WO2016112963A1 - Administration de biomolécules dans des cellules - Google Patents

Administration de biomolécules dans des cellules Download PDF

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WO2016112963A1
WO2016112963A1 PCT/EP2015/050519 EP2015050519W WO2016112963A1 WO 2016112963 A1 WO2016112963 A1 WO 2016112963A1 EP 2015050519 W EP2015050519 W EP 2015050519W WO 2016112963 A1 WO2016112963 A1 WO 2016112963A1
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composition
complex
cell
seq
oligonucleotide
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PCT/EP2015/050519
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Jacques Rohayem
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Riboxx Gmbh
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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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • C12N2310/151—Nucleic acids forming more than 2 strands, e.g. TFOs more than 3 strands, e.g. tetrads, H-DNA
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention generally relates to compositions or complexes, respectively, containing a biologically active substance or biomolecule and at least one nucleotide, oligonucleotide and polynucleotide forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions,
  • the present invention also relates to specific embodiments of G-quadruplex forming oligonucleotides or polynucleotides, respectively, per se.
  • Reagents for delivery of nucleic acids and other compounds into cells are known in the art as transfection agents and are based, e.g. on lipids (e.g. Lipofectamine ® and derivatives; see WO-A-00/27795), proteins (e.g. TurboFectTM), other polymers and/or cell penetrating peptides (e.g.
  • the technical problem underlying the present invention is to provide a novel system for delivery of biomolecules, in particular nucleic acid molecules peptides or proteins, into cells.
  • biomolecules in particular nucleic acid molecules peptides or proteins
  • compositions or complexes respectively, containing a biologically active substance or biomolecule, respectively, together with one or more nucleotide(s), oligonucleotide(s) and/or polynucleotide(s) forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures (i.e., in general, intermolecular or intramolecular complexes of or within one or more of such nucleotides, oligonucleotides or polynucleotides, typically based on interactions other than Watson-Crick base pairing) under appropriate conditions.
  • nucleotide(s) oligonucleotide(s) and/or polynucleotide(s) forming G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures (i.e., in general, intermolecular or intramolecular complexes of or within one or more of such nucle
  • the resent inventions also relates to the use or these compositions or complexes as well as the G-quadruplex forming entities as such for delivery of said biologically active substance or said biomolecule, respectively, in particular nucleic acids such as single or double stranded RNA and single or double stranded DNA, into cells in vitro or in vivo.
  • intramolecular and/or intermolecular structures and/or suprastructures under appropriate conditions relates to assemblies of said nucleotides (preferably G, C, U, T, or A or analogues thereof), oligonucleotides or polynucleotides through inter and/or intramolecular interactions between and/or within said species under permissive conditions including buffer, salt components, temperature etc., more preferably under physiological conditions.
  • the conditions required for the formation of said G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures are generally known in the art. Reference is given in this context, e.g. to Haider et al.
  • RNA Quadruplexes in Sigel (ed.): Structural and catalytic roles of metal ions in RNA, RSC Publishing, Cambridge, 201 1 , pp. 125-139) and also to Neidle and Balasubramanian (eds.): Quadruplex Nucleic Acids, RSC Publishing, 2005.
  • the quadruplex forming entities of the invention typically represent complexes of the mentioned nucleotide, oligonucleotide or polynucleotide species with monovalent and/or divalent cations, in particular monovalent cations of alkaline metals such as K + and Na + and/or divalent cations of alkaline earth metals such as Ca 2+ or Mg 2+ , and/or divalent cations of transition metals such as Mn 2 .
  • alkaline metals such as K + and Na + and/or divalent cations of alkaline earth metals such as Ca 2+ or Mg 2+
  • transition metals such as Mn 2 .
  • biomolecule are used synonymously and refer to any molecule present in biological or biochemical structures such as cells, tissues, organisms, viruses or viroids, which molecules include macromolecules such as proteins, polysaccharides, lipids and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites and other natural products. It is to be understood that the "biologically active substance” or “biomolecule” is not confined to naturally occurring molecules but also includes artificial substances not found in nature but being typically composed of naturally occurring building blocks and/or building blocks being chemically similar to the naturally occurring ones. According to the present invention, the biologically active substance or biomolecule is typically different from the G- quadruplex forming component of the inventive composition or complex, respectively.
  • oligonucleotides or polynucleotides respectively, represented by sequences containing two guanine repeats separated by non-guanine nucleotides, for example oligonucleotides or polynucleotides containing or having a sequence of the following formula (II):
  • oligonucleotides or polynucleotides are oligonucleotides or polynucleotides, respectively, represented by sequences forming intramolecular G-quadruplexes containing or having a sequence of the following formula (III):
  • G-repeats not only species comprising four G-repeats (present in one molecule as is the case for above formula (III) or provided by more than one molecule) but any number of G-repeats can be included into a run of guanines and fold into an
  • compositions or complexes especially useful as delivery agents for biomolecules into cells, including species that form self-association of Watson-Crick duplex sequences, G:C tetrads formed, e.g. from (CCC)n:CCG repeats or other tetrad forming sequences such as the human quadruplex structures formed from sequences such as (TAGGGTTAGGGT) 2 (SEQ ID NO: 1 ) repeats and GAGCCAGT.
  • nucleic acids in particular ribonucleic acids, contained in the compositions and complexes as disclosed herein such as those of the above and the below formulas have a length of up to 1 1 nucleotides.
  • compositions or complexes, respectively, according to the invention in particular useful as reagents for delivery of biomolecules into cells, include poly and oligonucleotides, respectively, having the following formula (IV) wherein n is an integer of at least 2 or at least 3, preferably 2 or 3 to 40, more preferably 2 or 3 to 20.
  • oligonucleotides molecules included in the complexes of the invention according to formula (I) will be referred to as "oligonucleotides", independent of their length.
  • the above nucleotide, oligonucleotide or polynucleotide species contain a free 5'-triphosphate group.
  • a "5'-triphosphated nucleotide, oligonucleotide or polynucleotide" as defined herein has a free triphosphate group bound to the 5' position of the (or at least one, in case of double stranded molecules) 5'-terminal ribose or deoxyribose, respectively. If embodiments of such species are initially produced, in particular through enzymatic synthesis using, e.g.
  • RNA polymerases typically as outlined below using RNA-dependent RNA-polymerases (RdRps) of caliciviruses, so that said species contain a free 5'-triphosphate group, it is desirable in certain embodiments of the invention to remove this 5'-triphosphate group. This can be done easily, if desired, especially for in vivo applications, e.g.
  • a suitable phosphatase such as calf intestinal alkaline phosphatase (CIAP), (preferably recombinant) shrimp alkaline phosphatase (rSAP) or Antarctic phosphatase under appropriate buffer, salt and temperature conditions in a known fashion (e.g. for CIAP: 37°C for 30 to 60 min in 50mM Tris-HCI (pH 9.3 at 25°C), 1 mM MgCI 2 , 0.1 mM ZnCI 2 and 1 mM spermidine).
  • a suitable phosphatase such as calf intestinal alkaline phosphatase (CIAP), (preferably recombinant) shrimp alkaline phosphatase (rSAP) or Antarctic phosphatase under appropriate buffer, salt and temperature conditions in a known fashion (e.g. for CIAP: 37°C for 30 to 60 min in 50mM Tris-HCI (pH 9.3 at 25°C), 1 mM MgCI 2 , 0.1
  • inventive G-quadruplex forming entities are very efficient delivery reagents for biomolecules, in particular nucleic acids, peptides and/or proteins, into cells, which need almost no incubation step with the cargo, i.e. the biomolecule, in particular nucleic acids such as RNA, to be delivered into the desired cell.
  • n in the above formula (IV) is 2, 3, 4, 5 or 6 to 14, particularly preferred 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14..
  • Especially preferred oligonucleotides are those in which n in the above formula is 6 to 14, most preferred 6 to 1 1 .
  • the nucleotides, poly or oligonucleotides as defined herein form G-quadruplexes and/or intramolecular and/or intermolecular structures and/or suprastructures, typically in complex with mono and/or divalent cations under appropriate conditions, (including buffer, salt components, temperature; see, e.g. the review by Haider et al. "RNA Quadruplexes" in Sigel (ed.): Structural and catalytic roles of metal ions in RNA, Royal Society of Chemistry, Cambridge, 201 1 , pp. 125-139).
  • compositions or complexes, respectively, of the invention are single stranded and at least comprise ribonucleotides.
  • Mixed RNA DNA species are contemplated as well, but especially preferred oligonucleotides are RNA oligonucleotides.
  • the invention also relates to compositions or complexes, respectively, containing a biologically active substance or biomolecule and at least two different nucleotides, oligonucleotides or polynucleotides as defined above, more preferably of oligonucleotides according to above formula (I).
  • compositions contain at least two different oligonucleotides of above formula (IV), most preferred selected from the group consisting of (G) 6 , (G) 7 , (G) 8 , (G) 9 , (G)i 0 (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G) i2 (SEQ ID NO: 4), (G)i3 (SEQ ID NO: 5) and (G) i4 (SEQ ID NO: 6). It is to be understood that the present invention is expressly directed to compositions with all possible combinations of at least two of the above oligonucleotides.
  • the oligonucleotides in such compositions are, or, in other embodiments, comprise (G) 8 , (G) 9 and (G)i 0 (SEQ ID NO: 2).
  • the compositions contain, as oligonucleotide species (or at least as the majority of oligonucleotide species) (G) 6 , (G) 7 , (G) 8 , (G) 9 , (G)i 0 (SEQ ID NO: 2), (G)ii (SEQ ID NO: 3), (G) i2 (SEQ ID NO: 4), (G) i3 (SEQ ID NO: 5) and (G) i4 (SEQ ID NO: 6).
  • one ore more, preferably all of, the species (G) 6 to (G)i4 as outlined before contain a free 5'-triphosphate group.
  • the present invention also relates to compositions of at least two different compounds
  • nucleotides oligonucleotides or polynucleotides as defined above per se.
  • nucleotides, oligonucleotides or polynucleotides are preferably free of chemical modifications or other nucleotide analogues.
  • nucleotides, oligonucleotides or polynucleotides contained in the compositions or complexes, respectively, of the present invention can comprise at least one chemically modified and/or labeled nucleotide.
  • the chemical modification of the nucleotide analogue in comparison to the natural occurring nucleotide may be at the ribose, phosphate, and/or base moiety.
  • modifications at the backbone i.e. the ribose and/or phosphate moieties, are especially preferred.
  • ribose-modified ribonucleotides are analogues wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN with R being CrC 6 alkyl, alkenyl or alkynyl and halo being F, CI, Br or I.
  • modified ribonucleotide also includes 2'- deoxyderivatives, such as 2'-0-methyl derivatives, which may at several instances also be termed "deoxynucleotides”.
  • the at least one modified nucleotide may be selected from analogues having a chemical modification at the base moiety.
  • analogues include, but are not limited to, 5-aminoallyl-uridine, 6-aza-uridine, 8-aza-adenosine, 5-bromo-uridine, 7- deaza-adenine, 7-deaza-guanine, N 6 -methyl-adenine, 5-methyl-cytidine, pseudo-uridine, and 4-thio-uridine.
  • inosine can be used as base analogue in a ribonucleotide or deoxyribonucleotide contained in a composition or complex, respectively, according to the invention.
  • label that may be included in the nucleotide analogue can be any chemical entity which enables the detection of the oligonucleotide in question via physical, chemical, and all biological means.
  • label linked to or integrated into one or more of the nucleotides or added to one end of the molecules as disclosed herein are radioactive labels, chromophores, and fluorophores (e. g. fluorescein, TAM etc.).
  • nucleotides, oligonucleotides and polynucleotides for use in the present invention can be prepared chemically using appropriate DNA or RNA, respectively, solid- phase chemistry well known in the art.
  • 5'-triphosphated nucleotides, oligonucleotides and polynucleotides for use in the invention may be prepared chemically (see, e.g., Zlatev et al. (2010) Org. Lett. 12 (10), pp. 2190-2193).
  • oligonucleotides and polynucleotides contained in the compositions or complexes, respectively, of the present invention may be prepared enzymatically, or by a combination of chemical and enzymatic steps.
  • Methods for the enzymatic preparation of the oligonucleotides are preferably those that use RNA-dependent RNA polymerases (RdRps) of caliciviruses as disclosed in WO-A-2007/012329. With respect to enzymatic synthesis of chemically modified RNAs using such RdRps the methods referred to in WO-A-2009/150156 are preferred. Enzymes of this type are also capable of using DNA templates; see WO-A- 2012/038448.
  • a further aspect of the present invention relates to a method for preparing the above compositions containing oligonucleotides or polynucleotides comprising the steps of (i) incubating a corresponding oligonucleotide or polynucleotide template with an RNA polymerase of a calicivirus, preferably in the presence of mono and/or divalent cations, typically cations of alkaline metals such as Na + and/or K + , and/or of alkaline earth metals and/or of transition metals, preferably Mg 2+ and/or Mn 2+ , and in the presence of appropriate rNTPs, in particular rGTP, rATP, rCTP, rUTP, and/or modified analogues thereof including, but not limited to, rITP; and (ii) contacting the thus-obtained reaction mixture with a biologically active substance or biomolecule, respectively.
  • an RNA polymerase of a calicivirus
  • step (i) In case of the compounds according to above formula (IV) only GTP (and/or modified analogue thereof) is needed in step (i). Still a further aspect of the present invention relates to a corresponding method for preparing the above oligonucleotides or polynucleotides as such comprising the step of incubating a corresponding oligonucleotide or polynucleotide template with an RNA polymerase of a calicivirus, preferably in the presence of mono and/or divalent cations, typically cations of alkaline metals such as Na + and/or K + , and/or of alkaline earth metals and/or of transition metals, more preferably Mg 2+ and/or Mn 2+ , and in the presence of appropriate rNTPs, in particular rGTP, rATP, rCTP, rUTP, and/or modified analogues thereof including, but not limited to, rITP.
  • GTP rGTP
  • oligonucleotides selected from the group consisting of (G) 6, (G) 7 , (G) 8 , (G) 9 , (G)i 0 (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G) i2 (SEQ ID NO: 4), (G) i3 (SEQ ID NO: 5), and (G)i4 (SEQ ID NO: 6), more preferred a composition containing (as oligonucleotide species) (G) 8 , (G) 9 and (G) 10 (SEQ ID NO: 2), or (G) 6 , (G) 7 , (G) 8 , (G) 9 , (G) 10 (SEQ ID NO: 2), (G)n (SEQ ID NO: 3), (G)i 2 (SEQ ID NO: 4
  • the result is a mixture of the above preferred oligonucleotides and, depending on the amount of template used, the (C) > 5 template, optionally with minor contributions of the type (C)> 5 (G)x (x being an integer of about 1 to 5), and complexes of the aforementioned oligonucleotides with mono and/or divalent cations.
  • oligonucleotides prepared in this manner can be controlled by the reaction conditions and reagent concentrations used, in particular the reaction time.
  • complexes or compositions of the invention containing individual oligonucleotide species, as well as the individual oligonucleotides as such can also be isolated from these compositions by gel electrophoresis or chromatography, especially HPLC, in a known manner.
  • the mechanism underlying this preferred manufacturing method for certain oligonucleotide compositions as disclosed herein is that the calicivirus RNA polymerase transcribes the (C)> 5 template into a corresponding homopolymeric polyG and then adds one or several G residues to the 3' end of template and/or the homopolymeric polyG though its terminal transferase activity (see WO-A-2007/012329; WO-A- 2012/038448). Since, according to preferred embodiments of the invention, the template is rather short, the melting temperature of the oligo(G:C) product of the transcription process is rather low so that the strands separate easily and a new round of transcription of the template can start.
  • the co-production of by-products such as, e.g. (C)> 5 (G) X can be avoided by adjusting, preferably lowering, the concentration of the template.
  • the oligonucleotides/polynucleotides, complexes and compositions thereof according to the present invention are particularly useful as reagents for delivery of biomolecules into cells (also referred to herein as "cell delivery reagents” or “delivery reagents", respectively).
  • the invention therefore generally relates to the use of the compositions or complexes, respectively, containing a biologically active substance or biomolecule, respectively, and the nucleotides, oligonucleotides and/or polynucleotides as well as their compositions as disclosed herein, as well as the use of the nucleotides, oligonucleotides or polynucleotides and their compositions as such, for introducing said biologically active substance or biomolecule, respectively, preferably nucleic acids, peptides and/or proteins, into a cell.
  • the complexes or compositions, respectively of the invention are referred to as "transfection reagents".
  • transfection reagents The same applies tor the nucleotides, oligonucleotides and polynucleotides as such as well as their compositions as defined herein.
  • compositions comprising one or more of the above-described compositions containing a biologically active substance or biomolecule, respectively, together with at least one nucleotide, oligonucleotide or polynucleotide species as described herein and at least one additional component, preferably selected from cells, cell culture media, and one ore more transfection enhancers and/or cell delivery enhances.
  • additional component preferably selected from cells, cell culture media, and one ore more transfection enhancers and/or cell delivery enhances.
  • oligonucleotides or polynucleotides as defined herein and the cargo molecule, in particular a biomolecule such as a nucleic acids, peptide or protein, are present in the form of complexes
  • the invention also relates to corresponding compositions comprising one or more of the above described nucleotides, oligonucleotides and/or polynucleotides as such, as well as compositions thereof as outlined above, and at least one additional component as outlined before.
  • Cells into which a biomolecule, preferably a nucleic acid, peptide or protein, can be introduced by using the reagents disclosed herein are prokaryotic cells such as bacteria, or eukaryotic cells.
  • Preferred target cells are eukaryotic cells, e.g. cells of epithelial, neuronal, hematopoietic or any type originating or derived from primary cells, tumor and/or cancer cells or immortalized culture cell lines.
  • the cell may be from any source including bacteria, animals (e.g. canine, mouse, hamster, cattle, sheep, goat, pig, guinea pig, fish, insect etc.), humans, fungi and plants.
  • compositions according to the invention comprising a delivery or transfection, respectively, reagent as disclosed herein together with the desired cargo, e.g. present in the form of compositions or complexes as described above, may take the form of liquid compositions such as an aqueous solution containing the components, preferably in a suitable buffer or medium (e.g. a cell culture medium), but also extend to solid, typically dried compositions or complexes, respectively, e.g. a lyophilized mixture of the delivery reagent with the cargo, e.g. a nucleic acid, preferably examples as outlined below, or a lyophilized complex of these components.
  • a suitable buffer or medium e.g. a cell culture medium
  • Suitable mass ratios of the delivery or transfection, respectively, reagent according to the invention to the cargo can be determined easily using routine experimentation using the desired cell.
  • Examples of useful ratios with respect to nucleic acid transfection using the transfection reagents according to the invention range from 1 :1 to 3: 1 , preferably 1.5:1 to 2.5:1 , most preferred 2:1 .
  • Polyanions preferably nucleic acids, which may be delivered or transfected, respectively, into cells using the delivery reagents of the invention (which may therefore be part of the compositions or complexes, respectivelym comprising the delivery reagent and the cargo, as described above), are preferably selected from DNA and RNA which may each be single stranded or double stranded as well as mixed forms thereof, i.e. DNA RNA hybrids.
  • Particularly preferred examples include, but are not limited to, plasmids, cosmids, oligonucleotides of DNA or RNA type, siRNA, sgRNA (small-guide RNA) (RNAs and DNAs useful in the CRISPR/Cas system; cf. US Patent No. 8,697,359), microRNA and microRNA mimetics, long non-coding RNAs as well as any combination thereof.
  • nucleic acids to be delivered or transfected, respectively, according to the invention may contain nucleotide analogues as outlined before, and/or may be coupled to entities such as dyes, fluorophores, and proteins such as antibodies as further described below for the transfection agents of the invention. Proteins such as antibodies or other macromolecules may be equally used for delivery into cells using the complexes according to the invention.
  • Biomolecule delivery enhancers and/or transfection enhancers useful in the present invention include any known aids for delivery of cargo molecules into cells, such as proteins, peptides, growth factors, lipids, (poly)saccharides and the like enhancing cell-targeting, uptake, internalization, nuclear targeting and expression.
  • Such cell delivery and/or transfection enhancers include specific ligands such as insulin, transferrin, fibronectin for targeting cell surfaces.
  • Further cell delivery and/or transfection enhancers include molecules or substances targeting cellular receptors, e.g. peptides targeting integrin receptors.
  • Further transfection enhancers useful in the present invention include polycationic compounds such as protamine or poly-L-lysine, and liposomes, in particular cationic liposomes.
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one composition or complex containing a nucleotide, oligonucleotide or polynucleotide together with at least one pharmacologically active ingredient to be delivered into a cell or cells, respectively, of a, preferably mammalian, more preferably human, patient, optionally in combination with at least one pharmaceutically acceptable carrier, excipient, and/or diluent.
  • the pharmaceutical composition of the invention thus includes a complex as described above in combination with at least one pharmaceutically acceptable carrier, excipient, and/or diluent.
  • compositions according to the invention are particularly applied for treatment and/or prevention of cancer, infections, degenerative diseases, and are foreseen for application in humans or animals.
  • the invention also relates to corresponding botanical formulations for application in plants such as for the use of siRNAs as pesticides or agents used for the treatment of plant diseases.
  • nucleotides, oligonucleotides and polynucleotides as disclosed herein may also be conjugated with other chemical entities, in particular with chemical groups which can be used to target the nucleotide, oligonucleotide or polynucleotide, respectively, to a cell in which said nucleotide, oligonucleotide or polynucleotide, or the cargo (biologically active substance or biomolecule) is to exert its properties.
  • Chemical entities may be coupled to the inventive species through any kind of bonds such as covalent bonds, hydrogen bonds or Van der Waals bonds.
  • Chemical entities to which the inventive species can be coupled include aptamers, polyethylenglycol such as PEG groups having an average molecular weight of from about 500 to about 1000 Da, e.g. about 750 Da, peptides, a palmitoyi group, cholesterol groups, phospholipids, proteins such as antibodies, and partners of non-covalent binding pairs such as biotin or digoxigenin.
  • biotin it can serve for associating the construct to a streptavidin group present on an antibody, or the biotin may serve as the ligand of an anti-biotin antibody which may, e.g. in turn have a second affinity to a target cell or tissue such as by having an affinity to a target receptor and/or ligand expressed intracellulary and/or on the surface of the target cell and/or tissue.
  • Antibodies in the context of the present invention may be selected from polyclonal, monoclonal, humanized, chimeric, single-chain antibodies and antibody fragments which antibodies or antibody fragments may be single-specific or bispecific species.
  • the antibody fragment may be a Fab fragment, a F(ab') 2 fragment, or any fragment that retains the antigen-binding specificity of the intact antibody.
  • Especially preferred antibody ligands that may be coupled to the molecules as disclosed herein are selected from antibodies or fragments thereof directed against cancer and/or tumor antigens, cancer- and/or tumor- associated antigens or oncoproteins, or antigens present on non-cancer and/or non-tumoral cells.
  • Further subject matter of the present invention relates methods for the delivery of molecules, in particular biomolecules, into cells in vitro and in vivo.
  • the cargo to be delivered into a cell is a nucleic acid
  • such methods are also referred to as "transfection methods”.
  • the present invention provides a method for introducing a biologically active substance into a cell in vitro comprising the step of contacting a complex or composition, respectively, as defined above comprising a nucleotide, oligonucleotide or polynucleotide as disclosed above and the biologically active substance (preferably as defined above) with the cell.
  • the cell is present in culture medium in a reaction container and the composition or complex, respectively, according to the inventions is added to said culture medium.
  • the composition of cell delivery reagent and biologically active substance is present in a suitable container, and the cell, preferably in culture medium, is added thereto.
  • a solid, i.e. dry or substantially dry, e.g. lyophilized, mixture of delivery regent of the invention and the desired biologically active substance (representing, in certain embodiments, a complex as described above), e.g.
  • a nucleic acid may be present in a suitable reaction or culture container, e.g. a well of a multiple-well plate (e.g. dry mixtures of transfection reagent with different molecules to be transfected such as nucleic acids of different sequence, and each different mixture present in an individual well).
  • a suitable reaction or culture container e.g. a well of a multiple-well plate (e.g. dry mixtures of transfection reagent with different molecules to be transfected such as nucleic acids of different sequence, and each different mixture present in an individual well).
  • the invention therefore also extends to such reaction or culture containers containing a delivery
  • composition delivery reagent of the invention plus cargo, or, in certain embodiments of the invention, the complex of delivery reagent with cargo either in liquid or dry state.
  • the invention also provides a method for introducing a biologically active substance (preferably as outlined above) into a cell in vivo comprising the step of
  • a composition comprising a biologically active substance or biomolecule, respectively, together with at least one nucleotide, oligonucleotide or polynucleotide as defined above to a tissue or organism, preferably a mammalian, more preferably a human, containing said cell.
  • a cell into which a biologically substance is to be delivered by the embodiments according to the invention may be taken from said tissue or organism, the biologically active substance is delivered into the cell in vitro as outlined above, and the thus-treated cell is re-introduced into the tissue or organism.
  • compositions as outlined herein are able to deliver cargo substances such as biologically active substances, preferably nucleic acids, peptides and/or proteins, into a target cell is currently under investigation.
  • cargo substances such as biologically active substances, preferably nucleic acids, peptides and/or proteins
  • the G- quadruplex forming entities according to the invention represent negatively charged moieties which quite tightly attract cations which in turn can bind to negatively charged positions of the cargo (see Figs. 20 and 22 for nucleic acid as cargo molecule).
  • These assemblies of G- quadruplex/cations with the cargo are presumably taken up by the target cell through a clathrin-dependent pathway and transported to endosomes.
  • compositions of the invention i.e. the reagents for delivery of molecules, typically biomolecules such as nucleic acids peptides and proteins, are not only
  • the invention also provides the use of delivery reagents as disclosed herein for protecting biologically active substances or molecules, respectively, against destabilizing and/or degrading environments such as solutions containing enzymes that can degrade or otherwise destabilize the biologically active substances or biomolecules, respectively (generally referred to herein as the "cargo” or “cargo substance” or “cargo molecule”).
  • a preferred application of this kind is the use of the delivery agents as disclosed herein for protecting a nucleic acid against nucleic acid degrading enzymes such as exo- and endonucleases, e.g. DNAses and RNAses, more preferred the use of the delivery agents of the invention for protecting RNA against RNAses, e.g. present in an RNAse-containing solution or medium such as a cell culture medium or an animal, preferably mammalian, more preferably human, body fluid such as blood serum.
  • RNAse-containing solution or medium such as a cell culture medium or an animal, preferably mammalian, more preferably human, body fluid such as blood serum.
  • Fig. 1 Synthesis of a delivery reagent of the invention using an RNA polymerase (RdRp) of a calicivirus.
  • RdRp RNA polymerase
  • An ssRNA template (rC5; 5 ' -CCCCC-3') or an ssDNA template (dC6; 5'-CCCCCC-3') was incubated with an RdRp of a calicivirus for 15 min, 45 min or 90 min. After purification of the reaction products using size exclusion chromatography, the amount of single stranded RNA was measured.
  • A Graphical representation of the amount of ssRNA produced using the ssRNA template.
  • B Amount of ssRNA produced using the ssDNA template.
  • Fig. 2 Synthesis of a delivery reagent of the invention using an RdRp of a calicivirus and a single stranded RNA (rC5; 5 ' -CCCCC-3 ' ) as a template. All reactions were performed in a total volume of 100 ⁇ at 37°C. The reaction mix contained 8 ⁇ g of the template, 10 ⁇ RdRp, 2 mM GTP, 20 ⁇ reaction buffer (HEPES 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 ⁇ . The reaction was stopped at 15 min, 45 min and 90 min by adding 10 ⁇ EDTA 0.3 M.
  • reaction products were analyzed using ion exchange HPLC and the area under the curve (AUC) of the eluted peak was measured.
  • A Elution profile of the product of reaction using IEX-HPLC at 15 min, 45 min and 90 min.
  • B Plot of AUC of eluted product as a function of time.
  • Fig. 3 Analysis of oligonucleotides produced according to the invention. All reactions were performed in a total volume of 100 ⁇ at 37°C using a single stranded RNA (rC5; 5 ' - CCCCC-3') or a single stranded DNA (dC6; 5 ' -CCCCCC-3 ' ) as a template.
  • the reaction mix contained 8 ⁇ g of the template, 10 ⁇ RdRp, 2 mM GTP, 20 ⁇ reaction buffer (HEPES 250 mM, MnCI2 25 mM, DTT 5 mM, pH 7.6), and RNAse-DNAse free water to a total volume of 100 ⁇ .
  • RNA marker corresponds to double stranded RNA of 19 bp, 21 bp and 25 bp, respectively, in length, as well as a single stranded RNA of 24 nt in length, as indicated.
  • the visible products are isolated single stranded 5'-triphosphated poly(G) and/or single stranded 5'-triphosphated poly(G) forming complexes and G- quadruplexes (PPPrGx) as well as suprastructures.
  • the single stranded RNA template has been used as a marker (rC5; 5 ' -CCCCC-3 ' ).
  • Fig. 4 Characterisation of the synthesized products according to the reaction as described for Fig. 3B using a single stranded RNA (rC5; 5 -CCCCC-3 ) as a template by
  • Fig. 5 Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS.
  • the single stranded RNA template (rC5; 5 ' -CCCCC-3 ' ) was used for the reaction as described above (see Fig. 3B).
  • the product was denatured and characterized by ESI/MS.
  • the elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a molecular weight (MW) of 1463.22 Da corresponding to the template rC5 (RNA molecule; 5 ' -CCCCC-3 ' ).
  • Fig. 6 Characterisation of the products synthesized as described above for Fig. 4B by ESI/MS.
  • the single stranded RNA template (rC5; 5 ' -CCCCC-3 ' ) was used for the reaction as described above (see Fig. 3B).
  • the product was denatured and characterized by ESI/MS.
  • the elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a MW of 1808.27 Da corresponding to the MW of the product rC5G1 (RNA molecule; 5 ' -CCCCCG-3 ' ).
  • This product is expected to result from a terminal transferase activity of the RdRp on the rC5 template.
  • Fig. 7 Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS.
  • the single stranded RNA template (rC5; 5 ' -CCCCC-3 ' ) was used for the reaction as described above (see Fig. 3B).
  • the product was denatured and characterized by ESI/MS.
  • the elution product of the peak as indicated in panel A was analyzed by ESI/MS, as shown in panel B. It displays a MW of 2499.43 Da corresponding to the MW of the product rC5G3 (i.e. RNA molecule 5 ' -CCCCCGGGG-3 ' ).
  • This product is also expected to result from a terminal transferase activity of the RdRp on the rC5 template.
  • Fig. 8 Characterisation of the products synthesized as described above for Fig. 3B by ESI/MS.
  • the single stranded RNA template (rC5; 5 ' -CCCCC-3 ' ) was used for the reaction as described above (see Fig. 3B).
  • the product was denatured and characterized by ESI/MS.
  • the elution products of the peak as indicated in panel A were analyzed by ESI/MS, as shown in panel B. They display MWs of 2249 Da, 2594 Da, 2939 Da, 3284 Da, 3630 Da, 3975 Da, 4320 Da, and 4665 Da, respectively, as indicated, corresponding to the respective MW of the following products:
  • PPPG6 i.e. RNA molecule 5 ' -GGGGGG-3 ' , with a triphosphate bound to the 5' end of the molecule;
  • PPPG7 i.e. RNA molecule 5 ' -GGGGGGG-3 ' , with a triphosphate bound to the 5 ' end of the molecule;
  • PPPG8 i.e. RNA molecule 5 ' -GGGGGGGG-3 ' , with a triphosphate bound to the 5 ' end of the molecule;
  • PPPG9 i.e. RNA molecule
  • 5 ' -GGGGGGGGG-3 ' with a triphosphate bound to the 5 ' end of the molecule
  • PPPG10 i.e. RNA molecule 5 ' -GGGGGGGG-3 ' (SEQ ID NO: 2), with a triphosphate bound to the 5 ' end of the molecule;
  • PPPG1 1 i.e. RNA molecule 5 ' -GGGGGGGGG-3 ' (SEQ ID NO: 3), with a triphosphate bound to the 5 ' end of the molecule;
  • PPPG12 i.e. RNA molecule 5 ' -GGGGGGGGGG-3 ' (SEQ ID NO: 4), with a triphosphate bound to the 5 ' end of the molecule
  • PPPG13 i.e. RNA molecule 5 ' -GGGGGGGGGGG-3 ' (SEQ ID NO: 5), with a triphosphate bound to the 5 ' end of the molecule.
  • Fig. 9 Characterisation of the products synthesized as described above for Fig. 4B by ESI/MS.
  • the single stranded RNA template (rC5; 5 ' -CCCCC-3 ' ) was used for the reaction as described above (see Fig. 3B).
  • the product was denatured and characterized by ESI/MS.
  • the elution products of the peak as indicated in panel A were analyzed by ESI/MS, as shown in panel B. They display MWs of 3630 Da, 3975 Da, 4320 Da, 4665 Da, and 501 1 Da, respectively, as indicated, corresponding to the respective MW of the following products: PPPG10, i.e. RNA molecule 5 ' -GGGGGGGGGG-3 ' (SEQ ID NO: 2), with a triphosphate bound to the 5 ' end of the molecule;
  • PPPG1 1 i.e. RNA molecule 5 ' -GGGGGGGGG-3 ' (SEQ ID NO: 3), with a triphosphate bound to the 5 ' end of the molecule;
  • PPPG12 i.e. RNA molecule 5 ' -GGGGGGGGGG-3 ' (SEQ ID NO: 4), with a triphosphate bound to the 5 ' end of the molecule;
  • PPPG13 i.e. RNA molecule 5 ' -GGGGGGGGGGG-3 ' (SEQ ID NO: 5), with a
  • PPPG14 i.e. RNA molecule 5 ' -GGGGGGGGGGGG-3 ' (SEQ ID NO: 6), with a triphosphate bound to the 5 ' end of the molecule.
  • Fig. 10 Silencing of GAPDH gene expression in HeLa cells by an siRNA targeting GAPDH in the presence of a cell delivery reagent of the invention.
  • An siRNA iBONi ® siRNA -P4H; Riboxx GmbH, Radebeul, Germany; antisense strand: 5 ' - AUGAGUCCUUCCACGAUACCCCC-3 ' (SEQ ID NO: 7); sense strand:
  • transfection reagent are shown.
  • the silencing of GAPDH is evidenced in dose-dependent manner on the addition of the cell delivery reagent according to the invention to the siRNA.
  • the delivery reagent does not induce gene silencing by itself as shown in (B).
  • Fig. 11 Uptake of siRNA by HeLa cells in the presence of a cell delivery reagent according to the invention.
  • chromophore riboxx570 10 labelled with chromophore riboxx570 was added in a 24-well plate at a concentration of 20 nM in the presence of a cell delivery reagent of the invention (30 ⁇ of a 0.3 ⁇ g/ ⁇ l solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4 h. Uptake of the siRNA was visualized by confocal microscopy. The cell nuclei were stained with DAPI (in blue, here in dark gray). The siRNA was labelled with a red chromophore (here in white to light gray).
  • siRNA siRNA targeting GAPDH in the presence of a cell delivery reagent of the invention.
  • An siRNA iBONi ® siRNA -P4H; Riboxx GmbH, Germany; antisense strand: 5 ' -
  • Fig. 13 Uptake of siRNA by 293T cells in the presence of a cell delivery reagent according to the invention. Same experiment as in Fig. 1 1 , but using 293T cells.
  • Fig. 14 Uptake of ssDNA by HeLa cells in the presence of a cell delivery reagent according to the invention.
  • a fluorescein-labelled single stranded DNA was added to a 24- well plate at a concentration of 20 nM in the presence of a cell delivery reagent of the invention (10 ⁇ of a 0.3 Mg/ ⁇ solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4 h. Uptake was visualized by confocal microscopy. The cell nuclei are stained with DAPI (blue color; here in dark gray). The single stranded DNA bears a green chromophore (here in white to light gray).
  • Fig. 15 Uptake of ssDNA by 293 Tcells in the presence of a cell delivery reagent according to the invention. Same experiment as in Fig. 14, but using 293T cells.
  • Fig. 16 Uptake of dsDNA by HeLa cells in the presence of a cell delivery reagent according to the invention.
  • a fluorescein-labelled double stranded DNA was added to a 24-well plate at a concentration of 20 nM in the presence of a cell delivery reagent of the invention (10 ⁇ of a 0.3 Mg/ ⁇ solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4h. Uptake was visualized by confocal microscopy. The cell nuclei are stained with DAPI (blue color; here in dark gray). The double stranded DNA bears a green chromophore (here in white to light gray).
  • Fig. 17 Uptake of dsDNA by 293T cells in the presence of a cell delivery reagent according to the invention. Same experiment as in Fig. 16, but using 293T cells.
  • An exemplary delivery reagent of the invention was synthesized as previously described (see Fig. 3B using the rC5 ssRNA template).
  • the complex consisting of the delivery reagent and siRNA was mixed as previously described (see above with respect to Fig. 10; 56 ⁇ g of delivery reagent with 28 ⁇ g of siRNA) and analysed by HPLC (A) and (C).
  • PPPG8 RNA molecule; 5 ' -GGGGGGGG-3 ' , with a triphosphate bound to the 5 ' end of the molecule
  • RNA molecule 5 ' -GGGGGGGGG-3 ' , with a triphosphate bound at 5 ' -End of the molecule
  • PPPG10 RNA molecule; 5 ' -GGGGGGGGGG-3 ' (SEQ ID NO: 2), with a triphosphate bound to the 5 ' end of the molecule
  • PPPG1 1 RNA molecule; 5 ' -GGGGGGGGGGG-3 ' (SEQ ID NO: 3), with a triphosphate bound to the 5 ' end of the molecule
  • PPPG12 RNA molecule; 5 ' -GGGGGGGGGG-3 ' (SEQ ID NO: 4), with a triphosphate bound to the 5 ' end of the molecule
  • PPPG13 RNA molecule; 5 ' -GGGGGGGGGGGGG-3 ' (SEQ ID NO: 5), with a triphosphate bound to the 5 ' end of the molecule.
  • PPPG13 RNA molecule; 5 ' -GGGGGGGGGGGGG-3 ' (SEQ ID NO: 5), with a triphosphate bound to the 5 ' end of the molecule
  • RNA molecule 5 ' -GGGGGGGGGGGGG-3 ' (SEQ ID NO: 6), with a triphosphate bound to the 5 ' end of the molecule ssRNA (antisense strand); 5 ' -AUGAGUCCUUCCACGAUACCCCC-3 ' (SEQ ID NO: 7) ssRNA (sense strand); 5 ' -GGGGGUAUCGUGGAAGGACUCAU-3 ' (SEQ ID NO: 8), with a triphosphate bound to the 5 ' end of the molecule.
  • Fig. 19 Serum stability of complexes comprising a delivery reagent of the invention and siRNA.
  • siRNA iBONi ® siRNA -P4H; Riboxx GmbH, Germany; antisense strand: 5 ' - AUGAGUCCUUCCACGAUACCCCC-3 ' (SEQ ID NO: 7); sense strand: 5 - pppGGGGGUAUCGUGGAAGGACUCAU-3 ' (SEQ ID NO: 8)) was incubated at a
  • siRNA concentration of 2 ⁇ for 37°C for the time as indicated in panel (A) in mouse serum (80%).
  • the siRNA was mixed as previously described (see above with respect to Fig. 10; 56 ⁇ g of delivery reagent with 28 ⁇ g of siRNA) and incubated at a concentration of 2 ⁇ for 37°C for the indicated time (h) as indicated in panel (B) in mouse serum (80%).
  • the siRNA incubated alone and in admixture with a delivery reagent of the invention were run on 20% native PAGE and visualized by UV transillumination.
  • FIG. 20 Schematic representation of the putative interaction of quadruplex-forming delivery reagents with nucleic acids.
  • G-quadruplex forming delivery reagents of the invention are represented by gray dots.
  • A Interaction of G-quadruplex forming delivery reagents with double stranded RNA or DNA.
  • B Interaction of G-quadruplex forming delivery reagents with single stranded RNA or DNA.
  • C Interaction of G-quadruplex forming delivery reagents with double stranded RNA or DNA bearing a functional group such as a chromo-, fluorophore, peptide, or a tag.
  • Fig. 21 Schematic representation of a putative mechanism of uptake and release of nucleic acids associated to G-quadruplex forming delivery reagents of the invention into cells.
  • a double-stranded RNA or DNA formulated with G-quadruplex forming delivery reagents of the invention is taken up through a clathrin-dependent pathway, and
  • Fig. 22 Schematic representation of the putative interaction of nucleic acid with G- quadruplex forming delivery reagents of the invention. Interaction occurs through cations (mono- or divalent, e.g. K + , Na + , Mg 2+ and/or Mn 2+ ) present in the formulation.
  • G-quadruplex forming reagents of the invention are present in complex with cations which in turn attract nucleic acid cargo molecule (negatively charged), thus forming a ternary complex G- quadruplex-cation-cargo nucleic acid.
  • the present invention is further illustrated by the following non-limiting examples:
  • Example 1 Synthesis of cell delivery reagents of the invention by RNA-dependent
  • reaction products were also analyzed using ion exchange HPLC and the area under the curve (AUC) of the eluted peak was calculated.
  • Results for the reaction using the rC5 template are shown in Fig. 2A (elution profile of reaction stopped after 15 min), Fig. 2B (elution profile of reaction stopped after 45 min) and Fig. 2C (elution profile of reaction stopped after 90 min).
  • Fig. 2D shows the plot of the results of the AUC
  • Example 2 Analysis of reaction products by PAGE RdRp reactions were set up ad carried out as in Example 1 using the rC 5 template or the dC 6 template, and the reactions were stopped at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min by adding 10 ⁇ EDTA 0.3 M. After purification of the reaction products by size exclusion chromatography using the riboxxPURE kit according to the instructions of the manufacturer (RiboxX GmbH, Radebeul, Germany), 15 ⁇ of each reaction were analyzed by 20% PAGE and visualized through UV transillumination. Result of the time course experiment using the rC5 ssRNA template is shown in Fig. 3A.
  • Fig, 3B shows the products of reactions using the rC 5 or the rC 6 template each allowed to proceed for 45 min.
  • the bands visible correspond to isolated single stranded 5'-triphosphated poly(G) and/or single stranded 5'-triphosphated poly(G) forming complexes and G-quadruplexes (PPPrGx) as well as higher suprastructures.
  • Example 2 The reaction product of Example 2 using the rC 5 template was subjected to HPLC.
  • the elution profile is shown in Fig. 4.
  • the peak fractions as indicated in Figs. 5A, 6A, 7A, 8A and 9A were denatured and further analyzed by ESI/MS.
  • the corresponding mass spectra are shown in Fig. 5B, 6B, 7B, 8B and 9B, respectively.
  • the peak(s) in the mass spectra correspond to the molecular weight of the species indicated in Fig. 5B, 6B, 7B, 8B and 9B, respectively.
  • Example 4 Gene-silencing in eukaryotic cells by siRNA using transfection with a delivery reagent of the invention
  • siRNA iBONi ® siRNA-P4H; RiboxX GmbH, Radebeul, Germany; antisense strand: 5 ' - AUGAGUCCUUCCACGAUACCCCC-3 ' (SEQ ID NO: 7); sense strand:
  • 5'-pppGGGGGUAUCGUGGAAGGACUCAU-3 ' (SEQ ID NO: 8)) targeting the GAPDH gene was added in a 24-well plate at a concentration of 40 nM in the presence of 20 ⁇ , 10 ⁇ , 5 ⁇ or 2.5 ⁇ of a 0.3 ⁇ g/ ⁇ l solution of the reaction of Example 1 using the rC5 ssRNA template.
  • HeLa or 293T cells were added at 25.000 cells/well, and the plates incubated at
  • Example 5 Transfection of RNA and DNA into eukaryotic cells using a delivery reagent of the invention An siRNA (iBONi ® siRNA transfection control T1 -H; antisense strand: 5 ' - AUGAGUCCUUCCACGAUACCCCC-3 ' (SEQ ID NO: 7); sense strand:
  • chromophore riboxx570 (RiboxX GmbH, Radebeul, Germany) was added to a 24-well plate at a concentration of 20 nM in the presence of the cell delivery reagent of the invention (30 ⁇ of a 0.3 Mg/ ⁇ solution), then added to the cells seeded at 25.000 cells/well, and the plates were incubated at 37°C/5%C02 for 4h. Uptake was visualized by confocal microscopy. The cell nuclei were stained with DAPI (blue color). The transfected siRNA is visible through its bound red fluorophore at 570 nm. Fig.
  • FIG. 1 1 shows a photograph of a representative section of the plate wherein the cell nuclei are visible as darker gray structures, and the uptake of the siRNA is clearly visible as white to light gray dots. Larger areas in white to light gray indicate larger amounts of cell delivery reagent/siRNA complexes within the cells.
  • 293T cells epidermal cells which are not easy to transfect with hitherto known transfection agents
  • oligonucleotides of the invention are potent cell delivery reagents for all kinds of nucleic acids. As shown by the siRNA experiments, the cell delivery reagent of the invention does not interfere with the function of the transfected nucleic acid in the cell.
  • Example 6 Cell delivery reagents of the invention form complexes with a nucleic acid cargo
  • a cell delivery reagent was produced as outlined in Example 1 using the rC5 ssRNA template. 56 ⁇ g of cell delivery reagent were mixed with 28 ⁇ g of an siRNA (iBONi ® siRNA- P4H; RiboxX GmbH, Radebeul, Germany; antisense strand: 5 ' - AUGAGUCCUUCCACGAUACCCCC-3 ' (SEQ ID NO: 7); sense strand:
  • Example 7 Cell delivery reagent of the invention protects RNA from degradation in mouse serum
  • siRNA iBONi ® siRNA -P4H; Riboxx GmbH, Germany; antisense strand: 5 ' - AUGAGUCCUUCCACGAUACCCCC-3 ' (SEQ ID NO: 7); sense strand:
  • 5'-pppGGGGGUAUCGUGGAAGGACUCAU-3 ' (SEQ ID NO: 8) was incubated at a concentration of 2 ⁇ for 37°C for 48 h in mouse serum (80%).
  • the siRNA was mixed with a cell delivery reagent of the invention as described in Example 6 and incubated at a concentration of 2 ⁇ for 37°C for 48 h mouse serum (80%). Samples were taken from both reactions before incubation and after 0.5 h, 2 h, 4 h, 24 h and 48 h of incubation. The samples were run on 20% native PAGE and visualized by UV

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Abstract

La présente invention concerne généralement des compositions ou des complexes, respectivement, contenant une substance ou biomolécule biologiquement active et au moins un nucléotide, oligonucléotide et polynucléotide formant des quadruples hélices de G, et/ou des structures intramoléculaires et/ou intermoléculaires et/ou des suprastructures dans des conditions appropriées, des compositions contenant ceux-ci, des procédés pour leur production et leur utilisation en tant que réactifs d'administration et/ou réactifs de transfection in vitro et in vivo. La présente invention concerne en outre des modes de réalisation spécifique d'oligonucléotides ou polynucléotides formant des quadruples hélices de G, respectivement, en tant que tels.<i />
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