneren des Kapsids oder kapsidartigen Gebildes gewandt ist. Ferner ist es von Vorteil, dass das/die Oligoribonukleotid/e zum primären oder prozessierten RNA-Transkript des Zielgens kom plementär ist/sind. Die Zelle kann eine Vertebratenzelle oder eine menschliche Zelle sein.Has to transport the oligoribonucleotides according to the invention it has also proven to be advantageous for this to apply at least one derived from, derived from or bound a synthetically produced viral coat protein, associated with or surrounded by it. The coat protein can be derived from polyoma virus. The coat protein can in particular virus protein 1 and / or virus protein 2 of the polyoma virus included. According to a further embodiment it is provided that when a capsid or capsid is formed structure from the coat protein one side to the inside of the capsid or capsid-like structure. Further it is advantageous that the oligoribonucleotide (s) is primary or processed RNA transcript of the target gene com is / are complementary. The cell can be a vertebrate cell or be a human cell.
Erfindungsgemäß ist weiterhin die Verwendung der vorgenannten ersten und zweiten Oligoribonukleotide mit den vorgenannten Merkmalen zur Hemmung der Expression eines Zielgens in einer Zelle vorgesehen. Es wird insoweit auf die vorangegangenen Ausführungen verwiesen.The use of the aforementioned is also in accordance with the invention first and second oligoribonucleotides with the aforementioned Features for inhibiting the expression of a target gene in a Cell provided. In this respect, it is based on the previous ones Executions referenced.
Nach weiterer Maßgabe der Erfindung wird die Aufgabe gelöst durch einen Stoff zur Hemmung der Expression eines Zielgens, umfassend mindestens ein erstes und ein zweites Oligoribonu kleotid in einer zur Hemmung der Expression des Zielgens aus reichenden Menge, wobei das erste und das zweite Oligoribonu kleotid jeweils eine doppelsträngige aus höchstens 49 aufein anderfolgenden Nukleotidpaaren gebildete Struktur aufweisen, und wobei ein Strang oder zumindest ein Abschnitt eines Strangs der doppelsträngigen Struktur des ersten Oligoribonu kleotids komplementär zu einem ersten Bereich des Zielgens ist, und wobei ein Strang oder zumindest ein Abschnitt eines Strangs der doppelsträngigen Struktur des zweiten Oligoribo nukleotids komplementär zu einem zweiten Bereich des Zielgens ist.According to another aspect of the invention, the object is achieved by a substance for inhibiting the expression of a target gene, comprising at least a first and a second oligoribonu kleotide in one to inhibit expression of the target gene sufficient amount, the first and the second oligoribonu Kleotid each a double-stranded from 49 at most have a structure formed on the following nucleotide pairs, and wherein a strand or at least a portion of one Strands of the double-stranded structure of the first oligoribonu kleotids complementary to a first region of the target gene is, and wherein a strand or at least a portion of a Strand of the double-stranded structure of the second oligoribo nucleotides complementary to a second region of the target gene is.
Nach einem weiteren Ausgestaltungsmerkmal weist zumindest ein Ende des ersten und/oder zweiten Oligoribonukleotids zumin dest ein nicht nach Watson & Crick gepaartes Nukleotid auf. Wegen der weiteren vorteilhaften Ausgestaltung des ersten und zweiten Oligoribonukleotids wird auf die vorangegangenen Aus führungen verwiesen.According to a further design feature, at least one End of the first and / or second oligoribonucleotide at least at least one nucleotide not paired according to Watson & Crick. Because of the further advantageous embodiment of the first and second oligoribonucleotide is based on the previous Aus guided tours.
Die Erfindung wird nachfolgend anhand der Zeichnungen bei spielhaft erläutert. Es zeigen:The invention is described below with reference to the drawings explained in a playful way. Show it:
Fig. 1a-c schematisch ein erstes, zweites und drittes Oligoribonukleotid und Fig. 1a-c schematically shows a first, second and third oligoribonucleotide and
Fig. 2 schematisch ein Zielgen. Fig. 2 shows schematically a target gene.
Die in den Fig. 1a bis c gezeigten Oligoribonukleotide dsRNA I, dsRNA II und dsRNA III weisen jeweils ein erstes Ende E1 und ein zweites Ende E2 auf. Das erste Oligoribonukleotid dsRNA I und das zweite Oligoribonukleotid dsRNA II weisen an ihren Enden E1 und E2 einzelsträngige aus etwa 1 bis 4 unge paarten Nukleotiden gebildete Abschnitte auf. Beim dritten Oligoribonukleotid dsRNA III handelt es sich um ein langes Oligoribonukleotid mit mehr als 49 Nukleotidpaaren.The oligoribonucleotides dsRNA I, dsRNA II and dsRNA III shown in FIGS . 1a to c each have a first end E1 and a second end E2. The first oligoribonucleotide dsRNA I and the second oligoribonucleotide dsRNA II have at their ends E1 and E2 single-stranded sections formed from about 1 to 4 unpaired nucleotides. The third oligoribonucleotide dsRNA III is a long oligoribonucleotide with more than 49 nucleotide pairs.
In Fig. 2 ist schematisch ein auf einer DNA befindliches Zielgen gezeigt. Das Zielgen ist durch einen schwarzen Balken kenntlich gemacht. Es weist einen ersten Bereich B1, einen zweiten Bereich B2 und einen dritten Bereich B3 auf. A target gene located on a DNA is shown schematically in FIG . The target gene is identified by a black bar. It has a first area B1, a second area B2 and a third area B3.
Jeweils ein Strang S1, S2 und S3 des ersten dsRNA I, zweiten dsRNA II und dritten Oligoribonukleotids dsRNA III ist kom plementär zum entsprechenden Bereich B1, B2 und B3 auf dem Zielgen.One strand each S1, S2 and S3 of the first dsRNA I, second dsRNA II and third oligoribonucleotide dsRNA III is com complementary to the corresponding area B1, B2 and B3 on the Target gene.
Die Expression des Zielgens wird dann besonders wirkungsvoll gehemmt, wenn die kurzkettigen ersten dsRNA I und zweiten Oligoribonukleotide dsRNA II an ihren Enden E1, E2 einzel strängige Abschnitte aufweisen. Die einzelsträngigen Ab schnitte können sowohl am Strang S1, S2 als auch am Ge genstrang oder am Strang S1, S3 und am Gegenstrang ausgebil det sein. Es hat sich weiter gezeigt, dass ab einer bestimm ten Länge der Oligoribonukleotide, z. B. ab einer Länge von mehr als 49 Nukleotidpaaren, eine einzelsträngige Ausbildung der Enden E1, E2 weniger stark zur Unterdrückung der Expres sion des Zielgens beiträgt. Bei langen Oligoribonukleotiden, hier beim dritten Oligoribonukleotid dsRNA III, ist eine ein zelsträngige Ausbildung an den Enden E1, E2 nicht unbedingt erforderlich.The expression of the target gene then becomes particularly effective inhibited when the short chain first dsRNA I and second Oligoribonucleotides dsRNA II at their ends E1, E2 individually have stringy sections. The single-stranded Ab cuts can be made on strand S1, S2 as well as on Ge trained in the opposite strand or on strand S1, S3 and on the opposite strand det be. It has also been shown that from a certain th length of the oligoribonucleotides, e.g. B. from a length of more than 49 nucleotide pairs, a single-stranded formation the ends E1, E2 less strong to suppress the express sion of the target gene. With long oligoribonucleotides, here with the third oligoribonucleotide dsRNA III, is a single-stranded training at the ends E1, E2 not necessarily required.
Die Bereiche B1, B2 und B3 können, wie in Fig. 2 gezeigt, von einander beabstandet sein. Sie können aber auch an einander grenzen oder überlappen.The areas B1, B2 and B3 can, as shown in FIG. 2, be spaced apart from one another. But you can also border or overlap each other.
Im Falle der einzelsträngigen Ausbildung der Enden E1, E2 sind alle denkbaren Permutationen möglich, d. h. es können ein Ende oder beide Enden des Strangs S1, S2, S3 oder ein Ende oder beide Enden des Gegenstrangs überstehen. Der einzel strängige Abschnitt kann 1 bis 4 gepaarte Nukleotide aufwei sen. Es ist auch möglich, dass ein Ende oder beide Enden E1, E2 mindestens ein nicht nach Watson & Crick gepaartes Nukleo tidpaar aufweisen. In the case of single-strand formation of the ends E1, E2 all possible permutations are possible, d. H. it can be a End or both ends of the string S1, S2, S3 or one end or protrude both ends of the opposite strand. The single stranded section can have 1 to 4 paired nucleotides sen. It is also possible that one end or both ends E1, E2 at least one nucleo not paired according to Watson & Crick tidpaar exhibit.
Es wurden aus Sequenzen des Grün-fluoreszierenden Proteins (GFP) der Alge Aequoria victoria abgeleitete doppelsträngige RNAs (dsRNAs) hergestellt und zusammen mit dem GFP-Gen in Fi broblasten mikroinjiziert. Anschließend wurde die Fluores zenzabnahme gegenüber Zellen ohne dsRNA ausgewertet.It was made up of sequences of the green fluorescent protein (GFP) of the alga Aequoria victoria derived double-stranded RNAs (dsRNAs) are produced and together with the GFP gene in Fi microblown broblasts. Then the fluorescence Decrease in cells compared to cells without dsRNA was evaluated.
Mittels eines RNA-Synthesizers (Typ Expedite 8909, Applied Biosystems, Weiterstadt, Deutschland) und herkömmlicher che mischer Verfahren wurden die aus den Sequenzprotokollen SQ141 SQ144 ersichtlichen RNA-Einzelstränge und die zu ihnen kom plementären Einzelstränge synthetisiert. Die Hybridisierung der komplementären Einzelstränge zum Doppelstrang erfolgte für jede einzelne dsRNA durch Aufheizen des stöchiometrischen Gemischs der Einzelstränge in 10 mM Natriumphosphatpuffer, pH 6,8, 100 mM NaCl, auf 90°C und nachfolgendes langsames Abküh len über 6 Stunden auf Raumtemperatur. Anschließend erfolgte Reinigung mit Hilfe der HPLC. Die so erhaltenen deRNAs wurden einzeln oder gemeinsam in die Testzellen mikroinjiziert. Als Testsystem für diese in-vivo-Experimente diente die muri ne Fibroblasten-Zellinie NIH/3T3. Mit Hilfe der Mikroinjekti on wurde das GFP-Gen in die Zellen eingebracht. Die Expressi on des GFP wurde unter dem Einfluß gleichzeitig mittransfi zierter sequenzhomologer dsRNA untersucht. Die Auswertung un ter dem Fluoreszenzmikroskop erfolgte 3 Stunden nach Injekti on anhand der grünen Fluoreszenz des gebildeten GFP.Using an RNA synthesizer (type Expedite 8909, Applied Biosystems, Weiterstadt, Germany) and conventional che Mixing methods were those from the sequence listing SQ141 SQ144 visible RNA single strands and the com complementary single strands synthesized. The hybridization the complementary single strands to the double strand took place for each individual dsRNA by heating the stoichiometric Mix the single strands in 10 mM sodium phosphate buffer, pH 6.8, 100 mM NaCl, to 90 ° C and subsequent slow cooling len over 6 hours to room temperature. Then followed Cleaning with the help of HPLC. The deRNAs thus obtained were micro-injected individually or together into the test cells. The muri was used as the test system for these in vivo experiments ne fibroblast cell line NIH / 3T3. With the help of micro-injections the GFP gene was introduced into the cells. The Expressi on the GFP was under the influence of transfi graced sequence homologous dsRNA examined. The evaluation un The fluorescence microscope was taken 3 hours after injecting on based on the green fluorescence of the GFP formed.
Die Zellen wurden in DMEM mit 4,5 g/l Glucose, 10% fötalem Rinderserum unter 7,5% CO2-Atmosphäre bei 37 W in Kultur schalen inkubiert und vor Erreichen der Konfluenz passagiert. Das Ablösen der Zellen erfolgte mit Trypsin/EDTA. Zur Vorbe reitung der Mikroinjektion wurden die Zellen in Petrischalen überführt und bis zu Bildung von Mikrokolonien weiter inku biert.The cells were incubated in DMEM with 4.5 g / l glucose, 10% fetal bovine serum under 7.5% CO 2 atmosphere at 37 W in culture dishes and were passaged before reaching confluence. The cells were detached using trypsin / EDTA. To prepare the microinjection, the cells were transferred to Petri dishes and incubated until microcolonies were formed.
Die Kulturschalen wurde zur Mikroinjektion für ca. 10 Minuten aus dem Inkubator genommen. Es wurde in ca. 50 Zellen pro An satz innerhalb eines markierten Bereiches unter Verwendung des Mikroinjektionssystems FemtoJet der Firma Eppendorf, Deutschland, einzeln injiziert. Anschließend wurden die Zel len weitere drei Stunden inkubiert. Für die Mikroinjektion wurden Borosilikat-Glaskapillaren der Firma Eppendorf mit ei nem Spitzeninnendurchmesser von 0,5 µm verwendet. Die Mikro injektion wurde mit dem Mikromanipulator 5171 der Firma Ep pendorf durchgeführt. Die Injektionsdauer betrug 0,8 Sekun den, der Druck ca. 80 hPa. Die in die Zellen injizierten Pro ben enthielten 0,01 µg/µ1 pGFP-C1 (Clontech Laboratories GmbH, Heidelberg, Deutschland) sowie an Dextran-70000 gekop peltes Texas-Rot in 14 mM NaCl, 3 mM KCl, 10 mM KP04, pH 7,5. Zusätzlich wurden in ca. 100 pl folgende dsRNAs zugegeben: Ansatz 1: 100 µM dsRNA (Sequenzprotokoll SQ141); Ansatz 2: 100 µM dsRNA (Sequenzprotokoll SQ142); Ansatz 3: 100 µM dsRNA (Sequenzprotokoll SQ143); Ansatz 4: 100 µM dsRNA (Sequenzpro tokoll SQ144); Ansatz 5: Gemisch von je 25 µM dsRNA (nach Se quenzprotokoll SQ141, SQ142, SQ143 und SQ144); Ansatz 6: ohne RNA.The culture dishes were microinjected for about 10 minutes taken out of the incubator. It was in about 50 cells per type sentence within a marked area using the micro injection system FemtoJet from Eppendorf, Germany, injected individually. Then the Zel len incubated for a further three hours. For microinjection were borosilicate glass capillaries from Eppendorf with egg nem inner diameter of 0.5 microns used. The micro injection was carried out with the micromanipulator 5171 from Ep pendorf carried out. The injection duration was 0.8 seconds den, the pressure about 80 hPa. The Pro injected into the cells ben contained 0.01 µg / µ1 pGFP-C1 (Clontech Laboratories GmbH, Heidelberg, Germany) as well as dextran-70000 peltes Texas red in 14mM NaCl, 3mM KCl, 10mM KP04, pH 7.5. In addition, the following dsRNAs were added in about 100 μl: Batch 1: 100 μM dsRNA (sequence listing SQ141); Approach 2: 100 µM dsRNA (sequence listing SQ142); Batch 3: 100 µM dsRNA (Sequence listing SQ143); Approach 4: 100 µM dsRNA (sequence pro tokoll SQ144); Approach 5: Mixture of 25 µM dsRNA (according to Se sequence protocol SQ141, SQ142, SQ143 and SQ144); Approach 6: without RNA.
Die Zellen wurden bei Anregung mit Licht der Anregungswellen länge von Texas-Rot, 568 nm, bzw. von GFP, 513 nm, mittels eines Fluoreszenzmikroskops untersucht. Die Fluoreszenz aller Zellen im Gesichtsfeld wurde bestimmt und in Relation zur Zelldichte (ausgedrückt durch deren Gesamtproteinkonzentrati on) gesetzt.The cells were excited with light from the excitation waves length of Texas red, 568 nm, or of GFP, 513 nm, by means of of a fluorescence microscope. The fluorescence of everyone Cells in the visual field were determined and in relation to the Cell density (expressed by their total protein concentration on).
Sowohl bei einer Gesamtkonzentration von 10 als auch von 100 µM dsRNA konnte bei gleichzeitiger Verwendung von vier unter schiedlichen dsRNAs ein deutlich stärkerer hemmender Effekt auf die Expression des GFP-Gens in Fibroblasten beobachtet werden als mit einer dsRNA allein (Tabelle 1). Darüber hinaus war bei gleichzeitiger Verwendung von vier unterschiedlichen dsRNAs eine starke Hemmung bereits bei einer Konzentration von 10 µM zu erreichen, was mit nur einer dsRNA nicht möglich war.Both at a total concentration of 10 and 100 µM dsRNA could be used with the simultaneous use of four different dsRNAs a significantly stronger inhibitory effect observed for the expression of the GFP gene in fibroblasts are considered to be with a dsRNA alone (Table 1). Furthermore was while using four different ones dsRNAs show strong inhibition even at a concentration of 10 µM, which is not possible with just one dsRNA was.
Die Verwendung mehrerer, gegen das selbe Zielgen gerichteten
dsRNAs ermöglicht somit eine stärkere Hemmung der Genexpres
sion in Säugerzellen bereits bei niedrigeren Konzentrationen
als dies mit nur einer dsRNA erreichbar ist.
The use of several dsRNAs directed against the same target gene thus enables a greater inhibition of gene expression in mammalian cells even at lower concentrations than can be achieved with only one dsRNA.
Tabelle 1: Die Symbole geben den relativen Anteil an nicht oder schwach fluoreszierende Zellen an (+++ < 90%; ++ 60-90%; + 30-60%; - < 10%). Table 1: The symbols do not indicate the relative proportion of or weakly fluorescent cells on (+++ <90%; ++ 60-90%; + 30-60%; - <10%).
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Claims (100)
Einführen mindestens eines ersten (dsRNA I) und eines zweiten Oligoribonukleotids (dsRNA II) in einer zur Hemmung der Ex pression des Zielgens ausreichenden Menge,
wobei das erste (dsRNA I) und das zweite Oligoribonukleotid (dsRNA II) jeweils eine doppelsträngige aus höchstens 49 auf einanderfolgenden Nukleotidpaaren gebildete Struktur aufwei sen,
wobei ein Strang (S1) oder zumindest ein Abschnitt eines Strangs (S1) der doppelsträngigen Struktur des ersten Oligo ribonukleotids (dsRNA I) komplementär zu einem ersten Bereich (B1) des Zielgens ist,
und wobei ein Strang (S2) oder zumindest ein Abschnitt eines Strangs (S2) der doppelsträngigen Struktur des zweiten Oligo ribonukleotids (dsRNA II) komplementär zu einem zweiten Be reich (B2) des Zielgens ist.1. A method for inhibiting expression of a target gene in a cell comprising the following steps:
Introducing at least a first (dsRNA I) and a second oligoribonucleotide (dsRNA II) in an amount sufficient to inhibit the expression of the target gene,
wherein the first (dsRNA I) and the second oligoribonucleotide (dsRNA II) each have a double-stranded structure formed from a maximum of 49 successive nucleotide pairs,
wherein a strand (S1) or at least a portion of a strand (S1) of the double-stranded structure of the first oligo ribonucleotide (dsRNA I) is complementary to a first region (B1) of the target gene,
and wherein one strand (S2) or at least a portion of one strand (S2) of the double-stranded structure of the second oligo ribonucleotide (dsRNA II) is complementary to a second region (B2) of the target gene.
wobei das erste (dsRNA I) und das zweite Oligoribonukleotid (dsRNA II) jeweils eine doppelsträngige aus höchstens 49 auf einanderfolgenden Nukleotidpaaren gebildete Struktur aufwei sen,
wobei ein Strang (S1) oder zumindest ein Abschnitt eines Strangs (S1) der doppelsträngigen Struktur des ersten Oligo ribonukleotids (dsRNA I) komplementär zu einem ersten Bereich (B1) des Zielgens ist,
und wobei ein Strang (S2) oder zumindest ein Abschnitt eines Strangs (S2) der doppelsträngigen Struktur des zweiten Oligo ribonukleotids (dsRNA II) komplementär zu einem zweiten Be reich (B2) des Zielgens ist. 36. Use of a first (dsRNA I) and a second oligoribonucleotide (dsRNA II) in an amount sufficient to inhibit the expression of the target gene,
wherein the first (dsRNA I) and the second oligoribonucleotide (dsRNA II) each have a double-stranded structure formed from a maximum of 49 successive nucleotide pairs,
wherein a strand (S1) or at least a portion of a strand (S1) of the double-stranded structure of the first oligo ribonucleotide (dsRNA I) is complementary to a first region (B1) of the target gene,
and wherein one strand (S2) or at least a portion of one strand (S2) of the double-stranded structure of the second oligo ribonucleotide (dsRNA II) is complementary to a second region (B2) of the target gene.
wobei das erste (dsRNA I) und das zweite Oligoribonukleotid (dsRNA II) jeweils eine doppelsträngige aus höchstens 49 auf einanderfolgenden Nukleotidpaaren gebildete Struktur aufwei sen,
und wobei ein Strang (S1) oder zumindest ein Abschnitt eines Strangs (S1) der doppelsträngigen Struktur des ersten Oligo ribonukleotids (dsRNA I) komplementär zu einem ersten Bereich (B1) des Zielgens ist,
und wobei ein Strang (S2) oder zumindest ein Abschnitt eines Strangs (S2) der doppelsträngigen Struktur des zweiten Oligo ribonukleotids (dsRNA II) komplementär zu einem zweiten Be reich (B2) des Zielgens ist.72. Substance for inhibiting the expression of a target gene, comprising at least a first (dsRNA I) and a second oligonucleotide (dsRNA II) in an amount sufficient to inhibit expression of the target gene,
wherein the first (dsRNA I) and the second oligoribonucleotide (dsRNA II) each have a double-stranded structure formed from a maximum of 49 successive nucleotide pairs,
and wherein a strand (S1) or at least a portion of a strand (S1) of the double-stranded structure of the first oligo ribonucleotide (dsRNA I) is complementary to a first region (B1) of the target gene,
and wherein one strand (S2) or at least a portion of one strand (S2) of the double-stranded structure of the second oligo ribonucleotide (dsRNA II) is complementary to a second region (B2) of the target gene.
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