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WO2010133369A1 - Procédé de transfection pour systèmes de transfert de gènes à efficacité améliorée par blocage du système immunitaire inné - Google Patents

Procédé de transfection pour systèmes de transfert de gènes à efficacité améliorée par blocage du système immunitaire inné Download PDF

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WO2010133369A1
WO2010133369A1 PCT/EP2010/003105 EP2010003105W WO2010133369A1 WO 2010133369 A1 WO2010133369 A1 WO 2010133369A1 EP 2010003105 W EP2010003105 W EP 2010003105W WO 2010133369 A1 WO2010133369 A1 WO 2010133369A1
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receptor
antibody
delivery system
protein
kinase
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Roland KLÖSEL
Stephan KÖNIG
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Biontex Laboratories GmbH
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the innate immune system The innate immune system
  • 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.
  • the innate immune defense is the first defense front against pathogens and represents an evolutionarily very old system.
  • PRRs pattern recognition receptors
  • PAMPs pathogen-associated molecular patterns
  • DAMPS danger-associated molecular patterns
  • the PRRs can be divided into two groups, the so-called “endocytic PRRs” and the “signaling PRRs".
  • the “endocityc PRRs” do not trigger signal transduction but endocytosis and phagocytosis.
  • “Signaling PRRs” include the membrane-bound toll-like receptors (TLRs) (Heine H. et al., Int. Arch. Allergy Immunol. 2003; 130; 180-192 and Uematsu S. et al., J. Biol. Chem. 2007, May 25; 282 (21); 15319-23), the cytoplasmic NOD-like receptors and RNA helicases that specifically recognize PAMPs and initiate corresponding inflammatory and immune responses, thereby preventing the organism between "self” and “not.” NOD-like receptors and RNA helicases are ubiquitously expressed in the cytosol.
  • TLRs membrane-bound toll-like receptors
  • NOD-like receptors Inohara N, Nu ⁇ ez G., Nat Rev Immunol., 2003 May; 3 (5): 371-82)
  • NODs Approximately 20 of these proteins were found in the genome of mammals and can be divided into two major groups, so-called NODs and NALPs. Others are CIITA, IPAF and BIRC1. These receptors recognize endogenous and microbial molecules or stress signals and form oligomers that activate inflammatory molecules such as NF-kB, cytokines or caspases. The NOD-like receptors are also known under other names (eg CATERPILLER or CLR). NODs
  • NOD1 and NOD2 are the ligands currently known.
  • NOD1 detects a peptidoglycan of Gram-negative bacteria (meso-DAP).
  • NOD2 detects a muramyldipetide (MDP) of Gram-positive and Gram-negative bacteria.
  • MDP muramyldipetide
  • NODs feed the signal transduction pathway of NF-kB pathway and MAP kinases via the kinase RIP2. They got their name because they contain a nucleotide-binding oligomerization domain that binds Nukteotidtriphosphate. Nalps
  • NALPs Like NODs, NALPs also have a nucleotide-binding oligomerization domain. Currently, 14 members of this family are known. Some of these receptors also know what they detect. For example, NALP3 detects bacterial DNA, MDP, ATP, toxins, dsRNA, paramyoxy viruses and uric acid crystals. RNA helicases
  • RNA helicases such as RIG-I and MDA5. These helicases control antiviral programs.
  • RIG-I retinoic inducible gene I
  • Mda5 melanoma-differentiation-associated gene 5
  • the receptors are able to detect single-stranded RNA with a triphosphate residue at the 5 'position as well as dsRNA.
  • RIG-l-like helicases A group with similar properties as RIG-I is termed RIG-l-like helicases summarized.
  • the membrane-bound PRRs include, in particular, the mannose receptor and the toll-like receptors.
  • the mannose receptor is found mainly on the surface of macrophages and dendritic cells. It binds carbohydrates found on the surface of infectious microorganisms and triggers endocytosis and phagocytosis of the same.
  • 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. These are transmembrane proteins with an extracellular, "leucine-rich repeat" domain (LRR) as well as a cytoplasmic domain that is homologous to that of the IL-1 R family may respond to extracellular or endosomal PAMPs.
  • LRR leucine-rich repeat
  • the different TLRs selectively respond to different molecular viral and bacterial components and control gene activation through a signal transduction cascade. This is done first via so-called adapter molecules and subsequently via kinases which finally activate transcription factors (eg NF-kB and the IRF families) by phosphorylating the same or corresponding intracellular inhibitors of these transcription factors. Finally, in addition to a large number of specific genes that have an antimicrobial effect, so-called cytokines are produced. Cytokines are again necessary stimulators for the acquired immune defense and thus also a link between innate and acquired immune defense.
  • transcription factors eg NF-kB and the IRF families
  • TLRs 13 different TLRs are known (11 of them in mammals), the number of which 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
  • 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. When 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 Virtually every body cell has TLRs depending on their differentiation. In immune cells, they are often expressed very strongly. TLR2, 4, 5 and 6 are located, for example: in particular in the plasma membranes of monocytes, natural killer cells, mast cells or myotonic dendritic cells, 7, 8, and 9 are in particular 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. signal transduction:
  • 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 the TLRs is highly similar to that of the interleukin-1 receptor and is therefore also referred to as the Toll / IR-1 receptor domain (TIR).
  • MyD88-deficient splenocytes showed 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), TRAF6 (TLR1 to 9) and TRAM (TLR4).
  • a corresponding signal transduction cascade usually begins with a receptor on the cell surface with a cytosolic domain, which passes its signal on assignment with a suitable ligand via cytosolic adapter molecules to kinases that activate transcription factors via cascade.
  • 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, the especially lead 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.
  • the adapter molecules MyD88 and TRAF6 are involved in the signal transmission.
  • As kinases at least IRAK1, IRAK4, TAK1, IKKalpha, IKKbeta, IKKgamma and MKKs are involved.
  • 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, 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.
  • G protein-coupled receptors can also be attributed in part to the innate immune system, since they have a strong regulating effect on it. They are found only in eukaryotes and have as ligands a variety of substances, including hormones, lipids, proteins, pheromones, but also small peptides. When a ligand binds to the receptor, a so-called G protein is activated by a change in the conformation of the receptor. The further course of the signal transduction depends on the G-protein. Significantly, many components of the signal transduction pathways of the TLRs and the GPCRs are identical.
  • 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 secreted by the cells into the surrounding medium and stimulate other cells if they have an appropriate receptor. Often, the cytokine expression is controlled by the PRR signal transduction cascades. There are five main groups of cytokines:
  • IFN Interferons
  • 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 therefore stems from that 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, often resulting in the release of cytokine-derived TLR signaling cascades, and, in particular, type I interferons also not infected directly by a virus cells in an antiviral status, so "warned”.
  • Part of the antiviral response of the cells is the enhanced expression of 2 ', 5'-oligoadenylate synthase (OAS) and protein kinase R (PKR). Both are activated by dsRNA.
  • OFAS 2 ', 5'-oligoadenylate synthase
  • PSR protein kinase R
  • OAS then generates from ATP 2 ', 5' oligoadenylate, which in turn activates the ribonuclease RNaseL, which degrades the cell's own RNA.
  • the PKR in turn, phas- phorylates the translation-essential initiation factor elF2, which then undergoes an inactive complex with elF2B to stop transcription. Both effects counteract viral proliferation.
  • ribonuclease Rnasen
  • deoxyribonucleases Dnasen
  • Dnasen deoxyribonucleases
  • Dnasen his especially the human Dnasen Dnase 1, Dnase 2, Dnase1 / L2, Dnase1 / L3 and Dnase 2like acid Dnase called.
  • 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.
  • interleukins and tumor necrosis factors also have a strong influence on the innate immune defense and therefore represent cytosolic constituents of the innate immune defense according to the invention.
  • Stress can also trigger signal transduction pathways leading to antiviral status. These signal transduction cascades cross the signal transduction cascades of the TLR. Stress-signaling pathways:
  • Cellular stress can be triggered by:
  • TNFalpha biological and chemical agents
  • chemotherapeutic agents eg TNFalpha, chemotherapeutic agents
  • Cell is used in the present application in both the singular and plural, i. "Cells”, uses and defines in all cases a single cell, multiple cells or a cell population.
  • 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 Ros / Rho family
  • 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
  • there is an intercellular response in which the cell affected by contact with a pathogen "informs" other cells, which were not exposed to direct contact with the corresponding pathogen, of the "infection" with the pathogen Contact with a pathogen released cell cytokines, which are detected by cytokine receptors, which are located on the other cells not coming into contact with the pathogen.
  • Binding of the cytokines to the cytokine receptors triggers a signal transduction cascade in the cells that were not exposed to direct contact with the pathogen, with the result that their physiological status and expression profile also change, although they are not directly in contact with the pathogen came.
  • the change of the physiological status and the expression profile of the cells is intended to ward off the pathogenic attack and to ensure the survival of the cells.
  • Transfection ie the introduction of genetic material into a eukaryotic cell, in particular a mammalian cell, 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, a clarification of the function of different genes would be much more difficult. Not to be forgotten is the possibility to faithfully produce proteins of eukaryotic origin, as the correct posttranslational modification by the eukaryotic cell, 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.
  • cationic polymers are, for example, poly-L-lysine (PLL), (EP 388758) polyethylenimine (PEI), (JP Behr et al., Proc. Natl. Acad. Sci. USA; 1995; 92; 7297 (WO 9602655), diethylaminoethyldextran (DEAE), (SC De Smedt et al., Phar Res .; 2000; 17; 113), Starburst Dendrimer (PAMAM), (Szoka FC et al., Bioconjug. Chem., 1996; 7; 703; WO 9502397), chitosan derivatives (Wu Guang Liu et al., J.
  • cationic polymers are e.g. Superfect, Polyfect (Qiagen), ExGen ⁇ OO (Biomol) and jetPEI (Qbiogene).
  • known 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 nucleic acid 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 ratio of genetic material to cationic component in the preparation of the lipo / polyplexes, ionic strength during lipo / polyplexe production, absolute amount of lipo / polyplexes per cell, cell type, cell proliferation status, cell physiological status, cell division rate, incubation time etc. These Influence parameters are an expression of a complicated transfection process in which the lipo / polyplexes or the genetic material contained must overcome a multitude of cellular barriers.
  • the first barrier is the outer negatively charged cell membrane. It is believed that transfection-active lipoplexes must have a net positive charge and reach the inside of the cell by adsorptive endocytosis or liquid phase endocytosis.
  • endocytosis which is an active transport process of the cell
  • material on the cell surface with cell membrane is encased and internalized 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 is released from the lipoplex in the cytosol because attempts to achieve protein expression by microinjection of lipoplexes directly into the nucleus failed. It appears that the DNA bound in the lipoplexes is inaccessible to the transcriptional apparatus.
  • antisense molecules or siRNA directed against mRNA the biological site of action is reached and the duration of the effect depends essentially on the concentration of cytosolic RNases and the rate of release from the lipo / polyplexes.
  • 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.
  • non-viral methods based on chemical methods include systems which carry a DNA-binding moiety as well as a ligand capable of triggering receptor-mediated endocytosis (example transferrin infection).
  • 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.
  • the cells are allowed for a short time Regeneration of the cell membrane. Subsequently, the cells are sown in a culture vessel and cultivated as usual.
  • 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.
  • non-viral carrier systems or their synthetic components such as cationic lipids can by the innate immune system by various Receptors are recognized as foreign and change the state of the cell.
  • Such an antiviral or altered state of a cell also provides a barrier to transfection with a non-viral gene delivery system that is difficult or impossible to overcome.
  • a traditional method of injecting molecules of all kinds, including proteins, into a cell is to insert the molecule (s) into the inner aqueous environment of
  • liposomes that are taken up by cells by endocytosis.
  • the lipids used are one
  • HIV-1 TAT the Drosophila Antennapedia homeodomain or the "DNA binding
  • penatratin 1 is a commercial product derived from a 16
  • Amino acid peptide exists. It corresponds to the third helix of the homeodomain of the
  • Antennapediaproteins In order to covalently attach it to a protein or peptide, it is provided with an N-terminal pyrydyl disulfide group that reacts with a free one
  • Peptides or lipids form non-covalent complexes that can penetrate into the cell.
  • An example of such a peptide is the commercialized Chariot TM from Active Motif (US 6841535). It is a 2843 dalton peptide that complexes with peptides, proteins and antibodies. This complex formation is based on hydrophilic and hydrophobic interactions. The underlying peptide has a mainly positively charged amino acids existing hydrophilic domain and a hydrophobic domain, so similar to amphiphilic compounds. After intemalization, this complex dissociates and releases the biologically active macromolecule. Since also works at low temperatures, for example 4 0 C, the process is assumed that the recording process as in the PTDs of endocytosis is independent. This is a gentle method because the macromolecule is not exposed to the harsh conditions of this uptake mechanism, which is reflected in the high transfer rate of up to 95% of the macromolecule used. The delivery process takes less than 2 hours.
  • BioPORTER TM is the lipid composition BioPORTER TM from Gene Therapy Systems (O. Zelphati et al., J. Biol. Chem., 2001, 276, 35103-35110, US 2003/0008813, US 2003/0054007 and EP 1133465). This is based on a cationic lipid and a colipid (DOPE). Even with BioPORTER TM, large proteins and antibodies can be introduced into cells while retaining their full biological activity. It is assumed that postively charged bioporter / protein complexes are formed, which are taken up by cells via endocytosis. Similar products are Pro-Deliverl TM or AB-Deliverln TM from OZ Biosciences or Pulsin TM from Polyplus.
  • Electroporation and microinjection are physical methods by which biologically active peptides and proteins can be introduced into cells.
  • the object of the invention is to provide a method which allows a more efficient transfection. Furthermore, the task is to influence the physiological status of the cell population as little as possible, ie the protein Ideally, the expression profile of the cell population should only change with respect to proteins whose genes have been introduced into the cell or whose expression should be reduced or blocked by the introduced genetic material.
  • transfection comprising the steps: (a) introduction of at least one antibody in the
  • a cytosol of a eukaryotic cell wherein the at least one antibody restricts, neutralizes or blocks at least one biological function of a cytosolic component of the innate immune system;
  • the term "at least" or "at least” herein means one or more.
  • the introduction of at least one antibody into the cytosol of a eukaryotic cell is to be understood so that one antibody or several different antibodies can be introduced into the cytosol of the eukaryotic cell. If various antibodies are introduced into the cytosol of the eukaryotic cell, these various antibodies may restrict, neutralize or block a biological function of a single cytosolic component or each of different cytosolic components of the innate immune system.
  • the step (a) and / or the step (b) of the transfection method can be carried out in vivo or in vitro.
  • the step (a) of the transfection method can be performed in a time interval of 0.01 to 48 hours before the step (b), preferably in a time interval of 0.01 to 12 hours, most preferably in a time interval of 2 to 5 hours before step (b).
  • step (a) at least one antibody can be introduced into the cytosol of a eukaryotic cell which is directed against at least one domain of a transmembrane receptor projecting into the cytosol, the transmembrane receptor being selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, IFN type I receptor, IFN receptor, TNF receptor, IL receptor, IL-1 receptor, G protein-coupled receptor and mannose Receptor.
  • step (a) at least one antibody can be introduced into the cytosol of a eukaryotic cell which is directed against at least one cytoplasmic receptor, the receptor being selected from NOD-like receptors, NODS, NOD1, NOD2, NALPS, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, CIITA, IPAF, BIRC-1, RNA helicases, RIG-I, RIG-I-like receptors, and Mda5.
  • NOD-like receptors NODS, NOD1, NOD2, NALPS, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, CIITA, IPAF, BIRC-1, RNA helicases, RIG-I, RIG-I-like receptors, and
  • step (a) at least one antibody can be introduced into the cytosol of a eukaryotic cell which is directed against at least one adapter molecule, the adapter molecule being selected from MyD88, TRAF1, TRAF2, TRAF3, TRAF6, TRAM, TIRAP, TRIF, NAP-1, RAC-1, RIP-1 and FADD.
  • the adapter molecule being selected from MyD88, TRAF1, TRAF2, TRAF3, TRAF6, TRAM, TIRAP, TRIF, NAP-1, RAC-1, RIP-1 and FADD.
  • step (a) at least one antibody can be introduced into the cytosol of a eukaryotic cell which is directed against at least one kinase, wherein the kinase is selected from kinase PKR, IKKalpha, IKKbeta, IKKgamma, IKKdelta, IKKepsilon, IKKi, IKKg, IRAK1, IRAK4, PI3K, JNK, JNK1, JNK2, p28MAPK, MKKs, ERK-1, ERK2, ERK3, ERK4, ERK5, ERK6, ERK7, ERK8, MEK1, MEK2, MEK5, MSK1, RIP-2, TBK-1, TAK-1, IRF kinase, MAP kinase, MAPK kinase, MAPKK kinase, MAPKKK kinase, MKK4 / SEK, MKK5, MKK7, p38
  • step (a) at least one antibody into the cytosol of a eukaryotic cell which is directed against at least one transcription factor, wherein the transcription factor is selected from NF-kB, AP-1, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, STAT1 and STAT2.
  • step (a) at least one antibody can be introduced into the cytosol of a eukaryotic cell resistant to IkB, 2'5'-oligoadenylate synthase, G proteins, Raf, Ras, Rnase, RnaseL, Dnasen, Dnase 1, Dnase 2 , Dnasei / L2, Dnasei / L3 or Dnase 2like acid Dnase.
  • the at least one antibody in step (a), may preferably be used in an amount of 0.01 to 5 picograms, preferably in an amount of 0.01 to 0.5 picograms, most preferably in an amount of 0.01 to 0, 05 picogram into the cytosol of the eukaryotic cell.
  • a protein delivery system to be used in step (a) for introducing the at least one antibody into the cytosol of a eukaryotic cell.
  • the protein delivery system may comprise a cationic lipid, a cationic polymer, a cationic protein, a cationic protein having hydrophobic moieties, or a compound having an antibody binding domain capable of inducing receptor-mediated endocytosis and / or membrane transfer; or it may be used as a protein delivery system electroporation, microinjection, magnetoprotection, ultrasound, ballistic method or hydrodynamic method.
  • the protein delivery system according to the invention may comprise a cationic protein having hydrophobic moieties, the cationic protein having hydrophobic moieties Proportions may comprise a peptide of 16 to 30 amino acids having a hydrophobic portion of at least 4 amino acids and a hydrophilic portion of up to 12 amino acids having a plurality of cationic amino acids, optionally separated by a spacer sequence of up to 10 amino acids.
  • Chariot can be used as the protein delivery system.
  • the protein delivery system according to the invention may comprise a cationic lipid carrying a polylysine head group whose primary amino groups are optionally derivatized by an acid to amides.
  • the protein delivery system may be Bioporter, BioTrek, ABdeliverin or prodeliverin.
  • the non-viral gene delivery system in step (b) may comprise a cationic lipid, a cationic polymer, a cationic protein, or a compound having a DNA and / or RNA binding domain and receptor-mediated endocytosis and / or membrane transfer can trigger; or a physical method such as electroporation, microinjection, magnetofection, ultrasound, ballistic method or hydrodynamic method may be employed as the non-viral gene delivery system.
  • the non-viral gene delivery system may comprise a cationic polymer selected from a linear or branched polyethyleneimine and / or a cationic dendrimer.
  • the non-viral gene delivery system comprises a cationic lipid with a polyamine head group and / or the colipid DOPE.
  • the non-viral gene delivery system in step (b) Lipofectin, Lipofectamine, Lipofectamine 2000, Lipofectamine RNAiMAX, Freestyle MAX, Optifect, DMRIE-C, 293fect, Oligofectamine, Metafectene, Metafectene Pro, Metafectene Easy, Fugene, DOTAP , Cellfectin, Cytofectene, CellPhect, Gene Limo, Clonfectin, ExGen®, Gene Juice, a member of the TransIT series, Transfast, a member of the Tfx series, Gene Shuttle, Duofect, Superfect, Effectene, Polyfect, Dosper, X-treme genes Q2, Extreme Gene siRNA, escort series representative, Lipotaxi, Geneporter, Geneporter 2, Genesil
  • the protein delivery system may be identical to the non-viral gene delivery system.
  • modified or unmodified ssDNA modified or unmodified dsDNA, modified or unmodified ssRNA, modified or unmodified dsRNA and / or modified or unmodified siRNA can be used.
  • the invention further provides a composition comprising at least three of the following components (1) to (4):
  • a protein delivery system capable of transporting a biologically active antibody into the cytosol of a cell; (4) at least one antibody which restricts, neutralizes or blocks the biological function of a cytosolic component of the innate immune system.
  • composition according to the invention can be used to carry out the transfection method proposed here.
  • kit of parts which comprises at least three of the following components (1) to (4):
  • composition or kit of parts of the invention may contain as component (4) an antibody directed against at least one cytosolic domain of a transmembrane receptor, wherein the transmembrane receptor is selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6 , TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, IFN type I receptor, IFN receptor, TNF receptor, IL receptor, IL-1 receptor, G protein coupled receptor and mannose receptor ,
  • composition according to the invention or the kit of parts according to the invention may also contain as component (4) an antibody which is directed against at least one cytoplasmic receptor, the receptor being selected from NOD-like receptors, NODS, NOD1, NOD2, NALPS, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, CIITA, IPAF, BIRC-1, RNA helicases, RIG-I, RIG-I-like receptors, and Mda5.
  • an antibody which is directed against at least one cytoplasmic receptor, the receptor being selected from NOD-like receptors, NODS, NOD1, NOD2, NALPS, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, CIITA, IPAF, BIRC-1, RNA
  • composition according to the invention or the kit of parts according to the invention as component (4) can contain an antibody which is directed against at least one adapter molecule, the adapter molecule being selected from MyD88, TRAF1, TRAF2, TRAF3, TRAF6, TRAM, TIRAP, TRIF, NAP-1, RAC-1, RIP-1 and FADD.
  • the adapter molecule being selected from MyD88, TRAF1, TRAF2, TRAF3, TRAF6, TRAM, TIRAP, TRIF, NAP-1, RAC-1, RIP-1 and FADD.
  • composition according to the invention or the kit of parts according to the invention as component (4) can contain an antibody which is directed against at least one kinase, wherein the kinase is selected from kinase PKR, IKKalpha, IKKbeta, IKKgamma, IKKdelta, IKKepsilon, IKKi, IKKg, IRAK1, IRAK4, PI3K, JNK, JNK1, JNK2, p28MAPK, MKKs, ERK-1, ERK2, ERK3, ERK4, ERK5, ERK6, ERK7, ERK8, MEK1, MEK2, MEK5, MSK1, RIP-2, TBK-1, TAK-1, IRF kinase, MAP kinase, MAPK kinase, MAPKK kinase, MAPKKK kinase, MKK4 / SEK, MKK5, MKK7, p38 MAP kinase, wherein
  • composition according to the invention or the kit of parts according to the invention can also contain as component (4) an antibody which is directed against at least one transcription factor, the transcription factor being selected from NF-kB, AP-1, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, STAT1 and STAT2.
  • composition according to the invention or the kit of parts according to the invention can also contain, as component (4), an antibody which is resistant to IkB, 2'5'-oligoadenylate synthase, G proteins, Raf, Ras, Rnase, RnaseL, Dnasen, Dnase 1, Dnase 2 , Dnasei / L2, Dnasei / L3 or Dnase 2like acid Dnase.
  • the composition of the invention or the kit of parts as component (3) may contain a protein delivery system comprising a cationic lipid, a cationic polymer, a cationic protein, a cationic protein having hydrophobic moieties, or a compound has an antibody binding domain and can induce receptor-mediated endocytosis and / or membrane transfer.
  • a protein delivery system comprising a cationic lipid, a cationic polymer, a cationic protein, a cationic protein having hydrophobic moieties, or a compound has an antibody binding domain and can induce receptor-mediated endocytosis and / or membrane transfer.
  • composition of the invention or the kit of parts according to the invention as component (3) may contain a protein delivery system comprising a peptide of 16 to 30 amino acids with a hydrophobic portion of at least 4 amino acids and a hydrophilic portion of up to 12 amino acids with a plurality of cationic Amino acids optionally separated by a spacer sequence of up to 10 amino acids.
  • composition according to the invention or the kit of parts according to the invention as component (3) may preferably contain the protein delivery system Chariot.
  • composition or kit of parts of the invention may contain as component (3) a protein delivery system comprising a cationic lipid bearing a polylysine head group whose primary amino groups are optionally derivatized by an acid to amides.
  • composition according to the invention or the kit of parts according to the invention as component (3) may contain the protein delivery system Bioporter, BioTrek, ABdeliverin or prodeliverin.
  • composition according to the invention or the kit of parts according to the invention as component (1) may preferably contain a non-viral gene delivery system which comprises a cationic lipid, a cationic polymer, a cationic protein and / or a compound comprising a DNA and / or RNA. having binding domain and can induce receptor-mediated endocytosis and / or membrane transfer.
  • the composition according to the invention or the kit of parts according to the invention may preferably contain as component (1) a non-viral gene delivery system comprising a linear or branched polyethyleneimine; and / or a cationic dendrimer; and / or a cationic lipid having a polyamine head group and / or colipid DOPE.
  • the composition according to the invention or the kit of parts as component (1) may contain a non-viral gene delivery system comprising Lipofectin, Lipofectamine, Lipofectamine 2000, Lipofectamine RNAiMAX, Freestyle MAX, Optifect, DMRIE-C, 293fect, Oligofectamine, Metafectene, Metafectene Pro, Metafectene Easy, Fugene, DOTAP, Cellfectin, Cytofectene, CellPhect, Gene Limo, Clonfectin, ExGen500, Gene Juice, a TransIT range member, Transfast, a Tfx series member, Gene Shuttle, Duofect, Superfect , Effectene, Polyfect, Dosper, X-treme Gene Q2, Extreme Gene siRNA, escort series representative, Lipotaxi, Geneporter, Geneporter 2, Genesilencer, Neuroporter, jetPEI, jetSI, Interferin, Fecturin, Perfectin, Dharmafect, siPort Amine, siPort Li
  • component (1) and the component (3) in the composition according to the invention or the kit of parts according to the invention are identical.
  • composition according to the invention or the kit of parts according to the invention as component (2) may preferably contain genetic material which comprises modified or unmodified ssDNA, modified or unmodified dsDNA, modified or unmodified ssRNA, modified or unmodified dsRNA and / or modified or unmodified siRNA.
  • all components may be present separately or the components may be present as a combination of 2, 3 or 4 components, the remaining components of the combination being present separately or as a further combination of 2 components.
  • the components can either be separated from each other e.g. in glass or plastic containers that are packaged together, or the components can be provided in pairs or more in appropriate containers.
  • compositions comprising one of the above-mentioned inventive compositions.
  • kits of parts which comprises one of the abovementioned kit of parts according to the invention.
  • composition according to the invention or a kit of parts according to the invention can be used for the treatment of a disease by gene therapy.
  • the invention may be cystic fibrosis, muscular dystrophy, phenylketonuria, maple syrup disease, propionic acidemia, methylmalonic acidemia,
  • Adenosine deaminase deficiency hypercholesterolemia, hemophilia, beta-thalassemia, cancer, viral disease, degeneration of the macula, amyotrophic lateral sclerosis, and / or inflammatory disease.
  • several of the abovementioned antibodies for at least partial suppression of the immune defense can be combined with one another, ie it is possible to use two, three or more components of component (4). It is likewise possible to use several of the abovementioned components (1) and / or (2) and / or (3).
  • the innate immune defense can be neutralized or blocked, at least partially suppressed, by at least one antibody which restricts a component of the innate immune defense.
  • antibody as used herein includes polyclonal, monoclonal and all types of recombinant antibodies. It is crucial that the antibody binds to the component of the innate immune system in such a way that its biological function is eliminated as far as possible.
  • the cytosolic constituents of the innate immune system are all proteins
  • antibodies or antibodies derived from antibodies are used against these proteins.
  • the antibodies or derivatives derived therefrom with protein delivery systems, which are suitable for the cytosoplasmic transport of biologically active antibodies are introduced into the cells.
  • antibodies are suitable which are capable of blocking the cytosol components of the innate immune system in their biological activity. As a rule, these must therefore be directed against the cytosolic constituents of the innate immune system of the cells which are to be transfected. So if it is a human cell, then the antibody must usually be directed against the human cytosolic components of the innate immune system. In some cases, the antibodies are due to the great similarity of the cytosolic components of the innate It is also cross-reactive from different species, that is, although an antibody has been developed against a target from one species, it also exhibits its properties against a similar target from another species. Ideally, for in vivo applications, antibodies from cells of the same species to be transfected are used because they are little or not immunogenic in this case.
  • antibodies which have been obtained based on an immunization of organisms.
  • the antibodies may also have been prepared recombinantly and, for example, on the expression of recombinant antibody DNA or parts thereof, for example. in bacteria or bacteriophages (eg HuCaI technology, Morphosys).
  • the antibody is highly affine, the antibody can be polyclonal as well as monoclonal, or antibody-derived derivatives, ie, the antibody can be modified to its properties
  • the Fc fragment can be cleaved off, since the binding to the antigen is exclusively due to the Fab fragments (Fab antibodies), for example monovalent or bivalent mini-antibodies are obtained, and it can also be a trifunctional
  • the antibody may also be tagged for purification, detection or fusion with other molecules. If the antibody is to be used e.g. easier to detect, it can be labeled with a fluorescent dye.
  • the antibodies of the invention are not limited to any isoform.
  • the transfection result can be improved and / or undesired changes in the expression profile, such as. reduce or avoid the antiviral status of a transfected cell.
  • cytoplasmic components are blocked.
  • multiple antibodies to different cytoplasmic components can be combined to utilize additive effects and / or synergistic effects.
  • the method is combined with the blocking of components of the innate immune system, which is not based on antibodies.
  • blocking may also be based on siRNA, shRNA, inhibitors, ribozymes or aptamers.
  • the method is combined with the blocking of components of the innate immune system based on antibodies and non-cytoplasmic components in their biological function.
  • An example is the blockade of the extracellular and / or endosomal domains of TLR receptors by antibodies.
  • 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.
  • Anti-MyD88 antibody (rabbit, anti-human, polyclonal), USBiological Cat No .: M9755-
  • the antibody is purified before the experiment according to the instructions of the manufacturer with the Protein-G SpinTrap columns.
  • the eluate obtained is measured photometrically at 280 nm (Antibodies: A Laboratory Manual, Edward Harlow, David Lane, CSHL Press, 1988, p 673) and the antibody concentration is adjusted to 0.1 ⁇ g / ⁇ l by dilution with PBS (phospate buffered saline) ,
  • HeIa cells are sown in a 48 well plate. In this case, a cell count of 1, 0 x 10 5 is seeded in 250 ul complete culture medium (10% FCS) per well. The mixture is then incubated for 24 h in a CO 2 incubator (10%).
  • 0.5 ⁇ g (0.5 ⁇ l) pCMVIacZ plasmid solution is diluted in 15 ⁇ l PBS.
  • 2 ⁇ l Metafectene Pro are also diluted in 15 ⁇ l PBS.
  • the solutions are combined and incubated for 15 minutes at room temperature.
  • the lipoplex solution is then applied to the cells and incubated in a CO 2 incubator (10%).
  • the reporter gene assay ( ⁇ -galactosidase assay) and protein determination (BCA assay) are performed according to the manufacturer's instructions.
  • the cells are lysed with the lysis buffer of the ⁇ -galactosidase assay kit. Subsequently, a part is used in the BCA assay. The remainder is available for the ⁇ -galactosidase assay.
  • the plates are developed in the ⁇ -galactosidase assay until a yellow color with an absorbance of 1-2 with a microplate reader is measured and then immediately stopped. The incubation period is then noted. The values are read out again and the mean value is calculated for the double test.
  • the relative transfection efficiencies are determined by the amount of protein per
  • Galactosidase amount is normalized to a unit of time (second)
  • TLRs activation of 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. Further, different gene delivery systems 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 can lead to different results through stress signal transduction cascades. In summary, different blocking strategies may be 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,
  • 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 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
  • Other diseases specifically targeted for treatment with siRNA include age-related macular degeneration (eye disease), liver cancer, solid tumors, amyotrophic lateral sclerosis and inflammatory diseases.
  • transfection for example, for the production of cancer vaccines plays an increasingly important role. 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.
  • lipid / DNA complexes are 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.
  • exemplary but in no way limiting definitions are listed according to the invention:
  • 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 near the cell, in particular use physical Method, however, energy supply, in particular in the form of thermal, kinetic, electrical or other energy to mediate transport of the genetic material through the cell membrane.
  • Chemical methods are based either on a chemical modification or derivatization of the nucleic acids, which in particular make them cell-permeable, or consist in particular of substances which bind DNA and can mediate transport through the cell membrane. In particular, these use electrostatic forces or hydrogen bonds to bind the nucleic acids.
  • 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.
  • the substances can also be specially formulated, in particular as micelles or liposomes and also consist of several components, in particular with different functions.
  • a protein delivery system is capable of infecting proteins into eukaryotic cells.
  • the protein delivery methods are physical methods and chemical methods. Physical methods locate at least the protein in the vicinity of the cell, but in particular, physical methods use energy input, especially in the form of thermal, kinetic, electrical, or other energy to mediate transport of the protein across the cell membrane.
  • Chemical methods are based either on a chemical modification or derivatization of the proteins, which in particular make them cell-permeable, or consist in particular of substances which can not covalently bind proteins and mediate transport through the cell membrane. In particular, these use for binding the electrostatic forces, hydrohilic and / or hydrophobic interactions or also Hydrogen bonds.
  • the transport of proteins across the cell membrane occurs through an active transport mechanism of the cell, endocytosis.
  • Substances which exhibit these properties contain, in particular, cationic lipids, cationic polymers, cationic peptides, cationic peptides having a hydrophobic domain, or even molecules which have a domain capable of binding proteins and at the same time have a second domain which contains a ligand which belongs to a Receptor also the cell surface is detected and triggers endocytosis by this recognition process.
  • the substances can also be specially formulated, in particular as micelles or liposomes and also consist of several components, in particular with different functions.
  • the transport can also be done by a passive transport through the cell membrane, so a membrane transfer.
  • Membrane transfer is the passage of a molecule from one side of a cell membrane and the other
  • the antibody can be obtained by immunizing an organism or by recombinant methods.
  • the antibody can cleave off parts that are not necessary for binding. Such fragments can also be obtained by recombinant methods.
  • additional moieties may also be introduced into the antibody to facilitate purification, detection, or fusion with other molecules
  • the term "antibody” includes any of the above-listed types of antibodies, particularly polyclonal or monoclonal antibodies obtained by immunization, eg IgA, IgD, IdE, IgG, IgM, IgY and IgW, and all kinds of recombinant antibodies.
  • 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 defense is characteristic of most cell types.
  • the innate immune defense uses the recognition of pathogens attributable 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 about these messenger substances (cytokines) unaffected cells and trigger there a changed physiological status (eg "antiviral status"), whereby the cytokines dock to cytokine receptors of the other cells and in turn trigger a signal transduction cascade.
  • cytokines messenger substances
  • antiviral status e.g "antiviral status”
  • 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.
  • peptides or proteins into eukaryotic cells takes place in a living organism.
  • peptides or proteins into eukaryotic cells takes place outside a living organism, especially in vessels suitable for the cultivation of eukaryotic cells.
  • Natural nucleic acids that have been modified by modification in their properties. These modifications may be, in particular, chemical changes, which in particular relate to the phosphate scaffold, 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, which lead to new properties of the nucleic acids, in particular to optical traceability by fluorescence labels or labels, which determine the nucleic acids to a particular location eg. in the core in the cell conduct (localization elements) or labels, the Passage of nucleic acids through membranes mediate and so make nucleic acids cell-like example.
  • 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 that is able to detect a substance (agonist) and thus trigger a biological reaction.
  • receptors are um
  • 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.
  • AP-1 Activated protein-1
  • BIRC-1 Baculoviral IAP repeat-containing 1
  • ERK Extracellular-signal Regulated Kinase
  • FADD Fas Associated Protein with Death Domain
  • IkB inhibitory-binding protein kB
  • IKKi IKK epsilon
  • IL interleukin
  • IPAF Apaf-1-related protein
  • IRAK1 interleukin 1 receptor-associated kinase 1
  • IRAK4 interleukin-1 receptor-associated kinase 4
  • IRF interferon regulating factor
  • IRF kinase interferon-regulating factor kinase
  • JNK c-Jun N-terminal kinase
  • TIRAP MyD88-adapter-like
  • MAPK mitogen activated protein kinase
  • MAPKK mitogen activated protein kinase kinase
  • MAPKKK mitogen activated protein kinase kinase kinase
  • Mda5 melanoma differentiation-associated gene-5 protein
  • MEK MAPK / ERK kinase
  • MKK mitogen-activated protein kinase kinase
  • MSK mitogen and stress activated kinase
  • MyD88 myeloid differentiation factor 88
  • NALP-2 NIGHT-LRR and pyrin domain-containing protein 2
  • NALPS NIGHT-LRR and pyrin domain-containing proteins
  • NAP1 Nck-associated protein 1
  • NF-kB nuclear factor kappaB
  • NOD nucleotide-binding oligomerization domain containing protein
  • PDK1 phosphoinositide-dependent protein kinase 1
  • PDK2 phosphoinositide-dependent protein kinase 1
  • PI 3K phosphoinositol 3-kinase
  • Raf proteins "rapidly growing fibrosarcoma" proteins
  • RIG-I retinoic acid inducible gene I
  • R1 P1 receptor-interacting protein 1
  • Rnase ribonuclease
  • SEK stress-activated protein / Erk kinase
  • TAK1 Transforming growth factor- ⁇ -activated kinase
  • TLR toll-like receptor
  • TNF tumor necrosis factor
  • TRAF TNF receptor-associated factor
  • TRAF33 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 en mesure de détecter du matériel étranger à la cellule grâce aux récepteurs de reconnaissance de motifs (Pattern Recognition Receptors" (PRR)) et d'induire des stratégies de défense. L' état d'alerte qui en découle représente également une barrière pour des systèmes de transfert de gènes non viraux. En bloquant par un anticorps la fonction biologique de quelques voire de plusieurs composants cytoplasmiques du système immunitaire inné, il est possible d'augmenter l'efficacité de transfection de systèmes de transfert de gènes non viraux et d'éviter des modifications indésirables du profil d'expression. A cet effet, l'invention prévoit de transporter l'anticorps dans la cellule par un système de transfert de protéines avant ou durant la transfection.
PCT/EP2010/003105 2009-05-20 2010-05-20 Procédé de transfection pour systèmes de transfert de gènes à efficacité améliorée par blocage du système immunitaire inné Ceased WO2010133369A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102016113714A1 (de) 2016-07-26 2018-02-01 Rosa Karl Transfektionsverfahren mit nicht-viralen Genliefersystemen
WO2018019341A1 (fr) 2016-07-26 2018-02-01 Karl Rosa Procédé de transfection avec systèmes de transfert de gènes non viraux

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