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WO2023093960A1 - Conjugué peptide-oligonucléotide modifié - Google Patents

Conjugué peptide-oligonucléotide modifié Download PDF

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Publication number
WO2023093960A1
WO2023093960A1 PCT/DK2022/050248 DK2022050248W WO2023093960A1 WO 2023093960 A1 WO2023093960 A1 WO 2023093960A1 DK 2022050248 W DK2022050248 W DK 2022050248W WO 2023093960 A1 WO2023093960 A1 WO 2023093960A1
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oligonucleotide conjugate
peptide
oligonucleotide
aso
modified
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Inventor
Jesper Thagaard WENGEL
Sara Raquel do Souto PEREIRA
Rita Sobral Fernandes Machado dos SANTOS
Luis Miguel de Aquino MOREIRA
Nuno Filipe AZEVEDO
Nuno Miguel da Rocha GUIMARÃES
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Syddansk Universitet
Universidade do Porto
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Syddansk Universitet
Universidade do Porto
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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
    • C12N15/11—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
    • 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
    • C12N2310/11—Antisense
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • 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
    • C12N2320/50—Methods for regulating/modulating their activity

Definitions

  • the present invention relates to antisense oligonucleotides conjugated to cellpenetrating peptides for intracellular delivery.
  • Bacterial infections are a major problem worldwide. Especially as treatment of bacterial infections with current antibiotics is threatened by the bacterial resistance to antibiotics. Antimicrobial resistant infections are estimated to cause 33,000 annual deaths, only in Europe.
  • ASOs Antisense oligonucleotides
  • ASOs composed of nucleic acid mimics (NAMs), such as locked nucleic acids (LNA) and 2'-O-Methyl RNA (2'OMe), hold promise as antibacterial agents due to their improved target affinity and their resistance against nucleases compared to unmodified monomers. Additionally, their safety and efficiency are proved by their use in FDA-approved ASOs-based therapeutics for the treatment of non-infectious diseases.
  • ASOs can be designed to inhibit the expression of essential genes, thus inhibiting bacterial growth, or genes involved in resistance to antibiotics, thus restoring the susceptibility to antibiotics.
  • ASOs can easily be redesigned upon a point mutation on the bacteria, which renders resistance to the ASO.
  • ASOs To act as antimicrobials, ASOs must reach the cytosol to hybridize with the target mRNA. However, to be able to do so, they must be conjugated with a vector capable of penetrating the multilayered bacterial cell envelopes.
  • CPPs Cell-penetrating peptides
  • PNA neutrally charged ASOs
  • a technology enabling conjugation of CPPs with phosphate-based ASOs would be advantageous, and in particular a cell-penetrating peptide conjugated with ASOs containing negatively charged phosphate, e.g. phosphodiester or phosphorothioate, linkages with low cytotoxicity and improved affinity to the target RNA would be an advantage.
  • ASOs containing negatively charged phosphate e.g. phosphodiester or phosphorothioate
  • an object of the present invention relates to a cell-penetrating peptide conjugated to an antisense oligonucleotide.
  • one aspect of the invention relates to a modified peptide-oligonucleotide conjugate comprising:
  • ASO antisense oligonucleotide
  • modified peptide-oligonucleotide conjugate has been covalently modified by reducing the total negative charge of the peptide-oligonucleotide conjugate by addition of positively charged groups.
  • Another aspect of the present invention relates to a peptide-oligonucleotide conjugate according to the present invention, or the pharmaceutical composition according to the present invention, for use as a medicament.
  • Figure 1A-B shows chemical representation of the monomers inserted in the ASOs.
  • LNA monomers were replaced by (A) aminoalkyl-amino-LNA (AB-IT) in POC-3 and (B) zip nucleic acid (ZNA) was introduced in the 3'-end of the ASOs included in POC-4, POC-7 and POC9668.
  • the base in AB-IT and ZNA could be either adenine, thymine, uracil, guanine, cytosine or 5-methylcytosine.
  • Figure 1C-D shows chemical representation of the monomers inserted in the ASOs.
  • LNA monomers were replaced by (C) glycol 2'-amino-LNA-T (aT(AT)) in POC-5, POC9664, POC9665, POC9666 and POC9678, whereas LNA monomers were replaced by (D) glycyl 2'-amino-LNA-T (aT(gly)) monomers in POC-6 and POC-7.
  • the base in aT(AT) and aT(gly) could be either adenine, thymine, uracil, guanine, cytosine or 5-methylcytosine.
  • Figure 2 shows azide-alkyne click-chemistry protocol for synthesis of the peptideoligonucleotide conjugates (POCs).
  • Figure 3 shows far-ultraviolet CD spectra of POC-1, POC-2, POC-3 and POC-4 at 20 °C in 5.8 mM phosphate buffer pH 7.0 with 100 mM NaCI and 0.10 mM EDTA. The spectra were recorded using a 0.2 cm path-length cell. The corresponding reference (buffer solution) was subtracted from each spectrum.
  • Figure 4 shows time-kill curve of E. coli K12 in the presence of POC-1 and POC-2 at 30 and 60 ⁇ M.
  • the PS linkages present in POC-1 hindered an anti-bacterial effect and the growth inhibition of E. coli caused by POC-2 was still very poor.
  • the (KFF) 3 K CPP was used as a control at a concentration of 60 ⁇ M.
  • CB represents the bacterial growth control in culture medium without any supplementation. Results from three independent experiments (using duplicates in each) are presented as mean values and respective standard deviations.
  • Figure 5A shows time-kill curves of E. coli K12 in the presence of POC-3 to POC-7 (SEQ ID NO: 5-9) designed to target the acpP gene in E. coli.
  • the figure shows the POCs ability to inhibit E. coli K12 growth.
  • the positive charges added to the ASOs targeting E. coli benefited the antimicrobial activity of the POCs.
  • the peptide (KFF)sK SEQ ID NO: 1 was used as control at the concentration of 60 ptM, and bacteria in culture media was used as negative control (CB). Three independent assays were performed.
  • Figure 5B shows time-kill curves of S. aureus and P. aeruginosa in the presence of POC-3 (for P. aeruginosa) (SEQ ID NO: 5), POC-4 (SEQ ID NO: 6) and POC-5 ((SEQ ID NO: 7)) designed to target the acpP gene in E. coli.
  • the figure shows the specificity of the POCs (by the lack of activity against S. aureus and P. aeruginosa).
  • the peptide (KFF)sK (SEQ ID NO: 1) was used as control at the concentration of 60 ptM, and bacteria in culture media was used as negative control (CB). Three independent assays were performed.
  • Figure 6 shows the chemical structure of 5-azidopentanoyl-(KFF)3K.
  • Figure 7 shows results from the RT-PCR analysis of HRAS exon 2 splicing in T24 cells transfected with the HRAS ASOs with different modifications (BCN-CPP, aT(AT), LNA, ZNA) run on a TBE agarose gel.
  • BCN-CPP, aT(AT), LNA, ZNA modifications
  • a schematic representation of the HRAS exon 2 splicing is shown to the right of the gel picture. The lower bands correspond to the transcript with exclusion of HRAS exon 2 that is caused by transfection with the HRAS ASOs.
  • Figure 8 shows results from the RT-PCR analysis of HRAS exon 2 splicing in T24 cells transfected with the HRAS ASOs with different modifications (BCN-CPP, aT(AT), LNA, ZNA) and a non-targeting control ASO at 20 and 40 nM run on a TBE agarose gel.
  • a schematic representation of the HRAS exon 2 splicing is shown to the right of the gel picture. The lower band corresponds to the transcript with exclusion of HRAS exon 2, which is caused by transfection with the HRAS ASOs.
  • the present invention describes new antisense oligonucleotide (ASO) constructs, designed to target specific genes in bacterial as well as eukaryotic cells, like human and animal cells or living organisms. Further, it is an object of the present invention to make an efficient conjugation of ASOs with cationic cell-penetrating peptides (CPPs) to overcome the cytotoxicity issues, insufficient affinity to the target RNA and to potentiate their cellular, antimicrobial as well as in vivo activity, e.g. in human cells in a human.
  • ASO antisense oligonucleotide
  • ASO means an oligonucleotide complementary in full or in part with an RNA target.
  • the ASO can contain chemically modified nucleotides in full or in part and may also contain one or more DNA nucleotides.
  • conjugate means an atom or group of atoms bound to an oligonucleotide or oligomeric compound.
  • conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
  • hybridization means the pairing of complementary oligomeric compounds (e.g., an ASO and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding.
  • linker means any atom or group of atoms used to attach a conjugate to an oligonucleotide or an oligomeric compound.
  • LNA Locked nucleic acid nucleoside
  • locked nucleic acid nucleoside or "LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2'-bridge.
  • mRNA means an RNA molecule that encodes a protein.
  • non-natural monomer describes a nucleotide modification, i.e. a nucleotide that is chemically modified relative to the natural DNA or RNA nucleotides.
  • nucleotide means a nucleoside further comprising a phosphate linking group.
  • linked nucleosides may or may not be linked by phosphate linkages and thus include, but is not limited to “linked nucleotides”.
  • linked nucleosides are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are present between those that are linked).
  • nucleobase means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
  • nucleobase complementarity or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase.
  • adenine (A) is complementary to thymine (T).
  • adenine (A) is complementary to uracil (U).
  • complementary nucleobase means a nucleobase of an ASO that is capable of base pairing with a nucleobase of its target nucleic acid.
  • nucleobases at a certain position of an ASO are capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid
  • the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered complementary at that nucleobase pair.
  • Nucleobases comprising certain modifications, preferably covalent modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
  • complementary in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions.
  • Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.
  • complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary).
  • complementary oligomeric compounds or regions are 80% complementary.
  • complementary oligomeric compounds or regions are 90% complementary.
  • complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary. In another embodiment, the oligomeric compounds comprise up to 3 mismatches, such as up to 2 or 1 mismatches. Preferably, no mismatches are present.
  • oligonucleotide means a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • unmodified nucleobase or “naturally occurring nucleobase” mean the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
  • Modified nucleobase adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
  • modified nucleobase means any nucleobase that is not a naturally occurring nucleobase.
  • modified nucleoside means a nucleoside comprising at least one chemical covalent modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
  • subject comprises humans of all ages, other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals in general, including commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, cats and dogs, as well as birds. Preferred subjects are humans.
  • subject also includes healthy subjects of the population.
  • the modified peptide-oligonucleotide conjugate (POC) is composed of different elements.
  • the core design comprises a cell-penetrating peptide (CPP), followed by a linker, which again is followed by an antisense oligonucleotide (ASO).
  • CPP cell-penetrating peptide
  • ASO antisense oligonucleotide
  • the ASO can be followed by a conjugating group.
  • the different elements in the POC can be modified to reduce the net-charges of the POC. This stabilises the secondary structure and protects the POC from enzymatic degradation.
  • a modified peptide-oligonucleotide conjugate (POC)
  • the conjugate comprises
  • ASO antisense oligonucleotide
  • the POC contains an ASO part which has been covalently modified by reducing the total negative charge provided by the negatively charged phosphate internucleotide linkages by addition of positively charged groups.
  • Another aspect of the present invention relates to a modified peptideoligonucleotide conjugate, the conjugate comprises
  • peptide-oligonucleotide conjugate has been covalently modified by reducing the total negative charge of the peptide-oligonucleotide conjugate by addition of positively charged groups.
  • a further aspect of the present invention relates to a modified peptide- oligonucleotide conjugate (POC), the conjugate comprises
  • ASO antisense oligonucleotide
  • the ASO has been covalently modified by reducing the negative charge of the ASO by addition of positively charged groups.
  • the CPP is positively charged, whereas the ASO is negatively charged. Thus, if no modifications are made to the construct, interaction between CPP and ASO may affect the penetration or inhibitory potential of the peptide-oligonucleotide conjugate.
  • the positively charged groups are added either on the sugar moiety of the ASO, the nucleobase of the ASO, the internucleotide linkage, the linker, or the conjugating group.
  • the overall negative charge of the molecule is y with a standard phosphate-based ASO
  • at least one positively charged group is added to give a net overall molecular charge of y-1
  • two positively charged groups are added to give a net overall molecular charge of y-2
  • preferably three positively charged groups are added.
  • the modification provides the POC conjugate a charge in the range of -4 to -10, preferably -5 to -7.
  • modifications are applied to the different elements of the POC conjugate.
  • An effect of the modification is the shielding effect, which reduces the electrostatic interaction between the negatively charged ASO and the positively charged CPP by introducing a group that sterically hinders such electrostatic interaction. It could be through any groups that is added to a nucleotide, either via the base, sugar or internucleoside linkage.
  • modifications are added by introducing a group that sterically hinders electrostatic interaction between the CPP and ASO.
  • the group could be added to the nucleotide via the base, sugar or internucleoside linkage.
  • the CPP has a net positive charge and known to penetrate and the conjugating group, which is optional, has a net positive charge.
  • the ASO includes at least a non-natural monomer.
  • a non-natural monomer is locked nucleic acids (LNA), which improves the stability of the ASO and protects the oligonucleotide against enzymatic degradation.
  • LNA locked nucleic acids
  • Other examples of non-natural monomers are threose nucleic acid (TNA), hexitol nucleic acid (HNA), flexible nucleic acid (FNA), O2'-alkylated RNA nucleotides, 2'-MOE-RNA, 2'- Fluoro-RNA, glycerol nucleic acid (GNA), peptide nucleic acid (PNA).
  • the ASO comprises at least one non-natural monomer.
  • the non-natural monomer is selected from the list consisting of TNA, HNA, FNA, GNA, PNA and LNA.
  • the oligonucleotide comprises or consist of at least one LNA.
  • the modifications applied to increase the charge of the POC or the ASO as described above can be covalent modifications to the non-natural monomer, such as LNA within the ASO.
  • RNA targeting capability i.e. RNA hybridization affinity or specificity, or ASO stability, or the both.
  • the covalent modifications can be either polyamine-LNA (NH2-LNA), aminoalkyl-amino-LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) or glycyl 2 '- amino-LNA-T (aT(gly)).
  • the LNA polymer is modified to contain one or more monomers selected from the list consisting of polyamine-LNA, aminoalkyl-amino- LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)).
  • the non-natural monomer is modified to contain one or more monomers selected from the list consisting of polyamine-LNA, aminoalkyl- amino-LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)), wherein the LNA is replaced with another non-natural monomer.
  • monomers selected from the list consisting of polyamine-LNA, aminoalkyl- amino-LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)
  • the ASO is modified to contain a combination of monomers selected from the above lists of modifications.
  • the ASO is modified to contain a combination of monomers selected from the list consisting of polyamine-LNA, aminoalkyl-amino- LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)).
  • modified non-natural monomers like LNA in the ASO can vary dependent on the target.
  • the ASO comprises 1 modified non-natural monomer, such as 2 modified non-natural monomers, such as 3 modified non- natural monomers, preferably, the ASO comprises 4 modified non-natural monomers.
  • the ASO comprises at least 1 modified LNA, such as at least 2 modified LN As, such as at least 3 modified LN As, preferably the ASO comprises at least 4 modified LNAs, preferably the number of modified LNAs is an integer selected from the range of 1-25.
  • the ASO is a chimeric antisense oligonucleotide molecule containing affinity-enhancing modified nucleotides.
  • the affinity-enhancing modified nucleotides is selected from the list consisting of LNA, 2'-amino-LNA, 2'-N-acyl-2'-amino-LNA, 2'-N-alkyl-amino-LNA, a-L-LNA, BNA, CEt or O2'-alkylated RNA nucleotides.
  • the ASO comprises DNA, RNA and/or LNA nucleotides, preferably the ASO comprises an alternating mixture of said nucleotides.
  • the ASO comprises a mixture of O2'-alkylated RNA nucleotides and LNA nucleotides, preferably in an alternating constitution.
  • the ASO can comprise a predetermined part of modified non-natural monomers in relation to the total number of monomers in the ASO.
  • the ASO comprises 0%-100% modified non-naturel monomers of the total amount of monomers, such as 10-90%, such as 20-80%, such as 25-70%, such as 30-60%, preferably the ASO comprises 30-50% modified non-natural monomers of the total amount of monomers in the ASO.
  • the LNAs present in the ASO can be linked to each other through different linkages.
  • the linkage can be either phosphorothioate (PS) or phosphodiester (PO).
  • the length of the ASO can vary dependent on the target gene.
  • the length may vary between 5 and 40 monomers.
  • the oligonucleotide has PO and/or PS linkages and, preferably, has a length of 14-18 monomers
  • the ASO has PO linkages.
  • Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom.
  • Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous- containing linkages are well known.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as "base”) modifications or substitutions.
  • Nucleobase modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds.
  • Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-Me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C
  • the oligonucleotide has a phosphate-based linkage.
  • the oligonucleotide has a non-phosphate-based linkage.
  • any modification known by the skilled person can be applied to the ASO, such as any internucleoside linkage known by the skilled person, such as phosphate-based or non-phosphate-based modifications, nucleoside monomers with non-natural bases, and with positive charges placed on the sugar units, the base units or the internucleoside linkages.
  • any internucleoside linkage known by the skilled person such as phosphate-based or non-phosphate-based modifications, nucleoside monomers with non-natural bases, and with positive charges placed on the sugar units, the base units or the internucleoside linkages.
  • the modified oligonucleotide is a singlestranded or a double-stranded oligonucleotide.
  • the ASO has a length of 5-40 monomers, such as 8-30 monomers, such as 10-25 monomers, preferably the ASO has a length of 12-22 monomers, more preferably the ASO has a length of 14-18 monomers.
  • the ASO is selected from the group consisting of SEQ ID NO: 10, 11 and 13, preferably the ASO is SEQ ID NO: 10.
  • the ASO works by targeting RNA, for example messenger RNA (mRNA) within the cells and thereby altering mRNA expression, leading for example to gene silencing or decreased level of mRNA.
  • RNA messenger RNA
  • the ASO may work as a silencer of that specific gene.
  • the ASO is a gene silencer.
  • the ASO acts as a splice switching oligonucleotide.
  • the ASO acts and a gene enhancer, i.e. an ASO that leads to upregulation of the given gene.
  • the design of the ASO can be specified to genes only expressed by specific cells or genes only active in a certain situation. Thereby it is possible to control the exact target of the ASO.
  • the oligonucleotide is silencing a gene involved in the development of antibiotic resistance.
  • genes are the acpP gene, expressed by a high number of bacteria, which codes for an acryl carrier protein, involved in the biosynthesis of fatty acids.
  • acpP gene expressed by a high number of bacteria, which codes for an acryl carrier protein, involved in the biosynthesis of fatty acids.
  • fmhB gene which codes for an aminoacyl transferase involved in bacteria cell wall biosynthesis.
  • the ASO can be designed to target genes in either prokaryotic or eukaryotic cells or in animals or in humans.
  • the ASO is silencing a gene in prokaryotic cells. In another embodiment, the ASO is silencing a gene in eukaryotic cells.
  • the ASO is silencing a gene in human and/or animal cells.
  • the oligonucleotide is silencing a gene selected from the list consisting of: acpP, fmhB, gyrA, ftsZ, rpoD
  • the ASO is silencing the acpP gene.
  • the ASO can be modified by a conjugating group introduced in the 3 '-end or 5 '-end of the ASO sequence.
  • the conjugating group improves the hybridization properties of oligonucleotides due to increased affinity for their targets caused by a reduced electrostatic repulsion between nucleic acid strands.
  • a conjugating group is inserted at the 3 'end of the oligonucleotide.
  • the conjugating group is positively charged.
  • the conjugating group can be polyamine such as oligospermine.
  • the conjugating group is a polyamine, such as oligospermines.
  • ZNA zip nucleic acid
  • structure II is a monomer composed of a cationic moiety with a spermine derivate. ZNA improves the hybridization properties of oligonucleotides due to increased affinity for their targets caused by a reduced electrostatic repulsion between nucleic acid strands.
  • Another example of a conjugating group is a 4-spermine-substituted UNA having the following structure
  • the conjugating group is selected from the group consisting of: ZNA and 4-spermine-substituted UNA.
  • the conjugating group is ZNA.
  • the conjugating group modification can be combined with other modifications like modifications to the non-natural monomers as previously described, such as polyamine-LNA, aminoalkyl-amino-LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)).
  • modifications like modifications to the non-natural monomers as previously described, such as polyamine-LNA, aminoalkyl-amino-LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)).
  • polyamine such as oligospermine is combined with one or more modifications to the non-natural polymer according to the invention.
  • ZNA modification is combined with one or more modifications selected from the list consisting of: polyamine-LNA, aminoalkyl- amino-LNA (AB-IT), glycol 2 '-amino-LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)).
  • the conjugating group is introduced at the 5 'end of the ASO.
  • the POCs is as described above, composed, among others, of a CPP and an ASO linked to each other.
  • the linker has the structure according to structure I: (structure I)
  • CPPs are more efficient in penetrating eukaryotic cells, whereas others are preferred in relation to prokaryotic cells.
  • the peptides penetratin, TAT, R9-TAT, (KKF)sK and (RXR)4XB are known to be effective in penetrating bacterial cells.
  • the peptides penetratin, Octaarginine (R8), TAT, Transportan, Xentry, cR8, cTAT, Pep-1 and HA-TAT are known to be effective in penetrating eukaryotic cells.
  • the cell-penetrating peptide is selected from the group consisting of (KFF)sK and (RXR)4XB.
  • the cell-penetrating peptide (CPP) is selected from the list consisting of penetratin, Octaarginine (R8), TAT, Transportan, Xentry, cR8, CTAT, HA-TAT, R9-TAT, (KKF) 3 K and (RXR) 4 XB.
  • the CPP is selected from the list consisting of KLA, F-3, Pep3, Pentratin, Tat, TP10, R6-Pentratin, (RXR)4, MPG-alpha, Pip2a, CatLip, Fatty acid PNA-Arg9, RGD, Bombesin, Stearyl-(RXR)4, Stearyl-TPIO, PepFectl4, (RXR) 4 , B-peptide, B-MPS, Pip5e, Lys-n and PkkkRKV.
  • the CPP is (KFF) 3 K.
  • the CPP is positively charged.
  • the charge of the molecule can affect the structure as the CPP and ASO may bind to each other.
  • Circular dichroism (CD) spectroscopy is a measure of the secondary structure of the molecule (expressed in nm).
  • the POCs according to the present invention can be used in a composition comprising different amounts of the described POCs.
  • one aspect of the present invention relates to a composition comprising the POC according to the present invention.
  • the sequence of the POC is selected from SEQ ID NO: 12 and 14-17 or a sequence having at least 80%, preferably at least 90% sequence identity to any of SEQ ID NO: 12 and 14-17.
  • compositions for use according to the invention comprising the POC according to the invention, and one or more pharmaceutical acceptable excipients and/or carriers.
  • pharmaceutical acceptable excipients and carriers suitable for administration of the compounds provided herein include all such excipients and carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the POCs can be used to treat a large number of diseases. Due to the ability to design the POC against a specific gene, the molecule can be directed towards a specific disease.
  • an aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compounds according to the present invention or the composition according to the present invention.
  • the present invention can be used as a medicament.
  • the invention can be used in the treatment, prevention or alleviation of infectious diseases, such as bacterial or viral infection.
  • said infectious diseases include coronaviruses, such as SARS-CoV-2, Ebola, Influenza, Human immunodeficiency virus (HIV), Hepatitis, and Zika virus.
  • coronaviruses such as SARS-CoV-2, Ebola, Influenza, Human immunodeficiency virus (HIV), Hepatitis, and Zika virus.
  • the present invention can be used in the treatment, prevention or alleviation of bacterial infections.
  • said bacterial infection is resistant to antibiotic treatment.
  • the bacterial infection is caused by E.coli, P. aeruginosa, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii. Pseudomonas aeruginosa, and Enterobacter, preferably E. co/i.
  • the bacterial infection is caused by E.coli.
  • the invention can be used in the treatment of specific diseases.
  • the invention can be used in the treatment, prevention or alleviation of a disease selected from the group consisting of cancer, inflammatory diseases, neurodegenerative or neurological diseases, diseases in the CNS, metabolic conditions, chronic liver disease and inherited retinal dystrophies (IRDs), septicaemia, lung infection and cystic fibrosis.
  • a disease selected from the group consisting of cancer, inflammatory diseases, neurodegenerative or neurological diseases, diseases in the CNS, metabolic conditions, chronic liver disease and inherited retinal dystrophies (IRDs), septicaemia, lung infection and cystic fibrosis.
  • said cancer is selected from brain cancer, glioblastoma, lung cancer, colorectal cancer, skin cancer, malignant melanoma, pancreas cancer, bladder cancer, liver cancer, breast cancer, eye cancer and prostate cancer.
  • said cancer is a haematological cancer, such as selected from the group consisting of multiple myeloma, acute myeloblastic leukemia, chronic myelogenic leukemia, acute lymphoblastic leukemia and chronic lymphocytic leukemia.
  • said cancer is malignant melanoma, breast cancer, none-small cell lung cancer, pancreatic cancer, head and neck cancer, liver cancer, sarcoma, or B cell lymphoma.
  • the invention is for use in the treatment, prevention or alleviation of autoimmune disease.
  • said autoimmune disease is celiac disease, inflammatory bowel disease, Graves' disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
  • said autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, multiple schlerosis and psoriasis.
  • the present invention can be used in the treatment of Septicaemia, lung infection and cystic fibrosis.
  • the method of treatment comprises administering to the subject a POC or composition according to the invention.
  • Administration of the composition can be done in a number of ways as described in the following, non-limiting, examples.
  • intradermal injection which is a delivery into the dermis of the skin, located between epidermis and the hypodermis.
  • the composition can be administered intravenously, which is an administration directly into the blood stream of the subject.
  • administration of the composition intramuscular is an injection into the muscles of the subject.
  • the composition can be administered subcutaneously, which is under the skin, in the area between the muscle and the skin of the subject.
  • the composition can be administered intratracheally, which is administration directly into the trachea, and transdermal, which is administration across the skin.
  • Intracavity administration includes, but is not limited to administration into oral, vaginal, rectal, nasal, peritoneal, or intestinal cavities as well as, intrathecal, (i.e., into the spinal canal), intraventricular (i.e., into the brain ventricles or the heart ventricles), intraarterial (i.e., into the heart atrium) and subarachnoid (i.e., into the subarachnoid spaces of the brain) administration.
  • intrathecal i.e., into the spinal canal
  • intraventricular i.e., into the brain ventricles or the heart ventricles
  • intraarterial i.e., into the heart atrium
  • subarachnoid i.e., into the subarachnoid spaces of the brain
  • Any mode of administration can be used as long as the mode results in the delivery of the composition in the desired tissue, in an amount sufficient to treat the disease.
  • Administration means of the present invention includes; needle injection, catheter infusion, biolistic injections, particle accelerators, needle-free jet injection, osmotic pumps, oral tablets or topical skin cream. Further, Energy assisted plasmid delivery (EAPD) methods or such methods involving the application of brief electrical pulses to injected tissues, commonly known as electroporation, may be used to administer the composition according to the invention.
  • EAPD Energy assisted plasmid delivery
  • the "subject" as described herein is supposed to receive the composition and comprises humans of all ages, other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals in general, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, cats, dogs; and/or birds. Preferred subjects are humans.
  • the subject is selected from the group consisting of; humans of all ages, other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals in general, including commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, cats and dogs, as well as birds.
  • primates e.g., cynomolgus monkeys, rhesus monkeys
  • mammals in general including commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, cats and dogs, as well as birds.
  • the subject is a human.
  • One aspect of the present invention relates to a method for producing the modified peptide-oligonucleotide conjugate according to the present invention, the method comprises:
  • a further aspect relates to a method for treating or alleviating Septicaemia, lung infection, cystic fibrosis, in a subject in need thereof, which method comprises administering to the subject a POC, composition or pharmaceutical composition according to the invention.
  • An aspect of the invention relates to a method of treating a condition which is caused by, exacerbated by or associated with bacterial infections, which method comprises administering to the subject a POC, composition or pharmaceutical composition according to the invention.
  • the aim of this study was to introduce modifications into the ASO in order to increase the potency of the POC and decrease the interaction between the ASO and the CPP.
  • ASOs were designed to target acpP ⁇ an essential gene encoding a protein involved in fatty acid biosynthesis.
  • a region including the start codon of the targeted gene was selected based on previous studies showing higher susceptibility to inhibition by ASOs.
  • ASOs were synthesized under anhydrous conditions using a Perspective Biosystems Expedite 8909 nucleic acid synthesizer.
  • the synthesis was performed on a 1 pmol scale using a universal support with the following conditions: trichloroacetic acid in CH2CI2 (3:97) as detritylation reagent, 0.25 M 4,5-dicyanoimidazole (DCI) in CH3CN as an activator, acetic anhydride in THF (9:91, v/v) as cap A solution, N- methylimidazole in THF (1:9, v/v) as cap B solution, and a thiolation solution containing 0.0225 M xanthan hydrate in pyridine/CHsCN (20:90, v/v).
  • BCN phosphoramidite was added to the 5'-end in anhydrous CH3CN (0.1 M) and activated by tetrazole with a 15 min coupling time.
  • the stepwise coupling yields were determined by the UV absorbance (at 500 nm) of dimethoxytrityl cations (DMT + ) that were released after each coupling.
  • the resulting ASOs were purified by reverse-phase HPLC (RP-HPLC) using a Waters System 600 HPLC equipment equipped with a Waters XBridge BEH C18-column (5pm, 100 nm x 19 mm). Their composition and purity (>85%) were confirmed by MALDI-TOF MS and ion- exchange HPLC analysis, respectively. Concentrations of purified oligonucleotides were determined by UV absorption measurements at 600 nm.
  • Modifications were introduced in the ASO to decrease electrostatic interactions between the ASO and CPP.
  • Standard LNA monomers were replaced by either aminoalkyl-amino-LNA (AB-IT), glycol 2'-amino-LNA-T (aT(AT)) and glycyl 2'- amino-LNA-T (aT(gly)) monomers (figure 1). While maintaining the excellent duplex stability of an LNA-type oligonucleotide, these monomers introduce positively charged moieties and partial neutralization of the otherwise negatively charged ASO.
  • ZNA zip nucleic acid
  • ZNA is a monomer composed of a cationic moiety with a spermine derivate and is currently commercialized for PCR assays.
  • ZNAs improve the hybridization properties of oligonucleotides due to increased affinity for their targets caused by a reduced electrostatic repulsion between nucleic acid strands. Therefore, both kind of modifications were herein used to attenuate the electrostatic attraction between negatively charged LNA/2'OMe ASOs and the cationic CPP.
  • Modifications such as aminoalkyl-amino-LNA (AB-IT), glycol 2'-amino-LNA-T (aT(AT)) and glycyl 2'-amino-LNA-T (aT(gly)) monomers, were introduced in the ASOs to decrease electrostatic interactions between the ASO and CPP.
  • AB-IT aminoalkyl-amino-LNA
  • aT(AT) glycol 2'-amino-LNA-T
  • aT(gly) glycyl 2'-amino-LNA-T
  • the aim of this study was to synthesize a complete POC where ASOs were conjugated to CPP.
  • the reaction was carried out on a Biotage Initiator microwave synthesizer at 60 °C for 3 h, whereupon all solvents were removed in vacuo, and the residue was re-dissolved in Milli-Q water.
  • Analytical RP-HPLC and MALDI-TOF MS were performed to confirm their composition and purity.
  • the resulting solutions were de-salted by precipitation of the products by adding a solution of MeCN and cold ethanol (1 mL; -20 °C).
  • the resulting suspensions were stored at -20 °C for 1 h, and after centrifugation (16000 x g, 5 min, 4 °C), the supernatants were removed, and the pellet further washed with the same solution.
  • PS phosphorothioate linkages
  • PO phosphodiester linkages
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • 2'-OMe or m
  • AB-IT aminoalkyl-amino-LNA
  • ZNA or Z: zip nucleic acid
  • aT(AT) glycol 2'-amino-LNA-T
  • aT(Gly) glycyl 2'-amino-LNA-T
  • O glycosidic bond
  • BCN cyclooctyne linkage following application of the relevant phosphoramidite during POC synthesis.
  • the aim of this study was to test if the presence of the CPP affected the stability of the ASO hybridization with the target RNA sequence.
  • duplex melting temperatures were determined by UV melting studies on a Perkin Elmer Lambda 35 UV/VIS spectrometer.
  • the samples were prepared as followed: each ASO or POC and their complementary strand were mixed in a 1: 1 ratio (1 pM each strand) in a medium salt buffer (200 mM NaCI, 20 mM NaH2PO4, pH 7.4) to a total volume of 1 mL.
  • the mixtures were denatured by heating up to 85 °C for 5 min and cooled to 4 °C.
  • Samples were transferred to Hellma SUPRASIL synthetic quartz 1 cm path length cuvettes to record the UV absorbance at 275 nm as a function of time.
  • a temperature range of 10 °C to 80 °C and a ramp of 1.0 °C/min were used and controlled by a Peltier temperature controller.
  • Tm values were calculated using WinLab software using an average of Tm values from 3 independent experiments.
  • Table 2 shows the melting temperature (°C) of both PS and PO POCs and their unconjugated counterparts in a salt buffer.
  • the melting temperature and thus the stability of the oligo hybridization with the target RNA sequence was not affected by the presence of the CPP. Further, the higher melting temperature of the complexes with PO linkages compared to PS linkages indicates that PO linkages lead to a higher stabilisation than PS linkages.
  • the aim of this study was to test the effect of the backbone variation on the hybridization and antibacterial activity of the POCs.
  • E. coli K12 MG1655 and S. aureus Mu50 were grown overnight in tryptic soy broth (TSB) at 37 °C with shaking (180 rpm). The cell suspensions were diluted to achieve a cell concentration of about 10 6 cells/mL. Cells were washed, diluted in Muller- Hinton broth and aliquoted into individual low-adhesion polypropylene microtubes.
  • POCs were added to achieve a final concentration of 60 pM and 30 pM and the cells were incubated at 37 °C with constant stirring (180 rpm). Bacterial cultures were harvested at 0, 2, 4, 6, 8 and 24h and a tenfold serial dilution was prepared before the suspensions were plated on Muller-Hinton agar. The plates were incubated overnight at 37 °C to determine the CFU/ mL. (KFF)sK alone and the ASO alone were tested as controls at 60 pM. Three independent experiments were performed.
  • Such POC aggregation may prevent (KFF)sK peptide from promoting the bacterial envelope penetration, thus explaining the decreased inhibition of POC-1 compared to POC-2.
  • the observed higher Tm obtained for PO compared with the PS species (example 4) may be contributing to the higher antibacterial efficiency of POC-2.
  • FIG. 5 shows the growth curves of E. coli over a period of 24h in the presence of the POCs at a concentration of 30 pM and 60 pM.
  • POC-4 inhibited the growth of E. coli by 3-log (P ⁇ 0.0001), whilst the insertion of the AB-IT in the ASO in POC-3 resulted in a 4-log reduction (P ⁇ 0.0001).
  • POC-6 no inhibition was observed, while its combination with ZNA (POC-7) resulted in an inhibition of 2.5 log after 4h (P ⁇ 0.0001) (figure 5).
  • Example 7 The POCs are active in human cells
  • the POCs can penetrate bacterial envelopes and elicit an anti-bacterial effect directed against the acpP gene.
  • the inventors wanted to investigate whether a new set of POCs would be active in humane cells namely by mediating splice shifting.
  • the inventors wanted to induce splice shifting in the HRAS gene.
  • the HRAS gene is located on chromosome llpl5.5 and consist of 6 exons [Entrez Gene, 10 NCBI Reference Sequence: NG_007666.1].
  • HRAS-ASOs Splice shifting oligonucleotides targeting HRAS exon 2 in the pre-mRNA transcript of the HRAS gene that harbors cancer activating mutations should induce exon 2 skipping.
  • RNAiMAX Lipofectamine RNAiMAX
  • HRAS- ASO Lipofectamine RNAiMAX
  • PCR was carried out using TEMPase Hot Start DNA polymerase (Ampliqon) and 1 pl cDNA per reaction, and with primers located in exon 1 (SEQ ID NO: 18; HRASlsNhelS) and spanning the exon 3-4 junction (SEQ ID NO: 19; RasEx4Ex3AS) of the HRAS gene. 0.5 pmol/pl of each primer was used.
  • the PCR products were separated on a 1.5% Seakem LE (Lonza) TBE agarose gel, for Ih at 80V.
  • T24 bladder cancer cells were seeded in 12-well plates and reverse transfected at 60% confluence with 20 and 40 nM HRAS-ASOs using Lipofectamine RNAiMAX (Invitrogen). RNA was harvested after 48 h using Trizol (Invitrogen) and chloroform to isolate the RNA, followed by precipitation with isopropanol.
  • Complementary DNA cDNA was synthesized from 500 ng RNA using the High Capacity cDNA kit (Applied Biosystems). PCR was carried out using TEMPase Hot Start DNA polymerase (Ampliqon) and 1 pl cDNA per reaction, and with primers located in exon 1 and spanning the exon 3-4 junction of the HRAS gene. 0.5 pmol/pl of each primer was used. The PCR products were separated on a 1.5% Seakem LE (Lonza) TBE agarose gel, for Ih at 80V.
  • the splice-shifting oligonucleotides used in this study are listed in Table 3 and as SEQ ID NO: 10-17 in the sequence list.
  • PS phosphorothioate linkages
  • LNA locked nucleic acid
  • 2'-OMe or m
  • ZNA or Z
  • aT(AT) glycol 2'-amino-LNA-T
  • BCN cyclooctyne linkage following application of the relevant phosphoramidite during POC synthesis.
  • the standard LNA/2'-OMe-RNA mixmer NAC9632 mediates efficient splice shifting as shown by the strong lower band in figure 7.
  • the only other HRAS ASO able to mediate as efficient splice shifting as NAC9632 is POC9664 which contains the CPP ((KFF) 3 K) and three of the positively charged amino-LNA-based nucleotide monomers (aT(AT)).
  • POC9665 without the CPP unit
  • POC9666 with the CPP unit
  • the latter seems to be the more active of the two (evaluated by comparing the relative intensities of the upper and lower bands).
  • POC9667 and POC9668 also mediates an efficient splice shifting.
  • the standard LNA/2'-0Me-RNA mixmer NAC9632 mediates efficient splice shifting as shown by the strong lower band in figure 8 when tested at both 20 nM and 40 nM.
  • the HRAS POCs of the invention POC9664, POC9666, POC9668 and POC9678 all mediate efficient splice shifting at both 20 nM and 40 nM.
  • ASOs and the CPP-conjugated ASOs of the present invention can mediate splice shifting in humane cells, thus said ASOs and POCs are active in human cells and can be used as a medicament to treat diseases, such as cancer, in humans.
  • a modified peptide-oligonucleotide conjugate comprising:
  • ASO antisense oligonucleotide
  • modified peptide-oligonucleotide conjugate has been modified, preferably covalently modified, by reducing the total negative charge of the peptide-oligonucleotide conjugate by addition of positively charged groups.
  • modified peptide-oligonucleotide conjugate according to item 1 wherein the positively charged groups are added either on the sugar moiety of the antisense oligonucleotide, the nucleobase of the antisense oligonucleotide, the internucleotide linkage, the linker, or the conjugating group or wherein the peptide-oligonucleotide conjugate has been covalently modified by reducing the total negative charge provided by the negatively charged phosphate internucleotide linkages by addition of positively charged groups.
  • the LNA polymer is modified to contain one or more monomers selected from the list consisting of polyamine-LNA, aminoalkyl-amino-LNA (AB-IT), glycol 2 '-amino- LNA-T (aT(AT)) and glycyl 2 '-amino-LNA-T (aT(gly)).
  • CPP is selected from the group consisting of (KFF) 3 K, (RXR)4XB, Pepl, penetratin, Octaarginine (R8), TAT, Transportan, Xentry, cR8, CTAT, HA-TAT, R9-TAT, (KKF) 3 K and (RXR) 4 XB.
  • a pharmaceutical composition comprising the peptide-oligonucleotide conjugate according to anyone of the preceding items and further comprising one or more pharmaceutical acceptable excipients and/or carriers.
  • coronaviruses such as SARS-CoV-2, Ebola, Influenza, Human immunodeficiency virus (HIV), Hepatitis, and Zika virus.
  • inflammatory disease is celiac disease, inflammatory bowel disease, Graves' disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
  • peptide-oligonucleotide conjugate according to anyone of items 1-20 or the pharmaceutical composition according to item 21, for use according to anyone of the items 22-29, wherein the POC or pharmaceutical composition is administered to a subject selected from the group consisting of; humans of all ages, primates e.g., cynomolgus monkeys, rhesus monkeys; mammals in general, including commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, cats and dogs, as well as birds, preferably the subject is a human. Sequence list
  • PS phosphorothioate linkages
  • PO phosphodiester linkages
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • 2'-OMe or m
  • ZNA or Z
  • aT(AT) glycol 2'-amino-LNA-T
  • aT(Gly) glycyl 2'-amino-LNA-T
  • O glycosidic bond
  • BCN cyclooctyne linkage following application of the relevant phosphoramidite during POC synthesis.

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Abstract

La présente invention concerne un conjugué peptide-oligonucléotide (POC) modifié composé d'un peptide de pénétration cellulaire (CPP) conjugué à un oligonucléotide antisens. Le POC peut réguler l'expression génique dans les cellules cibles et peut ainsi être utilisé dans le traitement de différentes maladies telles que les infections bactériennes et le cancer.
PCT/DK2022/050248 2021-11-26 2022-11-25 Conjugué peptide-oligonucléotide modifié Ceased WO2023093960A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002079467A2 (fr) * 2001-03-29 2002-10-10 Københavns Universitet Selection de souche bacterienne sans antibiotique a l'aide de molecules antisens
EP2899197A1 (fr) * 2012-09-21 2015-07-29 Osaka University Oligonucléotide et nucléoside artificiel ayant un pont guanidine
WO2016128583A2 (fr) * 2015-02-15 2016-08-18 Ribo Task Aps Oligonucléotides acyl-amino-lna et/ou hydrocarbyl-amino-lna
WO2018033492A1 (fr) * 2016-08-16 2018-02-22 Steffen Panzner Nucléotides 2'-amino-lna carboxylés et oligonucléotides comprenant ceux-ci

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2002079467A2 (fr) * 2001-03-29 2002-10-10 Københavns Universitet Selection de souche bacterienne sans antibiotique a l'aide de molecules antisens
EP2899197A1 (fr) * 2012-09-21 2015-07-29 Osaka University Oligonucléotide et nucléoside artificiel ayant un pont guanidine
WO2016128583A2 (fr) * 2015-02-15 2016-08-18 Ribo Task Aps Oligonucléotides acyl-amino-lna et/ou hydrocarbyl-amino-lna
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HEGARTY JOHN P ET AL: "Advances in therapeutic bacterial antisense biotechnology", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 102, no. 3, 5 December 2017 (2017-12-05), pages 1055 - 1065, XP036390096, ISSN: 0175-7598, [retrieved on 20171205], DOI: 10.1007/S00253-017-8671-0 *
KEITH T. GAGNON ET AL: "Antisense and Antigene Inhibition of Gene Expression by Cell-Permeable Oligonucleotide-Oligospermine Conjugates", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 133, no. 22, 8 June 2011 (2011-06-08), pages 8404 - 8407, XP055033269, ISSN: 0002-7863, DOI: 10.1021/ja200312y *
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