DE102007056488A1 - Composition, useful e.g. transfection comprises a non-viral gene delivery system containing e.g. cationic lipid, and an agent for partial suppression and/or activation of innate intracellular and/or intercellular immune defense - Google Patents

Abstract

Composition for transfection, comprises: (a) a non-viral gene delivery system containing cationic lipid, cationic polymer or a cationic protein, a compound, which exhibits a DNA and/or RNA-binding domain, and/or a compound; and (b) an agent for partial suppression and/or activation of innate intracellular and/or intercellular immune defense comprising e.g. an antibody against toll-like receptor (TLR) 1 to TLR 13 and cytokine receptor or cytokine receptor-antagonist and an inhibitor of the kinase of mitogen-activated protein kinase 1 (MEK1) and/or MEK2. Composition for transfection, comprises: (a) a non-viral gene delivery system containing a cationic lipid, a cationic polymer or a cationic protein, a compound, which exhibits a DNA and/or RNA-binding domain and activates receptor-mediated endocytosis or a membrane transfer, and/or a compound, which is covalently bonded to DNA and/or RNA and activates receptor-mediated endocytosis or a membrane transfer; and (b) an agent for at least partial suppression and/or activation of the innate intracellular and/or intercellular immune defense comprising an antibody against toll-like receptor (TLR) 1 to TLR 13, an antibody against a cytokine receptor or a cytokine receptor-antagonist, an inhibitor of the kinase of mitogen-activated protein kinase/extracellular signal-regulated kinase 1 (MEK1) and/or MEK2, an agonist for TLR7 and/or TLR8 comprising bropirimine, imidazoquinoline, thiazoloquinoline and/or guanosine analogs. Independent claims are included for: (1) a kit of parts for transfection, comprising the composition; and (2) a procedure for improving the transfection output of non-viral gene delivery system comprising treating the cells with (b), before and/or during the transfection and inserting the transfection genetic material into the cells or treating the cells with (b), before, during and/or after and inserting modified or unmodified small interfering RNA (siRNA) into the cells. ACTIVITY : CNS-Gen.; Respiratory-Gen.; Muscular-Gen.; Metabolic; Antilipemic; Hemostatic; Antianemic; Cytostatic; Virucide; Ophthalmological; Neuroprotective; Antiinflammatory. MECHANISM OF ACTION : Gene therapy.

Description

State of the art

In immune responses of the body ( Luke A. et al .; Spectrum of Science, August 2005, pages 68-75 ) against an infectious or immunological challenge, a distinction is made between the innate (innate immunity) and the acquired immune response (antigen-specific acquired immunity). The acquired immune defense is trained only when an infection with pathogens. It has a kind of memory, so that in a second infection by the same pathogen usually no longer comes to the onset of the disease. Vaccines are based on this principle.

would it would only be this type of immune defense that the organism would have before the first infection completely unprotected. This is however, not the case, as there is another very original one Immune defense exists, which is called innate immune defense and from the fly Drosophila to mammals, yes even is found in plants. This immune defense is the first defense front against pathogens and represents an evolutionary very old system

In innate immune defense, so-called toll-like receptors (TLRs) ( Heine H. et al .; Int. Arch. Allergy Immunol. 2003; 130; 180-192 and Uematsu S. et al., J. Biol. Chem. 2007, May 25; 282 (21); 15319-23) and RIG-I-like helicases (RLH), the disease-associated molecular patterns, so-called PAMPs (phatogenically associated molecular patterns), recognized and initiated appropriate inflammatory and immune responses. This allows the organism to distinguish between "self" and "not self". RLHs are ubiquitously expressed in the cytosol, where they are able to recognize the dsRNA resulting from viral infection. TLRs and RLHs belong to a group of receptors, also known as RPPs (pattern recognition receptors).

RIG-I-like helicases (RLH)

RIG-I (retinoic inducible gene I) is an intracellular receptor of the innate immune system ( Perry AK et al; Cell Research 2005; 15 (6); 407-422 and Kawai T. et al .; J. Biochem; 2007; 141; 137-145) , The receptor belongs to the helicases and is able to detect single- and double-stranded RNA with a triphosphate residue at the 5'-position. He is thus able to distinguish between cellular and viral ribonucleic acids. If foreign RNA is detected, an immune response is triggered via a signal transduction cascade and finally produced type I interferon. How this signal transduction cascade works is not fully understood. However, the involvement of the transcription factors NF-kB, IRF3, IRF7 and the kinases TBK1 and IKK-i is suspected. A group with similar properties is summarized under the term RIG-I-like helicases.

Toll-like receptors (TLR)

Toll-like receptors were first discovered in the mid-1990s ( Zimmer A. et al .; PNAS, 1999; 96 (10), 5780-5785 ). The name is derived from a protein found in Drosophila melanogaster by Christiane Nusslein-Volhard, which you called "Toll." TLR proteins are similar to this type and are referred to as "toll-like" proteins. These are transmembrane proteins with an extracellular, "leucine-rich repeat" domain (LRR) as well as a cytoplasmic domain homologous to that of the IL-1R family.The various TLRs selectively respond to and direct various molecular viral and bacterial components This is done first via so-called adapter molecules and subsequently via kinases which finally transcription factors by phosphorylation of the same or corresponding intracellular inhibitors of these transcription factors (eg NF-kB and the IRF families). Finally, in addition to a large number of specific genes that have an antimicrobial effect, so-called cytokines are produced, which in turn are necessary stimulators of the acquired immune defense and thus also a link between innate and acquired immune defenses However, ligand recognition, signal transduction and signal forwarding are only partially understood.

• TLR1: bildet mit TLR2 ein Heterodimer, ist der Rezeptor von triacyliertem Lipoprotein und Zymosan aus Hefen.
• TLR2: ist der Rezeptor für bestimmte Peptidoglykane, Lipopeptide, Glykolipide und verschiedener Bakterien.
• TLR3: erkennt lange dsRNA, wie Sie bei einer Virusreplikation in infizierten Zellen vorkommt.
• TLR4: ist der Rezeptor für Lipopolysaccharide (LPS, auch Endotoxine), verschiedene Hüllglykoproteine (auch von Viren) und Taxol. LPS sind Bestandteile von Bakterienzellwänden.
• TLR5: ist der Rezeptor von Flagellin, einem Hauptbestandteil der Geiseln (Flagellae), mit welchen sich Bakterien fortbewegen.
• TLR6: bildet mit TLR4 ein Heterodimer, ist der Rezeptor von diacyliertem Lipoprotein und bestimmten Peptidoglykanen.
• TLR7&TLR8: beide codierenden Gene liegen auf dem X-Chromosom und weisen ein hohe Homologie auf, es sind Rezeptoren für kurze ssRNA/dsRNA z. B. von RNA-Viren. Es werden letztendlich auch dendritische Zellen und T-Zellen aktiviert.
• TLR9: Ist der Rezeptor für bakterielle DNA, bzw. für nicht methylierte CpG Motive, die in bakterieller DNA gehäuft (20 x häufiger als in Säugerzellen) auftritt. Das CpG Motiv ist in Säugerzellen stark methyliert, wodurch es unterschieden werden kann. Ähnliches wie für bakterielle DNA gilt auch für virale DNA. Es konnte gezeigt werden, dass TLR9 IL-12 aktiviert, das wiederum T-Helferzellen vom Typ I stimuliert. Weiter wird die Induktion von Interferonen vermittelt.

So far, 13 different TLRs are known (10 of them in humans), whose number is sufficient for the detection of all pathogens, ranging from bacteria to fungi to the virus. The receptors recognize all the pathogens common structures, and sometimes also several components at the same time, without these structurally similar. For example, the TLR4 recognizes lipopolysaccharides, but also taxol. So far it is not known how the TLRs can do that. The TLRs differ only a little from species to species.

• TLR1: forms a heterodimer with TLR2, is the receptor of triacylated lipoprotein and yeast zymosan.
• TLR2: is the receptor for certain peptidoglycans, lipopeptides, glycolipids and various bacteria.
• TLR3: Detects long dsRNA, as occurs in virus replication in infected cells.
• TLR4: is the receptor for lipopolysaccharides (LPS, also endotoxins), various envelope glycoproteins (also of viruses) and taxol. LPS are components of bacterial cell walls.
• TLR5: is the receptor of flagellin, a major constituent of hostages (flagellae), with which bacteria move.
• TLR6: forms a heterodimer with TLR4, is the receptor of diacylated lipoprotein and certain peptidoglycans.
• TLR7 & TLR8: both coding genes are located on the X chromosome and have a high homology, they are receptors for short ssRNA / dsRNA z. B. of RNA viruses. Finally, dendritic cells and T cells are also activated.
• TLR9: Is the receptor for bacterial DNA, or for non-methylated CpG motifs that accumulates 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. It has been shown that TLR9 activates IL-12, which in turn stimulates Type I helper T cells. Furthermore, the induction of interferons is mediated.

• TLR10: Ligand noch nicht bekannt
• TLR11: Ist Rezeptor für das urpathogene Bakterium Escherichia coli und dem Profilinähnlichen Protein des Urtierchens Toxoplasma gondii
• TLR12: Funktion und Ligand noch unbekannt
• TLR13: Funktion und Ligand noch unbekannt

About the immunostimulatory property of bacterial DNA reported in the early 80s, the group around Dr. med. Tokunaga. As an associated receptor was from the group around Dr. med. Shizuo Akira identified the TLR9 receptor (elucidating the roles of Toll receptors and their signal transduction cascades by gene targeting, Robert Koch lecture by Dr. Shizuo Akira, General Press Release 2002 ; www.robertkoch.stiftung.de ).

• TLR10: ligand not yet known
• TLR11: is the receptor for the pathogenic bacterium Escherichia coli and the profilin-like protein of the predator Toxoplasma gondii
• TLR12: Function and ligand still unknown
• TLR13: Function and ligand still unknown

Localization of the TLR:

TLR2, 4, 5 and 6 are particularly located in the plasma membranes of monocytes, natural killer cells, mast cells or myotonic dendritic cells., 7, 8, and 9 are particularly in endosomes of immune cells ( Siegmund-Schultze N., www.aerzteblatt.de ). Activation of the immune response therefore requires intracellular uptake via endocytosis and maturation of the endosomes. Signal transmission begins here in an endosomal compartment. For TLR3 there is evidence that it is in the plasma membranes, but there are also representations in the literature that assume endosomal localization. TLRs often act in pairs and occur in different combinations of different cell types.

signal transduction:

The signal transduction pathways of the different TLRs ( Perry AK et al; Cell Research 2005; 15 (6); 407-422 and Kawai T. et al .; J. Biochem; 2007; 141; 137-145 ) are similar in some cases, but also have larger differences, so that it comes in the end to a different gene expression and thus different biological reactions. With the exception of TLR3, all TLRs pass their signal to the adapter protein MyD88. MyD88 plays a crucial role in signal transmission the TLR / interleukin-1 receptor. The cytosolic domain of TLRs is highly similar to that of the interleukin-1 receptor and is therefore also referred to as the Toll / IR-1 receptor domain (TIR). MyD88-deficient splenocytes showed z. B. no reactions to II-1, LPS or CpG DNA. In addition, no activation of signaling molecules such as NF-kB or MAP kinases was observed in MyD88 deficient cells in response to TLR2, TLR7, TLR9 ligands. This is a clear indication of the complete dependency of the TLRs (except TLR3 and TLR4) of MyD88 for their signal forwarding. Other adapter molecules include TIRAP (Toll Interleukin-1 Receptor (TIR) domain-containing adapter protein (TLR2 and TLR4), Mal (MyD88 adapter-like), TRIF (TLR3 and TLR4), and TRAM (TLR4).

Which Proteins next to the adapter molecules still participate hangs from the respective TLR.

Signal transduction cascade over TLR3:

enabled after binding of appropriate ligands, the transcription factors "interferons regulating factor 3 "(IRF3) and NF-kB by the adapter molecule TRIF. The further signal transduction cascade according to TRIF is now known. TRIF recruits the IRF kinase TBK1 or PKR (dsRNA activated Protein kinase). After phosphorylation of the transcription factor "interferone regulatory factor 3 "(IRF3) dimerizes this and can as such into the cell nucleus, where it releases cytokines (Type I interferons) or interferon-beta) (L. Martinez-Sobrido et al.). to full activity of IRF are additionally the phosphatidylinositol 3-kinase and other downstream kinases necessary PKR Incidentally, it belongs to a class of 20 dsRNA binding proteins. It is also activated by this bond and can therefore also independent of TLR3 to an immune response to lead.

Signal transduction cascades over TLR7, TLR8 and TLR9

The Induction of type I interferons by TLR7 / 8 and TLR9 runs mainly via the adapter molecule MyD88. Finally, the transcription factor "interferons regulatory factor 7 "(IRF7) via phosphorylation by Kinases (IRAK-1, IRAK-4, IKK-alpha) activated, whereupon he in the Nucleus migrates and the transcription of interferons of the type I induced. Another variant is via the transcription factor IRF1, but the exact mechanism is not known yet.

TLRs are of great therapeutic interest. TLR agonists be z. As adjuvants in vaccination strategies or in cancer therapy used. Examples are the treatment of basal cell carcinoma by the TLR7 / 8 agonists imiquimod / resiquimod or from bladder cancer through a TLR2 agonist. The TLR9 receptor is made by a synthetic CpG-containing oligonucleotide (CpG 7909 and CpG 10101) for the therapy of autoimmune diseases, cancer and infectious diseases driven. TLR9-based therapeutic strategies are being commercialized from the company Coley Pharmaceuticals.

But also by an overreaction of the innate immune system many diseases can be triggered For example, autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here, TLRs react with decomposition products the body's own cells and thus cause the immune system to fail.

The TLRs are even suspected of causing cardiovascular disease to be related. Inflammatory reactions in the heart can contribute to the formation of arteriosclerotic plaques, which ultimately by vascular occlusion to the infarction being able to lead.

Cytokines / Cytokines:

cytokines are multifunctional signaling substances. These are sugary Proteins that have a regulatory function for growth and have the differentiation of body cells. Some They are therefore also called growth factors. Lots Cytokines also play an important role in immunological reactions and are therefore also called mediators. Cytokines are used by the Secretions release cells into the surrounding medium and stimulate them other cells, if they have a suitable receptor. you distinguishes 5 main groups of cytokines:

1. Interferons (IFN)

interferons cells indicate to form proteins that cause a viral infection complicate or prevent. Also, interferons may be antitumoral Have effect.

2. Interleukins (IL)

interleukins serve the communication of immune cells with each other and thereby increase the coordination in the defense against pathogens and the fight against tumors

3. Colony stimulating factors

colony stimulating Factors are formed in the kidney. These are growth factors for blood corpuscle

4. Tumor Necrosis Factors (TNF)

The most important function of TNFs is the activity of different To regulate immune cells. They are mainly of macrophages distributed. TNFs can cause cell death (apoptosis), Cell proliferation, cell differentiation and secretion stimulate other cytokines.

5. Chemokines

chemokines are chemoattractants that cause cells with matching receptors to migrate through chemotaxis to the source of chemokines

Of particular importance as mediators for immunological processes are the interferons (IFN), in particular the type I interferons. The first interferon of this type was found in 1957 by Isaacs and Lindemann ( Isaacs, A. et al .; J. Proc. R. Soc. Lond. B. Biol. Sci. 147, 258-267 ). The name comes from the fact that this protein interferes with the replication of viruses. Type I interferons are key cytokines that elicit antiviral cell responses, establish "antiviral status," and stimulate immune system cells to produce an antiviral response that binds to a receptor known as the IFN-alpha receptor (IFNAR) There are several different subtypes called IFN-alpha, IFN-beta, IFN-kappa, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega and IFN-zeta. Of particular importance again are alpha and beta interferons, which are secreted by many cells, such as lymphocytes, macrophages, fibroblast endothelial cells, osteoblasts and others.

The IFN-alpha proteins are present in 13 subtypes known as IFNAX (x = 1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17 and 21). All of their genes are clustered on chromosome 9.

From Two of the IFN-beta proteins are described. It is a matter of IFNB1 and IFNB3. A protein described as IFNB2 could later be identified as a known interleukin.

about a signal transduction cascade is formed after binding to the in the outer cell membrane lying interferon type 1 receptor the transcription factor "interferon stimulated gene factor 3 "(ISGF3), a heterotrimer and activator of the transcription of the Transcription factors STAT1, STAT2 and "IFN regulatory factor 9 (IRF9), which migrate into the cell nucleus and there the transcription of hundreds of effector molecules (via so-called IFN inducible genes). These effector molecules directly affect protein synthesis, cell growth and survival in the process of establishing the so-called "antiviral Status "(antiviral state) .In this status becomes infectiousness the virus z. B. repelled by reduced replication rates or at least reduced.

additionally The adaptive immune system is activated by the maturation of dendritic Cells elicited the antibody response of B Cells and the T cell response is activated. There are lymphocytes and monocytes recruited by induced chemokines to the site of infection.

Non-viral gene delivery methods

The introduction of genetic material into eukaryotic cells (transfection), especially mammalian cells, is today a method that is indispensable in modern research ( Domb AJ; Review in Molecules; 2005; 10; 34 and Xiang G; Keun-Sik K .; Dexi L .; Review in The AAPS Journal; 2007 ; 9 (1) Article 9; http://www.aapsj.org )). Without this method, a clarification of the function of different genes would be much more difficult. Not to be forgotten is the possibility to produce in this way proteins of eukaryotic origin true to the original, as the correct posttranslational modification by the eukaryotic Cells, in contrast to previously commonly used prokaryotic cells, is ensured. Furthermore, it is expected in the near future that, in particular, the introduction of genetic material into human cells, ie gene therapy, will find its way into modern medicine in the form of clinically tested procedures and therapies. The introduction of genetic material allows z. B. in eukaryotic cells destroyed DNA to replace areas and thus to fix malfunction. In addition, suicide genes can be introduced, for example, to force cancer cells to "suicide." But the knockdown of genes can be achieved by, for example, siRNA (small interfering RNA) or antisense molecules are used. To be able to access the genetic control apparatus of the cell, humans therefore have a valuable means at their disposal, but also to increase their understanding of their influence on the naturally occurring processes in a cell.

The enormous opportunities involving the introduction of genetic Material in eukaryotic cells brings with it an arsenal of methods that only have unsatisfactory performance exhibit. The specific occurring defects until existing methods essentially concern the important ones Parameters efficiency, toxicity, immunogenicity, Targeting, restriction on the size of the genetic Materials, possibilities of in vivo / in vitro application, Possibility of high-throughput applications, potential dangers, Simplicity of the method and cost of the method. No procedure is able to meet all these parameters adequately and so is attributed to the lack of a suitable gene carrier system, that until today despite considerable research expenditures no could establish gene therapy based medical therapy.

In In recent years, research into so-called gene delivery methods has been achieved (Gene Delivery Systems) used both in vitro and in vivo be enormous, because there are great opportunities be granted to help gene therapy to a breakthrough. One focus of gene therapy research is on viruses as a To use carrier systems. As the introduction of DNA or RNA into foreign cells is an integral part of the multiplication cycle of viruses, this ability was provided by a natural, evolutionary process in the developmental history of the virus so far refines that to date there are no more effective gene carriers. The naturally occurring viruses are genetically engineered that manipulates your ability to reproduce and lose their pathogenicity, but a cell with recombinant introduced can infect genetic material. Because viruses except consisting of genetic material consisting essentially of proteins, However, they offer the immune system a large attack surface. In doing so, the immune system has an equally evolutionary one Adaptation strategies are developed to meet these invaders to defend. Therefore, the immune response of the body as a particularly significant factor regarding failed Called gene therapy studies.

The currently available gene delivery methods can be divided into the two main groups viral systems and non-viral Systems are divided. The non-viral systems can again be differentiated into chemical and physical methods.

From Non-viral systems based on chemical methods In particular, those worth mentioning are those on cationic Lipids (so-called lipofection) or cationic polymers (so-called polyfection) are based. Their efficiency is usually far behind the viral systems.

Well-known cationic polymers are, for example, poly-L-lysine (PLL), ( EP 388758 ) Polyethyleneimine (PEI), ( JP Behr et al .; Proc. Natl. Acad. Sci. USA; 1995; 92; 7297 ( WO 9602655 ), Diethylaminoethyldextran (DEAE), ( SC De Smedt et al .; Phar. Res .; 2000; 17; 113 ), Starburst Dendrimere (PAMAM), ( FC Szoka et al .; Bioconjug. Chem .; 1996; 7; 703 ; WO 9502397 ), Chitosan derivatives ( W. Guang Liu et al .; J. Control. release; 2002; 83; 1 ) or polydimethylaminoethyl methacrylates ( P. van de Wetering et al .; J. Gene Med .; 1999; 1; 156 ; WO 9715680 ). Also, the widely used Ca-phosphate precipitation method broadly uses a "cationic polymer" and thus can be counted among this group.

Commercially available products of such cationic polymers z. Superfect, Polyfect (Qiagen), ExGen500 (Biomol), and jetPEI (Qbiogene).

Likewise known cationic lipids ( JP Behr; Bioconjugate Chem .; 1994; 5; 382 ) are, for example, DOTMA ( US 4946787 ), DOTAP ( Leventis et al .; Biochim. Biophys. Acta; 1990; 1023; 124 ), DOGS ( EP 394111 ), DOSPA ( WO 9405624 ), DOSPER ( WO 97002419 ), DMRIE ( US 5264618 ) or DC-Chol ( Huang et al; Biochem. Biophys. Res. Commun .; 1991; 179; 280 ; WO 9640067 ). Such or similar lipids are formulated as such or in combination with so-called colipids (eg DOPE) usually in ethanolic, aqueous buffer solutions as micelles or liposomes. As such, or as an oil or solid substance to the egg gene formulation, they are available as commercially available reagents such as Lipofectin, Lipofectamin, Lipofectamine 2000 (Invitrogen), Fugene (Roche), Effectene (Qiagen), Transfectam (Promega), Metafectene (Biontex) etc.

cationic Lipids and cationic polymers form in the presence of DNA or RNA due to the opposite charge conditions spontaneously called lipoplexes or polyplexes. The DNA will be there condensed by the compensation of the negative charge on the phosphate radical, so minimized in size. In general depends on the transfection efficiency of lipoplexes or polyplexes from a variety of parameters. The most important are the quantity ratio from genetic material to cationic component in manufacture the lipo / polyplexes, ionic strength during production the lipo / polyplexes, absolute amount of lipo / polyplexes per cell, Cell type, proliferation status of cells, physiological status cells, cell division rate, incubation time, etc. These influence parameters are an expression of a complicated Transfektionsgeschehens, in which the lipo / polyplexes or the contained genetic materials overcome a variety of cellular barriers have to.

The first barrier represents the outer negatively charged one Cell membrane. It is believed that transfection-active lipoplexes, which must have a net positive charge, by adsorptive Endocytosis or liquid phase endocytosis in the interior of the Cell. By endocytosis, which is an active transport process represents the cell becomes material on the cell surface encased with cell membrane and internalized as a vesicle (endosome). By merging with so-called lysosomes, which is a complex Containing a mixture of enzymes, those contained in the endosomes Substances degraded.

There for this degradation, a low pH is necessary possess Endosomes proton pumps that protons into the endosomes pump until a suitable pH is reached. To charge neutrality to maintain chloride ions flow to the same extent into the endosomes.

Lots modern cationic lipids or polymers possess this reason Buffering properties. In this way, the low pH does not reached and there is an entry of ions into the endosomes, the endosomes burst due to the resulting osmotic pressure brings. In this way, these lipo / polyplexes enter the cytosol. As a number of transfection-active lipids and polymers without Buffer properties are known, another mechanism must be exist that allows the lipo / polyplexes to enter the cytosol. At least in the case of lipids, one suspects a fusion of the involved ones Membranes and concomitant destabilization. Whether there predominantly the lipoplex or the containing DNA / RNA as such got into the cytosol is unclear. However, it is believed that the DNA in the cytosol is released from the lipoplex, as experiments failed by microinjection of lipoplexes directly into the nucleus to achieve protein expression. It seems like those in the Lipoplexes bound DNA not accessible to the transcription apparatus is.

These it is antisense molecules directed against mRNA or siRNA, the biological action site is reached and the duration of the effect depends essentially on the concentration of cytosolic RNases and the rate of release from the lipo / polyplexes. As such, DNA can not penetrate the nucleus, what is called "nuclear Barrier ", but it does arrive during the cell division at their site of action and thus leads to the expression of proteins.

When other non-viral methods based on chemical methods, be called systems that a DNA-binding moiety and carry a ligand, receptor-mediated endocytosis can trigger (examples Transferrinfektion).

The most important example of a non-viral method based on a physical process is electroporation. The cells to be transfected are placed between two electrodes, to which a typical voltage profile is applied. In this way, the cells are exposed to an intense electrical impulse (pulse), which leads to a reversible opening (pores) of the cell membrane. Through these pores substances such. B. genetic material, which are located in the immediate vicinity of the pores penetrate into the cell. The pulse (ie voltage curve) as one of the most important success parameters must be optimized for each cell type. There are now several commercial providers of electroporators (eg Eppendorf / Multiporator, US 6008038 , Biorad / Genpulser, US 4750100 Genetronics Inc., US 5869326 , BTX / ECM series), which were developed especially for eukaryotic cells and allow adaptation of the pulse parameters to the respective cell type. In fact, there are now devices that make an in vivo application possible. In the in vitro application, the cells are suspended in an electroporation buffer, together with the DNA / RNA to be transfected into an electrode ne electroporation cuvette and exposed to one or more pulses. In addition to the voltage curve, other important parameters are the nature of the buffer, the temperature, the cell concentration and the DNA concentration. After the cells have been exposed to the pulse, they are left for a short time to regenerate the cell membrane. Subsequently, the cells are sown in a culture vessel and cultivated as usual.

When other physical methods are microinjection, hydrodynamic Methods, ballistic methods (Genegun) or methods that use ultrasound called use or even the injection of naked DNA into different Organs that lead to low expression of the corresponding genes.

To the methods the physical methods as well as chemical methods In particular, magnetofection, the Uses DNA-binding molecules on magnetic nanoparticles in order to have a magnetic field gradient DNA also the surface enriched cells and cause endocytosis

Description of the invention

With repetitive Lipofektionsversuchen, where initially a Transfection with a specific targeted against a specific protein siRNA was performed and then a Plasmid transfection followed with a reporter gene was found that the transfection success of plasmid transfection did not occur, although the cells made a healthy impression. Only at very low siRNA levels could detect a small amount of the reporter protein become.

Around exclude that it is an off-target effect the specific siRNA, the experiment was conducted with a nonspecific repeated siRNA, which "bred" against the human genome has been. The result, however, remained the same.

There It is known that the proliferation of cells also has an influence When lipofection has, it has been studied whether the cells in your Proliferation behavior affected by pre-transfection with siRNA were. It was found that the proliferation rates at higher siRNA levels decrease, but sufficient Proliferation was given in the experiments, than that of pre-transfection following plasmid transfection failed. It seemed as if the cells surprisingly against the second transfection can fight back.

With repetitive transfection experiments involving two plasmid transfections could be switched in a row, could a similar Result will be obtained, though not with the above Clarity. The second transfection step was usually either very inefficient or counterproductive. Also here were similar Investigations, as mentioned above, carried out to ensure that they are not toxic effects. It turns, therefore the task, the transfection efficiency, especially with repeated To increase transfection.

These The object is achieved by a method to increase the transfection efficiency of a non-viral gene delivery system in which the non-viral gene delivery system in particular a cationic lipid, a cationic polymer, a cationic one Protein or a compound comprising a DNA and / or RNA-binding Domain and induce receptor-mediated endocytosis or in which the non-viral gene delivery system in particular on a physical method like electroporation, microinjection, Ultrasound or a ballistic or hydrodynamic method based, characterized in that the innate immune defense is reduced.

That is, in particular, the intracellular signal transduction cascade from the TLRs, via the adapter molecules, via the corresponding kinases that phosphorylate the transcription factors, which in turn induce the interferons, can be disrupted. Furthermore, the signal transmission through messenger substances between the cells can be interrupted. Since these are all proteins, preferably activity-increasing or activity-reducing effectors such as antibodies, aptamers, antagonists or inhibitors against these proteins are used according to the invention. In this case, the corresponding active ingredients can be spent as such or with appropriate auxiliary molecules on or in the cells, depending on cell permeability or destination. So z. B. agents are transported via liposomal carrier into the endosomes. If the target site is the cytosol, particular preference is given to electroporation or special peptide sequences capable of permeabilizing the cell walls. If the active substances are peptides or proteins, then according to the invention they can also be genetically modified by transfection material are formed intracellularly and, if necessary, with localization sequences to the appropriate cell compartments are directed. If one wishes to shut down siRNA genes which code for crucial proteins of the sign transduction apparatus, the known transfection systems are available according to the invention. The method according to the invention or the use according to the invention can be used or take place both in vitro and in vivo.

at the process of the invention can, for. At least one of the TLR3, 7, 8, 9, one or more RIG-I helicases and / or one or more RIG-I-like helicases are blocked.

So are antibodies against the Toll Like receptors TLR7, TLR8 and TLR9 are known and commercially available. These could together with endocytosis-inducing lipo- or polyplexes as such or packed in liposomes into the endosomes and so block the receptors. Also against TLR3 is a Antibodies are known and commercially available. As TLR3 according to previous findings on the surface the cells is in this case, an addition to the extracellular is sufficient Room. The choice of the receptor to be blocked of course depends on the invention on the type of genetic material to be transfected.

However, inhibitors or antagonists for various TLRs are also known and are used according to the invention in a preferred manner. Thus, it has been found that certain DNA sequences of viral origin can block the TLR9 receptor ( War AM et al; Proc. Natl. Acad. Sci. USA; 1998; 95 (21); 12631-6 ); the corresponding disclosure is incorporated by reference. This DNA sequence usually contains the motif TTAGGG in repeated form. Corresponding antisense molecules as nuclease-resistant phosphorothioates are likewise commercially available from InvivoGen (San Diego, USA). The same company also offers plasmids that encode TLRs and TLR-related genes shRNA or various inhibitors of kinases such as 2-amino purines ( Kaufman RJ et al., Proc. Natl. Acad. Sci. USA; 1999; 96 (21); 11693-5 ) directed against PKR (dsRNA activated protein kinase) or LY294002, a cell-permeable inhibitor of phosphatidylinositol 3-kinase, which is also involved in the signal transduction cascade after TLR3.

Also it is possible by knocking down with siRNA at least one Turn off the gene necessary for a signal transduction necessary Protein encoded.

However, inhibitors which attack the important adapter molecule MyD88 and / or TRIF are known and are also commercially available and are used according to the invention in a preferred manner. For example, IMGENEX (San Diego, USA) offers a peptide that blocks the homodimerization necessary for the action of MyD88. The preferred peptide motif is RDVLPGT ( Loiarro M. et al .; J. Biol. Chem. 2005; 16; 15809-14 ). The commercially available peptide also contains a PTD sequence (protein transduction sequence), which makes the entire peptide cell-permeable ( Derossi D. at al. J. Biol. Chem. 1994, 269; 10,444 to 10,459 ). Incidentally, the same company also offers antibodies directed against MyD88, which can be used according to the invention.

Further can the intercellular signal transmission according to the invention z. B. be interrupted by antibodies or aptamers, which are directed against interferons or their receptors. Appropriate Antibodies are in turn commercially available.

According to the invention at least one of the kinases IRF kinase TBK1, phosphatidylinositol 3-kinase, IRAK-1, IRAK-4, IKK-alpha or PKR or at least one of the transcription factors IRF1, IRF3, IRF7, IRF9, IRF2, IRF4, IRF5, IRF6, IRF8, STAT1, STAT2, ISGF3 or NF-KB blocked.

Also it is possible according to the invention, at least a cytokine, at least one tumor necrosis factor, at least one interleukin and / or block at least one interferon. As to be blocked Interferon comes, for example, an interferon type I, in particular an interferon-alpha or interferon-beta into consideration.

Also can be at least one receptor for cytokines or at least a receptor for type I interferons are blocked.

• a) ein nicht virales Genliefersystem,
• b) einen Wirkstoff, und
• c) ein Mittel zur Verringerung der angeborenen Immunabwehr.

The invention further provides a composition comprising at least two of the following components:

• a) a non-viral gene delivery system,
• b) an active substance, and
• c) a means of reducing innate immune defense.

• a) ein nicht virales Genliefersystem,
• b) einen Wirkstoff, und
• c) ein Mittel zur Verringerung der angeborenen Immunabwehr.

Also according to the invention a kit of parts is provided which comprises at least two of the following components:

• a) a non-viral gene delivery system,
• b) an active substance, and
• c) a means of reducing innate immune defense.

there For example, the non-viral gene delivery system includes, in particular, a cationic one Lipid, a cationic polymer, a cationic protein and / or a compound having a DNA and / or RNA-binding domain and can induce receptor-mediated endocytosis.

According to the invention as an active ingredient, such as. B. an agent for repairing a gene defect, z. B. genetic material such as modified or unmodified ssDNA, modified or unmodified dsDNA, modified or unmodified ssRNA, modified or unmodified dsRNA and / or modified or unmodified siRNA can be used.

The Means for reducing innate immune defense can be an activity-reducing or activity-enhancing effector, or an antibody, Include intrabody, aptamer, antagonist, inhibitor and / or siRNA, wherein the antibody, intrabody, aptamer, antagonist, inhibitor and / or the siRNA preferably at least one signal transduction cascade of the innate immune system blocked.

According to one particularly preferred embodiment comprises the agent to reduce the innate immune system the TTATGG.

Also may be the means of reducing the innate immunity genetic Material that involves a knockdown of a signal transduction cascade protein of the innate immune system causes or the expression of a Protein that performs the activity of a protein blocked a signal transduction cascade of the innate immune system.

Especially a peptide with the structural motif RDVLPGT can be used.

Especially For example, the above-mentioned components may also be respectively be used individually or in groups: So two, three or more different components a) and / or b) and / or c) be used.

• – alle Komponenten getrennt voneinander vorliegen, oder
• – die Komponenten a) und b) gemeinsam, oder
• – die Komponenten a) und c) gemeinsam, oder
• – die Komponenten b) und c) gemeinsam vorliegen.

The kit of parts according to the invention can:

• - all components are separate, or
• - Components a) and b) together, or
• - the components a) and c) together, or
• - The components b) and c) are present together.

So The components can be separated from each other z. B. in glass or plastic container, which together are packaged, or the components can be twos or provided to several in respective containers become.

The Composition according to the invention and / or the Kit of Parts according to the invention can for carrying out a method according to the invention Procedure can be used.

According to the invention further the use of an inventive Composition or a kit according to the invention of Parts for the treatment of a gene defect Illness revealed.

at the disease can be, for. Cystic fibrosis, muscular dystrophy, Phenylketonuria, maple syrup disease, propionic acidemia, Methylmalonic acidemia, adenosine deaminase deficiency, hypercholesterolemia, hemophilia, β-thalassemia, Cancer, a viral disease, degeneration of the macula, amyotrophic Act lateral sclerosis and / or an inflammatory disease.

example 1

Material:

• 1. Hela cells
• 2. Metafectene Pro, Lot AD 2, Biontex Laboratories
• 3. 24-well plate, TPP, product number 92024
• 4. DMEM, PAA, cat. No. E15-883
• 5. FCS Mycoplex, PAA, cat. No. E15-773
• 6. pCMV-lacZ, Lot PF462-060207, Plasmid Factory, c = 1 mg / ml in WFI
• 7. Sheep Polyclonal Antibody Against Human IFNβ, FBI Biomedical Laboratory Stories, Product Number 31400-1, 0.25 mg / ml (estimate), 2 × 10 ⁴ units / ml.
• 8. β-galactosidase assay kit, Stratagene

Detailed experimental description:

1 day:

HeLa cells are seeded in a 24 well plate. A cell count of 2.3 × 10 ⁵ cells is plated out in a well in 500 μl of complete medium (10% FCS). It is then incubated for 24 h in a CO ₂ incubator (10%).

2 day:

First, the antibody (Sheep Polyclonal Antibody Against Human IFNβ) is thawed. It is then mixed with 400 μl of PBS (Phosphate Buffered Saline) and mixed gently. He now has a concentration of 0.05 μg / μl. Subsequently, the wells of the 24-well plate are supplied with the following amounts of antibody: 1 2 3 4 5 6 A 0 μg (0 μl) 0.25 μg (5 μl) 0.5 μg (10 μl) 1 μg (20 μl) 1.5 μg (30 μl) 3 μg (60 μl) B 0 μg (0 μl) 0.25 μg (5 μl) 0.5 μg (10 μl) 1 μg (20 μl) 1.5 μg (30 μl) 3 μg (60 μl) C 0 μg (0 μl) 0.25 μg (5 μl) 0.5 μg (10 μl) 1 μg (20 μl) 1.5 μg (30 μl) 3 μl (60 μl) D 0 μg (0 μl) 0.25 μg (5 μl) 0.5 μg (10 μl) 1 μg (20 μl) 1.5 μg (30 μl) 3 μg (60 μl)

Subsequently is incubated in the incubator for 0.5 h. Meanwhile the lipoplexes produced. For this purpose, 26 .mu.l of DNA (pCMV-lacZ) pipetted into 1300 μl of PBS and gently boosted Pipette mixed. 104 μl of Metafectene Pro will also be added pipetted into 1300 μl PBS and gently opened and closed Pipette mixed. Now both solutions are mixed and incubated for 15 min.

At the end, 100 μl of the lipoplex solution are added to each well. It is then incubated for 24 h in a CO ₂ incubator (10%).

3rd day:

On the third day, the medium of lines C and D is renewed. The steps of lipoplex production and transfection are repeated for these lines. It is then incubated for 24 h in a CO ₂ incubator (10%).

4th day

reporter gene:

The efficiency of the transfection is carried out with the β-galactosidase assay kit according to the manufacturer's instructions. The plates are developed as long as the microplate reader a yellowing with an Ab sorption of 1-2 is measured and then immediately stopped. The incubation period is then noted. The values are read out with the microplate reader, and the mean value is formed.

Result:

• Incubation time 9.5 min

Mean of 3 measurements: 1 2 3 4 5 6 A 0.717 0.689 0,650 0.652 0,635 0.509 B 0.668 0.672 0,690 0.679 0.714 0.525 C 0.858 0.823 1,228 1,690 1,852 0,975 D 1,501 1,805 1,518 1,886 2,017 1,909

Lines A and B represent duplicates of the results for a single transfection. Lines C and D are duplicates of the results for a repetitive transfection. The average is therefore formed: 1 2 3 4 5 6 STARTING AT 2 .6925 .6805 .6700 .6655 .6745 .5170 C & D / 2 1.1810 1.3140 1.3730 1.7880 1.9345 1.4420 ABS / 9.5 min Ab = Anti INFB Antibody

Example 2

Material:

• 1. Hela cells
• 2. Metafectene Pro, Lot AD 2, Biontex Laboratories
• 3. 24-well plate, TPP, product number 92024
• 4. DMEM, PAA, cat. No. E15-883
• 5. FCS Mycoplex, PAA, cat. No. E15-773
• 6. pCMV-lacZ, Lot PF462-060207, Plasmid Factory, c = 1 mg / ml in WFI
• 7. β-galactosidase assay kit, Stratagene
• 8. Mouse monoclonal antibody against human interferons alpha / beta Receptor Chain 2 (CD118), clone MMHAR-2, Isotype Ig2a, C = 0.5 mg / ml in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), PBL Biomedical Laboratory Stories, Product-No: 21385

Detailed experimental description:

1 day:

HeLa cells are sown in a 24 well plate. In this case, a cell count of 2.3 x 10 ⁵ cells were plated (10% FCS) in a well in 500 ul complete medium. It is then incubated for 24 h in a CO ₂ incubator (10%).

2 day:

First, the antibody is thawed (Mouse monoclonal Antibody Against Human Interferon Alpha / Beta Receptor Chain). It is then mixed with 400 μl of PBS (Phosphate Buffered Saline) and mixed gently. He now has a concentration of 0.1 μg / μl. Subsequently, the wells of the 24-well plate are supplied with the following amounts of antibody: 1 2 3 4 5 6 A 0 μg (0 μl) 0.5 μg (5 μl) 1 μg (10 μl) 2 μg (20 μl) 3 μg (30 μl) 6 μg (60 μl) B 0 μg (0 μl) 0.5 μg (5 μl) 1 μg (10 μl) 2 μg (20 μl) 3 μg (30 μl) 6 μg (60 μl) C 0 μg (0 μl) 0.5 μg (5 μl) 1 μg (10 μl) 2 μg (20 μl) 3 μg (30 μl) 6 μg (60 μl) D 0 μg (0 μl) 0.5 μg (5 μl) 1 μg (10 μl) 2 μg (20 μl) 3 μg (30 μl) 6 μg (60 μl)

Subsequently is incubated in the incubator for 5 h.

Subsequently, the lipoplexes are prepared. For this purpose, 26 μl of DNA (pCMV-lacZ) are pipetted into 1300 μl PBS and mixed by gently pipetting up and down. 104 μl Metafectene Pro are also pipetted into 1300 μl PBS and mixed by gently pipetting up and down. Subsequently, both solutions are mixed and incubated for 15 min. At the end, 100 μl of the lipoplex solution are added to each well. It is then incubated for 24 h in a CO ₂ incubator (10%).

3rd day:

At the third day, the medium of lines C and D is renewed. The steps Lipoplex preparation and transfection are used for these lines repeated.

It is then incubated for 24 h in a CO ₂ incubator (10%).

4th day

reporter gene:

The Efficiency of transfection is enhanced with the β-galactosidase Assay kit according to manufacturer's instructions. The Plates are developed until the microplate reader Yellow staining measured with an absorbance of 1-2 and then stopped immediately. The incubation period is then noted. The values are with the microplate reader read out, and the mean value formed.

Result:

• Incubation period: 4min

Mean of 3 measurements: 1 2 3 4 5 6 A 1,269 1,113 1,163 1,185 1,214 1,084 B 1,132 1,102 1,088 1,229 1,113 1,154 C 1,127 1,438 1,533 1,378 1,504 1,367 D 1,395 1,393 1,377 1,417 1,459 1,467

Lines A and B represent duplicates of the results for a single transfection. Lines C and D are duplicates of the results for a repetitive transfection. The average is therefore formed: 1 2 3 4 5 6 STARTING AT 2 1,2005 1.1075 1.1255 1.2070 1.1635 1.1190 C & D / 2 1.2610 1.4155 1.4550 1.3975 1.4815 1.4170 ABS / 4 min Ab = Anti INF receptor Type I Antibody

Example 3

Material:

• 1. Hela cells
• 2. Metafectene Pro, Lot AD 2, Biontex Laboratories
• 3. 24-well plate, TPP, product number 92024
• 4. DMEM, PAA, cat. No. E15-883
• 5. FCS Mycoplex, PAA, cat. No. E15-773
• 6. pCMV-lacZ, Lot PF462-060207, Plasmid Factory, c = 1 mg / ml in WFI
• 7. β-galactosidase assay kit, Stratagene
• 8. ODN (Full PTO = Phosphothioatoligo): Seq .: 5'TTT AGG GTT AGG GTT AGG GTT AGG G-3 '; 0.2 μmol synthesis scale, Purification: HPLC + NAP, lyophilized, 213.8 μg; TLR9 antagonist

Detailed experimental description:

1 day:

HeLa cells are sown in a 24 well plate. A cell count of 2.3 × 10 ⁵ cells is plated out in a well in 500 μl of complete medium (10% FCS). It is then incubated for 24 h in a CO ₂ incubator (10%).

2 day:

First becomes the TLR9 antagonist (ODN). Then it will be in Dissolve 956 μl sterile redistilled water. He has now a concentration of 0.25 μg / μl.

lipoplexes:

Lipoplexes of the following composition are prepared for the cells of the respective wells of the 24-well plate with the cells (DNA = pCMV-LacZ, ODN = TLR9 antagonist): 1 2 3 4 5 6 0.5 μg (0.5 μl) DNA 0.0 μg (0.0 μl) ODN 2.0 μl M. Pro 0.5 μg (0.5 μl) DNA 0.1 g (0.4) ODN 2.4 μl M. Pr 0.5 μg (0.5 μl) DNA 0.2 μg (0.8 μl) ODN 2.8 μl M. Pro 0.5 μg (0.5 μl) DNA 0.3 μg (1.2 μl) ODN 3.2 μl M. Pro 0.5 μg (0.5 μl) DNA 0.4 μg (1.6 μl) ODN 3.6 μl M. Pro 0.5 μg (0.5 μl) DNA 0.5 μg (2.0 μl) ODN 4.0 μl M. Pro 0.5 μg (0.5 μl) DNA 0.0 μg (0.0 μl) ODN 2.0 μl M. Pro 0.5 μg (0.5 μl) DNA 0.1 μg (0.4 μl) ODN 2.4 μl M. Pro 0.5 μg (0.5 μl) DNA 0.2 μg (0.8 μl) ODN 2.8 μl M. Pro 0.5 μg (0.5 μl) DNA 0.3 μg (1.2 μl) ODN 3.2 μl M. Pro 0.5 μg (0.5 μl) DNA 0.4 μg (1.6 μl) ODN 3.6 μl M. Pro 0.5 μg (0.5 μl) DNA 0.5 μg (2.0 μl) ODN 4.0 μl M. Pro 0.5 μg (0.5 μl) DNA 0.0 μg (0.0 μl) ODN 2.0 μl M. Pro 0.5 μg (0.5 μl) DNA 0.1 μg (0.4 μl) ODN 2.4 μl M. Pro 0.5 μg (0.5 μl) DNA 0.2 μg (0.8 μl) ODN 2.8 μl M. Pro 0.5 μg (0.5 μl) DNA 0.3 μg (1.2 μl) ODN 3.2 μl M. Pro 0.5 μg (0.5 μl) DNA 0.4 μg (1.6 μl) ODN 3.6 μl M. Pro 0.5 μg (0.5 μl) DNA 0.5 μg (2.0 μl) ODN 4.0 μl M. Pro 0.5 μg (0.5 μl) DNA 0.0 μg (0.0 μl) ODN 2.0 μl M. Pro 0.5 μg (0.5 μl) DNA 0.1 μg (0.4 μl) ODN 2.4 μl M. Pro 0.5 μg (0.5 μl) DNA 0.2 μg (0.8 μl) ODN 2.8 μl M. Pro 0.5 μg (0.5 μl) DNA 0.3 μg (1.2 μl) ODN 3.2 μl M. Pro 0.5 μg (0.5 μl) DNA 0.4 μg (1.6 μl) ODN 3.6 μl M. Pro 0.5 μg (0.5 μl) DNA 0.5 μg 2.0 μl) ODN 4.0 μl M. Pro

The DNA for each well is dissolved in 100 μl of PBS and the appropriate amount of ODN is pipetted in and mixed. The appropriate amounts of Metafectene Pro are then pipetted in and mixed again. It is then incubated for 15 min and the lipoplexes are added to the cells in the corresponding wells. It is then incubated for 24 h in a CO ₂ incubator (10%).

3rd day:

At the third day, the medium of lines C and D is renewed. The steps Lipoplex preparation and transfection are used for these lines repeated. Furthermore, in the case of lines A and B 500 μl each Medium added to the wells.

It is then incubated for 24 h in a CO ₂ incubator (10%).

4th day

reporter gene:

The Efficiency of transfection is enhanced with the β-galactosidase Assay kit according to manufacturer's instructions. The Plates are developed until the microplate reader Yellow staining measured with an absorbance of 1-2 and then stopped immediately. The incubation period is then noted. The values are with the microplate reader read out, and the mean value formed.

Result:

• Incubation time: 80 min

Mean of 3 measurements: 1 2 3 4 5 6 A 0,431 0,400 0,448 0.404 0.419 0.566 B 0.415 0.453 0.419 0,435 0.696 0.543 C 0.573 0.966 0,748 1,054 0,820 1,234 D 0.778 0,898 1,312 1,302 1,195 1,352

Lines A and B represent duplicates of the results for a single transfection. Lines C and D are duplicates of the results for a repetitive transfection. The average is therefore formed: 1 2 3 4 5 6 STARTING AT 2 .4230 .4265 .4335 .4195 .5570 .5545 C & D / 2 .6575 .9320 1.0300 1.1780 1.0075 1.2930 ABS / 80 min ODN = TLR9 antagonist

Example 4

Of the TLR7, 8 and 9 detect foreign RNA or DNA in the endosomes. On the cytosolic side of the adapter molecule binds MyD88. This protein consists of two protein chains, through Homodimerization form the biologically active adapter molecule. Peptides with the sequence motif RDVLPGT prevent this homodimerization, by themselves binding to the monomers. These peptides are, however usually not cell-like. But you can provided with a "protein transduction sequence" (PTO) become. Such a sequence is z. B. DRQIKIWFQNRRMKWKK. The complete Peptide is thus able to penetrate cells and signal transduction TLRs to stop. Such a complete peptide is used by the company Imgenex commercially offered.

Material:

• 9. Hela cells
• 10. Metafectene Pro, Lot AD 2, Biontex Laboratories
• 11. 24-well plate, TPP, product number 92024
• 12. DMEM, PAA, cat. No. E15-883
• 13. FCS Mycoplex, PAA, cat. No. E15-773
• 14. pCMV-lacZ, Lot FF462-060207, plasmid factory, c = 1 mg / ml in WFI
• 15. β-galactosidase assay kit, Stratagene
• 16. Imgenex, MyD88 Homodimerization Inhibitory Peptide Set, Product No. IMG, 2005-1, 2 mg, lyophilized, MW 3100, Sequence: DRQIKIWFQNRRMKWKK RDVLPGT

Detailed experimental description:

1 day:

HeLa cells are sown in a 24 well plate. In this case, a cell count of 1.5 × 10 ⁵ cells is plated in a well in 200 μl of complete medium (10% FCS). It is then incubated for 24 h in a CO ₂ incubator (10%).

Preparing the inhibitor:

From the MyD88 homodimerization inhibitor peptide becomes the data of the Following the manufacturer, prepared a 5 mM solution in PBS.

Subsequently, the wells are supplied with the following amounts of inhibitor to adjust the corresponding concentrations: 1 2 3 4 5 6 A 0 μl (0 μM) 2 μl (50 μM) 4 μl (100 μM) 8 μl (200 μM) 12 μl (300 μM) 16 μl (400 μM)

It is then incubated for 24 h in a CO ₂ incubator (10%).

2 day:

First Add 3 μl of complete medium to each well.

Subsequently, the lipoplexes are prepared. For this purpose, 13 μl of DNA (pCMV-lacZ) are pipetted into 650 μl PBS and mixed by gently pipetting up and down. 52 μl Metafectene Pro are also pipetted into 650 μl PBS and mixed by gently pipetting up and down. Subsequently, both solutions are mixed and incubated for 15 min. At the end, 100 μl of the lipoplex solution are added to each well. Followed by 24 h in a CO ₂ incubator (10%) is incubated.

4th day

reporter gene:

• Ergebnis: Inkubationszeit: 4 min

The efficiency of the transfection is carried out with the β-galactosidase assay kit according to the manufacturer's instructions. The plates are developed until a yellow color is measured in the Microplate reader with an absorption of 1-2 and then immediately stopped. The incubation period is then noted. The values are read out with the microplate reader and the mean value is formed.

• Result: incubation time: 4 min

Mean of all 3 measurements: 1 2 3 4 5 6 A 0,850 1,120 1,268 1.555 1,518 1,442 ABS / 4 min; MHI = MyD88 homodimerization inhibitor

As already stated, the invention for therapeutic purposes be used. In particular, the invention for the gene therapy of, for example, cystic fibrosis, muscular dystrophy, Phenylketonuria, maple syrup disease, propionic acidemia, Methylmalonic acidemia, adenosine deaminase deficiency and hypercholesterolemia, Hemophilia, β-thalassemia can be used. Gentherapeutic treatment methods are also interesting, when hormones, growth factors, cytotoxins or immunomodulatory acting proteins in the organism to be synthesized. For The purposes mentioned above can be DNA fragments by means of the invention be brought highly effective in cells in which these DNA should develop the desired effect. The desired Effect can be the replacement of missing or defective DNA areas or the inhibition of DNA regions (for example by antisense DNA / RNA or siRNA) that cause the disease in the diseased Be cell type. In this way, for. B. tumor suppressive Genes used in cancer therapy or by the introduction cholesterol-regulating genes contribute to the prevention of heart disease. and blood vessel diseases. Furthermore, may encode DNA, which ribozymes, siRNA or shRNA, or ribozymes or siRNA itself be introduced into diseased cells. The Translation of that DNA generates active ribozymes or siRNA that binds to specifically cleave m-RNA catalytically and in this way prevent transcription. That way, for example viral mRNA can be cleaved without any other cellular affect m-RNA. The multiplication cycle of viruses (HIV, Herpes, Hepatitis B and C, Respiratory Syncytial Virus) can be interrupted in this way. Other diseases, which are specifically cured by treatment with siRNA are the age-related degeneration of the macula (eye disease), Liver cancer, solid tumors, amyotrophic lateral sclerosis and inflammatory diseases are currently in the focus of clinical trials.

Also In cancer therapy, transfection plays a role in the production of cancer vaccines an ever-increasing role. With that is that also possible field of application for the invention.

A the invention can be used further, for example, in vaccination processes find, based on the expression of DNA, which immunogenic Peptides encoded in the body of humans and animals work. To be z. B. lipid / DNA complexes used as vaccines. The infiltration DNA into body cells leads to expression of the immunogenic peptide and thus triggers the adaptive immune response out.

in the The following are exemplary according to the invention, but by no means limiting definitions:

transfection:

infiltrate of genetic material into eukaryotic cells.

transfection

amount protein expression of a cell population as a result of transfection processes with genetic material, which among other things expressed this Protein encoded or extent of knockdown of protein expression a cell population as a result of transfection processes with genetic Material that can trigger such a knock-down, in particular siRNA or ribozymes or DNA coding for shRNA or ribozyme encodes or proportion of cells of a total population of cells that infiltrated the biological efficacy of the genetic material as a result of transfection processes.

Non-viral gene delivery system:

Non-viral gene delivery systems are not produced by recombination of genetic material from naturally occurring viruses. They are able to infect genetic material into eukaryotic cells. In particular, the non-viral gene delivery systems are physical methods and chemical methods. Physical methods locate at least the genetic material in the vicinity of the cell, but in particular physical methods use energy input, in particular in the form of thermal, kinetic, electrical or other energy to mediate transport of the genetic material through the cell membrane. Chemical methods are based either on a chemical modification or derivatization of the nucleic acids, which in particular make them cell-permeable, or consist in particular of substances which bind DNA and can mediate transport through the cell membrane. In particular, these use electrostatic forces or hydrogen bonds to bind the nucleic acids. Again, in particular, the transport of DNA through the cell membrane occurs through an active transport mechanism of the cell, endocytosis. Substances which exhibit these properties contain, in particular, cationic lipids, cationic polymers, cationic peptides or also molecules which have a domain which DNA or RNA can bind and at the same time have a second domain containing a ligand, which is recognized by a receptor of the cell surface and triggers endocytosis by this recognition process. The substances can also be specially formulated, in particular as micelles or liposomes and also consist of several components, in particular with different functions.

Gene Therapy

therapy to cure or alleviate disease when used as an active ingredient modified or unmodified nucleic acids used become.

Innate immune defense

The innate immune defense is different from the acquired or adaptive Immune defense depart from the fact that it fends off a pathogen, without that it had ever come to a contact with the pathogen before have to be. The innate immune system is the most common type of cell owns and uses the recognition of pathogens attributable molecular Structures through receptors. In particular, these receptors collide Signal transduction cascades, in particular by expression Many cellular genes in an "antiviral status" of the Cells open. Furthermore, the triggering of an "antiviral Status "in other, especially neighboring cells by messengers (especially cytokines), which in turn respond to receptors of others Dock cells and in turn trigger a signal transduction cascade, Part of the innate immune system.

Antiviral status

status of cells characterized by the cell trying the potential biological effectiveness of foreign genetic To prevent material by countermeasures.

Transfection in vivo

The Introducing genetic material into eukaryotic cells in a living organism.

Transfection in vitro

The Introducing genetic material into eukaryotic cells outside of a living organism, in particular in vessels that are used for the cultivation of eukaryotic Cells are suitable.

Genetic material

nucleic acids, in particular ribonucleic acids or deoxyribonucleic acids, in particular of two at least partially complementary Strands (double-stranded = ds) exist z. B. dsDNA and dsRNA, or in particular from one strand (single-stranded = ss), z. B. ssDNA and ssRNA, which is partially complementary May have regions that are via hydrogen bonding can be connected to each other.

Modified genetic material

natural Nucleic acids by modification in their properties were changed. These modifications can be made In particular, be chemical changes, in particular the phakat framework that affects sugar or bases, what in particular the stability of the nucleic acids against nucleases and ribonucleases. Furthermore, can molecules (labels) to the nucleic acids covalently or non-covalently attached to new properties the nucleic acids lead, in particular to optical Traceability through fluorescent labels or labels containing the nucleic acids to direct to a certain place in the cell (localization elements) or labels that detect the passage of nucleic acids through Communicate membranes and so nucleic acids, for example make it cell-like.

siRNA (short interfering RNA):

short dsRNA (up to 28 bp) knocked down by RNA interference Can cause protein.

shRNA (short hairpin RNA):

short ssRNA, the 3'-end and the 5'-end complementary regions owns and thereby over hydrogen bond can hybridize and form a hairpin structure. shRNA can cause the knock-down of a protein by RNA interference.

Receptor:

Molecule, which is able to detect a substance and thus a biological Reaction triggers, in particular, are receptors to proteins.

Blocking:

prevention the biological function, in particular of proteins.

DNA / RNA binding domain:

Area in a molecule, the DNA is covalent or not carries covalent interactions bound, in particular are the non-covalent interactions electrostatic forces and hydrogen bonds.

signal transduction

outgoing from a receptor, the presence of a substance by attachment This substance is detected at the receptor, the information is transmitted via the presence of this substance is converted into a signal and over a chain of molecules carried by signal transduction. At the end there is a biological reaction. In particular, this is through The signal generated by receptor molecules of adapter molecules and in particular via kinases, in particular to transcription factors carried on. The transcription factors stimulate expression of genes that mediate the biological response.

Knock-down

attenuation or elimination of the expression of a gene.

antibody

molecules especially proteins that are capable of molecular structures, especially other proteins, to recognize and by binding of it affect biological effect.

intrabodies

intracellularly expressed antibodies.

aptamers

RNA molecules, able by training a tertiary structure are similar to molecular structures, especially proteins to recognize an antibody and by binding its biological Influence effect. Aptamers can be modified from or unmodified RNA.

antagonist

Substance, the one agonist by blocking a corresponding receptor inhibits its effect without triggering an effect itself. In turn, an agonist is capable of binding to a receptor to achieve a biological effect.

inhibitors

molecules the biological effect of another molecule, in particular of proteins. In particular, the Inhibitors themselves proteins, modified or unmodified nucleic acids or small organic molecules.

TLRs, RIG-I helicase, RIG-I-like helicase

receptors cross-species in groups according to their function be summarized.

adapter molecules

molecules who take a signal from receptors and cross-species be summarized in groups according to their function.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - EP 388758 [0032]
• WO 9602655 [0032]
• WO 9502397 [0032]
• WO 9715680 [0032]
• US 4946787 [0034]
• - EP 394111 [0034]
• WO 9405624 [0034]
• - WO 97002419 [0034]
• US 5264618 [0034]
• WO 9640067 [0034]
• - US 6008038 [0041]
• US 4750100 [0041]
• - US 5869326 [0041]

Cited non-patent literature

• Luke A. et al .; Spectrum of Science, August 2005, pages 68-75 [0001]
• Heine H. et al .; Int. Arch. Allergy Immunol. 2003; 130; 180-192 [0003]
• Uematsu S. et al., J. Biol. Chem. 2007, May 25; 282 (21); 15319-23) [0003]
• - Perry AK et al; Cell Research 2005; 15 (6); 407-422 [0004]
• Kawai T. et al .; J. Biochem; 2007; 141; 137-145) [0004]
• - Zimmer A. et al .; PNAS, 1999; 96 (10), 5780-5785 [0005]
• - Dr. Shizuo Akira, General Press Release 2002 [0007]
• - www.robertkoch.stiftung.de [0007]
• - Siegmund-Schultze N., www.aerzteblatt.de [0008]
• - Perry AK et al; Cell Research 2005; 15 (6); 407-422 [0009]
• Kawai T. et al .; J. Biochem; 2007; 141; 137-145 [0009]
• Isaacs, A. et al .; J. Proc. R. Soc. Lond. B. Biol. Sci. 147, 258-267 [0022]
• - Domb AJ; Review in Molecules; 2005; 10; 34 [0027]
• - Xiang G; Keun-Sik K .; Dexi L .; Review in The AAPS Journal; 2007 [0027]
• - http://www.aapsj.org [0027]
• JP Behr et al .; Proc. Natl. Acad. Sci. USA; 1995; 92; 7297 [0032]
• - SC De Smedt et al .; Phar. Res .; 2000; 17; 113 [0032]
• - Szoka FC et al .; Bioconjug. Chem .; 1996; 7; 703 [0032]
• W. Guang Liu et al .; J. Control. release; 2002; 83; 1 [0032]
• P. van de Wetering et al .; J. Gene Med .; 1999; 1; 156 [0032]
• - JP Behr; Bioconjugate Chem .; 1994; 5; 382 [0034]
• Leventis et al .; Biochim. Biophys. Acta; 1990; 1023; 124 [0034]
• - Huang et al; Biochem. Biophys. Res. Commun .; 1991; 179; 280 [0034]
• - War AM et al; Proc. Natl. Acad. Sci. USA; 1998; 95 (21); 12631-6 [0052]
• Kaufman RJ et al., Proc. Natl. Acad. Sci. USA; 1999; 96 (21); 11693-5 [0052]
• Loiarro M. et al .; J. Biol. Chem. 2005; 16; 15809-14 [0054]
• - Derossi D. at al. J. Biol. Chem. 1994, 269; 10444-10459 [0054]

Claims (34)

A method for increasing the transfection efficiency of a non-viral gene delivery system, wherein the non-viral gene delivery system comprises, in particular, a cationic lipid, a cationic polymer, a cationic protein, or a compound having a DNA and / or RNA-binding domain capable of inducing receptor-mediated endocytosis, or in which the non-viral gene delivery system is based in particular on a physical method such as electroporation, microinjection, ultrasound or a ballistic or hydrodynamic method, characterized in that the innate immune defense is reduced. The method of claim 1, wherein the innate Immune defense by at least one antibody, Intrabody, Aptamer, antagonist, inhibitor and / or a siRNA decreased becomes. Method according to one of claims 1 or 2, wherein at least one of the TLR3, 7, 8, 9, RIG-I helicase and / or RIG-I-like helicase is blocked. The method of claim 3, wherein the TLR 9 with at least one modified or unmodified DNA sequence, especially with the turf sequence TTAGGG is blocked. The method of claim 2, wherein by knock-down With siRNA at least one gene is switched off, which for encodes a protein necessary for signal transduction. Method according to one of claims 1 or 2, where the adapter molecule blocks MyD88 and / or TRIF becomes. The method of claim 6, wherein the adapter molecule is blocked by a peptide with the structural motif RDVLPGT. Method according to one of claims 1 or 2, in which at least one of the kinases IRF kinase TBK1, phosphatidylinositol 3-kinase, IRAK-1, IRAK-4, IKK-alpha or PKR is blocked. Method according to one of claims 1 or 2, in which at least one of the transcription factors IRF1, IRF3, IRF7, IRF9, IRF2, IRF4, IRF5, IRF6, IRF8, STAT1, STAT2, ISGF3 or NF-kB is blocked. Method according to one of claims 1 or 2, in which at least one cytokine is blocked. Method according to one of claims 1 or 2, in which at least one tumor necrosis factor is blocked. Method according to one of claims 1 or 2, in which at least one interleukin is blocked. Method according to one of claims 1 or 2, in which at least one interferon is blocked. The method of claim 13, wherein at least one Interferon of type I is blocked. Method according to one of claims 13 or 14, in which at least interferon-alpha or interferon-beta is blocked. Method according to one of claims 1 or 2, in which at least one receptor for cytokines is blocked. The method of claim 16, wherein at least one Receptor for type I interferons is blocked. Method according to one of the preceding claims, in which the drug modifies modified or unmodified ssDNA or unmodified dsDNA, modified or unmodified ssRNA, modified or unmodified dsRNA and / or modified or includes unmodified siRNA. Composition containing at least two of the following Components includes: a) a non-viral gene delivery system, b) an active ingredient, and c) a means of reducing the innate Immune defense. Kit of Parts, which includes at least two of the following components includes: a) a non-viral gene delivery system, b) one Active ingredient, and c) a means of reducing the innate Immune defense. Composition or kit of parts according to one of Claims 19 or 20, wherein: a) the non-viral gene delivery system a cationic lipid, a cationic polymer, a cationic one Protein and / or a compound comprising a DNA and / or Having RNA-binding domain and receptor-mediated endocytosis can trigger. Composition or kit of parts according to one of Claims 19 to 21, wherein: b) the active substance genetic Material, in particular modified or unmodified ssDNA or dsDNA or ssRNA or dsRNA. Composition or kit of parts according to one of Claims 19 to 22, wherein: c) the means of reduction the innate immune system an activity-reducing or activity-enhancing effector. Composition or kit of parts according to one of Claims 19 to 23, wherein: c) the means of reduction the innate immune system an antibody, intrabody, Aptamer, antagonist, inhibitor and / or siRNA. Composition or kit of parts according to claim 24, wherein the antibody, intrabody, aptamer, antagonist, Inhibitor and / or the siRNA at least one signal transduction cascade of the innate immune system. Composition or kit of parts according to one of Claims 19 to 25, wherein: c) the means of reduction the innate immune system includes the turf sequence TTAGGG. Composition or kit of parts according to one of Claims 19 to 26, wherein: c) the means of reduction The innate immune system includes genetic material that causes a knock-down a protein of a signal transduction cascade of the innate Immune system causes. Composition or kit of parts according to one of Claims 19 to 23, wherein: c) the means of reduction The innate immune system includes genetic material that is used for Expression of a protein that results in the activity a protein of a signal transduction cascade of the innate Immune system blocked. Composition or kit of parts according to one of Claims 19 to 25, wherein: c) the means of reduction the innate immune system comprises a peptide with the structural motif RDVLPGT. Kit of parts according to one of claims 19 to 29, where either: - all components separated from each other, or - the components a) and b) together, or - the components a) and c) together, or - The components b) and c) are present together. Pharmaceutical composition containing a composition according to any one of claims 19 to 30. Pharmaceutical Kit of Parts, which is a kit of Parts according to one of claims 19 to 30. Use of a composition or kit of parts according to any one of claims 19 to 30 for treatment from a disease caused by a genetic defect. The use of claim 33, wherein the disease is cystic fibrosis, muscular dystrophy, phenylketonuria, maple syrup disease, propionic acidemia, methylmalonic acidemia, adenosine deaminase deficiency, hypercholesterolemia, hemophilia, β-thalassemia, cancer, viral disease, degeneration macula, lateral amyotrophic sclerosis and / or inflammatory disease.