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  • 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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  • 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
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
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  • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
• • C—CHEMISTRY; METALLURGY
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein

Definitions

• the present invention relates to a system for intracellular cargo delivery, named NickFect, comprising at least one component A, which is attached covalently to cell penetrating peptide B and/or peptide or non-peptide construct C.
• the said delivery system NickFect relates to chemically modified new cell-penetrating peptides (CPP) non-covalently or covalently complexed with cargo for efficient cellular.
• CPP cell-penetrating peptides
• the invention relates to constructs comprising non-toxic peptide delivery vector, with increased stability and more potent to escape from endosomal -lysosomal compartment and is capable to form stable complexes with cargo, which remain intact in the presence of serum.
• the invention relates on a delivery system, which has overall negative charge of formed nanoparticles for oligonucleotide delivery.
• the invention also relates on a delivery system, which comprises branched CPP, where fatty acid modified TP 10 or its segment are linked through , ⁇ , ⁇ , ⁇ - amino groups of lysine or their analogues to amphipatic or/and a-helical peptide or peptide segments.
• the invention relates to a method of delivering cargos (RNA, DNA, drugs, plasmids, minicircles etc.) into cytosol or nucleus of a target cell in vitro or in vivo. It also relates to the use of the system in diagnosis of deceases, as research tool and as a targeting system, a composition comprising the system and especially a pharmaceutical composition a material covered with the system and a material having the delivery systems into material. Finally also relates to novel cell-penetrating peptides.
• cargos RNA, DNA, drugs, plasmids, minicircles etc.
• the hydrophobic cell membrane is a lipid bilayer that encloses the cellular contents. It functions as barrier that normally only small and/or hydrophobic molecules can cross and prevent access to the interior of cells of other macromolecules like naked DNA, RNA or proteins. Although few strategies were designed to solve this problem in last decade, the inability to cross the plasma membrane is still one of the major hindrance to overcome in order to success current drug research and development (R&D).
• Viral vectors adenoviral, recombinant Endogenous recombination, oncogenic lentiviral vectors, etc. ) effects and immunological reactions
• CPPs Cell-penetrating peptides
• PTDs protein transduction domains
• CPPs In order to promote intracellular delivery CPPs should be conjugated with cargo (oligonucleotide, plasmid, minicicle etc.) in covalent or non-covalent manner. Covalent attachment of peptide to ON or any other biomolecule is laborious procedure and usually high concentrations of peptide conjugates are needed to obtain a significant biological response. In case of non-covalent complexes, the efficient concentrations are in nanomolar range and non-covalent complex formation can be reached by mixing together peptide and biomolecule solutions in water for 1 hour.
• the present invention provides a system for intracellular cargo delivery comprising a new series of cell-penetrating peptides that overcome the drawbacks of non-covalent gene- delivery, low and heterogeneous delivery as well as toxicity.
• Figure 1A Pre-mRNA of the modified luciferase gene.
• the intron 2 from the B-globin gene carrying a point mutation at nucleotide 705 is inserted into the luciferase gene. Blockage of this site with SCO redirects splicing towards the functional mRNA.
• Fig IB, 1C General schematic structures of NickFect delivery system, where solid line marked for covalent binding and dotted line is non-covalent conjugation.
• Hela pLuc 705 cells were treated with peptide:2'-OMe ON complexes at different molar ratios (5: 1, 7: 1, 10: 1) in serum free DMEM for 4 hours, after which media was replaced for full growth media and incubated additionally 20 hours.
• Lipofectamine 2000 was used according to manufactures protocol. The results clearly show that in addition to phosporylation modification in the backbone of the peptide is necessary to increase splice correction efficacy.
• Figure 4 Effect of the introduction of phosphoryl groups at tyrosine or threonine moieties in chemically modified Stearyl-TPlO analog (NF11) on the splice correction activity.
• the complexes were applied at molar ratios 3 : 1, 5: 1, 7: 1 to Hela pLuc 705 cells in SFM - A and in FM - B, at 200 nM ON concentration.
• Lipofectamine 2000 was used according to manufactures protocol.
• New phosphorylated CPPs induced a dramatic increase in splice correction and very low amounts of peptides and ONs are needed to gain a biological response.
• NFl acted more effectively in full media, compared to NF2.
• FIG. 5 The effect of the lysosomotropic reagent chloroquine on splice-correction activity.
• the peptide-ON complex NFl, NF2 and Stearyl-TPlO in SFM were applied to the cells at most efficient molar ratio (7: 1) at 200 nM ON concentration with and without the presence of chloroquine.
• Figure 6 The effect of oligonucleotide-peptide complexes on the viability of Hela cells as compared to LipofectamineTM 2000.
• the cell viability was measured by MTS assay after adding of peptide:2'-OMe ON complexes at different molar ratios after 24 hours. ON applied at was 200 nM concentration. NFl and NF2 were not toxic at most effective concentrations.
• FIG. 7 NFl, NF2 mediated siRNA delivery into EGFP-CHO cells, which are stably expressing GFP protein.
• Cells were treated with siRNA:pepteide complexes at MR 20, MR25, MR30, MR40 in serum free DMEM media for 4 hours and with HAM full media for 20 hours. After that cells were washed, trypsinized and analysed by flow cytometry.
• Figure 8 NF51, NF52, NF53 and NF61 as delivery vector for pGL3 plasmid transfection in HEK293 cells.
• Figure 9 NF51, NF52, NF53 and NF61 as delivery vector for pGL3 plasmid transfection in CHO cells.
• the present invention provides the series of new chemically modified delivery vectors, NickFects, that are based on TP 10 sequence (A G Y L L G K I N L K A L A A L A K K I L-NH 2 ) and that can be utilized for efficient delivery of different cargos (RNA, DNA, plasmid, minicircle, drugs, etc.) into cytosol and/or nucleus of a target cell in vitro or in vivo using a non-covalent or covalent approach.
• the new delivery system, NickFect is able to form stable, tightly packed, regular and spherical nanoparticles with cargo. Formed nanoparticles have better interaction properties with plasma and/or nucleus membrane and can more efficiently internalizes into cells.
• the abovementioned NickFects can be used in diagnosis of diseases, in gene therapy, in tumor treatment and for other pharmaceutical applications, as a r esearch tool and as a targeting system.
• the invention relates to constructs comprising non-toxic, with increased stability and more potent to escape from endosomal -lysosomal compartment and which are capable to form stable complexes with cargo, which remain intact in the presence of serum
• NickFect delivery vectors are more efficient than the commercial transfection reagent LipofectamineTM2000 in conveying various ONs and plasmids inside cells, while being less toxic.
• the low toxicity of NickFects renders them suitable for transfection in sensitive cell systems and in vivo, while LipofectamineTM2000 can be used only in vitro due to its high toxicity at concentrations needed.
• NickFects can be stored as a powder for a long period of time, while the producer guarantees the stability of LipofectamineTM2000 for only 6 months at +4 °C.
• the NickFect delivery vectors are more potent than conventionally used CPPs for RNA and DNA cellular delivery. Most important, only very low amounts of peptides and ONs are needed to gain a biological response.
• NFl is as active as Lipofectamine 2000 for the delivery of siRNAs into cells. While Lipofectamine/siRNA complexes rarely generates more than 80% down -regulation of gene expression at any given siRNA concentration, NickFectl complexed with siRNA confers almost complete RNAi at low siRNA concentrations.
• the present invention relates to a system for intracellular cargo delivery, named NickFect, comprising at least one cell penetrating peptide to B, which in some occasions comprises covalently linked component A and/or peptide or non-peptide construct C, which is targeting moiety.
• the said delivery system NickFect is capable of delivering cargo by covalent or non-covalent attachment (Fig. IB)
• the cell delivery system may comprise more than one peptide B, more than one component A and more than one targeting component C coupled to each other in any order without any cargo.
• the abovementioned delivery system may be linked to one or more cargoes which may be delivered into cells, tissues or across a cell layer.
• the invention also relates to novel cell -penetrating peptides as well as the method how to produce the NickFect constructs.
• the component C as a targeting moiety is capable of reaching specific cells or tissues of interest.
• the targeting moiety may be an aptamer or targeting peptide such as a homing peptide or a receptor ligand.
• Component A comprises phosphate group (P0 3 ) or any negatively charged moiety (Asp, Glu, carbohydrates, etc.). Component A can be even a peptide sequence with overall negative charge.
• One linear aliphatic component A may be conjugated to peptide B or two similar or different components A may be conjugated to peptide B via lysine branched spacer.
• Peptide B comprises chemical modifications of cell penetrating peptide TP 10 which has any fatty acid (e.g. stearic acid) covalently linked to the peptide backbone.
• fatty acid e.g. stearic acid
• Peptide B also comprises cell penetrating peptide, fatty acid modified TP 10, where in position 3, Tyr is replaced with Lys, Orn for subsequent addition of negatively charged molecules or/groups, with Thr, Ser for usage as tyrosine analogs and with Asp or Glu for insertion of carboxyl groups in peptide backbone.
• Peptide B also comprises cell penetrating peptide, fatty acid modified TP 10, where in position 8, He is replaced with Thr, Ser, Tyr, Asp or Glu any other hydrophilic amino acid to increase hydrophilic properties a-helix.
• Peptide B may also be fatty acid modified TP 10, where at least one Leucine is replaced with Leucine isomers and/or analogues (e.g. Norleucine).
• Peptide B may also be fatty acid modified TP 10, where at least one Lysine is replaced with Lysine isomers and/or derivatives or Ornitine isomers or/and analogues.
• Peptide B may be a branched peptide comprising of fatty acid modified TP 10 peptide or/and its segments (e.g. Galanin, Mastoparan) linked through ⁇ , ⁇ , ⁇ , ⁇ , ⁇ -amino groups of lysine or their analogues to amphipatic or/and a-helical peptides or/and peptide segments (e.g. PY, substance P, bradykinin, model sequences like (Ala-Leu) n , TP 10, Galanin, Mastoparan) (Fig. 1C)
• fatty acid modified TP 10 peptide or/and its segments e.g. Galanin, Mastoparan
• Peptide or non-peptide construct C comprises targeting component capable of reaching specific cells or tissues of interest and which is covalently or non-covalently linked with peptide B.
• Construct C is a c ell- or tumor homing peptide, aptamer, a receptor ligand, a spacer comprising a cleavable site coupled to an inactivating peptide, peptide ligand, cytotoxic peptide, bioactive peptide ligand for a known or unknown receptor, a peptide sequence which selectively binds to a certain tissue or cell type or nuclear localization sequence (NLS).
• NLS nuclear localization sequence
• Spacers may be used for the attachment of component A, C and cargoes to the component B.
• the spacer may be a linear or branched moiety comprising of one or several Lysine and/or Ornithine residues.
• Cargo is attached to the delivery system by covalent assembly or complex formation.
• the cargo may be selected from the group consisting of oligonucleotides and modified versions thereof, single-stranded oligonucleotides (DNA, RNA, PNA, LNA and synthetic oligonucleotides), double-strand oligonucleotides (siRNA, shRNA, decoy dsDNA etc), plasmids, minicircles and other varieties thereof, synthetic nucleotide analogs for the purpose of inhibition of viral replication or antiviral ONs.
• the cargo may be a detection marker imaging agent, labeling molecule, a fluorescent marker , aptamer, a receptor ligand, a s pacer comprising a cl eavable site coupled to an inactivating peptide, peptide ligand, cytotoxic peptide, bioactive peptide, antibody, diagnostic agent, protein, pharmaceutical e.g. anticancer drug or antibiotics.
• the anticancer drugs as a cargo may be chosen from an alkylating agent, an antimetabolite and a cytotoxic antibiotic.
• constructs according to the invention may be used in diagnosis of diseases, in gene therapy, as research tool, as targeting system and as pharmaceutical composition.
• Peptides were synthesized in stepwise manner at 0.1 mmol scale on an automated peptide synthesizer (Applied Biosystems, ABI433A,USA) using Fmoc (fluorenylmethyloxycarbonyl) solid-phase peptide synthesis strategy (Fields and Noble, 1990) with Rink-amide MBHA (methylbenzylhydrylamine) resin (Fluka) as solid phase to obtain C-terminally amidated peptides.
• the stearic acid was coupled manually to the N- terminus of the peptide by treatment of peptidyl -resins with 5 eq. stearic acid (Sigma, Germany), 3 eq. HOBt and 3 eq.
• FIBTU MultiSyntech, Germany
• DIEA dimethylformamide/dichloromethane (1 : 1) overnight at room temperature.
• phosphorylated peptides phosphothreonine Fmoc-Thr(PO(OBzl)OH)-OH (Fluka, Germany) and phosphotyrosine Fmoc-Tyr(PO(OBzl)OH)-OH (Merck, Germany) monomers were used and the coupling was carried out manually by treatment of peptidyl- resin with 3 eq. of a phosphomonomer, 3 eq. HOBt and 3eq. HBTU, 6 eq.
• the identity of peptides was analyzed by MALDI-TOF mass-spectroscopy (The Voyager-DETM PRO BiospectrometryTM System) in positive linear mode using a-cyano-4-hydroxycinnamic acid as matrix (Sigma-Aldrich). The molarity of the peptides was determined based on di lutions of accurately weighed substances. The sequences of the peptides are presented in Table 1.
• Cy5 labeled and unlabeled phosphorothioate 2'-0-methyl RNA oligonucleotides (CCU CUU ACC UCA GUU AC A) were purchased from Microsynth AG, Switzerland. Cell culture
• HeLa pLuc 705 cells kindly provided by R. Kole and B. Leblue, and HEK293 cells were grown at 37 °C, 5% C0 2 in Dulbecco's Modified Eagle's Media (DMEM) with glutamax supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10 % FBS, 100 U/ml penicillin, 100 mg/ml streptomycin.
• CHO cells were grown in DMEM-F12 media with glutamax supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 10 % FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were grown at 37 °C in 5% C0 2 atmosphere. All media and chemicals were purchased from PAA Laboratories GmbH (Germany).
• SCO/CPP complexes Phosphorothioate 2 -OMe oligonucleotides were mixed with CPPs at different molar ratios (1 :0-1 :20) in ddH 2 0 in 10% of the final treatment volume (i.e. 50 ⁇ ). Complexes were formed for 1 h at room temperature and meanwhile the cell medium was replaced in 24-well plates to fresh serum free DMEM (450 ⁇ ). Thereafter complexes were added to each well. When using LipofectamineTM 2000 (Invitrogen, USA), the complexes were prepared according to manufacturer's protocol in OPTFMEM medium (Invitrogen, USA).
• plasmid/CPP complexes 0.5 ⁇ g of pGL3 luciferase expressing plasmid or pEGFP green fluorescent protein expressing vector were mixed with CPPs at different charge ratios (1 : 1-1 :5) in ddH 2 0 in l/lO 111 of the final treatment volume (50 ⁇ ). After 1 hour, complexes were added to cells grown in 450 ⁇ of fresh serum free media. When using Lipofectamine 2000, complexes were prepared according to manufacturers protocol (Promega, USA).
• oligonucleotides/CPP complexes were analyzed using Cy5 labeled ON by Typhoon Variable Mode Imager (Amersham, Sweden) after electrophoresis of complexes in 2% agarose gel in Tris-acetate-EDTA (TAE) buffer for 30 min at 100V.
• TAE Tris-acetate-EDTA
• Plasmid/CPP complexes were analyzed electrophoresis on a 2% agarose gel in TAE buffer, containing ethidium bromide (Sigma, Germany), for 1 hour at 100V. Splice correction assay
• Cells (50,000) were seeded 24 h prior to experiments in 24-well plates to reach about 60 % confluence one day post seeding. Cells were treated with peptide: 2'-OMe ON complexes at six different molar ratios (1 : 1, 3 : 1, 5: 1, 7: 1, 10: 1, 15: 1) at 200nM oligonucleotide concentration for 4 h i n 500 ⁇ serum-free or serum-containing media, followed by the addition of 1 m 1 10% serum-containing medium and incubated for additional 20 h.
• inhibitor was added to the medium 30 min prior to treatment of cells with peptide: 2'-OMe ON complexes. 2 h after addition of the complexes to cells, medium was removed and replaced with fresh medium in order to avoid toxicity effects of inhibitors.
• chloroquine chloroquine (final concentration 100 ⁇ ) was added to cells along with peptide: 2'-OMe ON complexes in order to promote endosomal escape. 4 h after addition of the complexes and chloroquine to cells, medium was replaced with fresh medium in order to avoid toxic effects of chloroquine.
• CHO Choinese hamster ovary cell
• HEK293 Human Embryonic Kidney 293 cells
• EGFP-CHO cells (10 000) we seeded 24 h before experiment in 96-wellplate to reach 60% confluence on the day of the experiment.
• For complex formation peptide 100 ⁇ stock solution was mixed with siRNA (10 ⁇ stock solution) in MQ water in 1/lOth of final treatment volume (i.e. 10 ⁇ ).
• Final concentrations 100 nM siRNA, MR 20, MR25, MR30, MR40 in serum free DMEM were used.
• Flow cytometry analysis was carried out with BD FacsCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Population of viable cells was determined from a scatter plot: forward scattered light (FSC) vs. side scattered liht (SSC) plot. A minimum 10,000 events from the viable cell population per sample were analyzed.
• FSC forward scattered light
• SSC side scattered liht
• Cell proliferation was studied with CellTiter 96® A Queous Non-Radioactive Cell Proliferation Assay (MTS) according to the manufacturer's instructions. Briefly, 10000 Hela pLuc 705 cells per well were seeded 1 day prior to experiment on a 96-well plate in complete DMEM. Cells were treated with peptide: 2'-OMe ON complexes at four different molar ratios (5: 1, 7: 1, 10: 1 and 20: 1) for 4 hours in serum-free medium, followed by the addition of 10% serum containing medium and incubated for additional 20 hours. MTS was added according to the manufacturer's general protocol (Promega Biotech AB, Sweden).
• MTS Non-Radioactive Cell Proliferation Assay
• CPPs cell-penetrating peptides
• PNA antisense peptide nucleic acid
• Soomets U. et al. Deletion analogues of transportan. Biochim Biophys Acta 1467, 165-76 (2000).

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