Cell-penetrating peptides for antisense delivery

Abstract

To provide oligonucleotides, cell-penetrating peptides, and peptide-oligonucleotide conjugates.SOLUTION: Also provided herein are methods of treating a muscle disease, a viral infection or a bacterial infection in a subject in need thereof, the methods comprising administering to the subject the oligonucleotides, peptides and peptide-oligonucleotide conjugates described herein. Provided herein are peptide-oligonucleotide conjugates comprising an oligonucleotide covalently bound to a cell-penetrating peptide (CPP).SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 573,379, filed October 17, 2017, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Antisense technology provides a means for modulating the expression of one or more specific gene products, including alternative splice products, and is uniquely useful in several therapeutic, diagnostic, and research applications. The principle behind 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 one of several 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.

[0003] Although significant progress has been made in the field of antisense technology, there remains a need in the art for oligonucleotides and peptide - oligonucleotide conjugates having improved antisense or antigene performance.

Summary of the Invention

Means for Solving the Problems

[0004] Peptide - oligonucleotide conjugates are provided herein that include an oligonucleotide covalently linked to a cell - penetrating peptide (CPP). Also provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a peptide - oligonucleotide conjugate described herein.

[0005] Thus, in one aspect, the peptide-oligonucleotide conjugate of Formula I:

Chem.

Chem.

Chem.

[0006] In one embodiment, L is -C(O)(CH 2 ) 1~6 -triazole-(CH 2 ) 1~6 C(O).

[0007] In one embodiment, the peptide-oligonucleotide conjugate of Formula I is a peptide-oligonucleotide conjugate of Formula Ia:

Chem.

[0008] In another embodiment, the peptide-oligonucleotide conjugate of formula I is a peptide-oligonucleotide conjugate of formula Ib: [Chemical formula] or a pharmaceutically acceptable salt thereof [wherein, (J) t -G is a cell-permeable peptide as defined above] is as follows.

[0009] In yet another aspect, provided herein is a method of treating a muscle disorder, a viral infection, or a bacterial infection in a subject in need thereof, the method comprising administering to the subject a peptide-oligonucleotide conjugate of the present disclosure. [Brief Description of the Drawings]

[0010]

Fig. 1A - B

Fig. 1C - D

Fig. 1E

Fig. 2

Fig. 3

[0011] Efficient intracellular delivery of many pharmaceutically active compounds, such as proteins and nucleic acids, is an unsolved problem in the field of drug delivery. Many large macromolecules cannot cross the plasma membrane and often end up being trapped in endosomes and degraded within lysosomes. Over the past few decades, multiple approaches have been developed to facilitate the cytoplasmic delivery of large macromolecules, including overcharging the molecule with a high density of charges, complexing the molecule with delivery vehicles such as liposomes or nanoparticles, and conjugating the molecule to cell-penetrating peptides.

[0012] Since the discovery that a 20-amino acid fragment of the trans-activating transcription activator (TAT) from HIV-1 enabled proteins to cross the plasma membrane, hundreds of cell-penetrating peptides (CPPs) have been reported to improve cellular uptake. These peptides have been derived from many natural sources such as viral proteins, DNA-binding proteins, signal peptides, and antimicrobial peptides. In addition, CPPs have been rationally designed and identified from DNA-encoded peptide libraries. CPP sequences exhibit a wide variety of physicochemical properties, ranging from highly cationic to amphiphilic to hydrophobic. To confirm that a peptide is a cell-penetrating peptide, traditional experiments have included flow cytometry and live-cell confocal imaging using fluorophore-labeled CPPs (see, for example, FIGS. 1A, 1B, 1C, 1D, 1E, and 2C). These experiments provide evidence of cellular uptake, but they do not address the question of whether a CPP is suitable for the delivery of a particular macromolecular cargo. Nevertheless, CPPs have been utilized to improve the cellular delivery of peptides, enzymes, antibodies, oligonucleotides, nanoparticles, and chemotherapeutic agents.

[0013] One promising application of CPPs is for the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs). PMOs are charge-neutral antisense therapeutics in which the ribose sugars are replaced with methylene morpholine rings and the phosphodiester backbone is replaced with a phosphorodiamidate backbone. PMOs can bind to pre-mRNA and alter gene splicing through a process known as "exon skipping." Recently, the PMO eteplirsen became the first and only FDA-approved therapy for treating the underlying genetic cause of Duchenne muscular dystrophy (DMD) by skipping exon 51 of the dystrophin gene. Although PMO therapies such as eteplirsen show significant promise, the required dosages are often multiple grams per week due to limited intracellular delivery. Creating a conjugate between a CPP and a PMO has been an effective approach in improving delivery. O’Donovan et al. focused on a small library of 16 different CPP-PMO conjugates, and Moulton et al. identified an arginine-rich peptide that improved delivery of the PMO cargo for DMD. However, there has been no systematic investigation of the characteristics of CPPs that promote PMO delivery.

[0014] Peptide-oligonucleotide conjugates are provided herein that include an oligonucleotide covalently bound to a cell-permeable peptide. Also provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a peptide-oligonucleotide conjugate described herein. The oligonucleotides described herein and thus the peptide-oligonucleotide conjugates exhibit a stronger affinity for DNA and RNA without sacrificing sequence selectivity compared to native or unmodified oligonucleotides. In some embodiments, the oligonucleotides of the disclosure minimize or prevent cleavage by RNase H. In some embodiments, the antisense oligonucleotides of the disclosure do not activate RNase H.

[0015] The peptides described herein confer lower toxicity on their corresponding peptide-oligonucleotide conjugates, enhance the activity of the oligonucleotides, improve pharmacokinetics and tissue distribution, improve cell delivery, and confer a highly reliable and controllable in vivo distribution.

[0016] Definitions Definitions of the various terms used to describe the present disclosure are listed below. These definitions apply to the terms as used throughout this specification and the claims, either individually or as part of a larger group, unless otherwise limited in specific instances.

[0017] The term "about" is understood by those skilled in the art and varies somewhat depending on the context in which it is used. As used herein, when referring to measurable values such as amounts, time durations, etc., the term "about" is meant to encompass variations of ±20% or ±10% including ±5%, ±1%, and ±0.1% from the specified value, because such variations are appropriate for carrying out the disclosed methods.

[0018] The term "alkyl" in certain embodiments refers to a saturated, straight-chain or branched-chain hydrocarbon moiety containing from 1 to 6 or from 1 to 8 carbon atoms each. Examples of C 1~6 -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl moieties, and examples of C 1~8 -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

[0019] The number of carbon atoms in an alkyl substituent can be indicated by the prefix "C x~y ", where x is the minimum number of carbon atoms in the substituent and y is the maximum number. Similarly, Cx The alkyl chain contains x carbon atoms.

[0020] The term "heteroalkyl," whether used by itself or in combination with another term, means, unless otherwise specified, a stable straight-chain or branched-chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms are placed at any position of the heteroalkyl group including the position between the remainder of the heteroalkyl group and the moiety to which it is attached, and may be attached to the most distal carbon atom in the heteroalkyl group. Examples are -O-CH 2 -CH 2 -CH 3 -, -CH 2 -CH 2 -CH 2 -OH, -CH 2 -CH 2 -NH-CH 3 -, -CH 2 -S-CH 2 -CH 3 and -CH 2 -CH 2 -S(=O)-CH 3 are included. For example, -CH 2 -NH-OCH 3 or -CH 2 -CH 2 -S-S-CH 3 etc., up to two heteroatoms may be consecutive.

[0021] The term "aryl," when used alone or in combination with other terms, means, unless otherwise specified, a carbocyclic aromatic system containing one or more rings (typically 1, 2, or 3 rings), such rings may be attached together in a pendant fashion such as biphenyl, or may be fused such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. In various embodiments, examples of aryl groups include phenyl (e.g., C 6 -aryl) and biphenyl (e.g., C12 -aryl). In some embodiments, the aryl group has 6 to 16 carbon atoms. In some embodiments, the aryl group has 6 to 12 carbon atoms (e.g., C 6~12 -aryl). In some embodiments, the aryl group has 6 carbon atoms (e.g., C 6 -aryl).

[0022] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocyclic ring having aromatic characteristics. The heteroaryl substituent may be defined by the number of carbon atoms, e.g., C 1~9 -heteroaryl does not include the number of heteroatoms and indicates the number of carbon atoms contained in the heteroaryl group. For example, C 1~9 -heteroaryl will additionally contain 1 to 4 heteroatoms. The polycyclic heteroaryl may include one or more partially saturated rings. Non-limiting examples of heteroaryl include pyridyl, pyrazinyl, pyrimidinyl (including, e.g., 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, e.g., 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (including, e.g., 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.

[0023] Non-limiting examples of polycyclic heterocycles and heteroaryls include indolyl (e.g., including 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (e.g., including 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (e.g., including 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (e.g., including 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (e.g., including 3-, 4-, 5-, 6- and 7-benzothienyl), benzoxazolyl, benzothiazolyl (e.g., including 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (e.g., including 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolidinyl and quinolizinyl.

[0024] The term "protecting group" or "chemical protecting group" refers to a chemical moiety that blocks some or all of the reactive moieties of a compound and prevents such moieties from participating in chemical reactions until the protecting group is removed, for example, moieties listed and described in T.W. Greene, P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons (1999). When different protecting groups are used, it may be advantageous for each (different) protecting group to be removable by different means. Protecting groups that cleave under entirely different reaction conditions allow for the sequential 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 tert-butyldimethylsilyl can be used to protect carboxy and hydroxy reactive moieties in the presence of an amino group protected with an acid-labile Cbz group and a base-labile Fmoc group that are removable by hydrogenolysis. The carboxylic acid moiety may be blocked, without limitation, with a base-labile group such as methyl or ethyl, and the hydroxy reactive moiety may be blocked with a base-labile group such as acetyl or with a carbamate that is stable to both acid and base but hydrolytically removable, in the presence of an amine blocked with an acid-labile group such as tert-butyl carbamate.

[0025] The carboxylic acid and hydroxyl reactive moieties may be blocked with a hydrolytically removable protecting group such as a benzyl group, while the amine group may be blocked with a base-labile group such as Fmoc. An amine protecting group particularly useful in the synthesis of the compound of formula (I) is trifluoroacetamide. The carboxylic acid reactive moiety may be blocked with an oxidatively removable protecting group such as 2,4-dimethoxybenzyl, while a coexisting amino group may be blocked with a fluoride-labile silyl carbamate.

[0026] An allyl blocking group is useful in the presence of acid and base protecting groups, because the former is stable and can then be removed by a metal or palladium catalyst. For example, a carboxylic acid blocked with allyl can be deprotected by a palladium(0) catalyzed reaction in the presence of an acid-labile carbamic acid tert-butyl or a base-labile amine acetate protecting group. Yet another form of a protecting group is a resin to which a compound or intermediate can be attached. As long as the residue is attached to the resin, its functional group is blocked and cannot react. When released from the resin, the functional group becomes available for reaction.

[0027] The terms "nucleobase", "base pairing moiety", "nucleobase pairing moiety" or "base" refer to the heterocyclic ring moiety of a nucleoside, nucleotide and / or morpholino subunit. The nucleobase may be naturally occurring or may be modified or an analogue of these naturally occurring nucleobases, e.g., one or more nitrogen atoms of the nucleobase may independently at each occurrence be replaced by carbon. Exemplary analogues include hypoxanthine (the base component of the nucleoside inosine), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl modified pyrimidines, 10-(9-(aminoethoxy)phenoxazinyl) (G-clamp), etc.

[0028] Further examples of base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine, wherein their respective amino groups are protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacitidine, pyrimidine analogs such as pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purines, xanthine or hypoxanthine (the latter two being natural degradation products). Also contemplated are modified nucleobases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, the contents of which are incorporated herein by reference.

[0029] Further examples of base pairing moieties include, but are not limited to, enlarged-size nucleobases to which one or more benzene rings are attached. The contents of Glen Research catalog (www.glenresearch.com); Krueger AT et al., Acc. Chem. Res., 2007, 40, 141-150; Kool, ET, Acc. Chem. Res., 2002, 35, 936-943; Benner S.A., which are incorporated herein by reference. The nucleobase replacements described in 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 are contemplated as useful for the synthesis of the oligomers described herein. Examples of expanded-size nucleobases are shown below. [Chemical formula]

[0030] The term "oligonucleotide" or "oligomer" refers to a compound comprising a plurality of linked nucleosides, nucleotides, or a combination of both nucleosides and nucleotides. In the specific embodiments provided herein, the oligonucleotide is a morpholino oligonucleotide.

[0031] The phrase "morpholino oligonucleotide" or "PMO" refers to a modified oligonucleotide having morpholino subunits linked together by phosphoramidate or phosphorodiamidate linkages that join the morpholino nitrogen of one subunit to the 5'-exocyclic carbon of an adjacent subunit. Each morpholino subunit contains a nucleobase pairing moiety effective to bind to a nucleobase in a target by nucleobase-specific hydrogen bonding.

[0032] The terms "antisense oligomer", "antisense compound" and "antisense oligonucleotide" are used interchangeably and each has a base pairing moiety that hybridizes by Watson-Crick base pairing to a target sequence in a nucleic acid (typically RNA) to form a nucleic acid:oligomer heteroduplex within the target sequence, and refers to a sequence of subunits linked by inter-subunit linkages. The oligomer may have exact (perfect) or near (sufficient) sequence complementarity to the target sequence, and variations in the sequence near the ends of the oligomer are generally more preferred than those in the interior.

[0033] Such antisense oligomers can be designed to block or inhibit mRNA translation or to inhibit / alter normal or aberrant pre-mRNA splicing processing and can be said to be "directed to" or "targeted against" the target sequence to which it hybridizes. The target sequence is typically a region that includes the AUG start codon of the mRNA, a translation inhibitory oligomer, or a splice site of pre-processed mRNA, a splice inhibitory oligomer (SSO). The target sequence of the splice site may include an mRNA sequence having 1 to about 25 base pairs at its 5' end downstream of the normal splice acceptor junction in the pre-processed mRNA. In various embodiments, the target sequence may be any region of the pre-processed mRNA that includes a splice site or is completely contained within an exon coding sequence or extends to a splice acceptor or donor site. The oligomer is more generally said to be "targeted against" a biologically relevant target such as a protein, virus or bacterium when targeted to the target nucleic acid in the manner described above.

[0034] An antisense oligonucleotide and a target RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides capable of hydrogen bonding with each other, whereby a stable and specific bond appears between the oligonucleotide and the target. Thus, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or exact pairing such that a stable and specific bond appears between the oligonucleotide and the target. In the art, it is understood that the sequence of an oligonucleotide need not be 100% complementary to that of its target sequence to which it is specifically hybridizable. An oligonucleotide is specifically hybridizable when the binding of the oligonucleotide to its target molecule interferes with the normal function of the target RNA, and has a sufficient degree of complementarity to avoid non-specific binding to non-target sequences of the antisense oligonucleotide under desired conditions, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, and the conditions under which the assay is performed in the case of in vitro assays.

[0035] Oligonucleotides can also include nucleobase (often simply referred to as "base" in the art) modifications or substitutions. Oligonucleotides containing modified or substituted bases include oligonucleotides in which one or more of the purine or pyrimidine bases most commonly found in nucleic acids are replaced by less common or non-natural bases. In some embodiments, the nucleobase is covalently linked to the morpholine ring of a nucleotide or nucleoside at the N9 atom of a purine base or at the N1 atom of a pyrimidine base.

[0036] Purine bases have the general formula:

Chemical formula

[0037] Adenine and guanine are the two most common purine nucleobases found in nucleic acids. They may be substituted with other naturally occurring purines including, but not limited to, N6-methyladenine, N2-methylguanine, hypoxanthine and 7-methylguanine.

[0038] Pyrimidine bases have the general formula:

Chemical formula

[0039] Cytosine, uracil and thymine are the most common pyrimidine bases 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.

[0040] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 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-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, super G, super A and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil or derivatives thereof; and degenerate or universal bases such as 2,6-difluorotoluene or abasic sites such as abasic bases (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives (azaribose) in which the ring oxygen is replaced by nitrogen). Pseudouracil is a naturally occurring isomeric version of uracil that has a C-glycoside rather than the normal N-glycoside as in uridine.

[0041] Certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotides of the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. In various embodiments, the nucleobase may include a 5-methylcytosine substitution, which has been shown to increase the stability of nucleic acid duplexes by 0.6 to 1.2 °C.

[0042] In some embodiments, modified or substituted nucleobases are useful for facilitating the purification of antisense oligonucleotides. For example, in certain embodiments, an antisense oligonucleotide may contain 3 or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases. In certain antisense oligonucleotides, a series of 3 or more consecutive guanine bases can result in aggregation of the oligonucleotide, which complicates purification. In such antisense oligonucleotides, one or more of the consecutive guanines can be substituted with hypoxanthine. Substitution of one or more guanines with hypoxanthine in a series of 3 or more consecutive guanine bases can reduce aggregation of the antisense oligonucleotide, thereby facilitating purification.

[0043] The oligonucleotides provided herein are synthesized and do not contain antisense compositions of biological origin. The molecules of the disclosure may be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures or mixtures of compounds, such as liposomes, receptor targeting molecules, oral, rectal, topical or other formulations, to assist uptake, distribution or absorption or combinations thereof.

[0044] The terms "complementary" and "complementarity" refer to oligonucleotides (i.e., sequences of nucleotides) that are related by the base pairing rules. For example, the sequence "T-G-A (5'-3')" is complementary to the sequence "T-C-A (5'-3')". Complementarity may be "partial", where only some of the bases of the nucleic acid conform to the base pairing rules. Alternatively, there may be "perfect", "complete" or "full" (100%) complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. Perfect complementarity is often desirable, but some embodiments may include one or more, preferably 6, 5, 4, 3, 2 or 1 mismatch with respect to the target RNA. Such hybridization can occur with "near" or "substantial" complementarity and strict complementarity to the target sequence of the antisense oligomer. In some embodiments, the oligomer may hybridize to the target sequence with about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementarity. Variations at any location within the oligomer are included. In certain embodiments, variations in the sequence near the ends of the oligomer are generally more preferred than variations in the interior, and when present, are typically within about 6, 5, 4, 3, 2 or 1 nucleotide of the 5' end, 3' end or both ends.

[0045] The term "peptide" refers to a compound comprising a plurality of linked amino acids. The peptides provided herein may be considered cell permeable peptides.

[0046] 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 penetration within 100% of the cells of a given cell culture population and enable macromolecule translocation in multiple tissues in vivo upon systemic administration. In various embodiments, the CPP embodiments of the present disclosure may further include arginine-rich peptides, as described further below.

[0047] The term "treatment" refers to the application of one or more specific procedures used for the amelioration of a disease. In certain embodiments, the specific procedure is the administration of one or more pharmaceutically active agents. "Treatment" of an individual (e.g., a mammal such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be carried out either prophylactically or following the onset of a pathological event or following contact with a pathogen. Treatment includes any desirable effect on the symptoms or pathology of a disease or condition and may include, for example, minimal change or improvement in one or more measurable markers of the disease or condition being treated. Also included are "preventive" treatments that may be directed at reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.

[0048] "Effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic compound, such as an antisense oligomer, that is effective to produce a desired therapeutic effect when administered to a mammalian subject, either as a single dose or as part of a series of doses.

[0049] The term "improvement" means a reduction in the severity of at least one indicator of a condition or disease. In certain embodiments, improvement includes a delay or deceleration in the progression of one or more indicators of a condition or disease. The severity of an indicator can be determined by subjective or objective measures known to those of skill in the art.

[0050] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed oligonucleotides in which an existing acid or base moiety has been modified by converting it to its salt form. Lists of suitable salts can be 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.

[0051] Peptide-oligonucleotide conjugate Oligonucleotides chemically linked to a cell-penetrating peptide are provided herein. The cell-penetrating peptide enhances the activity, cellular distribution or cellular uptake of the oligonucleotide. In particular, the cell-penetrating peptide is a linear or acyclic peptide. In some embodiments, the CPP can be an arginine-rich peptide. The oligonucleotide can additionally be chemically linked to one or more heteroalkyl moieties (e.g., polyethylene glycol) that further enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. In one exemplary embodiment, a polypeptide, such as an arginine-rich polypeptide, is covalently coupled at its N-terminal or C-terminal residue to either or both termini of an oligonucleotide.

[0052] Thus, in one aspect, a peptide-oligonucleotide conjugate of Formula I:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0053] In one embodiment, E’ is H, -C 1~6 -alkyl, -C(O)C 1~6 -alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and

Chemical formula

[0054] In yet another embodiment, A’ is -N(C 1~6 -alkyl)CH 2 C(O)NH 2 、

Chemical formula

[0055] In another embodiment, E’ is H, -C(O)CH 3, benzoyl, stearoyl, trityl, 4 - methoxytrityl, and

Chem.

[0056] In yet another embodiment, A’ is -N(C 1~6 -alkyl)CH 2 C(O)NH 2 ,

Chem.

Chem.

[0057] In yet another embodiment, A’ is

Chem.

[0058] In another embodiment, E’ is selected from H, -C(O)CH 3 , trityl, 4 - methoxytrityl, benzoyl and stearoyl.

[0059] In yet another embodiment, E’ is selected from H and -C(O)CH 3 .

[0060] In yet another embodiment, the peptide - oligonucleotide conjugate of formula I is a peptide - oligonucleotide conjugate of formula Ia:

Chem.

[0061] In another embodiment, the peptide - oligonucleotide conjugate of formula I is a peptide - oligonucleotide conjugate of formula Ib:

Chem.

[0062] In certain embodiments of Formula I and Ib, E’ is selected from H, C 1~6 alkyl, -C(O)CH 3 , benzoyl and stearoyl.

[0063] In another embodiment of Formula I and Ib, E’ is selected from H and -C(O)CH 3 .

[0064] In certain embodiments of Formula I, Ia and Ib, each J is independently selected from glycine, alanine, leucine, methionine, phenylalanine, tryptophan, lysine, glutamine, glutamic acid, serine, proline, valine, arginine and threonine.

[0065] In another embodiment of Formula I, Ia and Ib, each J is independently selected from arginine, lysine and glutamine.

[0066] In another embodiment of Formula I, Ia and Ib, each J is independently selected from glycine, leucine, alanine, phenylalanine, serine, threonine, methionine, tryptophan, glutamine, proline, lysine, arginine and valine.

[0067] In another embodiment of Formula I, Ia and Ib, each J is independently selected from arginine, isoleucine, proline, leucine, phenylalanine and glycine.

[0068] In yet another embodiment of Formula I, Ia and Ib, each J is independently selected from glycine, arginine, proline, glutamic acid, serine, lysine and leucine.

[0069] In yet another embodiment of Formulas I, Ia and Ib, each J is independently selected from alanine, leucine, tryptophan, lysine, threonine, valine, proline and arginine.

[0070] In yet another embodiment of Formulas I, Ia and Ib, each R 1 is N(CH 3 ) 2 and is.

[0071] In yet another embodiment of Formulas I, Ia and Ib, each R 2 is a nucleobase, and the nucleobase independently at each occurrence is a C 4~6 -heterocyclic ring selected from pyridine, pyrimidine, triazinane, purine and deaza-purine.

[0072] In another embodiment of Formulas I, Ia and Ib, each R 2 is a nucleobase, and the nucleobase independently at each occurrence is a C 4~6 -heterocyclic ring selected from pyrimidine, purine and deaza-purine.

[0073] In yet another embodiment of Formulas I, Ia and Ib, each R 2 is, independently at each occurrence, a nucleobase selected from adenine, 2,6-diaminopurine, 7-deaza-adenine, guanine, 7-deaza-guanine, hypoxanthine, cytosine, 5-methyl-cytosine, thymine, uracil and hypoxanthine.

[0074] In yet another embodiment of Formulas I, Ia and Ib, each R 2 is, independently at each occurrence, a nucleobase selected from adenine, guanine, cytosine, 5-methyl-cytosine, thymine, uracil and hypoxanthine.

[0075] In another embodiment of Formulas I, Ia and Ib, L is -C(O)(CH 2 ) 1~6 -triazole-(CH 2 ) 1~6 C(O)-.

[0076] In another embodiment of Formulas I, Ia and Ib, L is [Chemical Formula] .

[0077] In another embodiment of Formulas I, Ia and Ib, G is selected from H, C(O)CH 3 , benzoyl and stearoyl.

[0078] In yet another embodiment of Formulas I, Ia and Ib, G is H or -C(O)CH 3 .

[0079] In still another embodiment of Formulas I, Ia and Ib, G is H,

[0080] In still another embodiment of Formulas I, Ia and Ib, G is -C(O)CH 3 .

[0081] In another embodiment of Formulas I, Ia and Ib, J is RRRRRRRRRRRR, GLAFLGFLGAAGSTMGAWSQPKKKRKV, RRIRPRPPRLPRPRPRPLPFPRPG, RKKRRQRRR, RRRRRRRRRR, GRPRESGKKRKRKRLKP, ALWKTLLKKVLKAPKKKRKV, RRIPNRRPRR, TRRQRTRRARRNR, HARIKPTFRRLKWKYKGKFW, GIGAVLKVLTTGLPALISWIKRKRQQ, LRRERQSRLRRERQSR, RRRRRRRRR, RQIKIWFQNRRMKWKK, KRARNTEAARRSRARKLQRMKQ, RHIKIWFQNRRMKWKK, RRRRRRRR, KMTRAQRRAAARRNRWTAR, RGGRLSYSRRRFSTSTGR, KQINNWFINQRKRHWK, KLWMRWYSPTTRRYG, RRWWRRWRR, SQIKIWFQNKRAKIKK, GAYDLRRRERQSRLRRRERQSR, TRRNKRNRIQEQLNRK, GKRKKKGKLGKKRDP, RQVTIWFQNRRVKEKK, RLRWR, PPRPPRPPRPPRPPR, CAYHRLRRC, SRRARRSPRHLGSG, PPRPPRPPRPPR, NAKTRRHERRRKLAIER, VKRGLKLRHVRPRVTRMDV, LYKKGPAKKGRPPLRGWFH, TAKTRYKARRAELIAERR, KGTYKKKLMRIPLKGT, PPRPPRPPR, RASKRDGSWVKKLHRILE, TRSSRAGLQWPVGRVHRLLRK, FKIYDKKVRTRVVKH, VRLPPPVRLPPPVRLPPP, GPFHFYQFLFPPV, PLILLRLLRGQF, YTAIAWVKAFIRKLRK, KETWWETWWTEWSQPKKRKV, LIRLWSHLIHIWFQNRRLKWKKK, VDKGSYLPRPTPPRPIYNRN, MDAQTRRRERRAEKQAQWKAAN, GSPWGLQHHPPRT, KLALKALKALKAALKLA, IPALK, VPALR, LLIILRRRIRKQAHAHSK, IAWVKAFIRKLRKGPLG, AAVLLPVLLAAPVQRKRQKLP, TSPLNIHNGQKL, VPTLK, or VSALK.

[0082] In yet another embodiment of Formulas I, Ia and Ib, J is RRRRRRRRRRRR, GLAFLGFLGAAGSTMGAWSQPKKKRKV, RRIRPRPPRLPRPRPRPLPFPRPG, RKKRRQRRR, RRRRRRRRRR, GRPRESGKKRKRKRLKP, ALWKTLLKKVLKAPKKKRKV, RRIPNRRPRR, TRRQRTRRARRNR, HARIKPTFRRLKWKYKGKFW, GIGAVLKVLTTGLPALISWIKRKRQQ, LRRERQSRLRRERQSR, RRRRRRRRR, RQIKIWFQNRRMKWKK, KRARNTEAARRSRARKLQRMKQ, RHIKIWFQNRRMKWKK, RRRRRRRR, KMTRAQRRAAARRNRWTAR, RGGRLSYSRRRFSTSTGR, KQINNWFINQRKRHWK, KLWMRWYSPTTRRYG, RRWWRRWRR, SQIKIWFQNKRAKIKK, GAYDLRRRERQSRLRRRERQSR, TRRNKRNRIQEQLNRK, GKRKKKGKLGKKRDP, RQVTIWFQNRRVKEKK, RLRWR, PPRPPRPPRPPRPPR, CAYHRLRRC, SRRARRSPRHLGSG, PPRPPRPPRPPR, NAKTRRHERRRKLAIER, VKRGLKLRHVRPRVTRMDV, LYKKGPAKKGRPPLRGWFH, TAKTRYKARRAELIAERR, or KGTYKKKLMRIPLKGT.

[0083] In yet another embodiment of Formulas I, Ia and Ib, J is RRRRRRRRRRRR, GLAFLGFLGAAGSTMGAWSQPKKKRKV, RRIRPRPPRLPRPRPRPLPFPRPG, RKKRRQRRR, RRRRRRRRRR, GRPRESGKKRKRKRLKP, or ALWKTLLKKVLKAPKKKRKV.

[0084] In yet another embodiment of Formula I, Ia and Ib, the oligonucleotide-peptide conjugate demonstrates at least a 2-fold improvement in uptake compared to the unconjugated oligonucleotide.

[0085] In one embodiment, the oligonucleotide-peptide conjugate demonstrates at least a 5-fold improvement in uptake compared to the unconjugated oligonucleotide.

[0086] In another embodiment, the oligonucleotide-peptide conjugate demonstrates an improvement in uptake compared to the corresponding Cy5.5-peptide conjugate.

[0087] In yet another embodiment, the oligonucleotide comprises a targeting sequence having sequence complementarity to an RNA target. In a specific embodiment, the RNA target is a cellular RNA target. In another specific embodiment, the targeting sequence has sufficient sequence complementarity to bind to the RNA target. In yet another specific embodiment, the targeting sequence has perfect sequence complementarity to the RNA target.

[0088] Representative peptide-oligonucleotide conjugates of the present disclosure include, among others, peptide-oligonucleotide conjugates of the following structure:

Chemical formula

[0089] In one embodiment of the peptide-oligonucleotide conjugate of the present disclosure, G is H.

[0090] In another embodiment of the peptide-oligonucleotide conjugate of the present disclosure, G is -C(O)CH 3 is.

[0091] In certain embodiments, L is covalently linked to the carboxy terminus of the peptide and G is covalently linked to the amino terminus of the peptide.

[0092] As used herein, "G is covalently linked to the carboxy terminus of J by an amide bond" indicates that the carboxy terminus of J(-COOH) is covalently bonded to variable G via an N(H) group, and the hydroxyl group at the carboxy terminus of J is replaced by N(H). For example, when G is H, the following structure is formed by J and G. [Chemical formula]

[0093] In some embodiments, the peptide-oligonucleotide conjugates described herein are not solvated. In other embodiments, one or more of the peptide-oligonucleotide conjugates are in a solvated form. As is known in the art, the solvate can be any of pharmaceutically acceptable solvents such as water, ethanol, etc.

[0094] The peptide-oligonucleotide conjugates of Formulas I, Ia, Ib and II are shown in their neutral forms, but in some embodiments, these peptide-oligonucleotide conjugates are used in pharmaceutically acceptable salt forms.

[0095] oligonucleotide Important properties of morpholino-based subunits include: 1) the ability to be linked in oligomeric form by stable uncharged or positively charged backbone linkages; 2) the ability to support nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil, 5-methyl-cytosine, and hypoxanthine), such that the resulting polymer can hybridize with a complementary base target nucleic acid containing the target RNA with a T greater than about 45° C. with relatively short oligonucleotides (e.g., 10-15 bases); 3) the ability of the oligonucleotide to be actively or passively transported into mammalian cells; and 4) the ability of the oligonucleotide and oligonucleotide:RNA heteroduplex to be resistant to RNase and RNase H degradation, respectively. M The stability of the duplex formed between the oligomer and the target sequence is a function of the binding T and the susceptibility of the duplex to enzymatic cleavage in cells. The T of the oligomer with respect to a complementary sequence RNA can be measured by conventional methods such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or as described in Miyada C. G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. In certain embodiments, the antisense oligomer can have a binding T higher than body temperature and, in one embodiment, higher than about 45° C. or 50° C. Ts in the range of 60-80° C. or higher are also included. According to well-known principles, with respect to a complementary base (based) RNA hybrid, the T of the oligomer

[0096] The stability of the double-strand formed between the oligomer and the target sequence is a function of the binding T M and the susceptibility of the double-strand to enzymatic cleavage in cells. The T of the oligomer with respect to the complementary sequence RNA M can be measured by conventional methods such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108, or as described in Miyada C. G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. In certain embodiments, the antisense oligomer has a binding T higher than body temperature and, in one embodiment, higher than about 45° C. or 50° C. M can have. Ts in the range of 60-80° C. or higher M are also included. According to well-known principles, with respect to a complementary base (based) RNA hybrid, the T of the oligomer Mcan be increased by increasing the ratio of C:G paired bases in the double strand, or by increasing the length of the heteroduplex (in base pairs), or both. At the same time, it may be advantageous to limit the size of the oligomer for the purpose of optimizing cellular uptake. For this reason, the compounds of the present disclosure have a high T at a length of 25 bases or shorter. M include compounds that exhibit (45 - 50 °C or higher).

[0097] The length of the oligonucleotide may vary as long as it can selectively bind to the intended location within the pre-mRNA molecule. The length of such sequences can be determined according to the selection procedures described herein. Generally, the oligonucleotide is from about 8 nucleotides in length to a maximum length of about 50 nucleotides. For example, the length (z) of the oligonucleotide can be 8 - 38, 8 - 25, 15 - 25, 17 - 21, or about 18. However, it will be appreciated that any length of nucleotides within this range can be used in the methods described herein.

[0098] In some embodiments, the antisense oligonucleotide contains a base modification or substitution. 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 0-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine and 2,6-diaminopurine. 5-Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6 - 1.2 °C and may be incorporated into the antisense oligonucleotides described herein. In one embodiment, at least one pyrimidine base of the oligonucleotide comprises a 5-substituted pyrimidine base, and 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 comprises an N-2, N-6 substituted purine base. In one embodiment, the N-2, N-6 substituted purine base is 2,6-diaminopurine.

[0099] Morpholino-based oligomers (including antisense oligomers) are described in, for example, U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, 5,506,337 and co-pending U.S. patent applications 12 / 271,036, 12 / 271,040, and PCT publications WO / 2009 / 064471 and WO / 2012 / 043730 and Summerton et al. 1997, Antisense and Nucleic Acid Drug Development, 7, 187-195, which are hereby incorporated by reference in their entirety.

[0100] Accordingly, in one aspect, an oligonucleotide of Formula II: [Chemical formula] or a pharmaceutically acceptable salt thereof [wherein, A is OH, -NHCH 2 C(O)NH 2 , -N(C 1~6 -alkyl)CH 2 C(O)NH 2 , [Chemical formula] selected from the group consisting of R 5 is -C(O)(O-alkyl) x OH, x is 3 to 10, and each alkyl group is, independently at each occurrence, -C 2~6 -alkyl, or R 5 is -C(O)C 1~6 -alkyl, trityl, monomethoxytrityl, -C 1~6 -alkyl-R 6 , -C 1~6 -heteroalkyl-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 selected from the group consisting of R 6 is selected from the group consisting of OH, SH and NH 2 , or R 6 is O, S or NH covalently linked to a solid support each R 1 is independently OH or -NR 3 R 4 , each R 3 and R 4 are, independently at each occurrence, -C 1~6 -alkyl each R 2is independently selected from the group consisting of H, a nucleobase, and a nucleobase functionalized with a chemical protecting group, and the nucleobase is independently, in each occurrence, selected from the group consisting of pyridine, pyrimidine, triazinane, purine, and deazapurine and contains a C 3~6 -heterocyclic ring, z is from 8 to 40, E is H, -C 1~6 -alkyl, -C(O)C 1~6 -alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and

Chemical formula

[0101] In one embodiment of Formula II, A is

Chemical formula

[0102] 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, and the nucleobase contains, independently at each occurrence, a pyrimidine or a purine.

[0103] In yet another embodiment, the oligonucleotide of Formula II is an oligonucleotide of Formula IIa:

Chemical Formula

[0104] In certain embodiments of Formulas II and IIa, R 2 is, independently at each occurrence, adenine, 2,6-diaminopurine, guanine, hypoxanthine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine, each R 1 is -N(CH 3 ) 2 as follows.

[0105] Various embodiments of the nucleotide moieties as described herein are provided in Table 1.

Table 1

[0106] In some embodiments, the oligonucleotides described herein are not solvated. In other embodiments, one or more of the oligonucleotides are in a solvated form. As is known in the art, the solvate can be any of pharmaceutically acceptable solvents such as water, ethanol, etc.

[0107] The oligonucleotides of Formulas II and IIa are shown in their neutral forms, but in some embodiments, these oligonucleotides are used in pharmaceutically acceptable salt forms.

[0108] Peptide The oligonucleotides provided herein include an oligonucleotide portion conjugated to a CPP. In particular, the cell-permeable peptide is a linear or acyclic peptide. In some embodiments, the CPP can have a theoretical net positive charge. In some embodiments, the positive net charge can be an integer from 1 to 12. Representatives of such CPPs are shown below:

Chemical formula

[0109] In some embodiments, the CPP can be an arginine-rich peptide transport moiety effective to enhance the transport of the compound into cells. The transport moiety is attached to the end of the oligomer in some embodiments. The peptide has the ability to induce cell permeation within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells of a given cell culture population, including all integers in between, and enables macromolecular translocation in multiple tissues in vivo upon systemic administration. In one embodiment, the cell-permeable peptide can be an arginine-rich peptide transporter.

[0110] The transport moiety as described above has been shown to significantly enhance the cellular uptake of the attached oligomers compared to the uptake of oligomers in the absence of the attached transport moiety. The uptake can be enhanced at least 2-fold, and in some embodiments up to 10-fold, compared to the unconjugated compound. In some embodiments, the uptake can be enhanced at least 2-fold, and in some embodiments up to 10-fold, compared to the CPP-Cy5.5 conjugate.

[0111] The use of arginine-rich peptide transporters (i.e., cell-penetrating peptides) is particularly useful in practicing the present disclosure. Certain peptide transporters have been shown to be highly effective in delivering antisense compounds to primary cells including muscle cells. Further, the peptide transporters described herein demonstrate enhanced ability to alter the splicing of several gene transcripts when conjugated to antisense PMOs.

[0112] Thus, in one aspect, a peptide of Formula III:

Chemical formula

[0113] In certain embodiments, each J is independently selected from glycine, alanine, leucine, methionine, phenylalanine, tryptophan, lysine, glutamine, glutamic acid, serine, proline, valine, arginine, and threonine.

[0114] In another embodiment, each J is independently selected from arginine, lysine, and glutamine.

[0115] In another embodiment, each J is independently selected from glycine, leucine, alanine, phenylalanine, serine, threonine, methionine, tryptophan, glutamine, proline, lysine, arginine, and valine.

[0116] In another embodiment, each J is independently selected from arginine, isoleucine, proline, leucine, phenylalanine, and glycine.

[0117] In yet another embodiment, each J is independently selected from glycine, arginine, proline, glutamic acid, serine, lysine, and leucine.

[0118] In yet another embodiment, each J is independently selected from alanine, leucine, tryptophan, lysine, threonine, valine, proline, and arginine.

[0119] In another embodiment, L is -C(O)(CH 2 ) 1~6 -C 1~6 -heteroaromatic-(CH 2 ) 1~6 C(O).

[0120] In another embodiment, L is

Chemical formula

[0121] In yet another embodiment, G is selected from the group consisting of H, C(O)CH 3 , benzoyl, and stearoyl.

[0122] In another embodiment, G is C(O)CH 3 or H.

[0123] In another embodiment, G is C(O)CH 3 is as follows.

[0124] In another embodiment, G is H.

[0125] In yet another embodiment, G is covalently linked to the carboxy terminus of J by an amide bond. In a further embodiment, L is covalently linked to the amino terminus of J by an amide bond.

[0126] In some embodiments, the peptides described herein are not solvated. In other embodiments, one or more of the peptides are in a solvated form. As is known in the art, the solvate can be any pharmaceutically acceptable solvent such as water, ethanol, etc.

[0127] The peptides of Formula III are shown in their neutral forms, but in some embodiments, these oligonucleotides are used in pharmaceutically acceptable salt forms.

[0128] Method A method for treating a muscle disease, viral infection, or bacterial infection in a subject in need thereof, the method comprising administering to the subject a peptide-oligonucleotide conjugate of Formula I, Ia, or Ib, is provided herein.

[0129] Accordingly, in one aspect, provided herein is a method of treating a muscle disease, viral infection, or bacterial infection in a subject in need thereof, the method comprising administering to the subject a peptide-oligonucleotide conjugate of the present disclosure.

[0130] In one embodiment, the muscle disease is Duchenne muscular dystrophy.

[0131] In another embodiment, the viral infection is caused by a virus selected from the group consisting of Marburg virus, Ebola virus, influenza virus, and dengue virus.

[0132] In another embodiment, the bacterial infection is caused by Mycobacterium tuberculosis.

[0133] Subjects contemplated herein are typically humans. However, a subject can be any mammal for which treatment is desired. Thus, the methods described herein can be applied to both human and veterinary applications.

[0134] Administration / Dosage The formulation of the therapeutic compositions and their subsequent administration (dosing) are within the skill of those in the art. Dosing depends on the severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months or until a sufficient reduction of the disease condition is achieved. The optimal dosing schedule can be calculated from measurements of drug accumulation in the patient's body.

[0135] One of ordinary skill in the art can readily determine the optimal dosage amount, dosing methodology, and repetition rate. The optimal dosage amount may vary depending on the relative potency of the individual oligomers and generally is in the EC that has been found to be effective in vitro and in vivo animal models 50It can be estimated based on this. Generally, the dosage can be 0.01 μg to 100 g per 1 kg of body weight, and it may be administered once or multiple times a day, once a week, once a month, or once a year, or even once every 2 to 20 years. A person skilled in the art can easily estimate the dosing repetition rate based on the measured residence time and drug concentration in body fluids or tissues. After treatment success, it may be desirable to subject the patient to maintenance therapy to prevent recurrence of the disease condition, and the oligomer is administered at a maintenance dose within the range of 0.01 μg to 100 g per 1 kg of body weight, once or multiple times a day to once every 20 years.

[0136] In some embodiments, the oligonucleotide (the oligonucleotide of formula II or IIa) is administered alone.

[0137] In some embodiments, the oligonucleotide is administered at a therapeutically effective amount or dosage. A "therapeutically effective amount" is the amount of the oligonucleotide of formula II or IIa that, when administered alone to a patient, effectively treats a muscle disease, viral infection, or bacterial infection. The amount determined to be a "therapeutically effective amount" for a particular subject in a given case may not be effective for 100% of subjects with the disease or condition being treated, even though such dosage is considered by a person skilled in the art to be a "therapeutically effective amount". The amount of the oligonucleotide corresponding to a therapeutically effective amount strongly depends on the type of disease, the stage of the disease, the age of the patient being treated, and other factors.

[0138] In different embodiments, depending on the oligonucleotide of formula II or IIa and the effective amount used, the oligonucleotide can modulate the expression of genes involved in muscle diseases, viral infections, or bacterial infections.

[0139] The amount of the oligonucleotide of Formula II or IIa should result in an effective treatment of a muscle disorder, viral infection or bacterial infection, but the amount should preferably not be overly toxic to the patient (i.e., the amount should preferably be within the toxicity limits as established by medical guidelines). In some embodiments, limitations are provided with respect to the total dosage administered, either to prevent excessive toxicity or to provide either or both of a more effective treatment of a muscle disorder, viral infection or bacterial infection. Typically, the amounts contemplated herein are per day, but half-day and two- or three-day cycles are also contemplated herein.

[0140] Different dosage regimens may be used to treat a muscle disorder, viral infection or bacterial infection. 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 3, 4, 5, 6, 7, 8, 9, or 10 days. Depending on the stage and severity of the disease being treated, a shorter treatment time (e.g., up to 5 days) may be used with a higher dosage, or a longer treatment time (e.g., 10 or more days, or weeks, or months, or longer) may be used with a lower dosage. In some embodiments, a dosage of once or twice a day is administered every other day.

[0141] Oligonucleotides of formula II and IIa or their pharmaceutically acceptable salts or solvate forms can be administered in pure form or in a suitable pharmaceutical composition via any of the acceptable modes of administration or agents known in the art. The oligonucleotide can be administered, for example, orally, nasally, parenterally (intravenously, intramuscularly or subcutaneously), topically, transdermally, intravaginally, intravesically, intracisternally or rectally. The dosage form can be, for example, a solid, semi-solid, lyophilized powder or liquid dosage form, such as tablets, pills, soft or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, etc., and can be a unit dosage form suitable for simple administration of an exact dosage. A particular route of administration is one that can adjust a convenient once-daily dosage regimen according to the severity of the disease being treated.

[0142] Excipients and adjuvant agents can include, for example, preservatives, wetting agents, suspending agents, sweetening agents, flavoring and odor-masking agents, perfuming agents, emulsifying agents and dispensing agents. Prevention of the activity of microorganisms is generally provided by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents such as sugars, sodium chloride, etc. may also be included. Sustained absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin. Excipients can also include wetting agents, emulsifying agents, pH buffering agents and antioxidants such as citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.

[0143] Solid dosage forms can be prepared using coatings and shells, such as enteric coatings and others well known in the art. These can contain opacifying agents and can be of a composition such that the active oligonucleotide(s) are released in a delayed manner in certain parts of the intestinal tract. Examples of encapsulating compositions that can be used are polymeric substances and waxes. The active oligonucleotide can, if appropriate, also be in microencapsulated form with one or more of the excipients mentioned above added.

[0144] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Such dosage forms can be prepared by dissolving, dispersing, etc., for example, the conjugates described herein or pharmaceutically acceptable salts thereof and, if necessary, pharmaceutical adjuvants in a carrier such as water, saline, aqueous dextrose, glycerol, ethanol, etc.; solubilizing and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butylene glycol, dimethylformamide; oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan; or mixtures of these substances, etc., thereby forming a solution or suspension.

[0145] Generally, depending on the intended mode of administration, the pharmaceutically acceptable composition will contain from about 1% to about 99% by weight of the oligonucleotide 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 be between about 5% and about 75% by weight of the oligonucleotide described herein or a pharmaceutically acceptable salt thereof, with the remainder being suitable pharmaceutical excipients.

[0146] The actual methods of preparing such dosage forms will be known or apparent to those of ordinary skill in the art. See, for example, Remington’s Pharmaceutical Sciences, 18th Ed. (Mack Publishing Company, Easton, Pa., 1990).

[0147] Kit In other embodiments, a kit is provided. A kit according to the present disclosure includes a package containing the oligonucleotides, peptides, peptide-oligonucleotide conjugates, or compositions of the present disclosure. In some embodiments, the kit includes a peptide-oligonucleotide conjugate according to Formula I, Ia, or Ib, or a pharmaceutically acceptable salt thereof. In other embodiments, the kit includes an oligonucleotide according to Formula II or IIa, or a pharmaceutically acceptable salt thereof. In yet other embodiments, the kit includes a peptide according to Formula III, or a pharmaceutically acceptable salt thereof.

[0148] The term “package” means any container that contains the oligonucleotides or compositions presented herein. In some embodiments, the package can be a box or wrapping paper. Packaging materials for use in packaging pharmaceutical products are well known to those of ordinary skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0149] The kit can also contain articles that are not contained within the package but are attached to the outside of the package, such as pipettes.

[0150] The kit can further contain instructions for administering the oligonucleotides or compositions of the present disclosure to a patient. The kit can also include instructions for the use of the oligonucleotides herein approved by a regulatory agency such as the US Food and Drug Administration. The kit can also contain labeling or product inserts for the oligonucleotides. The package or any product insert or both may themselves be approved by a regulatory agency. The kit can contain, within the package, the oligonucleotides in solid or liquid phase (such as a provided buffer). The kit can also include a buffer for preparing a solution for performing the method, and a pipette for transferring liquids from one container to another.

Examples

[0151] For purposes of illustration and to describe certain specific embodiments of the present disclosure, examples are set forth below. However, the claims are in no way limited by the examples set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications, including those related to the chemical structures, substituents, derivatives, formulations or methods of the present disclosure, can be made without departing from the spirit of the present disclosure and the scope of the appended claims. The definitions of variables in the structures within the schemes herein correspond to those at the corresponding positions in the formulas presented herein.

[0152] Cell-penetrating peptides (CPPs) can facilitate the intracellular delivery of therapeutically relevant macromolecules, including proteins and oligonucleotides. Hundreds of CPP sequences, derived from both natural and rational design, have been described in the literature, but the performance of any given sequence depends on its good fit to the cargo of interest. This experiment specifically focuses on CPPs for the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs), a compelling type of antisense therapeutic recently approved by the FDA for the treatment of Duchenne muscular dystrophy. A wide difference in the performance of known CPPs for the delivery of PMO cargo, as opposed to fluorophore cargo, is described herein. Thus, the use of a computational method to predict which peptide sequence most specifically performs PMO delivery would be an efficient way to determine active CPP-PMO conjugates.

[0153] Discovering or predicting effective CPPs for delivering specific cargo remains an unsolved problem. One way to predict CPP sequences is by constructing a computational model trained on an experimental dataset of CPPs. The use of support vector machines and N-to-1 neural networks for CPP prediction based on physicochemical properties, amino acid composition, and dipeptide composition has been described previously. These models are trained on CPP sequences collected from several papers, which use different experimental designs (cell lines, concentrations, etc.) and mainly use fluorescent cargo to measure cell penetration. As a result, these models do not acquire cargo-specific criteria that influence CPP selection for a given delivery problem.

[0154] To generate a library of computational models, 64 CPP-PMO conjugates were synthesized using a CPP array from the literature and evaluated for functional exon skipping in a fluorescence-based reporter assay. Next, a random decision forest classifier was fitted to predict whether a given peptide would enhance exon skipping by at least three-fold when conjugated to a PMO using exon skipping data from this library. Finally, seven previously unreported peptide sequences (referred to as "PPC") were evaluated in a reporter assay to validate the computational model. One of the computationally predicted sequences performed better than 80% of the CPPs from the literature tested for specific delivery of the PMO. These results highlight the importance of tailoring CPP arrays to the cargo of interest and the power of machine learning to discover peptide sequences with specific functions.

[0155] (Example 1) General methods for peptide preparation and purification High-speed flow peptide synthesis Peptides were synthesized on a 0.1 mmol scale using an automated flow peptide synthesizer. ChemMatrix Rink Amide HYR resin (200 mg) was loaded into a reactor maintained at 90 °C. All reagents were flowed at 80 mL / min using an HPLC pump through a stainless-steel loop maintained at 90 °C and then introduced into the reactor. For each coupling, a 10 mL solution containing 0.2 M amino acid and 0.2 M HATU in DMF was mixed with 200 μL of diisopropylethylamine and delivered to the reactor. Fmoc removal was performed using 10.4 mL of 20% (v / v) piperidine. Between each step, the reactor was flushed with 15 mL of DMF. The final coupling was with 4-pentynoic acid instead of an amino acid, but the same conditions were used. After completion of the synthesis, the resin was washed three times with DCM and dried under vacuum.

[0156] Peptide cleavage and deprotection Each peptide was subjected to simultaneous global side-chain deprotection and cleavage from the resin by treatment with 6 mL of Reagent K (82.5% trifluoroacetic acid, 5% phenol, 5% water, 5% thioanisole and 2.5% 1,2-ethanedithiol (EDT)). The cleavage was left at room temperature for 16 h to ensure complete removal of Pbf. The cleavage cocktail was filtered to remove the resin and evaporated by blowing N2 into the mixture. Then, about 35 mL of cold ether was added and the crude product was pelleted through 3 min of centrifugation. This ether trituration and centrifugation was repeated two more times. After the third wash, the pellet was redissolved in 50% water and 50% acetonitrile and lyophilized.

[0157] Peptide purification Solvent A: Water containing 0.1% TFA Solvent B: Acetonitrile containing 0.1% TFA The lyophilized peptide was dissolved in the minimum volume of mobile phase (95% A, 5% B). The solution was loaded onto a reverse-phase HPLC column (Agilent Zorbax SB C18 column: 9.4×250 mm, 5 μm or Agilent Zorbax SB C3 column: 9.4×250 mm, 5 μm) attached to a mass-based purification system. The linear gradient was run from 5% B to 55% B at 0.5% B / min. Using the mass data for each fraction from the instrument, only the pure fractions were pooled and lyophilized. The purity of the fraction pool was confirmed by LC-MS.

[0158] The peptides in Table 2 were synthesized using the protocol of Example 1.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0159] (Example 2) Peptide conjugation [Chemical formula] Procedure for coupling 5-azidopentanoic acid to PMO PMO IVS-654(R 2 =5’-GCT ATT ACC TTA ACC CAG-3’; z = 18) (200 mg, 32 μmol) was dissolved in 600 μL of DMSO. To the solution was added a solution containing 4 equivalents of 5-azidopentanoic acid (13.6 μL, 128 μmol) activated with HBTU (0.4 M HBTU in 320 μL of DMF, 128 μmol) and DIEA (22.3 μL, 128 μmol) in 244 μL of DMF (final reaction volume = 1.2 mL). After allowing the reaction to proceed for 25 minutes, it was quenched with 1 mL of water and 2 mL of ammonium hydroxide. Ammonium hydroxide hydrolyzes any esters formed during the reaction process. After 1 hour, the solution was diluted to 40 mL and purified using reverse-phase HPLC (Agilent Zorbax SB C3 column: 21.2 × 100 mm, 5 μm) and a linear gradient from 2 to 60% B over 58 minutes (1% B / min) (solvent A: water; solvent B: acetonitrile). Using the mass data for each fraction from the instrument, only the pure fractions were pooled and lyophilized. The purity of the fraction pool was confirmed by LC-MS. Lyophilization gave 171 mg of dry powder (84% yield).

[0160] General procedure for PMO-peptide conjugation by azide / alkyne Huisgen cycloaddition A 20 mL scintillation vial with a septum cap was charged with peptide alkyne (1.1 μmol), ISV2-654 azide (0.95 μmol), and copper bromide (0.05 mmol). The vial was purged with nitrogen for 5 minutes to ensure removal of oxygen, and then approximately 1 mL of DMF was added through the septum. The reaction mixture was vortexed for 1 minute. After 2 hours, the reaction mixture was diluted with 10 mL of 50 mM Tris (pH 8) and loaded onto a reverse-phase HPLC column (Agilent Zorbax SB C3 9.4×50 mm, 5 μm). Chromatography was performed using a linear gradient of 5 - 45% B over 20 minutes. Solvent A: 5 mM ammonium acetate in water, pH = 8; Solvent B: 90% acetonitrile 10% 5 mM ammonium acetate in water pH = 8. Using the mass data for each fraction from the instrument, only the pure fractions were pooled and lyophilized. The purity of the fraction pool was confirmed by LC-MS.

[0161] (Example 3) Calculated design Random forest classifier hyperparameters were optimized through a grid search with classification accuracy estimated using five-fold cross validation. The number of selected features, number of trees, and maximum tree depth were 5, 10, and 5, respectively. Performance metrics from classifier evaluation on a 20-sequence holdout test set are shown in Figure 2A.

[0162] Performance metrics are defined below, where TP refers to true positive, TN refers to true negative, FP refers to false positive, and FN refers to false negative.

Number

[0163] For each peptide sequence, 19 features were calculated, one of which was the peptide molecular weight. The remaining 18 features were derived from six amino acid physicochemical descriptors, which were generated by factor analysis of 384 molecular property factors calculated for 22 natural and 593 unnatural amino acids. For each peptide sequence, these six descriptors were averaged over five N-terminal residues, five C-terminal residues, and the entire peptide sequence.

[0164] The CPP sequences were classified as either positive or negative examples based on whether they exhibited a change greater than three-fold in eGFP fluorescence relative to the unconjugated PMO. Forty-four sequences were used as a training set for the random forest model, and 20 sequences were held out to serve as a test set for evaluating the extent to which the model could successfully predict exon skipping activity. The performance metrics of the model are shown in Figure 2A. To experimentally validate, random peptide sequences were generated by selecting peptide lengths and amino acid compositions that were likely to be proportional to the distributions observed in the training dataset from the CPP library. Among the random peptides, five positive sequences were predicted to lead to an increase greater than three-fold in eGFP fluorescence, and two were predicted to be negative sequences (NS). To develop novel peptide sequences for PMO delivery, more positive sequences were selected and these were designated as predicted PMO carriers (PPC). These PPCs were synthesized by solid-phase peptide synthesis, conjugated to PMO IVS2-654, and purified by RP-HPLC. The results are shown in Figure 2A and Figure 2B.

[0165] For experimental verification, random peptide sequences were generated by selecting peptide lengths and amino acid compositions that were likely to be proportional to the distributions observed in the training dataset from the CPP library. Among the random peptides, five positive sequences were predicted to lead to an increase greater than three-fold in eGFP fluorescence, and two were predicted to be negative sequences (NS). To develop novel peptide sequences for PMO delivery, more positive sequences were selected and these were designated as predicted PMO carriers (PPC). These PPCs were synthesized by solid-phase peptide synthesis, conjugated to PMO IVS2-654, and purified by RP-HPLC. The results are shown in Figure 2A and Figure 2B.

[0166] (Example 4) Flow cytometry To test the library of PMO-CPP conjugates, flow cytometry analysis of GFP fluorescence was performed. To test the CPP, HeLa654 cells were maintained in MEM supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin at 37 °C and 5% CO 2 2. Each stock of the PMO-CPP conjugate was prepared in phosphate buffered serum (PBS). The concentration of the stock was determined by measuring the absorbance at 260 nm and using an extinction coefficient of 168,700 Lmol -1 cm -1 2. The cells were incubated with each respective conjugate at a concentration of 5 μM in MEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C and 5% CO 2 2 for 22 h. Next, the treatment medium was aspirated, and the cells were incubated with 0.25% trypsin-EDTA at 37 °C and 5% CO 2 2 for 15 min, washed once with PBS, and resuspended in PBS supplemented with 2% FBS and 2 μg / mL propidium iodide.

[0167] Flow cytometry analysis was performed on a BD LSRII flow cytometer. Gates were applied to the data to exclude cells that were highly positive for propidium iodide or had forward / side scatter readings that were sufficiently different from the main cell population. Each histogram contained at least 10,000 gated events. The results are shown in Figure 1E, Figure 2C, and Table 3.

Table 3-1

Table 3-2

[0168] (Example 5) Inhibitor Experiment To inhibit the mechanisms of various endocytosis, pulse-chase experiments were performed. Briefly, HeLa654 cells were plated in 96-well plates at a density of 5,000 cells per well in MEM supplemented with 10% FBS and 1% penicillin-streptomycin. The next day, the cells were treated with each inhibitor at the indicated concentration. After 30 minutes, the PMO-peptide conjugate was added to each well at a concentration of 5 μM. After incubation at 37 °C and 5% CO 2 for 3 hours, the treatment medium was replaced with fresh medium (without inhibitor or PMO-peptide), and the cells were grown for another 22 hours at 37 °C and 5% CO 2 In the 4 °C experiment, on the day after plating, the cells were pre-incubated at 4 °C for 30 minutes, and subsequently, the PMO-peptide conjugate was added to each well at a concentration of 5 μM. After incubation at 4 °C for 3 hours, the treatment medium was replaced with fresh medium, and the cells were grown for another 22 hours at 37 °C and 5% CO 2 Then, sample preparation and flow cytometry were performed as described above. Each histogram contains at least 2,000 gated events, except for treatment with 20 μM cytochalasin D and 200 nM wortmannin. The results are shown in FIGS. 3A and 3B.

[0169] eGFP fluorescence did not change significantly after pre-incubation with most of the inhibitors, but pre-incubation with cytochalasin D led to a significant decrease in eGFP fluorescence. Cytochalasin D binds to the rapidly growing barbed ends of actin microfilaments, thereby preventing the assembly and dissociation of actin monomers. This affects not only the cell's cytoskeleton but also the ability of the membrane to disrupt and reorganize to facilitate macropinocytosis. The decrease in eGFP fluorescence may be due to downstream effects in the exon skipping pathway, but these results suggest that macropinocytosis plays an important role in the internalization of this conjugate.

[0170] Incorporation by reference The content of all references cited throughout this application (including bibliographic references, issued patents, published patent applications, and co-pending patent applications) is hereby incorporated by reference in its entirety. Unless otherwise defined, all technical and scientific terms used herein shall conform to the meanings commonly known to those of ordinary skill in the art.

[0171] Equivalents Those of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the disclosure described herein without undue experimentation. Such equivalents are intended to be encompassed by the following claims. In certain embodiments, for example, the following items are provided. (Item 1) A peptide-oligonucleotide conjugate of Formula I:

Chemical formula

Chemical formula

Chem.

Chem.

Chemical formula

Chemical formula

Chemical formula

Claims

[Claim 1] An object, method or system as described in this specification and drawings.

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