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WO2024069229A2 - Biologically active compounds - Google Patents

Biologically active compounds Download PDF

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Publication number
WO2024069229A2
WO2024069229A2 PCT/IB2023/000576 IB2023000576W WO2024069229A2 WO 2024069229 A2 WO2024069229 A2 WO 2024069229A2 IB 2023000576 W IB2023000576 W IB 2023000576W WO 2024069229 A2 WO2024069229 A2 WO 2024069229A2
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WIPO (PCT)
Prior art keywords
peptide
cell
moiety
molecule
stap
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/IB2023/000576
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French (fr)
Other versions
WO2024069229A3 (en
Inventor
Wouter EILERS
Keith Foster
Adam James Reginald GADD
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Sutura Therapeutics Ltd
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Sutura Therapeutics Ltd
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Publication of WO2024069229A2 publication Critical patent/WO2024069229A2/en
Publication of WO2024069229A3 publication Critical patent/WO2024069229A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C07K5/06095—Arg-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp

Definitions

  • the present invention relates to improvements in drug delivery.
  • CPPs Cell Penetrating Agents
  • StaPs Cell Penetrating Peptides
  • StiPs Cell Penetrating Peptides
  • These stabilized CPPs are conjugated to a drug or Biologically Active Compound (BAC) directly or via a BiFunctional Linker (BFL) so that the BAC can be carried though a cell membrane by the CPP.
  • BFL BiFunctional Linker
  • the resulting compounds may be referred to as drug carrying cell penetrating molecules.
  • the preferred BACs delivered in this manner are oligonucleotides (ONs), more preferably still electrically low charge carrying oligonucleotides (charge -3 to +3 at pH 7.5) and most preferably electrically neutral oligonucleotides (charge -1 to +1 at pH 7.5), such as, but not limited to, peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligonucleotides (PMOs) or modified derivatives thereof.
  • PNAs peptide nucleic acids
  • PMOs phosphorodiamidate morpholino oligonucleotides
  • Other BACs based on nucleic acids include, but are not limited mRNA, siRNA, miRNA, aptamers, and gapmers.
  • the preferred BFL may be PEGylated, comprising polyethylene glycol (PEG) groups including modifications such as an amine group, or incorporate a spacer, such as ⁇ -Ala. These modifications can improve solubilization or provide appropriate spacing between functional moieties.
  • PEG polyethylene glycol
  • the invention relates to a method of synthesizing the compounds.
  • the CPP can be synthesized by stepwise solid phase synthesis, which permits control over the exact order of amino acid monomers within the CPP.
  • the stepwise solid phase techniques can similarly be used to synthesize the ON with exact control over the sequence of the ON.
  • the techniques for stepwise synthesis of the CPP and the ON are interchangeable and the compounds can be synthesized in a one pot method prior to cleavage from the resin and subsequent collection for further application, thus streamlining the overall synthetic process and permitting complete control over the sequencing of both the CPP and the ON.
  • the stepwise solid phase synthetic techniques also permit the incorporation of handles comprising orthogonal reactive sites for further conjugation to occur on the compound, resulting in further functionalization of the compound.
  • the invention also relates to a method of facilitating the uptake of a BAC into a cell, the use of the compound in the treatment of a disease requiring alteration of an endogenous or exogenous gene, a method of improving the bioavailability of a drug or BAC, a method of introducing a drug or BAC to a site which is refractory to the drug or BAC in its native state, a method of treating a subject comprising administering the compounds of the invention and to a pharmaceutical composition comprising the compound and one or more pharmaceutically acceptable excipients.
  • oligonucleotides which term includes various ON analogs, which include, but are not limited to antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligonucleotides (PMOs), aptamers, and gapmers.
  • ONs oligonucleotides
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • miRNA microRNA
  • PNAs peptide nucleic acids
  • PMOs phosphorodiamidate morpholino oligonucleotides
  • ONs can target essential DNA, RNA and protein sequences and can modulate gene expression in a number of ways that includes steric blocking to suppress (i) RNA splicing, (ii) protein translation or (iii) other nucleic acidmucleic acid or nucleic acid:protein interactions.
  • the hybridization of ONs to specific RNA sequence motifs prevents correct assembly of the spliceosome, so that it is unable to recognize the target exon(s) in the pre- mRNA and hence excludes these exons in the mature gene transcript.
  • Exclusion of an in-frame exon can lead to a truncated yet functional gene product; exclusion of an out of frame exon results in a frame-shift of the transcript, potentially leading to a premature stop codon and a reduction in the target gene expression level.
  • CPAs It is desirable to facilitate the entry of BACs such as ONs into cells. This can be accomplished via the use of CPAs.
  • CPAs include CPPs, which can interact with and cross the cell membrane to enter the cell. CPPs can interact with cell membranes to participate in direct penetration, an energy independent process.
  • CPPs can also enter the cell via endocytosis, in particular, pinocytosis.
  • the BAC and CPP are encased in an endosome following cell entry via endocytosis.
  • endosomal escape that must be considered such that the CPP carrying the BAC can breach the endosome to reach the intended target for delivery of the BAC.
  • CPPs For effective clinical translation of ONs such as steric blocking antisense oligonucleotides, CPPs need to effectively deliver the BAC to either the cytoplasm or nucleoplasm whilst limiting any toxicity associated with cell entry.
  • the BAC must be able to retain its biological activity upon delivery into the cell via a CPP such that the delivered cargo can achieve its intended therapeutic effect.
  • providing compounds that are able to deliver a drug or BAC more efficiently or to a target site with lower toxicity and immunogenicity in which the biological activity of the BAC is preserved or retained would be highly desirable.
  • a method for synthesizing a compound comprising: an oligonucleotide moiety, covalently linked directly or covalently linked via a bifimctional linker moiety (BFL) to, ii.
  • BFL bifimctional linker moiety
  • the StaP or StiP when a molecule not part of the compound, is a stabilized peptide which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix, wherein synthesis of the compound comprises the steps of:
  • step (vi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), before or after step (iii), before or after step (iv), or before or after step (v);
  • step (xi) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs before or after step (vii), before or after step (viii), before or after step (ix), or after step (x); and
  • the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • the PMO comprises 5GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98).
  • the protecting group of the amino acid monomer is Fmoc.
  • the protecting group of the oligonucleotide monomer is Fmoc.
  • the oligonucleotide monomer is selected from the group consisting of:
  • the protecting group of the oligonucleotide monomer is Trt.
  • the oligonucleotide monomer is selected from the group consisting of:
  • the oligonucleotide is covalently linked to the StiP or the StAP.
  • the BFL comprising one or more handles is present in the compound, and the oligonucleotide is covalently linked via the BFL to the StaP or the StiP, whereby the BFL is covalently linked to the oligonucleotide, and the BFL is covalently linked to the StaP or the StiP.
  • BFL comprises:
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer; or,
  • (AMAS) a residue of N-a-maleimidoacet-oxysuccinimide ester, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer
  • (BMPS) a residue of N- ⁇ -maleimidopropyl-oxysuccinimide ester, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer
  • (GMBS) a residue of N- ⁇ -aleimidobutyryl-oxysuccinimide ester, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer
  • (GMBS) a residue of N- ⁇ -aleimidobutyryl-oxysuccinimide ester, where Z is and Y is a
  • Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
  • EMCS EMCS
  • Y N-terminus of the StaP or the StiP, or where n is a positive integer;
  • LC-SMCC a residue of succinimidyl 4-(N-maleimidomethyl) cyclohexane- l-carboxy-(6-amidocaproate), where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer; or, a residue of succinimidyl 4-(N- maleimidom ethyl) cyclohexane- 1 -carboxylate (polyethylene glycol) n , wherein n equals 1 to 10, Z is and, Y is either present or not present, and when Y is present, Y is where n is a positive integer, and when Y is not present, Y is a covalent bond to the N-terminus of the StaP or the StiP; or, (DSG), a residue of disuccinimidyl gluterate, where Z is not present, and instead there is a covalent bond to the N-
  • n is 5.
  • R is H.
  • R is NH 2 .
  • the forming step (v) comprises forming an olefin cross link between at least two unnatural amino acids.
  • the at least two unnatural amino acids are selected from the group consisting of:
  • S5 is (S)-pentenylalanine
  • R5 is (R)-pentenylalanine
  • S8 is (S)-octenylalanine
  • R8 is (R)- octenylalanine
  • B5 is ⁇ , ⁇ -di- substituted pentenylalanine
  • B8 is ⁇ , ⁇ -di-substituted octenylalanine
  • S-OAS and R-OAS are O-allylserine analogues.
  • the forming step (v) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
  • the lactam bridge is formed by cross linking a lysine and glutamic or aspartic acid residues.
  • a method for synthesizing a compound comprising: i. an oligonucleotide moiety, covalently linked to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids,
  • step (v) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), or before step (iii);
  • step (xi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs after step (vi), before or after step (vii), before or after step (viii), before or after step (ix), or before or after step (x); and
  • the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • the PMO comprises 5GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98).
  • the linker of step (i) comprises an amino acid monomer or a handle configured to couple with an oligonucleotide monomer.
  • the protecting group of the amino acid monomer is Fmoc.
  • the oligonucleotide monomer is selected from the group consisting of:
  • the protecting group of the oligonucleotide monomer is Trt.
  • the oligonucleotide monomer is selected from the group consisting of:
  • the forming step (x) comprises forming an olefin cross link between at least two unnatural amino acids.
  • the at least two unnatural amino acids are selected from the group consisting of:
  • S5 is (S)-pentenylalanine
  • R5 is (R)-pentenylalanine
  • S8 is (S)-octenylalanine
  • R8 is (R)- octenylalanine
  • B5 is ⁇ , ⁇ -di- substituted pentenylalanine
  • B8 is ⁇ , ⁇ -di-substituted octenylalanine
  • S-OAS and R-OAS are O-allylserine analogues.
  • the forming step (x) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
  • the lactam is formed by cross linking a lysine and glutamic or aspartic acid residues.
  • a comprising the compound made according to the present methods and one or more pharmaceutically acceptable excipients.
  • the composition is formulated for oral, parenteral, intravenous, or topical administration.
  • a method of administering the composition made by the present methods comprises the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
  • a molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the Sti
  • the molecule further comprises a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
  • the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
  • the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • PMO phosphorodiamidate morpholino oligonucleotide
  • the PMO is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
  • the bifunctional linker is SMCC.
  • the PMO has the sequence 5’- GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
  • the antisense oligonucleotide is a gapmer.
  • the gapmer is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
  • the bifunctional linker is SMCC.
  • the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 112).
  • a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule.
  • a method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule.
  • the cell is a cardiac muscle cell. [0069] In some embodiments, the cell is a skeletal muscle cell.
  • composition comprising the molecule and one or more pharmaceutically acceptable excipients.
  • the composition is formulated for oral, parenteral, intravenous, or topical administration.
  • a molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, z-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the Sti
  • the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
  • the molecule further comprises a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
  • the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
  • the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • the PMO is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
  • the bifunctional linker is SMCC.
  • the PMO has the sequence 5’- GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
  • the antisense oligonucleotide is a gapmer.
  • the gapmer is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
  • the bifunctional linker is SMCC.
  • the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
  • a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule.
  • a method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule.
  • the cell is a cardiac muscle cell.
  • the cell is a skeletal muscle cell.
  • composition comprising the molecule and one or more pharmaceutically acceptable excipients.
  • the composition is formulated for oral, parenteral, intravenous, or topical administration.
  • a molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the Sti
  • the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NOS: 103, 104, 105, 106, or 107.
  • the molecule further comprises a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
  • the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
  • the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • the PMO is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
  • the bifunctional linker is SMCC.
  • the PMO has the sequence 5’- GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
  • the antisense oligonucleotide is a gapmer.
  • the gapmer is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
  • the bifunctional linker is SMCC.
  • the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
  • a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule.
  • a method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule.
  • the cell is a cardiac muscle cell. [00105] In some embodiments, the cell is a skeletal muscle cell.
  • composition comprising the molecule and one or more pharmaceutically acceptable excipients.
  • the composition is formulated for oral, parenteral, intravenous, or topical administration.
  • Fig. 1A is a chemical reaction scheme showing the microwave solid phase synthesis of peptides using Fmoc protected reagents.
  • Fig. 1B is a chemical reaction scheme showing the ring closing metathesis reaction to obtain the cross link between two unnatural amino acids to obtain the StaP or StiP.
  • Fig. 2 is a scheme showing the various embodiments of handles that can be included on the N or C terminus of the peptide or the 5’ end or the 3’ end of the oligonucleotide.
  • the handles are orthogonal reactive sites for additional conjugation and functionalization of the compound.
  • Fig. 3A is a chemical reaction scheme demonstrating an additional coupling step of a handle containing a protected thiol group (S-trityl-3 -mercaptopropionic acid) to enable thiol-ene Michael addition.
  • a protected thiol group S-trityl-3 -mercaptopropionic acid
  • Fig. 3B is a chemical reaction scheme demonstrating the incorporation of a handle comprising a terminal alkyne group via nucleophilic substitution to enable Huisgen 1,3-dipolar cycloaddition.
  • Fig. 4A is a chemical reaction scheme demonstrating the incorporation of a handle at the C terminus of a peptide comprising a 4-methyl trityl protected lysine.
  • Fig. 4B is a chemical structure showing Universal PEGNovaTagTM resin.
  • Fig. 4C is a chemical structure showing incorporation of a hydrazine functional group.
  • Fig. 5 is a chemical reaction scheme showing the solid phase synthesis of the oligonucleotide utilizing Fmoc protected PMO nucleotide monomers.
  • Fig. 6 is a chemical reaction scheme showing the solid phase synthesis of the oligonucleotide utilizing trityl protected PMO nucleotide monomers.
  • Fig. 7 is a chemical reaction scheme showing the incorporation of a handle comprising a terminal alkyne group to the oligonucleotide via nucleophilic substitution to enable Huisgen 1,3- dipolar cycloaddition.
  • Fig. 8 is a chemical reaction scheme showing the integration of a handle at the 5’ end of the oligonucleotide via the addition of a 4-methyl trityl protected lysine linker to the resin followed by deprotection, functionalization with a 3-mercaptothiopropionic acid handle, and subsequent synthesis of the oligonucleotide.
  • Fig. 9 is a chemical reaction scheme showing the interchangeability of the synthetic techniques of synthesizing the peptide and oligonucleotide in a one pot synthesis. More in particular, in some embodiments, the peptide can be conjugated at the 3’ end of the oligonucleotide.
  • Fig. 10 is a chemical reaction scheme showing the integration of a 3- mercaptothiopropionic acid handle to a compound synthesized using the one pot synthetic method.
  • Fig. 11A is a general chemical structure of the compound in which the CPP forms a cross link via ring closing metathesis, the BAC is an anti-sense oligonucleotide (AON), and a series of handles, represented by a linker L, sulfur containing moiety Z, and spacer Y 3 act as a bifunctional linker to connect the BAC to the CPP.
  • AON anti-sense oligonucleotide
  • Fig. 11B is a chemical structure of a preferred embodiment of the invention.
  • Figs. 12A-12D show EGFP expression and cell toxicity of a first series of stabilized and unstabilized PEPTIGOs, PMO alone, and growth vehicle as a control.
  • Figs. 13A-13C show EGFP expression and cell toxicity of a second series of stabilized and unstabilized PEPTIGOs, PMO alone, and growth vehicle as a control.
  • Fig. 14 is a plot showing the body mass of IL1 ⁇ NLS (1,5)-PM0 conjugate- and PMO- treated D2-mdx mice during the dosing phase and the end of the study investigating the effect of dystrophin exon 23-skipping IL1 ⁇ -NLS-peptide based PMO conjugate and unconjugated PMO on dystrophin expression in the D2-mdx mouse.
  • Fig. 15 is a plot showing the body mass of IL1 ⁇ NLS (2,6)-PMO conjugate- and PMO- treated D2-mdx mice during the dosing phase and the end of the study.
  • Fig. 16A shows microscopy images of stained tissue with respect to dystrophin expression in heart muscle of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1 ⁇ -NLS (1,5)-PM0 conjugate.
  • the plot shows quantification (in the absolute number of fibres per muscle that stain positively for dystrophin. Data are shown as mean +/- SD and as individual data points.
  • Fig. 16B shows microscopy images of stained tissue with respect to dystrophin expression in skeletal muscle (gastrocnemius) of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1 ⁇ -NLS (1,5)-PM0 conjugate. The plot shows quantification of the dystrophin-positive fibres detected. Data are shown as mean +/- SD and as individual data points.
  • Fig. 17A shows microscopy images of stained tissue with respect to dystrophin expression in heart muscle of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1 ⁇ -NLS (2,6)-PMO conjugate. The plot shows quantification of the dystrophin-positive fibres detected. Data are shown as mean +/- SD and as individual data points.
  • Fig. 17B shows microscopy images of stained tissue with respect to dystrophin expression in skeletal muscle (gastrocnemius) of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1 ⁇ -NLS (1,5)-PM0 conjugate. The plot shows quantification of the dystrophin-positive fibres detected. Data are shown as mean +/- SD and as individual data points.
  • Fig. 18A is a chemical structure of the IL1 ⁇ NLS (1,5)-PM0 conjugate.
  • Fig. 18B is a chemical structure of the IL1 ⁇ NLS (2,6)-PMO conjugate.
  • Fig. 19 is a graph showing eGFP reporter expression after 24 hours incubation of
  • HeLaEGFP654 cells with ESE-1 NLS peptide-oligonucleotide conjugates or vehicle NH2- EGFP654 PMO is the unconjugated, NH2-modified PMO of the same batch used for preparing the conjugates. Data are shown as mean +/- SD.
  • Fig. 20 is a graph showing cytotoxicity after 24 hours incubation of HeLaEGFP654 cells with ESE-1 NLS peptide-oligonucleotide conjugates or vehicle.
  • NH2-EGFP654 PMO is the unconjugated, NH2-modified PMO of the same batch used for preparing the conjugates. Data are shown as mean +/- SD.
  • Oligonucleotides can be designed to target 5’ translation initiation start sites of viral gene transcript(s) to prevent binding of the translational machinery.
  • ASO anti-sense oligonucleotides
  • ONs can be designed to form aptamers such that the secondary and tertiary structures can bind proteins or other cellular targets thus impacting on specific gene expression levels or other cellular processes (e.g. post-translational modifications).
  • An advantage of steric blocking based suppression over that of siRNA/RNAi based RNase H-induction of the RNA Induced Silencing Complex is the reduced likelihood of off target side effects.
  • WO2013/150338 and WO 2014/053622 both disclose delivering negatively charged ONs of small size (typically smaller than 1.5KDa) by complexing them with positively charged linear or stapled peptides of equal or greater than 15 amino acids and in the range of 15- 27 amino acids.
  • IACS, Vol 136, 2014, GJ Hilnski et al describe stapled and stitched peptides that are able to penetrate cells. Reference is made to the possibility that these peptides could be used to deliver an oligonucleotide, presumably in the same manner as disclosed in the international applications disclosed above, i.e. by complexation. There is nothing to suggest creating new entities of much larger size (greater than 1.5KDa, through 2.5KDa, 5KDa, 7.5KDa, lOKDa, 12.5KDa or more) by covalently linking a BAC with a CPA, optionally via a BFL and indeed, the prior methodology requires the respective components to have opposite charges to facilitate complexing.
  • electrically low charge carrying oligonucleotides charge -3 to +3 at pH 7.5
  • electrically neutral oligonucleotides charge -1 to +1 at pH 7.5
  • PNAs peptide nucleic acids
  • PMOs phosphorodiamidate morpholino oligonucleotides
  • BFL BFL
  • WO2014/064258 is an example of the existing conjugating art. A negatively charged
  • the ON is coupled to a targeting peptide via a linker.
  • the targeting peptide is a receptor targeting moiety, and not a stapled or stitched peptide, and indeed considerable doubt exists as to whether DNA or RNA molecules can gain cell entry using a receptor targeting moiety as once a negatively charged ON is bound to such a moiety, non-covalent interactions alter its conformation 15 .
  • WO89/03849 discloses oligonucleotide-polyamide conjugates. There is no disclosure of the use of stitched or stapled peptides. The methodology described uses oligonucleotides as a scaffold for the chain extension of peptides and not as a conjugate for delivery, per se.
  • WO2011/131693 describes nucleic acid constructs which contain a nucleic acid specific for a given target gene and a selective inhibitor of a neurotransmitter transporter. There is no disclosure of the use of stitched or stapled peptides as a delivery agent.
  • a peptide capable of effecting peptide-mediated cell delivery may also be referred to as a Cell Delivery Peptide (CDP).
  • CDP Cell Delivery Peptide
  • Examples include: polyarginine, penetratin (based upon an antennapedia homeodomain), or PMO internalization peptides (PIPs).
  • FC formal charge
  • V valence electrons of the neutral atom in isolation
  • N the number of non-bonding valence electrons on the defined atom
  • B the total number of electrons shared in bonds.
  • the Applicant has overcome this major impediment by utilizing stabilized CPAs.
  • a drug or BAC to a stabilized CPA, including stitched and stapled peptides, they have surprisingly obtained enhanced cellular uptake dynamics, 10-20 fold better than current state of the art for CPAs 21,22 .
  • W 02017/109494 discloses methods of forming the cross link or bridge within the StiP and the StaP utilizing unnatural amino acids comprising terminal alkene side chains that undergo a ring closing olefin metathesis reaction.
  • W 02019/002875 discloses methods of forming the cross link or bridge within the StiP and the StaP via the cyclization of the amine functional groups of lysine and the carboxylic acid side chains of aspartic or glutamic acid.
  • one way to prepare stapled and stitched peptides is to incorporate amino acids into the peptide that are modified to bear e.g. an olefin (alkene) group (which may be incorporated at defined relative positions during solid-phase peptide synthesis).
  • an olefin (alkene) group which may be incorporated at defined relative positions during solid-phase peptide synthesis.
  • ring-closing metathesis is then used to close one (stapled [denoted as StaP herein]) or two or more (stitched [denoted as StiP herein]) all-hydrocarbon cross-links that induce the peptide to adopt a stabilized structure, typically, but not essentially an alpha helix.
  • StaPs it is preferred to use either one or both enantiomers of the un-natural amino acids, termed the S5 (S- pentenylalanine) or R5 (A-pentenylalanine), or the S8 (S-octenyl alanine) or R8 (R-octenyl alanine), depending on the stereo- chemi cal configuration.
  • S5 S- pentenylalanine
  • R5 A-pentenylalanine
  • S8 S-octenyl alanine
  • R8 R-octenyl alanine
  • Cross linking strategies are however not restricted to ring-closing metathesis of un-natural olefin-bearing ⁇ , ⁇ -di-substituted amino acids.
  • Other cross-linking chemistry’s may be used to stabilize the peptide, such as ring-closing metathesis between O-allylserine analogues (S-OAS or R-OAS).
  • WO2017/109494 utilizes conjugation of the BAC with the CPP.
  • discrete species of BAC and CPP undergo a coupling reaction with the assistance of a BFL to form the compound.
  • the coupling reaction between the two macromolecules resulted in a low overall yield (10%).
  • changing the identity of the CPP for the coupling reaction improved the overall yield to approximately 59%, more robust methods of synthesis are needed to achieve higher yields across all types of CPPs.
  • StaPs and StiPs Relative to their unmodified peptide precursors, StaPs and StiPs generally exhibit robust cellular uptake, significant resistance to proteolytic degradation, and in vivo stability that can support a half-life of more than 12 hours in non-human primates 25 . It is likely that this increase in drug-likeness stems from the highly rigidified structure and the burial of the backbone amide bonds in the core of e.g. the a-helix. This structural rigidity also decreases the likelihood that StiPs and StaPs will be immunogenic, as the design of major histocompatibility complexes is such that peptides must adopt an extended conformation to be presented. The potential reduced or lack of membrane toxicity and immunogenicity enhances the clinical translatability of compounds when conjugated to drugs and BACs such as ONs.
  • the BAC and CPP can be covalently conjugated directly, or covalently conjugated via a BFL. As discussed further below, many functional groups may be used to effect conjugation of the BAC to the CPP.
  • non-covalent affinities between molecules may be used to link the BAC and the CPP to each other.
  • streptavidin a tetrameric protein derived from Streptomyces avidinii has high affinity for biotin, also known as vitamin B7 or vitamin H. Streptavidin and biotin moieties can interact and form a strong, non-covalent complex.
  • ONs can be used to induce a steric block to any gene in humans, animals and lower order organisms and thus can be applied to natural disease (including genetic and age-related diseases) or acquired diseases in humans and animals.
  • VHFs viral hemorrhagic fevers
  • ASOs can be designed to target 5’ translation initiation start sites of viral gene transcript(s) to prevent binding of the translational machinery. Using ASO to suppress viral translation is a well-established technology 1 and has progressed into clinical trials for viral hemorrhagic fevers such as Marburg and Ebola 2,3 .
  • PMO, AVI-7537 was evaluated for human use in the West African Ebola outbreak in 2014-15.
  • Some tissues are particularly refractory to naked PMO transfection, e.g. heart, which may reflect differential vesicle-mediated PMO uptake mechanisms 23 .
  • direct intra-cardiac injection of naked PMO does not even lead to efficient transfection 26 , and refractory tissues tend to require repeat administration or high dose strategies 27 ' 29 .
  • CPP conjugation improves PMO bio-distribution and serum stability 30 ' 32 , toxicity is still a major roadblock for pipeline development 19 .
  • the compound of the invention comprises:
  • BAC biologically active compound
  • a cell penetrating agent which is a stitched (StiP) or stapled (StaP) peptide, wherein the StiP or StaP is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11 , the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix ; and [00175] optionally one or more handles wherein the one or more handles, alone or in combination, can
  • the CPP can be synthesized via solid phase synthesis, which provides absolute control over the peptide monomer addition sequence and high reaction yields. Surprisingly, it has been found that the methods used for solid phase synthesis of the CPP can be adapted for the synthesis of the BAC, in particular, an oligonucleotide. Similarly, solid phase synthesis techniques tolerate the reaction conditions necessary for the coupling of handles, which provide options for additional orthogonal chemistry and functionalization.
  • the present methods can be used to construct the CPP in a stepwise manner with each successive amino acid building block, as well combine these techniques in a successive procedure utilizing the same constructed CPP to construct the BAC via the addition of nucleic acid monomers onto the same, newly constructed CPP without altering reaction conditions and without intermediate isolation of the CPP (e.g., for ring closing metathesis to form staple or stitch) prior to addition of the first nucleic acid monomer in building the BAC.
  • These techniques can allow access to highly pure oligonucleotide-peptide conjugates at high yields.
  • the present methods also permit the construction of the BAC first via anchoring a modified nucleic acid monomer to a solid resin and successively adding nucleic acid monomer prior to the addition of the amino acid building blocks of the CPP.
  • This allows for a systematic study of a batch of synthesized BAC and subsequent modification of one or more amino acid residues in the peptide sequence of the cell penetrating peptide to obtain a series of oligonucleotide-peptide conjugates for further investigation utilizing a BAC synthesized from the same set of experiments and may facilitate the discovery of additional peptide moieties for intracellular delivery of the BAC.
  • the CPP is preferably synthesized stepwise utilizing solid phase synthesis techniques.
  • Solid phase peptide synthesis permits absolute control over the desired peptide sequence with adequate yields and recoveries.
  • Solid phase peptide synthesis is not limited to the proteingenic amino acids and the techniques may be adopted for incorporation of unnatural amino acids and other amino acid derivatives.
  • an amino protected amino acid is coupled to a solid phase material such as a resin via its carbonyl group.
  • the amino group is then deprotected, exposing a reactive amino group, which reacts with the carboxyl group of a successive amino acid monomer comprising a protected amino group, forming a dipeptide.
  • the process of deprotecting the terminal amino group and coupling of the successive amino acid monomer comprising a protected amino group is repeated until obtaining the desired peptide sequence after which the peptide is cleaved from the resin and recovered for further application.
  • a stapled peptide may be formed by, for example, stapling two conformationally adjacent amino acids together, and a stitched peptide (StiP) may be formed by, for example, stitching at least three conformationally adjacent amino acids to form a stitched peptide (StiP).
  • the stapling or stitching results in the formation of a cross link or bridge between two conformationally adjacent amino acids of the peptide.
  • the cross link or bridge comprises two components, a hydrocarbon bridge and a terminal methyl group.
  • the hydrocarbon bridge may be composed of a double hydrocarbon bond or a single hydrocarbon bond.
  • the cross link or bridge provides a cyclization between at least two amino acids formed by an olefin metathesis.
  • the CPP preferably comprises at least two unnatural amino acids bearing allhydrocarbon tethers (e.g. a-methyl,a-pentenyl glycine).
  • cross link and bridge is not limited to reacting functional side chains of unnatural amino acids and the cross link and bridge may be formed through reaction of the side chains of proteingenic amino acids.
  • the cross link or bridge provides a cyclization between at least two amino acids which are not formed by an olefin metathesis.
  • cyclization is meant that a staple or stitch is formed directly between conformationally adjacent amino acids, as opposed to by the introduction of a separate “bridging molecule”, such as, for example, an aryl group, such as an aromatic ring or a perfluroaryl group.
  • bridging molecule such as, for example, an aryl group, such as an aromatic ring or a perfluroaryl group.
  • a 5 membered heterocycle is formed between an azide and electron deficient nitrile containing amino acid or a propygyl containing amino acid.
  • the BAC is an oligonucleotide (ON). In some embodiments, the BAC is an anti-sense oligonucleotide (AON).
  • the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • a linker In solid phase synthesis of the BAC, a linker must first be coupled to a solid support resin wherein the linker is configured to couple to an oligonucleotide monomer.
  • the linker is an amino acid monomer.
  • the linker is a handle, which is a moiety comprising a functional group, further discussed below.
  • a BAC monomer can similarly be protected to prevent undesired successive coupling and subsequently deprotected to enable the addition of the next monomer.
  • the secondary amine of PMO at the 3’ end can be protected to permit coupling to occur at the phosphorodiamidate moiety located at the 5’ end and the secondary amine at the 3’ end deprotected to enable addition of a successive PMO monomer, which also comprises a protected secondary amine at its 3’ end.
  • the process of deprotecting the secondary amine at the 3 ’ end is repeated until the desired sequence is obtained.
  • the deprotected second amine may be used in a subsequent coupling reaction without further modification or with additional modification to incorporate other functional groups as described below.
  • the oligonucleotide monomers may be further configured to undergo a nucleophilic substitution reaction to facilitate the coupling of oligonucleotide monomers.
  • the phosphorodiamidate moiety may be modified at its 5’ end to have a halogen leaving group or another suitable organic leaving group to facilitate coupling with the linker of the solid support resin or the secondary amine of the 3’ end.
  • the phosphorodiamidate moiety may be modified with a primary amine or other functional group at its 5’ or 3’ end to facilitate coupling to the functional group of a handle.
  • the 5 ’ or 3 ’ end can be modified with a glycine whose amino group is protected with Fmoc or other suitable protecting group to provide a more reactive primary amine functionality to the phosphorodiamidate moiety.
  • the present methods permit the coupling of handles to the compound, which provide orthogonal reactive sites and permit the introduction of functionalities to tailor the application and properties of the compound.
  • the handle can be coupled anywhere on the CPP.
  • the handle is coupled to the N terminus of the CPP.
  • the handle is coupled to the C terminus of the CPP via functionalization of the amino acid at the C terminus (e g. deprotection of Mtt protected lysine).
  • the handle is coupled to an amino acid located internally between the C terminus and the N terminus.
  • the handle can be coupled anywhere on the BAC.
  • the handle is coupled to the 3’ end.
  • the handle is coupled to the 5’ or internally between the 5’ end and the 3’ end via addition of a residue that can be modified orthogonally (e.g. addition of Mtt protected lysine at the 5’ end and subsequent deprotection for coupling of handle).
  • the handle may be used to couple the CPP to the BAC.
  • one or more handles may be coupled together to form a bifunctional linker, which may be used to couple the CPP to the BAC.
  • the disclosed methods permit a one pot synthesis of the compound, obviating the need to carry out a coupling reaction between discrete species of CPP, BAC, and any associated linkers or handles, which have resulted in low overall yields.
  • the disclosed methods streamline the overall synthetic process of the compound with high yielding solid phase synthetic methods, improving the yield and purity of the compound and enabling complete control over the composition of the compound.
  • the compound can be synthesized in any order using the disclosed methods.
  • the CPP is synthesized first, followed by the BAC.
  • the BAC is synthesized first, followed by the CPP.
  • the compound is not limited to block copolymers of BAC and CPP and the monomers of both the BAC and the CPP can be random and arranged in any order or configuration.
  • BAC biologically active compound
  • BFL bifunctional linker
  • CPA cell penetrating agent
  • StaP stapled peptide
  • StiP stitched peptide
  • HNA hydrazynal nicotinic acid
  • the compound may be used in the treatment of a, for example, neuromuscular disease, metabolic disease, cancer, age-related degenerative disease or to treat an acquired viral infection.
  • a muscular dystrophy e g. Duchenne muscular dystrophy (DMD) although the skilled person will readily appreciate that the invention can be used to target a wide range of genes.
  • DMD Duchenne muscular dystrophy
  • the compound may comprise an anti-sense oligonucleotide (AON) targeting exon 51 of the dystrophin gene.
  • AON anti-sense oligonucleotide
  • a method of improving the bioavailability of a drug or BAC comprising linking the drug or BAC to a CPP, which is a stabilized peptide which has a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP).
  • a method of introducing a drug or BAC to a site which is refractory to a drug or BAC in its native state comprising linking the drug or BAC to a CPP, which is a stabilized peptide having a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP) and administering it to a subject.
  • a CPP which is a stabilized peptide having a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP) and administering it to a subject.
  • the compounds of the invention can be used to administer the drug or BAC to a target tissue.
  • the drug or BAC may be administered to any type of tissue, which include epithelial tissue, connective tissue, muscle tissue, and nervous tissue.
  • the compounds are specific to a type of muscle or the cells of a particular type of muscle.
  • cellular uptake and retention of biological activity of a BAC that is conjugated to a particular stabilized peptide may be improved for in one particular peptide conjugate in a specific muscle type (e.g., skeletal) whereas the same peptide conjugate carrying the same BAC may exhibit decreased cellular uptake and retained biological activity of the BAC in a different muscle type (e.g., cardiac) despite having a similar peptide sequence, identity of linker, BAC, hydrophobicity, location of stitch, and charge.
  • the compound is contacted with skeletal (striated) muscle or skeletal muscle cells.
  • the compound is contacted with smooth muscle or smooth muscle cells.
  • the compound is contacted with cardiac muscle or cardiac muscle cells.
  • a method of treating a subject to alter the expression of an endogenous or exogenous gene comprising administering a compound of the invention to a subject.
  • a method of delivering a biologically active moiety to a cell comprising contacting the cell with the compound of the invention.
  • the cell is a cardiac muscle cell or a cardiomyocyte.
  • the cell is a muscle cell.
  • the muscle cell is a smooth muscle cell.
  • the muscle cell is a skeletal (striated) muscle cell.
  • a composition comprising a compound of the invention and one or more pharmaceutically acceptable excipients enabling the composition to be administered orally, parenterally, intravenously or topically.
  • the pharmaceutical formulations of the present invention can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the pharmaceutical compositions comprise the compound and a pharmacologically acceptable excipient and the compositions have a pH of 7.0-7.5.
  • the excipient is selected from the group consisting of a tonicity agent, a pH adjuster, and a solvent.
  • the concentration of the compound in the pharmaceutical composition can vary and will depend on the method of administration and the needs of the patient. In some embodiments, the concentration of the pharmaceutical composition ranges between about 0.1 mM to about 10 mM.
  • Dosage will depend on the body mass of the patient and the concentration of the active ingredient in the pharmaceutical composition.
  • the dosage can range from about 0.1-10 ⁇ mol of the compound per kilogram of body mass. In some embodiments, the dosage of the compound is about 30 mg per kilogram of body mass.
  • Administration can occur at any frequency. In some embodiments, administration occurs daily, weekly, monthly, or bimonthly. Preferably, administration to the patient via an intravenous route occurs between once weekly and once monthly.
  • the compound of the invention has specificity for skeletal muscle.
  • the improved uptake and specificity for skeletal muscle renders the compounds of the invention good candidates for improved efficiency delivery of BACs that may be useful in therapeutic applications in skeletal tissue.
  • the three components forming the compound, the methods of synthesis, the formulation of the compound, and administration are discussed in more detail below.
  • the biologically active compound is any compound that can exert a biological effect within a biological cell.
  • the biologically active compound is conjugated to the cell -penetrating peptide and is delivered to the cell.
  • any compound that has biological activity can be used as the biologically active compound and may include, but is not limited to proteins, enzymes, antibodies and antibody fragments, nanoparticles, micelles, quantum dots, and organelles in organelle replacement therapy (e.g. mitochondria, lysosomes).
  • the BAC is one which will impact on the expression of one or more endogenous or exogenous genes.
  • BACs that can affect the expression of one or more endogenous or exogenous genes include nucleic acids, DNAzymes, ribozymes, aptamers, gapmers, and pharmaceuticals.
  • Preferred biologically active compounds for use in the present invention include electrically neutral oligonucleotides (charge -1 to +1 at physiological pH - about 7.5) such as peptide nucleic acids (PNAs) or PMOs or their modified derivatives that might impart a small electric charge (either positive or negative).
  • the biologically active compound may be used as a steric blocking compound to suppress or enhance: i) RNA splicing; ii) protein translation or iii) other nucleic acidmucleic acid or nucleic acid:protein interactions, altering the gene expression of endogenous or exogenous (pathogen derived) genes.
  • ONs can be designed to target 5’ translation initiation start sites of endogenous or viral gene transcript(s) to prevent binding of the translational machinery.
  • ASO to suppress viral translation is a well-established technology and has progressed into clinical trials for viral haemorrhagic fevers such as Marburg and Ebola.
  • ON can be designed to target 3’ untranslated region of an endogenous transcript that alters the stability of the transcript.
  • targets include, and are not limited to, poly adenylation and/or cleavage sites of the transcript.
  • ON can be designed to form aptamers such that the secondary and tertiary structures can bind proteins or other cellular targets thus impacting specific gene expression levels.
  • Non-limiting exemplary ON chemistries are illustrated in Table 1.
  • the preferred BAC is an oligonucleotide (ON), more preferably still an anti-sense oligonucleotide (AON).
  • ON oligonucleotide
  • AON anti-sense oligonucleotide
  • Different anti -sense oligonucleotide chemistries are illustrated in Table 1, with the use of low charge or neutral charged chemistries, such as, phosphorodiamidate morpholino oligonucleotides (PMOs) being preferred.
  • the length of a PMO can influence the observed cellular uptake by a cell and ability of the PMO to participate in inhibiting expression of its target gene.
  • PMOs typically range from 15 to 30 nucleotides in length although PMOs can be longer or shorter depending on the desired biological activity and application of the PMO.
  • the BAC may target and alter the expression of an endogenous or exogenous gene.
  • Endogenous gene targets include but are not limited to genes associated with neuromuscular disease, metabolic disease, cancer, age-related degenerative diseases, and exogenous gene targets include those of an acquired disease e.g. viral infections.
  • the BAC may comprise, but is not limited to, one of the following sequences (represented 5 ’ to 3 ’), which target exon 51 or the splice junction of intron 50/exon 51 of the human dystrophin gene:
  • sequences may also be represented as RNA sequences in which T is substituted with U.
  • the biologically active compound can be an siRNA (small interfering, short interfering, silencing RNA) that interferes with the expression of specific target genes and in particular, prevents mRNA translation.
  • the siRNA can comprise between about 15- 30 base pairs in length, preferably between 20-24 base pairs in length.
  • the target is exon 51 of the dystrophin gene and the BAC is a PMO that comprises the sequence: or its RNA analog:
  • the target is exon 23 of the dystrophin gene and the BAC is a PMO that comprises the sequence: or its RNA analog:
  • the oligonucleotides that are conjugated to the cell penetrating peptide are involved in antisense therapy, whose numerous functions include exon targeting and skipping.
  • Other forms of antisense therapy include, but are not limited to functions such as RNase H degradation of mRNA or pre-mRNA to knockdown the expression of a gene and the use of siRNA to interfere with and block the production of a target protein.
  • the antisense oligonucleotide is a gapmer, which recruits RNase H to degrade mRNA and pre-mRNA.
  • Gapmers are single stranded chimeric antisense oligonucleotides comprising a central DNA gap flanked by modified nucleotides on both ends. The modified nucleotides enhance stability and binding affinity, while the central DNA gap allows for RNase H-mediated degradation of the target RNA. Gapmers rely on the RNase Hl-dependent degradation of complementary RNA targets as the mode of action.
  • the RNA-DNA duplex is recognized by RNase H, which cleaves the RNA strand, resulting in the degradation of the target mRNA and subsequent reduction in protein expression.
  • RNase H-active gapmers can exert activity in both the nucleus, in which the gapmers interact with pre-mRNA, or in the cytoplasm via interacting with the mRNA.
  • Oligonucleotides such as gapmers, are susceptible to nucleolytic degradation, which adversely affects the function of the oligonucleotide.
  • nucleolytic degradation which adversely affects the function of the oligonucleotide.
  • nucleolytic degradation To increase their resistance to nucleases and nucleolytic degradation and to obtain higher half-lives in plasma, tissues and cells, various chemical structure modifications may be made.
  • Modifications to the chemical structure of the oligonucleotide to improve nuclease resistance include, but are not limited to including phosphorothiorate linkages between monomers on the oligonucleotide backbone, 2’ ribose modifications, which include, but are not limited to 2'-O-methyl (2'-0Me), 2 '-O-m ethoxy ethyl (2'- MOE), 2'-O-aminopropyl (2'-O-AP), and 2'-fluoro modifications, and utilizing a locked nucleic acid conformation (LNA) (also known as bridged nucleic acid), in which the ribonucleotides contain a methylene bridge that connects the 2’ oxygen and the 4’ carbon.
  • LNA locked nucleic acid conformation
  • the ribonucleotides contain a methylene bridge that connects the 2’ oxygen and the 4’ carbon.
  • LNA locked nucleic acid conformation
  • the gapmer may be for inhibition of a target pre-mRNA, a target mRNA, a target viral RNA, or a target long non-coding RNA.
  • gapmers Through the interaction and hybridization with either pre-mRNA or mRNA and the subsequent cleavage through recruitement of RNase H, gapmers halt the transcription process and can thus knockdown the expression of a target gene. This ability to knockdown and silence target genes renders gapmers attractive agents for therapy. Conjugation of a gapmer to the present CPPs can help to achieve specificity for a particular target tissue or a particular target cell to improve delivery of the therapeutic gapmer.
  • the cell penetrating agent which is a stabilized peptide
  • the cell penetrating agents of the invention are stabilized peptides.
  • the peptides may be stabilized by stapling, to form a stapled peptide (StaP), or by stitching to form a stitched peptide (StiP).
  • StaP includes one cross link that is formed between two amino acid residues on the peptide chain.
  • StiP includes more than one cross link formed.
  • All-hydrocarbon staples and stitches may confer a property, e.g. an a-helical structure, protease resistance, cellular penetrance, and biological activity.
  • the a-helix is used as a recognition motif in protein -protein interactions and the introduction of a staple or a stitch to lock lock down the conformation of the peptide can lead to improved cell penetrating ability in addition to improvements in stability against proteolytic degradation.
  • Non-limiting examples of stapled and stitched peptide sequences are illustrated in Table 3 and include peptide sequences including S5, S8 and B5 (as defined in Table 3).
  • Stabilization of e.g. the a-helical structure can be achieved by, for example, a ringclosing metathesis and may be catalysed by a variety of ruthenium catalysts including Grubbs generations 1 and 2 and Grubb s-Hoyveda generations 1 and 2.
  • All the peptide components may exist in specific geometric or stereoisomeric forms. All compounds include cis- and trans-isomers, (R)- and (S)-enantiomers, diastereoisomers and racemic mixtures thereof.
  • Preferred isomer/enantiomers will be enriched to give a greater proportion of one particular isomer or enantiomer. Embodiments thereof may be made of greater than 90%, 95%, 98% or 99%, by weight, of a preferred isomer/enantiomer.
  • Non-limiting examples of unnatural amino acids used in stabilizing a peptide structure are illustrated in Table 3.
  • the preferred stapled or stitched CPPs incorporate one or more of: a (S)- pentenylalanine (S5) or its enantiomer (R5), a S-octenylalanine (S8) or its enantiomer (R8) or combinations thereof (e.g. R-octenylalanine/S-pentenylalanine (R8/S5) or S-octenylalanine/R- pentenylalanine (S8/R5).
  • H can be substituted for R and K;
  • I, L, A, or V can be substituted for I, L, A, or V;
  • a PEG n group may be present before the handle, for example (S5-FLR-S5)FKR-PEG n - 3TPA;
  • the 3-(tritylthio)propionic acid moiety (3 TP A) can be substituted for any handle described in Table 5;
  • the parentheses enclosing the unnatural amino acids designate the location of the crosslink and the intermediate amino acids that span the crosslink;
  • amino acids utilized can be the D stereoisomer or the L stereoisomer, or combinations thereof;
  • the sequences can be synthesized in the reverse order.
  • the peptide of SEQ ID NO: 23, (S5-FLR-S5)FKR-3TPA can be synthesized as RFK-(S5-RLF-S5)-3TPA
  • the peptide of SEQ ID NO: 37, VKR-(S5-KKK-S5)-P-3TPA can be represented as 3TPA-PK(S5-KKR-S5)KV
  • the peptide of SEQ ID NO: 38, VK(S5-RKK-S5)KP-3TPA can be represented as 3TPA-P(S5-KKK-S5)RKV.
  • the present invention is not limited to the formation of a cross link or a bridge through the linking of unnatural amino acids.
  • a lactam can be formed between a free amine containing amino acid such as lysine and a carboxylic acid containing amino acid such as aspartic acid or glutamic acid.
  • the exemplified stabilized peptide comprises two or more olefin bearing side chains that are covalently formed, typically by means of a ring-closing metathesis.
  • the stabilized conformation typically comprises at least one alpha helix. It may however, in the alternative, comprise at least one turn (for example, but not limited to, ⁇ , ⁇ , ⁇ , ⁇ or ⁇ ), several turns to form a beta sheet, or a combination of one or more of: an alpha helix, turn, or beta sheet.
  • the applicant employs ⁇ , ⁇ -di substituted unnatural amino acids bearing all-hydrocarbon tethers (e.g. a-methyl,a-pentenyl glycine).
  • ⁇ , ⁇ -di substituted unnatural amino acids bearing all-hydrocarbon tethers e.g. a-methyl,a-pentenyl glycine.
  • S5 a (S)-pentenylalanine
  • R8/S5 S'-pentenyl alanine
  • S8/R5 S'-pentenyl alanine
  • S8/R5 S'-octenyl al anine/R-pentenyl alanine
  • S5 can be substituted at z, B5 at position z + 4 positions, and S8 can be substituted at i, i+4, i + 11 positions to generate stitched peptides.
  • peptides may comprise of one or more of the sequences in Table 3. Based upon the specific peptides shown in Table 3, a person skilled in the art can easily envisage peptides with 3, 4, 5 or more turn stabilizing staples.
  • the hydrocarbon bridge may be composed of a double hydrocarbon bond or a single hydrocarbon bond.
  • a combination of factors which include, but are not limited to, the overall hydrophobicity of the peptide chain, the location of the staple or stitch within the peptide chain, the overall charge of the peptide, the identity of the BAC, the identity of the target tissue or cell, and the identity of any bifunctional linker, are taken into account in determining a good candidate for intracellular delivery of a BAC such that the BAC retains its biological activity.
  • the cell penetrating agent has a stitched or stapled peptide comprising the amino acid sequence, (S5-FLR-S5)FKR-3TPA (SEQ ID NO: 23), or its reverse sequence RFK-(S5-RLF-S5)-3TPA.
  • the cell penetrating agent is an IL1 ⁇ -based stapled peptide having the sequence of S5-VLK-S5-RR (SEQ ID NO: 64) or K-S5-LKK-S5-R (SEQ ID NO: 60).
  • S5 in the IL1 ⁇ -based stapled peptide according to SEQ ID NO: 64 and SEQ ID NO: 60 is the unnatural amino acid (S)-pentenylalanine and wherein the olefin crosslink is formed between the (S)-pentenylalanine residues comprises olefin crosslinks formed by olefin metathesis.
  • ETS-related transcription factor Elf-3 ETS-related transcription factor Elf-3
  • NLS nuclear localization signal
  • the peptide is a branched stapled peptide.
  • the branched stapled peptide comprises 2 or more chains of peptides.
  • Branched peptides may be formed using any method known to the art. For example, in one embodiment, a lysine residue is used to branch two peptide chains.
  • Branched peptides allow for the presentation of multiple cell penetrating peptides to the cell membrane, which may improve the cell penetrating or uptake ability of the molecule due to the higher local concentration of cell penetrating peptides interacting with the cell membrane or via the synergy of the abilities and functions of the branched peptide.
  • a dendrimeric peptide formed by branching may include multiple copies of a peptide having adequate cell penetrating abilities to improve cellular uptake due to the increased and more localized presentation.
  • a dendrimic peptide formed by branching may include a combination of peptides that have adequate cell penetrating abilities and of peptides that exhibit improved endosomal escape abilities to improve delivery of the BAC both across the cell membrane and to the target via the improved abilities to both cross the cell membrane and break free from the endosome transporting the molecule comprising the dendrimeric peptide across the cell membrane.
  • Functional derivatives of disclosed peptide sequences could be used.
  • Functional derivatives may have representative fragments or homologues or peptides that include insertions to the original peptide. Typical derivative would have 70%, 80%, 90% or more of the original peptide sequence and may have up to 200% of the number of amino acids of the original peptide. The derivatives would be used to enhance the delivery of a biologically active compound.
  • Peptide sequence can include modified amino acids to include functional groups that permit the addition of other moieties.
  • moieties include an acetyl, a cholesterol, a fatty acid, a polyethylene glycol, a polysaccharide, an aminoglycan, a glycolipid, a polyphenol, a nuclear localizing signal, a nuclear export signal, an antibody and a targeting molecule.
  • the CPP can also comprise nuclear export signals (NES), which are short peptides that mediate the transport of proteins from the nucleus to the cytoplasm. Examples of NES include LCNCALEELRL or LWEFIRDILI, which are found in the transcription factor ESE-1.
  • the NES of the present invention have the following sequence motif: (L/I)X2-4(L/I)X1 -4(L/I)X(L/I) in which X is any amino acid and the numerical range modifying X indicate the range of possible X present.
  • NES include the sequence motifs:
  • the CPP can also be a nuclear localization signal (NLS), which are short peptides that mediate the transport of proteins from the cytoplasm into the nucleus of a cell.
  • NLS nuclear localization signal
  • KHGKRKR or HGKRKR are found in the transcription factor ESE-1.
  • NLS can have the following sequence motifs:
  • Handles can optionally be coupled to the compound to introduce additional functional groups that serve as orthogonal reactive sites, allowing for further functionalization and tailoring of physical properties of the compound.
  • the handle can be coupled to the BAC or the CPP during solid phase synthesis via an additional coupling reaction according to the invention.
  • the handle can be incorporated via a nucleophilic substitution reaction such as with a terminal amino group.
  • the handle serves as a linker between the BAC and the CPP.
  • the handle is not present and the BAC is covalently linked to the CPP.
  • the handle can comprise a PEGylated group such as a PEG peptide comprising amino and carboxyl groups and a PEG spacer to enhance solubility of the compound in aqueous environments.
  • a PEGylated group such as a PEG peptide comprising amino and carboxyl groups and a PEG spacer to enhance solubility of the compound in aqueous environments.
  • the handle can be an amino acid whose side chains introduce a functional group.
  • the amino acid handle may or not contain protecting groups.
  • the amino acid handle may be a proteingenic amino acid or the amino acid handle may be an unnatural amino acid.
  • the resin can similarly contain functional groups to enable orthogonal chemistry.
  • Fig. 4B shows the structure of Universal PEG NovaTagTM resin, which provide an Fmoc protected amine for growing the peptide chain and an Mtt protected amine for additional functionality at the C terminus.
  • Fig. 4C shows the structure of a resin having a hydrazine functionality.
  • handles utilized in coupling to the CPP or the BAC can be interchanged in coupling reactions to both the CPP and the BAC.
  • a handle used to couple to the CPP is not necessarily limited to coupling and functionalizing the CPP and may be used for coupling to and functionalizing the BAC.
  • the handles for the CPP of the present invention include but are not limited to those listed in Table 5:
  • handles for the BAC include but are not limited to those listed in Table 6: Table 6.
  • the handle can be incorporated at the N or C terminus of the peptide, internally within the peptide chain, or in combinations thereof.
  • the handle can be incorporated at the 5 ’ or 3 ’ end of the B AC, or internally, or in combinations thereof.
  • a handle can be used to introduce a label or a marker such that the bioactivity of the compound can be visualized and measured.
  • chemiluminescent labels such as a fluorescent label or a phosphorescent label.
  • the label is a fluorescent label that is fluorescein isothiocyanate (FITC).
  • FITC fluorescein isothiocyanate
  • the label or marker may be conjugated anywhere onto the compound and may be included at the C or N terminus of the peptide or internally within the peptide chain, within a handle or a series of handles linking the BAC to the CPP, or at the 5’ or 3’ end of the BAC or internally within the BAC chain.
  • a handle alone or in combination with other handles, can also serve as a bifunctional linker (BFL), which may be used to link the BAC to the CPP or the CPP to the BAC.
  • BFL bifunctional linker
  • Preferred linkers will link between, for example, an amine group on the BAC and a sulfhydryl (thiol) group (usually a cysteine residue) on the CPP terminus.
  • substrates to achieve this include, but are not limited to, SMCC (succinimidyl 4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate), AMAS (N- ⁇ -maleimidoacet-oxysuccinimide ester, BMPS (N- ⁇ -maleimidopropyl-oxysuccinimide ester), GMBS (N- ⁇ -aleimidobutyryl- oxysuccinimide ester), DMVS (N- ⁇ -maleimidovaleryl-oxysuccinimide ester, EMCS (N-s- malemidocaproyl-oxysuccinimide ester), and LC-SMCC (Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) as exemplified in Table 7.
  • SMCC succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-
  • a sulfur containing moiety e.g. cysteine and L is a handle that acts as a bifunctional linker such as SMCC, which introduces an alkene functionality to enable a thiol-ene Michael addition.
  • Z is a thiol- containing moiety that is covalently linked to the C-terminus of the StaP or StiP by a polyethylene glycol linker.
  • the thiol-containing moiety is a cysteine residue.
  • a preferred linker chemistry utilizes an amine to sulphydryl cross linker containing N- hydroxysuccinimide esters and maleimide reactive groups separated by a cyclohexane spacer namely succinimidyl 4-(N-maleimidom ethyl) cyclohexane- 1 -carboxylate (SMCC) to form a covalent bond between the bifunctional linker and the CPP.
  • SMCC succinimidyl 4-(N-maleimidom ethyl) cyclohexane- 1 -carboxylate
  • the CPP of the present invention are synthesized using solid phase synthesis in which each monomer is added to the growing peptide chain in a stepwise fashion.
  • Solid phase peptide synthesis is performed using resins as solid supports for the growing peptide chain. Any resin may be used for solid phase synthesis. The choice of synthetic strategy such as utilizing Boc or Fmoc amino acids as monomers will dictate the appropriate selection of resin. Resins that may be used include, but is not limited to PAM (4-hydroxymethyl- phenylacetamidom ethyl) resin, Wang resin, Rink amide resin, MBHA resin, PAL (4-alkoxy-2,6- dimethoxybenzylamine) resin, Sieber amide resin, trityl resins, DHP (dihydropyran) resin, and Weinreb aminomethyl resin.
  • PAM 4-hydroxymethyl- phenylacetamidom ethyl
  • Wang resin Wang resin
  • Rink amide resin Rink amide resin
  • MBHA resin PAL (4-alkoxy-2,6- dimethoxybenzylamine) resin
  • Sieber amide resin trityl resins
  • DHP dihydro
  • the amino acid monomers can be protected with any protecting group such that the monomer does not have the ability to react with another amino acid monomer or other reactive group such as a handle until completion of the current addition and deprotection. This ensures that one and only one monomer, handle, or other moiety is added to the growing peptide chain at a time.
  • protecting groups for amino acids include Boc (tert-butoxycarbonyl), Bn (benzyl), Fmoc (9-fluorenylmethoxycarbonyl), t-Bu (tert-butyl), Fm (9-fluorenylmethyl), Trt (trityl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Ddz (3,5- dimethoxyphenylisoproxycarbonyl), Bpoc (2-(4-biphenyl)isopropoxycarbonyl), Nps (2- nitrophenylsulfenyl), Nsc (2-(4-nitrophenylsulfonyl)ethoxycarbonyl), Bsmoc 1,1- dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl, Dmb (2,4-dimethyloxybenzyl), and pNZ (p- nitrobenzyloxycarbonyl).
  • the protecting group may be used to protect the amino or carboxyl group of the amino acid or a functional side chain.
  • the reactive amino group may be protected during monomer addition of successive amino acid monomers to the peptide chain to ensure that one and only one monomer is added at a time to the peptide chain.
  • the carboxylic acid side chain of aspartic or glutamic acid or the amino side chain of lysine can be protected during peptide synthesis to prevent unwanted side reactions to the side chains.
  • Deprotection procedure depends on the protecting group present on the amino acid. Deprotection can occur under acidic or basic conditions. For example, Boc is removed under acidic conditions, namely in the presence of trifluoroacetic acid in dichloromethane. In contrast, Fmoc is removed under basic conditions, namely with secondary amines such as piperidine.
  • the CPP is synthesized using Fmoc protected amino acids using MBHA resin.
  • Solid phase peptide synthesis can be carried out with or without the assistance of a microwave.
  • Microwave-assisted solid phase peptide synthesis can accelerate the rate of the amino acid coupling reaction as well as the deprotection reaction.
  • the CPP is synthesized with the assistance of a microwave.
  • Solid phase synthetic techniques are also applicable to the synthesis of the BAC.
  • the BAC cannot be directly coupled to the resin and prior to stepwise synthesis of the BAC, a linker, preferably an amino acid or a PEGylated linker, must be coupled to the resin.
  • the BAC monomers can similarly be protected with any protecting group such as those described for the protection of the amino group of the amino acid monomers of the CPP.
  • the protecting group is Fmoc or Trt.
  • the solid phase synthesis of the BAC preferably occurs under basic conditions.
  • the synthesis occurs in the present of N-ethylmorpholine (NEM) and 5- (ethylthio)-lH-tetrazole (ETT).
  • NEM N-ethylmorpholine
  • ETT 5- (ethylthio)-lH-tetrazole
  • DIPEA N,N-diisopropylethylamine
  • the BAC is phosphorodiamidate morpholino oligonucleotides (PMO).
  • the PMO monomers can have the following structures:
  • the secondary amine at the 3’ position is protected to prevent unwanted reactions and to ensure that only one monomer is added to the growing chain at a time.
  • the PMO monomers are preferably protected with Fmoc or Trt, which result in the following structures:
  • the Fmoc protecting groups in the PMO monomers can be removed under similar basic conditions (piperidine) to those utilized in the deprotection of amino acid monomers, removal of the Trt protecting group (detrityl ati on) is performed under acid conditions.
  • the acid used in detritylation is dichloroacetic acid (DCA) or trichloroacetic acid (TCA).
  • the PMO monomers may be further modified to comprise a functional group at the 5’ end to facilitate the coupling of the first PMO monomer to the functional group of a handle, which may be a terminal carboxylic acid, a carboxyl group comprising a N-hydroxysuccinimide group, a thiol, or an alkyne.
  • a handle which may be a terminal carboxylic acid, a carboxyl group comprising a N-hydroxysuccinimide group, a thiol, or an alkyne.
  • functional groups that may be attached include amines, thiols, alkynes.
  • the first PMO monomer is modified to have a primary amine at the 5’ terminus.
  • the PMO monomer can be coupled to Lys(mtt)-Fmoc, Universal PEG NovaTagTM resin, or a similar moiety allowing for a similar orthogonal deprotection.
  • the PMO can undergo selective chain extension and cleavage of the PMO monomer or chain extended PMO furnishes a final compound having the 5’ primary amine.
  • Techniques affording the 5’ amine modification can also be combined with other methods to selectively incorporate other functional groups for orthogonal chemistry onto the 5’ end.
  • the 5’ primary amine can undergo a subsequent nucleophilic substitution with a halo- alkyne to afford a PMO having a 5’ alkyne group.
  • the solid phase synthesis of the BAC can be carried out with or without the assistance of a microwave, which accelerates the rate of the coupling reaction and deprotection.
  • handles used to introduce additional functional groups into the compound comprise a variety of structures. While handles bearing structural similarity (i.e. amino acid based) or having similar functional groups (i.e. carboxyl and/or amino functional groups) can be coupled to the compound via similar techniques used to couple amino acid monomers (i.e. DIC/Oxyma), other applicable reactions may be used to couple handles to the compound, which include but are not limited to S N 2 reactions, SNI reactions, or reactions with N- hydroxysuccinimide activated esters or anhydrides.
  • structural similarity i.e. amino acid based
  • similar functional groups i.e. carboxyl and/or amino functional groups
  • other applicable reactions may be used to couple handles to the compound, which include but are not limited to S N 2 reactions, SNI reactions, or reactions with N- hydroxysuccinimide activated esters or anhydrides.
  • the solid phase synthetic techniques for the CPP, BAC, and handles enable a one pot synthesis of the compound as shown in Fig. 9.
  • the compound can be synthesized in any order. In other words, in some embodiments, the CPP is synthesized first on the resin followed by the BAC and in some embodiments, the BAC is synthesized first on the resin followed by the CPP.
  • handles can similarly be integrated using any coupling reaction as previously described.
  • the handle can be incorporated onto the PMO, the peptide, or anywhere internally within the polymeric chain of the compound.
  • the CPP comprises the sequence RKF-S5-RLF-S5 or its reverse sequence (S5-FER-S5)FKR and the BFE is a PEGylated SMCC
  • the resulting compound is termed CP8M.
  • the BAC which is a PMO
  • the resulting compound is termed PMO-CP8M.
  • the CPP comprises the sequence RKF-S5-RLF-S5 and the BFL is a PEGylated hydrazynal nicotinic acid (HNA)
  • HP8M PEGylated hydrazynal nicotinic acid
  • C8M the resultant compound is termed C8M.
  • Covalent linkage to the CPP may be via, for example, but not limited to, a ⁇ -ala or any other suitable moiety.
  • any form of the linker may be used.
  • any acceptable salt or derivative of the linker may be used for linking the BAC to the CPP.
  • the starting material to achieve linkage between the BAC to the CPP may be in a form that is soluble in organic solvent or in a salt form that is soluble in water.
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate
  • a sulfo salt CAS No. 92921-24- 9 for applications that require solubility in water.
  • HNA hydrazynal nicotinic acid
  • linkers such as DSG (disuccinimidyl gluterate) and DSCDS (disuccinimidyl- cyclohexl-l,4-diester) will include the ability to link the 5’-amino group of the BAC to the N- terminus of the CPP.
  • Handles may serve as linkers that include other elements that confer a desirable property on the compound e.g. spacer between BAC and CPP or an element that will enhance solubility, for example a PEGylated element. Non-limiting examples are shown in Tables 5 and 6.
  • the biologically active compound is covalently attached to the chimeric cell delivery peptide.
  • the cell delivery peptide is attached to the biologically active compound by means of a disulphide bridge or a thiol maleimide linker e.g. SMCC; the attachment may be by means of an amide linker or an oxime linker or a thioether linker.
  • a disulphide bridge or a thiol maleimide linker e.g. SMCC
  • the attachment may be by means of an amide linker or an oxime linker or a thioether linker.
  • the present invention provides a pharmaceutical composition comprising the compound and one or more pharmaceutically acceptable excipients for oral, parenteral, intravenous, or topical administration.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable.
  • compositions and formulations for parenteral or intravenous administration include sterile aqueous solutions, which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions and formulations for topical administration include solutions, gels, lotions, creams, oil-in water emulsions, water-in-oil emulsions, ointments, shampoos, sprays, sticks, powders, masks, pads, mouth rinses or washes, vaginal gels or suppositories, rectal gels or suppositories, urethral gels or suppositories, or other form acceptable for use on skin, nail, hair, oral mucosa, vaginal or anal mucosa, mouth, or gums.
  • the pharmaceutical composition may further include a substance having pharmaceutical activity other than the compound or a substance having no pharmaceutical activity.
  • the pharmaceutical composition can be produced using a commonly used excipient such as fdler, binder, wetting agent, disintegrator, surfactant, lubricant, dispersant, buffer, preservative, solubilizer, antiseptic, coloring agent, flavoring agent, stabilizer, tonicity agent, pH adjustor, solvent or vehicle, or combinations thereof.
  • the pharmaceutical composition comprises a tonicity agent, pH adjustor, and a solvent as excipients.
  • Tonicity agents include but are not limited to dextrose, glycerin, mannitol, potassium chloride, and sodium chloride.
  • Solvents or vehicles include, but are not limited to, water, alcohols, glycerin, propylene glycol, and polyethylene glycol 400.
  • pH adjusters include acidifying agents and alkalizing agents.
  • Acidifying agents include but are not limited to, acetic acid, citric acid, hydrochloric acid, nitric acid, propionic acid, tartaric acid, fumaric acid, lactic acid, phosphoric acid, sodium phosphate monobasic, potassium phosphate monobasic, citric acid, malic acid, and sulfuric acid.
  • Alkalizing agents include, but are not limited to, ammonia, monoethanol amine, sodium borate, sodium phosphate dibasic, ammonium carbonate, potassium hydroxide, sodium carbonate, trolamine, diethanolamine, sodium bicarbonate, and sodium hydroxide.
  • An exemplary pharmaceutical composition formulated for intravenous injection for the treatment of Duchenne’s muscular dystrophy comprises 500 mg of the compound, 80 mg sodium chloride, 2 mg potassium chloride, 2 mg potassium phosphate monobasic, and 11.4 mg sodium phosphate dibasic in 10 mL of water at a pH of 7.5 (50 mg/mL concentration of active ingredient).
  • the overall dosage of the compound, as administered intravenously to a patient can range from between about 0.1 ⁇ mol/kg and about 10 ⁇ mol/kg.
  • the intravenous dose of the pharmaceutical composition may be 1 mg or more, 5 mg or more, or 10 mg or more.
  • the dose of the pharmaceutical composition may be 150 mg or less, 100 mg or less, 50 mg or less, or 10 mg or less.
  • the number of times of administration of the pharmaceutical composition as described above may be adjusted as appropriate in accordance with the administration target, the body mass of the patient, the treatment stage, the type of pharmaceutical composition, or the like.
  • an intravenous dose of the pharmaceutical composition may be 25 mg/kg (2.1 ⁇ mol/kg) or more, 50 mg/kg (4.2 ⁇ mol/kg) or more, or 82.5 mg/kg (6.9 ⁇ mol/kg) or more, and may be 200 mg/kg (16.8 ⁇ mol/kg) or less, 175 mg/kg (14.6 ⁇ mol/kg) or less, or 125 mg/kg (10.4 ⁇ mol/kg) or less.
  • the dosage ranges between about 1.2 mg/kg and about 120 mg/kg (0.1 ⁇ mol/kg to 10 ⁇ mol/kg).
  • the composition will be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability.
  • aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, Dextrose injection, or combinations thereof
  • non-aqueous vehicles such as, but not limited to, ethyl oleate, peanut oil, corn oil, cottonseed oil, sesame oil, or isopropyl myristate, or combinations thereof
  • aqueous and non-aqueous isotonic sterile injection solutions which can contain bacteriostats, buffers, antioxidants, or solutes that render the formulation isotonic within the blood of the recipient, or combinations thereof; and non-aqueous and aqueous suspensions that can be sterile and can include solubility
  • the requisite volume of the pharmaceutical composition is withdrawn from the container and diluted in a vehicle to achieve a volume for administration.
  • the volume for administration can be 25 mL, 50 mb, 75 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 400 mL, or 500 mL.
  • a volume required for administration of 30 mg/kg of body mass is diluted to a volume between 100 and 150 mL in 0.9% sodium chloride injection, which can be administered to a patient via intravenous injection over a period a time.
  • Administration to the patient can take place over any period of time and may take place over a period of 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, or 180 minutes.
  • the rate of injection will depend on the total volume to be administered, following dilution in a vehicle, and the infusion time.
  • the rate of infusion can be, but is not limited to 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, or 5 mL per minute.
  • the pharmaceutical composition may be administered only once, or may be repeatedly administered.
  • the pharmaceutical composition may be administered once a day, twice or more a day, or once in every two or more days.
  • the administration term of the pharmaceutical composition may be 3 days or more and 7 days or less, or 7 days or more. Administration can occur monthly, bimonthly, quarterly, or semiannually.
  • additional vehicle may be used to flush the intravenous route of administration to ensure that the patient receives the entirety of the dose.
  • Also contemplated is a method for delivery of a biologically active moiety into a cell comprising contacting the cell with the compound of the present invention. Contacting the cell with the compound may occur utilizing neat compound or the compound may be contained within a composition or formulation.
  • the contacting may occur between the compound and any type of cell.
  • the cell is a muscle cell or a myocyte.
  • Smooth muscle lines the walls of a variety of different body cavities and hollow organs including, but not limited to: the gastrointestinal tract, hepatobiliary system, genitourinary tract, ureters, pharageal cavity, vascular spaces, arteries, veins, endolymphatic and cisternes in the intercranial space. Smooth muscle cells, unlike those of skeletal muscle cells, are not striated and do not contain myofibrils.
  • Cardiac muscle is striated muscle tissue that is found in the heart, which is under the control of the autonomic nervous system, i.e., the heart is involuntarily controlled.
  • the cells that constitute cardiac muscle are called cardiomyocytes or myocardiocytes.
  • the heart is an organ that is composed mostly of cardiac muscle and connective tissue. Disorders such as Duchenne’s muscular dystrophy initially affect skeletal muscle. However, later progression of the Duchenne’s muscular dystrophy spreads to cardiac muscle and can lead to death caused by respiratory or cardiac failure.
  • Skeletal muscle comprises striated muscle tissue which is under the control of the somatic nervous system, i.e., it is voluntarily controlled.
  • the term muscle refers to multiple bundles of muscle fibers held together by connective tissue.
  • Skeletal muscles may be attached to bones by tendons. Skeletal muscles may be attached to bones by tendons.
  • Non-limiting examples of skeletal muscles include, for example, the diaphragm, extensor digitorum longus, tibialis anterior, gastrocnemius, soleus, plantaris, biceps, triceps, deltoids, pectoralis major, pectoralis minor, rhomboids, trapezius, sartorius, knee flexors and extensors, elbow flexors and extensors, shoulder abductors, and abdominal muscles.
  • the cells lack the cytoskeletal protein dystrophin.
  • the dystrophin protein provides stability to the sarcolemma (i.e., the cell membrane of muscle cells) by linking the intracellular cytoskeletal network to the extracellular matrix. In the absence of dystrophin, muscle contraction mechanically stresses the cell membrane, inducing progressive damage to the myofibers initially in skeletal muscle.
  • the compound can be tissue specific for targeted delivery of the biologically active moiety.
  • the compound can have specificity for tissue including, but not limited to smooth muscle tissue, cardiac muscle tissue, and skeletal muscle tissue.
  • NOESY spectra were recorded with a 12626.263 Hz sweep width, 4096 complex points (DQD acquisition mode) in the direct dimension and 1024 indirect points (States-TPPI acquisition mode).
  • a NOESY mixing time of 250 ms was used to provide cross peaks with high signal to noise while largely avoiding spin diffusion.
  • TOC SY spectra were recorded with the same spectral width and resolution as the NOESY with a homonuclear Hartman-Hahn transfer using the MLEV17 sequence for an 80 ms mixing time (6). Two power levels were used for excitation (3 dB) and spinlock (12.2 dB). Water suppression was achieved as with the NOESY.
  • CD analysis was performed on an Applied Photophysics Chirascan Circular Dichroism spectrometer. Samples were dissolved in D 2 O at 0.125 w/w% and data acquired in triplicate at room temperature and subsequently averaged and smoothed using built in qCD software. Graphs were plotted by subtracting a blank D 2 O spectrum from the acquired data to provided blank correction.
  • Peptides were synthesized utilizing Fmoc-protected amino acids or reagents and microwave assisted solid phase peptide synthesis techniques.
  • the Fmoc-amino acid or reagent to be coupled (0.5 mmol, 0.2 M in DMF), N,N-Diisopropylcarbodiimide (DIC) (0.5 mmol, 0.5 M in DMF), Oxyma ((ethyl (E)-2-cyano-2-(hydroxyimino)acetate) (0.5 mmol, 1 M in DMF) and Fmoc- Rink Amide MBHA resin (ChemMatrix®) (0.1 mmol, preswelled in DMF) were added to a reaction vessel and heated in a microwave until the reaction was complete.
  • DIC N,N-Diisopropylcarbodiimide
  • Oxyma ((ethyl (E)-2-cyano-2-(hydroxyimino)acetate)
  • Fmoc- Rink Amide MBHA resin (ChemMatrix®) (0.1 mmol, preswelled in DMF)
  • reaction time and temperature depend on the particular Fmoc amino acid or reagent to be coupled.
  • the coupling of a hydrophobic amino acids or handles require at least 300 s at a reaction temperature of 90 °C to complete the coupling reaction.
  • the coupling of Fmoc-protected histidine requires at least 600 s at a reaction temperature of 50 °C to complete the coupling reaction.
  • Deprotection i.e. removal of the Fmoc protecting group
  • Deprotection occurs via reacting the resin containing the peptide with piperidine (20 v/v% in DMF) for at least 90 s at a reaction temperature of 90 °C.
  • the amount of resin utilized in the coupling and deprotection reactions determines the ultimate scale of the reaction and all other reagents are scaled according to the quantity of resin.
  • the excess quantity of reagent can range from a 3 -fold excess to a six -fold excess depending on the scale of the reaction.
  • Resins were transferred to a SPE tube, washed with DCM (4x), and subsequently dried by gentle vacuum via a vacuum manifold. Although it is preferred to conduct the ring closing metathesis while the peptide remains anchored to the resin, it is also possible to perform the ring closing metathesis following cleavage of the peptide from the resin.
  • a cleavage solution was freshly prepared containing TFA (Trifluoroacetic acid)/T!S (Triisopropylsilane)/H 2 O/DODT (3,6-dioxa-l,8- octanedithiol) (92.5/2.5/2.5/2.5 v/v%) at 5 mL per 250 mg of dried resin, but may be adjusted depending on the resin.
  • the cleavage solution and resin mixture were heated 40°C using a hot plate and mantel and stirred for a minimum of 30 minutes.
  • the mixture was then added to an SPE column and the filtrate added drop wise to a 50 mL falcon tube containing Et2O (3 x 35 mL, per 0.1 mmol synthesis).
  • Et2O 3 x 35 mL, per 0.1 mmol synthesis.
  • the crude peptide participate was collected by centrifugation (5 min, 1000 xg).
  • the supernatant was aspirated from the peptide pellet, MeCN (1 mL per 0.1 mmol crude peptide) added, and then diluted with H 2 O (5 mL).
  • the crude solution was transferred to a glass vial for subsequent lyophilization.
  • the mixture can be partitioned over water and the cleaved peptide can be extracted with subsequent washing of the organic layer with water (3x).
  • the aqueous layers can be combined and transferred to a vial for lyophilization.
  • the crude peptide is frozen on dry ice for a minimum of 1 hour. Vials are then transferred to a PTFE coated freeze dryer with a -105 °C condenser and primary lyophilization performed (72 hours 0.05 mbar).
  • the incorporation of a handle onto the N terminus of the peptide involves an additional coupling step. If the handle is contained on a carboxylic acid, the additional coupling step follows the procedure of Example 1 to couple the handle onto the N terminus of the peptide. Referring to Fig. 3 A, S-Trityl-3-mercaptopropionic acid can be coupled to the N terminus of a peptide utilizing the procedures and conditions of Example 1.
  • Coupling of the handle may occur prior to or after ring closing metathesis to form the cross link or bridge between amino acids. If the coupling of the handle to the peptide occurs after ring closing metathesis, the N terminus must remain protected with an Fmoc during the ring closing metathesis.
  • a handle can also be incorporated via nucleophilic addition to the nucleophilic N terminus.
  • 4-bromo-l -butyne (0.5 mmol) and trimethylamine (0.5 mmol) was added to a DMF solution of resin containing deprotected peptide (0.1 mmol).
  • the reaction was bubbled under nitrogen for 20 minutes between 25°C and 90°C and heated via microwave irradiation. After the modification the reaction mixture was washed with DMF (3x) and cleaved from the resin.
  • the reaction was monitored by performing a “mini cleavage” in which a small aliquot of resin was removed and the peptide cleaved, the crude sample was analyzed by LCMS to ascertain the molecular weight of the species present. If the LCMS results indicated that the reaction was incomplete, the coupling reaction was repeated and reanalyzed until the LCMS results indicated that the reaction was complete.
  • the nucleophilic addition can also occur using an N-hydroxysuccinimide (NHS) activated ester.
  • NHS N-hydroxysuccinimide
  • the amine component (1 equivalent) is stirred with the desired NHS ester (1.5 equivalents) in the presence of trimethylamine (2 equivalents) in DMF solvent at 30 °C for 24 hours.
  • the reaction was bubbled under nitrogen for 20 minutes between 25°C and 90°C and heated via microwave irradiation. After the modification the reaction mixture was washed with DMF (3x) and cleaved from the resin.
  • the reaction was monitored by performing a “mini cleavage” in which a small aliquot of resin was removed and the peptide cleaved, the crude sample was analyzed by LCMS to ascertain the molecular weight of the species present. If the LCMS results indicated that the reaction was incomplete, the coupling reaction was repeated and reanalyzed until the LCMS results indicated that the reaction was complete.
  • Incorporation of a handle at the C terminus of the peptide involves coupling an amino acid or another linker comprising a protected orthogonal group into the peptide chain using the procedure of Example 1.
  • the Mtt protecting group is removed via suspension of the resin containing the peptide in trifluoroacetic acid/triisopropylsilane/dichloromethane (1 :2:97 v:v:v) and gently shaken for 30 minutes after which the resin is filtered, washed twice with dichloromethane, twice with methanol, twice again with di chloromethane, twice with 1% diisopropylamine in DMF, and twice with DMF to obtain deprotected lysine to which S-Trityl-3-mercaptopropionic acid can be coupled to the exposed amine group of the deprotected lysine using the procedures of Example 1. Because removal of the Mtt group occurs under acidic conditions similar to those conditions used for cleaving the peptide as described in Example 3, the resin used is for solid phase synthesis is HMBA.
  • Fmoc-Rink Amide MBHA resin or PEG-HMBA resin (ChemMatrix®) is swelled in DMF for 30 mins prior to synthesis of the desired PMO.
  • a linker Prior to coupling the first PMO, a linker must be coupled to the resin.
  • the linker is typically an amino acid or Fmoc-PEG( n )-CO 2 H and coupling of the linker to the resin follows the procedure of Example 1.
  • PMO monomer 0.2 M in DMF
  • NEM 0.2 M in DMF
  • ETT 0.2 M in DMF
  • Fmoc-Rink Amide MBHA resin or PEG-HMBA resin (ChemMatrix®) is swelled in DMF for 30 mins prior to synthesis of the desired PMO.
  • a linker Prior to coupling the first PMO, a linker must be coupled to the resin.
  • the linker is typically an amino acid or Fmoc-PEG( n )-CO 2 H and coupling of the linker to the resin follows the procedure of Example 1.
  • PMO monomer 0.2 M in DMF
  • NEM 0.2 M in DMF
  • ETT 0.2 M in DMF
  • a handle can also be incorporated via nucleophilic addition to the 3’ end of the PMO.
  • 4-bromo-l -butyne (0.5 mmol) and trimethylamine (0.5 mmol) was added to aDMF solution (4 mL) of resin containing PMO (0. 1 mmol).
  • the reaction was bubbled under nitrogen for 20 minutes at a temperature range from room temperature to 90 °C and heated via microwave irradiation.
  • the reaction was monitored by performing a “mini cleavage” in which a small aliquot of resin was removed and the PMO cleaved.
  • the crude sample was measured by LCMS and the 3’ modification reaction repeated to achieve a higher completion. Following completion of the reaction, the reaction mixture was then washed with DMF (3x) and cleaved from the resin.
  • Example 10 Incorporation of handle at the 5 ’ end of the PMO or internally within the PMO chain [00415] Incorporation of a handle at the 5’ end of the PMO chain requires the addition of a residue that can be modified orthogonally to contain the functionality of the handle.
  • Fmoc-Lys-(Mtt)-OH was coupled to the resin according to the procedure of Example 1.
  • the Mtt protecting group is removed via suspension of the resin containing the peptide in trifluoroacetic acid/triisopropylsilane/dichloromethane (1 :2:97 v:v:v) and gently shaken for 30 minutes after which the resin is filtered, washed twice with dichloromethane, twice with methanol, twice again with dichloromethane, twice with 1% diisopropylamine in DMF, and twice with DMF to obtain deprotected lysine.
  • S-trityl-3-mercaptopropionic acid (0.05 mmol) was coupled to the exposed amine group according to the procedure of Example 1.
  • the thiol-ene Michael addition can also undergo reduction utilizing resin-bound tris(2- carboxyethyl) phosphine (TCEP) to reduce disulfide reactants.
  • TCEP resin-bound tris(2- carboxyethyl) phosphine
  • 10 equivalents of TCEP are added to a PBS solution of the disulfide-containing product in water with EDTA (2 mL) under stirring at room temperature. The solution is stirred at room temperature for approximately 10 minutes to complete the reaction.
  • the now reduced thiol compound is eluted from the resin, by transferring the reaction mixture to an empty SPE tube and washing the resin with water (3x). The eluate is collect directly into the reaction vessel containing the prepared Michael acceptor.
  • the Michael addition is monitored by LCMS and upon completion the desired product in isolated by preparative RP-HPLC.
  • the CuSO 4 solution is incubated with the THPTA solution prior to carrying out the cycloaddition reaction at a 1 :2 ratio of CuSOr solution to THPTA solution.
  • the solution can remain stable for several weeks in a frozen state.
  • the CPP having the sequence RKF-S5-RLF-S5 is synthesized via solid phase synthesis according to the procedures of Example 1 using Fmoc protected amino acids. Following the coupling of the terminal S5 residue, the peptide undergoes ring closing metathesis to crosslink the two S5 residues according to the procedures of Example 2. The Fmoc protecting group at the N terminus is subsequently removed, exposing a terminal amine and providing the CPP.
  • Fmoc-NH-PEG 5 -CO 2 H is coupled to the CPP via the procedures of Example 1 and the Fmoc group is removed.
  • a first PMO nucleotide monomer (C) is then directly reacted with the now exposed nucleophilic N terminus of the peptide via a nucleophilic substitution reaction as shown in Figure 5.
  • the Fmoc protecting group is removed from the 3’ secondary amine of the nucleotide and the PMO having the sequence is synthesized stepwise via the procedures of Example 7.
  • the compound can be recovered through cleavage from the Rink amide resin using the procedures of Example 3. If HMBA resin is used, cleavage is performed by exposure to nucleophile such as alkoxide, thiolate, amines or hydroxides.
  • a general procedure for cleavage is as follows. To the dried resin, in a round bottom flask, a cleavage mixture comprising a nucleophile was prepared in an appropriate solvent i.e. methoxide in methanol, hydroxide in water. The nucleophile solution is typically used at 5 mL per 250 mg of dried resin and contains lOOx excess of the nucleophile to the resin, but may be adjusted depending on the resin and nucleophile. The cleavage solution and resin mixture were stirred for a minimum of 15 mins (sequence specific cleavage times apply). The mixture was then added to an SPE column and the filtrate eluted using MeCN in H 2 O (25 v/v% MeCN) and collected. The crude solution was frozen and lyophilized before isolation of the desired compound by RP-HPLC.
  • an appropriate solvent i.e. methoxide in methanol, hydroxide in water.
  • the nucleophile solution is typically used at 5 mL per
  • amino acid “S5” is (S)-pentenylalanine and “S8” is S- octenylalanine.
  • the abbreviation 3TPA is 3-(tritylthio)propionic acid, which is substituted for the bifunctional linker in constructing the molecule.
  • Peptides were investigated as either stabilized species, in which there is a staple between the S5 amino acid residues, or unstabilized and thus, unstapled species. Stapling in stabilized peptides was accomplished via olefin metathesis with Grubbs’s catalyst. The crosslinked amino acid residues for a particular stapled peptide species are enclosed within the parentheses in Table 11.
  • the bifunctional linker utilized was succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (SMCC). The peptide, via a terminal cysteine residue, as part of the bifunctional linker, bonds via a thiol -ene coupling to the maleimide functional group of SMCC.
  • the biologically active oligonucleotide is a phosphordiamidate morpholino oligonucleotide (PMO) having the nucleotide sequence of 5’ATATTGCTATTACCTTAACCCAGAA3’ (SEQ ID NO: 101) in which the 5’ end was modified to have a primary amine functionality, which permits nucleophilic coupling at the N- hydroxysuccinimide functionality of SMCC.
  • the PMO targets an enhanced green fluorescent protein (EGFP) sequence that is interrupted by the intron of human ⁇ -globin encoding the position 654 splicing mutation. Upon splicing correction with the PMO, functional EGFP is expressed.
  • the molecules comprising the peptide (both stabilized via stapling and unstabilized) and PMO conjugated via bifunctional linker were designated PEPTIGOs.
  • a HeLa cell line expressing a mutated human ⁇ -globin intron, IVS2-654 was maintained at below 80% confluence in D-MEM supplemented with 10% fetal calf serum. Cells were seeded 24 hours before treatment in 96-well plates at ⁇ 10 4 cells per well in 100 ⁇ L of vehicle. For the uptake experiments, cells were plated in 96-well plates at 1 x 10 4 cells per well in 100 ⁇ L of vehicle. The growth vehicle was removed and replaced with 100 ⁇ L of fresh growth vehicle containing either only fresh vehicle or varying concentrations (in ⁇ M) of: i) unconjugated PMO, ii) unstabilized (i.e.
  • the stapled PEPTIGOs comprising the peptides TAT8M 4, 8, TAT8M1 4, 8, and TAT13M 5, 9 exhibited high EGFP expression at low doses, approximately 50,000 in fluorescence intensity at 10 ⁇ M, and low cell toxicity. These results show that there is cellular uptake of the PEPTIGOs and retention of the biological activity of the PMO. With respect to the stapled PEPTIGO utilizing TAT8M 4,8, the data demonstrated that the stapled PEPTIGO exhibited higher fluorescence intensity than that of the unstabilized (i.e.
  • the PEPTIGO utilizing stapled TAT13M1 in which the position of the glutamine and arginine residues at positions 6 and 7, respectively, are swapped compared to the peptide TAT13M, surprisingly demonstrated higher EGFP expression via higher fluorescence intensity than that of the PEPTIGO utilizing TAT13M (approximately 260,000 in TAT13M versus about 450,000 in TAT13M1 at 40 ⁇ M) and low cell toxicity.
  • the unstabilized PEPTIGO unexpectedly exhibited higher EGFP expression than the stabilized stapled PEPTIGO analog, indicating that the unstabilized PEPTIGO exhibits higher biological activity.
  • the sequence of SV40 3,7 may not be amenable to stapling and the conferred conformation may be disfavored, which resulted in the observed decreased biological activity. Further analysis of an unstabilized isomer of SV40 3,7 where the positions of the unnatural amino acids were shifted back one residue towards the C terminus (i.e.
  • SV40 2,6 indicated that despite the superior performance of the unstabilized PEPTIGO utilizing SV40 3,7 over the stapled, stabilized analog, shifting the positions of the unnatural amino acids resulted in a decrease in EGFP expression by more than half (about 400,000 in SV40 3,7 versus about 175,000 in SV40 2,6) despite SV40 2,6 exhibiting an improvement in cell viability.
  • the unifying factor appeared to be the specific sequence of the peptide in combination with stabilization via hydrocarbon crosslinking and the specific location of the crosslinking (i.e. positions of unnatural amino acids forming the crosslink) that contributed to the improvement in biological activity and cell viability.
  • the observed biological activity is sequence specific and cannot be predicted based solely on patterns of amino acid residues or a core amino acid structure. It is the specific combination of the particular amino acid sequence, the presence of stabilization via hydrocarbon crosslink, and the specific positions of the unnatural amino acids partaking in crosslinking that contributed to the observed biological activity of the PEPTIGO.
  • Example 15 Method of administration in treating Duchenne muscu1 ⁇ r dystrophy
  • An exemplary pharmaceutical composition formulated for intravenous injection comprises 500 mg of the compound, 80 mg sodium chloride, 2 mg potassium chloride, 2 mg potassium phosphate monobasic, and 11.4 mg sodium phosphate dibasic in 10 mL of water at a pH of 7.5 (50 mg/mL concentration of the compound as the active ingredient).
  • the utilized dosage is 30 mg per kilogram of body mass.
  • Example 16 Effect of a stapled inter leukin- la (IL1 ⁇ ) NLS peptide and PMO conjugate on dystrophin expression in heart and skeletal muscle
  • a compound comprising a CPP designated “ARE8M” ((S5-FLR- S5)FKR-3TPA) (SEQ ID NO: 23) conjugated to a splice-modifying PMO that was subsequently administered intravenously into D2-mdx mouse model of DMD, resulted in increased dystrophin expression in the heart and diaphragm compared to administration of PMO alone.
  • significant acute adverse effects upon intravenous administration were observed at dose levels required for this effect.
  • NLS Nuclear localization signals
  • Classical NLS sequences contain a cluster of basic amino acids (e.g. arginine or lysine) which are believed to be important for imparting cell-penetrating properties onto a molecule. The combination of these factors makes peptides based on a classical NLS candidates as peptide domains for the present compounds.
  • IL 1 ⁇ interleukin- 1 ⁇
  • the NLS is from pro-IL1 ⁇ , and cleaved upon activation of IL1 ⁇ .
  • Pro-IL1 ⁇ is a human NLS that has been shown to facilitate nuclear translocation in human cells and is not viral.
  • IL1 ⁇ NLS peptides including (S)-pentenylalanine (S5) monomers were synthesized and stabilized by olefin ring-closing metathesis by Peptide Protein Research Ltd (Hampshire, UK).
  • Bracketed numbers indicate the position of the S5 monomers relative to the (PEG)5 moiety.
  • S5 pentenylalanine
  • PEG polyethylene glycol.
  • PMO-FITC carboxyfluorescein
  • Conjugates utilizing an IL1 ⁇ NLS-based peptide as the CPP were produced by conjugating IL1 ⁇ NLS peptide to PMO-FITC via a SMCC linker to produce IL1 ⁇ NLS-PEG5-C- SMCC-PMO-FITC using methods described below.
  • PMO-FITC (280 mg, 30 ⁇ mol) was dissolved in PBS (9 mL, pH 7.4) and MeCN added (1 mL). This solution was incubated at room temperature after the addition of SMCC linker (100 mg, 229 ⁇ M, 10x excess) directly to the reaction vessel. After 6 hours the mixture was frozen and freeze-dried. The lyophilized powder was then reconstituted in 20% MeCN (1 mL) and desalted using sephadex g25 (medium) hydrated in H 2 O. SMCC-modified PMO-FITC was eluted and then freeze-dried to yield SMCC-PMO-FITC (201 mg, 21.6 ⁇ mol). Samples of SMCC-PMO-FITC were sent for mass spectrometry analysis. The initial purity of the SMCC-PMO-FITC was typically 70- 80% by HPLC analysis.
  • SMCC-PMO-FITC (55 mg, 5.8 ⁇ mol) was dissolved in conjugation buffer (3 mL, PBS pH 6.5) and MeCN was added (2 mL). Peptide (12 mg, 7.7 ⁇ mol) andEDTA solution (0.05 mL,100 mM) were mixed with immobilized TCEP (3 mL) for 1 h prior. The final concentration of EDTA was 2 mM. The reduced peptide was eluted from the immobilized TCEP into a glass vial containing the SMCC-modified PMO-FITC and stirred at room temperature for 2.5 hours after the last addition of peptide.
  • the reaction was diluted with H 2 O and batch-loaded onto an HPLC prep column (Waters). Using a 20 mL syringe, sample was batch-loaded in 4 mL aliquots onto the prep column using a 15% isocratic organic at 45 mL/min. The injection loop was washed with 15% MeCN solution prior to loading samples. The HPLC system was equilibrated for a minimum of 20 mins at 15 % MeCN, 1 mL/min.
  • D2-mdx Female D2.B 10-Dmd mdx /J (The Jackson Laboratory; strain: 013141; referred to here as D2-mdx) were used for this study. This line carries a nonsense mutation in exon 23 of the dystrophin gene which abrogates dystrophin expression. Removal of exon 23 from the dystrophin transcript restores the expression of a slightly truncated but functional dystrophin protein. These mice show muscle atrophy and increased fibrosis compared to wild-type mice (Coley et al., 2016), pathological features which are also observed in DMD patients.
  • the dose used for the repeated administrations was 2.0 ⁇ mol/kg.
  • the dosing frequency was once every two days.
  • Equimolar doses of conjugate and unconjugated PMO were administered to compare efficacy of delivery at the same dose.
  • Tables 13 and 14 show an overview of the mice dosed in this study.
  • mice were warmed in heat box for 10 minutes to induce vasodilation. Compounds were administered into the tail vein using a 30G insulin syringe. The mice were monitored in a separate cage following dosing before being returned to their home cage. Body mass was monitored during the dosing phase as an indicator of animal health. Tissue was recovered 13 or 14 days after the final injection. Mice were euthanized by exposure to rising carbon dioxide levels. Tissue was removed and snap-frozen in liquid nitrogen-cooled isopentane.
  • Dystrophin protein was detected in sections of heart muscle and gastrocnemius muscle by immunohistochemistry. Tissue sections (10 pm thickness) were cut on a microtome at -20°C, mounted on positively charged glass slides (SuperFrost Plus, VWR), air-dried and stored at -80°C until use. Sections from 6 different positions along the length of the tissue were mounted on the same slide.
  • Sections were washed in PBS-T again (3x3 minutes) before incubation with ABC reagent (Vector Laboratories) for 10 minutes. Horse-radish peroxidase- labelled antibodies were then detected by incubation of sections with DAB substrate solution (Vector Laboratories). All sections were incubated with DAB substrate solution for the same duration. Stained sections were washed in tap water for 5 minutes, dehydrated in 100% ethanol for 30 seconds, cleared in xylene for 10 minutes and permanently mounted in DPX, covered by a coverslip.
  • IL1 ⁇ NLS (1,5)- and IL1 ⁇ NLS (2,6)-PMO conjugates increased the number of fibers displaying membrane dystrophin expression in gastrocnemius muscle, but not heart muscle, compared to treatment with PMO-FITC and vehicle (Figs. 16A & 16B, Figs. 17A & 17B).
  • the average number of dystrophin-positive cells is slightly higher in heart sections of IL1 ⁇ NLS (1,5)-PM0 conjugate-treated mice compared to the PMO-FITC and vehicle controls (Figs. 16A & 16B), but as the percentage of dystrophin-positive cells to the total number heart cells is very low this difference is likely biologically negligible.
  • Intravenous IL1 ⁇ NLS (1,5)-PEG5-SMCC-PMO-FITC and IL1 ⁇ NLS (2,6)-PEG5- SMCC-PMO-FITC increased dystrophin expression in skeletal muscle, but not heart muscle of D2-mdx mice, compared to PMO-FITC.
  • Acute tolerance to intravenous dosing of IL1 ⁇ NLS (1,5)-PEG5-SMCC-PMO-FITC and IL1 ⁇ NLS (2,6)-PEG5-SMCC-PMO-FITC was improved compared to tolerance to of ARE8M-PEG5-SMCC-PMO-FITC at the same dose in the same animal model.
  • peptide-oligonucleotide conjugates comprising peptides based on the ETS-related transcription factor Elf-3 (ESE-1) nuclear localization signal (NLS).
  • ESE-1 NLS peptide variants which have been stabilized by olefin ring-closing metathesis between two (S)-pentenylalanine (S5) monomers placed in different positions along the length of the ESE- 1 NLS peptide (see Table 15 for amino acid sequences).
  • S5 olefin ring-closing metathesis between two (S)-pentenylalanine (S5) monomers placed in different positions along the length of the ESE- 1 NLS peptide (see Table 15 for amino acid sequences).
  • the biologically active oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO) having the nucleotide sequence of 5’ATATTGCTATTACCTTAACCCAGAA3’ (SEQ ID NO: 101) in which the 5’ end was modified to have a primary amine functionality, which permits nucleophilic coupling at the N- hydroxysuccinimide functionality of SMCC.
  • the PMO targets an enhanced green fluorescent protein (eGFP) sequence that is interrupted by the intron of human ⁇ -globin encoding the position 654 splicing mutation. Upon splicing correction with the PMO, functional eGFP is expressed.
  • eGFP expression is an indicator of the successful delivery of the PMO to the cell nucleus, and an improvement in eGFP expression is evidence of improved delivery to the nucleus.
  • 3TPA 3-(tritylthio)propionic acid
  • S5 (S)-pentenylalanine
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate
  • STP Stapled. Numbers in brackets indicate the position of the S5 monomers in the peptide.
  • EGFP654 PMO having the sequence 5’ATATTGCTATTACCTTAACCCAGAA3’ (SEQ ID NO: 101), was synthesized and supplied in lyophilized form by Gene Tools, LLC (Oregon, USA) with a 5’ NEL modification to allow conjugation to the peptides. A single batch was used for all conjugations and PMO-only controls in experiments.
  • Stapling in stabilized peptides was accomplished via olefin metathesis with Grubbs’s catalyst.
  • the crosslinked amino acid residues for a particular stapled peptide species are enclosed within the hyphens in Table 15, supra.
  • the bifunctional linker utilized was succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC).
  • SMCC succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate
  • HeLaEGFP654 expressing an eGFP gene containing the mutated human ⁇ -globin intron (IVS2-654) as described above, was maintained at below 80% confluence in D-MEM supplemented with 10% fetal calf serum. Cells were seeded 24 hours before treatment in 96-well plates at ⁇ 2 x 10 4 cells per well in 100 ⁇ L of vehicle.
  • the growth vehicle was removed and replaced with 100 ⁇ L of fresh growth vehicle containing either only fresh vehicle or varying concentrations (in ⁇ M) of: i) unconjugated PMO; ii) peptide-PMO conjugate comprising native ESE-1 NLS peptide; and iii) peptide-PMO conjugate comprising stabilized (i.e. stapled) ESE-1 NLS peptide.
  • Cells were assayed 24 hours later.
  • the treatment media was removed (used for the lactate dehydrogenase assay described below to measure cell toxicity) and replaced with HBSS and fluorescence measured using a fluorescence plate reader (Spectramax iD3) with monochromator set to 475/515 nm (ex/em).
  • EGFP fluorescence in live cells kept in HBSS was measured on a plate reader after 24 hours of incubation with the compounds at concentrations between 0.625 ⁇ M and 40 ⁇ M. Background fluorescence was measured in wells containing HBSS only and subtracted from fluorescence measurements in well containing cells.
  • eGFP expression was increased in HelaEGFP654 cells treated with peptide-PMO conjugate comprising the unmodified ESE-1 NLS peptide compared to vehicle- and unconjugated PMO-treated cells (Fig. 19).
  • eGFP expression was further increased in cells treated with conjugates comprising the stapled ESE-1 NLS peptide variants, in particular, at treatment concentrations of 10, 20 and 40 ⁇ M, and the extent of this increase depended on the position of the staple within the ESE-1 NLS peptide.
  • the present study utilizes the gapmer having the sequence 5’ AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108), which the ability to knockdown the pre- mRNA and mRNA of argonaute 2 (AGO2), whose knockdown induces apoptosis in cancer cells.
  • This gapmer is commercially available and can be procured with modifications to facilitate conjugation with a bifunctional linker such as SMCC.
  • the gapmer is modified with a C12 5’ amino modifier such that the 5'end of the gapmer bears a reactive amine group.
  • Fmoc amino acids and reagents are dissolved in DMF to give a 0.2 M amino acid solution, 1.0 M Oxyma solution, 0.5 M DIC solution and 20% piperidine in DMF.
  • the Fmoc amino acids are mixed with DIC/Oxyma and coupled in DMF. Coupling conditions are performed according to CEM manual recommendations: Amino acids A, V, L, I, F and S5 are coupled for 300 s at 90°C. All other amino acids coupled for 90 s at 90 °C with the exception of R which is double coupled at 75 °C for 300 seconds (i.e., 600 seconds in total).
  • Grubb’s catalyst (20 mol%, 6 mM, 4 mL, in DCE) is prepared immediately before utilization in the reaction. 4 mL of the Grubb’ s catalyst solution is transferred to the reaction vessel. The ring closing metathesis reaction proceeds at 25 °C for 30 min with nitrogen gas bubbling for 2 seconds and 5 seconds off. Nitrogen is bubbled throughout the RCM reaction to promote the removal of ethene and drive the formation of the peptide macrocycle. The reaction mixture is drained and a second aliquot of freshly prepared Grubb’s catalyst (4 mL, 6 mM, in DCE) is added and further reacted for 30 mins at 25 °C. The mixture is drained and transferred to a solid phase extraction tube, washed with DCM, and dried for continuation to the cleavage step.
  • the dried resin (100 ⁇ mol) is transferred to a 25 mL round bottom flask and a stirrer bar is added. A round bottom heating mantel is preheated to 40 °C. Addition of cleavage cocktail (6 mL, 92.5% TFA, 2.5% H2O, 2.5% TIS, 2.5% DODT) to the crude resin. The resin slurry is stirred at 40 °C for approximately 30-45 minutes depending on the sequence (higher arginine containing peptides require longer reaction times). This slurry is then transferred to a SPE tube and filtered into 50 mL falcon tubes containing approximately 35 mL of diethyl ether. The resin is washed a further 3 times with approximately 2 mL of cleavage cocktail. The ether slurry is centrifuged for 5 minutes at 1000 xg.
  • the etherial supernatant is decanted, and the crude pellet is isolated.
  • the pellet is dissolved using acetonitrile and water and combined in a 28 mL screw top vial and frozen on dry ice for a minimum of 30 minutes.
  • the screw cap is replaced with a porous membrane, which is fitted in place with rubber bands, and the vial is placed back on dry ice for approximately 5 to 10 minutes.
  • the vials are freeze-dried for a minimum of 72 hours at 0.01 mbar vacuum and -105°C collector temperature.
  • a scout LCMS analysis is run.
  • a sample of the crude lyophilized peptide is removed and diluted (H 2 O/MeCN 10%, 0.1 mL final volume) and 50 ⁇ L loaded on to a Xbridge BEH C18 4.6 x 150 mm column 186003580.
  • a gradient of 15% - 80% over 12 mins in H 2 O/MeCN +0.1% TFA is run.
  • a single wavelength of 220 nm is used and QDa is set up in positive mode with 1.5 kV capillary and 15 V cone voltage and a H 2 O/MeOH 50% + 0.01% formic acid.
  • Buffer A is H 2 O +0.1% TFA and buffer B is MeCN +0.1% TFA.
  • the crude sample is reconstituted in 2 ml of 10% MeCN in water and loaded onto the Xbridge BEH C18 5pm 30 ⁇ 150 mm column.
  • Fractions are identified through mass directed collection by ESI positive mode scans. Masses of the peptide are identified during the scout LCMS run and are manually inputted into a mass collection method used for purification. The chromatogram is monitored using the ESI+ spectrum in which compounds above EIC threshold of 1x10 6 are collected in 28 mL screw top vials and frozen on dry ice for freeze-drying. Mixed fractions or single compounds below 5x10 5 ESI threshold are discarded. Following from this, fractions collected were freeze-dried for a minimum of 72 hours.
  • QDa is set up in positive mode with 1.5 kV capillary and 15 V cone voltage and a H 2 O/MeOH 50% + 0.01% Formic acid.
  • the primary freeze drying was performed for 72 hours to obtain crude lyophilized peptide.
  • the bifunctional linker used to conjugate the 5 ’-amino modified gapmer to the peptide is SMCC.
  • Gapmer is dissolved in PBS (pH 7.2) and is incubated at room temperature after the addition of SMCC (5-fold excess relative to gapmer) dissolved in MeCN/H 2 O (1 : 1 , 1 mL). After 30 mins the mixture is desalted using sephadex g25 hydrated in conjugation buffer (PBS lx, pH 6.8) and is also used as the eluent. The final concentration of EDA is 2 mM.
  • the peptide is dissolved in milliQ water (4 mLO and EDTA solution (0.1 mL, 100 mM) and is mixed with TCEP (2.5 mL) for 1 h prior.
  • the final concentration of EDTA is 2 mM.
  • MeCN 8 mL
  • EDTA solution 0.1 mL, 100 mM
  • the reduced peptide is eluted from the immobilized TCEP into a tube containing the SMCC modified gapmer and is stirred at room temperature for 2 hours.
  • gapmer-peptide conjugates first involves investigation of the retention of the activity of the gapmer. Following confirmation that the gapmer retains its activity, gapmer-peptide conjugate and unconjugated gapmer are incubated with cells in the absence of a transfection agent to assess the ability of conjugated stapled peptides to improve cell/nuclear entry of the gapmer.
  • cytoplasmic and nuclear fractions of the HeLa cells are prepared.
  • a nuclear protein kit QIAGEN or a PARIS kit supplemented with RNase inhibitor is used to prepare the cytoplasmic and nuclear fractions from approximately 5 * 10 7 HeLa cells according to the kit instructions.
  • RNA preparation for qRT-PCR analysis utilizing an RNeasy (QIAGEN) kit according to the kit instructions.
  • qRT-PCR is performed using TaqMan primer probe sets with the StepOne real-time PCR system. Reverse transcription is performed at 48 °C for 30 mins, and 40 cycles of PCR reaction are carried out at 94 °C for 15 seconds and 52 °C for 15 seconds within each cycle.
  • mRNA levels in cytoplasmic or nuclear fractions are normalized to the levels of cytoplasmic 7SL RNA or nuclear MALAT1 RNAs, respectively, or to the levels of cytoplasmic 7SL RNA or the nuclear U16 RNA, respectively using a Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific).
  • Cytosolic and nuclear fractions are separated using a Cell Fractionation Kit- Standard (Abeam) according to the kit instructions. Cytosolic and nuclear proteins are dissolved in an equal volume of lysis buffer, and an equal volume of proteins from each fraction are analyzed by SDS- PAGE and western blot analyses.
  • cytoplasmic and nuclear fractions are separated in 4-12% gradient SDS-PAGE gels. Proteins are transferred to PVDF membrane using a semi-dry transfer apparatus. The membranes are blocked for 1 hr with block buffer (5% dry milk in 1 xTBS), and incubated with primary antibodies against AG02 (ab32381, 1 :1000) at 4 °C for overnight. After 3 washes with wash buffer (1 xTBS, 0.1 % Tween-20), membranes are incubated with anti-mouse or anti-rabbit secondary antibody in block buffer at room temperature for 1 hr. After 3 washes, proteins are detected using ECL (Abeam).
  • the gapmer utilized in the study is shown to knockdown both pre-mRNA and mRNA in the nucleus and cytoplasm, respectively via the recruitment of RNAse Hl.
  • qRT-PCR analysis shows a decrease in the level of the target RNA following transfection with the gapmer.
  • Western blot analysis of the cytoplasmic and nuclear fractions also shows a decrease in the level of the protein AGO2, correlating with the decrease in the level of the target RNA.
  • Control samples, which are not transfected with gapmer exhibit higher levels of target RNA and AGO2.
  • HeLa cells are exposed to equimolar amounts of gapmer-peptide conjugates, unconjugated gapmer, and growth vehicle as a control sample. Analysis and determination of RNA knockdown is performed as described above utilizing real time PCR.
  • the gapmer-peptide conjugates having the conjugated cell penetrating peptide exhibit improved cellular and nuclear entry compared to unconjugated gapmer alone.
  • the gapmer-peptide conjugate bearing a cell penetrating peptide exhibits improved cellular uptake compared to that of the unconjugated gapmer.
  • the gapmer cargo in retaining its biological activity following delivery into the cell, is able to recruit RNAse Hl to knockdown the pre-mRNA and mRNA encoding the protein AG02.
  • a method for synthesizing a compound comprising: i. an oligonucleotide moiety, covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to, ii.
  • BFL bifunctional linker moiety
  • the StaP or StiP when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix; wherein synthesis of the compound comprises the steps of:
  • step (vi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), before or after step (iii), before or after step (iv), or before or after step (v);
  • step (xi) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs before or after step (vii), before or after step (viii), before or after step (ix), or after step (x); and
  • the BFL if present, comprises one or more handles.
  • oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • SMCC succinimidyl 4-(N-maleimidomethyl) R cyclohexane- 1 -carboxylate, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer; or,
  • AAS N-a-maleimidoacet-oxysuccinimide ester, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer; or,
  • BMPS N- ⁇ -maleimidopropyl-oxysuccinimide ester
  • Z Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer
  • GMBS N-Y-aleimidobutyryl-oxysuccinimide ester, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer; or,
  • DMVS N- ⁇ -maleimidovaleryl-oxysuccinimide ester
  • n is a positive integer
  • EMCS EMCS
  • Y N-terminus of the StaP or the StiP, or where n is a positive integer;
  • LC-SMCC a residue of succinimidyl 4-(N-maleimidomethyl) cyclohexane- l-carboxy-(6-amidocaproate), where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer; or, a residue of succinimidyl 4-(N- maleimidom ethyl) cyclohexane- 1 -carboxylate (polyethylene glycol) n , wherein n equals 1 to 10, Z and, Y is either present or not present, and when Y is present, Y is where n is a positive integer, and when Y is not present, Y is a covalent bond to the N-terminus of the StaP or the StiP; or, (DSG), a residue of disuccinimidyl gluterate, where Z is not present, and instead there is a covalent bond to the N-termin
  • step (v) comprises forming an olefin cross link between at least two unnatural amino acids.
  • S5 is (S)-pentenylalanine
  • R5 is (R)-pentenylalanine
  • S8 is (S)-octenylalanine
  • R8 is (R)- octenylalanine
  • B5 is ⁇ , ⁇ -di- substituted pentenylalanine
  • B8 is ⁇ , ⁇ -di-substituted octenylalanine
  • S-OAS and R-OAS are O-allylserine analogues.
  • step (v) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
  • a method for synthesizing a compound comprising: i. an oligonucleotide moiety, covalently linked to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can
  • step (v) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), or before step (iii);
  • step (x) forming the cross link or the bridge between the at least two amino acids to obtain the compound; (xi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs after step (vi), before or after step (vii), before or after step (viii), before or after step (ix), or before or after step (x); and
  • oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
  • linker of step (i) comprises an amino acid monomer or a handle configured to couple with an oligonucleotide monomer.
  • (x) comprises forming an olefin cross link between at least two unnatural amino acids.
  • S5 is (S)-pentenylalanine
  • R5 is (R)-pentenylalanine
  • S8 is (S)-octenylalanine
  • R8 is (R)- octenylalanine
  • B5 is ⁇ , ⁇ -di- substituted pentenylalanine
  • B8 is ⁇ , ⁇ -di-substituted octenylalanine
  • S-OAS and R-OAS are O-allylserine analogues.
  • (x) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
  • composition comprising the compound made according to the method of any one of paragraphs 1-18 and one or more pharmaceutically acceptable excipients.
  • composition comprising the compound made according to the method of claim any one of paragraphs 19-31 and one or more pharmaceutically acceptable excipients.
  • composition of paragraph 32 formulated for oral, parenteral, intravenous, or topical administration.
  • composition of paragraph 33 formulated for oral, parenteral, intravenous, or topical administration.
  • a method of administering the composition of any one of paragraphs 32 or 34 intravenously to a subject comprising the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
  • a method of administering the composition of any one of paragraphs 33 or 35 intravenously to a subject comprising the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
  • a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of any one of paragraphs 38-48.
  • [00577] 50 A method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of any one of paragraphs 45-48.
  • composition comprising the molecule of any one of paragraphs 38-48 and one or more pharmaceutically acceptable excipients.
  • composition of paragraph 53 formulated for oral, parenteral, intravenous, or topical administration.
  • a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of any one of paragraphs 55-66.
  • composition comprising the molecule of any one of paragraphs 55-66 and one or more pharmaceutically acceptable excipients.
  • composition of paragraph 71 formulated for oral, parenteral, intravenous, or topical administration.
  • a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of any one of paragraphs 73-84.
  • a method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of any one of paragraphs 81-84.
  • composition comprising the molecule of any one of paragraphs 73-84 and one or more pharmaceutically acceptable excipients.
  • Gautam, A. et al. CPPsite a curated database of cell penetrating peptides. Database : the journal of biological databases and curation 2012, bas015, doi:10.1093/database/bas015 (2012).
  • Pip6-PM0 A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment. Molecu1 ⁇ r therapy. Nucleic acids 1, e38, doi: 10.1038/mtna.2012.30 (2012). Ivanova, G. D. el al. Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res 36, 6418-6428 (2008).

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Abstract

The invention relates to improvements in drug delivery and to the use of compounds comprising cell penetrating peptides (CPPs) stabilized by: i) stapling two amino acids to form Stapled CPPs (StaPs) or ii) stitching three or more amino acids to form stitched CPPs (StiPs), and a drug or Biologically Active Compound (BAC) directly bonded to or connected to the CPP via bifunctional linker. This enables the BAC to be carried though a cell membrane by the CPP. The preferred BAC is an electrically low charge carrying oligonucleotide such as a phosphorodiamidate morpholino oligonucleotide (PMO). The invention also relates to a method of synthesizing and further functionalizing the compound utilizing solid phase peptide synthesis techniques. Also provided is a method of treating a subject comprising administering a pharmaceutical composition comprising the compound and one or more pharmaceutically acceptable excipients.

Description

BIOLOGICALLY ACTIVE COMPOUNDS
RELATED APPLICATION AND INCORPORATION BY REFERENCE
[0001] This application claims priority to United States Provisional Patent Application No. 63/507,771, filed June 13, 2023, and to United States Provisional Patent Application No. 63/394,807, filed August 3, 2022, the contents of which are herein incorporated by reference in their entirety.
[0002] All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 3, 2023, is named Y9899-99004_SequenceListing_080323 and is 143,360 bytes in size.
FIELD OF THE INVENTION
[0004] The present invention relates to improvements in drug delivery.
[0005] More particularly it relates to the use of Cell Penetrating Agents (CPAs), and more particularly still to the use of Cell Penetrating Peptides (CPPs) which have been stabilized by, for example: i) stapling two amino acids to form Stapled CPPs (StaPs) or ii) stitching three or more amino acids to form stitched CPPs (StiPs).
[0006] These stabilized CPPs are conjugated to a drug or Biologically Active Compound (BAC) directly or via a BiFunctional Linker (BFL) so that the BAC can be carried though a cell membrane by the CPP. The resulting compounds may be referred to as drug carrying cell penetrating molecules. [0007] The preferred BACs delivered in this manner are oligonucleotides (ONs), more preferably still electrically low charge carrying oligonucleotides (charge -3 to +3 at pH 7.5) and most preferably electrically neutral oligonucleotides (charge -1 to +1 at pH 7.5), such as, but not limited to, peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligonucleotides (PMOs) or modified derivatives thereof. Other BACs based on nucleic acids include, but are not limited mRNA, siRNA, miRNA, aptamers, and gapmers.
[0008] The preferred BFL may be PEGylated, comprising polyethylene glycol (PEG) groups including modifications such as an amine group, or incorporate a spacer, such as β-Ala. These modifications can improve solubilization or provide appropriate spacing between functional moieties.
[0009] The invention relates to a method of synthesizing the compounds. The CPP can be synthesized by stepwise solid phase synthesis, which permits control over the exact order of amino acid monomers within the CPP. The stepwise solid phase techniques can similarly be used to synthesize the ON with exact control over the sequence of the ON. The techniques for stepwise synthesis of the CPP and the ON are interchangeable and the compounds can be synthesized in a one pot method prior to cleavage from the resin and subsequent collection for further application, thus streamlining the overall synthetic process and permitting complete control over the sequencing of both the CPP and the ON. The stepwise solid phase synthetic techniques also permit the incorporation of handles comprising orthogonal reactive sites for further conjugation to occur on the compound, resulting in further functionalization of the compound.
[0010] The invention also relates to a method of facilitating the uptake of a BAC into a cell, the use of the compound in the treatment of a disease requiring alteration of an endogenous or exogenous gene, a method of improving the bioavailability of a drug or BAC, a method of introducing a drug or BAC to a site which is refractory to the drug or BAC in its native state, a method of treating a subject comprising administering the compounds of the invention and to a pharmaceutical composition comprising the compound and one or more pharmaceutically acceptable excipients.
[0011] Still further aspects will be apparent from the detailed description.
BACKGROUND OF THE INVENTION [0012] In the treatment of diseases, it is desirable to deliver a drug or BAC into the body, and more preferably into a cell, at a target site, in a manner that ensures a maximal effect with minimal toxicity. This can be challenging.
[0013] An example of drugs or BACs which are delivered in a targeted manner are oligonucleotides (ONs), which term includes various ON analogs, which include, but are not limited to antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligonucleotides (PMOs), aptamers, and gapmers.
[0014] ONs can target essential DNA, RNA and protein sequences and can modulate gene expression in a number of ways that includes steric blocking to suppress (i) RNA splicing, (ii) protein translation or (iii) other nucleic acidmucleic acid or nucleic acid:protein interactions.
[0015] Specifically, the hybridization of ONs to specific RNA sequence motifs prevents correct assembly of the spliceosome, so that it is unable to recognize the target exon(s) in the pre- mRNA and hence excludes these exons in the mature gene transcript. Exclusion of an in-frame exon can lead to a truncated yet functional gene product; exclusion of an out of frame exon results in a frame-shift of the transcript, potentially leading to a premature stop codon and a reduction in the target gene expression level.
[0016] It is desirable to facilitate the entry of BACs such as ONs into cells. This can be accomplished via the use of CPAs. Examples of CPAs include CPPs, which can interact with and cross the cell membrane to enter the cell. CPPs can interact with cell membranes to participate in direct penetration, an energy independent process.
[0017] CPPs can also enter the cell via endocytosis, in particular, pinocytosis. The BAC and CPP are encased in an endosome following cell entry via endocytosis. Thus, there is also the consideration of endosomal escape that must be considered such that the CPP carrying the BAC can breach the endosome to reach the intended target for delivery of the BAC.
[0018] For effective clinical translation of ONs such as steric blocking antisense oligonucleotides, CPPs need to effectively deliver the BAC to either the cytoplasm or nucleoplasm whilst limiting any toxicity associated with cell entry.
[0019] Another consideration is that the BAC must be able to retain its biological activity upon delivery into the cell via a CPP such that the delivered cargo can achieve its intended therapeutic effect. [0020] Thus, providing compounds that are able to deliver a drug or BAC more efficiently or to a target site with lower toxicity and immunogenicity in which the biological activity of the BAC is preserved or retained would be highly desirable.
[0021] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
SUMMARY OF THE INVENTION
[0022] In accordance with a first aspect of the present invention, there is provided a method for synthesizing a compound, the compound comprising: an oligonucleotide moiety, covalently linked directly or covalently linked via a bifimctional linker moiety (BFL) to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix, wherein synthesis of the compound comprises the steps of:
(i) coupling an amino acid via its carboxyl group to a solid support, wherein the amino acid comprises a protecting group at its amino group;
(ii) removing the protecting group to expose a free amino group;
(iii) coupling, by stepwise solid phase synthesis, a successive amino acid monomer wherein the successive amino acid monomer comprises a protecting group at its amino group;
(iv) repeating steps (ii) and (iii) to obtain a peptide chain;
(v) forming the cross link or the bridge between the at least two amino acids;
(vi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), before or after step (iii), before or after step (iv), or before or after step (v);
(vii) coupling a first oligonucleotide monomer at its 5’ end to the N terminus of the peptide chain or the at least one functional group of the handle, wherein the oligonucleotide comprises a protecting group at its 3’ end;
(viii) removing the protecting group at the 3’ end to expose a free amino group;
(ix) coupling, by stepwise solid phase synthesis, a successive oligonucleotide monomer wherein the successive oligonucleotide monomer comprises a protecting group at its 3’ end;
(x) repeating steps (viii) and (ix) to obtain an oligonucleotide chain and the compound;
(xi) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs before or after step (vii), before or after step (viii), before or after step (ix), or after step (x); and
(xii) cleaving the compound from the solid support; wherein the BFL, if present, comprises one or more handles.
[0023] In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[0024] In some embodiments, the PMO comprises 5GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98). [0025] In some embodiments, the protecting group of the amino acid monomer is Fmoc.
[0026] In some embodiments, the protecting group of the oligonucleotide monomer is Fmoc.
[0027] In some embodiments, the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000007_0001
[0028] In some embodiments, the protecting group of the oligonucleotide monomer is Trt.
[0029] In some embodiments, the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000007_0002
[0030] In some embodiments, the oligonucleotide is covalently linked to the StiP or the StAP. [0031] In some embodiments, the BFL comprising one or more handles is present in the compound, and the oligonucleotide is covalently linked via the BFL to the StaP or the StiP, whereby the BFL is covalently linked to the oligonucleotide, and the BFL is covalently linked to the StaP or the StiP.
[0032] In some embodiments, BFL comprises:
(SMCC), a residue of succinimidyl 4-(N-maleimidomethyl)
Figure imgf000008_0001
cyclohexane- 1 -carboxylate, where Z is
Figure imgf000008_0002
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000008_0003
or,
(AMAS), a residue of N-a-maleimidoacet-oxysuccinimide ester, where Z is
Figure imgf000008_0004
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000008_0005
or,
Figure imgf000009_0001
(BMPS), a residue of N- β-maleimidopropyl-oxysuccinimide ester, where Z is
Figure imgf000009_0002
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000009_0003
Figure imgf000009_0004
(GMBS), a residue of N-γ-aleimidobutyryl-oxysuccinimide ester, where Z is
Figure imgf000009_0005
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000009_0006
Figure imgf000010_0001
(DMVS), a residue of N-δ-maleimidovaleryl-oxysuccinimide ester, where
Z
Figure imgf000010_0002
is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000010_0003
(EMCS), a residue of N- ε-malemidocaproyl-oxysuccinimide ester, and Y is a covalent bond to the N-terminus of the StaP or the StiP, or where n is a positive integer;
Figure imgf000010_0004
or,
Figure imgf000011_0001
(LC-SMCC), a residue of succinimidyl 4-(N-maleimidomethyl)
Figure imgf000011_0002
cyclohexane- l-carboxy-(6-amidocaproate), where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000011_0003
or, a residue of succinimidyl 4-(N-
Figure imgf000011_0004
maleimidom ethyl) cyclohexane- 1 -carboxylate (polyethylene glycol)n, wherein n equals 1 to 10, Z is
Figure imgf000011_0005
and, Y is either present or not present, and when Y is present, Y is where n is a positive integer, and when Y is not present, Y is a covalent
Figure imgf000011_0006
bond to the N-terminus of the StaP or the StiP; or,
Figure imgf000012_0001
(DSG), a residue of disuccinimidyl gluterate, where Z is not present, and instead there is a covalent bond to the N-terminus of the StaP or the StiP, or to the N of wherein n is a positive integer;
Figure imgf000012_0002
or,
Figure imgf000012_0003
(DSCDS), a residue of disuccinimidyl-cyclohexl-1,4-diester, where Z is not present, and instead there is a covalent bond to the N-terminus of the StaP or the StiP, or to the N of
Figure imgf000012_0004
wherein n is a positive integer, wherein R is H or NH2.
[0033] In some embodiments, in each Y moiety, n is 5.
[0034] In some embodiments, R is H.
[0035] In some embodiments, R is NH2.
[0036] In some embodiments, the forming step (v) comprises forming an olefin cross link between at least two unnatural amino acids.
[0037] In some embodiments, the at least two unnatural amino acids are selected from the group consisting of:
Figure imgf000013_0001
wherein S5 is (S)-pentenylalanine, R5 is (R)-pentenylalanine, S8 is (S)-octenylalanine, R8 is (R)- octenylalanine, B5 is α, α-di- substituted pentenylalanine, B8 is α, α-di-substituted octenylalanine, and S-OAS and R-OAS are O-allylserine analogues.
[0038] In some embodiments, the forming step (v) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
[0039] In some embodiments, the lactam bridge is formed by cross linking a lysine and glutamic or aspartic acid residues.
[0040] In accordance with another aspect of the invention, there is provided a method for synthesizing a compound, the compound comprising: i. an oligonucleotide moiety, covalently linked to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix; wherein synthesis of the compound comprises the steps of:
(i) providing a solid support comprising a linker configured to couple an oligonucleotide monomer at its 5’ end, wherein the oligonucleotide monomer comprises a protecting group at its 3’ end;
(ii) removing the protecting group to expose a free amino group;
(iii) coupling, by stepwise solid phase synthesis, a successive oligonucleotide monomer wherein the successive amino acid monomer comprises a protecting group at its 3’ end;
(iv) repeating steps (ii) and (iii) to obtain an oligonucleotide chain;
(v) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), or before step (iii);
(vi) coupling an amino acid via its carboxyl group to the 3’ end of the oligonucleotide chain, wherein the amino acid comprises a protecting group at its amino group,
(vii) removing the protecting group to expose a free amino group;
(viii) coupling, by stepwise solid phase synthesis, a successive amino acid monomer wherein the successive amino acid monomer comprises a protecting group at its amino group;
(ix) repeating steps (vii) and (viii) to obtain a peptide chain;
(x) forming the cross link or the bridge between the at least two amino acids to obtain the compound;
(xi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs after step (vi), before or after step (vii), before or after step (viii), before or after step (ix), or before or after step (x); and
(xii) cleaving the compound from the solid support. [0041] In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[0042] In some embodiments, the PMO comprises 5GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98).
[0043] In some embodiments, the linker of step (i) comprises an amino acid monomer or a handle configured to couple with an oligonucleotide monomer.
[0044] In some embodiments, the protecting group of the amino acid monomer is Fmoc.
[0045] In some embodiments, the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000015_0001
[0046] In some embodiments, the protecting group of the oligonucleotide monomer is Trt.
[0047] In some embodiments, the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000016_0001
[0048] In some embodiments, the forming step (x) comprises forming an olefin cross link between at least two unnatural amino acids.
[0049] In some embodiments, the at least two unnatural amino acids are selected from the group consisting of:
Figure imgf000016_0002
Figure imgf000017_0001
wherein S5 is (S)-pentenylalanine, R5 is (R)-pentenylalanine, S8 is (S)-octenylalanine, R8 is (R)- octenylalanine, B5 is α, α-di- substituted pentenylalanine, B8 is α, α-di-substituted octenylalanine, and S-OAS and R-OAS are O-allylserine analogues.
[0050] In some embodiments, the forming step (x) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
[0051] In some embodiments, the lactam is formed by cross linking a lysine and glutamic or aspartic acid residues.
[0052] According to another aspect of the invention, there is provided a comprising the compound made according to the present methods and one or more pharmaceutically acceptable excipients.
[0053] In some embodiments, the composition is formulated for oral, parenteral, intravenous, or topical administration.
[0054] In some embodiments, a method of administering the composition made by the present methods comprises the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
[0055] In accordance with another aspect of the invention, there is provided a molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acid sequence of SEQ ID NOS: 23, 24, 25, 28, 29, 30, 31, 32, 34, 37, 38, 59, or 60, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
[0056] In some embodiments, the molecule further comprises a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
[0057] In some embodiments, the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
[0058] In some embodiments, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[0059] In some embodiments, the PMO is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
[0060] In some embodiments, the bifunctional linker is SMCC.
[0061] In some embodiments, the PMO has the sequence 5’- GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
[0062] In some embodiments, the antisense oligonucleotide is a gapmer.
[0063] In some embodiments, the gapmer is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
[0064] In some embodiments, the bifunctional linker is SMCC.
[0065] In some embodiments, the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 112).
[0066] In some embodiments, there is provided a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule.
[0067] In some embodiments, there is provided a method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule.
[0068] In some embodiments, the cell is a cardiac muscle cell. [0069] In some embodiments, the cell is a skeletal muscle cell.
[0070] In some embodiments, there is provided a composition comprising the molecule and one or more pharmaceutically acceptable excipients.
[0071] In some embodiments, the composition is formulated for oral, parenteral, intravenous, or topical administration.
[0072] In accordance with another aspect of the invention, there is provided a molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, z-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acids arginine, leucine, and lysine, wherein at least one leucine residue and at least one lysine residue are located between positions z and i+4, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
[0073] In some embodiments, the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
[0074] In some embodiments, the molecule further comprises a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
[0075] In some embodiments, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
[0076] In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[0077] In some embodiments, the PMO is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
[0078] In some embodiments, the bifunctional linker is SMCC. [0079] In some embodiments, the PMO has the sequence 5’- GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
[0080] In some embodiments, the antisense oligonucleotide is a gapmer.
[0081] In some embodiments, the gapmer is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
[0082] In some embodiments, the bifunctional linker is SMCC.
[0083] In some embodiments, the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
[0084] In some embodiments, there is provided a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule.
[0085] In some embodiments, there is provided a method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule.
[0086] In some embodiments, the cell is a cardiac muscle cell.
[0087] In some embodiments, the cell is a skeletal muscle cell.
[0088] In some embodiments, there is provided a composition comprising the molecule and one or more pharmaceutically acceptable excipients.
[0089] In some embodiments, the composition is formulated for oral, parenteral, intravenous, or topical administration.
[0090] In accordance with another aspect of the invention, there is provided a molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acids arginine, glycine, histidine, and lysine, wherein at least one lysine residue is located between positions i and i+4, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
[0091] In some embodiments, the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NOS: 103, 104, 105, 106, or 107.
[0092] In some embodiments, the molecule further comprises a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
[0093] In some embodiments, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
[0094] In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[0095] In some embodiments, the PMO is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
[0096] In some embodiments, the bifunctional linker is SMCC.
[0097] In some embodiments, the PMO has the sequence 5’- GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
[0098] In some embodiments, the antisense oligonucleotide is a gapmer.
[0099] In some embodiments, the gapmer is linked to the thiol-containing moiety at the C- terminus of the StaP via a bifunctional linker.
[00100] In some embodiments, the bifunctional linker is SMCC.
[00101] In some embodiments, the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
[00102] In some embodiments, there is provided a method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule.
[00103] In some embodiments, there is provided a method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule.
[00104] In some embodiments, the cell is a cardiac muscle cell. [00105] In some embodiments, the cell is a skeletal muscle cell.
[00106] In some embodiments, there is provided a composition comprising the molecule and one or more pharmaceutically acceptable excipients.
[00107] In some embodiments, the composition is formulated for oral, parenteral, intravenous, or topical administration.
[00108] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.
[00109] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. [00110] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[00111] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings. [00112] Fig. 1A is a chemical reaction scheme showing the microwave solid phase synthesis of peptides using Fmoc protected reagents.
[00113] Fig. 1B is a chemical reaction scheme showing the ring closing metathesis reaction to obtain the cross link between two unnatural amino acids to obtain the StaP or StiP.
[00114] Fig. 2 is a scheme showing the various embodiments of handles that can be included on the N or C terminus of the peptide or the 5’ end or the 3’ end of the oligonucleotide. The handles are orthogonal reactive sites for additional conjugation and functionalization of the compound.
[00115] Fig. 3A is a chemical reaction scheme demonstrating an additional coupling step of a handle containing a protected thiol group (S-trityl-3 -mercaptopropionic acid) to enable thiol-ene Michael addition.
[00116] Fig. 3B is a chemical reaction scheme demonstrating the incorporation of a handle comprising a terminal alkyne group via nucleophilic substitution to enable Huisgen 1,3-dipolar cycloaddition.
[00117] Fig. 4A is a chemical reaction scheme demonstrating the incorporation of a handle at the C terminus of a peptide comprising a 4-methyl trityl protected lysine.
[00118] Fig. 4B is a chemical structure showing Universal PEGNovaTag™ resin.
[00119] Fig. 4C is a chemical structure showing incorporation of a hydrazine functional group.
[00120] Fig. 5 is a chemical reaction scheme showing the solid phase synthesis of the oligonucleotide utilizing Fmoc protected PMO nucleotide monomers.
[00121] Fig. 6 is a chemical reaction scheme showing the solid phase synthesis of the oligonucleotide utilizing trityl protected PMO nucleotide monomers.
[00122] Fig. 7 is a chemical reaction scheme showing the incorporation of a handle comprising a terminal alkyne group to the oligonucleotide via nucleophilic substitution to enable Huisgen 1,3- dipolar cycloaddition.
[00123] Fig. 8 is a chemical reaction scheme showing the integration of a handle at the 5’ end of the oligonucleotide via the addition of a 4-methyl trityl protected lysine linker to the resin followed by deprotection, functionalization with a 3-mercaptothiopropionic acid handle, and subsequent synthesis of the oligonucleotide.
[00124] Fig. 9 is a chemical reaction scheme showing the interchangeability of the synthetic techniques of synthesizing the peptide and oligonucleotide in a one pot synthesis. More in particular, in some embodiments, the peptide can be conjugated at the 3’ end of the oligonucleotide.
[00125] Fig. 10 is a chemical reaction scheme showing the integration of a 3- mercaptothiopropionic acid handle to a compound synthesized using the one pot synthetic method. [00126] Fig. 11A is a general chemical structure of the compound in which the CPP forms a cross link via ring closing metathesis, the BAC is an anti-sense oligonucleotide (AON), and a series of handles, represented by a linker L, sulfur containing moiety Z, and spacer Y3 act as a bifunctional linker to connect the BAC to the CPP.
[00127] Fig. 11B is a chemical structure of a preferred embodiment of the invention.
[00128] Figs. 12A-12D show EGFP expression and cell toxicity of a first series of stabilized and unstabilized PEPTIGOs, PMO alone, and growth vehicle as a control.
[00129] Figs. 13A-13C show EGFP expression and cell toxicity of a second series of stabilized and unstabilized PEPTIGOs, PMO alone, and growth vehicle as a control.
[00130] Fig. 14 is a plot showing the body mass of IL1αNLS (1,5)-PM0 conjugate- and PMO- treated D2-mdx mice during the dosing phase and the end of the study investigating the effect of dystrophin exon 23-skipping IL1α-NLS-peptide based PMO conjugate and unconjugated PMO on dystrophin expression in the D2-mdx mouse. The arrows indicate days on which mice were dosed. Mice were sacrificed and tissues were collected on day 19. n=2 per group. Data are shown as mean +/- SD.
[00131] Fig. 15 is a plot showing the body mass of IL1αNLS (2,6)-PMO conjugate- and PMO- treated D2-mdx mice during the dosing phase and the end of the study. The arrows indicate days on which mice were dosed. Mice were sacrificed and tissues were collected on day 20. n=2 per group. Data are shown as mean +/- SD.
[00132] Fig. 16A shows microscopy images of stained tissue with respect to dystrophin expression in heart muscle of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1α-NLS (1,5)-PM0 conjugate. The plot shows quantification (in the absolute number of fibres per muscle that stain positively for dystrophin. Data are shown as mean +/- SD and as individual data points.
[00133] Fig. 16B shows microscopy images of stained tissue with respect to dystrophin expression in skeletal muscle (gastrocnemius) of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1α-NLS (1,5)-PM0 conjugate. The plot shows quantification of the dystrophin-positive fibres detected. Data are shown as mean +/- SD and as individual data points. [00134] Fig. 17A shows microscopy images of stained tissue with respect to dystrophin expression in heart muscle of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1α-NLS (2,6)-PMO conjugate. The plot shows quantification of the dystrophin-positive fibres detected. Data are shown as mean +/- SD and as individual data points.
[00135] Fig. 17B shows microscopy images of stained tissue with respect to dystrophin expression in skeletal muscle (gastrocnemius) of treated D2-mdx mice after four intravenous doses of unconjugated PMO or IL1α-NLS (1,5)-PM0 conjugate. The plot shows quantification of the dystrophin-positive fibres detected. Data are shown as mean +/- SD and as individual data points. [00136] Fig. 18A is a chemical structure of the IL1α NLS (1,5)-PM0 conjugate.
[00137] Fig. 18B is a chemical structure of the IL1α NLS (2,6)-PMO conjugate.
[00138] Fig. 19 is a graph showing eGFP reporter expression after 24 hours incubation of
HeLaEGFP654 cells with ESE-1 NLS peptide-oligonucleotide conjugates or vehicle. NH2- EGFP654 PMO is the unconjugated, NH2-modified PMO of the same batch used for preparing the conjugates. Data are shown as mean +/- SD.
[00139] Fig. 20 is a graph showing cytotoxicity after 24 hours incubation of HeLaEGFP654 cells with ESE-1 NLS peptide-oligonucleotide conjugates or vehicle. NH2-EGFP654 PMO is the unconjugated, NH2-modified PMO of the same batch used for preparing the conjugates. Data are shown as mean +/- SD.
DETAILED DESCRIPTION OF THE INVENTION
[00140] Oligonucleotides can be designed to target 5’ translation initiation start sites of viral gene transcript(s) to prevent binding of the translational machinery. Using anti-sense oligonucleotides (ASO) to suppress viral translation is a well-established technology1 and has progressed into clinical trials for viral hemorrhagic fevers such as Marburg and Ebola2,3.
[00141] Also, ONs can be designed to form aptamers such that the secondary and tertiary structures can bind proteins or other cellular targets thus impacting on specific gene expression levels or other cellular processes (e.g. post-translational modifications). [00142] An advantage of steric blocking based suppression over that of siRNA/RNAi based RNase H-induction of the RNA Induced Silencing Complex is the reduced likelihood of off target side effects.
[00143] Modifications of an ON to produce a negatively charged backbone improve stability4' 7, but these backbone chemistries e.g. 2’0-Methyl Phosphothioate analogues, elicit membrane toxicity issues, cause thrombocytopaenia and injection site problems upon clinical translation8, such that efficacy is prevented by toxicity issues, even when administration protocols become increasingly intermittent9.
[00144] Indeed, WO2013/150338 and WO 2014/053622 both disclose delivering negatively charged ONs of small size (typically smaller than 1.5KDa) by complexing them with positively charged linear or stapled peptides of equal or greater than 15 amino acids and in the range of 15- 27 amino acids.
[00145] IACS, Vol 136, 2014, GJ Hilnski et al, describe stapled and stitched peptides that are able to penetrate cells. Reference is made to the possibility that these peptides could be used to deliver an oligonucleotide, presumably in the same manner as disclosed in the international applications disclosed above, i.e. by complexation. There is nothing to suggest creating new entities of much larger size (greater than 1.5KDa, through 2.5KDa, 5KDa, 7.5KDa, lOKDa, 12.5KDa or more) by covalently linking a BAC with a CPA, optionally via a BFL and indeed, the prior methodology requires the respective components to have opposite charges to facilitate complexing.
[00146] The use of electrically low charge carrying oligonucleotides (charge -3 to +3 at pH 7.5) and most preferably electrically neutral oligonucleotides (charge -1 to +1 at pH 7.5), such as, but not limited to, peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligonucleotides (PMOs), (covalently) conjugated directly or indirectly, using a BFL, was not apparent and indeed, limiting the charge on the ON further allows the use of smaller peptides (less than 15 amino acids in length, through 14, 13, 12, 11, 10, 9, 8, 7, 6 to as few as 4 or 5) as carriers.
[00147] The use of uncharged ON backbones, such as phosphorodiamidate morpholino oligonucleotides (PMOs), represent attractive BACs as they have an impeccable safety record in a preclinical and clinical setting.2 10'13
[00148] However, their ability to penetrate cells and access their targets is compromised due to their uncharged nature14. [00149] Over the last 20 years much research has been dedicated to developing CPAs that facilitate delivery of drugs and BACs to the biological site of action.
[00150] The approach has generally been to use charged peptides as non-covalent complexes to facilitate cell entry of a BAC. Conjugation has also been tried.
[00151] WO2014/064258 is an example of the existing conjugating art. A negatively charged
ON is coupled to a targeting peptide via a linker. The targeting peptide is a receptor targeting moiety, and not a stapled or stitched peptide, and indeed considerable doubt exists as to whether DNA or RNA molecules can gain cell entry using a receptor targeting moiety as once a negatively charged ON is bound to such a moiety, non-covalent interactions alter its conformation15.
[00152] WO89/03849 discloses oligonucleotide-polyamide conjugates. There is no disclosure of the use of stitched or stapled peptides. The methodology described uses oligonucleotides as a scaffold for the chain extension of peptides and not as a conjugate for delivery, per se.
[00153] WO2011/131693 describes nucleic acid constructs which contain a nucleic acid specific for a given target gene and a selective inhibitor of a neurotransmitter transporter. There is no disclosure of the use of stitched or stapled peptides as a delivery agent.
[00154] A peptide capable of effecting peptide-mediated cell delivery may also be referred to as a Cell Delivery Peptide (CDP). Examples include: polyarginine, penetratin (based upon an antennapedia homeodomain), or PMO internalization peptides (PIPs).
[00155] However, since their first description16 and given that many CPPs contain multiple arginines, β-alanine and 6-aminohexanoic acid residues, (e.g. poly-Arg12, TAT, Penetratin, Pip6a) [database maintained at http://crdd.osdd.net/raghava/cppsite/]17, it is surprising that no CPP- delivered drug has progressed through all phases of clinical trials. In part, this may be because the common arginine-rich core, which makes most CPPs effective, also causes membrane deformities18 and in higher mammals this manifests as prohibitive toxic side effects, such as tubular degeneration of the kidney19.
[00156] At a physiological pH, and based on pKa of amino acid R groups, a formal charge (FC) can be calculated based on the formula:
Figure imgf000027_0001
Where, V = valence electrons of the neutral atom in isolation; N = the number of non-bonding valence electrons on the defined atom; B = the total number of electrons shared in bonds. [00157] Indeed, based on this, the CPPs typically used to date harbor many positively charged residues. It has been shown that there is a correlation between this positive charge and membrane toxicity20.
[00158] Therefore, CPPs with a lower amount of positively charged residues within the amino acid sequence, whilst retaining the ability to cross a biological membrane, will be more clinically relevant.
[00159] The Applicant has overcome this major impediment by utilizing stabilized CPAs. By linking a drug or BAC to a stabilized CPA, including stitched and stapled peptides, they have surprisingly obtained enhanced cellular uptake dynamics, 10-20 fold better than current state of the art for CPAs21,22.
[00160] They have illustrated this by delivering an ON targeted to repair a gene producing dystrophin. Targeting specific genes with ON is of course in itself known, as illustrated by, for example, W02009/054725 and WO2010/123369. These publications however use a negatively charged backbone and deliver the cargo directly or using complexation.
[00161] W 02017/109494 discloses methods of forming the cross link or bridge within the StiP and the StaP utilizing unnatural amino acids comprising terminal alkene side chains that undergo a ring closing olefin metathesis reaction.
[00162] W 02019/002875 discloses methods of forming the cross link or bridge within the StiP and the StaP via the cyclization of the amine functional groups of lysine and the carboxylic acid side chains of aspartic or glutamic acid.
[00163] As described in WO2017/109494, one way to prepare stapled and stitched peptides, two linked amino acids (stapled) or three or more linked amino acids (stitched), is to incorporate amino acids into the peptide that are modified to bear e.g. an olefin (alkene) group (which may be incorporated at defined relative positions during solid-phase peptide synthesis). For example, on- resin ring-closing metathesis is then used to close one (stapled [denoted as StaP herein]) or two or more (stitched [denoted as StiP herein]) all-hydrocarbon cross-links that induce the peptide to adopt a stabilized structure, typically, but not essentially an alpha helix. For StaPs, it is preferred to use either one or both enantiomers of the un-natural amino acids, termed the S5 (S- pentenylalanine) or R5 (A-pentenylalanine), or the S8 (S-octenyl alanine) or R8 (R-octenyl alanine), depending on the stereo- chemi cal configuration. For StiPs, a further un-natural olefin-bearing a, a-di-substituted amino acid (B5 or B8) is utilized. Cross linking strategies are however not restricted to ring-closing metathesis of un-natural olefin-bearing α, α-di-substituted amino acids. Other cross-linking chemistry’s may be used to stabilize the peptide, such as ring-closing metathesis between O-allylserine analogues (S-OAS or R-OAS).
[00164] WO2017/109494 utilizes conjugation of the BAC with the CPP. In other words, discrete species of BAC and CPP undergo a coupling reaction with the assistance of a BFL to form the compound. The coupling reaction between the two macromolecules resulted in a low overall yield (10%). Though changing the identity of the CPP for the coupling reaction improved the overall yield to approximately 59%, more robust methods of synthesis are needed to achieve higher yields across all types of CPPs.
[00165] The cellular entry dynamics of existing CPAs and the StiPs and StaPs differ. Traditional CPPs enter cells via energy-independent direct plasma membrane translocation or via energy-dependent, clathrin and caveolin-mediated endocytosis; whereas the StiPs and StaPs utilized in the invention enter via an energy dependent, but clathrin and caveolin independent mechanism 21,23. Given that StiPs and StaPs uptake is abrogated with reduced cellular decoration of heparin sulphate21 a macropinocytotic entry mechanism is inferred24, suggesting this altered entry mechanism enables enhanced cellular uptake and bio-distribution compared to the state of the art.
[00166] Relative to their unmodified peptide precursors, StaPs and StiPs generally exhibit robust cellular uptake, significant resistance to proteolytic degradation, and in vivo stability that can support a half-life of more than 12 hours in non-human primates25. It is likely that this increase in drug-likeness stems from the highly rigidified structure and the burial of the backbone amide bonds in the core of e.g. the a-helix. This structural rigidity also decreases the likelihood that StiPs and StaPs will be immunogenic, as the design of major histocompatibility complexes is such that peptides must adopt an extended conformation to be presented. The potential reduced or lack of membrane toxicity and immunogenicity enhances the clinical translatability of compounds when conjugated to drugs and BACs such as ONs.
[00167] The BAC and CPP can be covalently conjugated directly, or covalently conjugated via a BFL. As discussed further below, many functional groups may be used to effect conjugation of the BAC to the CPP.
[00168] In some embodiments, non-covalent affinities between molecules may be used to link the BAC and the CPP to each other. For example, streptavidin, a tetrameric protein derived from Streptomyces avidinii has high affinity for biotin, also known as vitamin B7 or vitamin H. Streptavidin and biotin moieties can interact and form a strong, non-covalent complex.
[00169] ONs can be used to induce a steric block to any gene in humans, animals and lower order organisms and thus can be applied to natural disease (including genetic and age-related diseases) or acquired diseases in humans and animals.
[00170] For example, viral hemorrhagic fevers (VHFs) are animal-borne illnesses in which a prolonged inflammatory cytokine response leads to the gradual destruction of veins and arteries. Causes of VHF include Ebola and Marburg viruses and several Arena viruses; these diseases are presently considered unbeatable. Viral hemorrhagic fevers are characterized by high fever and bleeding disorders, and can cause death by shock and organ failure. ASOs can be designed to target 5’ translation initiation start sites of viral gene transcript(s) to prevent binding of the translational machinery. Using ASO to suppress viral translation is a well-established technology1 and has progressed into clinical trials for viral hemorrhagic fevers such as Marburg and Ebola2,3. One PMO, AVI-7537 was evaluated for human use in the West African Ebola outbreak in 2014-15.
[00171] Some tissues are particularly refractory to naked PMO transfection, e.g. heart, which may reflect differential vesicle-mediated PMO uptake mechanisms23. In fact, direct intra-cardiac injection of naked PMO does not even lead to efficient transfection26, and refractory tissues tend to require repeat administration or high dose strategies27'29. However, whilst CPP conjugation improves PMO bio-distribution and serum stability30'32, toxicity is still a major roadblock for pipeline development19.
[00172] The compound of the invention comprises:
[00173] a biologically active compound (BAC) - (see Table 1 for non-limiting examples);
[00174] a cell penetrating agent (CPA), which is a stitched (StiP) or stapled (StaP) peptide, wherein the StiP or StaP is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11 , the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix ; and [00175] optionally one or more handles wherein the one or more handles, alone or in combination, can form a bifunctional linker (BFL) (see Table 7 for non-limiting examples).
[00176] The CPP can be synthesized via solid phase synthesis, which provides absolute control over the peptide monomer addition sequence and high reaction yields. Surprisingly, it has been found that the methods used for solid phase synthesis of the CPP can be adapted for the synthesis of the BAC, in particular, an oligonucleotide. Similarly, solid phase synthesis techniques tolerate the reaction conditions necessary for the coupling of handles, which provide options for additional orthogonal chemistry and functionalization. The compatibility of solid phase synthesis for the stepwise synthesis of the CPP, BAC, and installation of handles enables the synthesis of the present compounds in a one pot synthesis, obviating the need for a low yielding coupling reaction between the CPP and BAC macromolecules, which may involve a bifunctional linker, and thus provides a general route of synthesis that can be applicable across all types of CPPs.
[00177] As demonstrated herein, the present methods can be used to construct the CPP in a stepwise manner with each successive amino acid building block, as well combine these techniques in a successive procedure utilizing the same constructed CPP to construct the BAC via the addition of nucleic acid monomers onto the same, newly constructed CPP without altering reaction conditions and without intermediate isolation of the CPP (e.g., for ring closing metathesis to form staple or stitch) prior to addition of the first nucleic acid monomer in building the BAC. These techniques can allow access to highly pure oligonucleotide-peptide conjugates at high yields.
[00178] The present methods also permit the construction of the BAC first via anchoring a modified nucleic acid monomer to a solid resin and successively adding nucleic acid monomer prior to the addition of the amino acid building blocks of the CPP. This allows for a systematic study of a batch of synthesized BAC and subsequent modification of one or more amino acid residues in the peptide sequence of the cell penetrating peptide to obtain a series of oligonucleotide-peptide conjugates for further investigation utilizing a BAC synthesized from the same set of experiments and may facilitate the discovery of additional peptide moieties for intracellular delivery of the BAC.
[00179] Although any method may be used to synthesize the CPP, the CPP is preferably synthesized stepwise utilizing solid phase synthesis techniques. Solid phase peptide synthesis permits absolute control over the desired peptide sequence with adequate yields and recoveries. Solid phase peptide synthesis is not limited to the proteingenic amino acids and the techniques may be adopted for incorporation of unnatural amino acids and other amino acid derivatives.
[00180] In solid phase synthesis, typically, an amino protected amino acid is coupled to a solid phase material such as a resin via its carbonyl group. The amino group is then deprotected, exposing a reactive amino group, which reacts with the carboxyl group of a successive amino acid monomer comprising a protected amino group, forming a dipeptide. The process of deprotecting the terminal amino group and coupling of the successive amino acid monomer comprising a protected amino group is repeated until obtaining the desired peptide sequence after which the peptide is cleaved from the resin and recovered for further application.
[00181] The ability to incorporate unnatural amino acids into the peptide sequence permits the introduction of functionalities that can used to obtain the stapled peptide (StaP) or stitched peptide (StiP) through the formation of a cross link or a bridge.
[00182] A stapled peptide (StaP) may be formed by, for example, stapling two conformationally adjacent amino acids together, and a stitched peptide (StiP) may be formed by, for example, stitching at least three conformationally adjacent amino acids to form a stitched peptide (StiP).
[00183] The stapling or stitching results in the formation of a cross link or bridge between two conformationally adjacent amino acids of the peptide.
[00184] In some embodiments, the cross link or bridge comprises two components, a hydrocarbon bridge and a terminal methyl group. The hydrocarbon bridge may be composed of a double hydrocarbon bond or a single hydrocarbon bond.
[00185] In some embodiments, the cross link or bridge provides a cyclization between at least two amino acids formed by an olefin metathesis.
[00186] The CPP preferably comprises at least two unnatural amino acids bearing allhydrocarbon tethers (e.g. a-methyl,a-pentenyl glycine).
[00187] Formation of the cross link and bridge is not limited to reacting functional side chains of unnatural amino acids and the cross link and bridge may be formed through reaction of the side chains of proteingenic amino acids.
[00188] In some embodiments, the cross link or bridge provides a cyclization between at least two amino acids which are not formed by an olefin metathesis.
[00189] By cyclization is meant that a staple or stitch is formed directly between conformationally adjacent amino acids, as opposed to by the introduction of a separate “bridging molecule”, such as, for example, an aryl group, such as an aromatic ring or a perfluroaryl group. This direct cyclisation may be achieved by one or more of:
[00190] i) condensation of an aldehyde or ketone with a hydrazine or protected hydrazine;
[00191] ii) a thiol-ene Michael addition;
[00192] iii) a di-sulfide formation;
[00193] iv) a Huisgen 1, 3 di-polar cycloaddition;
[00194] v) a reaction between an amine and carboxylic acid;
[00195] vi) a singlet or triplet based carbine reaction; or
[00196] vii) a Suzuki or Sonogashira coupling.
[00197] Particularly preferred cyclizations are obtained from chemistries iv) and v):
[00198] Using iv) a 5 membered heterocycle is formed between an azide and electron deficient nitrile containing amino acid or a propygyl containing amino acid.
[00199] Using v) a lactam is formed between a free amine containing amino acid and a carboxylic acid containing amino acid.
[00200] It has been found that solid phase synthetic techniques used to synthesize the exact sequence of the CPP can be applied to the synthesis of the biologically active compound (BAC).
[00201] In some embodiments, the BAC is an oligonucleotide (ON). In some embodiments, the BAC is an anti-sense oligonucleotide (AON).
[00202] In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[00203] In solid phase synthesis of the BAC, a linker must first be coupled to a solid support resin wherein the linker is configured to couple to an oligonucleotide monomer. In some embodiments, the linker is an amino acid monomer. In some embodiments, the linker is a handle, which is a moiety comprising a functional group, further discussed below.
[00204] Like amino acids comprising a protected amino group, a BAC monomer can similarly be protected to prevent undesired successive coupling and subsequently deprotected to enable the addition of the next monomer. For example, the secondary amine of PMO at the 3’ end can be protected to permit coupling to occur at the phosphorodiamidate moiety located at the 5’ end and the secondary amine at the 3’ end deprotected to enable addition of a successive PMO monomer, which also comprises a protected secondary amine at its 3’ end. The process of deprotecting the secondary amine at the 3 ’ end is repeated until the desired sequence is obtained. The deprotected second amine may be used in a subsequent coupling reaction without further modification or with additional modification to incorporate other functional groups as described below.
[00205] In some embodiments, the oligonucleotide monomers may be further configured to undergo a nucleophilic substitution reaction to facilitate the coupling of oligonucleotide monomers. In some embodiments, the phosphorodiamidate moiety may be modified at its 5’ end to have a halogen leaving group or another suitable organic leaving group to facilitate coupling with the linker of the solid support resin or the secondary amine of the 3’ end. In some embodiments, the phosphorodiamidate moiety may be modified with a primary amine or other functional group at its 5’ or 3’ end to facilitate coupling to the functional group of a handle. In some embodiments, the 5 ’ or 3 ’ end can be modified with a glycine whose amino group is protected with Fmoc or other suitable protecting group to provide a more reactive primary amine functionality to the phosphorodiamidate moiety.
[00206] In accordance with another aspect of the invention, the present methods permit the coupling of handles to the compound, which provide orthogonal reactive sites and permit the introduction of functionalities to tailor the application and properties of the compound.
[00207] Utilizing solid phase synthetic techniques, the handle can be coupled anywhere on the CPP. In some embodiments, the handle is coupled to the N terminus of the CPP. In some embodiments, the handle is coupled to the C terminus of the CPP via functionalization of the amino acid at the C terminus (e g. deprotection of Mtt protected lysine). In some embodiments, the handle is coupled to an amino acid located internally between the C terminus and the N terminus.
[00208] Utilizing solid phase synthetic techniques, the handle can be coupled anywhere on the BAC. In some embodiments, the handle is coupled to the 3’ end. In some embodiments, the handle is coupled to the 5’ or internally between the 5’ end and the 3’ end via addition of a residue that can be modified orthogonally (e.g. addition of Mtt protected lysine at the 5’ end and subsequent deprotection for coupling of handle).
[00209] In some embodiments, the handle may be used to couple the CPP to the BAC.
[00210] In some embodiments, one or more handles may be coupled together to form a bifunctional linker, which may be used to couple the CPP to the BAC.
[00211] In accordance with another aspect of the invention, the disclosed methods permit a one pot synthesis of the compound, obviating the need to carry out a coupling reaction between discrete species of CPP, BAC, and any associated linkers or handles, which have resulted in low overall yields. The disclosed methods streamline the overall synthetic process of the compound with high yielding solid phase synthetic methods, improving the yield and purity of the compound and enabling complete control over the composition of the compound.
[00212] The compound can be synthesized in any order using the disclosed methods. In some embodiments, the CPP is synthesized first, followed by the BAC. In some embodiments, the BAC is synthesized first, followed by the CPP. The compound is not limited to block copolymers of BAC and CPP and the monomers of both the BAC and the CPP can be random and arranged in any order or configuration.
[00213] In accordance with another aspect of the invention there is provided a method for facilitating the uptake of a biologically active compound (BAC) into a cell by the conjugation of the biologically active compound, directly or via a bifunctional linker (BFL), to a cell penetrating agent (CPA) which is a stabilized peptide which has a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP), to form a compound in the form of a drug carrying cell penetrating molecule and presenting said compound to said cell in a suitable vehicle.
[00214] Where hydrazynal nicotinic acid (HNA) has been incorporated into the terminal end of the CPP, to form a compound in which the BAC is an ON, the ON has been modified to incorporate 4-formyl benzoic acid to facilitate covalent conjugation.
[00215] According to another aspect of the present invention, there is provided a compound of for use in the treatment of a disease requiring alteration of the expression of an endogenous or exogenous gene.
[00216] The compound may be used in the treatment of a, for example, neuromuscular disease, metabolic disease, cancer, age-related degenerative disease or to treat an acquired viral infection. [00217] In one embodiment the compound is used in the treatment of a muscular dystrophy e g. Duchenne muscular dystrophy (DMD) although the skilled person will readily appreciate that the invention can be used to target a wide range of genes.
[00218] In the case of DMD the compound may comprise an anti-sense oligonucleotide (AON) targeting exon 51 of the dystrophin gene.
[00219] In accordance with another aspect of the present invention, there is provided a method of improving the bioavailability of a drug or BAC comprising linking the drug or BAC to a CPP, which is a stabilized peptide which has a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP).
[00220] In accordance with another aspect of the present invention, there is provided a method of introducing a drug or BAC to a site which is refractory to a drug or BAC in its native state comprising linking the drug or BAC to a CPP, which is a stabilized peptide having a conformation imposed upon it by stapling to form a stapled peptide (StaP) or stitching to form a stitched peptide (StiP) and administering it to a subject.
[00221] The compounds of the invention can be used to administer the drug or BAC to a target tissue. The drug or BAC may be administered to any type of tissue, which include epithelial tissue, connective tissue, muscle tissue, and nervous tissue.
[00222] In some embodiments, the compounds are specific to a type of muscle or the cells of a particular type of muscle. Thus, cellular uptake and retention of biological activity of a BAC that is conjugated to a particular stabilized peptide may be improved for in one particular peptide conjugate in a specific muscle type (e.g., skeletal) whereas the same peptide conjugate carrying the same BAC may exhibit decreased cellular uptake and retained biological activity of the BAC in a different muscle type (e.g., cardiac) despite having a similar peptide sequence, identity of linker, BAC, hydrophobicity, location of stitch, and charge.
[00223] In some embodiments, the compound is contacted with skeletal (striated) muscle or skeletal muscle cells.
[00224] In some embodiments, the compound is contacted with smooth muscle or smooth muscle cells.
[00225] In some embodiments, the compound is contacted with cardiac muscle or cardiac muscle cells.
[00226] In accordance with another aspect of the present invention, there is provided a method of treating a subject to alter the expression of an endogenous or exogenous gene comprising administering a compound of the invention to a subject.
[00227] In accordance with another aspect of the present invention, there is provided a method of delivering a biologically active moiety to a cell comprising contacting the cell with the compound of the invention.
[00228] In some embodiments, the cell is a cardiac muscle cell or a cardiomyocyte. [00229] In some embodiments, the cell is a muscle cell. In some embodiments, the muscle cell is a smooth muscle cell. In some embodiments, the muscle cell is a skeletal (striated) muscle cell. [00230] In accordance with another aspect of the present invention, there is provided a composition comprising a compound of the invention and one or more pharmaceutically acceptable excipients enabling the composition to be administered orally, parenterally, intravenously or topically.
[00231] The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. [00232] In some embodiments, the pharmaceutical compositions comprise the compound and a pharmacologically acceptable excipient and the compositions have a pH of 7.0-7.5.
[00233] In some embodiments, the excipient is selected from the group consisting of a tonicity agent, a pH adjuster, and a solvent.
[00234] The concentration of the compound in the pharmaceutical composition can vary and will depend on the method of administration and the needs of the patient. In some embodiments, the concentration of the pharmaceutical composition ranges between about 0.1 mM to about 10 mM.
[00235] Dosage will depend on the body mass of the patient and the concentration of the active ingredient in the pharmaceutical composition. The dosage can range from about 0.1-10 μmol of the compound per kilogram of body mass. In some embodiments, the dosage of the compound is about 30 mg per kilogram of body mass.
[00236] Administration can occur at any frequency. In some embodiments, administration occurs daily, weekly, monthly, or bimonthly. Preferably, administration to the patient via an intravenous route occurs between once weekly and once monthly.
[00237] In one aspect of the invention, the compound of the invention has specificity for skeletal muscle. The improved uptake and specificity for skeletal muscle renders the compounds of the invention good candidates for improved efficiency delivery of BACs that may be useful in therapeutic applications in skeletal tissue. [00238] The three components forming the compound, the methods of synthesis, the formulation of the compound, and administration are discussed in more detail below.
The Biologically Active Compound.
[00239] The biologically active compound is any compound that can exert a biological effect within a biological cell. The biologically active compound is conjugated to the cell -penetrating peptide and is delivered to the cell.
[00240] Any compound that has biological activity can be used as the biologically active compound and may include, but is not limited to proteins, enzymes, antibodies and antibody fragments, nanoparticles, micelles, quantum dots, and organelles in organelle replacement therapy (e.g. mitochondria, lysosomes).
[00241] Preferably, though not essentially, the BAC is one which will impact on the expression of one or more endogenous or exogenous genes. Examples of BACs that can affect the expression of one or more endogenous or exogenous genes include nucleic acids, DNAzymes, ribozymes, aptamers, gapmers, and pharmaceuticals. Preferred biologically active compounds for use in the present invention include electrically neutral oligonucleotides (charge -1 to +1 at physiological pH - about 7.5) such as peptide nucleic acids (PNAs) or PMOs or their modified derivatives that might impart a small electric charge (either positive or negative).
[00242] The biologically active compound may be used as a steric blocking compound to suppress or enhance: i) RNA splicing; ii) protein translation or iii) other nucleic acidmucleic acid or nucleic acid:protein interactions, altering the gene expression of endogenous or exogenous (pathogen derived) genes.
[00243] The hybridization of ONs to specific RNA sequence motifs prevents correct assembly of the spliceosome, so that it is unable to recognise the target exon(s) in the pre-mRNA and hence excludes these exons in the mature gene transcript. Exclusion of an in-frame exon can lead to a truncated yet functional gene product; exclusion of an out of frame exon results in a frame-shift of the transcript, potentially leading to a premature stop codon and a reduction in the target gene expression level.
[00244] Additionally, ONs can be designed to target 5’ translation initiation start sites of endogenous or viral gene transcript(s) to prevent binding of the translational machinery. Using ASO to suppress viral translation is a well-established technology and has progressed into clinical trials for viral haemorrhagic fevers such as Marburg and Ebola.
[00245] Also, ON can be designed to target 3’ untranslated region of an endogenous transcript that alters the stability of the transcript. Such targets include, and are not limited to, poly adenylation and/or cleavage sites of the transcript.
[00246] Also, ON can be designed to form aptamers such that the secondary and tertiary structures can bind proteins or other cellular targets thus impacting specific gene expression levels. [00247] Non-limiting exemplary ON chemistries are illustrated in Table 1.
[00248] The preferred BAC is an oligonucleotide (ON), more preferably still an anti-sense oligonucleotide (AON). Different anti -sense oligonucleotide chemistries are illustrated in Table 1, with the use of low charge or neutral charged chemistries, such as, phosphorodiamidate morpholino oligonucleotides (PMOs) being preferred.
[00249] The length of a PMO can influence the observed cellular uptake by a cell and ability of the PMO to participate in inhibiting expression of its target gene. PMOs typically range from 15 to 30 nucleotides in length although PMOs can be longer or shorter depending on the desired biological activity and application of the PMO.
Table 1. Anti-sense oligonucleotide structures
[00250] The BAC may target and alter the expression of an endogenous or exogenous gene. Endogenous gene targets include but are not limited to genes associated with neuromuscular disease, metabolic disease, cancer, age-related degenerative diseases, and exogenous gene targets include those of an acquired disease e.g. viral infections. [00251] The BAC may comprise, but is not limited to, one of the following sequences (represented 5 ’ to 3 ’), which target exon 51 or the splice junction of intron 50/exon 51 of the human dystrophin gene:
Figure imgf000041_0001
[00252] The above sequences may also be represented as RNA sequences in which T is substituted with U.
[00253] In some embodiments, the biologically active compound can be an siRNA (small interfering, short interfering, silencing RNA) that interferes with the expression of specific target genes and in particular, prevents mRNA translation. The siRNA can comprise between about 15- 30 base pairs in length, preferably between 20-24 base pairs in length.
[00254] In a preferred embodiment, the target is exon 51 of the dystrophin gene and the BAC is a PMO that comprises the sequence:
Figure imgf000041_0002
or its RNA analog:
Figure imgf000042_0001
[00255] In another preferred embodiment, the target is exon 23 of the dystrophin gene and the BAC is a PMO that comprises the sequence:
Figure imgf000042_0002
or its RNA analog:
Figure imgf000042_0003
[00256] The oligonucleotides that are conjugated to the cell penetrating peptide are involved in antisense therapy, whose numerous functions include exon targeting and skipping. Other forms of antisense therapy include, but are not limited to functions such as RNase H degradation of mRNA or pre-mRNA to knockdown the expression of a gene and the use of siRNA to interfere with and block the production of a target protein.
[00257] In some embodiments, the antisense oligonucleotide is a gapmer, which recruits RNase H to degrade mRNA and pre-mRNA. Gapmers are single stranded chimeric antisense oligonucleotides comprising a central DNA gap flanked by modified nucleotides on both ends. The modified nucleotides enhance stability and binding affinity, while the central DNA gap allows for RNase H-mediated degradation of the target RNA. Gapmers rely on the RNase Hl-dependent degradation of complementary RNA targets as the mode of action. When the gapmer hybridizes to its target RNA molecule, the RNA-DNA duplex is recognized by RNase H, which cleaves the RNA strand, resulting in the degradation of the target mRNA and subsequent reduction in protein expression. RNase H-active gapmers can exert activity in both the nucleus, in which the gapmers interact with pre-mRNA, or in the cytoplasm via interacting with the mRNA.
[00258] Oligonucleotides, such as gapmers, are susceptible to nucleolytic degradation, which adversely affects the function of the oligonucleotide. To increase their resistance to nucleases and nucleolytic degradation and to obtain higher half-lives in plasma, tissues and cells, various chemical structure modifications may be made. Modifications to the chemical structure of the oligonucleotide to improve nuclease resistance include, but are not limited to including phosphorothiorate linkages between monomers on the oligonucleotide backbone, 2’ ribose modifications, which include, but are not limited to 2'-O-methyl (2'-0Me), 2 '-O-m ethoxy ethyl (2'- MOE), 2'-O-aminopropyl (2'-O-AP), and 2'-fluoro modifications, and utilizing a locked nucleic acid conformation (LNA) (also known as bridged nucleic acid), in which the ribonucleotides contain a methylene bridge that connects the 2’ oxygen and the 4’ carbon. LNA can improve resistance to enzymatic degradation and improve affinity in base pairing because the conformation can be locked in an ideal position.
[00259] In some embodiments, the gapmer may be for inhibition of a target pre-mRNA, a target mRNA, a target viral RNA, or a target long non-coding RNA.
[00260] Through the interaction and hybridization with either pre-mRNA or mRNA and the subsequent cleavage through recruitement of RNase H, gapmers halt the transcription process and can thus knockdown the expression of a target gene. This ability to knockdown and silence target genes renders gapmers attractive agents for therapy. Conjugation of a gapmer to the present CPPs can help to achieve specificity for a particular target tissue or a particular target cell to improve delivery of the therapeutic gapmer.
The cell penetrating agent (CPA) which is a stabilized peptide
[00261] The cell penetrating agents of the invention are stabilized peptides.
[00262] The peptides may be stabilized by stapling, to form a stapled peptide (StaP), or by stitching to form a stitched peptide (StiP). A StaP includes one cross link that is formed between two amino acid residues on the peptide chain. A StiP includes more than one cross link formed.
[00263] All-hydrocarbon staples and stitches may confer a property, e.g. an a-helical structure, protease resistance, cellular penetrance, and biological activity. The a-helix is used as a recognition motif in protein -protein interactions and the introduction of a staple or a stitch to lock lock down the conformation of the peptide can lead to improved cell penetrating ability in addition to improvements in stability against proteolytic degradation.
[00264] Non-limiting examples of stapled and stitched peptide sequences are illustrated in Table 3 and include peptide sequences including S5, S8 and B5 (as defined in Table 3).
[00265] Stabilization of e.g. the a-helical structure can be achieved by, for example, a ringclosing metathesis and may be catalysed by a variety of ruthenium catalysts including Grubbs generations 1 and 2 and Grubb s-Hoyveda generations 1 and 2.
[00266] All the peptide components (amino acids, unnatural amino acids, unstapled/unstitched, partially stapled/stitched and stapled/stitched peptides) may exist in specific geometric or stereoisomeric forms. All compounds include cis- and trans-isomers, (R)- and (S)-enantiomers, diastereoisomers and racemic mixtures thereof. [00267] Preferred isomer/enantiomers will be enriched to give a greater proportion of one particular isomer or enantiomer. Embodiments thereof may be made of greater than 90%, 95%, 98% or 99%, by weight, of a preferred isomer/enantiomer.
[00268] Non-limiting examples of unnatural amino acids used in stabilizing a peptide structure are illustrated in Table 3.
[00269] The preferred stapled or stitched CPPs incorporate one or more of: a (S)- pentenylalanine (S5) or its enantiomer (R5), a S-octenylalanine (S8) or its enantiomer (R8) or combinations thereof (e.g. R-octenylalanine/S-pentenylalanine (R8/S5) or S-octenylalanine/R- pentenylalanine (S8/R5).
[00270] The preferred unnatural amino acids incorporated into the CPPs and reacted to form a cross link or bridge between them are illustrated in Table 2 and some exemplary and preferred resulting CPPs are illustrated in Table 3.
Table 2. Unnatural amino acids
Figure imgf000044_0001
Table 3. Peptide sequences
Figure imgf000044_0002
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
[00271] In referring to the above peptide sequences of Table 3:
[00272] H can be substituted for R and K;
[00273] I, L, A, or V can be substituted for I, L, A, or V;
[00274] a PEGn group may be present before the handle, for example (S5-FLR-S5)FKR-PEGn- 3TPA;
[00275] The 3-(tritylthio)propionic acid moiety (3 TP A) can be substituted for any handle described in Table 5;
[00276] the position of unnatural amino acids S5 and S8 can be changed within the sequence;
[00277] the parentheses enclosing the unnatural amino acids designate the location of the crosslink and the intermediate amino acids that span the crosslink;
[00278] the amino acids utilized can be the D stereoisomer or the L stereoisomer, or combinations thereof; and
[00279] the sequences can be synthesized in the reverse order. For example, the peptide of SEQ ID NO: 23, (S5-FLR-S5)FKR-3TPA can be synthesized as RFK-(S5-RLF-S5)-3TPA In some embodiments, the peptide of SEQ ID NO: 37, VKR-(S5-KKK-S5)-P-3TPA, can be represented as 3TPA-PK(S5-KKR-S5)KV and the peptide of SEQ ID NO: 38, VK(S5-RKK-S5)KP-3TPA, can be represented as 3TPA-P(S5-KKK-S5)RKV.
[00280] The present invention is not limited to the formation of a cross link or a bridge through the linking of unnatural amino acids. A lactam can be formed between a free amine containing amino acid such as lysine and a carboxylic acid containing amino acid such as aspartic acid or glutamic acid.
[00281] Alternative CPPs and their method of manufacture are disclosed in Chu et al, 2014 and associated supplementary information, and are incorporated by reference.21 [00282] The exemplified stabilized peptide comprises two or more olefin bearing side chains that are covalently formed, typically by means of a ring-closing metathesis.
[00283] The stabilized conformation typically comprises at least one alpha helix. It may however, in the alternative, comprise at least one turn (for example, but not limited to, α, β, γ, δ or π), several turns to form a beta sheet, or a combination of one or more of: an alpha helix, turn, or beta sheet.
[00284] The formal charge of a CPP is calculated at physiological pH (about 7.5) and is based on the pKa of amino acid R groups. These values (pKx) are represented in Table 4.
Table 4. Amino acid pKa values
Figure imgf000048_0001
[00285] CPPs typically used to date harbor many positively charged residues. Reducing the amount of positively charged residues within the amino acid sequence, whilst retaining the ability to cross a biological membrane, will be more clinically relevant.
[00286] Accordingly, it is possible to reduce the charge on the peptide sequences illustrated in Table 3.
[00287] In one embodiment the applicant employs α, α-di substituted unnatural amino acids bearing all-hydrocarbon tethers (e.g. a-methyl,a-pentenyl glycine). [00288] For single turn stapling, one embodiment could employ a (S)-pentenylalanine (S5) at, e.g. i, i + 4 positions, and in another embodiment, for double turn stapling, a combination of either R-octenyl alanine/, S'-pentenyl alanine (R8/S5) or , S'-octenyl al anine/R-pentenyl alanine (S8/R5) at e.g. i, i+ 7 positions can be used. The same pairings can be used to install more than one staple within a given peptide template. S5 can be substituted at z, B5 at position z + 4 positions, and S8 can be substituted at i, i+4, i + 11 positions to generate stitched peptides. The S5 configured amino acid and its enantiomer R5, or S8 configured amino acid and its enantiomer R8, differ only in the opposite stereochemical configuration of the staple they bear.
[00289] Based upon the inclusion of a single or a double turn staple, peptides may comprise of one or more of the sequences in Table 3. Based upon the specific peptides shown in Table 3, a person skilled in the art can easily envisage peptides with 3, 4, 5 or more turn stabilizing staples.
[00290] The hydrocarbon bridge may be composed of a double hydrocarbon bond or a single hydrocarbon bond.
[00291] Compared to a corresponding unmodified native peptide, the inclusion of a staple or a stitch to stabilize and lock down the conformation of the peptide has been shown to improve the cellular uptake of the stabilized peptide and BAC that is conjugated to to the stabilized peptide without any increase in cell toxicity. However, the mere presence of the staple or the stitch and associate low cytotoxicity is not indicative of a suitable candidate peptide for intracellular delivery of a BAC such that the BAC is able to retain its biological activity. A combination of factors, which include, but are not limited to, the overall hydrophobicity of the peptide chain, the location of the staple or stitch within the peptide chain, the overall charge of the peptide, the identity of the BAC, the identity of the target tissue or cell, and the identity of any bifunctional linker, are taken into account in determining a good candidate for intracellular delivery of a BAC such that the BAC retains its biological activity.
[00292] In some embodiments, the cell penetrating agent has a stitched or stapled peptide comprising the amino acid sequence, (S5-FLR-S5)FKR-3TPA (SEQ ID NO: 23), or its reverse sequence RFK-(S5-RLF-S5)-3TPA.
[00293] In some embodiments, the cell penetrating agent is an IL1α-based stapled peptide having the sequence of S5-VLK-S5-RR (SEQ ID NO: 64) or K-S5-LKK-S5-R (SEQ ID NO: 60). In some embodiments, S5 in the IL1α-based stapled peptide according to SEQ ID NO: 64 and SEQ ID NO: 60 is the unnatural amino acid (S)-pentenylalanine and wherein the olefin crosslink is formed between the (S)-pentenylalanine residues comprises olefin crosslinks formed by olefin metathesis.
[00294] In some embodiments, the peptide based on the ETS-related transcription factor Elf-3 (ESE-1) nuclear localization signal (NLS) having the amino acid sequence of SEQ ID NOS: 103- 107.
[00295] In another embodiment the peptide is a branched stapled peptide. The branched stapled peptide comprises 2 or more chains of peptides. Branched peptides may be formed using any method known to the art. For example, in one embodiment, a lysine residue is used to branch two peptide chains. Branched peptides allow for the presentation of multiple cell penetrating peptides to the cell membrane, which may improve the cell penetrating or uptake ability of the molecule due to the higher local concentration of cell penetrating peptides interacting with the cell membrane or via the synergy of the abilities and functions of the branched peptide. For example, in some embodiments, a dendrimeric peptide formed by branching may include multiple copies of a peptide having adequate cell penetrating abilities to improve cellular uptake due to the increased and more localized presentation. In some embodiments, a dendrimic peptide formed by branching may include a combination of peptides that have adequate cell penetrating abilities and of peptides that exhibit improved endosomal escape abilities to improve delivery of the BAC both across the cell membrane and to the target via the improved abilities to both cross the cell membrane and break free from the endosome transporting the molecule comprising the dendrimeric peptide across the cell membrane.
[00296] Functional derivatives of disclosed peptide sequences could be used. Functional derivatives may have representative fragments or homologues or peptides that include insertions to the original peptide. Typical derivative would have 70%, 80%, 90% or more of the original peptide sequence and may have up to 200% of the number of amino acids of the original peptide. The derivatives would be used to enhance the delivery of a biologically active compound.
[00297] Peptide sequence can include modified amino acids to include functional groups that permit the addition of other moieties. Non-limiting examples of such moieties include an acetyl, a cholesterol, a fatty acid, a polyethylene glycol, a polysaccharide, an aminoglycan, a glycolipid, a polyphenol, a nuclear localizing signal, a nuclear export signal, an antibody and a targeting molecule. [00298] The CPP can also comprise nuclear export signals (NES), which are short peptides that mediate the transport of proteins from the nucleus to the cytoplasm. Examples of NES include LCNCALEELRL or LWEFIRDILI, which are found in the transcription factor ESE-1.
[00299] In general, the NES of the present invention have the following sequence motif: (L/I)X2-4(L/I)X1 -4(L/I)X(L/I) in which X is any amino acid and the numerical range modifying X indicate the range of possible X present.
[00300] Other NES include the sequence motifs:
Class 1 :
Class 2:
Class 3:
Figure imgf000051_0001
in which [AP] represent any amino acid except proline and
Figure imgf000051_0002
represents any two or three amino acids except proline, F = L, I, V, M, F, C, W, A or T.
[00301] The CPP can also be a nuclear localization signal (NLS), which are short peptides that mediate the transport of proteins from the cytoplasm into the nucleus of a cell. Examples of NLS include KHGKRKR or HGKRKR, which are found in the transcription factor ESE-1.
[00302] NLS can have the following sequence motifs:
Class I : (K/R)4
Class 2: K(K/R)X(K/R)
Class 3: KRX(W/F/Y)XXAF
Class 4: (P/R)XXKR(K/R)
Bipartite: (K/R)(K/R)X10-12(K/R)3/5 in which X = any amino acid.
Handles and bifunctional linkers
[00303] Handles can optionally be coupled to the compound to introduce additional functional groups that serve as orthogonal reactive sites, allowing for further functionalization and tailoring of physical properties of the compound. In some embodiments, the handle can be coupled to the BAC or the CPP during solid phase synthesis via an additional coupling reaction according to the invention. In some embodiments, the handle can be incorporated via a nucleophilic substitution reaction such as with a terminal amino group. In some embodiments, the handle serves as a linker between the BAC and the CPP. In some embodiments, the handle is not present and the BAC is covalently linked to the CPP.
[00304] Referring to Fig. 2, examples of functional groups that can be introduced by handles of the present invention include, but are not limited to:
[00305] thio or an alkene groups to permit conjugation via a thiol-ene Michael addition;
[00306] alkyne or azido groups to permit conjugation via a Huisgen 1,3 dipolar cycloaddition; and
[00307] hydrazine or aldehyde groups to permit conjugation via hydrozone ligation.
[00308] In some embodiments, the handle can comprise a PEGylated group such as a PEG peptide comprising amino and carboxyl groups and a PEG spacer to enhance solubility of the compound in aqueous environments.
[00309] In some embodiments, the handle can be an amino acid whose side chains introduce a functional group. The amino acid handle may or not contain protecting groups. The amino acid handle may be a proteingenic amino acid or the amino acid handle may be an unnatural amino acid.
[00310] Referring to Figs. 4B and 4C, the resin can similarly contain functional groups to enable orthogonal chemistry. Fig. 4B shows the structure of Universal PEG NovaTag™ resin, which provide an Fmoc protected amine for growing the peptide chain and an Mtt protected amine for additional functionality at the C terminus. Fig. 4C shows the structure of a resin having a hydrazine functionality.
[00311] The handles utilized in coupling to the CPP or the BAC can be interchanged in coupling reactions to both the CPP and the BAC. In other words, a handle used to couple to the CPP is not necessarily limited to coupling and functionalizing the CPP and may be used for coupling to and functionalizing the BAC.
[00312] The handles for the CPP of the present invention include but are not limited to those listed in Table 5:
Table 5. Exemplary handles for cell penetrating peptide
Figure imgf000052_0001
Figure imgf000053_0001
[00313] The handles for the BAC include but are not limited to those listed in Table 6: Table 6. Exemplary handles for biologically active compound
Figure imgf000054_0001
Figure imgf000055_0001
[00314] In the CPP, the handle can be incorporated at the N or C terminus of the peptide, internally within the peptide chain, or in combinations thereof.
[00315] In the BAC, the handle can be incorporated at the 5 ’ or 3 ’ end of the B AC, or internally, or in combinations thereof.
[00316] A handle, alone or in combination with other handles, can be used to introduce a label or a marker such that the bioactivity of the compound can be visualized and measured. Examples include, but are not limited to chemiluminescent labels such as a fluorescent label or a phosphorescent label. Preferably, the label is a fluorescent label that is fluorescein isothiocyanate (FITC). The label or marker may be conjugated anywhere onto the compound and may be included at the C or N terminus of the peptide or internally within the peptide chain, within a handle or a series of handles linking the BAC to the CPP, or at the 5’ or 3’ end of the BAC or internally within the BAC chain.
[00317] A handle, alone or in combination with other handles, can also serve as a bifunctional linker (BFL), which may be used to link the BAC to the CPP or the CPP to the BAC. [00318] Preferred linkers will link between, for example, an amine group on the BAC and a sulfhydryl (thiol) group (usually a cysteine residue) on the CPP terminus. Examples of substrates to achieve this include, but are not limited to, SMCC (succinimidyl 4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate), AMAS (N-α-maleimidoacet-oxysuccinimide ester, BMPS (N-β-maleimidopropyl-oxysuccinimide ester), GMBS (N-γ-aleimidobutyryl- oxysuccinimide ester), DMVS (N-δ-maleimidovaleryl-oxysuccinimide ester, EMCS (N-s- malemidocaproyl-oxysuccinimide ester), and LC-SMCC (Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) as exemplified in Table 7.
[00319] Whilst the BAC may be linked to the CPP directly, the Applicant has found the use of a BFL desirable. Exemplary, non-limiting BFL chemistries are illustrated in Table 7.
Table 7. Bifunctional linkers
Figure imgf000056_0001
Figure imgf000057_0001
[003201 In the above Table 7, substituent R in the chemical structure of Z is H or NH2.
[00321] By way of a footnote to Table 7, the following should be noted:
[00322] In a preferred embodiment as shown in Fig. 11A, where AON is an anti-sense nucleotide such as that of SEQ ID NO: 6, Y1= Nitrogen, Y2 = Hydrogen, Y3 = spacer such as (PEG)n n=5, but not limited to those identified in Table 7, Z = a sulfur containing moiety e.g. cysteine and L is a handle that acts as a bifunctional linker such as SMCC, which introduces an alkene functionality to enable a thiol-ene Michael addition. In some embodiments, Z is a thiol- containing moiety that is covalently linked to the C-terminus of the StaP or StiP by a polyethylene glycol linker. In some embodiments, the thiol-containing moiety is a cysteine residue.
[00323] Other embodiments may utilize variations over the structure shown in Fig. 11 A. For example if another embodiment does not require a thiol for conjugation of the handle to the CPP, then Z = Y3, where Y3 is a spacer in Table 7. Where a sulfur is not required for conjugation of the BAC and CPP e.g. not limited to entries 9-11 in Table 7, Z= a covalent bond between L and Y3. [00324] Other embodiments may not require the use of a handle to link the BAC to the CPP and thus the following apply. If no spacer is utilized, then Y3 can represent a covalent bond between Y1 and the BAC in which case Z and L = Y1 where Y1 is an N terminus of the CPP.
[00325] These chemistries may be further expanded and Table 8 exemplifies modifications to amino acids via which functional groups can be introduced to provide desirable properties to the compound. These will include, but are not limited to, an acetyl, a cholesterol, a fatty acid, a polyethylene glycol, a polysaccharide, an aminoglycan, a glycolipid, a polyphenol, a nuclear localizing signal, a nuclear export signal, an antibody, and a targeting molecule.
Table 8. Coupling chemistries for linkers
Figure imgf000059_0001
[00326] A preferred linker chemistry utilizes an amine to sulphydryl cross linker containing N- hydroxysuccinimide esters and maleimide reactive groups separated by a cyclohexane spacer namely succinimidyl 4-(N-maleimidom ethyl) cyclohexane- 1 -carboxylate (SMCC) to form a covalent bond between the bifunctional linker and the CPP. Solid phase synthesis
[00327] The CPP of the present invention are synthesized using solid phase synthesis in which each monomer is added to the growing peptide chain in a stepwise fashion.
[00328] Typically, solid phase peptide synthesis is performed using resins as solid supports for the growing peptide chain. Any resin may be used for solid phase synthesis. The choice of synthetic strategy such as utilizing Boc or Fmoc amino acids as monomers will dictate the appropriate selection of resin. Resins that may be used include, but is not limited to PAM (4-hydroxymethyl- phenylacetamidom ethyl) resin, Wang resin, Rink amide resin, MBHA resin, PAL (4-alkoxy-2,6- dimethoxybenzylamine) resin, Sieber amide resin, trityl resins, DHP (dihydropyran) resin, and Weinreb aminomethyl resin.
[00329] The amino acid monomers can be protected with any protecting group such that the monomer does not have the ability to react with another amino acid monomer or other reactive group such as a handle until completion of the current addition and deprotection. This ensures that one and only one monomer, handle, or other moiety is added to the growing peptide chain at a time. Examples of protecting groups for amino acids include Boc (tert-butoxycarbonyl), Bn (benzyl), Fmoc (9-fluorenylmethoxycarbonyl), t-Bu (tert-butyl), Fm (9-fluorenylmethyl), Trt (trityl), Cbz (benzyloxycarbonyl), Alloc (allyloxycarbonyl), Ddz (3,5- dimethoxyphenylisoproxycarbonyl), Bpoc (2-(4-biphenyl)isopropoxycarbonyl), Nps (2- nitrophenylsulfenyl), Nsc (2-(4-nitrophenylsulfonyl)ethoxycarbonyl), Bsmoc 1,1- dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl, Dmb (2,4-dimethyloxybenzyl), and pNZ (p- nitrobenzyloxycarbonyl).
[00330] In the amino acid monomers, the protecting group may be used to protect the amino or carboxyl group of the amino acid or a functional side chain. For example, the reactive amino group may be protected during monomer addition of successive amino acid monomers to the peptide chain to ensure that one and only one monomer is added at a time to the peptide chain. On the other hand, the carboxylic acid side chain of aspartic or glutamic acid or the amino side chain of lysine can be protected during peptide synthesis to prevent unwanted side reactions to the side chains.
[00331] Deprotection procedure depends on the protecting group present on the amino acid. Deprotection can occur under acidic or basic conditions. For example, Boc is removed under acidic conditions, namely in the presence of trifluoroacetic acid in dichloromethane. In contrast, Fmoc is removed under basic conditions, namely with secondary amines such as piperidine.
[00332] In a preferred embodiment, the CPP is synthesized using Fmoc protected amino acids using MBHA resin.
[00333] Solid phase peptide synthesis can be carried out with or without the assistance of a microwave. Microwave-assisted solid phase peptide synthesis can accelerate the rate of the amino acid coupling reaction as well as the deprotection reaction. In a preferred embodiment, the CPP is synthesized with the assistance of a microwave.
[00334] In a typical solid phase synthesis, after the coupling of an amino acid monomer and deprotection, the coupling and deprotection steps are repeated with successive addition of monomers until the desired peptide sequence is obtained after which the terminal amino group is deprotected and the peptide cleaved from the solid support resin for recovery.
[00335] Solid phase synthetic techniques are also applicable to the synthesis of the BAC. However, the BAC cannot be directly coupled to the resin and prior to stepwise synthesis of the BAC, a linker, preferably an amino acid or a PEGylated linker, must be coupled to the resin.
[00336] The BAC monomers can similarly be protected with any protecting group such as those described for the protection of the amino group of the amino acid monomers of the CPP. Preferably, the protecting group is Fmoc or Trt.
[00337] The solid phase synthesis of the BAC preferably occurs under basic conditions. In a preferred embodiment, the synthesis occurs in the present of N-ethylmorpholine (NEM) and 5- (ethylthio)-lH-tetrazole (ETT). In another embodiment, N,N-diisopropylethylamine (DIPEA) may be used in place of NEM.
[00338] In a preferred embodiment, the BAC is phosphorodiamidate morpholino oligonucleotides (PMO).
[00339] Referring to the preferred embodiment, the PMO monomers can have the following structures:
Figure imgf000062_0001
[00340] As seen in the structures below and discussed previously, the secondary amine at the 3’ position is protected to prevent unwanted reactions and to ensure that only one monomer is added to the growing chain at a time. The PMO monomers are preferably protected with Fmoc or Trt, which result in the following structures:
Fmoc protected PMO monomers
Figure imgf000062_0002
Trt protected PMO monomers
Figure imgf000062_0003
[00341] While the Fmoc protecting groups in the PMO monomers can be removed under similar basic conditions (piperidine) to those utilized in the deprotection of amino acid monomers, removal of the Trt protecting group (detrityl ati on) is performed under acid conditions. Preferably, the acid used in detritylation is dichloroacetic acid (DCA) or trichloroacetic acid (TCA). [00342] In contemplation of the present solid phase synthetic methods, the PMO monomers may be further modified to comprise a functional group at the 5’ end to facilitate the coupling of the first PMO monomer to the functional group of a handle, which may be a terminal carboxylic acid, a carboxyl group comprising a N-hydroxysuccinimide group, a thiol, or an alkyne. Examples of functional groups that may be attached include amines, thiols, alkynes. In some embodiments, the first PMO monomer is modified to have a primary amine at the 5’ terminus. In achieving a 5’ primary amine functional group, the PMO monomer can be coupled to Lys(mtt)-Fmoc, Universal PEG NovaTag™ resin, or a similar moiety allowing for a similar orthogonal deprotection. As the first monomer addition, the PMO can undergo selective chain extension and cleavage of the PMO monomer or chain extended PMO furnishes a final compound having the 5’ primary amine. Techniques affording the 5’ amine modification can also be combined with other methods to selectively incorporate other functional groups for orthogonal chemistry onto the 5’ end. For example, the 5’ primary amine can undergo a subsequent nucleophilic substitution with a halo- alkyne to afford a PMO having a 5’ alkyne group.
[00343] Similar to synthesis of the CPP, the solid phase synthesis of the BAC can be carried out with or without the assistance of a microwave, which accelerates the rate of the coupling reaction and deprotection.
[00344] As seen in Tables 5 and 6 the handles used to introduce additional functional groups into the compound comprise a variety of structures. While handles bearing structural similarity (i.e. amino acid based) or having similar functional groups (i.e. carboxyl and/or amino functional groups) can be coupled to the compound via similar techniques used to couple amino acid monomers (i.e. DIC/Oxyma), other applicable reactions may be used to couple handles to the compound, which include but are not limited to SN2 reactions, SNI reactions, or reactions with N- hydroxysuccinimide activated esters or anhydrides.
[00345] Because the conditions for the coupling of the amino acid monomers of the CPP, the monomers of the BAC, and the coupling of handles are relatively mild and well-tolerated with little risk of unwanted side reactions occurring, the solid phase synthetic techniques for the CPP, BAC, and handles enable a one pot synthesis of the compound as shown in Fig. 9. The compound can be synthesized in any order. In other words, in some embodiments, the CPP is synthesized first on the resin followed by the BAC and in some embodiments, the BAC is synthesized first on the resin followed by the CPP. The ability to switch between the addition of an amino acid monomer or a monomer for the BAC freely between coupling steps does not limit the scope of the invention to block copolymers comprising the CPP and the BAC and the compound may be present as a random copolymer of amino acid and BAC repeat units.
[00346] Referring to Fig. 10, handles can similarly be integrated using any coupling reaction as previously described. The handle can be incorporated onto the PMO, the peptide, or anywhere internally within the polymeric chain of the compound.
[00347] In a particularly preferred embodiment the linker may incorporate polyethylene glycol in single or multiple units (PEG)n, where n=l to 10 PEG molecules.
[00348] Hereafter, where the CPP comprises the sequence RKF-S5-RLF-S5 or its reverse sequence (S5-FER-S5)FKR and the BFE is a PEGylated SMCC, the resulting compound is termed CP8M. When linked via the BFL to the BAC, which is a PMO, the resulting compound is termed PMO-CP8M.
[00349] Where the CPP comprises the sequence RKF-S5-RLF-S5 and the BFL is a PEGylated hydrazynal nicotinic acid (HNA), the resultant compound is termed HP8M.
[00350] Where the CPP comprises the sequence RKF-S5-RLF-S5 and the BFL is SMCC, the resultant compound is termed C8M.
[00351] Covalent linkage to the CPP may be via, for example, but not limited to, a β-ala or any other suitable moiety.
[00352] In coupling the BAC to the CPP, any form of the linker may be used. In other words, any acceptable salt or derivative of the linker may be used for linking the BAC to the CPP. More in particular, the starting material to achieve linkage between the BAC to the CPP may be in a form that is soluble in organic solvent or in a salt form that is soluble in water. For example, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate) can be in an unmodified form (CAS No. 64987-85-5), which is soluble in organic solvent, or as a sulfo salt (CAS No. 92921-24- 9) for applications that require solubility in water.
[00353] Another preferred linker system is hydrazynal nicotinic acid (HNA), however if the BAC is a PMO, the PMO is modified to incorporate 4-formyl benzoic acid.
[00354] Other linkers such as DSG (disuccinimidyl gluterate) and DSCDS (disuccinimidyl- cyclohexl-l,4-diester) will include the ability to link the 5’-amino group of the BAC to the N- terminus of the CPP. [00355] Handles may serve as linkers that include other elements that confer a desirable property on the compound e.g. spacer between BAC and CPP or an element that will enhance solubility, for example a PEGylated element. Non-limiting examples are shown in Tables 5 and 6. [00356] The biologically active compound is covalently attached to the chimeric cell delivery peptide. Again, this can be done using any method known in the art. Preferably, the cell delivery peptide is attached to the biologically active compound by means of a disulphide bridge or a thiol maleimide linker e.g. SMCC; the attachment may be by means of an amide linker or an oxime linker or a thioether linker.
Pharmaceutical compositions and administration
[00357] The present invention provides a pharmaceutical composition comprising the compound and one or more pharmaceutically acceptable excipients for oral, parenteral, intravenous, or topical administration.
[00358] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable.
[00359] Compositions and formulations for parenteral or intravenous administration include sterile aqueous solutions, which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
[00360] Compositions and formulations for topical administration include solutions, gels, lotions, creams, oil-in water emulsions, water-in-oil emulsions, ointments, shampoos, sprays, sticks, powders, masks, pads, mouth rinses or washes, vaginal gels or suppositories, rectal gels or suppositories, urethral gels or suppositories, or other form acceptable for use on skin, nail, hair, oral mucosa, vaginal or anal mucosa, mouth, or gums.
[00361] The pharmaceutical composition may further include a substance having pharmaceutical activity other than the compound or a substance having no pharmaceutical activity. For example, the pharmaceutical composition can be produced using a commonly used excipient such as fdler, binder, wetting agent, disintegrator, surfactant, lubricant, dispersant, buffer, preservative, solubilizer, antiseptic, coloring agent, flavoring agent, stabilizer, tonicity agent, pH adjustor, solvent or vehicle, or combinations thereof.
[00362] Preferably, the pharmaceutical composition comprises a tonicity agent, pH adjustor, and a solvent as excipients.
[00363] Tonicity agents include but are not limited to dextrose, glycerin, mannitol, potassium chloride, and sodium chloride.
[00364] Solvents or vehicles include, but are not limited to, water, alcohols, glycerin, propylene glycol, and polyethylene glycol 400.
[00365] pH adjusters include acidifying agents and alkalizing agents. Acidifying agents include but are not limited to, acetic acid, citric acid, hydrochloric acid, nitric acid, propionic acid, tartaric acid, fumaric acid, lactic acid, phosphoric acid, sodium phosphate monobasic, potassium phosphate monobasic, citric acid, malic acid, and sulfuric acid. Alkalizing agents include, but are not limited to, ammonia, monoethanol amine, sodium borate, sodium phosphate dibasic, ammonium carbonate, potassium hydroxide, sodium carbonate, trolamine, diethanolamine, sodium bicarbonate, and sodium hydroxide.
[00366] An exemplary pharmaceutical composition formulated for intravenous injection for the treatment of Duchenne’s muscular dystrophy comprises 500 mg of the compound, 80 mg sodium chloride, 2 mg potassium chloride, 2 mg potassium phosphate monobasic, and 11.4 mg sodium phosphate dibasic in 10 mL of water at a pH of 7.5 (50 mg/mL concentration of active ingredient). [00367] The overall dosage of the compound, as administered intravenously to a patient, can range from between about 0.1 μmol/kg and about 10 μmol/kg.
[00368] In defining an intravenous dose in terms of mass, the intravenous dose of the pharmaceutical composition may be 1 mg or more, 5 mg or more, or 10 mg or more. On the other hand, the dose of the pharmaceutical composition may be 150 mg or less, 100 mg or less, 50 mg or less, or 10 mg or less. The number of times of administration of the pharmaceutical composition as described above may be adjusted as appropriate in accordance with the administration target, the body mass of the patient, the treatment stage, the type of pharmaceutical composition, or the like. For example, when a patient is a human, an intravenous dose of the pharmaceutical composition may be 25 mg/kg (2.1 μmol/kg) or more, 50 mg/kg (4.2 μmol/kg) or more, or 82.5 mg/kg (6.9 μmol/kg) or more, and may be 200 mg/kg (16.8 μmol/kg) or less, 175 mg/kg (14.6 μmol/kg) or less, or 125 mg/kg (10.4 μmol/kg) or less. In some embodiments, the dosage ranges between about 1.2 mg/kg and about 120 mg/kg (0.1 μmol/kg to 10 μmol/kg).
[00369] For intravenous, cutaneous or subcutaneous injection, the composition will be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of skill in the art are well able to prepare suitable solutions using, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, Dextrose injection, or combinations thereof, and non-aqueous vehicles such as, but not limited to, ethyl oleate, peanut oil, corn oil, cottonseed oil, sesame oil, or isopropyl myristate, or combinations thereof aqueous and non-aqueous isotonic sterile injection solutions, which can contain bacteriostats, buffers, antioxidants, or solutes that render the formulation isotonic within the blood of the recipient, or combinations thereof; and non-aqueous and aqueous suspensions that can be sterile and can include solubilizers, stabilizers, thickening agents, suspending agents, and preservatives, or combinations thereof. Formulations of the compositions can be presented in unit-dose or multi -dose containers, such as bottles, ampules, syringes, tubes, capsules, and vials.
[00370] In some embodiments, the requisite volume of the pharmaceutical composition is withdrawn from the container and diluted in a vehicle to achieve a volume for administration. The volume for administration can be 25 mL, 50 mb, 75 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 400 mL, or 500 mL. For instance, a volume required for administration of 30 mg/kg of body mass is diluted to a volume between 100 and 150 mL in 0.9% sodium chloride injection, which can be administered to a patient via intravenous injection over a period a time.
[00371] Administration to the patient can take place over any period of time and may take place over a period of 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, or 180 minutes.
[00372] The rate of injection will depend on the total volume to be administered, following dilution in a vehicle, and the infusion time. The rate of infusion can be, but is not limited to 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, or 5 mL per minute.
[00373] There is no particular limitation on the frequency and the number of times of administration of the pharmaceutical composition according to one embodiment, the dose thereof, and the like. For example, the pharmaceutical composition may be administered only once, or may be repeatedly administered. Moreover, the pharmaceutical composition may be administered once a day, twice or more a day, or once in every two or more days. The administration term of the pharmaceutical composition may be 3 days or more and 7 days or less, or 7 days or more. Administration can occur monthly, bimonthly, quarterly, or semiannually.
[00374] After administration of the dosage, such as intravenously, additional vehicle may be used to flush the intravenous route of administration to ensure that the patient receives the entirety of the dose.
[00375] Also contemplated is a method for delivery of a biologically active moiety into a cell comprising contacting the cell with the compound of the present invention. Contacting the cell with the compound may occur utilizing neat compound or the compound may be contained within a composition or formulation.
[00376] The contacting may occur between the compound and any type of cell. In some embodiments, the cell is a muscle cell or a myocyte.
[00377] There are three types of muscles present in the human body, smooth muscle, cardiac muscle, and skeletal muscle, all of which comprise muscle tissue that include the requisite muscle cells.
[00378] Contacting with the compound for the delivery of the biologically active moiety may occur with smooth muscle cells. Smooth muscle lines the walls of a variety of different body cavities and hollow organs including, but not limited to: the gastrointestinal tract, hepatobiliary system, genitourinary tract, ureters, pharageal cavity, vascular spaces, arteries, veins, endolymphatic and cisternes in the intercranial space. Smooth muscle cells, unlike those of skeletal muscle cells, are not striated and do not contain myofibrils.
[00379] Contacting with the compound for the delivery of the biologically active moiety may occur with cardiac muscle cells. Cardiac muscle is striated muscle tissue that is found in the heart, which is under the control of the autonomic nervous system, i.e., the heart is involuntarily controlled. The cells that constitute cardiac muscle are called cardiomyocytes or myocardiocytes. The heart is an organ that is composed mostly of cardiac muscle and connective tissue. Disorders such as Duchenne’s muscular dystrophy initially affect skeletal muscle. However, later progression of the Duchenne’s muscular dystrophy spreads to cardiac muscle and can lead to death caused by respiratory or cardiac failure.
[00380] Contacting with the compound for delivery of the biologically active moiety may also occur with skeletal muscle. Skeletal muscle comprises striated muscle tissue which is under the control of the somatic nervous system, i.e., it is voluntarily controlled. The term muscle refers to multiple bundles of muscle fibers held together by connective tissue. Skeletal muscles may be attached to bones by tendons. Skeletal muscles may be attached to bones by tendons. Non-limiting examples of skeletal muscles include, for example, the diaphragm, extensor digitorum longus, tibialis anterior, gastrocnemius, soleus, plantaris, biceps, triceps, deltoids, pectoralis major, pectoralis minor, rhomboids, trapezius, sartorius, knee flexors and extensors, elbow flexors and extensors, shoulder abductors, and abdominal muscles. In muscular dystrophy, the cells lack the cytoskeletal protein dystrophin. The dystrophin protein provides stability to the sarcolemma (i.e., the cell membrane of muscle cells) by linking the intracellular cytoskeletal network to the extracellular matrix. In the absence of dystrophin, muscle contraction mechanically stresses the cell membrane, inducing progressive damage to the myofibers initially in skeletal muscle.
[00381] Based on the different and distinct types of muscle tissue present in the human body and the ability of the present compounds to deliver a biologically active moiety to the cells of the muscle tissue via contacting the respective cells of the muscle tissue with the compound, the compound can be tissue specific for targeted delivery of the biologically active moiety. For example, the compound can have specificity for tissue including, but not limited to smooth muscle tissue, cardiac muscle tissue, and skeletal muscle tissue.
[00382] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.
[00383] The present invention will be further illustrated in the following Examples, which are given for illustration purposes only and are not intended to limit the invention in any way.
Examples
Materials and methods
Nuclear Magnetic Resonance (NMR) Analysis
[00384] 1H NMR spectra were recorded using a Bruker Avance III 500 (500 MHz) spectrometer. Samples were dissolved in H2O with 10% D2O and 10 mM sodium acetate.
[00385] NOESY spectra were recorded with a 12626.263 Hz sweep width, 4096 complex points (DQD acquisition mode) in the direct dimension and 1024 indirect points (States-TPPI acquisition mode). A NOESY mixing time of 250 ms was used to provide cross peaks with high signal to noise while largely avoiding spin diffusion. A pre-saturation pulse on water and a 3-9-19 pulse sequence with 20% Z-gradients (4,5) aided solvent suppression. TOC SY spectra were recorded with the same spectral width and resolution as the NOESY with a homonuclear Hartman-Hahn transfer using the MLEV17 sequence for an 80 ms mixing time (6). Two power levels were used for excitation (3 dB) and spinlock (12.2 dB). Water suppression was achieved as with the NOESY.
High Resolution Mass Spectroscopy
[00386] High-resolution mass spectra were recorded on a Thermo scientific LQT Orbitrap XL under electron spray ionization conditions (ESI) or where indicated under Atmospheric Pressure Ionization (API) condition.
Circular Dichroism (CD) Spectroscopy
[00387] CD analysis was performed on an Applied Photophysics Chirascan Circular Dichroism spectrometer. Samples were dissolved in D2O at 0.125 w/w% and data acquired in triplicate at room temperature and subsequently averaged and smoothed using built in qCD software. Graphs were plotted by subtracting a blank D2O spectrum from the acquired data to provided blank correction.
Example 1 - Solid phase peptide synthesis
[00388] Peptides were synthesized utilizing Fmoc-protected amino acids or reagents and microwave assisted solid phase peptide synthesis techniques.
[00389] Referring to Fig. 1A, in a typical coupling step, the Fmoc-amino acid or reagent to be coupled (0.5 mmol, 0.2 M in DMF), N,N-Diisopropylcarbodiimide (DIC) (0.5 mmol, 0.5 M in DMF), Oxyma ((ethyl (E)-2-cyano-2-(hydroxyimino)acetate) (0.5 mmol, 1 M in DMF) and Fmoc- Rink Amide MBHA resin (ChemMatrix®) (0.1 mmol, preswelled in DMF) were added to a reaction vessel and heated in a microwave until the reaction was complete.
[00390] The reaction time and temperature depend on the particular Fmoc amino acid or reagent to be coupled.
[00391] The coupling of a hydrophobic amino acids or handles require at least 300 s at a reaction temperature of 90 °C to complete the coupling reaction. [00392] The coupling of Fmoc-protected histidine requires at least 600 s at a reaction temperature of 50 °C to complete the coupling reaction.
[00393] The coupling of all other amino acids requires at least 120 s at a reaction temperature of 90 °C to complete the coupling reaction.
[00394] Deprotection (i.e. removal of the Fmoc protecting group) occurs via reacting the resin containing the peptide with piperidine (20 v/v% in DMF) for at least 90 s at a reaction temperature of 90 °C.
[00395] The coupling steps described above are repeated with each successive amino acid monomer.
[00396] The reaction conditions for coupling of amino acids and other reagents (handles) and Fmoc deprotection via microwave assisted solid phase synthesis is summarized in Table 9 below:
Table 9. Reaction conditions for amino acid coupling and Fmoc deprotection
Figure imgf000071_0001
[00397] The amount of resin utilized in the coupling and deprotection reactions determines the ultimate scale of the reaction and all other reagents are scaled according to the quantity of resin. The excess quantity of reagent can range from a 3 -fold excess to a six -fold excess depending on the scale of the reaction.
Example 2 - Ring closing metathesis
[00398] Ring closing metathesis was performed with Grubb’s catalyst M102 (Benzylidene- bis(tricyclohexylphosphine)dichlororuthenium, 6 mM in DCE) (Fig. IB). The resin containing completed crude peptides was washed with DCE (4x) before addition of Grubb’s catalyst (6 mmol, 20 mol%). The mixture was then reacted at 25 °C for 30 minutes, washed again with DCE (4x), and a second aliquot or Grubb’s catalyst (6 mM, 20 mol% in DCE) added and reacted for a further 30 minutes. Resins were transferred to a SPE tube, washed with DCM (4x), and subsequently dried by gentle vacuum via a vacuum manifold. Although it is preferred to conduct the ring closing metathesis while the peptide remains anchored to the resin, it is also possible to perform the ring closing metathesis following cleavage of the peptide from the resin.
Example 3 - Cleavage of peptide from resin
[00399] To the dried resin, in a round bottom flask, a cleavage solution was freshly prepared containing TFA (Trifluoroacetic acid)/T!S (Triisopropylsilane)/H2O/DODT (3,6-dioxa-l,8- octanedithiol) (92.5/2.5/2.5/2.5 v/v%) at 5 mL per 250 mg of dried resin, but may be adjusted depending on the resin. The cleavage solution and resin mixture were heated 40°C using a hot plate and mantel and stirred for a minimum of 30 minutes. The mixture was then added to an SPE column and the filtrate added drop wise to a 50 mL falcon tube containing Et2O (3 x 35 mL, per 0.1 mmol synthesis). The crude peptide participate was collected by centrifugation (5 min, 1000 xg). The supernatant was aspirated from the peptide pellet, MeCN (1 mL per 0.1 mmol crude peptide) added, and then diluted with H2O (5 mL). The crude solution was transferred to a glass vial for subsequent lyophilization. Alternatively, after collection of the filtrate in Et2O, the mixture can be partitioned over water and the cleaved peptide can be extracted with subsequent washing of the organic layer with water (3x). The aqueous layers can be combined and transferred to a vial for lyophilization.
Example 4 - Lyophilization
[00400] In the subsequent lyophilization, the crude peptide is frozen on dry ice for a minimum of 1 hour. Vials are then transferred to a PTFE coated freeze dryer with a -105 °C condenser and primary lyophilization performed (72 hours 0.05 mbar).
Example 5 - Incorporation of handle at the N terminus of peptide
[00401] The incorporation of a handle onto the N terminus of the peptide involves an additional coupling step. If the handle is contained on a carboxylic acid, the additional coupling step follows the procedure of Example 1 to couple the handle onto the N terminus of the peptide. Referring to Fig. 3 A, S-Trityl-3-mercaptopropionic acid can be coupled to the N terminus of a peptide utilizing the procedures and conditions of Example 1.
[00402] Coupling of the handle may occur prior to or after ring closing metathesis to form the cross link or bridge between amino acids. If the coupling of the handle to the peptide occurs after ring closing metathesis, the N terminus must remain protected with an Fmoc during the ring closing metathesis.
[00403] A handle can also be incorporated via nucleophilic addition to the nucleophilic N terminus. In an exemplary coupling, referring to Fig. 3B, 4-bromo-l -butyne (0.5 mmol) and trimethylamine (0.5 mmol) was added to a DMF solution of resin containing deprotected peptide (0.1 mmol). The reaction was bubbled under nitrogen for 20 minutes between 25°C and 90°C and heated via microwave irradiation. After the modification the reaction mixture was washed with DMF (3x) and cleaved from the resin. The reaction was monitored by performing a “mini cleavage” in which a small aliquot of resin was removed and the peptide cleaved, the crude sample was analyzed by LCMS to ascertain the molecular weight of the species present. If the LCMS results indicated that the reaction was incomplete, the coupling reaction was repeated and reanalyzed until the LCMS results indicated that the reaction was complete.
[00404] The nucleophilic addition can also occur using an N-hydroxysuccinimide (NHS) activated ester. In a typical NHS reaction, the amine component (1 equivalent) is stirred with the desired NHS ester (1.5 equivalents) in the presence of trimethylamine (2 equivalents) in DMF solvent at 30 °C for 24 hours. The reaction was bubbled under nitrogen for 20 minutes between 25°C and 90°C and heated via microwave irradiation. After the modification the reaction mixture was washed with DMF (3x) and cleaved from the resin. The reaction was monitored by performing a “mini cleavage” in which a small aliquot of resin was removed and the peptide cleaved, the crude sample was analyzed by LCMS to ascertain the molecular weight of the species present. If the LCMS results indicated that the reaction was incomplete, the coupling reaction was repeated and reanalyzed until the LCMS results indicated that the reaction was complete.
Example 6 - Incorporation of handle at the C terminus of peptide
[00405] Incorporation of a handle at the C terminus of the peptide involves coupling an amino acid or another linker comprising a protected orthogonal group into the peptide chain using the procedure of Example 1. For example, referring to Fig. 4A, Fmoc-Lys-(Mtt)-OH (Mtt = 4-methyl trityl) may be the first amino acid coupled to the resin in the solid phase synthesis of the peptide using the procedure of Example 1. Following completion of all coupling reactions, the Mtt protecting group is removed via suspension of the resin containing the peptide in trifluoroacetic acid/triisopropylsilane/dichloromethane (1 :2:97 v:v:v) and gently shaken for 30 minutes after which the resin is filtered, washed twice with dichloromethane, twice with methanol, twice again with di chloromethane, twice with 1% diisopropylamine in DMF, and twice with DMF to obtain deprotected lysine to which S-Trityl-3-mercaptopropionic acid can be coupled to the exposed amine group of the deprotected lysine using the procedures of Example 1. Because removal of the Mtt group occurs under acidic conditions similar to those conditions used for cleaving the peptide as described in Example 3, the resin used is for solid phase synthesis is HMBA.
Example 7 - Solid phase synthesis of phosphorodiamidate morpholino oligonucleotides (PMO) comprising Fmoc protected PMO monomers
[00406] Referring to Fig. 5, Fmoc-Rink Amide MBHA resin or PEG-HMBA resin (ChemMatrix®) is swelled in DMF for 30 mins prior to synthesis of the desired PMO. Prior to coupling the first PMO, a linker must be coupled to the resin. The linker is typically an amino acid or Fmoc-PEG(n)-CO2H and coupling of the linker to the resin follows the procedure of Example 1.
[00407] PMO monomer (0.2 M in DMF), NEM (0.2 M in DMF) and ETT (0.2 M in DMF) are added to the microwave reactor with the preswelled resin and heated to the desired temperature. Once completed the resin is washed (3x, DMF) and the PMO is deprotected via the addition of piperidine (20 v/v% in DMF) to the resin and heated.
[00408] Following removal of the Fmoc protecting group from the PMO, the coupling procedure is repeated with successive PMO monomers.
Example 8 - Solid phase synthesis of phosphorodiamidate morpholino oligonucleotides (PMO) comprising trityl protected PMO monomers
[00409] Referring to Fig. 6, Fmoc-Rink Amide MBHA resin or PEG-HMBA resin (ChemMatrix®) is swelled in DMF for 30 mins prior to synthesis of the desired PMO. Prior to coupling the first PMO, a linker must be coupled to the resin. The linker is typically an amino acid or Fmoc-PEG(n)-CO2H and coupling of the linker to the resin follows the procedure of Example 1.
[00410] PMO monomer (0.2 M in DMF), NEM (0.2 M in DMF) and ETT (0.2 M in DMF) are added to the microwave reactor with the preswelled resin and heated to the desired temperature. Once completed the resin is washed (3x, DMF) and then trityl deprotection (detritylation) performed.
[00411] To accomplish detritylation, TCA or DCA (1-3 v/v% in DCM) was added to the resin, and the eluent monitored by UV-vis spectroscopy at 495 nm for the completion of the detritylation reaction. After removal of the trityl protecting group the monomer coupling step is repeated with each successive PMO monomer to be coupled. The reaction is monitored until there is a 95% reduction from the peak reading. An successful coupling can be ascertained from a lower or absent peak reading from prior coupling reactions.
[00412] The conditions utilized for coupling of PMO monomers and detritylation is shown in Table 10:
Table 10. Reaction conditions for coupling of PMO monomers via step-wise synthesis
Figure imgf000075_0001
Figure imgf000076_0001
Example 9 - Incorporation of handle at the 3 ’ end of the PMO
[00413] The incorporation of a handle onto the 3’ end of the PMO requires modification of the secondary amine on the morpholino ring of the PMO. If the handle is contained on a carboxylic acid, the additional coupling step follows the procedure of Example 1 to couple the handle onto the 3’ end of the PMO.
[00414] A handle can also be incorporated via nucleophilic addition to the 3’ end of the PMO. In an exemplary coupling, referring to Fig. 7, 4-bromo-l -butyne (0.5 mmol) and trimethylamine (0.5 mmol) was added to aDMF solution (4 mL) of resin containing PMO (0. 1 mmol). The reaction was bubbled under nitrogen for 20 minutes at a temperature range from room temperature to 90 °C and heated via microwave irradiation. The reaction was monitored by performing a “mini cleavage” in which a small aliquot of resin was removed and the PMO cleaved. The crude sample was measured by LCMS and the 3’ modification reaction repeated to achieve a higher completion. Following completion of the reaction, the reaction mixture was then washed with DMF (3x) and cleaved from the resin.
Example 10 - Incorporation of handle at the 5 ’ end of the PMO or internally within the PMO chain [00415] Incorporation of a handle at the 5’ end of the PMO chain requires the addition of a residue that can be modified orthogonally to contain the functionality of the handle.
[00416] Referring to Fig. 8, Fmoc-Lys-(Mtt)-OH was coupled to the resin according to the procedure of Example 1. The Mtt protecting group is removed via suspension of the resin containing the peptide in trifluoroacetic acid/triisopropylsilane/dichloromethane (1 :2:97 v:v:v) and gently shaken for 30 minutes after which the resin is filtered, washed twice with dichloromethane, twice with methanol, twice again with dichloromethane, twice with 1% diisopropylamine in DMF, and twice with DMF to obtain deprotected lysine. S-trityl-3-mercaptopropionic acid (0.05 mmol) was coupled to the exposed amine group according to the procedure of Example 1. Example 11 - Thiol-ene Michael addition
[00417] A typical thiol-ene Michael addition reaction between a thiol and a maleimide is illustrated below.
[00418] The thiol component (1.2 mmol) and the maleimide component (1 mmol) are added to a solution of acetonitrile containing PBS buffer (pH 6.8). The reaction is stirred at room temperature for up to 6 hours to complete the reaction. Aliquots of the reaction mixture were periodically removed for monitoring reaction progress by LCMS. If needed, a further aliquot of the thiol component can be added to promote completion of the reaction.
[00419] The thiol-ene Michael addition can also undergo reduction utilizing resin-bound tris(2- carboxyethyl) phosphine (TCEP) to reduce disulfide reactants. In a typical TCEP reduction, 10 equivalents of TCEP are added to a PBS solution of the disulfide-containing product in water with EDTA (2 mL) under stirring at room temperature. The solution is stirred at room temperature for approximately 10 minutes to complete the reaction. The now reduced thiol compound is eluted from the resin, by transferring the reaction mixture to an empty SPE tube and washing the resin with water (3x). The eluate is collect directly into the reaction vessel containing the prepared Michael acceptor. The Michael addition is monitored by LCMS and upon completion the desired product in isolated by preparative RP-HPLC.
Example 11 - Hydrazone ligation
[00420] In a typical hydrazone ligation, the hydrazine (1.1 mmol) and the aldehyde (1.0 mmol) components are added to a solution of PBS buffer containing 10 mM of aniline (pH 6.0) and the reaction is stirred at room temperature for 1-16 hours to complete the reaction. Aliquots were periodically removed from the reaction for monitoring reaction completion by LCMS. Upon completion, the desired product was isolated by preparative RP-HPLC.
Example 12 - Huisgen 1, 3 dipo1αr cycloaddition
[00421] The following stock solutions are prepared:
[00422] 1. 200 mM tris-hydroxypropyltriazolylmethylamine (THPTA) in water;
[00423] 2. 100 mM CuSO4 in water;
[00424] 3. 100 mM sodium ascorbate in water; [00425] 4. Alkyne component in water (2 mM); and
[00426] 5. 10 mM azide component in DMSO/tert-butyl alcohol, or water.
[00427] The CuSO4 solution is incubated with the THPTA solution prior to carrying out the cycloaddition reaction at a 1 :2 ratio of CuSOr solution to THPTA solution. The solution can remain stable for several weeks in a frozen state.
[00428] An excess (1-50 equivalents) of the azide component is added to the solution containing the alkyne component.
[00429] 25 equivalents of the THPTA/CuSO4 solution are subsequently added to the alkyne/azide solution and briefly mixed prior to the addition of sodium ascorbate.
[00430] 40 equivalents of the sodium ascorbate solution are added to the alkyne/azide solution containing THPTA/CuSO4 to imitate the coupling reaction between alkyne and azide. The reaction was stirred at room temperature for 30-60 minute and the reaction was monitored by LCMS. The conjugate can either be precipitated in EtOH, acetone or isolated by preparative RP-HPLC. The THPTA chelates and Cu are removed during isolation by RP-HPLC or by precipitation of the conjugate in EtOH or acetone.
Example 13 - One pot synthesis ofPMO-CP8M
[00431] The synthesis of PMO-CP8M according to FIG. 11B can occur in one pot starting from a single batch of resin, avoiding the need for a coupling reaction between discrete PMO and CPP species.
[00432] The CPP having the sequence RKF-S5-RLF-S5 is synthesized via solid phase synthesis according to the procedures of Example 1 using Fmoc protected amino acids. Following the coupling of the terminal S5 residue, the peptide undergoes ring closing metathesis to crosslink the two S5 residues according to the procedures of Example 2. The Fmoc protecting group at the N terminus is subsequently removed, exposing a terminal amine and providing the CPP.
[00433] Fmoc-NH-PEG5-CO2H is coupled to the CPP via the procedures of Example 1 and the Fmoc group is removed.
[00434] To construct the PMO, a first PMO nucleotide monomer (C), is then directly reacted with the now exposed nucleophilic N terminus of the peptide via a nucleophilic substitution reaction as shown in Figure 5. The Fmoc protecting group is removed from the 3’ secondary amine of the nucleotide and the PMO having the sequence is synthesized stepwise
Figure imgf000079_0001
via the procedures of Example 7.
[00435] Following the coupling of the last nucleotide residue, the Fmoc protecting group is removed to give PMO-CP8M that is anchored to the resin.
[00436] The compound can be recovered through cleavage from the Rink amide resin using the procedures of Example 3. If HMBA resin is used, cleavage is performed by exposure to nucleophile such as alkoxide, thiolate, amines or hydroxides.
[00437] A general procedure for cleavage is as follows. To the dried resin, in a round bottom flask, a cleavage mixture comprising a nucleophile was prepared in an appropriate solvent i.e. methoxide in methanol, hydroxide in water. The nucleophile solution is typically used at 5 mL per 250 mg of dried resin and contains lOOx excess of the nucleophile to the resin, but may be adjusted depending on the resin and nucleophile. The cleavage solution and resin mixture were stirred for a minimum of 15 mins (sequence specific cleavage times apply). The mixture was then added to an SPE column and the filtrate eluted using MeCN in H2O (25 v/v% MeCN) and collected. The crude solution was frozen and lyophilized before isolation of the desired compound by RP-HPLC.
Example 14 - Biological Activity of Peptide-Oligonucleotide Conjugates
[00438] To ascertain the biological activity of constructs comprising the peptide conjugated via a bifunctional linker to a biologically active oligonucleotide according to the present claims, experiments were conducted using peptides of varying amino sequences conjugated via a bifunctional linker to a biologically active oligonucleotide, in particular, an antisense oligonucleotide that acts to restore a reporter gene’s expression (EGFP) via correction of aberrant RNA splicing. The peptides in the following Table 11, were used as cell penetrating peptides in these experiments:
Table 11. Peptides used to study biological activity of PEPTIGOs
Figure imgf000079_0002
Figure imgf000080_0001
[00439] In the above table, the amino acid “S5” is (S)-pentenylalanine and “S8” is S- octenylalanine. The abbreviation 3TPA is 3-(tritylthio)propionic acid, which is substituted for the bifunctional linker in constructing the molecule.
[00440] Peptides were investigated as either stabilized species, in which there is a staple between the S5 amino acid residues, or unstabilized and thus, unstapled species. Stapling in stabilized peptides was accomplished via olefin metathesis with Grubbs’s catalyst. The crosslinked amino acid residues for a particular stapled peptide species are enclosed within the parentheses in Table 11. The bifunctional linker utilized was succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (SMCC). The peptide, via a terminal cysteine residue, as part of the bifunctional linker, bonds via a thiol -ene coupling to the maleimide functional group of SMCC.
[00441] The biologically active oligonucleotide is a phosphordiamidate morpholino oligonucleotide (PMO) having the nucleotide sequence of 5’ATATTGCTATTACCTTAACCCAGAA3’ (SEQ ID NO: 101) in which the 5’ end was modified to have a primary amine functionality, which permits nucleophilic coupling at the N- hydroxysuccinimide functionality of SMCC. The PMO targets an enhanced green fluorescent protein (EGFP) sequence that is interrupted by the intron of human β-globin encoding the position 654 splicing mutation. Upon splicing correction with the PMO, functional EGFP is expressed. The molecules comprising the peptide (both stabilized via stapling and unstabilized) and PMO conjugated via bifunctional linker were designated PEPTIGOs.
[00442] A HeLa cell line expressing a mutated human β-globin intron, IVS2-654 was maintained at below 80% confluence in D-MEM supplemented with 10% fetal calf serum. Cells were seeded 24 hours before treatment in 96-well plates at ~ 104 cells per well in 100 μL of vehicle. For the uptake experiments, cells were plated in 96-well plates at 1 x 104 cells per well in 100 μL of vehicle. The growth vehicle was removed and replaced with 100 μL of fresh growth vehicle containing either only fresh vehicle or varying concentrations (in μM) of: i) unconjugated PMO, ii) unstabilized (i.e. unstapled) PEPTIGO; and iii) stabilized (i.e. stapled) PEPTIGO. Cells were assayed 24 hours later. The treatment media was removed (used for the lactate dehydrogenase assay described below to measure cell toxicity) and replaced with HBSS and fluorescence measured using a fluorescence plate reader (spectramax iD3) with monochromator set to 475/515 nm (ex/em).
[00443] For assessment of cell toxicity Promega LDH-Glo Cytotoxicity assay was used. After 24 hour treatment, the cell supernatant was removed and 10 μL used in the LDH assay per manufacturer’s instruction. Ideal PEPTIGO candidates are those that demonstrate pharmacological dose-response with high EGFP expression levels, indicative of enhanced cell penetration and biological activity, in which cell toxicity is absent or minimal. The experimental results from the treatment of cells for the restoration of EGFP expression using the PEPTIGOs are provided in Figs 12A-D, 13 A-C. The data is shown as the mean value +/- the calculated standard deviation.
[00444] Additional experiments were performed with a different series of PEPTIGOs utilizing the peptide TAT8M. This data is shown in Figs. 13A-C. (cell toxicity expressed as fluorescence measured from LDH assay in which a higher fluorescence intensity corresponds to higher cell toxicity).
[00445] The stapled PEPTIGOs comprising the peptides TAT8M 4, 8, TAT8M1 4, 8, and TAT13M 5, 9 exhibited high EGFP expression at low doses, approximately 50,000 in fluorescence intensity at 10 μM, and low cell toxicity. These results show that there is cellular uptake of the PEPTIGOs and retention of the biological activity of the PMO. With respect to the stapled PEPTIGO utilizing TAT8M 4,8, the data demonstrated that the stapled PEPTIGO exhibited higher fluorescence intensity than that of the unstabilized (i.e. unstapled) PEPTIGO in addition to that of the PMO alone (unconjugated), and the delivery vehicle, which indicates an improved cellular uptake in the stapled PEPTIGO analog, compared to the unstabilized analog, and retention of the PMO’s biological activity. Surprisingly, however, the stapled PEPTIGO utilizing TAT8M1 4,8 exhibited a lower EGFP expression than that of the unstapled analog. Thus, the stapled PEPTIGO exhibited reduced biological activity, thereby decreasing the measured EGFP expression in the cells. TAT8M 4,8 and TAT8M1 4,8 are isomers, the only difference being the swapping of the positions of arginine and glutamine residues at positions 6 and 7. It was not expected that the unstabilized PEPTIGO analog utilizing the TAT8M1 4,8 isomer in which the positions of two adjacent residues are swapped would outperform the stabilized PEPTIGO using TAT8M 4,8. Also surprising, with respect to the PEPTIGO using TAT13M 5,9, while the PEPTIGOs outperformed the unconjugated PMO alone and the delivery vehicle, both the stabilized and unstabilized PEPTIGO exhibited comparable EGFP expression and cell toxicity at similar PEPTIGO concentrations. In other words, there was no increase or decrease in cellular uptake and resulting biological activity between stapling and not stapling the peptide, all else being equal.
[00446] While the stapled PEPTIGO utilizing ARE8M 4,8 exhibited higher fluorescence at higher doses compared to that of the unstapled analog, thus exhibiting increased cellular uptake and retention of biological activity, the stapled PEPTIGO exhibited higher cell toxicity than that of the unstabilized PEPTIGO, which was unexpected. Also identified and isolated was a minor product following olefin metathesis in the synthesis of ARE8M 4,8, which is believed to be the E- olefin isomer whereas the major product is the Z-olefin isomer. The activity of this minor product was investigated It was not expected that the E-olefin isomer, in which only the orientation of the olefin double bond differs when compared with the Z-olefin isomer, would both exhibit very little EGFP expression but improved cell viability as compared to the major Z-olefin product.
[00447] As can be seen in the data drawn to cMYC-PAL 4,8, cMYC 4,8, IL1 1,5, IL1 2,6, and Xentry PEPTIGOs, even though the stapled PEPTIGOs exhibited higher fluorescence intensities (higher EGFP expression) and minimal cell toxicity compared to those of the unconjugated PMO, delivery vehicle, and unstabilized analogs, the measured fluorescence intensity was significantly decreased from those of the TAT8M series (between about 20,000 to 30,000 at 40 μM in stapled cMYC-PAL 4,8, cMYC 4,8, IL1 1,5, IL1 2,6, and Xentry PEPTIGOs versus about 200,000 in the stapled TAT8M PEPTIGO), indicating a decrease in biological activity. Despite enhancement of biological activity of the PEPTIGO via stapling as compared to that of the unstapled PEPTIGO, it was not expected that the specific sequence of the peptide and the position of the unnatural amino acids in the crosslink, such as in the comparison of IL1 1,5 and IL1 2,6 in which the unnatural amino acid positions forming the crosslink were shifted one position towards the N-terminus in IL1 2,6 and increased EGFP expression in the stabilized PEPTIGO of the former compared to that of the latter, contributed to the observed biological activity of the PEPTIGO.
[00448] As shown in the data in Figs. 13A-C, the PEPTIGO utilizing stapled TAT13M1, in which the position of the glutamine and arginine residues at positions 6 and 7, respectively, are swapped compared to the peptide TAT13M, surprisingly demonstrated higher EGFP expression via higher fluorescence intensity than that of the PEPTIGO utilizing TAT13M (approximately 260,000 in TAT13M versus about 450,000 in TAT13M1 at 40 μM) and low cell toxicity. These results indicate that sequence specificity plays a role in the overall observed biological activity of the PEPTIGO. In studying the PEPTIGO utilizing the stapled J16-S8 peptide, which exhibited similar cell viability as both the vehicle and the conjugated PMO, it was observed that the unconjugated PMO exhibited higher EGFP expression compared to the stabilized peptide. This was surprising because the unconjugated PMO does not have any means or carrier to cross the cell membrane to enter a cell. Thus, the unconjugated PMO was not expected to exhibit any improved cellular uptake. These results demonstrate the poor cellular uptake of the stapled J16-S8 peptide. In comparing the activity of PEPTIGOs utilizing the stapled or unstabilized SV40 3,7, the unstabilized PEPTIGO unexpectedly exhibited higher EGFP expression than the stabilized stapled PEPTIGO analog, indicating that the unstabilized PEPTIGO exhibits higher biological activity. The sequence of SV40 3,7 may not be amenable to stapling and the conferred conformation may be disfavored, which resulted in the observed decreased biological activity. Further analysis of an unstabilized isomer of SV40 3,7 where the positions of the unnatural amino acids were shifted back one residue towards the C terminus (i.e. SV40 2,6) indicated that despite the superior performance of the unstabilized PEPTIGO utilizing SV40 3,7 over the stapled, stabilized analog, shifting the positions of the unnatural amino acids resulted in a decrease in EGFP expression by more than half (about 400,000 in SV40 3,7 versus about 175,000 in SV40 2,6) despite SV40 2,6 exhibiting an improvement in cell viability.
[00449] Based on the above data, increasing the rigidity of a peptide and stabilizing the secondary and tertiary structure of the peptide is the extent of what can be expected from stapling the peptide through formation of the hydrocarbon bridge through olefin metathesis. The resulting biological activity of the PEPTIGO comprising the cell penetrating peptide and biologically active PMO covalently linked via bifunctional linker being able to penetrate the cell membrane and retain the biological activity of the PMO cannot be predicted based simply on whether the cell penetrating peptide is stabilized via stapling or not. The unifying factor appeared to be the specific sequence of the peptide in combination with stabilization via hydrocarbon crosslinking and the specific location of the crosslinking (i.e. positions of unnatural amino acids forming the crosslink) that contributed to the improvement in biological activity and cell viability. Switching the positions of two adjacent amino acid residues as in TAT8M and TAT8M1, which are isomers and would be expected to have similar activity, resulted in the unstabilized and unstapled PEPTIGO exhibiting superior activity compared to the stapled and stabilized PEPTIGO analog. Shifting the amino acid positions of the crosslink between IL1 1,5 and IL1 2,6 continued to improve the EGFP expression of the stabilized PEPTIGO compared to that of the unstabilized analog, whose activity decreased with the shift in unnatural amino acid residue positions. Accordingly, the observed biological activity is sequence specific and cannot be predicted based solely on patterns of amino acid residues or a core amino acid structure. It is the specific combination of the particular amino acid sequence, the presence of stabilization via hydrocarbon crosslink, and the specific positions of the unnatural amino acids partaking in crosslinking that contributed to the observed biological activity of the PEPTIGO.
Example 15 - Method of administration in treating Duchenne muscu1αr dystrophy
[00450] An exemplary pharmaceutical composition formulated for intravenous injection comprises 500 mg of the compound, 80 mg sodium chloride, 2 mg potassium chloride, 2 mg potassium phosphate monobasic, and 11.4 mg sodium phosphate dibasic in 10 mL of water at a pH of 7.5 (50 mg/mL concentration of the compound as the active ingredient).
[00451] The utilized dosage is 30 mg per kilogram of body mass.
[00452] For a patient having a body mass of 33.3 kg, 1000 mg of the compound are required for a full dosage regimen. Based on a 10 mL vial containing 500 mg of the compound, a total of 20 mL of the pharmaceutical composition is needed.
[00453] Prior to administration to the patient, 20 mL of the pharmaceutical composition are withdrawn from the vials containing the composition and the 20 mL dosage is diluted in 0.9% sodium chloride injection, USP, to a total volume of 100-150 mL. The solution is then administered intravenously to the patient using an in-line 0.2 micron fdter over the course of 35 to 60 minutes at a rate of approximately 1.5-4.5 mL per minute. After completion of the infusion, the intravenous access line is flushed with additional 0.9% sodium chloride injection to ensure that the entire dosage is administered to the patient.
[00454] Administration to the patient occurs on a weekly basis.
Example 16 - Effect of a stapled inter leukin- la (IL1α) NLS peptide and PMO conjugate on dystrophin expression in heart and skeletal muscle [00455] In a prior study, a compound comprising a CPP designated “ARE8M” ((S5-FLR- S5)FKR-3TPA) (SEQ ID NO: 23) conjugated to a splice-modifying PMO that was subsequently administered intravenously into D2-mdx mouse model of DMD, resulted in increased dystrophin expression in the heart and diaphragm compared to administration of PMO alone. However, significant acute adverse effects upon intravenous administration were observed at dose levels required for this effect. These results suggested that improvements in the peptide domain are needed to achieve efficacy in both heart and skeletal muscle at doses that are safe.
[00456] Splice modification requires successful delivery of the compound to the cell nucleus. Nuclear localization signals (NLS) are short signal peptides which facilitate the transport of proteins from the cytoplasm to the nucleus through the nuclear pore complex. Classical NLS sequences contain a cluster of basic amino acids (e.g. arginine or lysine) which are believed to be important for imparting cell-penetrating properties onto a molecule. The combination of these factors makes peptides based on a classical NLS candidates as peptide domains for the present compounds.
[00457] In this study, the effect of two peptide-oligonucleotide conjugates comprising a stapled interleukin- 1α (IL 1α) NLS peptide on dystrophin expression in heart and skeletal muscle using the D2-mdx mouse model of DMD. In particular, the study compares the effects of dystrophin exon 23-skipping IL1α-NLS-PMO conjugate and unconjugated PMO on dystrophin expression in the D2-mdx mouse. The NLS is from pro-IL1α, and cleaved upon activation of IL1α. Pro-IL1α is a human NLS that has been shown to facilitate nuclear translocation in human cells and is not viral. Because of its role in immunology, pro-IL1α is processed differently in tissues acting as either a NLS or a nuclear export signal. This may be of importance in an inflammation driven disease [00458] IL1α NLS peptides including (S)-pentenylalanine (S5) monomers (see Table 12 for sequences) were synthesized and stabilized by olefin ring-closing metathesis by Peptide Protein Research Ltd (Hampshire, UK).
Table 12. IL1α NLS peptide sequences
Figure imgf000085_0001
Figure imgf000086_0001
Bracketed numbers indicate the position of the S5 monomers relative to the (PEG)5 moiety. S5: pentenylalanine; PEG: polyethylene glycol.
[00459] PMO with 5’ primary amine and 3’ carboxyfluorescein (PMO-FITC) targeting the splice donor site at the 5’ end of intron 23 of the mouse dystrophin gene (5’-NH2- GGCCAAACCTCGGCTTACCTGAAAT-FITC-3’) (SEQ ID NO: 99) (Gebski et al., 2003) was synthesized and supplied in lyophilized form by Gene Tools, LLC (Oregon, USA). FITC- conjugated PMO was obtained because the fluorescent label allows for localization of PMO in in vitro experiments and potentially in in vivo experiments.
[00460] Conjugates utilizing an IL1α NLS-based peptide as the CPP were produced by conjugating IL1α NLS peptide to PMO-FITC via a SMCC linker to produce IL1α NLS-PEG5-C- SMCC-PMO-FITC using methods described below.
Peptide-PMO conjugation
[00461] PMO-FITC (280 mg, 30 μmol) was dissolved in PBS (9 mL, pH 7.4) and MeCN added (1 mL). This solution was incubated at room temperature after the addition of SMCC linker (100 mg, 229 μM, 10x excess) directly to the reaction vessel. After 6 hours the mixture was frozen and freeze-dried. The lyophilized powder was then reconstituted in 20% MeCN (1 mL) and desalted using sephadex g25 (medium) hydrated in H2O. SMCC-modified PMO-FITC was eluted and then freeze-dried to yield SMCC-PMO-FITC (201 mg, 21.6 μmol). Samples of SMCC-PMO-FITC were sent for mass spectrometry analysis. The initial purity of the SMCC-PMO-FITC was typically 70- 80% by HPLC analysis.
[00462] SMCC-PMO-FITC (55 mg, 5.8 μmol) was dissolved in conjugation buffer (3 mL, PBS pH 6.5) and MeCN was added (2 mL). Peptide (12 mg, 7.7 μmol) andEDTA solution (0.05 mL,100 mM) were mixed with immobilized TCEP (3 mL) for 1 h prior. The final concentration of EDTA was 2 mM. The reduced peptide was eluted from the immobilized TCEP into a glass vial containing the SMCC-modified PMO-FITC and stirred at room temperature for 2.5 hours after the last addition of peptide. [00463] The reaction was diluted with H2O and batch-loaded onto an HPLC prep column (Waters). Using a 20 mL syringe, sample was batch-loaded in 4 mL aliquots onto the prep column using a 15% isocratic organic at 45 mL/min. The injection loop was washed with 15% MeCN solution prior to loading samples. The HPLC system was equilibrated for a minimum of 20 mins at 15 % MeCN, 1 mL/min.
[00464] The purity of the peptide-oligonucleotide conjugate was >98%, with the molecule with an n-1 PMO-FITC domain being the only detectable impurity.
[00465] The lyophilized conjugate and PMO-FITC were dissolved in DMSO at a concentration that permitted use of a maximum of 10% DMSO in the final injection solution.
Formulation
[00466] Injection solutions for in vivo experiments had the following composition:
- 10% v/v conjugate or PMO in DMSO stock
- 10% v/v 1M HC1
- 9.5% v/v IM NaOH
- 70.5% v/v HBSS (Sigma #H8264)
[00467] To prepare the solutions, HC1 was added to HBSS, conjugate or PMO stock in DMSO was added to this acidic solution and finally NaOH was added. The required concentration of unconjugated PMO or peptide-PMO conjugate in the injection solution was calculated as the dose (in μmol/kg) divided by the injection volume per body mass (4 ml/kg).
[00468] PMO concentrations in the injection solutions were verified by measuring UV absorbance at 260 nm wavelength. 2 μl of the injection solution was diluted 1/10 in 0.1 M HC1 and absorbance measured on a Nanodrop spectrophotometer (ThermoSci entific). Injection volumes were adjusted where needed to give the predetermined dose.
Animal model
[00469] Female D2.B 10-Dmdmdx/J (The Jackson Laboratory; strain: 013141; referred to here as D2-mdx) were used for this study. This line carries a nonsense mutation in exon 23 of the dystrophin gene which abrogates dystrophin expression. Removal of exon 23 from the dystrophin transcript restores the expression of a slightly truncated but functional dystrophin protein. These mice show muscle atrophy and increased fibrosis compared to wild-type mice (Coley et al., 2016), pathological features which are also observed in DMD patients.
Experimental protocol [00470] To determine whether dose tolerability of the IL1α-NLS-PMO conjugates was compatible with the dosing protocol used in a prior study with the compound comprising a CPP designated “ARE8M” (4 x 2.0 μmol/kg), D2-mdx mice received a single intravenous dose of 1.0 μmol/kg, 1.5 μmol/kg or 2.0 μmol/kg of the IL1α NLS (1,5)-PM0 conjugate, or 1.0 μmol/kg or 2.0 μmol/kg of the IL1α NLS (2,6)-PMO conjugate.
[00471] The dose used for the repeated administrations was 2.0 μmol/kg. The dosing frequency was once every two days. Equimolar doses of conjugate and unconjugated PMO were administered to compare efficacy of delivery at the same dose. Tables 13 and 14 show an overview of the mice dosed in this study.
Table 13. Cohort of D2-mdx mice used for IL1α NLS (1,5)-PM0 conjugate experiments
Figure imgf000088_0001
Table 14. Cohort of D2-mdx mice used for IL1α NLS (2,6)-PMO conjugate experiments
Figure imgf000088_0002
In vivo procedures
[00472] For intravenous administration, mice were warmed in heat box for 10 minutes to induce vasodilation. Compounds were administered into the tail vein using a 30G insulin syringe. The mice were monitored in a separate cage following dosing before being returned to their home cage. Body mass was monitored during the dosing phase as an indicator of animal health. Tissue was recovered 13 or 14 days after the final injection. Mice were euthanized by exposure to rising carbon dioxide levels. Tissue was removed and snap-frozen in liquid nitrogen-cooled isopentane.
Assays
[00473] Dystrophin protein was detected in sections of heart muscle and gastrocnemius muscle by immunohistochemistry. Tissue sections (10 pm thickness) were cut on a microtome at -20°C, mounted on positively charged glass slides (SuperFrost Plus, VWR), air-dried and stored at -80°C until use. Sections from 6 different positions along the length of the tissue were mounted on the same slide.
[00474] Immunohistochemistry to detect dystrophin protein on tissue sections was done at room temperature. Frozen sections were air-dried and endogenous avidin/biotin was blocked with an avidin/biotin blocking kit (Vector Laboratories). Sections were then incubated with an antidystrophin antibody targeting the C-terminus of the protein (Abeam, #15277) in PBS with 0.1% tween-20 (PBS-T) and 1% fetal calf serum for 1 hour. Unbound antibody was washed off with PBS-T (3x3 min) and sections were incubated with a biotinylated species-specific anti-IgG antibody (DAKO) for 30 minutes. Sections were washed in PBS-T again (3x3 minutes) before incubation with ABC reagent (Vector Laboratories) for 10 minutes. Horse-radish peroxidase- labelled antibodies were then detected by incubation of sections with DAB substrate solution (Vector Laboratories). All sections were incubated with DAB substrate solution for the same duration. Stained sections were washed in tap water for 5 minutes, dehydrated in 100% ethanol for 30 seconds, cleared in xylene for 10 minutes and permanently mounted in DPX, covered by a coverslip.
[00475] Images of whole stained tissue sections were obtained on a microscope and cells with membrane dystrophin staining were counted. [00476] Muscle tissues from vehicle-treated D2-mdx mice of a similar age (n=4-5) from another experiment were analyzed alongside tissues from this study as a baseline for dystrophin expression.
[00477] The data was graphed using GraphPad Prism 9.5.1.
Study results
[00478] No major changes in body mass were observed in IL1α NLS-PMO-treated mice or PMO-FITC-treated mice during the repeated dosing phase (Fig. 14 & Fig. 15). Temporary reductions in activity and some muscle weakness were observed acutely following intravenous dosing with 2 μmol/kg IL1α NLS (1,5)-PMO conjugate. These effects were notably milder than following dosing with the ARE8M-PM0 conjugate at the same dose in the same mouse model. No visible adverse effects of intravenous dosing of 2 p mol /kg IL1α NLS (2,6)-PMO conjugate were observed.
[00479] Four intravenous doses of IL1α NLS (1,5)- and IL1α NLS (2,6)-PMO conjugates increased the number of fibers displaying membrane dystrophin expression in gastrocnemius muscle, but not heart muscle, compared to treatment with PMO-FITC and vehicle (Figs. 16A & 16B, Figs. 17A & 17B). The average number of dystrophin-positive cells is slightly higher in heart sections of IL1α NLS (1,5)-PM0 conjugate-treated mice compared to the PMO-FITC and vehicle controls (Figs. 16A & 16B), but as the percentage of dystrophin-positive cells to the total number heart cells is very low this difference is likely biologically negligible.
Conclusions
[00480] Intravenous IL1α NLS (1,5)-PEG5-SMCC-PMO-FITC and IL1α NLS (2,6)-PEG5- SMCC-PMO-FITC increased dystrophin expression in skeletal muscle, but not heart muscle of D2-mdx mice, compared to PMO-FITC.
[00481] Acute tolerance to intravenous dosing of IL1α NLS (1,5)-PEG5-SMCC-PMO-FITC and IL1α NLS (2,6)-PEG5-SMCC-PMO-FITC was improved compared to tolerance to of ARE8M-PEG5-SMCC-PMO-FITC at the same dose in the same animal model.
[00482] Comparison of the effects of both IL1α NLS-PMO conjugates in this study and that of the ARE8M-PM0 conjugate as previously studied suggests that efficacy in different tissues depends on the stapled peptide domain of the peptide-oligonucleotide conjugate. Example 17 - In vitro efficacy of stapled and unmodified ESE-1 NLS peptide-oligonucleotide conjugates in He1αEGFP654 splice reporter cells
[00483] This study compared the efficacy and cytotoxicity of peptide-oligonucleotide conjugates comprising peptides based on the ETS-related transcription factor Elf-3 (ESE-1) nuclear localization signal (NLS). In particular, the peptide-oligonucleotide conjugates comprise ESE-1 NLS peptide variants, which have been stabilized by olefin ring-closing metathesis between two (S)-pentenylalanine (S5) monomers placed in different positions along the length of the ESE- 1 NLS peptide (see Table 15 for amino acid sequences). These stapled variants were compared to i) a conjugate comprising the native ESE-1 peptide (i.e. not comprising any non-natural amino acids) and ii) the unconjugated oligonucleotide.
[00484] The biologically active oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO) having the nucleotide sequence of 5’ATATTGCTATTACCTTAACCCAGAA3’ (SEQ ID NO: 101) in which the 5’ end was modified to have a primary amine functionality, which permits nucleophilic coupling at the N- hydroxysuccinimide functionality of SMCC. The PMO targets an enhanced green fluorescent protein (eGFP) sequence that is interrupted by the intron of human β-globin encoding the position 654 splicing mutation. Upon splicing correction with the PMO, functional eGFP is expressed. Thus, eGFP expression is an indicator of the successful delivery of the PMO to the cell nucleus, and an improvement in eGFP expression is evidence of improved delivery to the nucleus.
Table 15. ESE-1 NLS peptide sequences and compound names
Figure imgf000091_0001
[00485] In Table 15, 3TPA: 3-(tritylthio)propionic acid; S5: (S)-pentenylalanine; SMCC: succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate; STP: Stapled. Numbers in brackets indicate the position of the S5 monomers in the peptide. [00486] EGFP654 PMO, having the sequence 5’ATATTGCTATTACCTTAACCCAGAA3’ (SEQ ID NO: 101), was synthesized and supplied in lyophilized form by Gene Tools, LLC (Oregon, USA) with a 5’ NEL modification to allow conjugation to the peptides. A single batch was used for all conjugations and PMO-only controls in experiments.
[00487] Stapling in stabilized peptides was accomplished via olefin metathesis with Grubbs’s catalyst. The crosslinked amino acid residues for a particular stapled peptide species are enclosed within the hyphens in Table 15, supra. The bifunctional linker utilized was succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC). The peptide, via a terminal cysteine residue, as part of the bifunctional linker bonds via a thiol-ene coupling to the mal eimide functional group of SMCC.
[00488] A HeLa cell line (HeLaEGFP654), expressing an eGFP gene containing the mutated human β-globin intron (IVS2-654) as described above, was maintained at below 80% confluence in D-MEM supplemented with 10% fetal calf serum. Cells were seeded 24 hours before treatment in 96-well plates at ~2 x 104 cells per well in 100 μL of vehicle. The growth vehicle was removed and replaced with 100 μL of fresh growth vehicle containing either only fresh vehicle or varying concentrations (in μM) of: i) unconjugated PMO; ii) peptide-PMO conjugate comprising native ESE-1 NLS peptide; and iii) peptide-PMO conjugate comprising stabilized (i.e. stapled) ESE-1 NLS peptide. Cells were assayed 24 hours later. The treatment media was removed (used for the lactate dehydrogenase assay described below to measure cell toxicity) and replaced with HBSS and fluorescence measured using a fluorescence plate reader (Spectramax iD3) with monochromator set to 475/515 nm (ex/em). EGFP fluorescence in live cells kept in HBSS was measured on a plate reader after 24 hours of incubation with the compounds at concentrations between 0.625 μM and 40 μM. Background fluorescence was measured in wells containing HBSS only and subtracted from fluorescence measurements in well containing cells.
[00489] For assessment of cell toxicity Promega LDH-Glo Cytotoxicity assay was used. After 24 hour treatment, the cell supernatant was removed and 10 μL used in the LDH assay per manufacturer’s instruction. Ideal peptide-oligonucleotide candidates are those that demonstrate pharmacological dose-response with high eGFP expression levels, indicative of enhanced cell penetration and biological activity, in which cell toxicity is absent or minimal. The experimental results from the treatment of cells for the restoration of eGFP expression using the peptide- oligonucleotide conjugates are provided in Figs. 19 and 20. The data is shown as the mean value +/- the calculated standard deviation.
Results
[00490] eGFP expression was increased in HelaEGFP654 cells treated with peptide-PMO conjugate comprising the unmodified ESE-1 NLS peptide compared to vehicle- and unconjugated PMO-treated cells (Fig. 19). eGFP expression was further increased in cells treated with conjugates comprising the stapled ESE-1 NLS peptide variants, in particular, at treatment concentrations of 10, 20 and 40 μM, and the extent of this increase depended on the position of the staple within the ESE-1 NLS peptide.
[00491] No significant increase in cytotoxicity as measured by LDH release into the treatment medium was detected for any of the compounds compared to vehicle-treated cells (Fig. 20).
[00492] The data indicates that peptide stapling by olefin ring-closing metathesis can improve cell entry and nuclear delivery of peptide-oligonucleotide conjugates without increasing cytotoxicity.
Example 18 - Synthesis of gapmer-peptide conjugates
[00493] The present study utilizes the gapmer having the sequence 5’ AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108), which the ability to knockdown the pre- mRNA and mRNA of argonaute 2 (AGO2), whose knockdown induces apoptosis in cancer cells. This gapmer is commercially available and can be procured with modifications to facilitate conjugation with a bifunctional linker such as SMCC.
[00494] To optimize conjugation of the gapmer to the cell penetrating peptide, the gapmer is modified with a C12 5’ amino modifier such that the 5'end of the gapmer bears a reactive amine group.
[00495] The peptides that are used to generate the gapmer-peptide conjugates are shown in the below Table 16.
Table 16. Peptides for conjugation to gapmer
Figure imgf000093_0001
Figure imgf000094_0001
[00496] Synthesis of peptides is performed using a Liberty Blue Microwave Synthesiser. Peptides are synthesized using CEM ProTide® rink resin with a loading capacity of 0.8 mmol/g and coupled with a “High Swelling” program.
[00497] Fmoc amino acids and reagents are dissolved in DMF to give a 0.2 M amino acid solution, 1.0 M Oxyma solution, 0.5 M DIC solution and 20% piperidine in DMF.
[00498] At a 100 μmol scale, 5 equivalents of reactants are used.
[00499] In the desired order, the Fmoc amino acids are mixed with DIC/Oxyma and coupled in DMF. Coupling conditions are performed according to CEM manual recommendations: Amino acids A, V, L, I, F and S5 are coupled for 300 s at 90°C. All other amino acids coupled for 90 s at 90 °C with the exception of R which is double coupled at 75 °C for 300 seconds (i.e., 600 seconds in total).
[00500] Deprotection is performed at 90°C for 90 seconds with 20% Piperidine in DMF solution.
[00501] The crude resin bound peptide (100 μmol) is transferred to the reaction chamber as a DCM slurry and subsequently washed within the reaction vessel with DCE (3 x 5 mL).
[00502] Grubb’s catalyst (20 mol%, 6 mM, 4 mL, in DCE) is prepared immediately before utilization in the reaction. 4 mL of the Grubb’ s catalyst solution is transferred to the reaction vessel. The ring closing metathesis reaction proceeds at 25 °C for 30 min with nitrogen gas bubbling for 2 seconds and 5 seconds off. Nitrogen is bubbled throughout the RCM reaction to promote the removal of ethene and drive the formation of the peptide macrocycle. The reaction mixture is drained and a second aliquot of freshly prepared Grubb’s catalyst (4 mL, 6 mM, in DCE) is added and further reacted for 30 mins at 25 °C. The mixture is drained and transferred to a solid phase extraction tube, washed with DCM, and dried for continuation to the cleavage step.
[00503] The dried resin (100 μmol) is transferred to a 25 mL round bottom flask and a stirrer bar is added. A round bottom heating mantel is preheated to 40 °C. Addition of cleavage cocktail (6 mL, 92.5% TFA, 2.5% H2O, 2.5% TIS, 2.5% DODT) to the crude resin. The resin slurry is stirred at 40 °C for approximately 30-45 minutes depending on the sequence (higher arginine containing peptides require longer reaction times). This slurry is then transferred to a SPE tube and filtered into 50 mL falcon tubes containing approximately 35 mL of diethyl ether. The resin is washed a further 3 times with approximately 2 mL of cleavage cocktail. The ether slurry is centrifuged for 5 minutes at 1000 xg.
[00504] The etherial supernatant is decanted, and the crude pellet is isolated. The pellet is dissolved using acetonitrile and water and combined in a 28 mL screw top vial and frozen on dry ice for a minimum of 30 minutes. After an appropriate time, the screw cap is replaced with a porous membrane, which is fitted in place with rubber bands, and the vial is placed back on dry ice for approximately 5 to 10 minutes. The vials are freeze-dried for a minimum of 72 hours at 0.01 mbar vacuum and -105°C collector temperature.
[00505] To identify the masses of the peptide, a scout LCMS analysis is run. A sample of the crude lyophilized peptide is removed and diluted (H2O/MeCN 10%, 0.1 mL final volume) and 50 μL loaded on to a Xbridge BEH C18 4.6 x 150 mm column 186003580. A gradient of 15% - 80% over 12 mins in H2O/MeCN +0.1% TFA is run. A single wavelength of 220 nm is used and QDa is set up in positive mode with 1.5 kV capillary and 15 V cone voltage and a H2O/MeOH 50% + 0.01% formic acid. Buffer A is H2O +0.1% TFA and buffer B is MeCN +0.1% TFA.
Peptide analysis - Initial: A=85%, B=15%. 3 min: A=85%, B=15%. 15 min: A=20%, B=80%. 19 min: A=20%, B=80% 20 min: A=85%, B=15%. 30 min: A=85%, B=15%.
[00506] For reverse phase-HPLC, the crude sample is reconstituted in 2 ml of 10% MeCN in water and loaded onto the Xbridge BEH C18 5pm 30^ 150 mm column.
[00507] For purification, a flow rate of 45mL/min and a gradient of 15-80% is ran over 12 mins with buffer A being H2O + 0.1% TFA and buffer B being MeCN + 0.1% TFA.
The gradient changes are as followed:
Initial: A=85%, B=15%
3 min: A=85%, B=15%
15 min: A=20%, B=80%
19 min: A~20%, B~80%
20 min: A=85%, B=15%
[00508] Fractions are identified through mass directed collection by ESI positive mode scans. Masses of the peptide are identified during the scout LCMS run and are manually inputted into a mass collection method used for purification. The chromatogram is monitored using the ESI+ spectrum in which compounds above EIC threshold of 1x106 are collected in 28 mL screw top vials and frozen on dry ice for freeze-drying. Mixed fractions or single compounds below 5x105 ESI threshold are discarded. Following from this, fractions collected were freeze-dried for a minimum of 72 hours.
[00509] A single wavelength of 220 nm is used and QDa is set up in positive mode with 1.5 kV capillary and 15 V cone voltage and a H2O/MeOH 50% + 0.01% Formic acid.
[00510] The obtained fractions are stored in 28 mL screw top vials labelled with peptide identity, batch date and fraction number obtained from purification. Vials are frozen on dry ice for a minimum of 30 minutes, in which time the freeze dryer shelf is also cooled on dry ice. After freezing, the screw cap is removed and replaced with porous membrane, secured in place with elastic bands, and briefly replaced back on dry ice (5-10 minutes). After the final cooling period, the shelf and vials are rapidly placed in the freeze dryer to prevent thawing and the freeze dryer is operated at the following settings: Collector temperature = -105°C, vacuum pressure = 0.010 mbar.
The primary freeze drying was performed for 72 hours to obtain crude lyophilized peptide.
[00511] To characterize the final peptide following purification by LCMS, a sample of the purified lyophilized peptide is removed and diluted (H2O/MeCN 10%, 0.1 mL final volume) and 50 μL loaded on to a Xbridge BEH C18 4.6 x 150 mm column 186003580. A gradient is run of 15% - 80% over 12 mins in H2O/MeCN +0.1% TFA. A single wavelength of 220 nm is used and QDa was setup in positive mode with 1.5 kV capillary and 15 V cone voltage and a TEO/MeOH 50% + 0.01% Formic acid. Peptide analysis - Initial: A=85%, B=15%. 3 min: A=85%, B=15%. 15 min: A=20%, B=80%. 19 min: A=20%, B=80% 20 min: A=85%, B=15%. 30 min: A=85%, B=15%.
[00512] The bifunctional linker used to conjugate the 5 ’-amino modified gapmer to the peptide is SMCC. Gapmer is dissolved in PBS (pH 7.2) and is incubated at room temperature after the addition of SMCC (5-fold excess relative to gapmer) dissolved in MeCN/H2O (1 : 1 , 1 mL). After 30 mins the mixture is desalted using sephadex g25 hydrated in conjugation buffer (PBS lx, pH 6.8) and is also used as the eluent. The final concentration of EDA is 2 mM.
[00513] The peptide is dissolved in milliQ water (4 mLO and EDTA solution (0.1 mL, 100 mM) and is mixed with TCEP (2.5 mL) for 1 h prior. The final concentration of EDTA is 2 mM.
[00514] MeCN (8 mL) and EDTA solution (0.1 mL, 100 mM) is added to the freshly desalted SMCC modified gapmer prior to the addition of peptide. The reduced peptide is eluted from the immobilized TCEP into a tube containing the SMCC modified gapmer and is stirred at room temperature for 2 hours.
[00515] The solution is loaded on to 3 x 560 mg HLB columns, and washed with milliQ water to remove any salts, then 10% MeCN in water. Columns are washed with 20% MeCN until the eluent runs clear. Finally, the gapmer-peptide conjugate is eluted with 50% MeCN in water. The eluted products then undergo size exclusion chromatography using sephadex superfine g25 hydrated in milliQ water as the eluent.
Example 19 - Biological testing of gapmer-peptide conjugates
[00516] Study of the biological activity of the gapmer-peptide conjugates first involves investigation of the retention of the activity of the gapmer. Following confirmation that the gapmer retains its activity, gapmer-peptide conjugate and unconjugated gapmer are incubated with cells in the absence of a transfection agent to assess the ability of conjugated stapled peptides to improve cell/nuclear entry of the gapmer.
[00517] A procedure adopted from that of Liang et al. (Molecular Therapy, 2017, 25, 2075- 2092) is used. Briefly, HeLa cells used in the study are grown on plates in D-MEM supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C in a 5% or 8% CO2 incubator. To accomplish transfection of the cells with gapmer, the cells are seeded at 50%-70% confluence, grown overnight, and transfected using Lipofectamine 2000 at concentrations ranging from 4 nM to 120 nM of the gapmer. Control samples are transfected utilizing only the growth vehicle.
[00518] Following transfection, cytoplasmic and nuclear fractions of the HeLa cells are prepared. A nuclear protein kit (QIAGEN) or a PARIS kit supplemented with RNase inhibitor is used to prepare the cytoplasmic and nuclear fractions from approximately 5 * 107 HeLa cells according to the kit instructions. To confirm distribution of marker proteins, aliquots of the cytoplasmic and nuclear fractions are analyzed by SDS-PAGE and western blot.
[00519] The cytoplasmic and nuclear fractions are subjected to RNA preparation for qRT-PCR analysis utilizing an RNeasy (QIAGEN) kit according to the kit instructions. qRT-PCR is performed using TaqMan primer probe sets with the StepOne real-time PCR system. Reverse transcription is performed at 48 °C for 30 mins, and 40 cycles of PCR reaction are carried out at 94 °C for 15 seconds and 52 °C for 15 seconds within each cycle. mRNA levels in cytoplasmic or nuclear fractions are normalized to the levels of cytoplasmic 7SL RNA or nuclear MALAT1 RNAs, respectively, or to the levels of cytoplasmic 7SL RNA or the nuclear U16 RNA, respectively using a Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific).
[00520] Cytosolic and nuclear fractions are separated using a Cell Fractionation Kit- Standard (Abeam) according to the kit instructions. Cytosolic and nuclear proteins are dissolved in an equal volume of lysis buffer, and an equal volume of proteins from each fraction are analyzed by SDS- PAGE and western blot analyses.
[00521] The cytoplasmic and nuclear fractions are separated in 4-12% gradient SDS-PAGE gels. Proteins are transferred to PVDF membrane using a semi-dry transfer apparatus. The membranes are blocked for 1 hr with block buffer (5% dry milk in 1 xTBS), and incubated with primary antibodies against AG02 (ab32381, 1 :1000) at 4 °C for overnight. After 3 washes with wash buffer (1 xTBS, 0.1 % Tween-20), membranes are incubated with anti-mouse or anti-rabbit secondary antibody in block buffer at room temperature for 1 hr. After 3 washes, proteins are detected using ECL (Abeam).
[00522] The gapmer utilized in the study is shown to knockdown both pre-mRNA and mRNA in the nucleus and cytoplasm, respectively via the recruitment of RNAse Hl. qRT-PCR analysis shows a decrease in the level of the target RNA following transfection with the gapmer. Western blot analysis of the cytoplasmic and nuclear fractions also shows a decrease in the level of the protein AGO2, correlating with the decrease in the level of the target RNA. Control samples, which are not transfected with gapmer, exhibit higher levels of target RNA and AGO2.
[00523] Following the above study showing the retention of the activity of the gapmer in the present system, the activity of gapmer-peptide conjugates and unconjugated gapmer in the absence of a transfection agent is studied to assess the ability of conjugated stapled peptides to improve cellular and nuclear entry of the gapmer.
[00524] HeLa cells are exposed to equimolar amounts of gapmer-peptide conjugates, unconjugated gapmer, and growth vehicle as a control sample. Analysis and determination of RNA knockdown is performed as described above utilizing real time PCR.
[00525] The gapmer-peptide conjugates having the conjugated cell penetrating peptide exhibit improved cellular and nuclear entry compared to unconjugated gapmer alone. Thus, in the absence of a transfection agent to facilitate entry into the cell, the gapmer-peptide conjugate bearing a cell penetrating peptide exhibits improved cellular uptake compared to that of the unconjugated gapmer. The gapmer cargo, in retaining its biological activity following delivery into the cell, is able to recruit RNAse Hl to knockdown the pre-mRNA and mRNA encoding the protein AG02. Real time PCR analysis of the gapmer-peptide conjugate samples shows a decrease in the levels of target RNA and western blot analysis of the cytoplasmic and nuclear fractions shows a decrease in the protein level of AGO2. Samples utilizing gapmer, which does not contain a mechanism to facilitate cellular entry, and growth vehicle alone exhibit higher levels of the target RNA and AGO2 as measured by real time PCT and western blot analysis. These results demonstrate the ability of the gapmer-peptide conjugate to both cross the cellular membrane and deliver the gapmer cargo, which retains its biological activity in recruiting RNase Hl to knockdown the target RNA. [00526] In various embodiments, embodiments of the invention are further described by the following numbered paragraphs:
[00527] 1. A method for synthesizing a compound, the compound comprising: i. an oligonucleotide moiety, covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix; wherein synthesis of the compound comprises the steps of:
(i) coupling an amino acid via its carboxyl group to a solid support, wherein the amino acid comprises a protecting group at its amino group;
(ii) removing the protecting group to expose a free amino group;
(iii) coupling, by stepwise solid phase synthesis, a successive amino acid monomer wherein the successive amino acid monomer comprises a protecting group at its amino group;
(iv) repeating steps (ii) and (iii) to obtain a peptide chain;
(v) forming the cross link or the bridge between the at least two amino acids;
(vi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), before or after step (iii), before or after step (iv), or before or after step (v);
(vii) coupling a first oligonucleotide monomer at its 5’ end to the N terminus of the peptide chain or the at least one functional group of the handle, wherein the oligonucleotide comprises a protecting group at its 3 ’ end;
(viii) removing the protecting group at the 3’ end to expose a free amino group; (ix) coupling, by stepwise solid phase synthesis, a successive oligonucleotide monomer wherein the successive oligonucleotide monomer comprises a protecting group at its 3’ end;
(x) repeating steps (viii) and (ix) to obtain an oligonucleotide chain and the compound;
(xi) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs before or after step (vii), before or after step (viii), before or after step (ix), or after step (x); and
(xii) cleaving the compound from the solid support;
[00528] wherein the BFL, if present, comprises one or more handles.
[00529] 2. The method of paragraph 1, wherein the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[00530] 3. The method of any one of paragraphs 1 or 2, wherein the PMO comprises
5'GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98).
[00531] 4 The method of any one of paragraphs 1-3, wherein the protecting group of the amino acid monomer is Fmoc.
[00532] 5. The method of any one of paragraphs 1-4, wherein the protecting group of the oligonucleotide monomer is Fmoc.
[00533] 6. The method of any one of paragraphs 1-5, wherein the oligonucleotide monomer is selected from the group consisting of:
[00534] 7. The method of claim any one of paragraphs 1-6, wherein the protecting group of the oligonucleotide monomer is Trt.
[00535] 8. The method of claim any one of paragraphs 1-7, wherein the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000102_0001
[00536] 9. The method of any one of paragraphs 1-8, wherein the oligonucleotide is covalently linked to the StiP or the StAP.
[00537] 10. The method of any one of paragraphs 1-9, wherein the BFL comprising one or more handles is present in the compound, and the oligonucleotide is covalently linked via the BFL to the StaP or the StiP, whereby the BFL is covalently linked to the oligonucleotide, and the BFL is covalently linked to the StaP or the StiP.
[00538] 11. The method of any one of paragraphs 1-10, wherein BFL comprises:
Figure imgf000103_0001
(SMCC), a residue of succinimidyl 4-(N-maleimidomethyl)
Figure imgf000103_0002
R cyclohexane- 1 -carboxylate, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is
Figure imgf000103_0003
where n is a positive integer; or,
(AMAS), a residue of N-a-maleimidoacet-oxysuccinimide ester, where Z is
Figure imgf000103_0004
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000103_0005
or,
(BMPS), a residue of N-β-maleimidopropyl-oxysuccinimide ester, where Z is
Figure imgf000104_0001
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000104_0002
Figure imgf000104_0003
(GMBS), a residue of N-Y-aleimidobutyryl-oxysuccinimide ester, where Z is
Figure imgf000104_0004
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is
Figure imgf000104_0005
where n is a positive integer; or,
(DMVS), a residue of N-δ-maleimidovaleryl-oxysuccinimide ester, where nd Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is
Figure imgf000105_0001
, where n is a positive integer;
(EMCS), a residue of N- ε-malemidocaproyl-oxysuccinimide ester, and Y is a covalent bond to the N-terminus of the StaP or the StiP, or where n is a positive integer;
Figure imgf000105_0002
or,
Figure imgf000106_0001
(LC-SMCC), a residue of succinimidyl 4-(N-maleimidomethyl) cyclohexane- l-carboxy-(6-amidocaproate), where Z is
Figure imgf000106_0002
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000106_0003
or,
Figure imgf000106_0004
a residue of succinimidyl 4-(N- maleimidom ethyl) cyclohexane- 1 -carboxylate (polyethylene glycol)n, wherein n equals 1 to 10, Z
Figure imgf000106_0005
and, Y is either present or not present, and when Y is present, Y is
Figure imgf000106_0006
where n is a positive integer, and when Y is not present, Y is a covalent bond to the N-terminus of the StaP or the StiP; or,
Figure imgf000107_0001
(DSG), a residue of disuccinimidyl gluterate, where Z is not present, and instead there is a covalent bond to the N-terminus of the StaP or the StiP, or to the N of wherein n is a positive integer;
Figure imgf000107_0002
or,
Figure imgf000107_0003
(DSCDS), a residue of disuccinimidyl-cyclohexl-l,4-diester, where Z is not present, and instead there is a covalent bond to the N-terminus of the StaP or the StiP, or to the N of
Figure imgf000107_0004
wherein n is a positive integer, and wherein R is H or NH2.
[00539] 12. The compound of paragraph 11, wherein in each Y moiety, n is 5.
[00540] 13. The compound of any one of paragraphs 11-12, wherein R is H.
[00541] 14. The compound of any one of paragraphs 11-12, wherein R is NH2.
[00542] 15. The method according to any one of paragraphs 1-14, wherein the forming step (v) comprises forming an olefin cross link between at least two unnatural amino acids.
[00543] 16. The method according to any one of paragraphs 1-15, wherein the at least two unnatural amino acids are selected from the group consisting of:
wherein S5 is (S)-pentenylalanine, R5 is (R)-pentenylalanine, S8 is (S)-octenylalanine, R8 is (R)- octenylalanine, B5 is α, α-di- substituted pentenylalanine, B8 is α, α-di-substituted octenylalanine, and S-OAS and R-OAS are O-allylserine analogues.
[00544] 17. The method according to any one of paragraphs 1-16, wherein the forming step (v) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
[00545] 18. The method according to claim any one of paragraphs 1-17, wherein the lactam bridge is formed by cross linking a lysine and glutamic or aspartic acid residues.
[00546] 19. A method for synthesizing a compound, the compound comprising: i. an oligonucleotide moiety, covalently linked to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix; wherein synthesis of the compound comprises the steps of:
(i) providing a solid support comprising a linker configured to couple an oligonucleotide monomer at its 5’ end, wherein the oligonucleotide monomer comprises a protecting group at its 3’ end;
(ii) removing the protecting group to expose a free amino group;
(iii) coupling, by stepwise solid phase synthesis, a successive oligonucleotide monomer wherein the successive amino acid monomer comprises a protecting group at its 3’ end;
(iv) repeating steps (ii) and (iii) to obtain an oligonucleotide chain;
(v) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), or before step (iii);
(vi) coupling an amino acid via its carboxyl group to the 3’ end of the oligonucleotide chain, wherein the amino acid comprises a protecting group at its amino group;
(vii) removing the protecting group to expose a free amino group;
(viii) coupling, by stepwise solid phase synthesis, a successive amino acid monomer wherein the successive amino acid monomer comprises a protecting group at its amino group;
(ix) repeating steps (vii) and (viii) to obtain a peptide chain;
(x) forming the cross link or the bridge between the at least two amino acids to obtain the compound; (xi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs after step (vi), before or after step (vii), before or after step (viii), before or after step (ix), or before or after step (x); and
(xii) cleaving the compound from the solid support.
[00547] 20. The method of paragraph 19, wherein the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[00548] 21. The method of any one of paragraphs 19-20, wherein the PMO comprises
5GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98).
[00549] 22. The method of any one of paragraphs 19-21, wherein the linker of step (i) comprises an amino acid monomer or a handle configured to couple with an oligonucleotide monomer.
[00550] 23. The method of any one of paragraphs 19-22, wherein the protecting group of the amino acid monomer is Fmoc.
[00551] 24. The method of any one of paragraphs 19-23, wherein the protecting group of the oligonucleotide monomer is Fmoc.
[00552] 25. The method of claim any one of paragraphs 19-24, wherein the oligonucleotide monomer is selected from the group consisting of:
[00553] 26. The method of any one of paragraphs 19-25, wherein the protecting group of the oligonucleotide monomer is Trt. [00554] 27. The method of any one of paragraphs 19-26, wherein the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000111_0001
[00555] 28. The method according to any one of paragraphs 19-27, wherein the forming step
(x) comprises forming an olefin cross link between at least two unnatural amino acids.
[00556] 29. The method according to paragraph 28, wherein the at least two unnatural amino acids are selected from the group consisting of:
Figure imgf000112_0001
wherein S5 is (S)-pentenylalanine, R5 is (R)-pentenylalanine, S8 is (S)-octenylalanine, R8 is (R)- octenylalanine, B5 is α, α-di- substituted pentenylalanine, B8 is α, α-di-substituted octenylalanine, and S-OAS and R-OAS are O-allylserine analogues.
[00557] 30. The method according to any one of paragraphs 19-29, wherein the forming step
(x) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
[00558] 31. The method according to any one of paragraphs 19-30, wherein the lactam is formed by cross linking a lysine and glutamic or aspartic acid residues.
[00559] 32. A composition comprising the compound made according to the method of any one of paragraphs 1-18 and one or more pharmaceutically acceptable excipients.
[00560] 33. A composition comprising the compound made according to the method of claim any one of paragraphs 19-31 and one or more pharmaceutically acceptable excipients.
[00561] 34. The composition of paragraph 32, formulated for oral, parenteral, intravenous, or topical administration.
[00562] 35. The composition of paragraph 33, formulated for oral, parenteral, intravenous, or topical administration.
[00563] 36. A method of administering the composition of any one of paragraphs 32 or 34 intravenously to a subject, comprising the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
[00564] 37. A method of administering the composition of any one of paragraphs 33 or 35 intravenously to a subject, comprising the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
[00565] 38. A molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acid sequence of SEQ ID NOS: 23, 24, 25, 28, 29, 30, 31 , 32, 34, 37, 38, 59, or 60, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
[00566] 39. The molecule of paragraph 38, further comprising a thiol -containing moiety that is linked to the peptide moiety at the C-terminus by a polyethylene glycol linker.
[00567] 40. The molecule of any one of paragraphs 38 or 39, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
[00568] 41. The molecule of any one of paragraphs 38-40, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
[00569] 42. The molecule of any one of paragraphs 38-41, wherein the PMO is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker.
[00570] 43. The molecule of any one of paragraphs 38-42, wherein the bifunctional linker is
SMCC.
[00571] 44. The molecule of any one of paragraphs 38-43, wherein the PMO has the sequence
5’-GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
[00572] 45. The molecule of any one of paragraphs 38-40, wherein the antisense oligonucleotide is a gapmer.
[00573] 46. The molecule of any one of paragraphs 38-40, 45, wherein the gapmer is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker. [00574] 47. The molecule of any one of paragraphs 38-40, 45-46, wherein the bifunctional linker is SMCC.
[00575] 48. The molecule of any one of paragraphs 38-40, 45-47, wherein the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
[00576] 49. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of any one of paragraphs 38-48.
[00577] 50. A method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of any one of paragraphs 45-48.
[00578] 51. The method of any one of paragraphs 49-50, wherein the cell is a cardiac muscle cell.
[00579] 52. The method of any one of paragraphs 49-50, wherein the cell is a skeletal muscle cell.
[00580] 53. A composition comprising the molecule of any one of paragraphs 38-48 and one or more pharmaceutically acceptable excipients.
[00581] 54. The composition of paragraph 53, formulated for oral, parenteral, intravenous, or topical administration.
[00582] 55. A molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i 4, and/or i, i I 7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acids arginine, leucine, and lysine, wherein at least one leucine residue and at least one lysine residue are located between positions z and i+4, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
[00583] 56. The molecule of paragraph 55, wherein the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
[00584] 57. The molecule of any one of paragraphs 55 or 56, further comprising a thiol- containing moiety that is linked to the peptide moiety at the C-terminus by a polyethylene glycol linker.
[00585] 58. The molecule of any one of paragraphs 55-57, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
[00586] 59. The molecule of any one of paragraphs 55-58, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO)
[00587] 60. The molecule of any one of paragraphs 55-59, wherein the PMO is linked to the thiol -containing moiety at the C-terminus of the StaP via a bifunctional linker.
[00588] 61. The molecule of any one of paragraphs 55-60, wherein the bifunctional linker is
SMCC.
[00589] 62. The molecule of any one of paragraphs 55-61, wherein the PMO has the sequence
5’-GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
[00590] 63. The molecule of any one of paragraphs 55-58, wherein the antisense oligonucleotide is a gapmer.
[00591] 64. The molecule of any one of paragraphs 55-58, 63, wherein the gapmer is linked to the thiol -containing moiety at the C-terminus of the StaP via a bifunctional linker.
[00592] 65. The molecule of any one of paragraphs 55-58, 63-64, wherein the bifunctional linker is SMCC.
[00593] 66. The molecule of any one of paragraphs 55-58, 63-65, wherein the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
[00594] 67. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of any one of paragraphs 55-66.
[00595] 68. A method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of any one of paragraphs 63-66. [00596] 69. The method of any one of paragraphs 67-68, wherein the cell is a cardiac muscle cell.
[00597] 70. The method of any one of paragraphs 67-68, wherein the cell is a skeletal muscle cell.
[00598] 71. A composition comprising the molecule of any one of paragraphs 55-66 and one or more pharmaceutically acceptable excipients.
[00599] 72. The composition of paragraph 71, formulated for oral, parenteral, intravenous, or topical administration.
[00600] 73. A molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i-4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acids arginine, glycine, histidine, and lysine, wherein at least one lysine residue is located between positions i and i+4, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
[00601] 74. The molecule of paragraph 73, wherein the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NOS: 103, 104, 105, 106, or 107.
[00602] 75. The molecule of any one of paragraphs 73 or 74, further comprising a thiol- containing moiety that is linked to the peptide moiety at the C-terminus by a polyethylene glycol linker.
[00603] 76. The molecule of any one of paragraphs 73-75, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
[00604] 77. The molecule of any one of paragraphs 73-76, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO). [00605] 78. The molecule of any one of paragraphs 73-77, wherein the PMO is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker.
[00606] 79. The molecule of any one of paragraphs 73-78, wherein the bifunctional linker is
SMCC.
[00607] 80. The molecule of any one of paragraphs 73-79, wherein the PMO has the sequence
5’-GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
[00608] 81. The molecule of any one of paragraphs 73-76, wherein the antisense oligonucleotide is a gapmer.
[00609] 82. The molecule of any one of paragraphs 73-76, 81, wherein the gapmer is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker.
[00610] 83. The molecule of any one of paragraphs 73-76, 81-82, wherein the bifunctional linker is SMCC.
[00611] 84. The molecule of any one of paragraphs 73-76, 81 -83, wherein the gapmer has the sequence 5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
[00612] 85. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of any one of paragraphs 73-84.
[00613] 86. A method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of any one of paragraphs 81-84.
[00614] 87. The method of any one of paragraphs 85-86, wherein the cell is a cardiac muscle cell.
[00615] 88. The method of any one of paragraphs 85-86, wherein the cell is a skeletal muscle cell.
[00616] 89. A composition comprising the molecule of any one of paragraphs 73-84 and one or more pharmaceutically acceptable excipients.
[00617] 90. The composition of paragraph 89, formulated for oral, parenteral, intravenous, or topical administration. [00618] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.
References;
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Claims

WHAT IS CLAIMED IS:
1. A method for synthesizing a compound, the compound comprising: i. an oligonucleotide moiety, covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix; wherein synthesis of the compound comprises the steps of:
(i) coupling an amino acid via its carboxyl group to a solid support, wherein the amino acid comprises a protecting group at its amino group;
(ii) removing the protecting group to expose a free amino group;
(iii) coupling, by stepwise solid phase synthesis, a successive amino acid monomer wherein the successive amino acid monomer comprises a protecting group at its amino group;
(iv) repeating steps (ii) and (iii) to obtain a peptide chain;
(v) forming the cross link or the bridge between the at least two amino acids;
(vi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), before or after step (iii), before or after step (iv), or before or after step (v); (vii) coupling a first oligonucleotide monomer at its 5’ end to the N terminus of the peptide chain or the at least one functional group of the handle, wherein the oligonucleotide comprises a protecting group at its 3 ’ end;
(viii) removing the protecting group at the 3’ end to expose a free amino group;
(ix) coupling, by stepwise solid phase synthesis, a successive oligonucleotide monomer wherein the successive oligonucleotide monomer comprises a protecting group at its 3’ end;
(x) repeating steps (viii) and (ix) to obtain an oligonucleotide chain and the compound;
(xi) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs before or after step (vii), before or after step (viii), before or after step (ix), or after step (x); and
(xii) cleaving the compound from the solid support; wherein the BFL, if present, comprises one or more handles.
2. The method of claim 1, wherein the oligonucleotide is a phosphorodi ami date morpholino oligonucleotide (PMO).
3. The method of claim 2, wherein the PMO comprises 5GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98).
4. The method of claim 1, wherein the protecting group of the amino acid monomer is Fmoc.
5. The method of claim 1, wherein the protecting group of the oligonucleotide monomer is
Fmoc.
6. The method of claim 5, wherein the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000125_0001
7. The method of claim 1, wherein the protecting group of the oligonucleotide monomer is
8. The method of claim 7, wherein the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000126_0001
9. The method of claim 1, wherein the oligonucleotide is covalently linked to the StiP or the
StAP.
10. The method of claim 1, wherein the BFL comprising one or more handles is present in the compound, and the oligonucleotide is covalently linked via the BFL to the StaP or the StiP, whereby the BFL is covalently linked to the oligonucleotide, and the BFL is covalently linked to the StaP or the StiP.
11. The method of claim 10, wherein BFL comprises:
Figure imgf000127_0001
(SMCC), a residue of succinimidyl 4-(N-maleimidomethyl)
Figure imgf000127_0002
cyclohexane- 1 -carboxylate, where Z is and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y i
Figure imgf000127_0003
where n is a positive integer; or,
Figure imgf000127_0004
(AMAS), a residue of N-a-maleimidoacet-oxysuccinimide ester, where Z is
Figure imgf000127_0005
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n is a positive integer;
Figure imgf000127_0006
or,
Figure imgf000128_0001
(BMPS), a residue of N-β-maleimidopropyl-oxysuccinimide ester, where Z i
Figure imgf000128_0002
s and Y is a covalent bond to the N-terminus of the StaP or the StiP, or
Y is where n is a positive integer;
Figure imgf000128_0003
or,
Figure imgf000128_0004
(GMBS), a residue of N-γ-aleimidobutyryl-oxysuccinimide ester, where Z
Figure imgf000128_0005
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or
Y is where n is a positive integer;
Figure imgf000128_0006
Figure imgf000129_0001
(DMVS), a residue of N-δ-maleimidovaleryl-oxysuccinimide ester, where Z is
Figure imgf000129_0002
and Y is a covalent bond to the N-terminus of the StaP or the
StiP, or Y is
Figure imgf000129_0003
where n is a positive integer;
Figure imgf000129_0004
(EMCS), a residue of N- ε-malemidocaproyl-oxysuccinimide ester, where Z is
Figure imgf000129_0005
and Y is a covalent bond to the N-terminus of the StaP or the
StiP, or Y is where n is a positive integer;
Figure imgf000129_0006
or,
Figure imgf000130_0001
(LC-SMCC), a residue of succinimidyl 4-(N- maleimidomethyl) cyclohexane- l-carboxy-(6-amidocaproate), where Z is
Figure imgf000130_0002
and Y is a covalent bond to the N-terminus of the StaP or the StiP, or Y is where n
Figure imgf000130_0003
is a positive integer; or,
Figure imgf000130_0004
(SM(PEG)n), a residue of succinimidyl 4-(N- maleimidomethyl) cyclohexane- 1 -carboxylate (polyethylene glycol )„, wherein n equals 1 to 10, Z is
Figure imgf000130_0005
and, Y is either present or not present, and when Y is present, Y is
Figure imgf000130_0006
where n is a positive integer, and when Y is not present, Y is a covalent bond to the N-terminus of the StaP or the StiP; or,
Figure imgf000131_0001
(DSG), a residue of disuccinimidyl gluterate, where Z is not present, and instead there is a covalent bond to the N-terminus of the StaP or the StiP, or to the N of wherein n is a positive integer;
Figure imgf000131_0002
or,
Figure imgf000131_0003
(DSCDS), a residue of disuccinimidyl-cyclohexl-l,4-diester, where Z is not present, and instead there is a covalent bond to the N-terminus of the StaP or the StiP, or to the N of
Figure imgf000131_0004
wherein n is a positive integer wherein R is H or NH2.
12. The compound of claim 11, wherein in each Y moiety, n is 5.
13. The compound of claim 11, wherein R is H.
14. The compound of claim 11, wherein R is NH2.
15. The method according to claim 1, wherein the forming step (v) comprises forming an olefin cross link between at least two unnatural amino acids.
16. The method according to claim 15, wherein the at least two unnatural amino acids are selected from the group consisting of: wherein S5 is (S)-pentenylalanine, R5 is (R)-pentenylalanine, S8 is (S)-octenylalanine, R8 is (R)-octenylalanine, B5 is α, α-di-substituted pentenylalanine, B8 is α, α-di-substituted octenylalanine, and S-OAS and R-OAS are O-allylserine analogues.
17. The method according to claim 1, wherein the forming step (v) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
18. The method according to claim 17, wherein the lactam bridge is formed by cross linking a lysine and glutamic or aspartic acid residues.
19. A method for synthesizing a compound, the compound comprising: i. an oligonucleotide moiety, covalently linked to, ii. a stapled peptide moiety (StaP) or a stitched peptide moiety (StiP), wherein: the StaP or StiP, when a molecule not part of the compound, is a stabilized peptide, which has a conformation imposed upon it by a cross link or a bridge, wherein the StaP comprises a cross link or a bridge between two amino acids of the peptide at positions i, i+4, and/or i, i+7 and the StiP comprises a cross link or a bridge between at least two olefin cross links between at least three amino acids of the peptide at positions i, i+4, and i+ 11, the cross link or bridge provides a cyclization between the at least two amino acids, and wherein the StaP or StiP can penetrate a cell membrane, and said stabilized conformation comprises at least one alpha helix; wherein synthesis of the compound comprises the steps of:
(i) providing a solid support comprising a linker configured to couple an oligonucleotide monomer at its 5’ end, wherein the oligonucleotide monomer comprises a protecting group at its 3’ end;
(ii) removing the protecting group to expose a free amino group;
(iii) coupling, by stepwise solid phase synthesis, a successive oligonucleotide monomer wherein the successive amino acid monomer comprises a protecting group at its 3’ end;
(iv) repeating steps (ii) and (iii) to obtain an oligonucleotide chain; (v) optionally coupling a handle to the oligonucleotide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (v) occurs before or after step (i), before or after step (ii), or before step (iii);
(vi) coupling an amino acid via its carboxyl group to the 3’ end of the oligonucleotide chain, wherein the amino acid comprises a protecting group at its amino group;
(vii) removing the protecting group to expose a free amino group;
(viii) coupling, by stepwise solid phase synthesis, a successive amino acid monomer wherein the successive amino acid monomer comprises a protecting group at its amino group;
(ix) repeating steps (vii) and (viii) to obtain a peptide chain;
(x) forming the cross link or the bridge between the at least two amino acids to obtain the compound;
(xi) optionally coupling a handle to the peptide chain, wherein the handle comprises a moiety having at least one functional group, wherein the optional coupling step (xi) occurs after step (vi), before or after step (vii), before or after step (viii), before or after step (ix), or before or after step (x); and
(xii) cleaving the compound from the solid support.
20. The method of claim 19, wherein the oligonucleotide is a phosphorodi ami date morpholino oligonucleotide (PMO).
21. The method of claim 20, wherein the PMO comprises 5GUCCAACAUCAAGGAAGAUGGCAUUUCUAG3' (SEQ ID NO: 98).
22. The method of claim 19, wherein the linker of step (i) comprises an amino acid monomer or a handle configured to couple with an oligonucleotide monomer.
22. The method of claim 19, wherein the protecting group of the amino acid monomer is
Fmoc.
23. The method of claim 9 wherein the protecting group of the oligonucleotide monomer is
Fmoc.
24. The method of claim 23, wherein the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000135_0001
25. The method of claim 19, wherein the protecting group of the oligonucleotide monomer is Trt.
26. The method of claim 25, wherein the oligonucleotide monomer is selected from the group consisting of:
Figure imgf000136_0001
27. The method according to claim 19, wherein the forming step (x) comprises forming an olefin cross link between at least two unnatural amino acids.
28. The method according to claim 27, wherein the at least two unnatural amino acids are selected from the group consisting of
Figure imgf000136_0002
wherein S5 is (S)-pentenylalanine, R5 is (R)-pentenylalanine, S8 is (S)-octenylalanine, R8 is (R)-octenylalanine, B5 is α, α-di-substituted pentenylalanine, B8 is α, α-di-substituted octenylalanine, and S-OAS and R-OAS are O-allylserine analogues.
29. The method according to claim 19, wherein the forming step (x) comprises forming a lactam bridge between a free amine containing amino acid and a carboxylic acid containing amino acid.
30. The method according to claim 29, wherein the lactam is formed by cross linking a lysine and glutamic or aspartic acid residues.
31. A composition comprising the compound made according to the method of claim 1 and one or more pharmaceutically acceptable excipients.
32. A composition comprising the compound made according to the method of claim 19 and one or more pharmaceutically acceptable excipients.
33. The composition of claim 32, formulated for oral, parenteral, intravenous, or topical administration.
34. The composition of claim 33, formulated for oral, parenteral, intravenous, or topical administration.
35. A method of administering the composition of claim 33 intravenously to a subject, comprising the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
36. A method of administering the composition of claim 34 intravenously to a subject, comprising the steps of diluting a dosage of the composition into 0.9% sodium chloride injection to obtain a volume between 100 and 150 mL and administering the dosage to the subject via intravenous infusion over a period between 35 and 60 minutes.
37. A molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i+4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acid sequence of SEQ ID NOS: 23, 24, 25, 28, 29, 30, 31, 32, 34, 37, 38, 59, or 60, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
38. The molecule of claim 37, further comprising a thiol-containing moiety that is linked to the peptide moiety at the C-terminus by a polyethylene glycol linker.
39. The molecule of claim 38, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
40. The molecule of claim 39, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
41. The molecule of claim 40, wherein the PMO is linked to the thiol -containing moiety at the C-terminus of the StaP via a bifunctional linker.
42. The molecule of claim 41, wherein the bifunctional linker is SMCC.
43. The molecule of claim 42, wherein the PMO has the sequence 5’-
GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
44. The molecule of claim 39, wherein the antisense oligonucleotide is a gapmer.
45. The molecule of claim 44, wherein the gapmer is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker.
46. The molecule of claim 45, wherein the bifunctional linker is SMCC.
47. The molecule of claim 46, wherein the gapmer has the sequence
5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
48. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of claim 37.
49. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of claim 43.
50. A method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of claim 47.
51. The method of claim 48, wherein the cell is a cardiac muscle cell.
52. The method of claim 49, wherein the cell is a cardiac muscle cell.
53. The method of claim 50, wherein the cell is a cardiac muscle cell.
54. The method of claim 48, wherein the cell is a skeletal muscle cell.
55. The method of claim 49, wherein the cell is a skeletal muscle cell.
56. The method of claim 50, wherein the cell is a skeletal muscle cell.
57. A composition comprising the molecule of claim 37 and one or more pharmaceutically acceptable excipients.
58. The composition of claim 57, formulated for oral, parenteral, intravenous, or topical administration.
59. A molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i+4, and/or i, i+7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acids arginine, leucine, and lysine, wherein at least one leucine residue and at least one lysine residue are located between positions i and i+4, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
60. The molecule of claim 59, wherein the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
61. The molecule of claim 60, further comprising a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
62. The molecule of claim 61, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
63. The molecule of claim 62, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
64. The molecule of claim 63, wherein the PMO is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker.
65. The molecule of claim 64, wherein the bifunctional linker is SMCC.
66. The molecule of claim 65, wherein the PMO has the sequence 5’-
GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99)
67. The molecule of claim 62, wherein the antisense oligonucleotide is a gapmer.
68. The molecule of claim 67, wherein the gapmer is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker.
69. The molecule of claim 68, wherein the bifunctional linker is SMCC.
70. The molecule of claim 69, wherein the gapmer has the sequence
5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
71. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of claim 59.
72. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of claim 66.
73. A method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of claim 70.
74. The method of claim 71, wherein the cell is a cardiac muscle cell.
75. The method of claim 72, wherein the cell is a cardiac muscle cell.
76. The method of claim 73, wherein the cell is a cardiac muscle cell.
77. The method of claim 71, wherein the cell is a skeletal muscle cell.
78. The method of claim 72, wherein the cell is a skeletal muscle cell.
79. The method of claim 73, wherein the cell is a skeletal muscle cell.
80. A composition comprising the molecule of claim 59 and one or more pharmaceutically acceptable excipients.
81. The composition of claim 80, formulated for oral, parenteral, intravenous, or topical administration.
82. A molecule comprising a biologically active moiety and a peptide moiety, wherein the biologically active moiety is covalently linked directly or covalently linked via a bifunctional linker moiety (BFL) to the peptide moiety, wherein the peptide moiety is a stapled peptide (StaP) or a stitched peptide (StiP), wherein the StaP or StiP is a stabilized peptide which has a conformation comprising at least one alpha helix by olefin cross linking comprising in the StaP an olefin cross link between two unnatural amino acids of the peptide at positions i, i I 4, and/or i, i I 7 and in the StiP at least two olefin cross links between at least three unnatural amino acids of the peptide at positions i, i+4, and i+ 11, and the StaP or the StiP can penetrate a cell membrane, wherein the peptide moiety comprises the amino acids arginine, glycine, histidine, and lysine, wherein at least one lysine residue is located between positions i and i+4, wherein the molecule can penetrate a cell membrane, and has biological activity of the biologically active moiety.
83. The molecule of claim 82, wherein the peptide moiety is a StaP and comprises the amino acid sequence of SEQ ID NOS: 103, 104, 105, 106, or 107.
84. The molecule of claim 83, further comprising a thiol-containing moiety that is linked to the StaP at the C-terminus by a polyethylene glycol linker.
85. The molecule of claim 84, wherein the biologically active moiety is a biologically active siRNA or antisense oligonucleotide moiety.
86. The molecule of claim 85, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
87. The molecule of claim 86, wherein the PMO is linked to the thiol-containing moiety at the C-terminus of the StaP via a bifunctional linker.
88. The molecule of claim 87, wherein the bifunctional linker is SMCC.
89. The molecule of claim 88, wherein the PMO has the sequence 5’- GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 99).
90. The molecule of claim 85, wherein the antisense oligonucleotide is a gapmer.
91. The molecule of claim 90, wherein the gapmer is linked to the thiol -containing moiety at the C-terminus of the StaP via a bifunctional linker.
92. The molecule of claim 91, wherein the bifunctional linker is SMCC.
93. The molecule of claim 92, wherein the gapmer has the sequence
5’AGCCGGGTGTGGTGCCTCTT3’ (SEQ ID NO: 108).
94. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of claim 82.
95. A method of delivering a biologically active moiety into a cell and retaining biological activity of the biologically active moiety comprising contacting the cell with the molecule of claim 89.
96. A method of delivering a biologically active gapmer into a cell and retaining biological activity of the gapmer comprising contacting the cell with the molecule of claim 93.
97. The method of claim 94, wherein the cell is a cardiac muscle cell.
98. The method of claim 95, wherein the cell is a cardiac muscle cell.
99. The method of claim 96, wherein the cell is a cardiac muscle cell.
100. The method of claim 94, wherein the cell is a skeletal muscle cell.
101. The method of claim 95, wherein the cell is a skeletal muscle cell.
102. The method of claim 96, wherein the cell is a skeletal muscle cell.
103. A composition comprising the compound of claim 82 and one or more pharmaceutically acceptable excipients.
104. The composition of claim 103, formulated for oral, parenteral, intravenous, or topical administration.
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