EP3490608A1

EP3490608A1 - Transfection method comprising nonviral gene delivery systems

Info

Publication number: EP3490608A1
Application number: EP17751996.4A
Authority: EP
Inventors: Rosa KARL
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-07-26
Filing date: 2017-06-27
Publication date: 2019-06-05

Abstract

The invention relates to a transfection method for introducing nucleic acids in eukaryotic cells by means of a nonviral gene delivery system, wherein the cells are treated at least with at least one inhibitor for IKKe and/or TBK-1 and at least one inhibitor for nucleic-acid-detecting Toll-like receptors before and/or during the transfection. The invention further relates to a composition and to a modular system for such a method.

Description

Transfection procedure with non-viral gene delivery vstemen

Environment of the invention

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.

Background of the invention

The introduction of nucleic acids (DNA, RNA, etc.) into eukaryotic cells, also referred to as transfection, is 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, for example, 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

Research laboratories, in medicine and in the industrial production of proteins application. Last but not least, mention should be made of the fundamental application of transfection in genome editing according to the CRISPR / Cas9 method, which makes it possible to modify the genome of a cell in a very targeted way.

In particular, 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

Angiogenesis in cancerous tumors be shut down. 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.

How successful such a transfection is, among other things defined by the so-called transfection efficiency. A 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. Frequently, 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 processes shows, defined.

In order to be able to introduce nucleic acids into cells, gene delivery systems are generally required which make at least the membrane barrier of the cells passable for the nucleic acid. In the following, therefore, transfection is understood as meaning a treatment of eukaryotic cells by a gene delivery system and nucleic acids.

Usually, the existing genetic delivery systems are divided into two major groups, namely viral systems and non-viral systems. In the particular in gene therapy research preferred viral systems, 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.

A disadvantage of the viral systems, however, is that the viruses the

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

There are essentially no alternatives.

In 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

Method based systems.

The 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.

[0011] 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

Compensation of the negative charge condensed on the phosphate radical, so minimized in size. These complexes can be taken up by the cells by endocytosis. Also, sometimes mixed forms are used in which 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.

However, 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. In this case, 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,

in particular DNA and / or RNA, covalently or via non-covalent

Interactions, especially electrostatic forces and

Binds hydrogen bonds. 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

Detection process trigger endocytosis.

Alternatively, this ligand may be capable of initiating membrane transfer, ie mediating transport to the other side of the membrane. By membrane transfer is meant that a molecule from one side of the Membrane can get to the other side. However, 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. In this method, 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.

Other important physical methods are microinjection,

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. Here, 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, however, as already noted above, that their efficiency does not match that of the viral systems. However, since 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.

In the prior art, for example WO2009 / 065618 or WO2010 / 133369, it has already been found that the innate immune system can be a barrier to successful transfection, since the cells are able to detect nucleic acids via endosomal and cytosolic receptors and then change their physiological behavior, with the purpose of warding off a microbial attack. It has therefore been proposed in the art to target the immune system to increase transfection efficiency.

The problem with this proposal, however, is that the innate immune system has a redundant structure in large areas, which includes an immense number of interacting mechanisms, some of which have not been explored and their relationships are not yet clear. The redundancy is based on the evolutionary exchange of blows

especially between bacteria and viruses on the one hand and the eukaryotes on the other. If a signal strand is interrupted by a pathogen as an attack strategy, the cell is not defenseless due to the redundant structure of the innate immune system. As a consequence, the interruption of a signal train usually only provides modest increases in transfection efficiency.

The object of the present invention is therefore based on an immune system suppressing mechanism on a non-viral

Genetic delivery system based on transfection, which allows an increase in transfection efficiency.

Description of the invention This object is achieved by a method according to claim 1, a composition according to claim 9, and a modular system according to claim 18.

According to the invention, 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 (short TLR, from English, toll-like 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

Immune system initiated and modulated. Through the Toll-like receptors, the innate immune system between "self" and "not self" too

differ. More specifically, the TLRs are around

Transmembrane proteins with an extracellular, "leucine-rich repeat" domain (LRR) and a cytoplasmic domain homologous to those of the IL-1R family The different TLRs selectively respond to and control different molecular viral and bacterial components

Signal transduction cascade a corresponding activation of genes. This is done first via so-called adapter molecules and subsequently via kinases which finally activate transcription factors (eg NF-kappaB and the IRF families) by phosphorylating the same or corresponding intracellular inhibitors of these transcription factors. Ultimately, in addition to a variety of specific genes that have an antimicrobial effect, so-called cytokines produced. Cytokines are again necessary stimulators for the acquired immune system and thus also a link between the innate and acquired immune system.

So far, 13 different TLRs are known (of which 10 in humans), of which in turn only three in humans are currently recognized as nucleic acid-detecting: TLR3 (long dsRNA), TLR7 (ssRNA / dsRNA eg of RNA viruses) and 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.

According to the invention, 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. In this case, the siRNA must

if necessary by the gene running system be introduced. Also, 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. Although both the IKKe / TBK1 kinase and the TLRs are known as influencing factors for the effectiveness of the immune system. However, it has been found, quite unexpectedly, that cells treated with a combination of an inhibitor of the kinase IKKe and / or the kinase TBK-1 and an inhibitor of nucleic acid detecting Toll-like receptors, ie the effect of IKKe / TBK1 kinases and TLRs, show a synergistic increase in transfection efficiency in the application of nonviral gene delivery systems. The increase through the combination is greater than the sum of the increases in the individual components.

According to a further advantageous embodiment, the

Transfektionseffizienz this method especially by the fact that an inhibitor for the Toll-like receptor 9 (TLR9) is used. 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.

According to a further embodiment is used as an inhibitor for

IKKe / TBK-1 an inhibitor with an IC 50 value of less than 500 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,

preferably less than 500 nM, most preferably less than 200 nM. Inhibitors with such an IC 50 value or EC 50 value enable a particularly good inhibition, which significantly increases the transfection efficiency.

According to a further advantageous embodiment, 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. Among the group of 4-aminoquinoline, compounds and their salts will together have the following basic structure, wherein R 1 to R 7 may be any substituents.

Among the group of 9-aminoacridines, compounds and their salts are summarized, which show the following basic structure, wherein R1 to R4 may in turn be any desired substituents.

According to a preferred embodiment is used as an inhibitor for at least one Toll-like receptor quinacrine from the group of 9

Aminoacridine and / or chloroquine from the group of 4-aminoquinolines used.

quinacrine:

chloroquine:

Although chloroquine was previously used to increase the

Transfection used in transfection processes, but showed in the solitary use previously studied a very inconsistent behavior, so that the transfection efficiency could not be increased reliably. Only through the inventive combination with an inhibitor of IKKe / TBK-1 kinase was a reliable and significant increase in the

Transfektionseffizienz be achieved.

According to a further preferred embodiment, the following compounds from the group of 9-aminoacridines and 4-amino-uinolines are used as inhibitors:

These compounds, in combination with an inhibitor of IKKe / TBK-1 kinase show a significant increase in transfection efficiency.

Alternatively, as an inhibitor of a Toll-like receptor also a

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.

Here, further inhibitors for nucleic acid-detecting Toll-like receptors may be included in the scope of the invention, which are recognized later than such. In addition, 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.

In a particularly preferred embodiment of the invention, the inhibitor of IKKe / TBK-1 is one or more of the following inhibitors,

including their salts, used:

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);

Cay10576 (5- (5,6-dimethoxybenzimidazol-1-yl) -3- (2-methanesulfonyl-benzyloxy) -thiophene-2-carbonitrile, CAS 862812-98-4);

Cay10575 (5- (5,6-dimethoxybenzimidazol-1-yl) -3 - ((4-methylsulfonyl) phenyl) methoxy) -2-thiophenecarboxamide, CAS 916985-21 -2);

Amlexanox (2-amino-7- (1-methylethyl) -5-oxo-5H- [1] benzopyrano [2,3-b] pyridines-3-carboxylic acid, CAS 68302-57-8);

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).

In the advantageous use of these inhibitors, an increase in transfection efficiency is clearly seen. As other known inhibitors of IKKe / TBK-1, SU6668 (Sugen Inc.), MPI-0485520 (Myraid Pharma), MCCK1, and the Amgen TBK1 inhibitor (Compound II) (Ou et al., Molecular Cell; 201 1; 41; 458-470). In EP 1720864, WO 2009030890, WO 2010100431, WO 2012059171,

WO002012161877, WO002012161879, WO002013034238, WO002013024282, WO002013075785, WO0020131 17285, WO002014189806, WO002014128486 WO002014093936, WO002015134171, WO002016057338, US020150352108 and US20160015709 further IKKe / TBK-1 inhibitors are listed which can also be used.

In this case, other 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.

[0044] According to a further advantageous embodiment of the

The method according to the invention involves in particular the nucleic acid introduced by the transfection modified and / or

unmodified ssDNA, modified and / or unmodified dsDNA, modified and / or unmodified ssRNA, modified and / or unmodified dsRNA. In this case, dsDNA and ssRNA have proven to be particularly preferred. Various types of nucleic acids can also be used in combination, as is often necessary, for example, in "genome editing" according to the CRISPR / Cas9 method.

A nucleic acid is understood to mean a ribonucleic acid or a deoxyribonucleic acid which consists in particular of two at least partially complementary strands (double-stranded = ds), eg dsDNA and dsRNA, or which consists of one strand (single-stranded = ss), eg ssDNA and ssRNA, which may have partially complementary regions. 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. As 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

Increased ribonucleases and their recognition by nucleic acid-detecting receptors should reduce.

Furthermore, molecules (labels) can be covalently or non-covalently attached to the nucleic acids leading to new properties of the nucleic acids

Nucleic acids lead, in particular to optical traceability

Fluorescence labels or labels that direct the nucleic acids to a specific location in the cell (localization elements) 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 2ΌΗ 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) to achieve transport into the cell nucleus.

As a non-viral gene delivery system, any gene delivery system known to those skilled in the art can be selected. In particular, as another embodiment demonstrates, when preferred as a non-viral gene delivery system is preferred

Genliefersystem is used, which:

a cationic lipid, a cationic polymer, or a cationic protein; and or a compound having a DNA and / or RNA-binding domain and a receptor-mediated endocytosis or a

Can trigger membrane transfer; and or

a compound which is covalently bound to DNA and / or RNA and can induce receptor-mediated endocytosis or membrane transfer.

Alternatively or additionally, 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.

According to a further aspect of the present invention is further a composition of at least at least one inhibitor for IKKe and / or TBK-1 and at least one inhibitor for at least one

provided with nucleic acid detecting Toll-like receptor. Preferably, the composition may further comprise at least one non-viral gene delivery system, and / or one or more modified or unmodified nucleic acids.

In this case, preferably in the composition of one or more of the inhibitors discussed above, gene delivery systems and / or

Nucleic acids include.

According to a further aspect of the present invention there is further provided 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.

Alternatively, according to this aspect of the invention, the

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.

In addition, the modular system a

Genelfersystemzusammensetzung with at least one non-viral

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

Inhibitors as discussed above.

In another embodiment, in the

Modular system one or more of the compositions or

Partial compositions form a combination composition with one or more other compositions or partial compositions.

That is, in the modular system according to the invention, all components may be present separately from each other, or in all combinatorially possible combinations together as a composition. Thus, 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.

The composition according to the invention and / or the

modular system according to the invention can for carrying out the

inventive method can be used. Furthermore, the

Composition according to the invention as a pharmaceutical composition. Furthermore, a composition according to the invention or a modular system for the treatment of a disease by gene therapy, for "Genome Editing" eg by means of CRISPR-Cas9 or also to the repetitive ones

Transfection of cells are used.

Further advantages and advantageous embodiments are defined in the claims, the description and the drawings. Furthermore, the features described may be present individually or in combination. In addition, unless otherwise specified, features described in combination may be presented as individual features or in combinations other than the combination indicated.

Brief description of the drawings

In the following, the invention will be described in more detail with reference to exemplary embodiments shown in the figures. Here are the

Embodiments purely exemplary nature and are not the

Define protection area of login. This is defined solely by the appended claims.

Show it:

1 shows a graphic representation of transfection efficiencies according to a first embodiment; and

2 shows a graphic representation of transfection efficiencies according to a second exemplary embodiment.

Detailed description of the drawings

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.

As can be seen from the figures, 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

Reporter gene system. These are established systems for the detection of

Transfection efficiency. Thus, 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.

In the figures, the different treatments of the cells are plotted on the X-axis and the transfection efficiency in [%] achieved for the respective treatments on the Y-axis. The measurements were based on the

Transfection normalized without inhibitors by equating this 100%. In both examples, the treatment by the IKKe / TBK-1 inhibitor and an inhibitor for a nucleic acid-detecting Toll-like

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.

[0064] To the figures in detail:

As a general material, the following was used:

1 . HeLa cells; ATTC CCL-2

2. Rotifect Plus, Carl Roth; Cat No .: CL21 .2

3. 48 well plates; Cell Star; Greiner BioOne; Cat No .: 677180

4. DMEM; High glucose; Biowest, w / stable glutamine / sodium pyruvate, Cat No: L103-500

5. Serum FBS Gold; PAN Biotech; Lot.Nr. P130914; Cat No .: P40-37500 6. pCMV-luc, Plasmid Factory, Cat No .: PF461

7. Luciferase Assay Kit, Promega

8. dimethyl sulfoxide (DMSO for molecular biology), Fluka; Cat No .: 41639

9. (N - (3 - ((5-iodo-4 - ((3- (2-thienylcarbonyl) amino) propyl) amino) -2-pyrimidinyl) amino) phenyl) -1 -pyrrolidinecarboxamide (BX795),

Merck4Biosciences, Cat No .: 204001

10. chloroquine diphosphate; Fluka; Cat. No .: C6628

1 1. Quinacrine dichloride hydrate; TCI; Cat. No .: Q0056

Example 1 - Fig. 1

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.

Lipofection of HeLa cells was performed during a 3-day period:

1st day: HeLa cells were seeded in a 48 well plate. In this case, a cell count of 1 ^* 10 ⁵ cells per well in 250 μΙ complete DMEM medium was plated (10% FCS). It was then incubated for 24 h in a CO2 incubator (10%).

2 day:

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 μΜ and BX795 in a concentration of 0.5 μΜ. 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 μΙ 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%).

3rd day:

The efficiency of transfection was determined with a luciferase assay kit. The assay was performed according to the manufacturer's instructions.

Results in RLU (mean of three tests)

ST = standard transfection without BX795 and chloroquine

BX795: C = 0.5 μΜ

Chloroquine: C = 10 μΜ

The increase by the individual application of chloroquine or BX795 compared to the standard transfection is 19% or 256%. The increase by combined use, at 474%, is much more than the sum of the increases in single use, i. The combined application shows a synergistic effect.

Example 2 - Fig. 2

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.

Results in RLU / g protein (means) 100% 141% 385% 732%

ST = standard transfection without BX795 and quinacrine

BX795: C = 0.5 μΜ

Quinacrine: C = 2.5 μΜ

The increase by the single application of quinacrine or BX795 compared to the standard transfection is 41% and 285%, respectively. The increase by combined use, at 632%, is much more than the sum of the increases in single use, i. The combined application shows a synergistic effect.

In general, therefore, it can be summarized that advantageously by the treatment according to the invention of the cells before and / or during the

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.

Claims

Claims:

1 . Transfection method for introducing one or more

Nucleic acids in eukaryotic cells by means of a non-viral

gene delivery,

characterized in that

the cells are treated before and / or during transfection 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, TLR.

2. Transfection method according to claim 1, wherein at least one inhibitor for TLR 9, the Toll-like receptor 9, is used as an inhibitor for the nucleic acid-detecting Toll-like receptor.

3. transfection method according to claim 1 or 2, wherein as inhibitor of IKKe / TBK-1, an inhibitor with an IC 50 value of less than 500 nM,

preferably less than 200 nM, most preferably less than 100 nM; and or

as an inhibitor of the nucleic acid detecting Toll-like receptor, an inhibitor having an EC 50 value of less than 1000 nM, preferably less than 500 nM, most preferably less than 200 nM, is used.

4. transfection method according to any one of the preceding claims, wherein as an inhibitor for the nucleic acid-detecting Toll-like receptor at least one compound from the group of 4-aminoquinolines,

including their salts, the compound having the following basic structure

has, wherein R1 to R7 is arbitrary selectable.

5. transfection method according to claim 4, wherein at least chloroquine, including its salts, is used as an inhibitor for the nucleic acid-detecting Toll-like receptor with R5 = Cl and R7 =

NHCHMe CH2) 3NEt2 and, accordingly, the following structure

and / or as an inhibitor for the nukleinsauredetektierenden Toll-like receptor is at least including their salts, used with R5 and R7 = Cl NHCH2CH a compound (C6H ₄ OMe) (CH2) 2NEt2, and accordingly the following structure:

and / or as an inhibitor for the nucleic acid-detecting Toll-like receptor at least one compound, including its salts, is used with R5 = NHCHMe (CH2) 3NEt2, R1 = CeHs and accordingly following

6. Transfection method according to one of the preceding claims, wherein at least one compound from the group of 9-aminoacridines, including their salts, is used as inhibitor for the nucleic acid-detecting Toll-like receptor, wherein the compound has the following basic structure

has, wherein R1 to R4 are arbitrary selectable.

7. transfection method according to claim 6, wherein at least quinacrine, including its salts, is used as an inhibitor for the nucleic acid-detecting Toll-like receptor with R1 = OMe, R2 = H, R3 = Cl, R4 =

NHCHMe CH2) 3NEt2 and, accordingly, the following structure

8. transfection method according to any one of the preceding claims, wherein as inhibitor of IKKe and / or TBK-1 one or more from the group 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) -pyhmidin-4-yl) -amino) -propyl) -2,2-dimethyl-propane-diamine, CAS 702676-93 5);

Cay10576 (5- (5,6-dimethoxybenzimidazol-1-yl) -3- (2-methanesulfonyl-benzyloxy) -thiophene-2-carbonithle, CAS 862812-98-4);

MRT-67307 (N- [3 - [[5-Cyclopropyl-2 - [[3- (4-morpholinylmethyl) phenyl] amino] -4-pyrazidinyl] amino] propyl] cyclobutanecarboxamide, CAS 1 190378-57-4) ;

CYT387 (N- (cyanomethyl) -4- [2 - [[4- (4-morpholinyl) phenyl] amino] -4-py- imidinyl] -benzamide, CAS 1056634-68-4);

including their salts.

9. Composition for a transfection according to one of the preceding claims, characterized in that the

Composition comprises at least 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.

10. The composition of claim 9, wherein the composition further comprises:

a. a non-viral gene delivery system, and / or

b. one or more modified or unmodified nucleic acids.

1 1. A composition according to claim 9 or 10, wherein the at least one inhibitor for the nucleic acid-detecting Toll-like receptor is an inhibitor of TLR 9, the Toll-like receptor 9.

12. The composition of any one of claims 9 to 11, wherein the inhibitor for IKKe / TBK-1 has an IC 50 value of less than 500 nM,

preferably less than 200 nM, most preferably less than 100 nM; and or

the inhibitor for the nucleic acid detecting Toll-like receptor has an EC 50 value of less than 1000 nM, preferably less than 500 nM, on

most preferably less than 200 nM.

13. The composition according to any one of claims 9 to 12, wherein the inhibitor for the nucleic acid-detecting Toll-like receptor comprises at least one compound from the group of 4-aminoquinolines, including their salts, the following basic structure

has, with R1 to R7 arbitrary selectable.

14. The composition according to claim 13, wherein the inhibitor for the nucleic acid-detecting Toll-like receptor at least chloroquine,

including its salts, comprising with R5 = Cl and R7 = NHCHMe (CH2) 3NEt2 and having the following structure

and / or the inhibitor for the nucleic acid-detecting Toll-like receptor comprises at least one compound, including its salts, with R 5 = Cl and R 7 = NHCH 2 CH (C 6 H ₄ OMe) (CH 2) 2 NE 2 and correspondingly the following structure:

and / or the inhibitor for the nucleic acid-detecting Toll-like receptor comprises at least one compound, including its salts, with R5 = OMe, R7 = NHCHMe CH2) 3NEt2, R1 = CeHs and correspondingly the following structure:

15. The composition according to any one of claims 9 to 14, wherein the inhibitor for the nucleic acid-detecting Toll-like receptor comprises at least one compound from the group of 9-aminoacridines, including its salts, the following basic structure

has, wherein R1 to R4 are arbitrary selectable.

The composition of claim 15, wherein said inhibitor of nucleic acid detecting Toll-like receptor is at least quinacrine, inclusive its salts comprising, where R 1 = OMe, R ₂ = H, R 3 = Cl, R 4 = NHCHMe (CH ₂ ) 3NEt ₂ and, accordingly, folende structure

17. The composition according to any one of claims 9 to 16, wherein the inhibitor for IKKe and / or TBK-1 one or more from the group

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);

including their salts.

18. Modular system for a transfection according to one of

Claims 1 to 8, characterized in that the modular system

at least one first inhibitor sub-composition comprising at least one inhibitor for IKKe and / or TBK-1, and a second An inhibitor sub-composition comprising at least one inhibitor for at least one nuclear acid-detecting Toll-like receptor

or that the modular system

an inhibitor composition which comprises 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.

19. Modular system according to claim 18, wherein the

Inhibitor composition or at least one of

Inhibitor component compositions is a composition according to any one of claims 1 to 17.

20. Modular system according to one of claims 18 to 19, wherein the modular system further comprises:

a. a gene delivery system composition having at least one nonviral gene was run, and / or

b. a nucleic acid composition having at least one modified or unmodified nucleic acid.

21. A modular system according to any one of claims 18 to 20, wherein one or more of the compositions or sub-compositions comprises a

Combination composition with one or more others

Form compositions or partial compositions.