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WO2000008195A1 - Method and device for the transfer of oligonucleotides in cells - Google Patents

Method and device for the transfer of oligonucleotides in cells Download PDF

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Publication number
WO2000008195A1
WO2000008195A1 PCT/DE1999/002327 DE9902327W WO0008195A1 WO 2000008195 A1 WO2000008195 A1 WO 2000008195A1 DE 9902327 W DE9902327 W DE 9902327W WO 0008195 A1 WO0008195 A1 WO 0008195A1
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Prior art keywords
oligonucleotides
transferred
cell
cells
shock wave
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Ceased
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PCT/DE1999/002327
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German (de)
French (fr)
Inventor
Stefan Endres
Michael Delius
Friedrich Ueberle
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Dornier Medtech Holding International GmbH
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Dornier Medtech Holding International GmbH
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Application filed by Dornier Medtech Holding International GmbH filed Critical Dornier Medtech Holding International GmbH
Priority to AU63241/99A priority Critical patent/AU6324199A/en
Publication of WO2000008195A1 publication Critical patent/WO2000008195A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli

Definitions

  • the invention relates to a method for intracellular transfer of oligonucleotides according to method claim 1 and a device for carrying out the method according to device claim 4
  • oligonucleotides The intracellular transfer of oligonucleotides is used to specifically inhibit the synthesis of individual proteins in the cell.
  • a short-chain synthetic nucleic acid with a freely selectable sequence of bases is introduced into the cytoplasm of the cell as soon as the cell nucleus of the cell is used to synthesize a If the so-called mRNA, which is necessary for the protein, is released, the oligonucleotide in the cell can take the place of the mRNA, the sequence of which is complementary to the base sequence of the gonucleotide.
  • This “application” blocks the synthesis of exactly one protein. This cell is therefore absent an otherwise present protein The absence of this protein can result in a change in the cell's properties or mortality
  • carrier lipids are used, by means of which an improvement in the uptake of the gonucleotides into the cytoplasm of the cell is produced.
  • a disadvantage of this known method is the need for these relatively expensive carrier lipids in addition to the Having to apply oligonucleotides as consumable for intracellular transfer
  • Another known possibility of transferring oligonucleotides provides for direct introduction, for example by means of microinjections or electroporation.
  • direct introduction for example by means of microinjections or electroporation.
  • the effectiveness of the method of direct introduction is disadvantageous
  • cavitation occurs in liquids under the influence of shock waves.
  • This cavitation can be represented simply as the formation and movement of bubbles or cavities in a liquid.
  • a very fine, needle-like liquid jet is formed. This liquid jet penetrates the cell membrane and transfers a small amount of the liquid into the cell.
  • Oligonucleotides there is a probability, depending on the concentration of the oligonucleotides in the liquid, that oligonucleotides have been transferred into the cell.
  • FIG. 1 shows a schematic structure of a device 1 with which the method according to the invention can be carried out.
  • the device 1 is filled with a liquid 2.
  • the liquid 2 can be exposed to a shock wave by means of a shock wave generator 3.
  • a sample container 4 is partially immersed in the liquid 2.
  • the sample container 4 is attached to a holder, not shown for reasons of clarity.
  • This solution 5 contains the oligonucleotides and the target cells into which the oligonucleotides are to be transferred.
  • the material of the sample container 4 is continuous for shock waves, the essentially aqueous solution 5 in which the oligonucleotides and the target cells for the transfer of the oligonucleotides also pass on the shock waves.
  • Fluid jet hits the cell, it penetrates the cell membrane like a microinjection. A small amount of the liquid that makes up the fluid jet remains in the cell.
  • the oligonucleotides to be transferred are stochastically distributed in the liquid, the oligonucleotides are also transferred stochastically to individual cells.
  • Activating the shock wave source several times increases the probability that oligonucleotides will be transferred into the cell.
  • the target cells can be in a living being, and the oligonucleotides can be located near the target cells by suitable measures (such as blood circulation or injection) Extracorporeal shock waves in the body of the living being and focusing on the one area of the body in which the target cells are located, it is possible to introduce locally limited oligonucleotides into cells of the body and thus change the properties of the cells and / or their mortality or lethality.
  • suitable measures such as blood circulation or injection
  • shock wave source for the purpose of transferring oligonucleotides in cells both in vivo and in vitro represents a method in which harmful influences on the cells, for example through the currents during electroporation or side effects caused by the carrier lipids, are advantageously avoided.

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Mechanical Engineering (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Oligonucleotides are transferred in the cells under the influence of shock waves.

Description

VERFAHREN UND VORRICHTUNG ZUM TRANSFER VON OLIGONUKLEOTIDEN IN DIE ZELLEMETHOD AND DEVICE FOR THE TRANSFER OF OLIGONUCLEOTIDS INTO THE CELL

Die Erfindung betrifft ein Verfahren zum intrazellularen Transfer von Oligonukleotiden gemäß dem Verfahrensanspruch 1 und einer Vorrichtung zur Durchfuhrung des Verfahrens gemäß dem Vorrichtungsanspruch 4The invention relates to a method for intracellular transfer of oligonucleotides according to method claim 1 and a device for carrying out the method according to device claim 4

Der intrazellular e Transfer von Oligonukleotiden dient zur gezielten Synthesehemmung von einzelnen Proteinen in der Zelle Um die Synthese eines Proteins zu hemmen wird eine kurzkettige synthetische Nuklemsaure mit einer frei wahlbaren Abfolge von Basen in das Zytoplasma der Zelle eingebracht Sobald vom Zellkern der Zelle die zur Synthese eines Proteins notwendige sogenannte mRNS abgegeben wird, kann sich das in der Zelle befindliche Oligonukleotid an die Stelle der mRNS anlegen, deren Abfolge komplementär zu der Basenabfolge des O gonukleotids aufgebaut ist Durch dieses „Anlegen" wird die Synthese genau eines Proteins blockiert Folglich fehlt in dieser Zelle ein ansonsten vorhandenes Protein Das Fehlen dieses Proteins kann in einer Änderung der Zelleigenschaften oder Mortalität der Zelle resultierenThe intracellular transfer of oligonucleotides is used to specifically inhibit the synthesis of individual proteins in the cell.In order to inhibit the synthesis of a protein, a short-chain synthetic nucleic acid with a freely selectable sequence of bases is introduced into the cytoplasm of the cell as soon as the cell nucleus of the cell is used to synthesize a If the so-called mRNA, which is necessary for the protein, is released, the oligonucleotide in the cell can take the place of the mRNA, the sequence of which is complementary to the base sequence of the gonucleotide. This “application” blocks the synthesis of exactly one protein. This cell is therefore absent an otherwise present protein The absence of this protein can result in a change in the cell's properties or mortality

Bei einem bekannten Verfahren zum intrazellularen Transfer von Oligonukleotiden werden sogenannte Trager- Lipide verwendet, durch welche eine Verbesserung der Aufnahme der O gonukleotide in das Zytoplasma der Zelle erzeugt wu d Nachteilig bei diesem bekannten Verfahren ist die Notwendigkeit dieser relativ teuren Trager- Lipide zusätzlich zu den Oligonukleotiden als Verbrauchsmateπal für den intrazellularen Transfer aufbringen zu müssenIn a known method for the intracellular transfer of oligonucleotides, so-called carrier lipids are used, by means of which an improvement in the uptake of the gonucleotides into the cytoplasm of the cell is produced. A disadvantage of this known method is the need for these relatively expensive carrier lipids in addition to the Having to apply oligonucleotides as consumable for intracellular transfer

Eine andere bekannte Möglichkeit des Transfers von Oligonukleotiden sieht ein direktes Einbringen etwa durch Mikroinjektionen oder Elektroporation vor Nachteilig bei der Methode des direkten Einbringens ist jedoch die EffektivitätAnother known possibility of transferring oligonucleotides provides for direct introduction, for example by means of microinjections or electroporation. However, the effectiveness of the method of direct introduction is disadvantageous

Es ist daher Aufgabe der Erfindung ein Verfahren und eine Vorrichtung zu schaffen wodurch in effektiver Weise ein intrazellularer Transfer von Oligonukleotiden ohne den aufwendigen Zusatz von Trager- Lipiden möglich istIt is therefore an object of the invention to provide a method and a device whereby an intracellular transfer of oligonucleotides is possible in an effective manner without the complex addition of carrier lipids

Die erfmdungsgemaße Losung der Aufgabe erfolgt durch die kennzeichnenden Merkmale des Verfahrensanspruchs 1 und des Vorrichtungsanspruchs 4 Die erfindungsgemäße Lösung der Aufgabe erfolgt durch die kennzeichnendenThe solution to the problem according to the invention is achieved by the characterizing features of method claim 1 and device claim 4 The object of the invention is achieved by the characterizing

Merkmale des Verfahrensanspruchs 1 und des Vorrichtungsanspruchs 4.Features of process claim 1 and device claim 4.

Die jeweiligen Unteransprüche betreffen Weiterbildungen der Erfindung und/oder besonders vorteilhafte Ausgestaltungsformen.The respective subclaims relate to further developments of the invention and / or particularly advantageous embodiments.

Die Überlegungen, die zu der vorliegenden Erfindung führten gingen davon aus, daß unter dem Einfluß von Stoßwellen in Flüssigkeiten Kavitation entsteht. Diese Kavitation läßt sich vereinfacht darstellen als Bildung und Bewegung von Blasen oder Hohlräumen in einer Flüssigkeit. Zusätzlich zu den dadurch enstandenen Scherkräften kommt es zur Bildung eines sehr feinen, nadelartigen Flüssigkeitsstrahls. Dieser Flüssigkeitsstrahl durchdringt die Zellmembran und transferiert eine geringe Menge der Flüssigkeit in die Zelle.The considerations that led to the present invention assumed that cavitation occurs in liquids under the influence of shock waves. This cavitation can be represented simply as the formation and movement of bubbles or cavities in a liquid. In addition to the resulting shear forces, a very fine, needle-like liquid jet is formed. This liquid jet penetrates the cell membrane and transfers a small amount of the liquid into the cell.

Wenn sich in dieser Flüssigkeit stochastisch verteilt die zu transferierendenIf the liquid to be transferred is stochastically distributed in this liquid

Oligonukleotide befinden, besteht eine von der Konzentration der Oligonukleotide in der Flüssigkeit abhängige Wahrscheinlichkeit, daß Oligonukleotide in die Zelle transferiert wurden.Oligonucleotides, there is a probability, depending on the concentration of the oligonucleotides in the liquid, that oligonucleotides have been transferred into the cell.

Nachfolgend wird ein mögliches Ausführungsbeispiel der Erfindung anhand einer Zeichnung näher erläutert:A possible exemplary embodiment of the invention is explained in more detail below with reference to a drawing:

Die einzige Figur, im folgenden Fig. 1 genannt zeigt einen schematischen Aufbau einer Vorrichtung 1 mit welcher das erfindungsgemäße Verfahren durchführbar ist. Die Vorrichtung 1 ist mit einer Flüssigkeit 2 gefüllt.The only figure, referred to below as FIG. 1, shows a schematic structure of a device 1 with which the method according to the invention can be carried out. The device 1 is filled with a liquid 2.

Durch einen Stoßwellengenerator 3 kann die Flüssigkeit 2 einer Stoßwelle ausgesetzt werden.The liquid 2 can be exposed to a shock wave by means of a shock wave generator 3.

In die Flüssigkeit 2 ist ein Probenbehälter 4 teilweise eingetaucht. Der Probenbehälter 4 ist an einem aus Gründen der Übersichtlichkeit nicht gezeichneten Halter befestigt.A sample container 4 is partially immersed in the liquid 2. The sample container 4 is attached to a holder, not shown for reasons of clarity.

Im Probenbehälter 4 befindet sich eine im wesentlichen wässrige Lösung 5. In dieser Lösung 5 befinden sich die Oligonukleotide und die Ziel-Zellen in welche die Oligonukleotide transferiert werden sollen. Das Material des Probenbehälters 4 ist für Stoßwellen durchgängig, die im wesentlichen wässrige Lösung 5 in der sich die Oligonukleotide und die Ziel-Zellen für den Transfer der Oligonukleotide leitet die Stoßwellen ebenfalls weiter.There is an essentially aqueous solution 5 in the sample container 4. This solution 5 contains the oligonucleotides and the target cells into which the oligonucleotides are to be transferred. The material of the sample container 4 is continuous for shock waves, the essentially aqueous solution 5 in which the oligonucleotides and the target cells for the transfer of the oligonucleotides also pass on the shock waves.

Durch Aktivieren der Stoßwellenquelle 3 entsteht in der wässrigen Lösung 5 Kavitation.By activating the shock wave source 3, cavitation occurs in the aqueous solution 5.

Bei der Kavitation tritt ein sehr feiner, nadelartiger Fluidstrahl auf. Wenn dieserA very fine, needle-like fluid jet occurs during cavitation. If this

Fluidstrahl auf die Zelle trifft, durchdringt er ähnlich einer Microinjektion die Zellmembran. Eine geringe Menge der Flüssigkeit aus welcher der Fluidstrahl besteht verbleibt in der Zelle.Fluid jet hits the cell, it penetrates the cell membrane like a microinjection. A small amount of the liquid that makes up the fluid jet remains in the cell.

Da sich in der Flüssigkeit stochastisch verteilt die zu transferierenden Oligonukleotide befinden, werden ebenso stochastisch verteilt in einzelne Zellen die Oligonukleotide transferiert.Since the oligonucleotides to be transferred are stochastically distributed in the liquid, the oligonucleotides are also transferred stochastically to individual cells.

Durch mehrmaliges Aktivieren der Stoßwellenquelle erhöht sich die Wahrscheinlichkeit, daß Oligonukleotide in die Zelle transferiert werden.Activating the shock wave source several times increases the probability that oligonucleotides will be transferred into the cell.

Auf diese Weise ist es rationell möglich mit verhältnismäßig geringem Aufwand Oligonukleotide in eine größere Anzahl von Zellen zu transferieren.In this way it is economically possible to transfer oligonucleotides into a larger number of cells with relatively little effort.

Ebenso ist es auch möglich „in vivo" Oligonukleotide in Zellen zu transferieren. Dabei können sich die Ziel Zellen in einem Lebewesen befinden, und die Oligonukleotide können durch geeignete Maßnahmen (etwa Blutkreislauf oder Injektion ) in der Nähe der Ziel Zellen befinden. Durch Einleiten von extrakorporalen Stoßwellen in den Körper des Lebewesens und Fokusierung auf die einen Bereich des Körpers in welchem sich die Ziel Zellen befinden ist es möglich lokal begrenzt Oligonukleotide in Zellen des Körpers einzubringen und so die Eigenschaften der Zellen und/oder deren Mortalität bzw Letalität verändern.It is also possible to transfer “in vivo” oligonucleotides into cells. The target cells can be in a living being, and the oligonucleotides can be located near the target cells by suitable measures (such as blood circulation or injection) Extracorporeal shock waves in the body of the living being and focusing on the one area of the body in which the target cells are located, it is possible to introduce locally limited oligonucleotides into cells of the body and thus change the properties of the cells and / or their mortality or lethality.

Die Verwendung der Stoßwellenquelle zum Zwecke des Transfers von Oligonukleotiden in Zellen sowohl in vivo als auch in vitro stellt eine Methode dar, bei der schädliche Einflüsse auf die Zellen, etwa durch die Ströme bei der Elektroporation oder Nebenwirkungen durch die Träger-Lipide vorteilhaft vermieden werden. The use of the shock wave source for the purpose of transferring oligonucleotides in cells both in vivo and in vitro represents a method in which harmful influences on the cells, for example through the currents during electroporation or side effects caused by the carrier lipids, are advantageously avoided.

Claims

SchutzansprucheVerfahren und Vorrichtung zum intrazellularen Transfer von Oligonukleotiden. Process and device for intracellular transfer of oligonucleotides. 1 . Verfahren zum Transfer von Oligonukleotiden in eine Zelle, wobei die Zellmembran temporar und partiell durchlassig gemacht wird um ausserhalb der Zelle vorhandene Oligonukleotide in die Zelle einbringen zu können dadurch gekennzeichnet, daß die Zelle, in welche die Oligonukleotide transferiert werden sollen und die zu transferierenden Oligonukleotide gemeinsam einer Stoßwelle ausgesetzt werden1 . Process for the transfer of oligonucleotides into a cell, the cell membrane being made temporarily and partially permeable so that oligonucleotides present outside the cell can be introduced into the cell, characterized in that the cell into which the oligonucleotides are to be transferred and the oligonucleotides to be transferred together be exposed to a shock wave 2. Verfahren nach Anspruch 1 dadurch gekennzeichnet, daß die Zelle, in welche die Oligonukleotide transferiert werden sollen und die zu transferierenden Oligonukleotide sich gemeinsam in einer im wesentlichen wassngen Losung befinden wahrend sie der Stoßwelle ausgesetzt werden2. The method according to claim 1, characterized in that the cell into which the oligonucleotides are to be transferred and the oligonucleotides to be transferred are together in a substantially water solution while they are exposed to the shock wave 3. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die Zelle, in welche die Oligonukleotide transferiert werden sollen, sich in einem lebenden Zellverbund befinden und die zu transferierenden Oligonukleotide sich in der, die Zellen umgebenden Flüssigkeit befinden wahrend die Zellen und die Oligonukleotide der Stoßwelle ausgesetzt werden.3. The method according to claim 1, characterized in that the cell into which the oligonucleotides are to be transferred are in a living cell network and the oligonucleotides to be transferred are in the liquid surrounding the cells while the cells and the oligonucleotides of the shock wave get abandoned. 4. Vorrichtung zur Durchfuhrung des Verfahrens nach Anspruch 1 dadurch gekennzeichnet, daß durch einen Stoßwellengenerator eine oder mehrere Zellen, in welche die Oligonukleotide transferiert werden sollen sowie die zu transferierenden Oligonukleotide mit einer Stoßwelle beaufschlagbar sind. 4. A device for performing the method according to claim 1, characterized in that one or more cells into which the oligonucleotides are to be transferred and the oligonucleotides to be transferred can be subjected to a shock wave by means of a shock wave generator. 5. Vorrichtung nach Anspruch 4, dadurch gekennzeichnet, daß die Zellen in welche die Oligonukleotide transferiert werden sollen und die zu transferierenden Oligonukleotide in einem Stoßwellen weiterleitenden Medium innerhalb eines gemeinsamen Behälters angeordnet sind, der Behälter die Stoßwellen weiterleitet und seinerseits in ein Stoßwellen weiterleitendes Medium einsetzbar sind und in dieses Medium eine durch eine Stoßwellenquelle erzeugte Stoßwelle einleitbar ist.5. The device according to claim 4, characterized in that the cells into which the oligonucleotides are to be transferred and the oligonucleotides to be transferred are arranged in a shock wave transmitting medium within a common container, the container forwards the shock waves and in turn usable in a shock wave transmitting medium are and a shock wave generated by a shock wave source can be introduced into this medium. 6. Vorrichtung nach einem der Ansprüche 4 bis 5, dadurch gekennzeichnet, daß die Stoßwellen auf den Behälter, in welchem sich die Zellen und die Oligonukleotide befinden fokusierbar ist.6. Device according to one of claims 4 to 5, characterized in that the shock waves on the container in which the cells and the oligonucleotides are focusable. 7. Vorrichtung nach Anspruch 4, dadurch gekennzeichnet, daß sich die Zellen, in welche die Oligonukleotide transferiert werden sollen und die zu transferierenden Oligonukleotide innerhalb eines Körpers eines Lebewesens befinden und die Stoßwellen von aussen in den Körper einleitbar sind. 7. The device according to claim 4, characterized in that the cells into which the oligonucleotides are to be transferred and the oligonucleotides to be transferred are located within a body of a living being and the shock waves can be introduced into the body from the outside.
PCT/DE1999/002327 1998-07-31 1999-07-30 Method and device for the transfer of oligonucleotides in cells Ceased WO2000008195A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63241/99A AU6324199A (en) 1998-07-31 1999-07-30 Method and device for the transfer of oligonucleotides in cells

Applications Claiming Priority (2)

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DE19834612A DE19834612A1 (en) 1998-07-31 1998-07-31 Method for intracellular transfer of oligonucleotides and device for carrying out the same
DE19834612.3 1998-07-31

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Cited By (1)

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WO2001048181A3 (en) * 1999-12-23 2002-04-18 Dornier Medizintechnik Device for transferring molecules in cells

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DE10223196B4 (en) * 2002-05-24 2004-05-13 Dornier Medtech Systems Gmbh Method and device for transferring molecules into cells
JP7466199B2 (en) * 2018-10-26 2024-04-12 国立大学法人九州大学 Bubble jetting method, bubble jetting device, and bubble jetting apparatus

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Publication number Priority date Publication date Assignee Title
WO2001048181A3 (en) * 1999-12-23 2002-04-18 Dornier Medizintechnik Device for transferring molecules in cells

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DE19834612A1 (en) 2000-02-24

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