EP2212425A2 - Amélioration de résultats de transfection de systèmes de livraison de gènes non viraux par action sur le système immunitaire inné - Google Patents

Amélioration de résultats de transfection de systèmes de livraison de gènes non viraux par action sur le système immunitaire inné

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Publication number
EP2212425A2
EP2212425A2 EP08851432A EP08851432A EP2212425A2 EP 2212425 A2 EP2212425 A2 EP 2212425A2 EP 08851432 A EP08851432 A EP 08851432A EP 08851432 A EP08851432 A EP 08851432A EP 2212425 A2 EP2212425 A2 EP 2212425A2
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European Patent Office
Prior art keywords
tlr
transfection
receptor
cells
antibody
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EP08851432A
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German (de)
English (en)
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Roland KLÖSEL
Stefan König
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Biontex Laboratories GmbH
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Biontex Laboratories GmbH
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Priority claimed from DE102007056488A external-priority patent/DE102007056488A1/de
Priority claimed from DE102008016275A external-priority patent/DE102008016275A1/de
Priority claimed from DE102008023913A external-priority patent/DE102008023913A1/de
Application filed by Biontex Laboratories GmbH filed Critical Biontex Laboratories GmbH
Priority to EP11001260A priority Critical patent/EP2363494A1/fr
Publication of EP2212425A2 publication Critical patent/EP2212425A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • Immune responses of the body (Luke A. et al., Spectrum of Science, August 2005, pages 68-75) against an infectious or immunological challenge differentiate between innate immunity and acquired immune response. ,
  • 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. If only the acquired immune defense were present, the organism would be completely unprotected before the first infection. However, this is not the case, as there is another very original immune defense called innate immune defense, found from the fly Drosophila to mammals, and even plants.
  • TLRs Toll-like receptors
  • RIG-I-like helicases RIG-I-like helicases
  • PAMPs pathogen-associated molecular patterns
  • TLRs Toll-like receptors
  • 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 Nüsslein-Volhard, which you called "Toll.” TLR proteins are similar to this type and are referred to as "Toll-Iike" proteins.
  • LRR leucine-rich repeat domain
  • cytoplasmic domain homologous to that of the IL-1 R family, which selectively respond to different molecular viral and bacterial components and direct gene activation via a signal transduction cascade
  • adapter molecules and, subsequently, kinases that ultimately activate transcription factors eg NF-kB and the IRF families
  • Cytokines are once again necessary stimulators of the acquired immune defense and thus also a link between innate and acquired immune defense, although the principles of ligand recognition, signal transduction and signal transduction are still in the offing understood.
  • TLRs 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.
  • the TLR4 recognizes lipopolysaccharides, but also taxol. So far it is not known how the TLRs can do that.
  • the TLRs differ 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 lipopolysaccharides
  • TLR4 receptor requires an additional membrane-bound protein (TLR assisting protein) for its function.
  • CD14 binds eg. the LPS and deliver it to the TLR4 receptor. Binding to CD14 alone does not trigger a signal transduction cascade.
  • TLR5 is the receptor of flagellin, a major constituent of hostages (flagellae), with which bacteria move.
  • TLR6 forms a heterodimer with TLR2, is the receptor of diacylated lipoprotein and certain peptidoglycans.
  • CD36 membrane-bound protein CD36
  • TLR7 & TLR8 are receptors for imidazoquinolines and ssRNA / dsRNA, e.g. of RNA viruses.
  • 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, which is also detected by TLR9. 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 (elucidation of 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.robert-koch.stattung.de).
  • TLR2 4, 5 and 6 are located in particular 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.aeticianblatt.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 TLR 3 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.
  • TLR3 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 in part similar, but suggest There are also larger differences, so that in the end different gene expression and thus different biological reactions occur. With the exception of TLR3, all TLRs pass their signal to the adapter protein MyD88. MyD88 plays a critical role in signal transduction via 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).
  • TIR Toll / IR-1 Receptor Domain
  • MyD88-deficient splenocytes showed e.g. no reactions to interleukin-1, LPS or CpG DNA.
  • 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) of MyD88 for their signal forwarding.
  • Other adapter molecules are e.g.
  • TIRAP Toll Interleukin-i Receptor domain-containing adapter protein (TLR1, TLR2, TLR4 and TLR6), Mal (MyD88 adapter-like), TRIF (TLR3 and TLR4) and TRAM (TLR4).
  • a signal transduction cascade usually begins with a receptor on the cell surface with a cytosolic domain that, when mapped to a suitable ligand, transduces its signal through cytosolic adapter molecules to kinases that activate cascade transcription factors.
  • the activated transcription factors localize in the nucleus and trigger the expression of proteins, mostly cytokines.
  • TLR1 / TLR2, TLR2 / TLR2, TLR2 / TLR6 The receptors that occur in corresponding pairs trigger the same signal transduction cascades when occupied with suitable ligands.
  • the transcription factors NF-kB and AP-1 are activated, leading in particular to the expression of cytokines.
  • the adapter molecules RAC-1, TIRAP, MyD88 and TRAF6 are involved in the signal transmission.
  • the kinases involved are at least IRAK1, IRAK4, TAK1, PI3K, IKKalpha, IKKbeta, IKKgamma, JNK, p38 MAPK and MKKs.
  • the receptor requires the membrane-bound protein CD14 for its full function.
  • CD14 binds appropriate agonists and leads you to the receptor. This triggers the activation of the transcription factors NF-kB, AP-1, IRF3 and IRF7 in the assignment with suitable ligands and in turn leads in particular to the expression of cytokines.
  • the adapter molecules TIRAP, MyD88, TRAM, TRIF, TRAF3, TRAF6, NAP1 and RIP1 are involved in the signal transmission.
  • the kinases involved are at least IRAK1, IRAK4, TAK1, IKKalpha, IKKbeta, IKKgamma, IKKepsilon, TBK1, ERK1, ERK2, JNK, p38 MAPK, MEK1, MEK2 and MKKs.
  • the receptor ultimately solves the activation of the transcription factors NF-kB and AP-1, which lead in particular to the expression of cytokines.
  • the adapter molecules MyD88 and TRAF6 are involved in the signal transmission.
  • the kinases involved are at least IRAK1, IRAK4, TAK1, IKKalpha, IKKbeta, IKKgamma, JNK, p38 MAPK and MKKs.
  • the receptors finally resolve the activation of the transcription factors NF-kB and AP-1, which lead in particular to the expression of cytokines in the assignment with suitable ligands.
  • the adapter molecules MyD88 and TRAF6 are involved in the signal transmission.
  • the receptor triggers the activation of the transcription factors NF-kB, AP-1, IRF3 and IRF7 in the assignment with suitable ligands, in turn, in particular, cytokines are increasingly expressed.
  • the adapter molecules TRIF 1 TRAF6, TRAF3, NAP1 and RIP1 are involved in the signal transmission.
  • As kinases at least IRAK1, IRAK4, TAK1, IKKalpha, IKKbeta, IKKgamma, IKKepsilon, TBK1, PKR, PI K3, JNK, p38 MAPK and MKKs are involved.
  • the receptors in the assignment with suitable ligands ultimately trigger the activation of the transcription factors NF-kB, AP-1, IRF1, IRF5 and IRF7, again in particular cytokines are increasingly expressed ..
  • the adapter molecules MyD88, TRAF6 and TRAF3 involved.
  • IRAK1, IRAK4, TAK1, IKKalpha, IKKbeta, IKKgamma, JNK, p38 MAPK and MKKs are involved.
  • TLRs are of great therapeutic interest.
  • TLR agonists are used e.g. used as an adjuvant in vaccination strategies or in cancer therapy. Examples are the treatment of basal cell carcinoma by the TLR7 / 8 agonist imiquimod / resiquimod or bladder cancer by a TLR2 agonist.
  • the TLR9 receptor is driven by a synthetic CpG-containing oligonucleotide (CpG 7909 and CpG 10101) for the treatment of autoimmune diseases, cancer and infectious diseases.
  • CpG 7909 and CpG 10101 synthetic CpG-containing oligonucleotide
  • TLR7 and TLR8 receptors are generally immidazoquinolines (Schoen et al., Oncogene, 2008, 27, 190-199), such as imiquimod (R837, 1- (2-methylpropyl) -1H-imidazole [4, 5-c] quinoline-4-amine), resiquimod (R848, 4-amino-2- (ethoxymethyl) - ⁇ , ⁇ -dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol) and gardiquimod (1- (4-amino-2-ethylamino-methyl-imidazo [4,5-c] quinolin-1-yl) -2-methyl-propan-2-ol),
  • Guanosine analogs such as. Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) and others such as. Bropyrimines (2-amino-5-bromo-6-phenyl-4-pyrimidinones).
  • Other immidazoquinolines have been described in the literature (Miller et al Drug News & Perspectives, 2008, 21 (2), 69-87; Gorden et al., J. Immunol., 2005, 174, 1259-1268; Jurk et al. , Eur. J. Immunol., 36, 1815-1826; 2006; Gorden et al., J.
  • ssRNA especially single stranded poly-uridine and ssRNA with U or GU rich sequences
  • the ssRNA can also be present as phosphothioate. Particular mention should be made of the sequence motifs UGUGU and GUCCUUCAA (Hornung et al., Nat. Med., 2005, 11, 263-270, Judge et al., Nat.
  • ssRNA are stimulating from a length of 16bp.
  • the ssRNAs must be transported into the endosomes to reach the TLR8. In general, this is done with the usual transfection reagents.
  • the company InvivoGen offers a ssRNA40 complexed with a cationic lipid (Lyovec) as a TLR8 agonist.
  • TLRs react with decay products of the body's own cells and thus cause the immune system to fail.
  • the TLRs are even suspected to be causally related to cardiovascular disease. Inflammatory reactions in the heart can contribute to the formation of atherosclerotic plaques, which can ultimately lead to an infarct by vascular occlusion.
  • Cytokines are multifunctional signaling substances. These are sugary proteins that have a regulatory function for the growth and differentiation of body cells. Some of them are therefore also referred to as growth factors. Many cytokines also play an important role in immunological reactions and are therefore also called mediators. Cytokines are released from the cells into the surrounding medium by secretion and stimulate other cells if they have an appropriate receptor. There are five main groups of cytokines:
  • Interferons instruct cells to produce proteins that complicate or prevent viral infection. Also, interferons may have antitumoral activity.
  • Interleukins serve in particular the communication of immune defense cells with one another and thereby increase the coordination in the defense of pathogens and the fight against tumors
  • Colony stimulating factors are formed in the kidney. These are growth factors for blood cells
  • TNF Tumor Necrosis Factors
  • TNFs The most important function of TNFs is to regulate the activity of different immune cells. They are mainly released by macrophages. TNFs can stimulate cell death (apoptosis), cell proliferation, cell differentiation and release of other cytokines.
  • Chemokines are chemoattractants that cause cells with matching receptors to migrate through chemotaxis to the source of chemokines
  • interferons IFN
  • the first interferon of this type was found by Isaacs and Lindemann in 1957 (Isaacs, A. et al., J. Proc Lond B. Biol., 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)
  • IFN-alpha receptor IFN-alpha receptor
  • IFN-alpha receptor IFN-alpha receptor
  • IFN-beta IFN-kappa
  • IFN-delta IFN-epsilon
  • IFN-tau IFN-omega
  • IFN-zeta IFN-alpha receptor
  • IFN-beta proteins two are described. These are IFNB1 and IFNB3. A protein described as IFNB2 could later be identified as a known interleukin.
  • the so-called JAK / STAT pathway After binding to the interferon stimulated gene factor 3 (ISGF3), which is located in the outer cell membrane, the interferon stimulated gene factor 3, a heterotrimer of the transcription factors STAT1, STAT2 and " IFN regulatory factor 9 (IRF9), which migrates into the nucleus and induces the transcription of hundreds of effector molecules (known as IFN inducible genes), which directly influence protein synthesis, cell growth, and survival in the process of establishing the so-called "Antiviral status" (antiviral state). In this status, the infectivity of the viruses, e.g. prevented or at least reduced by reduced replication rates.
  • ISGF3 interferon stimulated gene factor 3
  • IRF9 IFN regulatory factor 9
  • the adaptive immune system is activated by triggering the maturation of dendritic cells, activating the B cell antibody response, and the T cell response. Lymphocytes and monocytes are recruited to the site of infection by induced chemokines.
  • Stress can also trigger signal transduction pathways leading to antiviral status. These signal transduction cascades cross the signal transduction cascades of the TLR.
  • Cellular stress can be triggered by:
  • inflammation biological and chemical agents eg TNFalpha, chemotherapeutic agents
  • the cells respond to stress with complex changes in signal chain activity involving specific MEK, MSK and MAP kinases and various transcription factors (eg NF-kB), apoptosis regulators and cell cycle regulators.
  • GTP-binding proteins (Ras / Rho family) bound to the membrane play a special role in the cell's response to cellular stress.
  • the innate immune response occurs both intracellularly and intercellularly.
  • a cell affected by contact with a pathogen triggers signal transduction cascades via PRRs, such as TLRs and RLHs, thereby altering the physiological status and expression profile of the cell.
  • PRRs such as TLRs and RLHs
  • the intercellular response is different from the intracellular response by the different receptors and agonists. Cytokine receptors at the intercellular response and PRRs at the intracellular response act as receptors.
  • the agonists of the intercellular response are cytokines and in the intracellular response the agonists are pathogenic patterns.
  • Transfection ie the introduction of genetic material into eukaryotic cells, especially mammalian cells, is today a method that has become 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, elucidating the function of different genes would be essential difficult. Not to be forgotten is the possibility to faithfully produce proteins of eukaryotic origin, as the correct posttranslational modification by the eukaryotic cells, unlike previously used prokaryotic cells, is ensured.
  • viruses as carrier systems. Since the introduction of DNA or RNA into foreign cells is an integral part of the multiplication cycle of the viruses, this ability has been refined by a natural, evolutionary process in the history of the virus to such an extent that to date there are no more effective gene carriers.
  • the naturally occurring viruses are genetically engineered to lose their ability to reproduce and be pathogenic, yet infect a cell with recombinant genetic material. Because viruses, except for genetic material, consist essentially of proteins, they offer the immune system a large attack surface. The immune system has developed strategies to resist these invaders in an evolutionary adjustment process. Therefore, the body's immune response is cited as a particularly significant factor in failed gene therapy studies.
  • the currently available gene delivery methods can be divided into the two main groups of viral systems and non-viral systems.
  • the non-viral systems can in turn be differentiated into chemical and physical methods.
  • those which are based on cationic lipids (so-called lipofection) or cationic polymers (so-called polyfection) are particularly worth mentioning. Their efficiency is usually far behind the viral systems.
  • Well-known cationic polymers are, for example, poly-L-lysine (PLL), (EP 388758) polyethylenimine (PEI), (JP Behr et al., Proc. Natl. Acad., See, USA; 1995; 92; 7297; WO 9602655). , Diethylaminoethyldextran (DEAE), (SC De Smedt et al, Phar Res, 2000, 17, 113), Starburst Dendrimers (PAMAM), (FC Szoka et al, Bioconjug Chem, 1996; 7; 703; WO 9502397), chitosan derivatives (W.Gu Liu, Li et al, J.
  • cationic lipids 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 usually formulated in ethanolic aqueous buffer solutions as micelles or liposomes.
  • colipids e.g., DOPE
  • lipids are usually formulated in ethanolic aqueous buffer solutions as micelles or liposomes.
  • colipids e.g., DOPE
  • they are commercially available as reagents, such as Lipofectin, Lipofectamin, Lipofectamine 2000 (Invitrogen), Fugene (Roche), Effectene (Qiagen), Transfectam (Promega), Metafectene (Biontex) etc. available.
  • 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 condensed by the compensation of the negative charge on the phosphate radical, so minimized in size.
  • the transfection efficiency of lipoplexes or polyplexes depends on a variety of parameters. The most important are the quantitative ratio of genetic material to cationic component in the production of the lipo / polyplexes, ionic strength during the preparation of the lipo / polyplexes, absolute amount of lipo / polyplexes per cell, cell type, proliferation state of the cells, physiological status of the cells, cell division rate, Incubation time, etc. These influence parameters are an expression of a complicated Trans Stammionsholds, in which the lipo / polyplexes or the genetic material contained must overcome a variety of cellular barriers.
  • the first barrier is the outer negatively charged cell membrane. It is believed that transfection-active lipoplexes, which must have a net positive charge, enter the interior of the cell by adsorptive endocytosis or liquid phase endocytosis.
  • endocytosis which is an active transport process of the cell, material is encased on the cell surface with cell membrane and intemalized as a vesicle (endosome).
  • lysosomes which contain a complex mixture of enzymes, the substances contained in the endosomes are broken down. Because of the low pH required for this degradation, endosomes have proton pumps that pump protons into the endosomes until a proper pH is achieved. To maintain charge neutrality, chloride ions flow into the endosomes to the same extent.
  • DNA can not enter the cell nucleus, which is called the "nuclear barrier," but it arrives at its site of action during cell division, resulting in the expression of proteins.
  • Other non-viral methods based on chemical methods include systems carrying a DNA-binding moiety and a ligand capable of triggering receptor-mediated endocytosis (for example, transferinfection, Wagner et al., Proc. Natl. Acad. 1990, 87, 3410).
  • RNA and / or RNA-binding domains consist of a DNA and / or RNA-binding domain, as well as a ligand that can trigger membrane transfer; e.g. Penetratin, Derossi et al .; Trends in Cell Biology; 1998; 8th ; 84 or HIV Tat Petid, Gratton et al .; Nature Medicine; 2003; 9 (3); 357.
  • membrane transfer is meant that a molecule can pass from one side of the membrane to the other side.
  • Suitable DNA and / or RNA-binding domains are all structural elements of a compound capable of binding DNA and / or RNA via electrostatic interactions (eg, cations) or hydrogen bonds (eg, peptide nucleic acids, PNAs).
  • Intercalating compounds eg acridine
  • the compounds 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 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.
  • the cells are exposed to an intense electrical impulse (pulse), which leads to a reversible opening (pores) of the cell membrane.
  • pulse ie voltage curve
  • the pulse ie voltage curve as one of the most important success parameters must be optimized for each cell type.
  • electroporators eg, Eppendorf / Multiporator, US 6008038, Biorad / Genpulser, US 4750100, Genetronics Inc., US 5869326, BTX / ECM series
  • electroporators eg, Eppendorf / Multiporator, US 6008038, Biorad / Genpulser, US 4750100, Genetronics Inc., US 5869326, BTX / ECM series
  • the cells are suspended in an electroporation buffer, transferred to an electroporated electroporation cuvette along with the DNA / RNA to be transfected, and exposed to one or more pulses.
  • other important parameters are the nature of the buffer, the temperature, the cell concentration and the DNA concentration. After the cells are exposed to the pulse they are left to regenerate the cell membrane for a short time. Subsequently, the cells are sown in a culture vessel and cultivated as usual.
  • Other physical methods include microinjection, hydrodynamic methods, ballistic methods (Genegun) or methods that use ultrasound or the injection of naked DNA into various organs, which leads to low expression of the corresponding genes.
  • magnetofection which uses DNA-binding molecules on magnetic nanoparticles to enrich DNA on the surface of cells via a magnetic field gradient and to trigger endocytosis
  • the innate immune system of eukaryotes may present a significant barrier to non-viral gene delivery systems.
  • the reason is that the eukaryotic innate immune system is able to recognize foreign genetic material via Toll Like receptors and initiate signal transduction cascades that trigger an antiviral state of cell populations.
  • Such an antiviral state of a cell also provides a barrier to transfection with a non-viral gene delivery system that is difficult or impossible to overcome.
  • genes can be selectively switched off if the mRNA has a sequence homology to the introduced dsRNA.
  • RNA interference Fire et al., Nature, 1998, 391, 806-8111 and usually proceeds as follows. An inserted into the cell dsRNA with homologous RNA interference (Fire et al., Nature, 1998, 391, 806-811) and usually proceeds as follows. An inserted into the cell dsRNA with homologous RNA interference (Fire et al., Nature, 1998, 391, 806-811) and usually proceeds as follows. An inserted into the cell dsRNA with homologous
  • the sequence of a cell-specific mRNA is converted by the enzyme Dicer into many small dsRNA
  • Dicer is an ATP-dependent ribonuclease.
  • the resulting nucleotide fragments have at the 3 'end 2-3 nucleotides, which survive.
  • the small pieces of RNA are called "small interfering RNA” (siRNA) (Elbashir et al., Nature, 2001, 411, 494-498).
  • RNA interference RNA interference
  • RNA interference is found in protozoa, fungi, plants and animals, although the individual mechanisms differ slightly. Archaebacteria and prokaryotes do not have this ability.
  • RNA viruses serve to repel RNA viruses.
  • virus-infected plants can recover, and newly developed leaves weaken the symptoms. Symptom-free leaves can no longer be infected with related viruses.
  • Complementary copies of the invading virus or RNA are used to serve as a template for the synthesis of the original RNA. It forms virus-specific dsRNA, which triggers the PTGS mechanism. Since initially the concentration of dsRNA is too low, the plant can only gradually recover and gain control of the viruses. It is thought that a cell-specific RNA-dependent RNA polymerase (RdRP) recognizes the single-stranded viral genome, converts it into dsRNA and then starts the RNAi process.
  • RdRP cell-specific RNA-dependent RNA polymerase
  • RNA interference has gained immense importance in recent years because it offers the possibility of eliminating unwanted genes or proteins and thus of combating viral and other diseases. Furthermore, it has also become an indispensable tool in the discovery of gene-function relationships.
  • RNA interference is the transfection of synthetically produced siRNA molecules, ie double-stranded RNA with a 3'-overhang of 2-3 nucleotides.
  • siRNA molecules ie double-stranded RNA with a 3'-overhang of 2-3 nucleotides.
  • more than 30bp of introduced dsRNA causes enzymatic destruction of all mRNAs and stops protein synthesis (Kaufmann, Proc Natl Acad., USA, 1999, 96, 11693-11695).
  • some higher eukaryotes can react with the production of interferons, which can inhibit the expression of viral genes and direct the cell into apoptosis.
  • the length of the siRNA must be below 30 bp for mammalian cells (Tuschl et al., Genes Dev., 2001, 15, 188-200).
  • transfection methods known from the DNA transfection methods are available. The difference to DNA transfection consists only in the site of action of the introduced genetic material. This is the core of DNA transfection. In siRNA transfection it is the cytosol.
  • reagents suitable for siRNA transfection are commercially available. Examples are interferon (Polyplus), X-treme genes siRNA (Roche), siPort (Ambion), Silentfect (Biorad), Dharmafect (Dharmacon) and Lipofectamine RNAiMax (Invitrogen).
  • siRNA transfection the relative knockdown of a protein or a gene compared to an untreated sample or with a non-specific siRNA (siRNA without target) transfected sample.
  • a problem in siRNA transfection is so-called OFF-target effects. This includes, for example, the unimpaired impairment of the expression of a gene that is not the target of knockdown. This can eg. come in sequence similarities.
  • OFF-target effects This includes, for example, the unimpaired impairment of the expression of a gene that is not the target of knockdown. This can eg. come in sequence similarities.
  • a requirement for potential siRNA transfection systems is to achieve the highest possible knockdown with the lowest possible amount of siRNA used.
  • Another preferred requirement, particularly for in vivo applications is to change the expression profile of the cells as little as possible, apart from the expression of the target protein.
  • the object of the invention is to provide a method which allows a more efficient transfection. This applies both to a simple transfection and to a repeated, ie at least two or more transfections. Furthermore, the task is to influence the physiological status of the cell population as little as possible, ie Ideally, the protein expression profile of the cell population should only change with respect to proteins whose genes have been introduced into the cell or whose expression is reduced or blocked by the introduced genetic material. In the case of transfection of siRNA, however, it may also be advantageous to purposely alter the physiological status of the cell in order to bring the cells into an "antiviral status", since RNA interference is particularly efficient here
  • a kit of parts is provided containing the components for performing more efficient transfection of eukaryotic cells with non-viral gene delivery systems.
  • This object is achieved by a method for improving the transfection of non-viral gene delivery systems, characterized in that
  • the method according to the invention for improving the transfection result can be carried out in vitro and / or in vivo.
  • the transfection result can be improved and / or unwanted changes in the expression profile of a transfected cell can be avoided.
  • 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.
  • 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.
  • drugs can be delivered to the endosomes via liposomal carriers.
  • the target site is the cytosol
  • electroporation or special peptide sequences capable of permeabilizing the cell walls are particularly preferred.
  • the active ingredients are peptides or proteins, then according to the invention they can also be formed intracellularly by transfection of suitable genetic material and, if necessary, be directed with localization sequences to the corresponding cell compartments. 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. It may be used to prevent or even prevent the formation of "antiviral status" of cells during transfection, as the cells may also be associated with biological material, such as serum or trypsin, which may contain substances during cultivation If one or more TLRs (eg, DNA, RNA, LPS, etc.) are responsive, then cells may already be in an antiviral state before transfection is started, and considering this background, it becomes clear why The quality of transfection results strongly depends on the initial immunological state of the cells, which in turn depends on the pretreatment (eg subcultivation).
  • TLRs eg, DNA, RNA, LPS, etc.
  • the non-viral gene delivery system may comprise a cationic lipid, a cationic polymer, or a cationic protein; and / or comprise a compound having a DNA and / or RNA-binding domain capable of inducing receptor-mediated endocytosis or membrane transfer; and / or include a compound covalently linked to DNA and / or RNA capable of inducing receptor-mediated endocytosis or membrane transfer; and / or on a physical method such as electroporation, microinjection, magnetofection, ultrasound or a ballistic or hydrodynamic method.
  • transfection may be performed at least twice, i. two or more times (3, 4, 5, 6, etc.).
  • a cationic lipid may preferably be present in the non-viral gene delivery system, in particular a cationic lipid according to formula (I):
  • R 2 and R 3 independently of one another are dodecyl, dodecenyl, tetradecyl, tetradecenyl, hexadecyl, hexadecenyl, octadecyl, octadecenyl or other alkyl radicals which in all possible combinations may be saturated, unsaturated, branched, unbranched, fluorinated or non-fluorinated, and from 5 to 30 carbon atoms can be constructed;
  • R 2 and R 3 independently of one another, are dodecyl, dodecenyl, te
  • the innate intracellular and / or intercellular immune defense can be blocked by at least one antibody, intrabody, aptamer, antagonist, inhibitor and / or an siRNA which blocks the transmission of an intracellular and / or intercellular signal of the innate immune defense (en ), at least partially suppressed.
  • en an intracellular and / or intercellular signal of the innate immune defense
  • Corresponding antibodies, intrabodies, aptamers, antagonists and inhibitors are commercially available.
  • siRNA short hairpin RNA
  • TLRs TLRs
  • kinases transcription factors
  • transcription factors are partially commercially available (Imgenex / Invivogen).
  • TLR 1, TLR 2, TLR 4 and / or TLR 9 TLR 10, TLR 11, TLR 12, TLR 13, CD14, CD38, RIG-I helicase and / or RIG-I-like helicase.
  • antibodies are suitable which are able to block the TL receptors.
  • antibodies against the Toll Like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and CD14 are known and commercially available.
  • Antibodies capable of blocking TLR can be channeled into the endosomes to block the receptors. As a rule, however, an addition to the extracellular space is sufficient.
  • the choice of the receptor to be blocked also depends on the nature of the genetic material to be transfected.
  • the transfecting agent can also determine the choice of the receptor to be blocked.
  • the TLRs that detect genetic material i. TLR 3, TLR 7, TLR 8 and TLR 9 are usually located in the endosomes. The remaining TLR sit on the plasma membrane. If one wants to transfect DNA, for example, TLR 9 can preferably be blocked. If the DNA is of bacterial origin, it is preferable to block TLR 9, TLR 4 and / or TLR 5 in addition to TLR.
  • an antibody concentration of 0.01-100 ⁇ g / ml is used by addition to the culture medium.
  • the concentration of the antibody is 0.01-1 ⁇ g / ml of culture medium and in the most preferred embodiment 0.01-5 ⁇ g / ml.
  • the antibody may also be incorporated into the transfection-active complex, e.g. Lipoplex, integrated.
  • the ratio of antibody: genetic material is from 0.01: 1 ( ⁇ g / ⁇ g) to 10: 1 ( ⁇ g / ⁇ g).
  • the amount is 0.01-1 ⁇ g / ⁇ g and in the most preferred embodiment 0.01-0.3 ⁇ g / ⁇ g genetic material.
  • the antibody or antibodies used must / must generally be directed against the target molecule to be blocked, eg. a receptor such as a TLR, cytokine receptor, interferon receptor, etc. of the cells to be transfected.
  • a receptor such as a TLR, cytokine receptor, interferon receptor, etc.
  • the antibody must typically target the human TLR receptor that is to be blocked.
  • the antibodies are because of the high similarity of the target molecules from different species, eg.
  • TLRs also cross-reactive; that is, although an antibody to a target molecule has been developed by one species, it also exhibits its properties against a similar target molecule from another species.
  • antibodies from cells are preferred of the same species to be transfected, since in this case they are little or not immunogenic.
  • the antibodies may also have been produced recombinantly.
  • the antibodies used may be polyclonal or monoclonal, with monoclonal antibodies being preferred, or a modified antibody may be used, for example
  • a modified antibody may lack the Fc fragment because binding to the target molecule / antigen is solely through the Fab fragments
  • a modified antibody may also be modified with hydrophobic groups such as lipids to facilitate its anchoring in membranes and antigens If it is desired to detect the antibody used more easily, it can also be labeled with a fluorescent dye, or several modifications can be made to an antibody forbid such as certain preservatives.
  • the kinase MEK1 and / or MEK2 can be inhibited by a compound which has an IC50 value of less than 100 nM. Furthermore, according to the invention, the kinase MEK1 and / or MEK2 can be blocked by 1,4-diamino-2,3-dicyano-1,4-bis (o-amionophenylmercapto) butadiene (U0126). In a preferred embodiment, a concentration of 1-500 ⁇ M is used. In a still more preferred embodiment, the concentration is 1-100 ⁇ M, and in the most preferred embodiment 1-30 ⁇ M.
  • Interferon to be blocked is an interferon of type I, in particular at least one of the interferons, selected from interferon-alpha, interferonbeta, interferon-gamma or interferon-omega.
  • interleukin-1 is considered as the interleukin to be blocked.
  • At least one receptor for cytokines can be blocked, in particular at least one receptor for interferons, in particular of type I, at least one receptor for interleukins, and / or at least one receptor for tumor necrosis factors.
  • the receptor for interleukin-1 may be blocked by an antibody or an antagonist such as IL-ra (Human Interleukin-1 Receptor Antagonist, Biomol, Cat. No .: 54592).
  • IL-ra Human Interleukin-1 Receptor Antagonist, Biomol, Cat. No .: 54592
  • Another preferred target is the interferon-gamma receptor.
  • the antagonist (s) used must generally be active against the target molecule to be blocked, eg. a receptor such as an interleukin receptor, cytokine receptor, interferon receptor, etc. that targets cells to be transfected.
  • a receptor such as an interleukin receptor, cytokine receptor, interferon receptor, etc. that targets cells to be transfected.
  • the antagonist will usually have to target the human receptor that is to be blocked.
  • the antagonists are because of the great similarity of the target molecules from different species, eg. TLRs, also cross-reactive; i.e.
  • an antagonist has been developed against a target molecule from one species, it also exhibits its properties against a similar target molecule from another species.
  • antagonists from cells of the same species to be transfected are preferably used, since they are little or not immunogenic in this case.
  • the antagonists may also have been produced recombinantly or synthetically.
  • the antagonist used binds high affinity to its target molecule.
  • Antagonists used according to the invention may also be modified; analogous to the modified antibodies.
  • a preferred embodiment of the invention several of the aforementioned receptors and / or proteins involved in the signal transduction cascade to induce innate intracellular and / or intercellular immune defense are blocked.
  • several antibodies to TLR receptors can be combined to to use additive effects and / or synergy effects.
  • inhibitors and / or antibodies, and / or intrabodies, and / or aptamers, and / or antagonists, and / or siRNA against TLR-receptors and / or TLR-assisting proteins and / or adapter molecules and / or kinases and / or Transcription factors and / or cytokines and / or cytokine receptors are combined to at least partially interrupt the innate immune signal transduction cascade.
  • the method of the invention can be further used to improve the transfection results of siRNA transfections with non-viral gene delivery systems in a number of ways.
  • the siRNA may be modified or unmodified.
  • an agonist in the method according to the invention, can be used as a means for activating the innate immune defense.
  • the method according to the invention can be used to activate the RNA interference machinery by stimulating the intracellular part of the innate immune system. Activation is achieved by occupying various TLR receptors with appropriate agonists, but especially by occupancy (and thus activation) of the receptors TLR7 and TLR8.
  • the receptors TLR7 and TLR8 are located in the endosomes and detect ssRNA as expected in the case of an infection of an RNA virus. The activation of these receptors leads to a particularly strong "antiviral status" to which an active RNA interference machinery can be assigned. The assumption that RNA interference is a mechanism for the defense against viruses fits easily into the overall picture.
  • Improving the transfection results of siRNA transfections differs from the transfection with other genetic material in that, for good transfection success, an active RNAi machinery that is part of the innate immune system must be available, whereby the active RNAi machinery overcompensates the negative influence of the innate immune system to the uptake of siRNA.
  • the innate intracellular and / or intercellular immune defense can be at least partially activated by at least one agonist for a TL receptor, in particular by at least one agonist for TLR7 and / or TLR8.
  • the at least one agonist for TLR7 and / or TLR8 can be selected from the group comprising bropirimines (2-amino-5-bromo-6-phenyl-4-pyrimidinones), imidazoquinolines, thiazoloquinolines, guanosine analogs and ssRNA.
  • the at least one agonist imiquimod (R837, 1- (2-methylpropyl) -1H-imidazole [4,5-c] quinolin-4-amine), resiquimod (R848, 4-amino-2-) (ethoxymethyl) - ⁇ , ⁇ -dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol) or gardiquimod (1 - (4-amino-2-ethylamino-methyl-imidazo [4,5-c] quinoline) -1 -yl) -2-methylpropan-2-ol); or CL075 or CL097; or loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) or isolibine (7-thia-8-oxoguanosine); or ssRNA with U and / or GU rich sequences, in particular ssRNA with the sequence motifs UGUGU and / or GUCCUUCAA
  • Imiquimod can be used at a concentration of 0.1-100 ⁇ g / ml, preferably at a concentration of 0.1-20 ⁇ g / ml, and most preferably at a concentration of 0.1-10 ⁇ g / ml.
  • the addition preferably occurs at the same time as the transfection and / or later.
  • the ssRNA has a length of at least 15 bases.
  • the ssRNA may have a phosphothioate backbone.
  • an ssRNA of 20 nucleotides in length may be employed at a concentration of 0.1-100 ⁇ g / ml, preferably 0.1-20 ⁇ g / ml, and most preferably 0.1-10 ⁇ g / ml.
  • the addition of the ssRNA preferably takes place at the same time as the transfection and / or after the transfection.
  • the immunostimulatory ssRNA may be included in the transfection-active complex of non-viral gene delivery system and siRNA or plasmid DNA encoding shRNA.
  • the cell-permeable agonists can be added directly to the medium of the cells before, during or after the actual siRNA transfection step.
  • the ssRNA and its analogues must be complexed with suitable transfection reagents, since they are not cell-friendly by nature and thus do not reach the TLRs in the endosomes.
  • the agonists can be covalently bound to cationic polymers.
  • the inventive method can be used to activate the RNA interference machinery by stimulating the intercellular part of the innate immune system. Activation is achieved by occupying receptors of antiviral cytokines with suitable agonists.
  • interferon beta and / or interferon gamma are used in a concentration of 1-10000 U / ml. In a still more preferred embodiment, the concentration is 1-5000 and in the most preferred embodiment 1-2000 U / ml. The addition preferably occurs at the same time as the transfection and / or later.
  • the inventive method can be used to stimulate parts of the adaptive immune system, which are suitable for the activation of the siRNA machinery and at the same time to block other parts, eg. by regulating the level of endocytosis influence the amount of siRNA entered. Synergy effects can be exploited.
  • the cells can be treated for up to 4 days, preferably up to 18 hours, in particular up to 6 hours before transfection with the at least one agent for at least partial activation of the innate intracellular and / or intercellular immune defense.
  • the cells can be treated for up to 2 days, preferably up to 12 hours, in particular up to 6 hours after transfection, with the at least one agent for at least partial activation of the innate intracellular and / or intercellular immune defense.
  • the cells can be treated simultaneously with the at least one means for at least partially suppressing or activating the innate intracellular and / or intercellular immune defense and be brought into contact with the non-viral gene delivery system.
  • the method according to the invention can furthermore be used to prevent an undesired reaction of the innate immune system and thus an altered gene expression of the cell. This is of particular importance in in vivo applications and in the elucidation of protein functions by siRNA transfections in complex signal pathways. Often, different signal transduction pathways of the cell are also coupled with the signal transduction pathways of the innate immune system. In the case of a knockdown of a protein, which at the same time massively influences the physiological status of the cell, the assignment of the function to the protein is difficult. In order to avoid this, the method according to the invention can be used to partially or completely block the response of the innate immune system. The method according to the invention is suitable for the transfection of eukaryotic cells.
  • the cells When the eukaryotic cells are transfected in vitro, the cells may be adherent or suspended in a suitable culture medium. Preferably, at the time of transfection, the cells are in the logarithmic phase of proliferation in the case of transfection with DNA. If RNA is to be transfected, the cells are preferably in the lag or log phase at the time of transfection. If a transfection is to be carried out with the aim of expressing a protein, comes as a genetic material dsDNA with an expressible as RNA or peptide / protein portion or ssRNA with a peptide / protein expressible portion (either modified or unmodified) into consideration.
  • modified or unmodified dsDNA with a portion expressible as a small hairpin RNA (shRNA) or modified or unmodified siRNA can be used; only in this case is an activation of the TLR 7 and / or TLR 8 useful.
  • shRNA small hairpin RNA
  • additional blocking of TLR and / or cytokine receptors and / or interruption of signal transduction by blocking MEK1 and / or MEK2 may be advantageous, particularly so as not to affect the expression profile of the cells as much as possible.
  • a method according to the invention may preferably comprise the following method steps:
  • step (d) incubating the mixture of step (c);
  • the first solution may be an antagonist or antibody to a cytokine receptor or TLR 1, TLR 2, TLR 3, TLR 4, TLR 5, TLR 6, TLR 7, TLR 8, or TLR 9 as the target molecule; an inhibitor for the target molecule MEK 1 and / or MEK 2; or an agonist for TLR 7 and / or TLR 8; in particular, an above-mentioned same.
  • An antagonist or antibody may preferably be in a buffered saline solution, eg PBS, basal medium, etc., having a physiological pH of 6.8-7.45 and a physiological osmolality of 270-310 mosmol / kgH 2 O, or also in water or in unbuffered saline solution having a pH of 5-9.
  • the inhibitor can also be diluted in solubility problems in a suitable physiologically acceptable solvent, eg ethanol / DMSO.
  • the concentration of the antagonist, antibody or inhibitor in the first solution may be 0.01-1 ⁇ g / ⁇ l.
  • the first solution contains an MEK 1 and / or MEK 2 inhibitor, or an antibody or antagonist directed against TLR 1, TLR 2, TLR 3, TLR 4, TLR 5, or TLR 6 or cytokine receptor.
  • the addition is made by adding an appropriate amount of the first solution to the culture medium of the cells to be transfected.
  • the resulting concentration of the antibody or antagonist in the culture medium of the cells to be transfected may be from 0.01 ⁇ g / ml to 50 ⁇ g / ml, preferably 0.05-12 ⁇ g / ml, in particular 0.1-5 ⁇ g / ml.
  • the resulting concentration of the inhibitor in the culture medium of the cells to be transfected may be 1 nM to 500 ⁇ M, preferably 5-100 ⁇ M, in particular 10-50 ⁇ M.
  • the mixing of the second solution with the non-viral gene delivery system and the third solution with the genetic material to be transfected can be carried out by pipetting.
  • Per transfected cell can be used on genetic material to be transfected 0.1 picogram / cell to 300 picograms / cell, preferably 0.2-50 picograms / cell, in particular 1-10 picograms / cell to be transfected genetic material.
  • the amount of non-viral gene delivery system depends on the amount of genetic material. In the case of an electrostatic binding of the non-viral gene delivery system to the genetic material, the amount is defined by the charge ratio (+/-) between gene delivery system and genetic material.
  • the charge ratio (+/-) Gencastersystem: genetic material can be 0.1: 1 to 100: 1, preferably 1: 1 to 20: 1, in particular 4: 1 to 10: 1. In the event of a non-electrostatic bond defines the amount of non-viral
  • Gene delivery system on the stoichiometric ratio to the genetic material may be from 0.1: 1 to 1000: 1, preferably 1: 1.
  • the mixture of the second solution with the non-viral gene delivery system and the third solution with the genetic material to be transfected may be incubated for a period of 1 minute to 30 minutes, preferably 10-15 minutes.
  • a non-viral gene delivery system comprising a cationic lipid, in particular an abovementioned cationic lipid according to the formula (I), can be used.
  • steps (a) to (e) can be repeated after 24 h in order to transfect the cells once again with genetic material.
  • the culture medium with additives in which the cells to be transfected are to be replaced by fresh culture medium.
  • a first solution with an antagonist or antibody to TLR 3, TLR 7, TLR 8 or TLR 9 can be mixed with a second solution with a non-viral gene delivery system and a third solution with the genetic material to be transfected.
  • the first solution with an antibody / antagonist is added to the second solution with a non-viral gene delivery system and mixed.
  • the amount of antibody used is 0.1% by weight to 50% by weight, based on the amount of the non-viral gene delivery system, preferably 0.1 to 10% by weight.
  • the amount of non-viral gene delivery system depends on the amount of genetic material to be used.
  • the amount is defined by the charge ratio (+/-) between gene delivery system and genetic material.
  • the charge ratio (+/-) Gencastersystem: genetic material can be 0.1: 1 to 100: 1, preferably 1: 1 to 20: 1, in particular 4: 1 to 10: 1.
  • the amount of non-viral gene delivery system is defined by the stoichiometric ratio to the genetic material.
  • the stoichiometric ratio of non-viral gene delivery system: genetic material may be from 0.1: 1 to 1000: 1, preferably 1: 1.
  • the mixture of first and second solution is preferably incubated for at least 5 minutes.
  • the third solution of genetic material to be transfected is added to the incubated mixture of first and second solutions and mixed.
  • Each cell to be transfected can be transfected with genetic material 0.1 Picogram / cell to 300 picograms / cell, preferably 0.2-50 picograms / cell, especially 1-10 picograms / cell of genetic material to be transfected.
  • the antibody / antagonist is incorporated into the transfection-active complex of non-viral gene delivery system and genetic material.
  • antibodies / antagonists are used which are modified with a lipophilic part of the molecule to facilitate the binding of the antibody / antagonist into the lipoplex.
  • TLR 3, TLR 7, TLR 8 or TLR 9 may be covalently linked to the non-viral gene delivery system.
  • cationic immunoliposomes from liposomes comprising cationic lipids or lipoplexes i.
  • Complexes containing DNA and cationic lipids to covalently label with antibodies or antibody fragments are known.
  • the methods were developed to allow targeting of the gene delivery systems towards the target cells in the body.
  • Most methods use so-called cross-linkers.
  • Cross-linkers are bifunctional molecules that create a covalent link between two molecules with corresponding functional groups.
  • Pierce offers a broad range of water-soluble and water-insoluble heterobifunctional (that is, more suitable for linking two different functional groups) and homobifunctional (more suitable for linking two like functional groups) cross-linkers.
  • carboxyl and amino groups can be used; in the case of Fab fragments, the thiol group can also be used for linking.
  • Cationic lipids and polymers contain amino functions for linking.
  • carboxyl and amino groups can be used for linking.
  • Cationic immunoliposomes can be formulated by derivatizing antibodies with lipids and adding them to the cationic lipids (possibly with colipids) prior to formulation.
  • homobifunctional cross-linkers such as. DSP (dithiobis [succinimidyl propionate]
  • amino functions can be used to covalently link a lipid with an amino function with an antibody.
  • a thiol function and the cross-linker N-succinimidyl-4- (p-maleimidophenyl) butyrate (Martin et al., J. Biol.
  • lipoplexes with modified antibodies covalently bound to a lipid are known to those skilled in the art.
  • the lipoplexes then absorb the modified antibodies (Lee et al., J. Biomed., Sei, 2003, 10, 337).
  • methods for cationic polymers such as. PEI or dendrimers or cationic proteins, such as. Polylysine (Chen et al., FEBS Letters; 1994; 338; 167; Suh et al; J. Controlled Release; 2001; 72; 171) covalently link to antibodies or antibody fragments.
  • PEI is eg. reacted with DPS in DMSO and added to an antibody solution in PBS. After dialysis, the antibody-coupled non-viral gene delivery system is available (Chiu et al., J. Controlled Release, 2004); 97; 357).
  • the first solution may contain an agonist for activating the TLR 7 and / or TLR 8, in particular one of the abovementioned.
  • the agonist may be dissolved in a buffered saline solution, eg, PBS, basal medium, etc., having a physiological pH of 6.8-7.45 and a physiological osmolality of 270-310 mosmol / kgH 2 O, in water or in an unbuffered saline solution pH of 5-9 be solved.
  • the concentration of the agonist may be 0.01-1 ⁇ g / ⁇ l.
  • This first solution with a TLR 7 and / or TLR 8 agonist may be added before, during or after transfection to the cells to be transfected in the culture medium.
  • the time of agonist treatment should be chosen so that an innate immune system as active as possible encounters the highest possible number of siRNAs.
  • the addition of the first solution with the agonist is carried out simultaneously with the addition of the transfection complex to the cells to be transfected by adding a corresponding amount of first solution to the culture medium.
  • the amount of agonists depends on the amount of cells. Preference is given to using 0.1 ⁇ g of agonist / cell to 25 ⁇ g of agonist / cell, preferably 1-500 ⁇ g of agonist / cell, in particular 10 to 250 ⁇ g of agonist / cell.
  • the amount of genetic material depends on the type of genetic material, the target compartment, eg blood, extracellular fluid of the tissues, cell tissue, etc., the form of administration and the type of addition, eg infusion, injection, or inhalation, and is 0.01-500 mg / kg body weight.
  • the amount of antibody, antagonist or agonist upon direct administration of a first solution to an individual may be from 0.1 mg / kg to 500 mg / kg of body weight, preferably 1-100 mg / kg, especially 5-10 mg / kg.
  • the amount of inhibitor in a direct administration of a first solution to an individual may be 0.1 mg / kg to 500 mg / kg body weight, preferably 10-300 mg / kg, especially 100-200 mg / kg.
  • the time and duration of the addition of a first solution depends on the target compartment, eg blood, extracellular fluid of the tissue, cell tissue, etc., the form of administration and the mode of administration, eg infusion, injection, inhalation.
  • the time interval between two transfections may be up to 6 weeks.
  • the invention further provides a composition comprising at least two of the following components: a) a non-viral gene delivery system, c) genetic material, and b) a means for at least partially suppressing or activating the innate intracellular and / or intercellular immune defense.
  • kits of parts which comprises at least two of the following components: a) a non-viral gene delivery system, c) genetic material, and b) a means for at least partially suppressing or activating the innate intracellular and / or intercellular immune defense.
  • the non-viral gene delivery system comprises a cationic lipid, a cationic polymer, a cationic protein; and / or a compound having a DNA and / or RNA-binding domain capable of inducing receptor-mediated endocytosis or membrane transfer; and / or a compound which is covalently bound to DNA and / or RNA and can induce receptor-mediated endocytosis or membrane transfer.
  • genetic material for example, genetic material for repairing a gene defect, e.g. genetic material, in particular modified or unmodified ssDNA, modified or unmodified dsDNA, modified or unmodified ssRNA, modified or unmodified dsRNA and / or modified or unmodified siRNA.
  • genetic material for example, genetic material for repairing a gene defect, e.g. genetic material, in particular modified or unmodified ssDNA, modified or unmodified dsDNA, modified or unmodified ssRNA, modified or unmodified dsRNA and / or modified or unmodified siRNA.
  • At least one activity-reducing or activity-enhancing effector can be used as a means for at least partially suppressing and / or activating the innate intracellular and / or intercellular immune defense.
  • an antibody, intrabody, aptamer, antagonist, inhibitor and / or a siRNA can be used according to the invention as a means for at least partially suppressing the innate intracellular and / or intercellular immune defense.
  • the antibody (s), intrabody, aptamer, antagonist, inhibitor and / or siRNA used as a means for at least partially suppressing the innate intracellular and / or intercellular immune defense may preferably block at least one signal transduction cascade of the innate immune system.
  • an agonist can be used as a means for at least partial activation of the innate intracellular and / or intercellular immune defense.
  • the agonist used as a means for at least partially activating the innate intracellular and / or intercellular immune defense can activate at least one signal transduction cascade of the innate immune system.
  • the agent according to the invention for the at least partial suppression of the innate intracellular and / or intercellular immune defense can also be genetic material which can cause knockdown of a protein of a signal transduction cascade of the innate immune system.
  • the means according to the invention for the at least partial suppression of the innate intracellular and / or intercellular immune defense can comprise genetic material which can lead to the expression of a protein which can block the activity of a protein in a signal transduction cascade of the innate immune system.
  • a transfection composition of the invention may comprise (a) a non-viral gene delivery system and (b) a means for at least partially suppressing and / or activating the innate intracellular and / or intercellular immune defense, wherein the non-viral gene delivery system comprises:
  • (i) may comprise a cationic lipid, a cationic polymer, or a cationic protein; and / or (ii) may comprise a compound containing a DNA and / or RNA-binding
  • Can trigger membrane transfer and / or (iii) may comprise a compound covalently bound to DNA and / or RNA capable of inducing receptor-mediated endocytosis or membrane transfer; and the means for at least partially suppressing and / or activating the innate intracellular and / or intercellular immune defense, may be selected from:
  • TLR 1 an antibody to TLR 1, TLR 2, TLR 3, TLR 4, TLR 5, TLR 6, TLR 7,
  • TLR 8 TLR 9, TLR 10, TLR 11, TLR 12, or TLR 13;
  • an antibody to a cytokine receptor or a cytokine receptor an antibody to a cytokine receptor or a cytokine receptor
  • an inhibitor of kinase MEK1 and / or MEK2 an inhibitor of kinase MEK1 and / or MEK2;
  • an agonist for TLR7 and / or TLR8 selected from the group comprising bropyrimines (2-amino-5-bromo-6-phenyl-4-pyrirnidinones),
  • Imidazoquinolines thiazoloquinolines, and guanosine analogs; and (v) a combination thereof.
  • a transfection kit of parts according to the invention may preferably comprise (a) a non-viral gene delivery system and (b) a means for at least partial suppression and / or Activation of the innate intracellular and / or intercellular immune defense, wherein the non-viral gene delivery system comprises:
  • (i) may comprise a cationic lipid, a cationic polymer, or a cationic protein; and or
  • (ii) may comprise a compound having a DNA and / or RNA-binding domain capable of inducing receptor-mediated endocytosis or membrane transfer;
  • (iii) may comprise a compound covalently linked to DNA and / or RNA capable of inducing receptor-mediated endocytosis or membrane transfer; and the means for at least partially suppressing and / or activating the innate intracellular and / or intercellular immune defense, may be selected from:
  • TLR 1 an antibody to TLR 1, TLR 2, TLR 3, TLR 4, TLR 5, TLR 6, TLR 7,
  • TLR 8 TLR 9, TLR 10, TLR 11, TLR 12, or TLR 13;
  • an antibody to a cytokine receptor or a cytokine receptor an antibody to a cytokine receptor or a cytokine receptor
  • an agonist for TLR7 and / or TLR8 selected from the group comprising bropirimines (2-amino-5-bromo-6-phenyl-4-pyrimidinones), imidazoquinolines, thiazoloquinolines, and guanosine analogs; and (v) a combination thereof.
  • composition of the invention or a kit of parts according to the invention comprises a non-viral gene delivery system with a cationic lipid.
  • composition according to the invention or a kit of parts according to the invention may comprise a non-viral gene delivery system with a cationic lipid according to the following formula:
  • R 2 and R 3 are independently dodecyl, dodecenyl, tetradecyl, tetradecenyl, hexadecyl, hexadecenyl, octadecyl, octadecenyl or other alkyl radicals, which in all possible combinations may be saturated, unsaturated, branched, unbranched, fluorinated or non-fluorinated, and from 5 can be built up to 30 carbon atoms;
  • composition or the kit of parts may contain modified or unmodified genetic material, in particular modified or unmodified ssDNA, modified or unmodified dsDNA, modified or unmodified ssRNA, modified or unmodified dsRNA and / or modified or unmodified siRNA.
  • the composition or the kit of parts may be used as a means for at least partially suppressing and / or activating the innate intracellular and / or intercellular immune defense 1, 4-diamino-2,3-dicyano-1,4-bis (o-amionophenylmercapto) - butadiene (U0126); Imiquimod (R837, 1- (2-methylpropyl) -1 H -imidazole [4,5-c] quinoline-4-amine); Resiquimod (R848, 4-amino-2- (ethoxymethyl) - ⁇ , ⁇ -dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol); Gardiquimod (1- (4-amino-2-ethylamino-methyl-imidazo [4,5-c] quinolin-1-yl) -2-methyl-propan-2-ol); CL075; CL097; Loxoribine (7-allyl-7,8-dihydro-8-oxo-guamino
  • composition or kit of parts as an agent for at least partially suppressing and / or activating the innate intracellular and / or intercellular immune defense may contain an antibody to TLR 3, TLR 7, TLR 8, or TLR 9.
  • composition or the kit of parts according to the invention as an agent for at least partial suppression and / or activation of the innate intracellular and / or intercellular immune defense an antibody or antagonist against interleukin-1 receptor, in particular IL-ra; Interferon type I receptor; Interferon-gamma receptor; or tumor necrosis factor receptor.
  • interleukin-1 receptor in particular IL-ra; Interferon type I receptor; Interferon-gamma receptor; or tumor necrosis factor receptor.
  • several of the above-mentioned means for at least partially suppressing and / or activating the immune defense may be combined with each other, i.
  • Two, three or more components can be used in the agent according to the invention for the at least partial suppression and / or activation of the innate intracellular and / or intercellular immune defense. It is likewise possible to use a plurality of the abovementioned components a) and / or b) and / or c).
  • kit of parts according to the invention can:
  • all components may be completely separate from one another; the components a) and b) are present separately from one another; or components a) and b) are present together.
  • the components may either be separate from one another, for example in glass or plastic containers, which are packaged together, or the components may be provided in pairs or more in corresponding containers.
  • the kit of parts may comprise the non-viral gene delivery system in the form of salts, in particular formulated as liposomes / micelles; as a solution, in particular in the form of an aqueous solution, saline solution, buffered saline solution, eg saline solution with HEPES buffer, or solution in a physiologically acceptable solvent, eg ethanol / DMSO; in lyophilised form; as a solid or as a film.
  • Solutions of the non-viral gene delivery system preferably have a pH of 5 to 9.
  • the final concentration of the non-viral gene delivery system in the culture medium is preferably 0.01-10 mg / ml.
  • the means for at least partially suppressing and / or activating the innate intracellular and / or intercellular immune defense may be lyophilized in a kit of parts according to the invention; dissolved in water, saline, buffered saline, e.g. Saline with PBS buffer, or a suitable organic solvent, e.g. Ethanol, DMSO, etc, be included. Solutions preferably have a pH of from 5 to 9. Optionally, stabilizers may be included.
  • the agent for at least partial suppression and / or activation of the innate intracellular and / or intercellular immune defense is preferably present in a concentration of 0.01 to 10 mg / ml.
  • Genetic material kits can lyophilize this genetic material, dissolved in water, saline or buffered saline, e.g. Saline solution containing TE buffer. Solutions of the genetic material preferably have a pH of 5 to 9. The final concentration of the genetic material in the culture medium is preferably 0.01-10 mg / ml.
  • composition according to the invention and / or the kit of parts according to the invention can be used for carrying out a method according to the invention.
  • composition according to the invention can be present as a pharmaceutical composition and the kit of parts according to the invention as a pharmaceutical kit of parts.
  • a composition or kit of parts according to the invention can be used for the treatment of a disease by gene therapy.
  • the disease may be, for example, cystic fibrosis, muscular dystrophy, phenylketonuria, maple syrup disease, propionic acidemia, methylmalonic acidemia, adenosine deaminase deficiency, hypercholesterolemia, hemophilia, ⁇ -thalassemia, cancer, viral disease, macular degeneration, amyotrophic lateral sclerosis, and / or inflammatory disease act.
  • the present invention can also be used to perform transfections without altering the expression profile of the cells to an undesired extent. This is of particular interest in in vivo applications because activation of the immune system is often a problem here.
  • FCS Mycoplex PAA, cat. No. E15-773
  • HeIa Luc cells stably transfected with luciferase cells
  • siRNA desalted, 30 pmol / ⁇ l in Universal Buffer (siMAX from MWG) directed against Luciferase GL3 (anti-luciferase siRNA): Sense Sequence: CUUACGCUGAGUACUUCGAtt Antisense Sequence: U CG AAG U AC UC AG CG U AAGtt Non-specific control : Sense Sequence: AGGUAGUGUAAUCGCCUUGtt Antisense Sequence: CAAGGCGAU UACACUACCUtt
  • HeLa cells are seeded in a 24 well plate. A cell count of 2.3 * 10 5 cells is plated in a well in 500 ⁇ l complete medium (10% FCS). The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • the antibody Sheep Polyclonal Antibody against human IFN ⁇
  • PBS Phosphate Buffered Saline
  • the efficiency of the transfection is performed with the ß-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.
  • 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:
  • FIG. 1 A first figure.
  • the antibody (Mouse monoclonal Antibody Against Human Interferon Alpha / Beta Receptor Chain) is mixed with 400 ⁇ l PBS (Phosphate Buffered Saline) and mixed gently. He now has a concentration of 0.1 ⁇ g / ⁇ l.
  • PBS Phosphate Buffered Saline
  • the lipoplex addition takes place after 5 h incubation time with the antibody
  • 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:
  • HeLa cells are seeded in a 48 well plate. A cell count of 1, 2 * 10 5 cells is plated in a well in 250 ⁇ l complete medium (10% FCS). The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • the antibody is adjusted with PBS to a concentration of 0.05 ⁇ g / ⁇ l. Subsequently, the wells of the 48-well plate are supplied with the following amounts of antibody:
  • the mixture is then incubated in the incubator for 5 or 0.5 h, depending on the antibody. Subsequently, the lipoplexes are produced. For this, 13 ⁇ l of DNA (pCMV-lacZ) are pipetted into 650 ⁇ l of PBS and mixed by gently pipetting up and down. 52 ⁇ l Metafectene Pro are also pipetted into 650 ⁇ l PBS and mixed by gently pipetting up and down. Now both solutions are mixed and incubated for 15 min. At the end, add 50 ⁇ l of the lipoplex solution to each well. The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • Antibody Mouse anti human TLR1 antibody (monoclonal, 0.05% sodium azide, 100 ⁇ g lyophilisate); Invivogen, No. Mab htlr1.
  • the antibody was dialyzed against PBS: dialysis membrane: Spectra / Por Dispo Dialyser (500 ⁇ l, cellulose ester membrane, 25,000 Da Molecular Weight Cut-Off); Spectrum Laboratories; No. 135492, Lot 3224004.
  • the antagonist is adjusted to a concentration of 0.025 ⁇ g / ⁇ l with PBS. Subsequently, the wells of the 48-well plate are supplied with the following amounts of antibody:
  • Taq HeLa cells are seeded in a 48 well plate. A cell count of 0.8 * 10 5 cells is plated in a well in 250 ⁇ l complete medium (10% FCS). The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • inhibitor 1 mg is dissolved in 100 ⁇ l DMSO (stock solution). Then dilute with PBS 1:20 (working solution). Subsequently, the wells of the 48-well plate are supplied with the following amounts of inhibitor:
  • the antibody was dialyzed against PBS: dialysis membrane: Spectra / Por Dispo Dialyser (500 ⁇ l, cellulose ester membrane, 25,000 Da Molecular Weight Cut-Off); Spectrum Laboratories; No. 135492, Lot 3224004.
  • HeLa cells are seeded in a 48 well plate. In this case, a cell count of 1, 0 * 10 5 cells in a well in 250 ul complete medium (10% FCS) plated. The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • the antibody is adjusted with PBS to a concentration of 0.1 ⁇ g / ⁇ l.
  • 15 ⁇ g DNA (pCMV ⁇ Gal) and 60 ⁇ l Metafectene Pro are each dissolved in 300 ⁇ l serum-free DMEM. By gently pipetting up and down, the individual solutions are mixed. Subsequently, lipoplexes of the following composition are prepared.
  • the Metafectene Pro solution is combined with the antibody solution and incubated for five minutes at room temperature (RT). Subsequently, the DNA solution and the serum-free DMEM are added and incubated for a further 15 minutes at RT. In each case 50 .mu.l of the DNA-antibody-lipid complexes are pipetted into the wells. The solution 1 is pipetted into four wells of column 1. Solution 2 in four wells of column 2 and so on. The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • Lanes 1 and 2 are duplicates of results for a single transfection.
  • Cols. 3 and 4 are duplicates of the results for a repetitive transfection. The average is therefore formed:
  • the added amount of antibody corresponds to the following amounts of antibody per well:
  • HeLa-Luc cells are seeded in a 48 well plate. A cell count of 1.5 ⁇ 10 4 cells is plated in a well in 250 ⁇ l of complete medium (10% FCS). The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • the wells of the 48 well plate are supplied with the following amounts of inhibitor:
  • the lipoplexes are prepared.
  • 1 ⁇ l of anti-luciferase siRNA stock solution is pipetted into 300 ⁇ l of PBS and mixed by gently pipetting up and down. Only 240 ⁇ l of this solution will be used.
  • 1 ⁇ l of non-specific-control siRNA is pipetted into 300 ⁇ l PBS and mixed by gently pipetting up and down. Only 40 ⁇ l of this solution will be used.
  • 3 ⁇ l of Metafectene Pro are pipetted into 225 ⁇ l of PBS and mixed by gently pipetting up and down. Now 190 ⁇ l are pipetted to the anti-luciferase siRNA working solution and 35 ⁇ l to the non-specific-control-siRNA working solution. The solutions are mixed and incubated for 15 min. This results in lipoplexes with an siRNA reagent ratio of 1: 5 ⁇ g / ⁇ l At the end, 15 ⁇ l each of the anti-luciferase siRNA lipoplex solution are added to the first 6 rows of the plate. 15 ⁇ l of the non-specific-control-siRNA-lipoplex solution are added to the 7th row of the plate. This results in a siRNA amount of 1 pmol / well. Row 8 remains untransfected. The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • FIG. 11 Example 9 siRNA transfection in the presence of the TLR7 / 8 agonists:
  • a cell count of 2 ⁇ 10 4 cells / cm 2 should be plated in a well in 250 ⁇ l complete medium.
  • the first 5 wells of the first 3 rows of a 48 well plate are filled with 30 ⁇ l PBS each.
  • the stock solution of anti-luciferase siRNA and non-specific-control siRNA is adjusted to a concentration of 1 pmol / 10 ⁇ l with PBS. Then each 10 .mu.l of the
  • the Metafectene Pro reagent is diluted 1: 300 with PBS. In each case, 20 ⁇ l of this dilution are added to each well with siRNA.
  • the lipoplexes are incubated for 20 minutes at room temperature. Thereafter, 250 ⁇ l of cell suspension are added per well.
  • the two 48-well plates are incubated for 48 hours in CO 2 .
  • the cells of a plate are trypsinized and, using a Neubauer counting chamber, a vitality determination is carried out with trypan blue for each well.
  • Luciferase assay RLU / ⁇ l cell extract / sec
  • TLR 4 detects traces of LPS in the plasmid solution.
  • TLR9 and TLR3 detect genetic Material, in this case plasmids or corresponding impurities.
  • TLR1 and TLR2 detect the transfection reagent.
  • TLRs before and during transfection depends on many factors. Depending on the expression profile of the target cell can eg. a particular sensitivity of the cells to be given to certain PAMPs. Furthermore, different reagents and different genetic material can activate different TLRs. Even different DNA sequences can influence the transfection results depending on the CpG content. Last but not least, contaminants also play a role that should not be underestimated. For example, DNA of bacterial origin may be contaminated with LPS or flagellin or RNA. ssRNA can be contaminated with dsRNA and thus, in addition to TLR7 / 8, also respond to TLR3 and vice versa. Different treatment of the cells by stress signal transduction cascades can lead to different results. In summary, it can be said that different blocking strategies are required for different transfection experiments.
  • the invention can be used for therapeutic purposes.
  • the invention may be useful for gene therapy of, for example, cystic fibrosis, muscular dystrophy, phenylketonuria, maple syrup disease, propionic acidemia,
  • Methylmalonic acidemia adenosine deaminase deficiency
  • hypercholesterolemia hemophilia
  • ß-thalassemia ß-thalassemia
  • Gene therapy methods are also of interest if hormones, growth factors, cytotoxins or immunomodulating proteins are to be synthesized in the organism.
  • DNA fragments can be effectively brought into cells by means of the invention, in which this DNA can develop the desired effect without leading to undesired side effects.
  • the desired effect may be the replacement of missing or defective DNA regions or the inhibition of DNA regions (for example by antisense DNA / RNA or siRNA) which cause the disease in the diseased cell type.
  • tumor-suppressing genes can be used in cancer therapy or by introducing cholesterol-regulating genes, a contribution to the prevention of heart and blood vessel diseases are made.
  • DNA encoding ribozymes, siRNA or shRNA, or ribozymes or siRNA itself can be introduced into diseased cells.
  • the translation of the DNA produces active ribozymes or siRNA, which catalytically cleave m-RNA at specific sites and thus prevent transcription.
  • viral mRNA can be cleaved without affecting another cellular mRNA.
  • the multiplication cycle of viruses HAV, herpes, hepatitis B and C, respiratory syncytial virus
  • siRNA diseases specifically targeted for treatment with siRNA include age-related macular degeneration (eye disease), liver cancer, solid tumors, amyotrophic lateral sclerosis and inflammatory diseases.
  • Eye disease age-related macular degeneration
  • liver cancer solid tumors
  • amyotrophic lateral sclerosis and inflammatory diseases.
  • Transfection also plays a role in cancer therapy, for example an increasingly important role in the production of cancer vaccines. Thus, that is also possible application for the invention.
  • Another application may be found, for example, in vaccination methods which function on the basis of the expression of DNA encoding immunogenic peptides in the human and animal body.
  • DNA encoding immunogenic peptides in the human and animal body.
  • Lipid / DNA complexes used as vaccines.
  • the introduction of DNA into the body cells leads to the expression of the immunogenic peptide and thus triggers the adaptive immune response.
  • Amount of protein expression of a cell population as a result of transfection processes with genetic material encoding, inter alia, this expressed protein or extent of knockdown of protein expression of a cell population as a result of transfection processes with genetic material capable of triggering such knockdown in particular siRNA or Ribozymes or DNA encoding shRNA or ribozymes or proportion of cells of a total population of cells that demonstrates the biological activity of the introduced genetic material as a result of transfection processes.
  • the physiological status of the cell population should be influenced as little as possible, ie the protein expression profile of the cell population should ideally only change with respect to the proteins whose genes have been introduced into the cell or whose expression is reduced or prevented by the introduced genetic material should.
  • Non-viral gene delivery system is a 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.
  • 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, in particular, use physical methods, however, energy supply especially in shape 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.
  • the transport of DNA through the cell membrane occurs through an active transport mechanism of the cell, endocytosis.
  • Substances having these properties contain, in particular, cationic lipids, cationic polymers, cationic peptides or also molecules which have a domain which can bind DNA or RNA and at the same time have a second domain which contains a ligand recognized by a receptor also on the cell surface and causes endocytosis by this recognition process.
  • this ligand is able to initiate a membrane transfer, ie mediate transport to the other side of the membrane.
  • the substances can also be specially formulated, in particular as micelles or liposomes and also consist of several components, in particular with different functions.
  • the innate immune defense sets itself apart from the acquired or adaptive immune defense in that it fends off a pathogen, without ever having to come into contact with the pathogen to train the immune system.
  • the innate immune response is most cell types to n intrinsically and consists of two parts, the intracellular and intercellular fraction.
  • the intracellular portion utilizes the recognition of pathogen-associated molecular structures by receptors.
  • these receptors initiate signal transduction cascades, which in particular result in expression of many cell-owned genes and phosphorylation of important proteins in a changed physiological status (eg, "antiviral status") of the cells directly affected Cytokines are also secreted.
  • affected cells notify unaffected cells via these messenger substances (cytokines) and trigger a changed physiological status (eg "antiviral status"), whereby the cytokines dock onto and release cytokine receptors of the other cells
  • cytokines messenger substances
  • antiviral status eg "antiviral status”
  • the intercellular portion is differentiated from the intracellular portion by the different receptors (cytokine receptors instead of PRRs (pattern recognition receptors) and agonists (cytokines instead of pathogenic patterns).
  • the introduction of genetic material into eukaryotic cells takes place outside of a living organism, especially in vessels suitable for the cultivation of eukaryotic cells.
  • 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 by RNA interference.
  • nucleic acids Natural nucleic acids that have been altered by modification in their properties. These modifications may in particular be chemical changes, which relate in particular to the phosphate skeleton, the sugars or bases, which should in particular increase the stability of the nucleic acids to nucleases and ribonucleases.
  • molecules labels
  • molecules can be covalently or non-covalently attached to the nucleic acids leading to new properties of the nucleic acids, in particular to optical traceability by fluorescence labels or labels directing the nucleic acids to a specific location in the cell (localization elements) or labels which mediate the passage of nucleic acids through membranes and thus make nucleic acids cell-like, for example.
  • a modification is the exchange of oxygen for sulfur in the phosphate backbone, the methylation of 2'-OH groups of the ribose in the case of RNA, or the complete removal of 2'OH groups by fluorine to increase the stability against nucleases.
  • Another example is the attachment of FITC (fluorescein isothiocyanate) as a fluorescent label to follow the path of the genetic material in the cell microscopically, or the attachment of so-called NLS (nuclear localization signal, eg PKKKRKVG) to a transport into the cell nucleus to reach.
  • FITC fluorescein isothiocyanate
  • NLS nuclear localization signal
  • siRNA short interfering RNA
  • Short dsRNA (up to 28 bp) that can cause the knock-down of a protein through RNA interference.
  • shRNA short hairpin RNA
  • Short ssRNA that has complementary regions at the 3'-end and the 5'-end and thereby hybridizes via hydrogen bonding and forms a hairpin structure.
  • shRNA can cause the knock-down of a protein by RNA interference.
  • Molecule capable of detecting a substance (agonist) and thus a biological substance
  • receptors are proteins.
  • Suppression Interruption of the communication network of the immune defense, ie of one or more immune response inducing signal transduction cascade (s), in particular the innate intra- and / or intercellular immune system. This interruption weakens the immune system, reducing the immune response.
  • s immune response inducing signal transduction cascade
  • Non-covalent interactions are electrostatic forces and hydrogen bonds.
  • the information on the presence of this substance is converted into a signal and transmitted via a chain of molecules by signal transduction.
  • the signal generated by the receptor is taken up by adapter molecules and, in particular, carried on via kinases, in particular to transcription factors.
  • the transcription factors stimulate the expression of genes that mediate the biological response.
  • Molecules in particular proteins, which are capable of recognizing molecular structures, in particular other proteins, and influencing their biological action by binding.
  • blocking antibody ie in the case of the receptors of the innate immune system signal transmission is reduced or prevented.
  • the antibodies can be polyclonal or monoclonal.
  • the antibodies can be humanized. As a rule, you must be directed against the receptor of a species whose cells are to be transfected. Due to the similarity of the receptors, it can also happen that an antibody is cross-reactive.
  • the antibodies can be modified, ie it can, for example. Parts of the molecule (eg Fc Fragments so that only Fab fragments remain) may be cleaved, provided that the biological effect is not significantly reduced.
  • Residues eg, phospholipids, A. Schnyder et al., J. Am. Soc., For Exp. NeuroTherapeutics;
  • Intracellularly expressed antibodies Intracellularly expressed antibodies.
  • RNA molecules which, by forming a tertiary structure, are able to recognize molecular structures, in particular proteins, similar to an antibody and influence their biological activity by binding.
  • Aptamers may consist of modified or unmodified RNA or DNA.
  • An agonist is able to achieve a biological effect by binding to a receptor.
  • Cytokines that are expressed in an infection of a eukaryotic cell with viruses. Examples are interferon alpha, interferon beta, interferon gamma, TNF alpha, TFN beta, interleukin 6, 8, 15, 28 and 29. inhibitors
  • Molecules that can inhibit the biological activity of another molecule, in particular of proteins.
  • the inhibitors themselves are proteins, modified or unmodified nucleic acids or small organic molecules.
  • Receptors that are grouped across species according to their function.
  • Groups are grouped according to their function.
  • AP-1 Activated protein-1
  • ERK Extracellular-signal Regulated Kinase
  • IkB inhibitory-binding protein kB
  • IKKi IKK epsilon
  • IRAK1 interleukin 1 receptor-associated kinase 1
  • IRAK4 interleukin-1 receptor-associated kinase 4
  • IRF interferon regulating factor
  • JNK c-Jun N-terminal kinase
  • TIRAP MyD88-adapter-like
  • MAPK mitogen activated protein kinase
  • MEK MAPK / ERK kinase
  • MKK MAP kinase kinase
  • MKK mitogen-activated protein kinase kinase
  • MSK mitogen and stress activated kinase
  • MyD88 myeloid differentiation factor 88
  • NAP 1 Nck-associated protein 1
  • NF-kB nuclear factor kappaB
  • P1 3K phosphoinositol 3-kinase
  • PDK1 phosphoinositide-dependent protein kinase 1
  • PDK2 phosphoinositide-dependent protein kinase 1
  • RIP1 receptor-interacting protein 1
  • TAKI transforming growth factor- ⁇ -activated kinase
  • TRAF3 TNF receptor-associated factor 3
  • TRAF6 TNF receptor-associated factor 6
  • TRAM TRIF-related adapter molecule
  • TRIF Toll / IL-1 receptor domain-containing adapter inducing interferon-b adapter protein

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Abstract

Le système immunitaire inné d'eucaryotes est capable d'identifier au moyen de récepteurs de type Toll du matériau génétique étranger et de lancer des cascades de transduction de signal qui induisent via une réponse interféron un état antiviral de populations cellulaires. Cet état antiviral constitue également une barrière pour des systèmes de livraison de gènes non viraux. Si la cascade de transduction de signal est interrompue de façon intracellulaire ou intercellulaire, on peut augmenter l'efficacité de transfection de systèmes de livraison de gènes non viraux, et éviter des modifications indésirables du profil d'expression. Comme l'interférence ARN est imputable à l'état antiviral, la machinerie ARNi est également activée à la suite d'une activation du système immunitaire inné. On peut ainsi augmenter l'efficacité du knock-down lors de la transfection de siRNA.
EP08851432A 2007-11-22 2008-11-24 Amélioration de résultats de transfection de systèmes de livraison de gènes non viraux par action sur le système immunitaire inné Withdrawn EP2212425A2 (fr)

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DE102007056488A DE102007056488A1 (de) 2007-11-22 2007-11-22 Steigerung von Transfektionseffizienzen nicht-viraler Genliefersysteme durch Blockierung des angeborenen Immunsystems
DE102008016275A DE102008016275A1 (de) 2008-03-28 2008-03-28 Verbesserung von Transfektionsergebnissen nicht-viraler Genliefersysteme durch Blockierung des angeborenen Immunsystems
DE102008023913A DE102008023913A1 (de) 2008-05-16 2008-05-16 Verbesserung von Transfektionsergebnissen nicht-viraler Genliefersysteme durch Beeinflussung des angeborenen Immunsystems
PCT/EP2008/009947 WO2009065618A2 (fr) 2007-11-22 2008-11-24 Amélioration de résultats de transfection de systèmes de livraison de gènes non viraux par action sur le système immunitaire inné

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EP11001260A Withdrawn EP2363494A1 (fr) 2007-11-22 2008-11-24 Amélioration de résultats de transfection de systèmes de livraison de gènes non-viraux par l'influence du système immunitaire inné

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US10837020B2 (en) 2009-04-22 2020-11-17 Massachusetts Institute Of Technology Innate immune suppression enables repeated delivery of long RNA molecules
EP3248618A1 (fr) * 2009-04-22 2017-11-29 Massachusetts Institute Of Technology Suppression de l'immunité innée permettant l'administration répétée de longues molécules d'arn
WO2010133369A1 (fr) * 2009-05-20 2010-11-25 Biontex Laboratories Gmbh Procédé de transfection pour systèmes de transfert de gènes à efficacité améliorée par blocage du système immunitaire inné
ES2698076T3 (es) 2011-10-07 2019-01-30 Isd Immunotech Aps Identificación de los dominios inmunosupresores en las proteínas de fusión de los virus de ARN con envoltura
CN102349995B (zh) * 2011-10-24 2013-06-05 武汉大学 一种广谱抗病毒的药物及其制备方法和应用
HUE054698T2 (hu) * 2013-04-10 2021-09-28 Isd Immunotech Aps Immunszuppresszív domének gyógyszerként való felhasználása
EP2984171A2 (fr) * 2013-04-10 2016-02-17 Skau Aps Peptides ayant des domaines immunosuppresseurs pour la transfection
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WO2009065618A3 (fr) 2009-09-24
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WO2009065618A2 (fr) 2009-05-28
CN101918566A (zh) 2010-12-15

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