EP3490608A1 - Transfektionsverfahren mit nicht-viralen genliefersystemen - Google Patents
Transfektionsverfahren mit nicht-viralen genliefersystemenInfo
- Publication number
- EP3490608A1 EP3490608A1 EP17751996.4A EP17751996A EP3490608A1 EP 3490608 A1 EP3490608 A1 EP 3490608A1 EP 17751996 A EP17751996 A EP 17751996A EP 3490608 A1 EP3490608 A1 EP 3490608A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- inhibitor
- receptor
- nucleic acid
- composition
- amino
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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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
Definitions
- the present invention relates to a method for improving the transfection efficiency of non-viral gene delivery systems, as well as a
- Composition for it and an associated modular system Composition for it and an associated modular system.
- nucleic acids DNA, RNA, etc.
- transfection a key technology in modern biotechnology because it provides access to the central
- Control apparatus of a cell and thus, for example, the production or switching off certain proteins allowed.
- Transfection with DNA or mRNA allows the expression (production) of any protein that allows transfection with siRNA, ribozymes or antisense molecules, for example by RNA interference, the "knockdown" of a gene or a protein MicroRNA can be introduced into cells and influence regulatory functions, a key technology found in both
- this key technology makes it possible to replace genes destroyed in human cells by mutation and thus to heal malfunctions. It is also possible, for example, to force cancer cells to commit suicide by means of suicidal genes. "Knock-down" of a gene is another way of acting in a healing manner, for example by producing important genes for
- Knock-down is understood as a weakening or elimination of the translation of an mRNA into a protein during protein biosynthesis. Many diseases are triggered by a mismanagement of cells that could be abrogated by microRNA.
- transfection efficiency is understood to be the amount of protein expression of a cell population as a consequence of transfection processes with genetic material which, inter alia, encodes this expressed protein or also the extent of a knockdown of a protein expression of a cell population as a consequence of
- Transfection processes with genetic material that can trigger such a knockdown In particular siRNA, antisense RNA, ribozymes, antisense DNA or DNA coding for siRNA or ribozymes is used as the genetic material.
- the transfection efficiency is also determined by the proportion of cells of a total population of cells that determines the biological effectiveness of the introduced genetic material as a result of
- transfection is understood as meaning a treatment of eukaryotic cells by a gene delivery system and nucleic acids.
- viruses are used as gene delivery systems. Since the introduction of nucleic acids, particularly DNA or RNA, into foreign cells is an integral part of the viral propagation cycle, this capability has been refined by a natural, evolutionary process in the history of the virus to make it a highly effective gene delivery system. The viruses used are genetically manipulated so that they no longer possess pathogenic properties and can no longer reproduce.
- Immune system offer a large attack surface, since the immune system has developed strategies in an evolutionary adaptation process, to defend the viruses.
- the immune defense and the activation of oncogenes by random integration of genetic material into the genome are unsolved problems, so that there are only a few approved gene therapies worldwide despite decades of research.
- Viruses are often used in basic research, but due to the security risk and the complex handling, the viral systems are only used where it is needed
- non-viral gene delivery systems generally no naturally occurring viruses are used and they are not produced by recombination of genetic material from naturally occurring viruses.
- These non-viral systems can in turn be subdivided into two sub-groups, the chemical-based systems and the physical ones
- non-viral gene delivery systems based on chemical methods are based either on a chemical modification or derivatization of the nucleic acids themselves, which make them cell-like, or comprise substances which, for example, via electrostatic forces or
- Hydrogen bonds, bind nucleic acids and transport can mediate through the cell membrane.
- the transport of the nucleic acid through the cell membrane is usually carried out by an active transport mechanism of the cell, the so-called endocytosis.
- Substances which allow binding of the nucleic acids include, for example, cationic lipids, cationic polymers, cationic peptides. These cationic lipids and cationic polymers spontaneously form so-called lipoplexes or polyplexes in the presence of DNA or RNA due to the opposing charge ratios. The DNA is thereby through the
- nucleic acid is "precondensed" by cationic polymers and then complexed by cationic lipids to form a mixture of lipoplexes and polyplexes, for example, polylysine, polyarginine or polyethylenimine, of course cationic lipids, polymers and peptides are used.
- the substances suitable for a chemically-based non-viral gene delivery method may also be molecules having at least a first and a second domain / moiety.
- the first domain is formed as a nucleic acid binding domain / moiety.
- a nucleic acid-binding moiety is understood as meaning an area in a molecule containing a nucleic acid,
- DNA and / or RNA covalently or via non-covalent
- the second domain / moiety preferably contains a ligand.
- This ligand can be recognized, for example, by a receptor on the cell surface and by this
- this ligand may be capable of initiating membrane transfer, ie mediating transport to the other side of the membrane.
- membrane transfer is meant that a molecule from one side of the Membrane can get to the other side.
- the ligands which can induce receptor-mediated endocytosis or membrane transfer can also be covalently bound to the genetic material, if the biological effect is not or only little affected.
- the substances may also be specially formulated, in particular as micelles or liposomes, or also comprise a plurality of components having different functions.
- Non-viral gene delivery systems based on physical methods localize the genetic material in the vicinity of the cell and utilize energy, in particular in the form of thermal, kinetic, electrical or other energy, to mediate transport of the genetic material through the cell membrane.
- An important example of a non-viral method based on a physical method is called electroporation.
- the cells to be transfected are placed between two electrodes to which a suitable voltage waveform is applied. In this way, the cells are exposed to an electrical pulse, which leads to a reversible opening (pores) of the cell membrane. These pores allow nucleic acids to enter the cell.
- hydrodynamic methods ballistic methods (Genegun) or methods that use ultrasound. This includes methods in which the
- Nucleic acid is injected nude into various organs or muscles, which in special cases can lead to low expression of the corresponding genes.
- Combination methods such as magnetofection combine chemical and physical methods.
- nucleic acids are chemically immobilized on magnetic nanoparticles to enrich them by a magnetic field gradient on the surface of cells and trigger endocytosis.
- a disadvantage of all non-viral systems that their efficiency does not match that of the viral systems.
- the viral method is very limited to gene therapy can be used due to the many disadvantages, is looking for non-viral methods for correspondingly powerful alternatives.
- the object of the present invention is therefore based on an immune system suppressing mechanism on a non-viral
- a transfection method for introducing one or more nucleic acids into eukaryotic cells by means of a non-viral gene delivery system is proposed in which the non-viral gene delivery system is improved in its performance in that the cells before and / or during transfection at least with at least one inhibitor for IKKe and / or TBK-1 and at least one inhibitor for at least one
- Nucleic acid-detecting toll-like receptor can be treated.
- a Toll-like (similar) receptor refers to proteins of the innate immune system. They belong to a group of receptors that serve to recognize pathogenic structures and to control the corresponding activation of genes. As a result, in particular, the activation of the antigen-specific acquired
- the TLRs are around
- TLR Transmembrane proteins with an extracellular, "leucine-rich repeat” domain (LRR) and a cytoplasmic domain homologous to those of the IL-1R family
- LRR leucine-rich repeat domain
- cytoplasmic domain homologous to those of the IL-1R family
- cytokines are again necessary stimulators for the acquired immune system and thus also a link between the innate and acquired immune system.
- TLR3 long dsRNA
- TLR7 ssRNA / dsRNA eg of RNA viruses
- TLR9 bacterial / viral DNA
- IKKe and TBK-1 are kinases that play an essential role in a innate immune signal transduction cascade downstream of a variety of cytosolic receptors that terminate in the activation of transcription factors IRF3 and IRF7.
- the kinase IKKe is also referred to as Ikkepsilon, Ikapa kepsilon, Ikappa kinase epsilon or IkK-3.
- TBK-1 is also referred to as TANK binding kinase 1. Since IKKe and TBK-1 are two closely related kinases, inhibitors of IKKe usually also act against TBK-1 and vice versa.
- an inhibitor is understood as meaning a molecule which can reduce or inhibit the biological action of another molecule, in particular of a protein.
- the inhibitors themselves are proteins, modified or unmodified nucleic acids or small organic molecules, whereby also suitable siRNA, which suppress the expression of a protein, can be regarded as inhibitors.
- the siRNA must be regarded as inhibitors. In this case, the siRNA must be regarded as inhibitors.
- the inhibitory effect can be achieved by masking a molecule that is normally recognized by a protein and thereby induces a biological effect.
- the effectiveness of an inhibitor is indicated by means of the so-called IC 50 value or EC 50 value.
- the IC 50 value indicates the concentration of an inhibitor necessary to block a target (eg enzyme, cell, cell receptor, microorganism etc.) in vitro by 50%.
- the ECso value, the effective concentration indicates this required concentration in vivo.
- TLR9 is a receptor for bacterial DNA, or non-methylated CpG motifs that accumulate in bacterial DNA (20 times more abundant than in mammalian cells).
- the CpG motif is highly methylated in mammalian cells, allowing it to be distinguished. Similar to bacterial DNA also applies to viral DNA, which is also detected by TLR9.
- IKKe / TBK-1 an inhibitor with an IC 50 value of less than 500 nM
- nM preferably less than 200 nM, most preferably less than 100 nM, and / or an inhibitor having an ECso value of less than 1000 nM as an inhibitor of the nucleic acid-detecting Toll-like receptor,
- Inhibitors with such an IC 50 value or EC 50 value enable a particularly good inhibition, which significantly increases the transfection efficiency.
- a compound from the group of 9-aminoacridines and / or 4-aminoquinolines, including salts thereof is used as an inhibitor for the nucleic acid-detecting Toll-like receptor.
- 4-aminoquinoline compounds and their salts will together have the following basic structure, wherein R 1 to R 7 may be any substituents.
- Oligonucleotide whose sequence is suitable, toll-like It is also possible to use antibodies which are directed against Toll-like receptors or to inhibit receptors. Of course, an inhibition can also be achieved with a combination of the mentioned exemplary embodiments.
- nucleic acid-detecting Toll-like receptors may be included in the scope of the invention, which are recognized later than such.
- other inhibitors may be used in addition to the combination of an inhibitor of IKKe / TBK-1 and an inhibitor of a toll-like receptor.
- the inhibitor of IKKe / TBK-1 is one or more of the following inhibitors,
- BX795 N - (3 - ((5-iodo-4 - ((3- (2-thienylcarbonyl) amino) propyl) amino) -2-pyrimidinyl) amino) phenyl) -1-pyrrolidine carboxamide, CAS 702675-74-9 );
- BX320 N - (3 - ((5-bromo-2- (3- (pyrrolidine-1-carbonylamino) anilino) pyrimidin-4-yl) amino) propyl) -2,2-dimethyl propane diamine, CAS 702676-93 5);
- MRT-67307 N- [3 - [[5-cyclopropyl-2 - [[3- (4-morpholinylmethyl) phenyl] amino] -4-pyrimidinyl] amino] propyl] -cyclobutanecarboxamide, CAS 1 190378-57-4) ; CYT387 (N- (cyanomethyl) -4- [2 - [[4- (4-morpholinyl) phenyl] amino] -4-pyrimidinyl] benzamide, CAS 1056634-68-4).
- inhibitors can be used, which are not listed above, if they have an inhibitory effect on IKKe and / or TBK-1. Also included are inhibitors whose effect on IKKe and / or TBK-1 will be recognized at a later date.
- the method according to the invention involves in particular the nucleic acid introduced by the transfection modified and / or
- dsDNA and ssRNA have proven to be particularly preferred.
- nucleic acids can also be used in combination, as is often necessary, for example, in "genome editing" according to the CRISPR / Cas9 method.
- the genetic material is used in the case of DNA for the production of RNA and / or proteins. In the case of ssRNA, it serves to produce proteins. In the case of dsRNA, the genetic material serves to knock down a gene through RNA To achieve interference or to act as a microRNA.
- antisense DNA or antisense RNA the nucleic acid serves to inhibit the translation of mRNA.
- Modified nucleic acids are natural nucleic acids which have been modified by modification in their properties. These modifications may be, in particular, chemical changes which relate to the phosphate skeleton, and / or the sugars and / or the bases, in particular the stability of the nucleic acids to nucleases and
- molecules can be covalently or non-covalently attached to the nucleic acids leading to new properties of the nucleic acids
- Fluorescence labels or labels that direct the nucleic acids to a specific location in the cell or labels that mediate the passage of nucleic acids through membranes and thus make nucleic acids cell-like, for example. Examples of modifications are the exchange of
- Oxygen to sulfur in the phosphate scaffold in the case of RNA the methylation of 2'-OH groups of the ribose, or the methylation of the bases.
- Another example is the complete substitution of 2 ⁇ groups of RNA by fluorine to increase stability against nucleases.
- Yet another example is the attachment of FITC (fluorescein isothiocyanate) as a fluorescent label to follow microscopically the path of the genetic material in the cell or the attachment of so-called NLS (nuclear localization signal, eg.
- PKKKRKVG PKKKRKVG
- any gene delivery system known to those skilled in the art can be selected.
- any gene delivery system known to those skilled in the art can be selected.
- another embodiment demonstrates, when preferred as a non-viral gene delivery system is preferred
- Gencastersystem is used, which:
- a cationic lipid a cationic polymer, or a cationic protein
- the non-viral gene running system can also be based on a physical method such as electroporation, microinjection, magnetofection, ultrasound or a ballistic or hydrodynamic method.
- composition may further comprise at least one non-viral gene delivery system, and / or one or more modified or unmodified nucleic acids.
- Nucleic acids include.
- a modular system for carrying out the above-discussed transfection method comprising at least a first inhibitor sub-composition comprising at least one of the inhibitors of IKKe and / or TBK-1 discussed above and a second inhibitor sub-composition comprising at least one of the inhibitors discussed above for at least one nucleic acid detecting Toll-like receptor.
- Modular system also provide an inhibitor composition, the at least one inhibitor for IKKe and / or TBK-1 and at least one inhibitor for at least one nucleic acid-detecting Toll-like receptor.
- Genetic delivery system and / or provide a nucleic acid composition having at least one modified or unmodified nucleic acid.
- the inhibitor composition or at least one of
- Inhibitor component compositions is a composition
- Modular system one or more of the compositions or
- Partial compositions form a combination composition with one or more other compositions or partial compositions.
- all components may be present separately from each other, or in all combinatorially possible combinations together as a composition.
- the components can either be separated from each other e.g. in glass or plastic containers, which are packaged together, or the components can be provided in pairs or more as a composition in respective containers.
- composition according to the invention and / or the
- modular system according to the invention can for carrying out the
- composition according to the invention as a pharmaceutical composition.
- FIG. 2 shows a graphic representation of transfection efficiencies according to a second exemplary embodiment.
- Figures 1 and 2 show schematically comparative representations of transfection efficiencies achieved with the method of the invention and without the method of the invention.
- transfection efficiency a significant increase in the transfection efficiency can be achieved by the treatment according to the invention of the cells before and / or during the transfection.
- the transfection efficiency was measured indirectly via a luciferase enzyme encoded by the introduced nucleic acid. It is a so-called
- Transfection efficiency the higher the amount of luciferase that can be detected in a culture vessel after lysis of the transfected cells, the greater the transfection efficiency.
- the detection of the amount of luciferase takes place via an enzyme-substrate reaction in which a light quantum is released. These quanta of light can be obtained from suitable measuring devices, so-called
- Luminometers are measured. Since the number of light quanta measured depends in particular on the time interval in which the measurement took place, this quantity of light quanta is also referred to as "relative light units.” For comparative studies, the measurement conditions must therefore be the same.
- Receptor can be recorded a significant increase, which can not be explained by mere addition of the individual rates of increase for IKKe / TBK-1 inhibitor and an inhibitor for a nucleic acid-detecting Toll-like receptor alone. It is clearly a synergistic effect.
- the first embodiment shown in Figure 1 relates to lipofection of HeLa cells with a commercially available transfection reagent (Rotifect Plus) treated before and during transfection with inhibitor BX795 for IKKe and TBK-1 and with chloroquine as inhibitor for TLR9.
- Rotifect Plus commercially available transfection reagent
- HeLa cells were seeded in a 48 well plate. In this case, a cell count of 1 * 10 5 cells per well in 250 ⁇ complete DMEM medium was plated (10% FCS). It was then incubated for 24 h in a CO2 incubator (10%).
- Stock solutions were prepared from chloroquine and BX795 in a mixture of DMSO and water. Two hours before transfection, 4 wells each were filled with chloroquine or BX795 and 4 wells with both substances. With an appropriate amount of the two stock solutions, the concentration of the inhibitors in the culture medium of the cells was adjusted so that chloroquine was present in a concentration of 10 ⁇ and BX795 in a concentration of 0.5 ⁇ . Further, care was taken that the DMSO concentration did not exceed 1% v / v.
- the transfection of all cells in the 48 well plate was 0.3 g pCMV-Luc and 1, 2 ⁇ Rotifect Plus were performed according to the manufacturer's instructions for the transfection reagent. It was then incubated for 24 h in a CO2 incubator (10%).
- the efficiency of transfection was determined with a luciferase assay kit.
- the assay was performed according to the manufacturer's instructions.
- the second embodiment shown in Figure 2 relates to lipofection of HeLa cells treated before and during transfection with inhibitor BX795 for IKKe and TBK-1 and with quinacrine as inhibitor of TLR9.
- the lipofection of the HeLa cells was carried out analogously to Example 1.
- Transfection at least with at least one inhibitor for IKKe and / or TBK-1 and at least one inhibitor for at least one nucleic acid-detecting Toll-like receptor a significant increase in the transfection efficiency is recorded, which is based on a synergistic effect.
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Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102061013714 | 2016-07-26 | ||
PCT/DE2017/200056 WO2018019341A1 (de) | 2016-07-26 | 2017-06-27 | Transfektionsverfahren mit nicht-viralen genliefersystemen |
Publications (1)
Publication Number | Publication Date |
---|---|
EP3490608A1 true EP3490608A1 (de) | 2019-06-05 |
Family
ID=66284937
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP17751996.4A Withdrawn EP3490608A1 (de) | 2016-07-26 | 2017-06-27 | Transfektionsverfahren mit nicht-viralen genliefersystemen |
Country Status (1)
Country | Link |
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EP (1) | EP3490608A1 (de) |
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2017
- 2017-06-27 EP EP17751996.4A patent/EP3490608A1/de not_active Withdrawn
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