WO2015069665A1

WO2015069665A1 - Intracellular delivery system for oligomers having low renal clearance rate

Info

Publication number: WO2015069665A1
Application number: PCT/US2014/063945
Authority: WO
Inventors: Christian ECKHOFF; Brian LEPPERT
Original assignee: Rain Bioscience, Inc.
Priority date: 2013-11-06
Filing date: 2014-11-04
Publication date: 2015-05-14

Abstract

Cell-penetrating peptides are described that promote delivery of molecular cargo such as phosporodiamidate morpholino oligomers to vertebrae target cells. Cell penetrating peptides are linked to oligomers by an amide bond. Select primary amines within the peptide are acylated with fatty acids so that the resulting compositions bind non-covalently to serum albumin and thus will not be subject to rapid renal filtration. The peptide sequence can be designed to provide rapid and/or targeted proteolytic degradation inside a living organism. Compositions so designed have a considerably greater therapeutic window compared with compositions that are proteolytically more stable and/or have little or no binding to serum albumin.

Description

INTRACELLULAR DELIVERY SYSTEM FOR OLIGOMERS HAVING LOW RENAL

CLEARANCE RATE

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 C.F.R. § 1.57.

FIELD OF THE INVENTION

[0001] 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.

BACKGROUND OF THE INVENTION

[0002] Some chemical compounds of medicinal interest are known to have poor ability to cross cell membranes and/or high renal toxicity. There are several reasons why compounds may experience poor cellular uptake. Passive diffusion across lipid bilayer membranes such as cell membranes is favored when the compound is small (low molecular mass), has few hydrogen bond donor and hydrogen bond acceptor groups, and is somewhat lipophilic. Antisense nucleic acids such as PMO meet none of these conditions.

[0003] 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.

SUMMARY OF THE INVENTION

[0004] The disclosure is summarized as relating to the embodiments listed below:

[0005] Some embodiments of the present application provide a molecule comprising a saturated fatty acid moiety, a polypeptide moiety and a nucleic acid moiety. In some embodiments, 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. In some embodiments, the polypeptide moiety comprises a sequence of residues comprising (XA)_nB, 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. In some embodiments, 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. In some embodiments, the PMO is covalently bound at its 3' end to the beta-alanine of said polypeptide.

[0006] 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.

[0007] 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. In some embodiments, practice of said method results disrupting a signaling pathway comprising CD47 and in does not result in lethal renal necrosis.

[0008] Some embodiments of the present application provide a molecule comprising a fatty acid moiety, a polypeptide moiety and a nucleic acid moiety. In some embodiments, the polypeptide moiety directs localization of the molecule to an interior space of a cell. In some embodiments, the nucleic acid moiety comprises nucleic acid base sequence that is reverse-complementary to a nucleic acid sequence of said cell.

[0009] In some embodiments, 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.

[0010] In some embodiments, 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. In some embodiments, Z is a neutral amino acid. In some embodiments, Z is selected from L- alanine, glycine, or beta-alanine. In some embodiments, Z is selected from L-alanine and beta-alanine. In some embodiments, Z is L-alanine. In some embodiments, said polypeptide moiety comprises a beta-alanine residue. In some embodiments, said polypeptide moiety comprises a beta-alanine residue at its carboxy-terminus. In some embodiments, said polypeptide moiety comprises a glycine residue at its carboxy-terminus.

[0011] In some embodiments, 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.

[0012] In some embodiments, 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.

[0013] In some embodiments, said nucleic acid comprises sequence that is reverse complementary to a pathogen sequence. In some embodiments, said pathogen is a viral pathogen. In some embodiments, wherein said pathogen is a eubacterial pathogen. In some embodiments, said pathogen is a eukaryotic pathogen.

[0014] In some embodiments, 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.

[0015] In some embodiments, 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.

[0016] In some embodiments, 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.

[0017] In some embodiments, 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.

[0018] In some embodiments, 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.

[0019] 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. In some embodiments, the method comprises conjugating a second fatty acid moiety to said polypeptide-PMO molecule. In some embodiments, the method comprises conjugating a third fatty acid moiety to said polypeptide-PMO molecule. In some embodiments, the method comprises comprising conjugating a second fatty acid moiety to said polypeptide-PMO molecule.

[0020] In some embodiments, the method comprises contacting said molecule to albumin. In some embodiments, said renal clearance rate upon administration of said conjugated polypeptide-PMO molecule to a mammal is reduced. In some embodiments, 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.

[0021] In some embodiments, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] FIG. 2 is the chemical structure of N-terminal L-arginine.

[0024] FIG. 3 is the chemical structure of N-terminal L-lysine.

[0025] FIG. 4 is the chemical structure of non-N-terminal L-lysine.

[0026] FIG. 5 is the chemical structure of the beta-alanine linkage bound to the 3 ' end of PMO.

DETAILED DESCRIPTION

[0027] Cell -penetrating peptides having 4-14 basic amino acids are disclosed. In some embodiments basic amino acids are selected independently from L-arginine or L-lysine. In some embodiments nonbasic amino acids are selected independently from L-alanine or beta-alanine. In some embodiments 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. In some embodiments 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.

[0028] 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. Without being bound by theory, 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] It has been recognized that one particular area of the kidney comprising the cells lining the proximal tubules (proximal tubule epithelial cells or RPTEC) is particularly sensitive to the toxic insult of many drugs. 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. 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.

[0033] Reducing or eliminating the renal toxicity associated with the CPP delivery system is very desirable in order to be able to employ CPP for the intracellular delivery of impermeable molecules of high pharmaceutical interest such as PMO. With the known potential of CPP conjugates for inducing renal toxicity, it is therefore of the greatest importance to design these conjugates in ways that will reduce renal exposure, and in particular the exposure of RPTEC from the luminal side.

[0034] Without being bound to theory, it is generally believed that fatty acid acylation enhances the composition's affinity to serum albumin. Binding to serum albumin reduces renal clearance rate of the compositions. Low renal clearance rate enhances proteolytic degradation of the cell-penetrating peptide prior to elimination of the composition by renal filtration. Compositions so designed can enter cells with much greater efficiency than unconjugated oligomer but have attenuated potency in renal tubules.

[0035] 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 ε-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.

[0036] 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. The CPP (XY)_n consists of 8-28 amino acid residues (n=4-14). The total number of amino acids in the localization motif of the CPP is an even number. Basic amino acids (L-arginine or L-lysine) and nonbasic amino acids (L- alanine or beta-alanine) alternate in the peptide sequence. 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.

[0037] FIG. 2 is the chemical structure of L-arginine at the N-terminus of the peptide. The alpha amino group of N-terminal L-arginine (indicated by the letter R in the chemical drawing) may be unmodified or conjugated with acetoyl (n=0), myristoyl (n=12), palmitoyl (n=14), or stearoyl (n=16). [0038] FIG. 3 is the chemical structure of L-lysine at the N-terminus of the peptide. The alpha amino group of N-terminal L-arginine (indicated by the letter R in the chemical drawing) may be unmodified or conjugated with acetoyl (n=0), myristoyl (n=12), palmitoyl (n=14), or stearoyl (n=16). The ε amino group of N-terminal L-arginine (indicated by the letter X in the chemical drawing) may be unmodified or conjugated with myristoyl (n=12), palmitoyl (n=14), or stearoyl (n=16).

[0039] FIG. 4 is the chemical structure of L-lysine at positions in the peptide chain other than the N-terminal position. The ε amino group of N-terminal L-arginine (indicated by the letter X in the chemical drawing) may be unmodified or conjugated with myristoyl (n=12), palmitoyl (n=14), or stearoyl (n=16).

[0040] 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.

[0041] The 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.

[0042] An exemplary polypeptide sequence disclosed herein comprises the sequence (XZ)_nB, 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), RARARARARARARARARARAB (SEQ ID NO: 10), RARARARARARARARARARARAB (SEQ ID NO: 11), RARARARARARARARARARARARAB (SEQ ID NO: 12) and RARARARARARARARARARARARARAB (SEQ ID NO: 13), wherein R is L-arginine, A is L-alanine and B is beta-alanine. In some embodiments one or more arginine residues is replaced with lysine. In some embodiments one or more alanine residues is replaced with beta-alanine. In some embodiments an exemplary polypeptide sequence is RARARARARARARARARARBB (SEQ ID NO: 14). In some embodiments an exemplary polypeptide sequence is (RA)₉RBB (SEQ ID NO: 14).

[0043] In some embodiments 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. In some embodiments the carboxy-terminal amino acid is beta-alanine. In some embodiments the carboxy-terminal amino acid is glycine.

[0044] In some embodiments the polypeptide comprises sequence of the formula (XZ)n, where XZ is represented by SEQ ID NO: 15; (XYX)_nX, where XYX is represented by SEQ ID NO: 16; (XY)„X, wherein XY is represented by SEQ ID NO: 17; (XYY)_nX, where XYY is represented by SEQ ID NO: 18; (XYYY)_nX, where XYYY is represented by SEQ ID NO: 19;or U(XY)_nB, 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. 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.

[0045] Additional exemplary sequences include the following: RARARARARARARARARB (SEQ ID NO: 21), RARARARARARARARAB (SEQ ID NO: 22), ARARARARARARARARBB (SEQ ID NO: 23), RARARARARARARARABB (SEQ ID NO: 24); KARARAKARARAKARARAKB (SEQ ID NO: 25); (R)₈B (SEQ ID NO: 26); (R)₇ARB (SEQ ID NO: 27); (R)₃A(R)₅B (SEQ ID NO: 28); RA(R)₃A(R)₃ARB (SEQ ID NO: 29); (RA)nB (SEQ ID NO: 30); RXRRBRRXRRBRXB (SEQ ID NO: 31); RXRRXRRXRRXRXB (SEQ ID NO: 32); (RA)₅B (SEQ ID NO: 33); (RB)₅B (SEQ ID NO: 34); (RG)₄B (SEQ ID NO: 35); ARAKARAKARAKARAB (SEQ ID NO: 36); KGRGRGKGRGRB (SEQ ID NO: 37); AKAKARARARAKAKARARAKB (SEQ ID NO: 38).

[0046] In some embodiments 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. In some embodiments the polypeptide is fused to the 3' end of the nucleic acid molecule at a beta-alanine. In some embodiments the polypeptide is fused to the 3' end of the nucleic acid molecule at a glycine. In some embodiments the polypeptide is fused at the 3' end of the molecule at a residue other than beta-alanine. In some embodiments 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.

[0047] In some embodiments 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.

[0048] Other 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.

[0049] In some embodiments 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. In preferred embodiments, 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. In some embodiments the polypeptide expression level is to be reduced upon administration of a molecule consistent with the disclosure herein. In some embodiments 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. In some embodiments 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.

[0050] In preferred embodiments, 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.

[0051] Without being bound by theory, 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. Alternatively, 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.

[0052] A number of nucleic acid molecules may be targeted through administration of a molecule consistent with the disclosure herein. In some embodiments 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. In some embodiments the nucleic acid molecule targeted is produced by a eubacterial or eukaryotic pathogen host such as tuberculosis, a Plasmodium species or a cholera bacterium.

[0053] In some embodiments 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's lymphoma (NHL), multiple myeloma (MM), and other solid tumors; although other cancer types are additionally contemplated.

[0054] A nonlimiting list of 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.

[0055] 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.

[0056] In some embodiments 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. In some exemplary embodiments the molecule comprises 10-50 bases reverse complimentary to its target. In some exemplary embodiments the molecule comprises 15-30 bases reverse complimentary to its target.

[0057] In some embodiments 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, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 350, 400, 450, or within 500 bases of a translation start "ATG" codon at the start of a coding open reading frame.

[0058] 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. In some embodiments the reverse complementarity may be less that 100%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments the reverse complementarity may be less than 90%, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%. In some embodiments the sequence identity may be sufficient to allow double-strand formation independent of sequence identity. In some embodiments the reverse complementarity may be at least 85%.

[0059] 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.

[0060] For example, 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. In this example, 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.

[0061] In some embodiments 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. In some embodiments, 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. In some embodiments 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. In some embodiments 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. Thus, 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.

[0062] In some embodiments the molecule targets transcripts encoding CD47. In some embodiments the molecule base-pairs with a region of the CD47 transcript that is common to all known CD47 transcript splice variants. In some embodiments the molecule targets one, two, or three specific splice variant classes of CD47. In some embodiments the molecule targets CD47 isoform 1. In some embodiments the molecule targets CD47 isoform 2. In some embodiments the molecule targets CD47 isoform 3. In some embodiments the molecule targets CD47 isoform 4. In some embodiments 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. 0001 ggggagcagg cgggggagcg ggcgggaagc agtgggagcg cgcgtgcgcg cggccgtgca 0061 gcctgggcag tgggtcctgc ctgtgacgcg cggcggcggt cggtcctgcc tgtaacggcg 0121 gcggcggctg ctgctccaga cacctgcggc ggcggcggcg accccgcggc gggcgcggag 0181 atgtggcccc tggtagcggc gctgttgctg ggctcggcgt gctgcggatc agctcagcta 0241 ctatttaata aaacaaaatc tgtagaattc acgttttgta atgacactgt cgtcattcca 0301 tgctttgtta ctaatatgga ggcacaaaac actactgaag tatacgtaaa gtggaaattt 0361 aaaggaagag atatttacac ctttgatgga gctctaaaca agtccactgt ccccactgac 0421 tttagtagtg caaaaattga agtctcacaa ttactaaaag gagatgcctc tttgaagatg 0481 gataagagtg atgctgtctc acacacagga aactacactt gtgaagtaac agaattaacc 0541 agagaaggtg aaacgatcat cgagctaaaa tatcgtgttg tttcatggtt ttctccaaat 0601 gaaaatattc ttattgttat tttcccaatt tttgctatac tcctgttctg gggacagttt 0661 ggtattaaaa cacttaaata tagatccggt ggtatggatg agaaaacaat tgctttactt 0721 gttgctggac tagtgatcac tgtcattgtc attgttggag ccattctttt cgtcccaggt 0781 gaatattcat taaagaatgc tactggcctt ggtttaattg tgacttctac agggatatta 0841 atattacttc actactatgt gtttagtaca gcgattggat taacctcctt cgtcattgcc 0901 atattggtta ttcaggtgat agcctatatc ctcgctgtgg ttggactgag tctctgtatt 0961 gcggcgtgta taccaatgca tggccctctt ctgatttcag gtttgagtat cttagctcta 1021 gcacaattac ttggactagt ttatatgaaa tttgtggctt ccaatcagaa gactatacaa 1081 cctcctagga aagctgtaga ggaacccctt aatgcattca aagaatcaaa aggaatgatg 1141 aatgatgaat aactgaagtg aagtgatgga ctccgatttg gagagtagta agacgtgaaa 1201 ggaatacact tgtgtttaag caccatggcc ttgatgattc actgttgggg agaagaaaca 1261 agaaaagtaa ctggttgtca cctatgagac ccttacgtga ttgttagtta agtttttatt 1321 caaagcagct gtaatttagt taataaaata attatgatct atgttgtttg cccaattgag 1381 atccagtttt ttgttgttat ttttaatcaa ttaggggcaa tagtagaatg gacaatttcc 1441 aagaatgatg cctttcaggt cctagggcct ctggcctcta ggtaaccagt ttaaattggt 1501 tcagggtgat aactacttag cactgccctg gtgattaccc agagatatct atgaaaacca 1561 gtggcttcca tcaaaccttt gccaactcag gttcacagca gctttgggca gttatggcag 1621 tatggcatta gctgagaggt gtctgccact tctgggtcaa tggaataata aattaagtac 1681 aggcaggaat ttggttggga gcatcttgta tgatctccgt atgatgtgat attgatggag 1741 atagtggtcc tcattcttgg gggttgccat tcccacattc ccccttcaac aaacagtgta 1801 acaggtcctt cccagattta gggtactttt attgatggat atgttttcct tttattcaca 1861 taaccccttg aaaccctgtc ttgtcctcct gttacttgct tctgctgtac aagatgtagc 1921 accttttctc ctctttgaac atggtctagt gacacggtag caccagttgc aggaaggagc 1981 cagacttgtt ctcagagcac tgtgttcaca cttttcagca aaaatagcta tggttgtaac 2041 atatgtattc ccttcctctg atttgaaggc aaaaatctac agtgtttctt cacttctttt 2101 ctgatctggg gcatgaaaaa agcaagattg aaatttgaac tatgagtctc ctgcatggca 2161 acaaaatgtg tgtcaccatc aggccaacag gccagccctt gaatggggat ttattactgt 2221 tgtatctatg ttgcatgata aacattcatc accttcctcc tgtagtcctg cctcgtactc 2281 cccttcccct atgattgaaa agtaaacaaa acccacattt cctatcctgg ttagaagaaa 2341 attaatgttc tgacagttgt gatcgcctgg agtactttta gacttttagc attcgttttt 2401 tacctgtttg tggatgtgtg tttgtatgtg catacgtatg agataggcac atgcatcttc 2461 tgtatggaca aaggtggggt acctacagga gagcaaaggt taattttgtg cttttagtaa 2521 aaacatttaa atacaaagtt ctttattggg tggaattata tttgatgcaa atatttgatc 2581 acttaaaact tttaaaactt ctaggtaatt tgccacgctt tttgactgct caccaatacc 2641 ctgtaaaaat acgtaattct tcctgtttgt gtaataagat attcatattt gtagttgcat 2701 taataatagt tatttcttag tccatcagat gttcccgtgt gcctctttta tgccaaattg 2761 attgtcatat ttcatgttgg gaccaagtag tttgcccatg gcaaacctaa atttatgacc 2821 tgctgaggcc tctcagaaaa ctgagcatac tagcaagaca gctcttcttg aaaaaaaaaa 2881 tatgtataca caaatatata cgtatatcta tatatacgta tgtatataca cacatgtata 2941 ttcttccttg attgtgtagc tgtccaaaat aataacatat atagagggag ctgtattcct

3001 ttatacaaat ctgatggctc ctgcagcact ttttccttct gaaaatattt acattttgct

3061 aacctagttt gttactttaa aaatcagttt tgatgaaagg agggaaaagc agatggactt

3121 gaaaaagatc caagctccta ttagaaaagg tatgaaaatc tttatagtaa aattttttat

3181 aaactaaagt tgtacctttt aatatgtagt aaactctcat ttatttgggg ttcgctcttg

3241 gatctcatcc atccattgtg ttctctttaa tgctgcctgc cttttgaggc attcactgcc

3301 ctagacaatg ccaccagaga tagtggggga aatgccagat gaaaccaact cttgctctca

3361 ctagttgtca gcttctctgg ataagtgacc acagaagcag gagtcctcct gcttgggcat

3421 cattgggcca gttccttctc tttaaatcag atttgtaatg gctcccaaat tccatcacat

3481 cacatttaaa ttgcagacag tgttttgcac atcatgtatc tgttttgtcc cataatatgc

3541 tttttactcc ctgatcccag tttctgctgt tgactcttcc attcagtttt atttattgtg

3601 tgttctcaca gtgacaccat ttgtcctttt ctgcaacaac ctttccagct acttttgcca

3661 aattctattt gtcttctcct tcaaaacatt ctcctttgca gttcctcttc atctgtgtag

3721 ctgctctttt gtctcttaac ttaccattcc tatagtactt tatgcatctc tgcttagttc

3781 tattagtttt ttggccttgc tcttctcctt gattttaaaa ttccttctat agctagagct

3841 tttctttctt tcattctctc ttcctgcagt gttttgcata catcagaagc taggtacata

3901 agttaaatga ttgagagttg gctgtattta gatttatcac tttttaatag ggtgagcttg

3961 agagttttct ttctttctgt tttttttttt tgtttttttt tttttttttt tttttttttt

4021 ttttgactaa tttcacatgc tctaaaaacc ttcaaaggtg attatttttc tcctggaaac

4081 tccaggtcca ttctgtttaa atccctaaga atgtcagaat taaaataaca gggctatccc

4141 gtaattggaa atatttcttt tttcaggatg ctatagtcaa tttagtaagt gaccaccaaa

4201 ttgttatttg cactaacaaa gctcaaaaca cgataagttt actcctccat ctcagtaata

4261 aaaattaagc tgtaatcaac cttctaggtt tctcttgtct taaaatgggt attcaaaaat

4321 ggggatctgt ggtgtatgta tggaaacaca tactccttaa tttacctgtt gttggaaact

4381 ggagaaatga ttgtcgggca accgtttatt ttttattgta ttttatttgg ttgagggatt

4441 tttttataaa cagttttact tgtgtcatat tttaaaatta ctaactgcca tcacctgctg

4501 gggtcctttg ttaggtcatt ttcagtgact aatagggata atccaggtaa ctttgaagag

4561 atgagcagtg agtgaccagg cagtttttct gcctttagct ttgacagttc ttaattaaga

4621 tcattgaaga ccagctttct cataaatttc tctttttgaa aaaaagaaag catttgtact

4681 aagctcctct gtaagacaac atcttaaatc ttaaaagtgt tgttatcatg actggtgaga

4741 gaagaaaaca ttttgttttt attaaatgga gcattattta caaaaagcca ttgttgagaa

4801 ttagatccca catcgtataa atatctatta accattctaa ataaagagaa ctccagtgtt

4861 gctatgtgca agatcctctc ttggagcttt tttgcatagc aattaaaggt gtgctatttg

4921 tcagtagcca tttttttgca gtgatttgaa gaccaaagtt gttttacagc tgtgttaccg

4981 ttaaaggttt ttttttttat atgtattaaa tcaatttatc actgtttaaa gctttgaata

5041 tctgcaatct ttgccaaggt acttttttat ttaaaaaaaa acataacttt gtaaatatta

5101 ccctgtaata ttatatatac ttaataaaac attttaagct attttgttgg gctatttcta

5161 ttgctgctac agcagaccac aagcacattt ctgaaaaatt taatttatta atgtattttt

5221 aagttgctta tattctaggt aacaatgtaa agaatgattt aaaatattaa ttatgaattt

5281 tttgagtata atacccaata agcttttaat tagagcagag ttttaattaa aagttttaaa 5341 tcagtc

[0063] 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. [0064] In some embodiments 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.

[0065] In some embodiments 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. Similarly, 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.

[0066] Acylation with one or more saturated fatty acids enhances the composition's non-covalent binding to serum albumin due to affinity of the composition for the free fatty acid binding site present on the albumin molecule. The greater binding to serum albumin protects the CPP-PMO conjugate from rapid renal filtration. The reduced renal clearance rate protects RPTEC from a rapid influx of the composition and thus reduces risk of injury to RPTEC. Furthermore, reduced renal clearance rate provides proteolytic enzymes in blood more time to deconstruct the CPP which reduces the number of basic amino acids present in the composition at the time of renal filtration. This will reduce the potency of the composition in the proximal renal tubule.

[0067] In some embodiments 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 number of fatty acids are contemplated herein, such as Butyric acid (Butanoic acid; CH₃(CH₂)₂COOH; C₄:0); Caproic acid (Hexanoic acid; CH₃(CH₂)₄COOH; C6:0); Caprylic acid (Octanoic acid; CH₃(CH₂)₆COOH; Cs:0); Capric acid (Decanoic acid; CH₃(CH₂)₈COOH; Ci₀:0); Laurie acid (Dodecanoic acid; CH₃(CH₂)i₀COOH; Ci₂:0); Myristic acid (Tetradecanoic acid; CH₃(CH₂)i₂COOH; Ci₄:0); Palmitic acid (Hexadecanoic acid; CH₃(CH₂)i₄COOH; Ci₆:0); Stearic acid (Octadecanoic acid; CH₃(CH₂)i₆COOH; Ci₈:0); Arachidic acid (Eicosanoic acid; CH₃(CH₂)isCOOH; C₂o:0); Behenic acid (Docosanoic acid; CH₃(CH₂)₂₀COOH; C₂₂:0); Lignoceric acid (Tetracosanoic acid; CH₃(CH₂)₂₂COOH; C₂₄:0); Cerotic acid (Hexacosanoic acid; CH₃(CH₂)₂₄COOH; C₂₆:0); Montanic acid (Octacosanoic acid; CH₃(CH₂)₂₆COOH; C₂₈:0); Melissic acid (Triacontanoic acid; CH₃(CH₂)₂₈COOH; C₃₀:0); Lacceroic acid (Dotriacontanoic acid; CH₃(CH₂)₃oCOOH; C₃₂:0); Geddic acid (Tetratriacontanoic acid; CH₃(CH₂)₃₂COOH; C₃₄:0); and Hexatriacontylic acid; Hexatriacontanoic acid; CH₃(CH₂)₃₄COOH; C₃₆:0).

[0068] 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. In some embodiments the fatty acid comprises an even-number of carbon atoms. In some embodiments the fatty acid conjugate comprises myristoyl. In some embodiments the fatty acid conjugate comprises palmitoyl. In some embodiments the fatty acid conjugate comprises stearoyl. In some embodiments the fatty acid conjugate is selected from the group consisting of myristoyl, palmitoyl, and stearoyl. In some embodiments the fatty acid conjugate is an acetoyl moiety.

[0069] 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 C₁₄, C₁₆ or C₁₈ fatty acid at a terminal (alpha) amino group, "#" indicates that the residue immediately preceding (lysine, K) is acylated with a C₁₄, Ci₆ or Ci₈ 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. [0070] 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.

[0071] Molecules consistent with the disclosure herein are observed to impact the expression levels of polypeptides. In particular, 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.

[0072] 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.

[0073] 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.

[0074] Disclosed herein are methods of reducing the renal toxicity associated with administering CPP-PMO molecules as disclosed herein, said methods comprising conjugating at least one fatty acid moiety to said molecules. Without being bound by theory, the 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.

[0075] While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.

EXAMPLES EXAMPLE 1: PEPTIDE SEQUENCE

[0076] A peptide of the sequence acetyl-(RA)₉RBB (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.

[0077] This example demonstrates the efficacy of peptide of the sequence acetyl- (RA)₉RBB (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

[0078] 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.

[0079] 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

[0080] 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.

[0081] 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

[0082] 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.

[0083] 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 5: NON-N-TERMINAL STEAROYL CONJUGATED PEPTIDE- LOCALIZED ANTI-CD47 MOLECULE DIRECTS REDUCED EXPRESSION OF CD47 PROTEIN

[0084] 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. [0085] 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 6: TESTS USING SECOND PEPTIDE-LOCALIZATION SEQUENCE

[0086] The Experiments of Examples 1-5 are repeated using a second polypeptide localization sequence having the sequence (RAKA)(RA)₇RBB (SEQ ID NO: 45). K indicates L-Lysine. The second polypeptide localization signal is found to perform similarly to the first localization signal of Example 1 , above, in assays reported above.

EXAMPLE 7: TESTS USING SECOND PEPTIDE-LOCALIZATION SEQUENCE

[0087] The Experiments of Examples 4-5 are repeated wherein the fatty acid to be tested is linked to the epsilon amino group of the Lysine of the localization polypeptide. The Lysine-conjugated fatty acyl molecule is found to perform similarly to the N-terminal and non-N-terminal fatty acyl conjugated molecules in assays reported above.

EXAMPLE 8: TESTS USING A SECOND ANTI-CD47 SEQUENCE

[0088] The Experiments of Examples 2-7 are repeated using a second morpholino oligomer reverse-complementary to CD47 encoding RNA. The second morpholino oligomer reverse-complementary to CD47 is found to perform similarly to the first localization signal of Example 2, above, in assays reported above.

EXAMPLE 9: TESTS USING A SEQUENCE REVERSE-COMPLEMENTARY TO AN ADDITIONAL TARGET

[0089] The Experiments of Examples 2-8 are repeated using a morpholino oligomer reverse-complementary to RNA encoding a protein associated with a disorder. The morpholino oligomer reverse-complementary to RNA encoding a protein associated with a disorder is found to perform similarly to the morpholino oligomers reported above, above, in assays to determine the ED50 for reduction of the polypeptide expression levels related to the a protein associated with said disorder, and the conjugated compound is observed to perform in renal cell viability assays similarly to that reported above.

EXAMPLE 10: MOLECULES CONSISTENT WITH THE DISCLOSURE

[0090] 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 C₁₄ 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

[0091] 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 Ci₆ fatty acid at a terminal (alpha) amino group, "#" indicates that the residue immediately preceding (lysine, K) is acylated with a C₁₆ 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

[0092] 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 Ci₈ fatty acid at a terminal (alpha) amino group, "#" indicates that the residue immediately preceding (lysine, K) is acylated with a C₁₈ 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

[0093] 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.

[0094] 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

[0095] 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.

[0096] 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

[0097] 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.

[0098] 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

[0099] 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.

[0100] This example demonstrates the efficacy of fatty acyl conjugation to reduce toxicity of a morpholino oligomer polypeptide.

[0101] The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0102] The term "about" as disclosed herein in the context of a numerical value refers to that value plus or minus 10%.

[0103] Not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0104] 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. [0105] 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.

[0106] Single capital letters may be used as representative of amino acids or amino acid residues in polypeptide molecules consistent with standard usage known to one of skill in the art or as specifically indicated herein.

[0107] The term '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.

[0108] Unless otherwise indicated, terms are given their standard meaning to one of skill in the art as represented, for example, by usage in Buchanan et al., eds., "Biochemistry and Molecular Biology of Plants" (2000) Rockville, Maryland; American Society of Plant Physiologists, which is hereby incorporated by reference in its entirety, or by Griffiths et al,., eds., "An Introduction to Genetic Analysis, 7^th edition" (2000) New York; W. H. Freeman, which is hereby incorporated by reference in its entirety.

[0109] A "nucleic acid moiety" as contemplated herein refers to any molecule or molecular component that presents bases capable of base-pairing with a target molecule.

[0110] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

[0111] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A molecule comprising a saturated fatty acid moiety, a polypeptide moiety and a nucleic acid moiety, wherein

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)_nB, 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; and

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, and wherein the PMO is covalently bound at its 3 ' end to the beta-alanine of said polypeptide.

2. A method of reducing CD47 protein expression in a mammalian subject comprising the step of administering the molecule of claim 1.

3. The method of claim 2, wherein practice of said method results in reducing CD47 protein expression and does not result in lethal renal necrosis.

4. A method of disrupting a signaling pathway comprising CD47 in a mammalian subject, said method comprising the step of administering the molecule of claim 1.

5. The method of claim 4, wherein practice of said method results disrupting a signaling pathway comprising CD47 and does not result in lethal renal necrosis.

6. A molecule comprising a fatty acid moiety, a polypeptide moiety and a nucleic acid moiety, wherein

the polypeptide moiety directs localization of the molecule to an interior space of a cell; and

the nucleic acid moiety comprises nucleic acid base sequence that is reverse- complementary to a nucleic acid sequence of said cell.

7. The molecule of claim 6, wherein said polypeptide moiety comprises a sequence of at least 8 amino acid residues, of which 50% are basic residues.

8. The molecule of any of claims 6-7 wherein said polypeptide moiety comprises a sequence of 28 or fewer amino acid residues, of which 50% are basic residues.

9. The molecule of any of claims 6-8, wherein said polypeptide comprises basic and neutral residues.

10. The molecule of claim 9, wherein said basic residues and said neural residues alternate in said polypeptide.

11. The molecule of any one of claims 6-10, wherein said polypeptide comprises amino acid residues having the sequence (XZ)_n, wherein X is independently selected from L- arginine and L-lysine, and Z is a non-basic amino acid.

12. The molecule of claim 11, wherein Z is a neutral amino acid.

13. The molecule of claim 12, wherein Z is selected from L-alanine, glycine, or beta- alanine.

14. The molecule of claim 13, wherein Z is selected from L-alanine and beta-alanine.

15. The molecule of claim 14, wherein Z is L-alanine.

16. The molecule of any of claims 6-15, wherein said polypeptide moiety comprises a beta-alanine residue.

17. The molecule of claim 16, wherein said polypeptide moiety comprises a beta- alanine residue at its carboxy- terminus.

18. The molecule of any of claims 6-15, wherein said polypeptide moiety comprises a glycine residue at its carboxy-terminus.

19. The molecule of any of claims 6-15, wherein said polypeptide moiety comprises a glycine residue at its carboxy-terminus.

20. The molecule of any of claims 6-17, wherein said nucleic acid moiety comprises at least 10 bases.

21. The molecule of claim 20, wherein said nucleic acid moiety comprises at least 15 bases.

22. The molecule of any of claims 20-21, wherein said nucleic acid moiety comprises no more than 50 bases.

23. The molecule of any of claims 20-22, wherein said nucleic acid moiety comprises no more than 30 bases.

24. The molecule of any of claims 20-23, wherein said nucleic acid moiety comprises sequence that is at least 85% reverse complementary to a target sequence within an RNA sequence.

25. The molecule of claim 24, wherein said target sequence comprises at least 15 bases of said RNA sequence.

26. The molecule of claim 24, wherein said target sequence comprises 30 bases of said RNA sequence.

27. The molecule of claim 24, wherein said target sequence comprises 50 bases of said RNA sequence.

28. The molecule of claim 24, wherein said RNA sequence is an mRNA sequence.

29. The molecule of claim 24, wherein said RNA sequence is a miRNA sequence.

30. The molecule of any of claims 24-29, wherein said nucleic acid comprises sequence that is at least 90% reverse-complementary to said target sequence.

31. The molecule of claim 30, wherein said nucleic acid comprises sequence that is at least 95% reverse-complementary to said target sequence.

32. The molecule of claim 31, wherein said nucleic acid comprises sequence that is 100% reverse-complementary to said target sequence.

33. The molecule of any of claims 20-31, wherein said nucleic acid comprises sequence that is reverse complementary to a sequence of a nucleic acid implicated in a disease.

34. The molecule of any of claims 20-31, wherein said nucleic acid comprises sequence that is reverse complementary to a pathogen sequence.

35. The molecule of claim 34, wherein said pathogen is a viral pathogen.

36. The molecule of claim 34, wherein said pathogen is a eubacterial pathogen.

37. The molecule of claim 34, wherein said pathogen is a eukaryotic pathogen.

38. The molecule of claim 33, wherein said disease is cancer.

39. The molecule of any of claims 24-33 or 38, wherein said target sequence is selected from a fragment of a CD47 transcript sequence.

40. The molecule of claim 39, wherein 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.

41. The molecule of claim 39, wherein 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.

42. The molecule of any of claims 20-41, wherein said nucleic acid moiety is covalently bound to said polypeptide sequence at the 3 ' end of said nucleic acid moiety.

43. The molecule of any of claims 20-41, wherein said nucleic acid moiety is covalently bound to said polypeptide sequence at the 5 ' end of said nucleic acid moiety.

44. The molecule of any of claims 20-43, wherein said nucleic acid moiety is covalently bound to said polypeptide moiety at a beta-alanine residue.

45. The molecule of any of claims 20-43, wherein said nucleic acid moiety comprises a phosphorodiamidate morpholino oligo.

46. The molecule of any of claims 6-45, wherein said fatty acid moiety is a saturated fatty acid moiety.

47. The molecule of claim 46, wherein said fatty acid moiety consists of an even number of carbon atoms.

48. The molecule of any of claims 46-47, wherein said fatty acid moiety comprises a myristoyl moiety.

49. The molecule of any of claims 46-47, wherein said fatty acid moiety comprises a palmitoyl moiety.

50. The molecule of any of claims 46-47, wherein said fatty acid moiety comprises a stearoyl moiety.

51. The molecule of any of claims 46-50, wherein said fatty acid moiety is covalently bound to said polypeptide moiety.

52. The molecule of claim 51 , wherein said fatty acid moiety is covalently bound to said polypeptide moiety at an amino moiety.

53. The molecule of claim 51, wherein said fatty acid moiety is covalently bound to said polypeptide moiety at an amino-terminal alpha amine.

54. The molecule of claim 51, wherein said fatty acid moiety is covalently bound to said polypeptide moiety at an epsilon amine.

55. The molecule of claim 51, wherein said fatty acid moiety is covalently bound to said polypeptide moiety at a non-amine position.

56. The molecule of any of claims 46-55, wherein said molecule comprises a second fatty acid moiety.

57. The molecule of any of claims 46-56, wherein said molecule comprises a third fatty acid moiety.

58. The molecule of any of claims 6-57, wherein said fatty acid moiety binds albumin.

59. 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.

60. The method of claim 59, further comprising conjugating a second fatty acid moiety to said polypeptide-PMO molecule.

61. The method of claim 60, further comprising conjugating a third fatty acid moiety to said polypeptide-PMO molecule.

62. The method of any of claims 59-61, further comprising contacting said molecule to albumin.

63. The method of any of claims 59-62, wherein renal clearance rate upon administration of said conjugated polypeptide-PMO molecule to a mammal is reduced.

64. The method of any of claims 59-62, further comprising administering to said mammal 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.

65. The method of any of claims 59-64, wherein said fatty acid moiety is a saturated fatty acid moiety.

66. The method of any of claims 59-64, wherein said fatty acid moiety consists of an even number of carbon atoms.

67. The method of any of claims 59-64, wherein said fatty acid moiety comprises a myristoyl moiety.

68. The method of any of claims 59-64, wherein said fatty acid moiety comprises a palmitoyl moiety.

69. The method of any of claims 59-64, wherein said fatty acid moiety comprises a stearoyl moiety.