EA039716B1 - Peptide-oligonucleotide conjugates - Google Patents

Abstract

The invention relates to a peptide-oligonucleotide conjugate of Formula I and variants thereof:The peptide-oligonucleotide conjugates of the invention have improved antisense or antigene performance.

Description

Sequence listing

The application for this invention is filed along with a sequence listing in a machine-readable format. The sequence listing is provided in a file called 581432_SPT-0012_sequence_listing_ST25.txt, created on May 13, 2016, which is 4227 bytes in size. Information in machine-readable format from the sequence listing is incorporated herein by reference in its entirety.

Background of the invention

Antisense technology provides a means to modulate the expression of one or more specific gene products, including alternative splicing products, and is uniquely useful in a number of therapeutic, diagnostic, and research applications. The principle of antisense technology is that an antisense compound, such as an oligonucleotide that hybridizes to a target nucleic acid, modulates gene expression activities such as transcription, splicing, or translation through any of a number of antisense mechanisms. The sequence specificity of antisense compounds makes them attractive as tools for target validation and gene functionalization, as well as therapeutic agents for selectively modulating the expression of genes involved in disease.

Although significant progress has been made in the field of antisense technology, there remains a need to develop the direction of oligonucleotides and peptide-oligonucleotide conjugates with improved antisense or antigenic characteristics. Such improved antisense or antigenic characteristics include, at least for example: lower toxicity, stronger affinity for DNA and RNA without compromising sequence selectivity, improved pharmacokinetics and tissue distribution, improved cellular delivery, and reliable and controlled distribution. in vivo.

Brief description of the invention

This document provides peptides, oligonucleotides and peptide-oligonucleotide conjugates. Also provided herein are methods of treating a disease in a subject in need thereof, comprising administering to the subject a peptide oligonucleotide conjugate as described herein.

Accordingly, in one aspect, provided herein is a peptide-oligonucleotide conjugate of formula (I)

E'(I), or a pharmaceutically acceptable salt thereof.

In one embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Ia)

(la), or a pharmaceutically acceptable salt thereof.

In another embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Ib)

(lb), or a pharmaceutically acceptable salt thereof.

In another embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Ic)

(Ic), or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the peptide oligonucleotide conjugate is a peptide oligonucleotide conjugate of formula (Id) of formula (I) pre-

(Id), or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the peptide oligonucleotide conjugate is a peptide oligonucleotide conjugate of formula (Ie) formula (I) pre-

(Ie), or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a peptide-oligonucleotide conjugate of formula (IV)

- 2 039716

E'(IV), or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a peptide-oligonucleotide conjugate of the formula V

(V) or a pharmaceutically acceptable salt thereof.

In yet another aspect, this document provides a method of treating a muscle disease, viral infection, or bacterial infection in a subject in need thereof, comprising administering to the subject a peptide oligonucleotide conjugate in accordance with the present invention.

Brief description of the figures

In FIG. 1 shows the degree of penetration of selected oligonucleotides, peptides and peptidoligonucleotide conjugates into HeLa cells based on experiments using flow cytometry.

In FIG. 2A is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 5 administration.

In FIG. 2B is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 6 administration.

In FIG. 2C is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 8 administration.

In FIG. 2D is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 10 administration.

In FIG. 3A is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 5 administration.

In FIG. 3B is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 7 administration.

In FIG. 3C is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 9 administration.

In FIG. 3D is a Western blot showing dystrophin levels in mouse quadriceps tissue following PPMO 11 administration.

In FIG. 4A shows quantification of dystrophin levels in mouse quadriceps tissue following PPMO 5 administration (in duplicate of the same assay).

In FIG. 4B shows quantification of dystrophin levels in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 6.

In FIG. 4C shows the quantification of exon 23 skipping (%) in mouse quadriceps tissue after PPMO 5 administration (in duplicate of the same assay).

In FIG. 4D shows the quantification of exon 23 skipping (%) in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 6; while wild type and mdx mice were used as controls.

In FIG. 5A shows quantification of dystrophin levels in mouse quadriceps tissue following administration of PPMO 5 (in duplicate of the same assay) or PPMO 8.

In FIG. 5B shows quantification of dystrophin levels in mouse quadriceps tissue following administration of PPMO 5 (in duplicate of the same assay) or PPMO 10.

In FIG. 5C shows quantification of exon 23 skipping (%) in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 8; while in

- 3 039716 wild-type and mdx mice were used as controls.

In FIG. 5D shows the quantification of exon 23 skipping (%) in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 10; while wild-type and mdx mice were used as controls.

In FIG. 6A shows quantification of dystrophin levels in mouse quadriceps tissue following PPMO 5 administration (in duplicate of the same assay).

In FIG. 6B shows the quantification of dystrophin levels in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 7.

In FIG. 6C shows the quantification of exon 23 skipping (%) in mouse quadriceps tissue following PPMO 5 administration (in duplicate of the same assay).

In FIG. 6D shows the quantification of exon 23 skipping (%) in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 7; while wild-type and mdx mice were used as controls.

In FIG. 7A shows quantification of dystrophin levels in mouse quadriceps tissue following administration of PPMO 5 (in duplicate of the same assay) or PPMO 9.

In FIG. 7B shows quantification of dystrophin levels in mouse quadriceps tissue following administration of PPMO 5 (in duplicate of the same assay) or PPMO 11.

In FIG. 7C shows the quantification of exon 23 skipping (%) in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 9; while wild-type and mdx mice were used as controls.

In FIG. 7D shows the quantification of exon 23 skipping (%) in mouse quadriceps tissue following administration of RRMO 5 (in duplicate of the same assay) or RRMO 11; while wild-type and mdx mice were used as controls.

Detailed description

This document provides peptides, oligonucleotides and peptide-oligonucleotide conjugates. Also provided herein are methods of treating a disease in a subject in need thereof, comprising administering to the subject a peptide oligonucleotide conjugate as described herein. The oligonucleotides and, accordingly, the peptide-oligonucleotide conjugates described herein exhibit stronger DNA and RNA affinity without compromising sequence selectivity over native or unmodified oligonucleotides. In some embodiments, the oligonucleotides of the present invention minimize or prevent RNase H cleavage. In some embodiments, the antisense oligonucleotides of the present invention do not activate RNase H.

The peptides described herein impart lower toxicity to their respective peptidoligonucleotide conjugates, increase oligonucleotide activity, improve pharmacokinetics and tissue distribution, improve cell delivery, and provide both reliable and controlled distribution in vivo.

Definitions

What follows is a definition of various terms used to describe the present invention. These definitions refer to terms that are used in the description and in the claims, unless they are otherwise limited to specific meanings, either individually or as part of a larger group.

The term will be approximately understood by those skilled in the art and will vary to some extent depending on the context in which it is used. For the purposes of this document, when referring to a measurable quantity such as quantity, duration, etc., the term approximately is intended to cover deviations of ±20 or ±10%, including ±5, ±1 and ±0.1% of the specified value, since such deviations are suitable for the implementation of the disclosed methods.

The term alkyl refers to straight or branched chain saturated hydrocarbon moieties containing, in some embodiments, one to six or one to eight carbon atoms, respectively. Examples C ₁ . The ₆ alkyl moieties include, without limitation, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl, n-hexyl moieties; and examples C ₁ . The ₈ alkyl moieties include, without limitation, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

The number of carbon atoms in an alkyl substituent may be denoted by the prefix C _x _ _y , where x is the minimum number and y is the maximum number of carbon atoms in the substituent. Similarly, Cx chain means an alkyl chain containing x carbon atoms.

The term heteroalkyl, by itself or in combination with other terms, means, unless otherwise indicated, a stable straight or branched chain alkyl group consisting of the specified number of carbon atoms and one or three heteroatoms selected from the group consisting of O, N and S ,

- 4 039716 wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternary. The heteroatom(s) may be placed at any position in the heteroalkyl group, including between the remainder of the heteroalkyl group and the moiety to which it is attached, and may also be attached to the outermost carbon atom in the heteroalkyl group. Examples include: -O-CH ₂ -CH2-CH ₃ , -CH ₂ -CH ₂ -CH ₂ -OH, -CH2-CH2-NH-CH3, -CH ₂ -S-CH ₂ -CH ₃ and -CH ₂ -CH ₂ -S(=O)-CH ₃ . No more than two heteroatoms may be consecutive, such as -CH2-NH-OCH3 or -CH ₂ -CH ₂ -SS-CH ₃ .

The term aryl, used alone or in combination with other terms, means, unless otherwise indicated, a carbocyclic aromatic system containing one or more rings (usually one, two or three rings), and such rings can be connected together in a pendant manner, for example, as in biphenyl, or may be condensed, for example, as in naphthalene. Examples of aryl groups include phenyl, anthracyl and naphthyl. In various embodiments, examples of the aryl group may include phenyl (eg, C ₆ aryl) and biphenyl (eg, C ₁₂ aryl). In some embodiments, the implementation of the aryl groups contain from six to sixteen carbon atoms. In some embodiments, the implementation of the aryl groups contain from six to twelve carbon atoms (for example, C ₆ _ ₁₂ aryl). In some embodiments, the implementation of the aryl groups contain six carbon atoms (for example, C ₆ aryl).

As used herein, the term heteroaryl or heteroaromatic radical refers to a heterocycle having an aromatic character. Substituents on heteroaryl can be defined by the number of carbon atoms, eg C ₁ . ₉ heteroaryl indicates the number of carbon atoms contained in a heteroaryl group without including the number of heteroatoms. For example, _C1 . ₉ heteroaryl will include an additional one to four heteroatoms. The polycyclic heteroaryl may include one or more rings that are partially saturated. Non-limiting examples of heteroaryls include pyridyl, pyrazinyl, pyrimidinyl (including, for example, 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, for example, 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (including, for example, , 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3 -oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

Non-limiting examples of polycyclic heterocycles and heteroaryls include indolyl (including, for example, 3-, 4-, 5-, 6-, and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (including, for example, 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (including, for example, 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5- naphthyridinyl, benzofuryl (including, for example, 3-, 4-, 5-, 6-, and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (including, for example, 3-, 4-, 5 -, 6- and 7-benzothienyl), benzoxazolyl, benzothiazolyl (including, for example, 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (including, for example, 2-benzimidazolyl), benzothriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinil and quinolizidinil.

The term protecting group or chemical protecting group refers to chemical moieties that block some or all of the reactive moieties of a compound and prevent such moieties from participating in chemical reactions until the protecting group is removed, such as the moieties listed and described in TW Greene, PGM Wuts, Protective Groups in Organic Synthesis, 3rd ^ed . John Wiley & Sons (1999). It may be advantageous to use different protecting groups such that each (different) protecting group can be removed by different means. Protecting groups that cleave under completely different reaction conditions allow differential removal of such protecting groups. For example, protecting groups can be removed by acid, base and hydrogenolysis. Groups such as trityl, monomethoxytrityl, dimethoxytrityl, acetal, and t-butyldimethylsilyl are acid-labile and can be used to protect reactive carboxy and hydroxy moieties in the presence of amino groups protected by Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are unstable with respect to bases. Carboxylic acid moieties can be capped with base-labile groups such as but not limited to methyl or ethyl, and hydroxy-reactive moieties can be capped with base-labile groups such as acetyl in the presence of acid-labile capped amines. groups such as t-butyl carbamate; or carbamates which are resistant to both acids and bases but are hydrolytically removable.

Reactive carboxylic acid moieties and hydroxyl moieties can also be capped with hydrolytically removable protecting groups such as a benzyl group, while amino groups can be capped with base-labile groups such as Fmoc. A particularly useful amino protecting group in the synthesis of compounds of formula (I) is trifluoroacetamide. Reactive carboxylic acid moieties can be blocked by oxidatively removable protecting groups such as

- 5 039716

2,4-dimethoxybenzyl, while the nearby amino groups can be blocked by silyl carbamates that are unstable with respect to fluorides.

Allyl blocking groups are useful in the presence of acid and base protecting groups because the former are stable and can then be removed with metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid can be deprotected by a palladium(0) catalyzed reaction in the presence of acid-labile t-butyl carbamate or base-labile acetate protecting groups for amino groups. Another form of protecting group is a resin, to which a compound or intermediate may be attached. As long as the residue is attached to the resin, such a functional group is blocked and cannot react. After being released from the resin, the functional group is capable of reacting.

The terms nucleobase, base-pairing moiety, nucleobase-pairing moiety, or base refer to the heterocyclic ring moiety of a morpholino-based nucleoside, nucleotide, and/or subunit. Nucleic bases may be naturally occurring bases, may be modified naturally occurring nucleobases, or may be analogues thereof, for example, one or more nitrogen atoms in the nucleic base may be independently replaced by carbon atoms in each case. Illustrative analogues include hypoxanthine (the main component of the nucleoside inosine); 2,6-diaminopurine; 5-methylcytosine; C ₅ -propynyl-modified pyrimidines; 10-(9-(aminoethoxy)phenoxazinil) (G-clamp) and the like.

Additional examples of base pairing moieties include, without limitation, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine having their respective amino groups protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-ioduracil, 2 ,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two are naturally occurring degradation products). Modified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196, and also discussed in Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, the contents of which are incorporated herein by reference.

Additional examples of base pairing moieties include, without limitation, oversized nucleobases to which one or more benzene rings have been added. Nucleic base substitutions described in the Glen Research catalog (www.glenresearch.com); Krueger A.T. et al., Acc. Chem. Res., 2007, 40, 141-150; Kool, E.T., Acc. Chem. Res., 2002, 35, 936-943; Benner S.A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F.E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627, the contents of which are incorporated herein by reference, are considered applicable to the synthesis of the oligomers described herein. Examples of oversized nucleic bases are shown below.

The terms oligonucleotide or oligomer refers to a compound containing a plurality of linked nucleosides, nucleotides, or a combination of nucleosides and nucleotides. In specific embodiments, provided herein is an oligonucleotide that is a morpholine

- 6 039716 oligonucleotide.

The expression morpholino oligonucleotide or PMO refers to a modified oligonucleotide containing morpholino-based subunits linked together by phosphoramidate or phosphorodiamidate bridges connecting the nitrogen atom in the morpholino from one subunit to the 5'-exocyclic carbon atom in the adjacent subunit. Each morpholino-based subunit contains a nucleobase-pairing moiety effective to bind to the nucleobase in the target via nucleobase-specific hydrogen bonding.

The terms antisense oligomer, antisense compound and antisense oligonucleotide are used interchangeably, they refer to a sequence of subunits, each of which carries a base-pairing moiety linked by intersubunit bonds, which allows the base-pairing moieties to hybridize to a target sequence in a nucleic acid (usually RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex in the target sequence. The oligomer may have exact (perfect) or close (sufficient) sequence complementarity to the target sequence; however, sequence deviations near the ends of the oligomer are generally favored over variability in the interior.

Such an antisense oligomer may be designed to block or inhibit mRNA translation or to inhibit/alter natural or abnormal splicing of pre-processed mRNA and may be referred to as targeting or targeting the target sequence to which it hybridizes. The target sequence is typically a region in the mRNA containing the start codon AUG for a translation suppression oligomer or a preprocessed mRNA splicing site for a splicing suppression oligomer (SSO). The splice site target sequence may include an mRNA sequence having 1 to about 25 bp at its 5' end in a 5' to 3' direction of the normal splice acceptor in the preprocessed mRNA. In various embodiments, the target sequence can be any region of preprocessed mRNA that includes a splice site, or is entirely contained within an exon encoding the sequence, or spans a splice acceptor site or a donor site. An oligomer more generally is generally referred to as targeting a biologically relevant target, such as a protein, virus, or bacteria, when it is targeted to a nucleic acid in the target in the manner described above.

An antisense oligonucleotide and an RNA in a target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can form a hydrogen bond with each other so that stable and specific binding occurs between the oligonucleotide and the target. Thus, specifically hybridizable and complementary are terms that are used to refer to such a sufficient degree of complementarity or exact pairing that stable and specific binding occurs between the oligonucleotide and the target. From the prior art it is clear that it is not necessary that the sequence of the oligonucleotide was 100% complementary to that of the target sequence for it, which is subject to specific hybridization. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to a target molecule disrupts the normal function of the target RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions where specific binding is necessary , i.e. under physiological conditions in the case of in vivo testing or therapeutic treatment, and in the case of in vitro testing, under the conditions of testing.

Oligonucleotides may also include modifications or substitutions of the nucleobase (often referred to in the art simply as base). Oligonucleotides containing a modified or substituted base include oligonucleotides in which one or more purine or pyrimidine bases most commonly found in nucleic acids have been replaced with less common or artificial bases. In some embodiments, the nucleobase is covalently linked to the morpholine ring of the nucleotide or nucleoside at the N9 atom of the purine base or at the N1 atom of the pyrimidine base.

Purine bases contain a pyrimidine ring fused to an imidazole ring as described by the general formula

- 7 039716

Purine

Adenine and guanine are the two purine nucleic bases most commonly found in nucleic acids. They may be substituted with other naturally occurring purines including, but not limited to, N ⁶ -methyladenine, N ² -methylguanine, hypoxanthine, and 7-methylguanine.

Pyrimidine bases contain a 6-membered pyrimidine ring as described by the general formula

Pyrimidine

Cytosine, uracil, and thymine are the pyrimidine bases most commonly found in nucleic acids. They may be substituted with other naturally occurring pyrimidines including, but not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain thymine bases instead of uracil.

Other modified or substituted bases include, without limitation, 2,6-diaminopurine, orotic acid, agmatidine, lyzidine, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine ( e.g. 5-halogenuracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6- diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenin, 8-aza-7-deaza-2,6-diaminopurine, G, Super A and N ⁴ -ethylcytosine or derivatives thereof; N ² -cyclopentylguanine (cPent-G), N ² -cyclopentyl-2-aminopurine (cPent-AP) and N ² -propyl-2-aminopurine (Pr-AP), pseudouracil or derivatives thereof; as well as degenerate or universal bases, such as 2,6-difluorotoluene, or missing bases, such as sites with nitrogenous bases removed (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the oxygen in the ring has been replaced by a nitrogen atom (azaribose)). Pseudouracil is a naturally occurring isomerized version of uracil with a C-glycoside instead of the normal N-glycoside as in uridine.

Certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of antisense oligonucleotides in accordance with the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2-, N-6-, and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. In various embodiments, the nucleic bases may include 5-methylcytosine substitutions that have been shown to increase the stability of the nucleic acid duplex by 0.6-1.2°C.

In some embodiments, modified or substituted nucleobases are useful to facilitate the purification of antisense oligonucleotides. For example, in some embodiments, antisense oligonucleotides may contain three or more (eg, 3, 4, 5, 6 or more) consecutive guanine bases. In some antisense oligonucleotides, a strand of three or more consecutive guanine bases can cause the oligonucleotides to aggregate, making purification difficult. In such antisense oligonucleotides, one or more consecutive guanines may be substituted with hypoxanthine. Substitution of hypoxanthine for one or more guanines in a strand of three or more consecutive guanine bases can reduce the aggregation of the antisense oligonucleotide, thus facilitating purification.

The oligonucleotides provided herein are synthesized and do not include antisense combinations of biological origin. Molecules of the present invention may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, such as, for example, liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations. for the purpose of promoting digestion, distribution or absorption, or a combination thereof.

The terms complementary and complementarity refer to oligonucleotides (ie, a sequence of nucleotides) associated with base pairing rules. For example, the sequence T-G-A (5'-3') is complementary to the sequence T-C-A (5'-3'). Complementarity can be partial, in which only some of the nucleic acid bases match according to

- 8 039716 base pairing rules, or between nucleic acids there can be complete, absolute or perfect (100%) complementarity. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often required, some embodiments may include one or more, but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the RNA in the target. Such hybridization can occur when the antisense oligomer has close or significant complementarity to the target sequence, as well as exact complementarity. In some embodiments, the oligomer may hybridize to the target sequence at about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementarity. Deviations at any location in the oligomer are included. In some embodiments, sequence deviations towards the ends of the oligomer are generally preferred over deviations in the interior and, if present, are typically within about 6, 5, 4, 3, 2, or 1 nucleotides at the 5' end, 3 '-end or both ends.

The term peptide refers to a compound containing a plurality of linked amino acids. The peptides provided herein can be considered as cell-penetrating peptides.

The terms cell-penetrating peptide and CPP are used interchangeably and refer to cationic cell-penetrating peptides, also referred to as transport peptides, carrier peptides, or peptide transduction domains. The peptides provided herein have the ability to induce cell entry in 100% of cells in a defined cell culture population and provide macromolecular translocation in a number of tissues in vivo when administered systemically. In various embodiments, an embodiment of a CPP according to the present invention may comprise an arginine-rich peptide, described further below.

The term treatment refers to the application of one or more specific procedures used to reduce the intensity of the disease. In some embodiments, the specific procedure is the administration of one or more pharmaceutical agents. Treatment of an individual (eg, a mammal, such as a human) or treatment of a cell is any type of intervention used in an attempt to alter the natural course of a disease in an individual or cell. Treatment includes, but is not limited to, administering a pharmaceutical composition and can be either prophylactic, after the onset of a pathological event, or after exposure to an etiological factor. Treatment includes any desired effect on the symptoms or pathology of the disease or condition and may include, for example, minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Also included are prophylactic treatments that may be directed to slowing the rate of progression of the disease or condition being treated, delaying the onset of such disease or condition, or lessening the severity of its onset. An effective amount or therapeutically effective amount refers to the amount of a therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, that is effective to achieve the desired therapeutic effect.

The term reduction in intensity means a decrease in the severity of at least one symptom of a condition or disease. In some embodiments, reducing the intensity includes delaying or slowing the progression of one or more signs of the condition or disease. The severity of the signs can be determined by subjective or objective indicators, which are known to specialists in this field of technology.

As used herein, pharmaceutically acceptable salts refer to derivatives of the disclosed oligonucleotides wherein the parent oligonucleotide is modified by converting an existing acid or base moiety into its salt form. A list of suitable salts is found in Remington's Pharmaceutical Sciences, 17th ^ed ., Mack Publishing Company, Easton, Pa., 1985, p. 1418, and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

Peptide-oligonucleotide conjugates.

Provided herein are oligonucleotides chemically linked to one or more moieties, such as cell-penetrating peptides, that enhance the activity, cell distribution, or cell uptake of the oligonucleotide. The oligonucleotides may additionally be chemically linked to one or more heteroalkyl moieties (eg, polyethylene glycol) that further enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. In one illustrative embodiment, an arginine-rich polypeptide is covalently linked at an N-terminal or C-terminal residue to one or both ends of an antisense compound.

Thus, in one aspect, provided herein is a peptide oligonucleotide

- 9 039716 conjugate according to the formula (I)

E'(I), or a pharmaceutically acceptable salt thereof, he

where A' is selected from -NHCH2C(O)NH ₂ , -N(C _1-6 alkyl)CH ₂ C(O)NH ₂ , ^and ' where R ⁵ is -C(O(O-alkyl)x-OH , x is 3-10 and each alkyl group is independently at each occurrence C2-6alkyl or R ⁵ is selected from -C(O)C1-6alkyl, trityl, monomethoxytrityl, -(C _1-6 alkyl)R ⁶ , -( C1-6heteroalkyl)-R ⁶ , aryl-R ⁶ , heteroaryl-R ⁶ , -C(O)O-(C1-6alkyl)-R ⁶ , C(O)O-aryl-R ⁶ ,

(J)t_ . 6 I 6 , _.......6 __ _

-C(O)O-heteroaryl-R and ^G ; R is selected from OH, SH and NH2 or R is O, S or NH covalently attached to the solid support;

each R ¹ is independently selected from OH and —NR ³ R ⁴ , wherein R ³ and R ⁴ are each independently at each occurrence —C _1-6 alkyl;

each R ² is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein the nucleobase independently at each occurrence contains a _C3-6 heterocyclic ring selected from pyridine, pyrimidine, triazinan, purine, and deazapurine;

z is in the range 8-40;

E' is selected from H, -C _1-6 alkyl, -C(O)C _1-6 alkyl, benzoyl, stearoyl,

trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

Q is -C(O)(CH)6C(O)- or C^CCH^CHNO)-;

R ⁷ is -(CH2)2OC(O)N(R ⁸ )2, where R ⁸ is -(CH ₂ ) ₆ NHC(=NH)NH ₂ ; and

R ¹¹ is selected from OH and -NR ³ R ⁴ ;

L is covalently linked via an amide bond to the carboxy terminus in J and L is selected from

-NH(CH2)i-6C(O)-, -NH(CH2)i-6C(O)NH(CH2)i-6C(O)- and t is in the range 4-9;

each J is independently at each occurrence selected from an amino acid structure

where each of r and q is independently 0, 1, 2, 3 or 4;

each R ⁹ is independently at each occurrence selected from H, an amino acid side group, and an amino acid side group functionalized with a chemical protecting group, wherein two or more amino acid side groups from R ⁹ independently at each occurrence contain sulfur, where two sulfur atoms together with atoms, to which they are attached form the structure s—(M) _d —S _K where d is 0 or 1 and M is selected from:

- 10 039716

where each R ¹⁰ independently in each case represents H or halogen;

G is covalently bonded to the amino terminus in J and G is selected from H, C(O)C _1-6 alkyl, benzoyl and stearoyl, and wherein at least one of the following is true:

1) A' represents

G(J)t

L

2) E' represents

or

3) E' represents

In one embodiment, E' is selected from H, -C _1-6 alkyl, -C(O)C _1-6 alkyl, benzoyl, stearoyl,

- 11 039716 trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl and

In another embodiment, only one of A' is

represents

or E' is

(J)t

In yet another embodiment, A' is ; or E' is

In another variant

implementation A' is selected from -N(C _1-6 alkyl)CH ₂ C(O)NH ₂ ,

In another embodiment, E' is selected from H, -C(O)CH ₃ , trityl, 4-methoxytrityl, benzoyl, stearoyl and

in another embodiment, A' is selected from -N(C _1-6 alkyl)CH ₂ C(O)NH ₂ , he

'n η ^{ζ ;} and E' represents

In yet another embodiment, A' is

In another embodiment, E' is selected from H, -C(O)CH ₃ , trityl, 4-methoxytrityl, benzoyl and stearoyl.

In yet another embodiment, E' is selected from H and -C(O)CH ₃ .

In yet another embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Ia)

- 12 039716

G

In one embodiment of formulas (I) and (Ia), R ⁵ is -C(O)(O-alkyl) _x OH, where each alkyl group is independently C _2-6 alkyl.

In another embodiment of formulas (I) and (Ia), R ⁵ is -C(O)(O-CH ₂ CH ₂ )3OH.

In another embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Ib)

In one embodiment of formulas (I) and (Ib), E' is selected from H, C _1-6 alkyl, -C(O)CH ₃ , benzoyl and stearoyl.

In another embodiment of formulas (I) and (Ib), E' is selected from H and -C(O)CH3.

In one embodiment of formulas (I), (Ia) and (Ib), each R ¹⁰ is independently a halogen selected from fluorine, chlorine, bromine and iodine.

In another embodiment of formulas (I), (Ia) and (Ib), each R ¹⁰ is fluoro.

In yet another embodiment of formulas (I), (Ia) and (Ib), M is

In another embodiment of formulas (I), (Ia) and (Ib), the two amino acid side group groups are independently at each occurrence cysteine or homocysteine amino acid side group groups.

In yet another embodiment of formulas (I), (Ia) and (Ib), each J is independently at each occurrence selected from an α-amino acid, a 2-amino acid, and a 3-amino acid.

In yet another embodiment of formulas (I), (Ia) and (Ib), each of r and q is 0.

In another embodiment of formulas (I), (Ia) and (Ib), J is independently selected from cysteine and arginine.

In yet another embodiment of formulas (I), (Ia) and (Ib), the two J groups are cysteine.

In another embodiment of formulas (I), (Ia) and (Ib) z is in the range of 8-25.

In another embodiment of formulas (I), (Ia) and (Ib) z is in the range of 15-25.

In another embodiment of formulas (I), (Ia) and (Ib) z is in the range of 10-20.

In another embodiment of formulas (I), (Ia) and (Ib), each R ¹ is independently NR3R ⁴ where R3 and R ⁴ are each independently C _1-3 alkyl.

In yet another embodiment of formulas (I), (Ia) and (Ib), each R ¹ is N(CH ₃ ) ₂ .

In yet another embodiment of formulas (I), (Ia) and (Ib), each R ² is a nucleobase, wherein the nucleobase independently at each occurrence contains a C _4-6 heterocyclic ring selected from pyridine, pyrimidine, triazinan, purine, and deazapurine.

In another embodiment of formulas (I), (Ia) and (Ib), each R ² is a nucleobase, wherein the nucleobase independently at each occurrence contains a C _4-6 heterocyclic ring selected from pyrimidine, purine and deazapurine.

In yet another embodiment of formulas (I), (Ia) and (Ib), each R ² is a nucleobase independently at each occurrence selected from adenine, 2,6-diaminopurine, 7-deazaadenine, guanine, 7-deaza-guanine , hypoxanthine, cytosine, 5-methylcytosine, thymine, uracil, and hypoxanthine.

In yet another embodiment of formulas (I), (Ia) and (Ib), each R ² is a nucleobase, independently at each occurrence selected from adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, and hypoxanthine.

In another embodiment of formulas (I), (Ia) and (Ib), L is selected from -NH(CH ₂ ) _1-6 C(O)-,

-NH(CH2)5C(O)NH(CH2)2C(O)- and

In another embodiment of formulas (I), (Ia) and (Ib) L is selected from glycine and ·

In yet another embodiment of formulas (I), (Ia) and (Ib), L is glycine.

In another embodiment of formulas (I), (Ia) and (Ib), G is selected from H, C(O)CH ₃ , benzoyl and stearoyl.

In yet another embodiment of formulas (I), (Ia) and (Ib), G is H or -C(O)CH ₃ .

In yet another embodiment of formulas (I), (Ia) and (Ib), G is -C(O)CH ₃ .

In another embodiment of formulas (I), (Ia) and (Ib), d is 1.

In yet another embodiment of formulas (I), (Ia) and (Ib), d is 0.

In yet another embodiment of formulas (I), (Ia) and (Ib), ^{G is} selected from:

- 14 039716

In another embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Ic)

In another embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Id)

In another embodiment, the peptide-oligonucleotide conjugate of formula (I) is a peptide-oligonucleotide conjugate of formula (Ie)

- 15 039716

In another aspect, provided herein is a peptide-oligonucleotide conjugate of formula (IV)

or a pharmaceutically acceptable salt thereof, he .R ²

Ν' where A' is selected from -NHCH2C(O)NH2, -N(C _1-6 alkyl)CH ₂ C(O)NH ₂ ,

R1

Ν η ^r where R ⁵ is -C(O)(O-alkyl)x-OH, where x is in the range of 3-10, and each alkyl group is independently at each occurrence C2.6 alkyl or R ⁵ is selected from - C(O)C1. ₆ alkyl, trityl, monomethoxytrityl, -(C 1.6 alkyl)R ₆ , -(C1.6heteroalkyl)-R ₆ , aryl-R ⁶ , heteroaryl-R ⁶ , -C(O)O- ^{( C1.6} _alkyl ) -R ⁶ ,

-C(O)O-aryl-R ⁶ , -C(O)O-heteroaryl-R ⁶ and

R ⁶ is selected from OH, SH and NH2, or R ⁶ is O, S, or NH covalently attached to a solid support;

each R ¹ is independently selected from OH and —NR ³ R ⁴ , wherein R ³ and R ⁴ are each independently at each occurrence —C _1-6 alkyl;

each R ² is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, wherein the nucleobase independently at each occurrence contains a _C3-6 heterocyclic ring selected from pyridine, pyrimidine, triazinan, purine, and deazapurine;

z is in the range 8-40;

E' is selected from H, -C _1-6 alkyl, -C(O)C _1-6 alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

where Q represents -C(O)(CH ₂ )6C(O)- or -C(O)(CH2)2S2(CH2)2C(O)-;

R ⁷ is -(CH2)2OC(O)N(R ⁸ )2, where R ⁸ is -(CH2) ₆ NHC(=NH)NH2; and R ¹¹ is selected from OH and -NR ³ R ⁴ ;

L is covalently linked via an amide bond to the carboxy terminus in J and L is selected from

-NH(CH2)i-6C(O)-, -NH(CH2)i-6C(O)NH(CH2)i-6C(O)- and

t is in the range 4-9;

- 16 039716 each J is independently selected at each occurrence from the amino acid structure

where each of r and q is independently 0, 1, 2, 3 or 4;

each R ⁹ is independently at each occurrence selected from H, an amino acid side group, and an amino acid side group functionalized with a chemical protecting group, wherein two or more amino acid side groups from R ⁹ independently at each occurrence contain sulfur, where two sulfur atoms together with atoms, to which they are attached form a structure

G is covalently linked to the amino terminus in J, and G is selected from H, C(O)C _1-6 alkyl, benzoyl, and stearoyl, and wherein at least one of the following is true:

1) A' represents

or

2) E' represents

3) E' represents

In one embodiment of formula (IV), d is 1. In another embodiment of formula (IV), E' is selected from H, -C(O)CH ₃ and

in one embodiment of formula (IV) A' is selected from -N(C _1-6 alkyl)CH ₂ C(O)NH ₂ ,

In still

E' represents

- 17 039716 (J)t .N.

N

In yet another embodiment of formula (IV), A' is —— ·

In another embodiment of formula (IV), E' is selected from H and -C(O)CH ₃ .

In yet another embodiment of formula (IV), M is

In another embodiment of formula (IV), each J is independently at each occurrence selected from an α-amino acid, a 2-amino acid, and a p ³ -amino acid.

In yet another embodiment of formula (IV), r and q are each 0.

In another embodiment of formula (IV), J is independently selected from cysteine and arginine.

In another embodiment of formula (IV) z is in the range of 15-25.

In yet another embodiment of formula (IV), z is in the range of 10-20.

In yet another embodiment of formula (IV), each R ¹ is N(CH ₃ ) ₂ .

In yet another embodiment of formula (IV), each R ² is a nucleobase, independently at each occurrence selected from adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, and hypoxanthine.

In yet another embodiment of formula (IV), L is glycine.

In another embodiment of formula (IV), G is selected from H, C(O)CH ₃ , benzoyl and stearoyl.

In yet another embodiment of formula (IV), G is -C(O)CH ₃ .

In yet another embodiment, the oligonucleotide contains a targeting sequence characterized by sequence complementarity to the target RNA. In a specific embodiment, the target RNA is a cellular target RNA. In another specific embodiment, the targeting sequence has sufficient sequence complementarity to bind to the target RNA. In yet another specific embodiment, the targeting sequence has perfect sequence complementarity to the target RNA.

Characteristic fragments are fragments of the following formulas:

in accordance with the present invention include, among others,

- 18 039716

- 19 039716

- 20 039716

- 21 039716

- 22 039716

Representative peptide oligonucleotide conjugates according to the present invention include, among others, peptide oligonucleotide conjugates with the following structures:

- 23 039716

- 24 039716

- 25 039716

- 26 039716

- 27 039716

- 28 039716

- 29 039716

- 30 039716

- 31 039716

- 32 039716

- 33 039716

or a pharmaceutically acceptable salt thereof, wherein G is selected from H and -C(O)CH ₃ and E' is selected from H and -C(O)CH ₃ .

In one embodiment of the peptide-oligonucleotide conjugates of the present invention, G is H.

In another embodiment of the peptide-oligonucleotide conjugates of the present invention, G is —C(O)CH ₃ .

In yet another embodiment of the peptide-oligonucleotide conjugates of the present invention, E' is H.

In yet another embodiment of the peptide-oligonucleotide conjugates of the present invention, E' is —C(O)CH ₃ .

In yet another embodiment of the peptide-oligonucleotide conjugates of the present invention, E' and G are -C(O)CH ₃ .

In yet another embodiment of the peptide oligonucleotide conjugates of the present invention, G is -C(O)CH ₃ and E' is H.

In some embodiments, the peptide-oligonucleotide conjugates described herein are non-solvated. In other embodiments, one or more of the peptide-oligonucleotide conjugates are in solvated form. As is known in the art, the solvate may be formed by any pharmaceutically acceptable solvent such as water, ethanol, and the like.

Although the peptide-oligonucleotide conjugates of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), and (IV) are depicted in their neutral forms, in some embodiments, such a peptide

- 34 039716 oligonucleotide conjugates are used in the form of a pharmaceutically acceptable salt.

Oligonucleotides.

Important properties of morpholino-based subunits include 1) the ability to bind in oligomeric form via stable uncharged or positively charged backbone bridges; 2) the ability to maintain a nucleotide base (for example, adenine, cytosine, guanine, thymidine, uracil, 5-methylcytosine and hypoxanthine) so that the resulting polymer can hybridize with the complementary base of the target nucleic acid, including the target RNA, while T values _m above about 45°C in relatively short oligonucleotides (eg, 10-15 bases); 3) the ability of the oligonucleotide to actively or passively transport into mammalian cells; and 4) the ability of the oligonucleotide and the oligonucleotide:RNA heteroduplex to resist RNase and RNase H cleavage, respectively.

The stability of the duplex formed by the oligomer and the target sequence is a function of Tm binding and the sensitivity of the duplex to enzymatic degradation of the cell. The TM of an oligomer with respect to the complementary sequence RNA can be measured by conventional methods, such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, p. 107-108, or as described in Miyada CG and Wallace RB, 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol. 154, p. 94-107. In some embodiments, the implementation of antisense oligomers can be characterized by _Tm binding in relation to the RNA of the complementary sequence in excess of body temperature and in some embodiments, the implementation of the implementation of more than about 45 or 50°C. Also included are T _m values in the range of 60-80° C. or higher. According to well-known principles, the Tm of an oligomer against a RNA hybrid based on a complementary sequence can be increased by increasing the C:G base pair ratio of the duplex, or by increasing the length (in base pairs) of the heteroduplex, or both. Along with this, in order to optimize cell uptake, it may be advantageous to limit the size of the oligomer. Therefore, compounds in accordance with the present invention include compounds that exhibit high T _m (45-50°C or higher) at a length of 25 bases or less.

The length of the oligonucleotide can be changed as long as it retains the ability to selectively bind to the intended location in the pre-mRNA molecule. The length of such sequences can be determined according to the selection procedures described herein. Typically, the oligonucleotide will be from about 8 nucleotides in length to no more than about 50 nucleotides in length. For example, the oligonucleotide length (z) may be 8-38, 8-25, 15-25, 17-21, or about 18. However, it is understood that any nucleotide length in this range may be applicable in the methods described herein.

In some embodiments, the antisense oligonucleotides contain base modifications or substitutions. For example, certain nucleobases may be selected to increase the binding affinity of the antisense oligonucleotides described herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2-, N-6-, and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, and 2,6-diaminopurine. 5-methylcytosine substitutions have been shown to increase the stability of the nucleic acid duplex by 0.61.2° C. and can be included in the antisense oligonucleotides described herein. In one embodiment, at least one pyrimidine base of the oligonucleotide contains a 5-substituted pyrimidine base, wherein the pyrimidine base is selected from the group consisting of cytosine, thymine, and uracil. In one embodiment, the 5-substituted pyrimidine base is 5-methylcytosine. In another embodiment, at least one purine base of the oligonucleotide contains an N-2-, N-6-substituted purine base. In one embodiment, the N-2-,N-6-substituted purine base is 2,6-diaminopurine.

Morpholino-based oligomers (including antisense oligomers) are described in detail in, for example, US Pat. Nos. 5,698,685; 5217866; 5142047; 5034506; 5166315; 5185444; 5521063; 5,506,337 and pending US patent applications No. 12/271036; 12/271040; and in PCT Application Publication Nos. WO 2009/064471 and WO 2012/043730 and Summerton et al. 1997, Antisense and Nucleic Acid Drug Development, 7, 187-195, which are incorporated herein by reference in their entirety.

Accordingly, in one aspect, provided herein is an oligonucleotide of formula (II)

- 35 039716

(ii) or a pharmaceutically acceptable salt thereof,

Rt x - n η where A is selected from the group consisting of OH, -NHCH2C(O)NH2, -N(C _1-6 alkyl)CH ₂ C(O)NH ₂ and '

R ⁵ is -C(O)(O-alkyl) _x OH, where x is in the range of 3-10 and each alkyl group is independently at each occurrence -C _2-6 alkyl, or R ⁵ is selected from the group consisting of from -C(O)C1-6alkyl, trityl, monomethoxytrityl, -C1-6alkyl-R ⁶ , -C1-6heteroalkyl-R ⁶ , -aryl-R ⁶ , -heteroaryl-R ⁶ , -C(O)O-C _1-6 alkyl-R ⁶ , -C(O)O-aryl-R ⁶ and -C(O)O-heteroaryl-R ⁶ ;

R ⁶ is selected from the group consisting of OH, SH and NH ₂ , or R ⁶ is O, S or NH covalently attached to a solid support;

each R ¹ is independently OH or —NR ³ R ⁴ ;

each of R ³ and R ⁴ independently at each occurrence is -C _1-6 alkyl;

each R ² is independently selected from the group consisting of H, a nucleobase and a nucleobase functionalized with a chemical protecting group, wherein the nucleobase independently each occurrence contains a _C3-6 heterocyclic ring selected from the group consisting of pyridine, pyrimidine, triazinane , purine and deazapurine;

z is in the range 8-40;

E is selected from the group consisting of H, -C _1-6 alkyl, -C(O)C _1-6 alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

Q is -C(O)(CH2)6C(O)- or -COXCH^CHhQO)-;

R ⁷ is -(CH2)2OC(O)N(R ⁸ )2;

R ⁸ is -(CH2)6NHC(=NH)NH2;

R ¹² is -C(O)NHC _1-6 alkyl or -C(O)OC _1-6 alkyl; and

R ¹³ is selected from the group consisting of trityl, monomethoxytrityl, dimethoxytrityl and sitrityl trimets.

In one embodiment of formula (II)

A represents

E is selected from the group consisting of H, -C(O)CH ₃ , benzoyl and stearoyl;

R ⁵ is -C(O)(O-alkyl) _x -OH, where each alkyl group is independently at each occurrence -C _2-6 alkyl, trityl and 4-methoxytrityl; and each R ² is independently a nucleobase, wherein the nucleobase independently at each occurrence contains a C _4-6 heterocyclic ring selected from the group consisting of pyridine, pyrimidine, purine and deazapurine.

In another embodiment of formula (II), R ⁵ is C(O)(O-CH ₂ CH ₂ ) ₃ -OH and each R ² is independently a nucleobase, wherein the nucleobase independently each occurrence contains pyrimidine or purine.

In yet another embodiment, the oligonucleotide of formula (II) is an oligonucleotide of formula (IIa)

- 36 039716

In one embodiment of formulas (II) and (IIa), R ² is independently at each occurrence adenine, 2,6-diaminopurine, guanine, hypoxanthine, cytosine, 5-methylcytosine, thymine, uracil, and hypoxanthine; and each R ¹ is -N(CH ₃ ) ₂ .

In table. 1 shows various embodiments of the nucleotide fragments described herein.

Table 1

Various embodiments of nucleotide fragments

In some embodiments, the oligonucleotides described herein are non-solvated. In other embodiments, one or more of the oligonucleotides are in solvated form. As is known in the art, the solvate may be formed from any pharmaceutically acceptable solvent such as water, ethanol, and the like.

Although the oligonucleotides of formulas (II) and (IIa) are depicted in their neutral forms, in some embodiments, such oligonucleotides are used in the form of a pharmaceutically acceptable salt.

Peptides.

Oligonucleotides provided herein include an oligonucleotide fragment conjugated to CPP. In some embodiments, the CPP may be an arginine-rich peptide transport moiety effective to increase the transport of a compound into cells. The transport fragment, in some embodiments, is attached to the end of the oligomer. The peptides have the ability to induce cell entry in 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the cells in a given cell culture population, including all integers in between, and provide high molecular weight translocation in a range of tissues in vivo upon systemic administered. In one embodiment, the cell-penetrating peptide may be an arginine-rich peptide transporter. In various embodiments, the peptide oligonucleotide conjugate of the present invention may use glycine as a linker between the CPP and the antisense oligonucleotide.

For the transport fragments described above, a significant increase in cell entry of attached oligomers was shown compared to oligomer uptake in the absence of an attached transport fragment. The capture can be increased at least 10 times and in some embodiments up to 20 times compared to the unconjugated compound.

The use of carriers based on arginine-rich peptides (ie, cell-penetrating peptides) is particularly useful in the practice of the present invention. Some peptide-based transporters have been shown to be highly effective in delivering antisense compounds to primary cells, including muscle cells. In addition, compared to other known peptide-based transporters such as Penetratin and the Tat peptide, the peptide-based transporters described herein, when conjugated to an antisense PMO, exhibit an increased ability to alter the splicing of multiple gene transcripts.

Thus, in one aspect, provided herein is a peptide of formula (III)

G—(J) _t ---L (III), or a pharmaceutically acceptable salt thereof, wherein G is selected from the group consisting of H, C(O)C _1-6 alkyl, benzoyl and stearoyl;

each J is independently at each occurrence selected from an amino acid structure

R ⁹ O each R ⁹ is independently at each occurrence selected from the group consisting of H, an amino acid side group, and an amino acid side group functionalized with a chemical protecting group, wherein two or more amino acid side groups in R ⁹ independently at each occurrence contain a thiol or a thiol , functionalized with a chemical protecting group;

r and q are independently 0, 1, 2, 3, or 4;

L is selected from the group consisting of -NH(CH ₂ ) _1-6 C(O)OH,NH(CH ₂ ) ₅ C(O)NH(CH ₂ ) ₂ C(O)OH and ' each of which may be covalently bound to a solid support; and t is in the range 4-9.

In one embodiment, two amino acid side group groups, where each of the two amino acid side group groups independently contain sulfur, together with the atoms to which they are attached, form the structure

M is selected from the group consisting of

- 38 039716

d is 0 or 1.

In another embodiment, M is

In yet another embodiment, the two amino acid side group groups are independently at each occurrence cysteine or homocysteine amino acid side group groups.

In yet another embodiment, each J is independently at each occurrence selected from an α-amino acid, a 2-amino acid, and a 3-amino acid.

In another embodiment, each of r and q is 0.

In another embodiment, J is independently selected from cysteine and arginine.

In yet another embodiment, the two J groups are cysteine.

is he

In yet another embodiment, L is selected from -NH(CH ₂ ) _1-6 C(O)OH and In another embodiment, L is

In yet another embodiment, L is NHCH ₂ C(O)OH.

In another embodiment, G is selected from the group consisting of H, C(O)CH ₃ , benzoyl and stearoyl.

In yet another embodiment, G is C(O)CH ₃ or stearoyl.

In another embodiment, G is C(O)CH ₃ .

In yet another embodiment, G is covalently linked to the amino terminus in J. In a further embodiment, L is covalently linked via an amide bond to the carboxy terminus in J.

In another embodiment, d is 0.

In yet another embodiment, d is 1.

In table. 2 shows representative peptides described herein.

- 39 039716

Illustrative Peptides

table 2

Structure Compound SEQID NO 1 1 Ac-Cys-R ₃ -Cys-R-Gly-OH 1AC one 1AD 1AE IBC 1BD 1BE 1 1 Ac-Cys-R ₃ -Cys-R ₂ -Gly-OH 2AC 2AD 2 2AE 2BC 2BD 2BE 1 1 Ac-Cys-R ₃ -Cys-R ₃ -Gly-OH 3AC 3 3AD 3AE 3BC 3BD 3BE 1 1 Ac-Cys-R ₆ -Cys-G ly-O H 4AC 4 4AD 4AE 4BC 4BD 4BE 1 1 Ac-Cys-R ₅ -Cys-Gly-OH 5AC 5 5AD 5AE 5BC 5BD 5BE 1 1 Ac-Cys-R ₄ -Cys-Gly-OH 6AC 6 6AD 6AE 6BC 6BD 6BE 1 1 Ac-Cys-R ₃ -Cys-Gly-OH 7AC 7 7AD 7AE 7BC 7BD 7BE 1 1 Ac-Cys-R ₂ -Cys-Gly-OH 8AC eight 8AD 8AE 8BC 8BD 8BE Gly = glycinyl or Gly-P3P (A) (B) R=Arg

- 40 039716

In some embodiments, the peptides described herein are unsolvated. In other embodiments, one or more of the peptides are in solvated form. As is known in the art, the solvate may be formed by any pharmaceutically acceptable solvent such as water, ethanol, and the like.

While the peptides of formula (III) are depicted in their neutral forms, in some embodiments, such oligonucleotides are used in a pharmaceutically acceptable form.

salt. General synthesis schemes. Scheme Ia Peptide crosslinking ΙΠΓ, hexaphthoebenzene, S TRIS Ac-Cys-R-R-R-R-R-R-Cys-G-OH --------------------► Scheme Ib F F /Arn A c-Cys-R- R-R-R-R- R-Cy s-G- 0 H

Conjugation of the peptide to the 3' end of the PMO

1 Γφ H HATU, DIPEA, DMSO + ____________________________ ^F \ ^F / FF \ Ac-Cys-RRRRRR-Cys-G-OH 1 (1/7^3)2 0 CO ►O— £___-s o - ώ

- 41 039716

Scheme Ic

Conjugation of the peptide to the 5' end of the PMO

Scheme IIa

Peptide conjugation via disulfide bonds to the 3' end of PMO

The method in scheme IIa is applicable for conjugation of the peptide to the 5' end of the PMO under similar conditions (see scheme Ic).

Scheme IIb

Cross-linking peptide-PMO-conjugate

The method in scheme IIb is applicable to conjugation of the peptide to the 5' end of the PMO under similar conditions.

- 42 039716

Scheme IIIa

Peptide and PMO conjugation and crosslinking in one reaction vessel

1) HATU. DIPEA. DMSO

Scheme IIIb

Peptide and PMO conjugation and crosslinking in one reaction vessel

1) HATU. DIPEA, DMSO

2) [I T. hexaphthosebenzene, |

Ways.

Provided herein are methods for treating a muscle disease, viral infection, or bacterial infection in a subject in need thereof, comprising administering to the subject a peptidoligonucleotide conjugate of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie ) or (V).

Accordingly, in one aspect, this document provides a method of treating a muscle disease, a viral infection, or a bacterial infection in a subject in need thereof, comprising administering to the subject a peptide oligonucleotide conjugate in accordance with the present invention.

In one embodiment, the muscle disease is Duchenne muscular dystrophy.

In another embodiment, the viral infection is caused by a virus selected from the group consisting of Marburg disease virus, Ebola virus, influenza virus, and dengue virus.

In another embodiment, the bacterial infection is due to Mycobacterium tuberculosis.

The subject in this document, as a rule, means a person. However, the subject may be any mammal for which treatment is necessary. Thus, the methods described herein can be used in both human and veterinary applications.

Introduction / dose.

The composition of the therapeutic compositions and their subsequent administration (dosing) are within the competence of those skilled in the art. Dosing depends on the severity and susceptibility of the disease state to be treated during a course of treatment lasting from several days to several months, or until a sufficient reduction in the disease state is achieved. Optimal dosing regimens can be calculated from measurements of drug accumulation in the patient's body.

Those skilled in the art can readily determine optimal dosages, dosing techniques, and repetition rates. Optimal dosages may vary depending on the relative activity of the individual oligomers and can generally be estimated based on EC ₅₀ values that have been shown to be effective in in vitro and in vivo animal models. In general, the dosage ranges from 0.01 µg to 100 g/kg of body weight and may be administered one or more times a day, a week, a month or a year, or even once every 2-20 years. Those skilled in the art can easily estimate dosing repetition rates from measured retention times and drug concentrations in body fluids or tissues. After successful treatment, the patient may need to undergo maintenance therapy to prevent recurrence of the disease state, in which the oligomer is administered at maintenance doses in the range from 0.01 μg to 100 g/kg of body weight from one or more times a day to once every 20 years.

In some embodiments, the implementation of the oligonucleotide (oligonucleotide according to formula (II) or (IIa)) is administered separately.

In some embodiments, the oligonucleotide is administered in a therapeutically effective amount or dosage.

A therapeutically effective amount is an amount of an oligonucleotide of formula (II) or (IIa) which, when administered alone to a patient, provides effective treatment for a muscle disease, viral infection, or bacterial infection. An amount that proves to be a therapeutically effective amount on a given occasion for a given subject may not be 100% effective for subjects being similarly treated for the disease or condition in question, even if such a dosage is considered to be a therapeutically effective amount by those skilled in the art. The amount of oligonucleotide that corresponds to a therapeutically effective amount is highly dependent on the type of disease, the stage of the disease, the age of the patient being treated, and other factors.

In various embodiments, depending on the oligonucleotide of formula (II) or (IIa) and the effective amounts used, the oligonucleotides can modulate the expression of a gene involved in a muscle disease, viral infection, or bacterial infection.

If an amount of an oligonucleotide of formula (II) or (IIa) would result in effective treatment of a muscle disease, viral infection, or bacterial infection, the amount is preferably not excessively toxic to the patient (i.e., the amount is preferably within the toxicity range established by medical guidelines ). In some embodiments, either in order to prevent excessive toxicity, or to provide more effective treatment, or both, in relation to a muscle disease, a viral infection, or a bacterial infection, a restriction on the total administered dosage is envisaged. As a rule, the quantities considered in this document are presented in the form of a number per day; however, this document also covers half-day cycles, as well as two-day or three-day cycles.

For the treatment of muscle disease, viral infection, or bacterial infection, various dosing regimens may be used. In some embodiments, a daily dosage, such as any of the exemplary dosages described above, is administered once, twice, three times, or four times a day for three, four, five, six, seven, eight, nine, or ten days. Depending on the stage and severity of the disease being treated, a shorter treatment time (eg, no more than five days) may be used along with a high dosage, or a longer treatment time may be applied (eg, ten or more days, or weeks, or a month or longer) along with a low dosage. In some embodiments, the once or twice daily dosage is administered every other day.

The oligonucleotides of formula (II) and (IIa), or their pharmaceutically acceptable salts or solvate forms, in pure form or in a suitable pharmaceutical composition, may be administered by any of the acceptable modes of administration or means known in the art. The oligonucleotides can be administered, for example, orally, nasally, parenterally (intravenously, intramuscularly, or subcutaneously), topically, transdermally, intravaginally, intravesically, intracystemially, or rectally. The dosage form may be, for example, solid, semi-solid, lyophilized powder or liquid dosage forms such as, for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols or the like, for example, in unit dosage forms suitable for easy administration of precise dosages. A particular route of administration is oral, in particular one that can adjust a convenient daily dosage regimen depending on the severity of the disease being treated.

The adjuvant and adjuvants may include, for example, preserving, moisturizing, suspending, sweetening, flavoring, flavoring, emulsifying and dosing agents.

- 44 039716

Prevention of the action of microorganisms is generally provided by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents such as sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin. Adjuvants may also include wetting agents, emulsifying agents, pH buffering agents, and antioxidants such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.

Solid dosage forms can be prepared with coatings and shells such as enteric coatings and others well known in the art. They may contain opacifying agents, and may also be formulated to release the active oligonucleotide or oligonucleotides in a particular region of the intestine with a delay. Examples of encapsulating compositions that can be used are polymeric materials and waxes. If desired, the active oligonucleotides may also be in microencapsulated form with one or more of the above excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and strong infusions. Such dosage forms are obtained, for example, by dissolution, dispersion, etc. the HDAC or retinoic acid inhibitors described herein or their pharmaceutically acceptable salts and optional pharmaceutical auxiliaries in a carrier such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like; solubilizing agents and emulsifiers such as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide; oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters and sorbitol; or mixtures of these substances, etc., to obtain a solution or suspension.

Typically, depending on the intended route of administration, pharmaceutically acceptable compositions will contain from about 1 to about 99% by weight of the oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, and from 99 to 1% by weight of a pharmaceutically acceptable excipient. In one example, the composition will contain from about 5 to about 75% by weight of the oligonucleotide described herein, or a pharmaceutically acceptable salt thereof, with the remainder being suitable pharmaceutical excipients.

Existing methods for preparing such dosage forms are known or will be apparent to those skilled in the art. Reference is made to, for example, Remington's Pharmaceutical Sciences, 18th ^Ed . (Mack Publishing Company, Easton, PA, 1990).

Sets.

In other embodiments, kits are provided. Kits in accordance with the present invention include packages containing oligonucleotides, peptides, peptidoligonucleotide conjugates or compositions in accordance with the present invention. In some embodiments, the kits contain a peptide-oligonucleotide conjugate according to formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), or (V) or a pharmaceutically acceptable salt thereof. In other embodiments, the kits contain an oligonucleotide according to formulas (II) or (IIa) or a pharmaceutically acceptable salt thereof. In still other embodiments, the kits contain a peptide according to formula (III) or a pharmaceutically acceptable salt thereof.

The expression package means any container containing the oligonucleotides or compositions presented in this document. In some embodiments, the package may be a box or wrapper. Packaging materials for use in the packaging of pharmaceutical products are well known to those skilled in the art. Examples of pharmaceutical packaging materials include, without limitation, bottles, vials, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

The kit may also contain items that are not contained in the package but are attached to the outside of the package, such as pipettes.

The kits may further contain instructions for administering the oligonucleotides or compositions of the present invention to a patient. The kits may also contain instructions for the use of the oligonucleotides provided herein approved by regulatory authorities such as the US Food and Drug Administration. The kits may also contain a label or package inserts for the oligonucleotides. Packages or any package inserts or both may themselves be approved by regulatory authorities. Kits may include solid phase or liquid phase oligonucleotides (eg, with buffers) in a package. Kits may also include buffers for preparing solutions for performing methods and pipettes for transferring liquids from one container to another.

Examples

The examples are given below to illustrate and describe some specific embodiments of the present invention. However, the scope of the claims is in no way limited by the examples provided herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications, including, without limitation, changes and modifications related to chemical structures, substituents, derivatives, compositions, or methods in accordance with the present invention, may be made without departing from the spirit of the present invention and the scope of the appended claims. The definitions of variables in the structures in the schemas in this document correspond to the definitions of the corresponding positions in the formulas presented in this document.

Example 1 Peptide Crosslinking Protocol Using Hexafluorobenzene

F ^F

S----SA)--S

1^4

Ac-Cys-Arg-Arg-Arg-Cys-Arg-Arg-Arg-Gly-OH

To a cyclic disulfide peptide Ac-CRRRCRRRG-OH (74.8 mg, 38.5 µmol) (SEQ ID NO: 3) in a glass vial was added a Tris base solution (8 ml, 50 mM in DMF) and hexafluorobenzene (111 µl, 962 µmol). To this solution is added a solution of TCEP in H ₂ O (136 μl, 0.71 M, pH 8) to restore the disulfide bond and initiate the reaction. The reaction mixture is stirred at room temperature for 7 hours and monitored by LC-MS (LC-MS). The resulting mixture was diluted with 40 ml of 0.1% TFA in water, centrifuged, filtered and purified by HPLC (HPLC). Fractions containing the peptide product (analyzed by LC-MS) are combined and lyophilized to give the desired perfluoroaryl peptide (19.9 mg, 8.9 μmol, 23% yield).

Example 2 Peptide Crosslinking Protocol Using Decafluorobiphenyl

F F Vs—Vs I I

Ac-Cys-Arg-Arg-Arg-Cys-Arg-Arg-Arg-Gly-OH

In a glass vial, dissolve the cyclic disulfide peptide Ac-CRRRCRRRG-OH (74.5 mg, 38.4 µmol) (SEQ ID NO: 3) and decafluorobiphenyl (25.6 mg, 76.7 µmol) in Tris base solution in DMF (8 ml, 50 mm). To this solution is added a solution of TCEP in H2O (135 µl, 0.71 M, pH ~8) to reduce the disulfide bond and initiate the reaction. The reaction mixture was stirred at room temperature for 7 hours and monitored by LC-MS. The resulting mixture was diluted with TFA in water (40 ml 0.1%), centrifuged, filtered and purified by HPLC. Fractions containing the peptide product were analyzed by LC-MS, pooled and lyophilized to give the final perfluoroaryl peptide (20 mg, 8.9 μmol, 23% yield).

Example 3 Peptide Conjugation Protocol

(PPMO 1)

To a mixture of PMO (nucleic base sequence (R ² ) representing GCT ATT ACC TTA ACC CAG (SEQ ID NO: 9); 10.7 mg, 1.72 µmol) and decafluorobiphenyl peptide Ac-CRRRCRRRG-OH (5, 9 mg, 2.64 µmol) in a plastic Eppendorf tube add DMSO

- 46 039716 (0.4 ml), HATU in DMSO (6.6 μl, 2.64 μmol, 0.4 M) and DIPEA (diisopropylethylamine) (2.3 μl, 13.2 μmol). The contents of the tube are mixed and left to stand at room temperature for 2 hours. The resulting mixture is diluted with water (8 ml) and sequentially subjected to weak cation exchange (CM Sepharose) and solid phase extraction (Amberchrom CG 300M). The resulting product was analyzed by LC-MS and MALDI-TOF MS and lyophilized to obtain a PMO-peptide conjugate (9.8 mg, 1.26 μmol, 73% yield).

Example 4 One-pot Conjugation Protocol of a PMO with a Disulfide Bonded Cyclic Peptide Followed by Crosslinking of the Peptide but

Ac-Cys-R-R-R-R-R-R-Cys-G-OH

HATU, DIPEA, DMSO

2) DTT, hexafluorobenzene, TRIS

(RRMO 2)

To a solution of PMO (85 mg, 0.01 mmol, 1 eq.; nucleic base sequence (R ² ): GCT ATT ACC TTA ACC CAG (SEQ ID NO: 9)), peptide (Ac-Cys-Arg-Arg-Arg -Arg-Arg-Arg-Cys-Gly-OH or Ac-Cys-Arg-Arg-Arg-Cys-Arg-Arg-Arg-Gly-OH, 37 mg, each with a disulfide bridge, 2 eq) and HATU ( 8 mg, 2 eq.) in DMSO (2 ml) was added diisopropylethylamine (9 μl, 5 eq.). The reaction mixture was stirred at room temperature for 5 hours. After adding 50 mM dithiothreitol (DTT) to a solution of DMSO (1 ml, 5 eq.), 50 mM Tris in a solution of DMSO (1 ml, 5 eq.), then hexafluorobenzene (60 µl, 25 eq) the reaction solution was stirred for another 5 hours. The desired product was obtained by weak cation exchange chromatography (CM-Sepharose) followed by solid phase extraction (Amberchrome CG300M) for desalting and finally lyophilization. Mass spectrum obtained using MALDI/TOF (mass/charge): calc.: 9975; measured value: 9976 (M+H).

Example 5 Analysis in HeLa cell line.

On the graph in Fig. 1 (see also Table 3) the y-axis shows the average fluorescence intensity of green fluorescence emission from the e-GFP protein, read for each individual cell with an Accuri C6 flow cytometer and summed with the processing software. The height of the green fluorescent signal bars indicates the different intensity based on the level of exon skipping that occurs in the nucleus of the HeLa-654 cell line whether or not the cells are treated with test compounds. The signal intensity or bar size also corresponds to the efficiency of cell uptake (ie, delivery to the cell). The x-axis shows test compounds tested using a number of different processing modes. Various processing modes include continuous processing of HeLa-654 cells in time or variants of processing with displacement of the label, where the test compound is incubated with the cells, and then after a certain period of time (3 hours) is washed out of the cell culture and fresh medium containing no test compound is added.

Results.

Baseline activity is shown for unconjugated PMO and untreated cells. All peptide/PMO conjugates (PPMOs) of the present invention show cell uptake that is higher than 1) unconjugated PMO and 2) untreated cells (which exhibit a green fluorescent background signal). This difference in fluorescence intensity between the peptide/PMO conjugates of the present invention and background or unconjugated PMO is indicative of significant pharmacological activity of the cross-linked peptide/PMO conjugates. AT

- 47 039716 each case of the RMO is represented by the following structure:

where the nucleobase sequence (comprising each R ² variant independently selected from a nucleobase selected from A, G, C, or T) is: GCT ATT ACC TTA ACC CAG (SEQ ID NO: 9).

Table 3

Delivery of peptide-oligonucleotide conjugates into the cell (Fig. 1)

but

where the nucleobase sequence (comprising each R ² variant independently selected from a nucleobase selected from A, G, C, or T) is: GCT ATT ACC TTA ACC CAG (SEQ ID NO: 9).

RRMO 4 refers to the following structure:

- 48 039716 where the nucleobase sequence (comprising each R ² variant independently selected from a nucleobase selected from A, G, C or T) is

GCTATTACCTTAACCCAG (SEQ ID NO: 9).

Example 6 In vivo assay for protein and exon skipping.

14 groups of 5 mice each (total 70 mice; 65 mdx mice (X-linked muscular dystrophy mice), 5 wild type) were treated as described below and then analyzed for dystrophin protein and exon skipping.

Mice received 200 μl of the compound as a tail vein bolus injection and were then euthanized 8 days after injection. Quadriceps, diaphragm, heart, and brain tissues (individual structures) were harvested, lysed, analyzed for dystrophin protein using conventional Western blot, and analyzed for exon skipping using reverse transcription PCR and the Caliper protocol (see FIG. 2 -7).

Embodiments of the compounds of the present invention were tested for their in vivo ability to affect mRNA and protein remodeling. These test compounds were found to be active in mdx mice, causing a significant increase in exon 23 skipping and dystrophin protein expression as shown by reverse transcription PCR and Western blotting, respectively. This activity is particularly pronounced when measured against the backdrop of negative control experiments in which mdx mice were dosed with saline: a condition that results in near-zero exon 23 skipping and dystrophin expression (FIGS. 4D, 5C, 5D, 6D, 7C, and 7D ). When compared with two positive controls, in wild-type and mdx mice dosed with the known active compound Ac-R6-Gly-M23D(+7-18) (PPMO 5), the test compounds showed clear activity. In some cases, the activity exceeded that of the control; for example, PPMO 11 (Table 4) elicited greater exon skipping and dystrophin expression responses at lower doses than the positive control compound (FIGS. 7B and 7D). Thus, the test compounds meet the requirement for positive pharmacological responses in the context of a seven day in vivo screening study.

Embodiments of the compounds (EPMO) according to the present invention, used in such studies, are characterized by the structure in accordance with formula (V) about

(V);

where the nucleobase sequence (comprising each R ² variant independently selected from a nucleobase selected from A, G, C, or T) is 5'-GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO: 14) and R ¹⁴ is defined in Table 1. 4.

Connections from the table. 4 was prepared according to examples 1-4 above.

Table 4

Compound of formula (V) R ¹⁴ (* see table 2) RRMO 5 Ac-R ₆ -Gly- (SEQ ID NO:15) RRMO 6 4AD* RRMO 7 3AD* RRMO 8 4 AC* RRMO 9 3AC* RRMO 10 4AE* RRMO 11 3AE*

Western blotting protocol for dystrophin.

1. Tissue was removed from -80°C conditions and manually cut into small pieces, 400-800 µl of RIPA lysis buffer was added depending on tissue type.

a) TA: 400 µl, QC: 800 µl, heart: 400 µl;

b) lysis buffer: RIPA buffer (Pierce, cat. no. 89901) and protease inhibitor cocktail (Roche, cat. no. 04693124001).

2. Approximately ten 2.0 mm diameter zirconium beads (Biospec, cat. no. 11079124zx) were added to the tube and tissues were lysed with a MagNA Lyser (Roche) as follows:

a) 5000 rpm, 30-40 s, cooling for 2 min on ice. The cycles were repeated until

- 49 039716 tissues were not completely lysed.

As an alternative to zirconium balls, metal balls (3-5 balls) were used,

3000 rpm, 20 seconds per cycle (chilled on ice between cycles for 3-5 minutes or until cold to the touch).

3. After lysis, the samples were centrifuged at 12,000 rpm for 10 min and the supernatant was transferred to an unused tube, followed by BCA analysis to quantify protein levels.

4. Gel electrophoresis;

a) load a marker (Invitrogen, cat. no. P/NLC5699), load 100 µg of protein per lane and run samples on a 3-8% Tris-acetate gel (Midi Gel, Biorad, cat. no. 345-0130) at 50 V, 5 minutes; 150 V for 1 h, then 200 V for 1.5 h (the 71 kDa marker was at the bottom of the gel).

5. Transfer.

Wet transfer overnight (16-18 hours) at 4°C, DC 100 mA (~25 V), 0.45-µm PVDF membrane (Biorad, cat. no. 1620261).

6. The membrane was washed in TBST for 5 minutes and blocked in 10% milk (Biorad, cat. no. 170-6404) in TBST for 1-2 hours at room temperature (RT).

7. PVDF membrane was rinsed with TBST 2-3 times and then incubated with primary antibody in 5% BSA in PBS overnight at 4°C:

a) Dys2 (Novocastra, Leica Biosystem, cat. no. NCL-DYS2) 1:30;

b) Dys1 (Novocastra, Leica Biosystem, cat. no. NCL-DYS1) 1:1000;

c) Mandys8 (Sigma cat. no. D8168) 1:3000.

8. The membrane was washed with TBST (three times, 10 min each time), horseradish peroxidase conjugated goat anti-mouse IgG (BioRad, cat. no. 1706516, 1:10000) was added, incubated for 1 h at k.t. and then washed with TBST (three times, 10 min each time).

9. Clarity Western ECL substrate (Biorad) was added, incubated for 5 min at rt. and then visualized using the ChemiDoc Touch imaging system.

10. The membrane was cleaned using 0.2N. NaOH for 7 min, then equilibrated in TBST for at least 10 min. The blot was blocked with 10% milk in TBST for 1 hour at RT.

11. Anti-α-actinin-2 antibody (Abcam, rabbit cat. no. ab68168, 1:15000) was added and incubated for 1 h at rt.

12. Washed with TBST (three times, 10 min each) and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (BioRad, cat. no. 1706515, 1:10,000) for 1 h at k.t.

13. The membrane was thoroughly washed with TBST and then visualized as described above.

A method for isolating RNA for mouse tissues.

1. Whole tissue pieces (half quadriceps, half heart, whole BBM...) were homogenized using MagNA Lyser and metal beads at 3000 rpm, 20 per cycle until the tissues were sufficiently lysed (sample cooled on ice between cycles for 3-5 minutes or until cold to the touch). Transfer 50-100 µl of the whole lysate to a new 96-well plate and mix with the same volume of RA4 buffer (from the GE RNAspin kit), then the lysate-based mixture was loaded into the 96-well RNAspin plate for the rest of the purification steps. The unused lysate (in RA1 buffer) was stored at -80°C for one year according to the kit's protocol.

2. Samples were centrifuged for 2 min at 5200xg. 500 μl of RA3 was added to each well of the RNA binding plate and the plate was centrifuged for 2 min at 5200xg. The filtrate was discarded.

3. Wash the membrane by adding 500 μl of RA2 to each well of the RNA binding plate and centrifuge for 2 min at 5200xg.

4. Add 800 µl of RA3 to each well and centrifuge for 2 min at 520xg.

5. Add 500 µl of RA4 to each well and centrifuge for 10 min at 5200xg.

6. Samples were eluted into a PCR plate (with conical wells) by pipetting 50 µl of RNase-free water to the bottom of each well, ensuring complete wetting of the membrane. Samples were incubated for 2 min at rt. and centrifuged at 5200xg for 3 min. If liquid remained in the well, the samples were incubated for an additional 2 min at rt. and centrifuged again at 5200xg for 3 min.

7. The RNA concentration was then measured on a Nanodrop 2000 spectrophotometer.

PCR protocol with reverse transcription.

The primers used for reverse transcription PCR were as follows: outer forward for dystrophin: 5'-CAATGTTTCTGGATGCAGACTTTGTGG-3' (SEQ ID NO: 10);

- 50 039716 external reverse for dystrophin: 5'-GTTCAGCTTCACTCTTTATCTTCTGCC-3' (SEQ ID NO: 11);

internal forward for dystrophin: 5'-CACATCTTTGATGGTGTGAGG-3' (SEQ ID NO: 12) and internal reverse for dystrophin: 5'-CAACTTCAGCCATCCATTTCTG-3' (SEQ ID NO: 13).

Received 25 μl of reaction mixtures (table. 5) for PCR with reverse transcription and primary amplification. The program for PCR with reverse transcription was carried out according to table. 6.

Table 5

Reaction mix for reverse transcription PCR

2x reaction mixture 12.5 µl Internal Forward Primer for Dys (10 µM) 0.75 µl Internal reverse primer for Dys (10 µM) 0.75 µl Blend Superscript ΙΙΙ/Platinum Tad 1 µl Messenger RNA (RNA sample) 3 µl Water up to 25 µl total volume 7 µl

Table 6

Program for reverse transcription PCR and primary amplification

Temperature Time reverse transcription 55°C 30 minutes Reverse transcription inactivation 94°C 2 minutes Denaturing 94°C 45 seconds 45 cycles Annealing 59°С 45 seconds Extension 68°C 1 minute 4°C OO

Caliper protocol.

After completion of reverse transcription PCR, 25 μl of PCR buffer was added and a total volume of 50 μl of the reaction mixture was transferred to a Caliper plate. Bioassay using the Caliper LabChip was performed based on the protocol recommended by the manufacturer. The PCR product of the full length dystrophin transcript is 445 bp. and 232 b.p. from mRNA skipping exon 23.

Inclusion by reference.

The contents of all references (including references to literature, issued patents, published patent applications and pending patent applications) cited throughout this specification are expressly incorporated herein in their entirety. Unless otherwise indicated, all technical and scientific terms used in this document have the meaning generally known to a person skilled in the art.

Equivalents.

Those skilled in the art will understand or be able to determine many equivalents to the specific embodiments of the present invention described herein using no more than standard experimentation. Such equivalents are intended to be included within the scope of the following claims.

CLAIM

Claims (15)

CLAIM 1) A' represents - 52 039716 1. Peptide-oligonucleotide conjugate of formula (I) (I) or a pharmaceutically acceptable salt thereof, in which A' represents > where R ⁵ represents -C(O)(O-alkyl) _x -OH, where x is in the range of 3-10, and each alkyl group independently at each occurrence represents (J)t I G C _2-6 alkyl or R ⁵ is selected from trityl, monomethoxytrityl and - 51 039716 each R ¹ is independently selected from OH and -NR ³ R ⁴ where each of R ³ and R 4 independently at each occurrence is -C _1-6 alkyl; each R ² is independently selected from H, a nucleobase, and an acylated nucleobase, wherein the nucleobase independently at each occurrence contains a _C3-6 heterocyclic ring selected from pyridine, pyrimidine, triazinan, purine, and deazapurine; z is in the range 8-40; E' is selected from H, -C _1-6 alkyl, -C(O)C _1-6 alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, where R ¹¹ is selected from OH and -NR ³ R ⁴ ; L is covalently linked via an amide bond to the carboxy terminus in J and L is selected from -NH(CH2)i-6C(O)-, -NH(CH2)1-6C(O)NH(CH2)1-6C(O)- and t is 4-9; each J is independently in each occurrence selected from cysteine and arginine, where two or more side groups of amino acids J independently in each occurrence contain sulfur, where two sulfur atoms, together with the atoms to which they are attached, form the structure where d is 0 or 1 and M is selected from: where each R ¹⁰ independently in each case represents H or halogen; and G is covalently bonded to the amino terminus in J and G is selected from H, C(O)C _1-6 alkyl, benzoyl and stearoyl, and wherein at least one of the following is true: 2. Peptide-oligonucleotide conjugate according to claim 1 or its pharmaceutically acceptable salt, where E' is selected from H, -C(O)CH ₃ , trityl, 4-methoxytrityl, benzoyl, stearoyl and 2) E' represents 3. Peptide-oligonucleotide conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, n< (J)t where A'represents; and E' is ^G 3) E' represents L or (J)t G. 4. Peptide-oligonucleotide conjugate according to claim 1 or its pharmaceutically acceptable salt, where G (J) t A' represents a roila. ' and E' is selected from H, -C(O)CH ₃ , trityl, 4-methoxytrityl, benzoyl and stea- 5. The peptide oligonucleotide conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the peptide oligonucleotide conjugate of formula (I) is a peptide oligonucleotide conjugate selected from: - 53 039716 6. The peptide-oligonucleotide conjugate according to claim 1 or 5, or a pharmaceutically acceptable salt thereof, wherein the peptide-oligonucleotide conjugate is represented by formula (Ia) and R ⁵ is -C(O)(O-CH ₂ CH ₂ )3OH. 7. A peptide oligonucleotide conjugate according to claim 1 or 5, or a pharmaceutically acceptable salt thereof, wherein the peptide oligonucleotide conjugate is represented by formula (Ib) and E' is selected from H, C _1-6 alkyl, -C(O)CH3, benzoyl, and stearoyl. 8. Peptide-oligonucleotide conjugate according to any one of claims 1-7 or its pharmaceutically acceptable e f ^F \ ^FF \ ^F salt, where M represents ^{FF or FFFF} · 9. A peptide-oligonucleotide conjugate according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein the two J groups are cysteine. 10. A peptide-oligonucleotide conjugate according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein z is 8-25. 11. A peptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein each R ¹ is N(CH3)2. 12. A peptide-oligonucleotide conjugate according to any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein L is selected from glycine and 13. A peptide-oligonucleotide conjugate according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, wherein L is glycine. 14. A peptide-oligonucleotide conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein G is -C(O)CH ₃ . 15. A peptide oligonucleotide conjugate according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, wherein the peptide oligonucleotide conjugate is selected from: