WO2015069665A1 - Système d'administration intracellulaire d'oligomères présentant une faible vitesse d'élimination rénale - Google Patents

Système d'administration intracellulaire d'oligomères présentant une faible vitesse d'élimination rénale Download PDF

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WO2015069665A1
WO2015069665A1 PCT/US2014/063945 US2014063945W WO2015069665A1 WO 2015069665 A1 WO2015069665 A1 WO 2015069665A1 US 2014063945 W US2014063945 W US 2014063945W WO 2015069665 A1 WO2015069665 A1 WO 2015069665A1
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molecule
moiety
sequence
polypeptide
fatty acid
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Christian ECKHOFF
Brian LEPPERT
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Rain Bioscience, Inc.
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
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    • C12N2310/3513—Protein; Peptide
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
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    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the subject matter disclosed herein relates to cell-penetrating peptides linked to antisense nucleic acids such as phosphorodiamidate morpholino oligomers (PMO) and to fatty acid moieties.
  • PMO phosphorodiamidate morpholino oligomers
  • Renal toxicity often results from an excessive uptake of a given compound into kidney cells.
  • a major challenge in the development of new pharmaceuticals is to develop molecules that are readily able to cross the membranes of therapeutic target cells, but that do not overload the renal cells to an extent that kidney cell viability or kidney function is jeopardized.
  • Some embodiments of the present application provide a molecule comprising a saturated fatty acid moiety, a polypeptide moiety and a nucleic acid moiety.
  • the fatty acid moiety comprises at least one myristoyl, palmitoyl or stearoyl element covalently bound to said polypeptide moiety at an amino-terminal alpha amine or an epsilon amine.
  • the polypeptide moiety comprises a sequence of residues comprising (XA) n B, where XA is represented by SEQ ID NO: 1, wherein X is independently selected from L-arginine and L-lysine, A is L-alanine, B is beta- alanine and n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
  • the nucleic acid moiety comprises a phosphorodiamidate morpholino oligo (PMO) having a sequence that is at least 85% reverse-complementary to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases of a CD47 transcript.
  • the PMO is covalently bound at its 3' end to the beta-alanine of said polypeptide.
  • Some embodiments of the present application provide a method of reducing CD47 protein expression in a mammalian subject comprising the step of administering a molecule as described herein. In some embodiments, practice of said method results in reducing CD47 protein expression and does not result in lethal renal necrosis.
  • Some embodiments of the present application provide a method of disrupting a signaling pathway comprising CD47 in a mammalian subject, said method comprising the step of administering a molecule as described herein.
  • practice of said method results disrupting a signaling pathway comprising CD47 and in does not result in lethal renal necrosis.
  • Some embodiments of the present application provide a molecule comprising a fatty acid moiety, a polypeptide moiety and a nucleic acid moiety.
  • the polypeptide moiety directs localization of the molecule to an interior space of a cell.
  • the nucleic acid moiety comprises nucleic acid base sequence that is reverse-complementary to a nucleic acid sequence of said cell.
  • the polypeptide moiety comprises a sequence of at least 8 amino acid residues, of which 50% are basic residues. In some embodiments, said polypeptide moiety comprises a sequence of 28 or fewer amino acid residues, of which 50% are basic residues. In some embodiments, said polypeptide comprises basic and neutral residues. In some embodiments, said basic residues and said neural residues alternate in said polypeptide.
  • said polypeptide comprises amino acid residues having the sequence (XZ) n , where XZ is represented by SEQ ID NO: 2, wherein X is independently selected from L-arginine and L-lysine, and Z is a non-basic amino acid.
  • Z is a neutral amino acid.
  • Z is selected from L- alanine, glycine, or beta-alanine.
  • Z is selected from L-alanine and beta-alanine.
  • Z is L-alanine.
  • said polypeptide moiety comprises a beta-alanine residue.
  • said polypeptide moiety comprises a beta-alanine residue at its carboxy-terminus.
  • said polypeptide moiety comprises a glycine residue at its carboxy-terminus.
  • said nucleic acid comprises at least 10 bases. In some embodiments, said nucleic acid comprises at least 15 bases. In some embodiments, said nucleic acid comprises no more than 50 bases. In some embodiments, said nucleic acid comprises no more than 30 bases. In some embodiments, said nucleic acid comprises sequence that is at least 85% reverse complementary to a target sequence within an RNA sequence. In some embodiments, said target sequence comprises at least 15 bases of said RNA sequence. In some embodiments, said target sequence comprises 30 bases of said RNA sequence. In some embodiments, said target sequence comprises 50 bases of said RNA sequence. In some embodiments, said RNA sequence is an mRNA sequence. In some embodiments, said RNA sequence is a miRNA sequence.
  • said nucleic acid comprises sequence that is at least 90% reverse-complementary to said target sequence. In some embodiments, said nucleic acid comprises sequence that is at least 95% reverse-complementary to said target sequence. In some embodiments, said nucleic acid comprises sequence that is 100% reverse- complementary to said target sequence. In some embodiments, said nucleic acid comprises sequence that is reverse complementary to a sequence of a nucleic acid implicated in a disease.
  • said nucleic acid comprises sequence that is reverse complementary to a pathogen sequence.
  • said pathogen is a viral pathogen.
  • said pathogen is a eubacterial pathogen.
  • said pathogen is a eukaryotic pathogen.
  • said target sequence is selected from a fragment of a CD47 transcript sequence. In some embodiments, said target sequence is selected from a fragment of a CD47 transcript sequence within 100 bases of a translation initiation site of said CD47 transcript. In some embodiments, said target sequence is selected from a fragment of a CD47 transcript sequence within 100 bases of an initial start codon of a CD47 protein- encoding open reading frame of said CD47 transcript.
  • said nucleic acid moiety is covalently bound to said polypeptide sequence at the 3' end of said nucleic acid moiety. In some embodiments, said nucleic acid moiety is covalently bound to said polypeptide sequence at the 5' end of said nucleic acid moiety. In some embodiments, wherein said nucleic acid moiety is covalently bound to said polypeptide moiety at a beta-alanine residue. In some embodiments, said nucleic acid moiety comprises a phosphorodiamidate morpholino oligomer.
  • said fatty acid moiety is a saturated fatty acid moiety. In some embodiments, said fatty acid moiety consists of an even number of carbon atoms. In some embodiments, said fatty acid moiety comprises a myristoyl moiety. In some embodiments, said fatty acid moiety comprises a palmitoyl moiety. In some embodiments, said fatty acid moiety comprises a stearoyl moiety.
  • said fatty acid moiety is covalently bound to said polypeptide moiety. In some embodiments, said fatty acid moiety is covalently bound to said polypeptide moiety at an amino moiety. In some embodiments, said fatty acid moiety is covalently bound to said polypeptide moiety at an amino-terminal alpha amine. In some embodiments, said fatty acid moiety is covalently bound to said polypeptide moiety at an epsilon amine. In some embodiments, said fatty acid moiety is covalently bound to said polypeptide moiety at a non-amine position.
  • said molecule comprises a second fatty acid moiety. In some embodiments, said molecule comprises a third fatty acid moiety. In some embodiments, said fatty acid moiety binds albumin.
  • Some embodiments of the present application provide a method of decreasing renal clearance rate upon administration of a polypeptide-PMO molecule to a mammal comprising conjugating at least one fatty acid moiety to said polypeptide-PMO molecule.
  • the method comprises conjugating a second fatty acid moiety to said polypeptide-PMO molecule.
  • the method comprises conjugating a third fatty acid moiety to said polypeptide-PMO molecule.
  • the method comprises comprising conjugating a second fatty acid moiety to said polypeptide-PMO molecule.
  • the method comprises contacting said molecule to albumin.
  • said renal clearance rate upon administration of said conjugated polypeptide-PMO molecule to a mammal is reduced.
  • a dose of said polypeptide-PMO molecule which is above a level sufficient to cause renal toxicity when said polypeptide-PMO molecule is administered in without conjugating at least one fatty acid moiety is administered to said mammal.
  • said fatty acid moiety is a saturated fatty acid moiety. In some embodiments, said fatty acid moiety consists of an even number of carbon atoms. In some embodiments, said fatty acid moiety comprises a myristoyl moiety. In some embodiments, said fatty acid moiety comprises a palmitoyl moiety. In some embodiments, said fatty acid moiety comprises a stearoyl moiety.
  • FIG. 1 is the general structure of cell-penetrating peptides (CPP) linked to the 3' position of a phosphorodiamidate morpholino oligomer, where XZ is represented by SEQ ID NO: 3.
  • CPP cell-penetrating peptides
  • FIG. 2 is the chemical structure of N-terminal L-arginine.
  • FIG. 3 is the chemical structure of N-terminal L-lysine.
  • FIG. 4 is the chemical structure of non-N-terminal L-lysine.
  • FIG. 5 is the chemical structure of the beta-alanine linkage bound to the 3 ' end of PMO.
  • Cell -penetrating peptides having 4-14 basic amino acids are disclosed.
  • basic amino acids are selected independently from L-arginine or L-lysine.
  • nonbasic amino acids are selected independently from L-alanine or beta-alanine.
  • basic and nonbasic amino acids alternate in the localization region of the polypeptide sequence so that the total number of amino acids is approximately or exactly equivalent to twice the number of basic amino acids in that region of the polypeptide.
  • the N-terminal amino acid is a basic amino acid.
  • the C-terminal amino acid may form an amide bond with an additional beta-alanine or glycine amino acid, which may link the cell-penetrating peptide to an oligomeric nucleic acid such as a phosphorodiamidate morpholino oligomer by an amide bond, which is in some embodiments formed between the beta-alanine or glycine carboxyl group and the secondary amine at the 3 ' end of the oligomer.
  • Phosphorodiamidate morpholino oligomers are a type of RNA targeting antisense oligonucleotide that resemble short strands (typically 15-30 nucleotides) of single- stranded RNA or DNA but have a modified backbone with no negative charges at physiologically relevant pH.
  • PMO are of high interest as compounds of potential medicinal value because their pharmacological target is a specific section of a nucleic acid such as mRNA, pre-mRNA, or miRNA. By having a base sequence that is reverse-complementary to the RNA target sequence, for example, PMO hybridize to the intended section of single- stranded RNA.
  • the resulting duplex then interferes with the activity of the native RNA molecule, which may initiate a biological response resulting in altered expression of an encoded protein.
  • the response may result from the duplex interfering with the ability of a ribosome to recognize and bind an mRNA molecule to direct translation of an encoded protein, for example, or the response may result from the duplex interfering with the ability of a miRNA molecule to bind to its target.
  • PMO have large molecular mass, typically greater than 5,000 Da, a high number of hydrogen bond donors and hydrogen bond acceptor groups, and little lipophilicity. Therefore, PMO diffuse poorly across biological membranes and have a very limited ability to reach intracellular mRNA targets.
  • Other base-pairing molecular options such as peptide nucleic acids or phosphorothioate nucleic acids, suffer similar challenges in diffusing across biological membranes.
  • Cell-penetrating peptides are rich in the basic amino acids arginine and lysine.
  • the HIV Tat protein was found to be internalized by cells (Frankel and Pabo, Cell 55: 1189-1193, 1988), and it was later demonstrated that a short basic domain of the Tat protein was responsible for the translocation through the plasma membrane (Vives et al., J Biol Chem 272:16010-16017, 1997).
  • Examples of synthetically derived CPP have been described in U.S. Pat. No. 7,468,418 to Iversen (2008) and U.S. Pat. No. 7,585,834 to Wender (2009), as well as in U. S. Pat. No. 7,084,248 to Summerton (2004), U.S. Pat. No. 8,053,420 to Iverson (2011), U.S. Publication No. 2010/0234280 to Geller, and U.S. Publication No. 2012/0058946, to Moulton, each of which is hereby incorporated by reference in its entirety.
  • CPP are known to effectively transduce across biological membranes by a not yet fully understood mechanism. It has been shown that conjugation of CPP with other chemical species of variable size, including molecules much larger than CPP itself, maintains membrane transduction and thus represents an opportunity to carry impermeable molecular cargo into cells (Futaki, Advanced Drug Del Rev. 57, 547-558, 2005). Due to the uncharged backbone, PMO is ideally suited for assisted membrane transduction by CPP. However, systemic administration of CPP-PMO conjugates has produced unacceptable toxicity in laboratory animals that has thus far precluded advancement of such conjugates into clinical candidates (Amantana et al., Bioconjugate Chem. 18, 1325-1331, 2007; Sazani et al., poster presentation at Treat-NMD/NIH Conference 2009, Brussels, Belgium, 2009). The kidney appeared to be the most sensitive organ for the toxicity of CPP-PMO conjugates.
  • proximal tubule epithelial cells proximal tubule epithelial cells
  • RPTEC proximal tubule epithelial cells
  • Cisplatin and other chemotherapeutic agents, aminoglycoside antibiotics, amphotericin B, and NSAIDs such as diclofenac have been found to induce necrosis of RPTEC.
  • a main function of RPTEC is the re-absorption of organic materials (mainly small proteins, particularly albumin, and nucleic acids) and electrolytes found in the filtrate of the glomerulae.
  • RPTEC Like the cells lining the small intestine, RPTEC have villi on the luminal (apical) side that greatly increase the effective surface area available for re-uptake of potentially valuable materials for the body, and are rich in transporter systems and pumps that can direct the flow of organic substances or ions.
  • compositions so designed can enter cells with much greater efficiency than unconjugated oligomer but have attenuated potency in renal tubules.
  • a saturated fatty acid such as myristoyl, palmitoyl, or stearoyl may be covalently bound to the N-terminal alpha amino group of the peptide, or the N-terminal alpha amino group may be acetylated or left unmodified. If lysine is present in the peptide, one or more lysine amino acids may be bound at the ⁇ -amino group with a saturated fatty acid such as myristoyl, palmitoyl, or stearoyl.
  • a saturated fatty acid may optionally be bound elsewhere throughout the peptide, and multiple fatty acids may be added to a polypeptide. Addition of one or more fatty acids may increase retention of the molecule in the blood and thereby decrease the maximal dose to which a renal cell is exposed.
  • FIG. 1 is the general structure of an exemplary CPP linked to the 3' position of a phosphorodiamidate morpholino oligomer.
  • a conjugate of this general structure is capable of delivering its PMO cargo to therapeutic target cells with greater efficiency than PMO alone, i.e., PMO that is not linked to a CPP.
  • Basic amino acids L-arginine or L-lysine
  • nonbasic amino acids L- alanine or beta-alanine
  • the N-terminal amino acid is either L-arginine or L-lysine.
  • Beta-alanine provides for the linkage to the 3' position of the PMO.
  • FIG. 2 is the chemical structure of L-arginine at the N-terminus of the peptide.
  • FIG. 3 is the chemical structure of L-lysine at the N-terminus of the peptide.
  • FIG. 4 is the chemical structure of L-lysine at positions in the peptide chain other than the N-terminal position.
  • FIG. 5 is the chemical structure of the beta-alanine linkage bound to the 3 ' end of PMO by an amide bond between the carboxyl group of beta-alanine and the secondary nitrogen of the morpholino ring.
  • polypeptide component of the polypeptide-conjugated molecules disclosed herein may be synthesized by any number of methods know to those of skill in the art. Polypeptides may be custom-synthesized, for example by a commercial polypeptide synthesis company such as United Biosystems, of Herndon, VA, American Peptide Company, of Sunnyvale, CA, or Bachem, of Torrance, CA. Any number of chemical synthesis chemistries or suppliers may be used compatibly with the disclosure herein.
  • An exemplary polypeptide sequence disclosed herein comprises the sequence (XZ) n B, where X is selected from L-arginine and L-lysine, Z is selected from L- alanine and beta-alanine, B is beta-alanine, and n is an integer ranging from 4 to 14. In some embodiments, n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20.
  • Exemplary polypeptide sequences presented in the N-terminal to C-terminal direction conventional for polypeptide nomenclature, include RARARARAB (SEQ ID NO: 4), RARARARARAB (SEQ ID NO: 5), RARARARARARAB (SEQ ID NO: 6), RARARARARARARAB (SEQ ID NO: 7), RARARARARARARARAB (SEQ ID NO: 8), RARARARARARARARARAB (SEQ ID NO: 9), RARARARARARARARAB (SEQ ID NO: 10), RARARARARARARARARARAB (SEQ ID NO: 11), RARARARARARARARARARAB (SEQ ID NO: 12) and RARARARARARARARARARAB (SEQ ID NO: 13), wherein R is L-arginine, A is L-alanine and B is beta-alanine.
  • one or more arginine residues is replaced with lysine. In some embodiments one or more alanine residues is replaced with beta-alanine.
  • an exemplary polypeptide sequence is RARARARARARARARARARBB (SEQ ID NO: 14). In some embodiments an exemplary polypeptide sequence is (RA) 9 RBB (SEQ ID NO: 14).
  • the polypeptide comprises less than 50% basic residues. In some embodiments the polypeptide comprises up to 50% basic residues. In some embodiments the polypeptide comprises about 50% basic residues. In some embodiments the polypeptide comprises 50% basic residues. In some embodiments the basic residues alternate with neutral residues, such as alanine residues.
  • the carboxy-terminal amino acid is beta-alanine. In some embodiments the carboxy-terminal amino acid is glycine.
  • the polypeptide comprises sequence of the formula (XZ)n, where XZ is represented by SEQ ID NO: 15; (XYX) n X, where XYX is represented by SEQ ID NO: 16; (XY) abuseX, wherein XY is represented by SEQ ID NO: 17; (XYY) n X, where XYY is represented by SEQ ID NO: 18; (XYYY) n X, where XYYY is represented by SEQ ID NO: 19;or U(XY) n B, where XY is represented by SEQ ID NO: 20; where X is L-arginine or L-lysine as above, Y is any amino acid, U is any amino acid other than arginine or lysine, B is beta-alanine, R is arginine, K is lysine, and G is glycine.
  • n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • Additional exemplary sequences include the following: RARARARARARARARARB (SEQ ID NO: 21), RARARARARARAB (SEQ ID NO: 22), ARARARARARARARARBB (SEQ ID NO: 23), RARARARARARABB (SEQ ID NO: 24); KARARAKARARAKARARAKB (SEQ ID NO: 25); (R) 8 B (SEQ ID NO: 26); (R) 7 ARB (SEQ ID NO: 27); (R) 3 A(R) 5 B (SEQ ID NO: 28); RA(R) 3 A(R) 3 ARB (SEQ ID NO: 29); (RA)nB (SEQ ID NO: 30); RXRRBRRXRRBRXB (SEQ ID NO: 31); RXRRXRRXRRXRXB (SEQ ID NO: 32); (RA) 5 B (SEQ ID NO: 33); (RB) 5 B (SEQ ID NO: 34); (RG) 4 B (SEQ ID NO: 35);
  • the polypeptide component of a polypeptide- conjugated molecules disclosed herein is fused to a nucleic acid molecule, such as a nucleic acid molecule having a 5 '-3' orientation.
  • the polypeptide is fused to the 3' end of the nucleic acid molecule at a beta-alanine.
  • the polypeptide is fused to the 3' end of the nucleic acid molecule at a glycine.
  • the polypeptide is fused at the 3' end of the molecule at a residue other than beta-alanine.
  • the polypeptide is fused at the 5' end of the molecule at a beta-alanine residue, glycine residue, or at a residue other than beta-alanine.
  • the nucleic acid molecule is a morpholino oligomer.
  • Morpholino oligomers may be synthesized by any number of methods known to those of skill in the art. Morpholino oligomers may be custom-synthesized, for example by a commercial morpholino oligomer synthesis company such as GeneTools, of Philomath, OR. Any number of chemical synthesis chemistries or suppliers may be used compatibly with the disclosure herein.
  • nucleic acid analogue chemistries presenting bases capable of base- pairing with a target nucleic acid sequence and entering a cell are contemplated and are consistent with the disclosure herein.
  • the nucleic acid molecule such as a morpholino oligomer comprises a sequence of nucleic acid bases.
  • Embodiments consistent with the disclosure herein may have a number of distinct nucleic acid sequences.
  • the sequence of bases on the nucleic acid molecule such as a morpholino oligomer is the reverse complement of an RNA transcript, such as an mRNA molecule encoding a polypeptide of which the expression level is to be targeted for perturbation and annealing near or at the translation initiation site or at or near the start of the encoded open reading frame.
  • the polypeptide expression level is to be reduced upon administration of a molecule consistent with the disclosure herein.
  • the polypeptide expression level is to be enhanced upon administration of a molecule consistent with the disclosure herein, for example due to administration of a molecule that interferes with a miRNA molecule that otherwise negatively impacts protein expression.
  • the polypeptide expression is altered to produce a different polypeptide upon administration of a molecule consistent with the disclosure herein than in the absence of such administration.
  • the sequence of bases on the nucleic acid molecule such as a morpholino oligomer is the reverse complement of an RNA transcript, such as an mRNA molecule or miRNA molecule, the function of which is to be perturbed upon administration of a molecule consistent with the disclosure herein.
  • antisense oligonucleotides comprising reverse-complement nucleic acid sequences, including PMO, may function by blocking the ability of the cellular translation machinery to access its target RNA. These steric -blocking oligonucleotides can prevent the ribosome from recognizing the translation start codon in an mRNA molecule. As a result, translation of a polypeptide from that transcript is reduced or eliminated. See, for a review of this and other RNA interference (RNAi) mechanisms, Kole et al., (2012) "RNA therapeutics: beyond RNA interference and antisense oligonucleotides", Nature Reviews Drug Discovery 11: 125, which is hereby incorporated by reference in its entirety.
  • antisense oligonucleotides comprising reverse-complement nucleic acid sequences, including PMO may function by blocking the ability of a miRNA molecule to bind to its target.
  • nucleic acid molecules may be targeted through administration of a molecule consistent with the disclosure herein.
  • the nucleic acid molecules are of a pathogenic origin, such as viral nucleic acid molecules. Examples include respiratory syncytial virus, an influenza virus such as parainfluenza virus, herpes simplex family viruses such as herpes simplex-2, a hepatitis virus, or an immunodeficiency virus such as HIV.
  • the nucleic acid molecule targeted is produced by a eubacterial or eukaryotic pathogen host such as tuberculosis, a Plasmodium species or a cholera bacterium.
  • the molecule to be targeted may encode a protein the presence of which or the misregulation of which may be correlated with unregulated cell growth or proliferation such as that seen in cancer.
  • a nonlimiting list of cancers that may be targeted through administration of a molecule consistent with the disclosure herein includes glioblastomas, fibrosarcomas, osteosarcomas, carcinomas, melanomas, breast cancer, gastrointestinal stromal tumors, lung cancer including small-cell and non-small-cell lung cancer, pancreatic cancer, colorectal cancer, bladder cancer, ovarian cancers, gastric cancers, head and neck cancers, brain cancers, blood cancers, malignant melanoma, papillary thyroid cancer, adenocarcinomas of the pancreas and colon, thyroid tumors, malignant T-cell lymphomas retinoblastoma, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), non-Hodgkin'
  • transcripts which may be targeted so as to reduce the accumulation levels of their encoded proteins includes transcripts encoding c-Sis, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR), HER2/neu, Src-family, Syk-ZAP-70 family, and BTK family of tyrosine kinases, the Abl gene in CML - Philadelphia chromosome, Raf kinase, and cyclin-dependent kinases, Ras protein, myc and CD47.
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • HER2/neu HER2/neu
  • Src-family Src-family
  • Syk-ZAP-70 vascular endothelial growth factor receptor
  • BTK BTK family of tyrosine kinases
  • transcripts encoding proteins which interact in the same pathway as the transcripts recited above are also contemplated, as are transcripts encoding proteins implicated in cancer but not listed herein.
  • the molecule comprises a sequence which is the reverse complement of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 90, 100, or more than 100 bases of its target transcript.
  • the molecule comprises 10-50 bases reverse complimentary to its target.
  • the molecule comprises 15-30 bases reverse complimentary to its target.
  • the molecule anneals to a target transcript in the proximity of a translation start "ATG" codon at the start of a coding open reading frame. In some embodiments the molecule anneals to a target transcript within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
  • the molecule may demonstrate complete reverse complementarity with its target such that complete base pairing may occur across the length of the molecule and its reverse complement sequence in its target.
  • the reverse complementarity may be less that 100%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the reverse complementarity may be less than 90%, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%.
  • the sequence identity may be sufficient to allow double-strand formation independent of sequence identity.
  • the reverse complementarity may be at least 85%.
  • Reverse complementarity may be calculated across the nucleic acid sequence of an entire molecule in comparison to a target region of a transcript or microRNA molecule. Sequence identity may be calculated across a subset of the nucleic acid sequence of an entire molecule in comparison to a target region of a transcript or microRNA molecule.
  • the nucleic acid sequence of an entire molecule may comprise both a first region of 30 nucleic acid bases which are 90% reverse complementary (i.e., 27 of 30) to a target region of a target transcript, and a second region of nucleic acid bases, such as a contiguous region to the first, for which nucleic acid sequence identity is not substantial enough to constitute base pairing.
  • a sequence identity of 90% with the reverse complement would be reported for the full length first region, and a sequence identity of greater than 90% with the reverse complement would be reported for subsets of the first region, even though the nucleic acid sequence of the entire molecule may comprise less than 90% identity, perhaps substantially less than 90% identity, to a target region of a target molecule.
  • reverse complementarity id based upon sequence identity and is determined to reflect standard nucleic acid double stranded base pairing, such that G pairs with C and A pairs with T.
  • account is taken of the fact known to one of skill in the art that the ribonucleic acid base U may base pair with either A or G.
  • sequence identity is defined to be present if at least one base of a target region matches the reverse complement of at least one base of a molecule as contemplated herein.
  • reverse-complementarity is defined to be present if at least one base of a target region is able to base-pair with at least one base of a target molecule such as an mRNA transcript or a miRNA molecule as contemplated herein.
  • a poly-U transcript sequence may be considered to have complete reverse-complementarity to a molecule as disclosed herein having a sequence of bases which is 50% G and 50% A over a span of similar length due to the fact that the two strands are able to base pair to form a double-helix.
  • the molecule targets transcripts encoding CD47.
  • the molecule base-pairs with a region of the CD47 transcript that is common to all known CD47 transcript splice variants.
  • the molecule targets one, two, or three specific splice variant classes of CD47.
  • the molecule targets CD47 isoform 1.
  • the molecule targets CD47 isoform 2.
  • the molecule targets CD47 isoform 3.
  • the molecule comprises a sequence which is the reverse complement of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 90, 100, or more than 100 bases of the CD47 isoform 1 sequence corresponding to the reverse-transcribed cDNA molecule generated from CD47 mRNA isoform 1, having the sequence of SEQ ID NO: 39 below: >CD47 Homo sapiens CD47 molecule (CD47), transcript variant 1, mRNA NCBI Reference Sequence: NM_001777.3, reverse transcribed into cDNA.
  • the corresponding CD47 isoform 1 mRNA sequence differs from that above by the replacement of "t" (thymidine) with "u” (uracil) throughout the sequence, having the effect that the mRNA molecule is able to base-pair with substantially more molecules than would be determined to have sequence that is identical to the reverse complement of the cDNA sequence.
  • the molecule targets a transcript encoding or regulating a CD47 interacting protein, such as Thrombospondin (TSP), Signal-regulatory protein-alpha (SIRPalpha), or an Integrin such as avb3.
  • TSP Thrombospondin
  • SIRPalpha Signal-regulatory protein-alpha
  • Integrin such as avb3.
  • a morpholino oligomer having the desired nucleotide sequence and optionally a beta-alanine or glycine attached at the 3' position may be synthesized by a supplier.
  • a cell penetrating polypeptide of the desired amino acid sequence with or without fatty acid acylation may be synthesized by a supplier.
  • a morpholino oligomer and cell penetrating peptide may be conjugated through a number of chemistries known to one of skill in the art, such as a simple amide bond formation between the carboxylate at the C terminus of the peptide and the alpha-amino group of beta-alanine or glycine using common amide bond formation chemistry, for example that taught by Abes et al. (2006), J Controlled Release 116 304-313 (morpholino and peptide) or Wesolowski et al. (2011) PNAS, 108(40): 16582-16587, each of which is hereby incorporated by reference in its entirety.
  • fatty acyl groups such as acetoyl groups, stearoyl groups or other fatty acyl groups may be conjugated, for example to the polypeptide component at an N-terminal amine moiety, at an internal epsilon-amino group of a Lysine reside, or at another internal position on a polypeptide.
  • Other chemistries and methods of conjugation or bond formation are known to one of skill in the art and compatible with the disclosure herein.
  • the polypeptide-nucleic acid conjugate such as a polypeptide morpholino oligomer conjugate is further conjugated to a fatty acid, such as a saturated fatty acid.
  • a fatty acid such as a saturated fatty acid.
  • fatty acids such as Butyric acid (Butanoic acid; CH 3 (CH 2 ) 2 COOH; C 4 :0); Caproic acid (Hexanoic acid; CH 3 (CH 2 ) 4 COOH; C6:0); Caprylic acid (Octanoic acid; CH 3 (CH 2 ) 6 COOH; Cs:0); Capric acid (Decanoic acid; CH 3 (CH 2 ) 8 COOH; Ci 0 :0); Why acid (Dodecanoic acid; CH 3 (CH 2 )i 0 COOH; Ci 2 :0); Myristic acid (Tetradecanoic acid; CH 3 (CH 2 )i 2 COOH; Ci 4 :0); Palmitic acid (Hexade
  • the fatty acid such as a saturated fatty acid, may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more than 36 carbon atoms.
  • the fatty acid comprises an even-number of carbon atoms.
  • the fatty acid conjugate comprises myristoyl.
  • the fatty acid conjugate comprises palmitoyl.
  • the fatty acid conjugate comprises stearoyl.
  • the fatty acid conjugate is selected from the group consisting of myristoyl, palmitoyl, and stearoyl.
  • the fatty acid conjugate is an acetoyl moiety.
  • Embodiments consistent with the disclosure herein include but are not limited to the following: R*ARARARARARARARARB-PMO (SEQ ID NO: 40); RAK# ARAR AR ARAR AR ARB -PMO (SEQ ID NO:41); R AK#AR AR ARAK# ARAR ARB - PMO (SEQ ID NO: 42) and R* AK#ARARARARARARARB -PMO (SEQ ID NO: 43); wherein PMO indicates a PMO moiety of unspecified structure (optionally comprising sequence reverse-complementary to a fragment of CD47), "*” indicates that the residue immediately preceding is acylated with a C 14 , C 16 or C 18 fatty acid at a terminal (alpha) amino group, "#” indicates that the residue immediately preceding (lysine, K) is acylated with a C 14 , Ci 6 or Ci 8 fatty acid at an epsilon amino group, and "-" indicates a bond between the C- terminal
  • Molecules consistent with the disclosure herein are observed to cross cell membranes such that they localize to the interior of human cells such as the human U937 cell line. Without being limited by theory, the polypeptide moiety is thought to direct this localization. Localization is not hindered substantially by the PMO moiety covalently bound to the polypeptide, nor by the presence of a fatty acyl moiety covalently bound to the polypeptide, either at an N-terminal alpha amino moiety, at an internal epsilon amino moiety, or elsewhere on the polypeptide.
  • Molecules consistent with the disclosure herein are observed to impact the expression levels of polypeptides.
  • polypeptides encoded by nucleic acid molecules comprising nucleic acid sequence of which the PMO moiety on the molecule has the reverse complement sequence demonstrate reduced expression levels in cells contacted with molecules consistent with the disclosure herein.
  • These polypeptides may be, for example, CD47. This activity is not negatively impacted by the presence of a fatty acyl moiety such as a stearoyl moiety on the molecules consistent with the disclosure herein, independent of the fatty acyl conjugation site.
  • the molecules consistent with the disclosure herein have reduced renal toxicity while demonstrating a strong ability to cross cell membranes and to influence the accumulation levels and/or signaling activity of pathways associated with proteins encoded by or influenced by nucleic acids reverse complementary to the nucleic acid sequence of the molecules consistent with the disclosure herein.
  • Cancer cell model cell lines for which aberrant CD47 activity is implicated are treated with molecules consistent with the disclosure herein comprising nucleic acid sequence reverse complementary to CD47.
  • the morpholino oligomer polypeptide conjugate is applied to human U937 monocytes.
  • the cell surface expression level of the CD47 molecule is monitored by flow cytometry using a commercial antibody that recognizes CD47.
  • the dose at which half the protein expression at the cell surface has been inhibited is determined.
  • Administration of the anti-CD47 morpholino oligomer is demonstrated to result in reduced expression of membrane-localized CD47 in treated U937 cells.
  • fatty acid moiety may bind albumin in the blood of a patient administered a molecule consistent with the method, such that the molecule is taken up by the kidneys of said patient at a substantially reduced rate, thereby reducing renal toxicity without reducing availability of the molecule to target cells.
  • a peptide of the sequence acetyl-(RA) 9 RBB (SEQ ID NO: 44) is synthesized.
  • R, A, and B refer to L-Arginine, L- Alanine and beta- Alanine, respectively, and amino acids are joined by peptide bonds.
  • the alpha amino group of the N-terminal L- Arginine residue is acetylated.
  • the peptide is applied to human monocyte cell line U937 and is demonstrated to localize into the cytosol of cells of said cell line.
  • This example demonstrates the efficacy of peptide of the sequence acetyl- (RA) 9 RBB (SEQ ID NO: 44) to localize into the cytosol of a mammalian cell line.
  • EXAMPLE 2 PEPTIDE SEQUENCE DIRECTS LOCALIZATION OF AN ANTI CD47 MOLECULE
  • a morpholino oligomer reverse-complementary to CD47 encoding RNA is identified and conjugated to the peptide of Example 1.
  • the molecule is applied to human monocyte cell line U937.
  • the anti-CD47 morpholino oligomer is demonstrated to localize into the cytosol of cells of said cell line.
  • This example demonstrates the efficacy of a peptide of the sequence (RA)gRBB (SEQ ID NO: 44) to direct localization of a morpholino oligomer into the cytosol of a mammalian cell line.
  • EXAMPLE 3 PEPTIDE-LOCALIZED ANTI-CD47 MOLECULE DIRECTS REDUCED EXPRESSION OF CD47 PROTEIN
  • the morpholino oligomer peptide conjugate of Example 2 is applied to human monocyte cell line U937.
  • CD47 protein levels are monitored by flow cytometry using a commercially available antibody to CD47.
  • the ED50 for knockdown of CD47 protein is determined.
  • Administration of the peptide conjugated anti-CD47 morpholino oligomer is demonstrated to result in reduced CD47 protein levels.
  • This example demonstrates the efficacy of peptide conjugated morpholino oligomer polypeptide conjugate of Example 2 to direct the reduced expression of CD47 protein in a cell contacted with the morpholino oligomer polypeptide conjugate.
  • EXAMPLE 4 N-TERMINAL STEAROYL CONJUGATED PEPTIDE- LOCALIZED ANTI-CD47 MOLECULE DIRECTS REDUCED EXPRESSION OF CD47 PROTEIN
  • the morpholino oligomer polypeptide conjugate of Example 2 is acylated with a stearoyl moiety at the N-terminus of its polypeptide component instead of an acetyl moiety.
  • the stearoyl conjugated morpholino oligomer polypeptide conjugate is applied to human monocyte cell line U937.
  • CD47 protein levels are monitored by flow cytometry using a commercially available antibody to CD47.
  • the ED50 for knockdown of CD47 protein is determined.
  • Administration of the anti-CD47 morpholino oligomer is demonstrated to result in reduced CD47 protein levels.
  • EXAMPLE 5 NON-N-TERMINAL STEAROYL CONJUGATED PEPTIDE- LOCALIZED ANTI-CD47 MOLECULE DIRECTS REDUCED EXPRESSION OF CD47 PROTEIN
  • the morpholino oligomer polypeptide conjugate of Example 2 is conjugated with a stearoyl moiety at a site on its polypeptide component other than the N- terminus.
  • the stearoyl conjugated morpholino oligomer polypeptide conjugate is applied to human monocyte cell line U937.
  • CD47 protein levels are monitored by flow cytometry using a commercially available antibody to CD47.
  • the ED50 for knockdown of CD47 protein is determined.
  • Administration of the anti-CD47 morpholino oligomer is demonstrated to result in reduced CD47 protein levels.
  • This example demonstrates that the addition of a fatty acid moiety to the polypeptide localization signal of the morpholino oligomer polypeptide conjugate of Example 2 does not materially impact the ED50 for knockdown of CD47 protein.
  • EXAMPLE 8 TESTS USING A SECOND ANTI-CD47 SEQUENCE
  • EXAMPLE 9 TESTS USING A SEQUENCE REVERSE-COMPLEMENTARY TO AN ADDITIONAL TARGET
  • EXAMPLE 10 MOLECULES CONSISTENT WITH THE DISCLOSURE
  • Molecules are synthesized consistent with the disclosure herein.
  • the molecules have the chemical structure as follows: R* ARAR AR ARAR AR ARARB -PMO (SEQ ID NO: 46); RAK# ARAR AR ARAR ARARB -PMO (SEQ ID NO: 47); R AK#AR AR ARAK# ARAR ARB -PMO (SEQ ID NO: 48) and
  • R*AK#ARARARARARARARB-PMO (SEQ ID NO: 49); wherein PMO indicates a PMO moiety comprising 20 bases that are reverse-complementary to a 20 base section common to the CD47 transcripts, "*" indicates that the residue immediately preceding is acylated with a C M fatty acid at a terminal (alpha) amino group, "#” indicates that the residue immediately preceding (lysine, K) is acylated with a C 14 fatty acid at an epsilon amino group, and "-" indicates a bond between the C-terminal beta-alanine and the 3' end of the PMO moiety.
  • EXAMPLE 11 MOLECULES CONSISTENT WITH THE DISCLOSURE
  • Molecules are synthesized consistent with the disclosure herein.
  • the molecules have the chemical structure as follows: R* ARAR AR ARAR AR ARARB -PMO (SEQ ID NO: 50); R AK#AR ARAR AR ARAR ARB -PMO (SEQ ID NO: 51); RAK# ARAR ARAK#AR ARARB -PMO (SEQ ID NO: 52) and
  • R* AK#AR ARAR AR ARAR ARB -PMO (SEQ ID NO: 53); wherein PMO indicates a PMO moiety comprising 20 bases that are reverse-complementary to a 20 base section common to the CD47 transcripts, "*" indicates that the residue immediately preceding is acylated with a Ci 6 fatty acid at a terminal (alpha) amino group, "#” indicates that the residue immediately preceding (lysine, K) is acylated with a C 16 fatty acid at an epsilon amino group, and "-" indicates a bond between the C-terminal beta-alanine and the 3' end of the PMO moiety.
  • EXAMPLE 12 MOLECULES CONSISTENT WITH THE DISCLOSURE
  • Molecules are synthesized consistent with the disclosure herein.
  • the molecules have the chemical structure as follows: R* ARAR AR ARAR AR ARARB -PMO (SEQ ID NO: 54); R AK#AR ARAR AR ARAR ARB -PMO (SEQ ID NO: 55); RAK# ARAR ARAK#AR ARARB -PMO (SEQ ID NO: 56) and
  • R* AK#AR ARAR AR ARAR ARB -PMO (SEQ ID NO: 57); wherein PMO indicates a PMO moiety comprising 20 bases that are reverse-complementary to a 20 base section common to the CD47 transcripts, "*" indicates that the residue immediately preceding is acylated with a Ci 8 fatty acid at a terminal (alpha) amino group, "#” indicates that the residue immediately preceding (lysine, K) is acylated with a C 18 fatty acid at an epsilon amino group, and "-” indicates a bond between the C-terminal beta-alanine and the 3' end of the PMO moiety.
  • EXAMPLE 13 PEPTIDE-LOCALIZED ANTI-CD47 MOLECULE DIRECTS REDUCED CD47 PROTEIN SIGNALING ACTIVITY
  • the morphohno oligomer polypeptide conjugate of Example 4 is applied to human monocyte cell line U937.
  • CD47 protein signaling pathways are monitored for reporters of activity.
  • the ED50 for knockdown of CD47 protein signaling pathways is determined.
  • Administration of the anti-CD47 morphohno oligomer is demonstrated to result in reduced CD-47 protein signaling pathways activity.
  • This example demonstrates the efficacy of morphohno oligomer polypeptide conjugate of Example 4 to direct the reduced signaling pathway activity of a pathway comprising a protein encoded by an RNA molecule complementary to the sequence of its conjugated morphohno oligomer in a cell contacted with the morphohno oligomer polypeptide conjugate.
  • EXAMPLE 14 PEPTIDE-LOCALIZED ANTI-CD47 MOLECULE DIRECTS REDUCED CD47 PROTEIN SIGNALING ACTIVITY
  • the morphohno ohgomer polypeptide conjugate of Example 4 is applied to human monocyte cell line U937. PI3K/Akt signaling is monitored for reporters of activity. The ED50 for knockdown of PI3K/Akt signaling pathways is determined. Administration of the anti-CD47 morphohno oligomer is demonstrated to result in reduced PI3K/Akt signaling pathways activity.
  • This example demonstrates the efficacy of morphohno oligomer polypeptide conjugate of Example 5 to direct the reduced signaling pathway activity of a pathway comprising a protein encoded by an RNA molecule complementary to the sequence of its conjugated morphohno oligomer in a cell contacted with the morphohno oligomer polypeptide conjugate.
  • EXAMPLE 15 PEPTIDE-LOCALIZED ANTI-CD47 MOLECULE ENABLES ANTI-TUMOR ACTIVITY AGAINST A TREATED LEUKEMIA CELL LINE
  • the morphohno ohgomer polypeptide conjugate of Example 5 is applied to human U937 cells. Treated cells are co-incubated with untreated wild-type preactivated macrophages, and proliferation of U937 cells is monitored. The ED50 to block cell proliferation is determined. Administration of the anti-CD47 morphohno oligomer is demonstrated to result in reduced U937 cell proliferation in the presence of activated macrophages.
  • This example demonstrates the efficacy of morpholino oligomer polypeptide conjugate of Example 5 to inhibit the proliferation of cancer cells via enhanced anti-tumor activity of wild-type macrophages against a cancer cell line.
  • EXAMPLE 16 FATTY ACYL CONJUGATED PEPTIDE-LOCALIZED ANTI- CD47 MOLECULE SHOWS REDUCED RENAL TOXICITY IN VIVO
  • the morpholino oligomer polypeptide conjugate of Example 5 and the analogous molecule lacking a fatty acyl conjugate are applied to C57 mice. Mice administered the morpholino oligomer polypeptide conjugate of Example 5 are demonstrated to tolerate much higher doses of administration than mice administered and the analogous molecule lacking a fatty acyl conjugate.
  • the terms 'reverse complement' and 'reverse complementary' refer to nucleic acid sequences having bases which, when oriented in antiparallel orientation with regard to their 5' and 3' ends (that is, such that the 5' direction of one nucleic acid strand is parallel to the 3' direction of the second strand) the bases of the nucleic acids are able to form standard DNA or RNA base pairs through part or all of the length of at least one of the molecules.
  • the term ED50 refers to the 'effective dose' that produces a quantal effect (all or nothing) in 50% of the population exposed it (median referring to the 50% population base). The ED50 is commonly used as a measure of reasonable expectance of a drug effect.
  • amino acid' is used to refer to an amino acid molecule, either as a free molecule or upon undergoing a reaction such as a dehydration reaction to form one or more polypeptide bonds, as a constituent of a polypeptide.
  • nucleic acid moiety refers to any molecule or molecular component that presents bases capable of base-pairing with a target molecule.

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Abstract

La présente invention concerne des peptides capables de pénétrer dans les cellules et qui favorisent l'administration d'une cargaison moléculaire constituée, par exemple, d'oligomères phosphorodiamidate morpholino en direction de cellules cibles de vertébrés. Les peptides capables de pénétrer dans les cellules sont liés aux oligomères par une liaison amide. Des amines primaires sélectionnées au sein du peptide sont acylées au moyen d'acides gras de façon à ce que les compositions résultantes se lient de façon non-covalente à la sérumalbumine et qu'elles ne fassent donc pas l'objet d'une filtration rapide par le rein. La séquence peptidique peut être conçue pour assurer une dégradation protéolytique rapide et/ou ciblée à l'intérieur d'un organisme vivant. Les compositions ainsi conçues sont caractérisées par une fenêtre thérapeutique considérablement plus large que celle des compositions plus stables d'un point de vue protéolytique et/ou peu ou pas liées à la sérumalbumine.
PCT/US2014/063945 2013-11-06 2014-11-04 Système d'administration intracellulaire d'oligomères présentant une faible vitesse d'élimination rénale WO2015069665A1 (fr)

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WO2020028254A1 (fr) * 2018-07-30 2020-02-06 Sarepta Therapeutics, Inc. Peptides trimères pour administration antisens

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US20110293725A1 (en) * 2008-10-08 2011-12-01 De Los Rios Miguel Chimeric therapeutics, compositions, and methods for using same
US20120289457A1 (en) * 2011-05-05 2012-11-15 Avi Biopharma, Inc. Peptide oligonucleotide conjugates
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