CN117957022A

CN117957022A - Antisense compounds and methods for targeting CUG repeats

Info

Publication number: CN117957022A
Application number: CN202280056925.0A
Authority: CN
Inventors: 沈秀龙; 钱自清; P·多尔蒂; M·凯拉迪; 李翔
Original assignee: Anzhuoda Treatment Co ltd
Current assignee: Anzhuoda Treatment Co ltd
Priority date: 2021-06-23
Filing date: 2022-06-22
Publication date: 2024-04-30

Abstract

Compounds comprising cyclic peptides such as cyclic cell penetrating peptides and antisense compounds are provided. The antisense compound binds to a gene having an amplified CTG repeat or a gene transcript having an amplified CUG repeat. The compounds can be delivered to a subject to treat diseases associated with amplified CTG-CUG repeats, such as type 1 myotonic dystrophy (DM 1), spinocerebellar ataxia-8 (SCA 8), and huntington's disease-like-2 (HDL 2).

Description

Antisense compounds and methods for targeting CUG repeats

Cross Reference to Related Applications

The present application claims U.S. provisional application Ser. No. 63/213,900 filed on day 23 of 6.2021; U.S. provisional application Ser. No. 63/239,847, filed on 1/9/2021; U.S. provisional application Ser. No. 63/290,892 filed on 12 months and 17 days 2021; U.S. provisional application Ser. No. 63/305,071, filed on 1/31 of 2022; U.S. provisional application Ser. No. 63/314,369 filed on 2/26 of 2022; U.S. provisional application Ser. No. 63/316,634, filed 3/4 at 2022; U.S. provisional application Ser. No. 63/317,856 filed on 3/8 of 2022; U.S. provisional application Ser. No. 63/326,201, filed 3/31 at 2022; U.S. provisional application Ser. No. 63/327,179 filed on 4/2022; U.S. provisional application Ser. No. 63/339,250, filed 5/6 at 2022; U.S. provisional application Ser. No. 63/362,295 filed on 3/2022; U.S. provisional application Ser. No. 63/239,671, filed on 1 at 9/2021; U.S. provisional application Ser. No. 63/290,960 filed on 12/17 of 2021; U.S. provisional application Ser. No. 63/298,565 filed on 1/11 of 2022; and U.S. provisional application Ser. No. 63/268,577 filed on 25/2/2022.

Technical Field

The present disclosure relates to compounds, compositions, and methods for modulating the activity and/or level of genes comprising amplified nucleotide repeats, particularly amplified ctg.cug repeats. The compounds and compositions comprising the compounds are useful in the treatment of diseases associated with genes comprising amplified nucleotide repeats, particularly amplified ctg.cug repeats.

Introduction to the invention

Some diseases are associated with genes having amplified nucleotide repeats, that is, a greater number of nucleotide repeats than are observed in a healthy phenotype. Amplified repeat sequences can cause aggregation and/or nucleation of transcripts containing amplified repeat sequences and/or nucleation of proteins that bind to transcripts containing amplified repeat sequences. The amplified repeat sequence may cause some proteins, such as pre-mRNA processing proteins, to be sequestered on the repeat sequence, thereby inhibiting the protein from performing its normal function, such as processing pre-mRNA transcripts of other genes that do not contain the amplified repeat sequence.

There are several diseases associated with genes having amplified ctg.cug trinucleotide repeats (CTG means DNA repeats, CUG means corresponding RNA repeats occurring upon transcription). Diseases associated with genes having amplified ctg.cugtrinucleotide repeats include, but are not limited to, type 1 myotonic dystrophy (DM 1), spinocerebellar ataxia-8 (SCA 8), huntington-like chorea-2 (HDL 2), and Fuchs' endothelial corneal dystrophy, FECD.

Type 1 myodystrophy (DM 1), the most common cause of myodystrophy in adults, is the most common cause of myodystrophy in individuals worldwide, and is related to genes with amplified trinucleotide repeats (Lee and Cooper.(2009)"Pathogenic mechanisms of myotonic dystrophy,"Biochem Soc Trans.37(06):10.1042/BST0371281).DM1 are a disorder affecting skeletal and smooth muscle and the eye, heart, endocrine system and central nervous system; DM1 is a disorder caused by abnormal amplification of CTG trinucleotide repeats in the non-coding region of genes encoding myotonic dystrophy protein kinase (DMPK). CTG amplification is located in the region corresponding to the 3 'untranslated region (3' -UTR) of DMPK mRNA. DMPK genes contain 5 to 40 CTG trinucleotide repeats whereas DM1 patients have 50 to thousands of CTG trinucleotide repeats. Since some regulatory RNA binding proteins nucleate in CUG amplification in the 3 'untranslated region (3' -UTR), CTG-trinucleotide repeats cause deregulation of gene expression in the affected individuals, cause the transcription of RNA-like proteins such as membrane-specific RNA binding proteins (membrane-specific RNA) in the 3 'untranslated region (3' -UTR) and the number of which can cause the formation of transcription factor-like RNA binding proteins in the other families (membrane-specific mRNA) are also increased with the increased number of the mRNA-like restricted RNA binding proteins (86.8664) in healthy individuals, the severity of the disease increased and the age of onset decreased (Pettersson et al (2015) "Molecular MECHANISMS IN DM-a focus on foci." Nucleic Acids Res.43 (4): 2433-2441).

The CUG-trinucleotide repeats in the 3' untranslated region of DMPK mRNA form imperfect stable hairpin structures that accumulate in the nucleus in the form of small ribonucleophiles or microscopic inclusions and impair the function of proteins involved in transcription, splicing or RNA export. Although the DMPK gene with CUG repeat sequence was transcribed into mRNA, the mutant transcript was sequestered as an aggregate (foci) in the nucleus, which resulted in a decrease in cytoplasmic DMPK mRNA levels. Due to the isolation of the two RNA binding proteins, these aggregates lead to alternative splicing deregulation of many different transcripts: MBNL1 (myoblindness-like protein 1) and CUGBP1 (CUG-binding protein 1), leading to loss of MBNL1 function and up-regulation of CUGBP1 (Lee and Cooper.(2009)"Pathogenic mechanisms of myotonic dystrophy,"Biochem Soc Trans.37(06):10.1042/BST0371281).MBNL1 and CUGBP-ETR-3-like factor 1 (CELF 1) are developmental regulators of splicing events during fetal to adult transition, their changes in activity in DM1 lead to expression of fetal splicing patterns in adult tissues, reducing the downstream effects of MBNL1 and increasing CELF1 levels includes disruption of alternative splicing, mRNA translation and mRNA attenuation in proteins such as cardiac troponin T (cTNT), insulin receptor (INSR), muscle-specific chloride channel (CLCN 1) and sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP 2A 1) transcripts, MBNL1 (Konieczny et al) (2017)"Myotonic dystrophy:candidate small molecule therapeutics."Drug Discov Today.22(11):1740-174).

Possible therapeutic methods for treating DM1 or other diseases associated with the amplified ctg.cugrepeats include the use of compounds containing therapeutic oligonucleotides. However, one major problem associated with the use of oligonucleotide compounds in therapeutic agents is their limited ability to enter the intracellular compartment when administered systemically. Intracellular delivery of oligonucleotide compounds may be facilitated by the use of carrier systems such as polymers, cationic liposomes, or by chemical modification of the construct, for example by covalent attachment of cholesterol molecules. However, the intracellular delivery efficiency of oligonucleotide compounds is still low. There remains a need for improved delivery systems to increase the efficacy of these compounds.

There is an unmet need for effective compositions for delivering therapeutic oligonucleotide compounds to intracellular compartments for the treatment of diseases caused by amplified CTG-CUG repeats, such as DM 1.

Disclosure of Invention

Described herein are compounds, compositions, and methods for treating diseases associated with amplified ctg.cug repeats. In embodiments, the disclosure relates to compounds comprising an Antisense Compound (AC) and a cyclic peptide, such as a cyclic cell penetrating peptide (cCPP). In embodiments, the AC binds to a gene or gene transcript comprising the amplified CUG repeat. In embodiments, the cyclic peptide promotes intracellular localization of AC. These compounds may comprise an Endosomal Escape Vector (EEV). EEV may comprise cyclic peptides and exocyclic peptides.

In an embodiment, provided herein is a compound comprising: (a) At least one cyclic peptide and (b) an Antisense Compound (AC) complementary to a target nucleotide. In embodiments, the target nucleotide comprises at least one amplified CUG or CTG repeat. In embodiments, the target nucleotide is a gene comprising at least one amplified CTG repeat. In embodiments, the target nucleotide is an RNA comprising at least one amplified CUG repeat. In embodiments, the RNA comprising at least one amplified CUG repeat sequence is a pre-mRNA sequence. In embodiments, the amplified CUG repeat corresponds to an amplified CTG repeat in a gene that transcribes a pre-mRNA. In embodiments, the antisense compound binds to an amplified CTG repeat or an amplified CUG repeat. In embodiments, the AC comprises 5-40 CAG repeats (e.g., ,5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39 or 40 repeats). In embodiments, the AC comprises the nucleotide sequences listed in table 2, table 10 or table 11. In embodiments, the AC comprises the nucleotide sequences listed in table 2.

In embodiments, the AC comprises at least one modified nucleotide or nucleic acid selected from the group consisting of: phosphorothioate (PS) nucleotides, phosphorodiamidate Morpholino (PMO) nucleotides, locked Nucleic Acids (LNA), peptide Nucleic Acids (PNA), nucleotides comprising a 2' -O-methyl (2 ' -OMe) modified backbone, 2' -O-methoxy-ethyl (2 ' -MOE) nucleotides, 2',4' restricted ethyl (cEt) nucleotides, 2' -deoxy-2 ' -fluoro- β -D-arabinonucleic acid (2 ' f-ANA), and combinations thereof. In embodiments, the AC comprises a PMO nucleotide.

In an embodiment, compounds are provided comprising a cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids. In embodiments, the Antisense Compound (AC) is complementary to at least a portion of the amplified CUG repeat in the target mRNA sequence. In embodiments, AC is a Phosphorodiamidate Morpholino (PMO) nucleotide.

In embodiments, the at least two charged amino acids of the cyclic peptide are arginine. In embodiments, the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthalanine (3-naphthalen-2-yl-alanine), or a combination thereof. In embodiments, the at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, glycine, or a combination thereof. In embodiments, the compound is a cyclic peptide having from 6 to 12 amino acids, wherein two amino acids of the cyclic peptide are arginine, at least two amino acids are aromatic hydrophobic amino acids selected from phenylalanine, naphthylalanine, and combinations thereof, and at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine, and combinations thereof.

In embodiments, the compounds comprise an endosomal escape vector comprising a cyclic peptide and an Exocyclic Peptide (EP). In embodiments, the EP is conjugated to the linker at the amino group. The linker may be a linker as described herein. In embodiments, the EP is conjugated to the cyclic peptide via a linker. In embodiments, the EP is conjugated to AC via a linker. In embodiments, the EP is conjugated to a linker that conjugates the AC to the cyclic peptide.

In embodiments, the EP comprises 2 to 10 amino acids. In embodiments, the EP comprises 4 to 8 amino acid residues. In embodiments, the EP comprises 1 or 2 amino acids comprising a side chain comprising a guanidino group or a protonated form thereof. In embodiments, the EP comprises 1,2, 3 or 4 lysine residues. In embodiments, the amino group on the side chain of each lysine residue is substituted with trifluoroacetyl (-COCF ₃), allyloxycarbonyl (Alloc), 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl (Dde) or (4, 4-dimethyl-2, 6-dioxocyclon-1-hexyl-3) -methylbutyl (ivDde). In embodiments, the EP comprises at least 2 amino acid residues having a hydrophobic side chain. In embodiments, the amino acid residue having a hydrophobic side chain is selected from valine, proline, alanine, leucine, isoleucine and methionine. In embodiments, the exocyclic peptide comprises one of the following sequences ：PKKKRKV;KR;RR;KKK;KGK;KBK;KBR;KRK;KRR;RKK;RRR;KKKK;KKRK;KRKK;KRRK;RKKR;RRRR;KGKK;KKGK;KKKKK;KKKRK;KBKBK;KKKRKV;PGKKRKV;PKGKRKV;PKKGRKV;PKKKGKV;PKKKRGV; or PKKKRKG. In embodiments, the exocyclic peptide consists of one of the following sequences ：PKKKRKV;KR;RR;KKK;KGK;KBK;KBR;KRK;KRR;RKK;RRR;KKKK;KKRK;KRKK;KRRK;RKKR;RRRR;KGKK;KKGK;KKKKK;KKKRK;KBKBK;KKKRKV;PGKKRKV;PKGKRKV;PKKGRKV;PKKKGKV;PKKKRGV; or PKKKRKG. In embodiments, the cyclic exopeptide has the following structure: ac-P-K-K-K-R-K-V-.

In embodiments, the cyclic peptide comprises 4 to 12 amino acids. In embodiments, the cyclic peptide comprises 6 to 12 amino acids. In some embodiments, at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are non-charged non-aromatic amino acids. In some embodiments, the at least two charged amino acids of the cyclic peptide are arginine, the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine, or a combination thereof, and the at least two uncharged non-aromatic amino acids are citrulline, glycine, or a combination thereof.

In embodiments, the cyclic peptide has 4 to 12 amino acids, wherein at least two of the amino acids are arginine, and at least two of the amino acids comprise hydrophobic side chains, provided that the cyclic peptide is not a cyclic peptide having the sequence of SEQ ID NO: 89-117. In embodiments, the cyclic peptide is not a cyclic peptide having the sequence of SEQ ID NO. 89-117.

Wherein F is L-phenylalanine, F is D-phenylalanine,Is L-3- (2-naphthyl) -alanine,/>Is D-3- (2-naphthyl) -alanine, R is L-arginine, R is D-arginine, Q is L-glutamine, Q is D-glutamine, C is L-cysteine, U is L-selenocysteine, W is L-tryptophan, K is L-lysine, D is L-aspartic acid, and Ω is L-norleucine.

In embodiments, the cyclic peptide has the following structure:

Or (b)

In its protonated form, the precursor of the precursor,

Wherein:

R ₁、R₂ and R ₃ are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

At least one of R ₁、R₂ and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

R ₄、R₅、R₆、R₇ is independently H or an amino acid side chain;

At least one of R ₄、R₅、R₆、R₇ is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N, N-dimethylarginine, 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, lysine, N-methyllysine, N, N-dimethyllysine, N-ethyllysine, N, N, N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N, N-dimethyllysine, β -homoarginine, 3- (1-piperidinyl) alanine;

AA _SC is the amino acid side chain to which the antisense compound is conjugated; and

Q is 1, 2, 3 or 4.

In embodiments, at least one of R ₄、R₅、R₆、R₇ is independently an uncharged non-aromatic side chain of an amino acid. In embodiments, at least one of R ₄、R₅、R₆、R₇ is independently H or a side chain of citrulline.

In embodiments, the cyclic peptide has the structure of formula I:

Or a protonated form thereof,

Wherein:

R ₁、R₂ and R ₃ are each independently H or an amino acid residue having a side chain comprising an aromatic group;

R ₄ and R ₇ are independently H or an amino acid side chain;

AA _SC is the amino acid side chain to which the antisense compound is conjugated;

q is 1, 2, 3 or 4; and

Each m is independently an integer of 0, 1,2 or 3.

In an embodiment, the cyclic peptide of formula (I) has one of the following structures:

Or (b)

In its protonated form.

Or (b)

In its protonated form.

Or (b)

In its protonated form.

In embodiments, the cyclic peptide of formula (I) has the following structure:

Or (b)

In its protonated form.

In embodiments, the compound has the structure of formula C:

Or (b)

In its protonated form or in the form of a salt,

Wherein:

R ₁、R₂ and R ₃ are each independently H or a side chain comprising an aryl or heteroaryl group, wherein at least one of R ₁、R₂ and R ₃ is a side chain comprising an aryl or heteroaryl group;

R ₄ and R ₇ are independently H or an amino acid side chain;

EP is the cyclic exopeptide;

each m is independently an integer from 0 to 3;

n is an integer from 0 to 2;

x' is an integer from 1 to 23;

y is an integer from 1 to 5;

q is an integer from 1 to 4;

z' is an integer from 1 to 23, and

The cargo is said antisense compound.

In embodiments, the compound has one or the following structures:

/>

Or a protonated form or salt thereof,

Wherein EP is said cyclic exopeptide, and

Oligonucleotides are the antisense compounds.

In embodiments, the oligonucleotide of formula (C-1), formula (C-2), formula (C-3) or formula (C-4) comprises the following sequence: 5'-CAG CAG CAG CAG CAG CAG CAG-3'.

In embodiments, the EP of the compound of formula (C-1), formula (C-2), formula (C-3) or formula (C-4) comprises the following sequence: PKKKRKV.

Drawings

FIG. 1 is a schematic diagram showing various strategies for targeting CUG repeats in mRNA.

FIG. 2 shows modified nucleotides used in the antisense oligonucleotides described herein. Structures 1-3 (1=phosphorothioate; 2= (S _C5-R_p) - α, β -CAN; 3=pmo) are phosphate backbone modifications; structure 4 (2-thio-dT) is a base modification; structure 5-8 (5 = 2' -OMe-RNA;6 = 2' o-MOE-RNA;7 = 2' f-RNA;8 = 2' f-ANA) is a 2' sugar modification; structures 9-11 are restriction nucleotides; structures 12-14 (9=lna; 10= (S) -cET; 11=tcdna; 12=fhna; 13= (S) 5' -C-methyl; 14=una) are additional sugar modifications; and structures 15-18 (15=e-VP; 16=methylphosphonate; 17=5 ' phosphorothioate; 18= (S) -5' -C-methyl with phosphate) are 5' phosphate stable modifications; structure 19 is a morpholino sugar. Reformatting from Khvorova, a. Et al, nat. Biotechnol.2017, month 3; 35 (3):238-248.

Figures 3A-3D illustrate conjugation chemistry for attaching AC to a cyclic cell penetrating peptide. FIG. 3A shows the amide bond formation between a peptide having a carboxylic acid group or having a TFP activated ester and a primary amine residue at the 5' end of AC. FIG. 3B shows conjugation of a 3' end secondary or primary amine modified AC to a peptide-TFP ester via an amide linkage. Figure 3C shows conjugation of peptide-azide to 5' cyclooctyne modified AC via copper-free azide-alkyne cycloaddition. FIG. 3D shows another exemplary conjugation between a 3 'modified cyclooctene AC or a 3' modified azide AC and a CPP containing a linker-azide or linker-alkyne/cyclooctyne moiety via copper-free azide-alkyne cycloaddition or copper-catalyzed azide-alkyne cycloaddition (click reaction), respectively.

FIG. 4 shows the conjugation chemistry for linking AC and CPP with an additional linker form containing a polyethylene glycol (PEG) moiety.

FIGS. 5A-5D provide structures of adenine (5A), cytosine (5B), guanine (5C) and thymine (5D) morpholino subunit monomers that can be used to synthesize phosphorodiamidate-linked morpholino oligomers (PMOs).

FIGS. 6A-6F show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) using Endo-Porter transfection reagent (FIGS. 6A-6C) or without Endo-Porter transfection reagent (FIGS. 6E-6F), 24 hours (FIGS. 6A-6B) and 48 hours (FIGS. 6C-6D) after treatment of HeLa-48 cells with 1. Mu.M, 3. Mu.M or 10. Mu.M of various PMO or PMO-EEV compounds, MBNL1 (exon 5; FIGS. 6A, 6B, 6E) and CLASP1 (exon 19; FIGS. 6B, 6D, 6F). The parental HeLa cell line and the HeLa-480 cell line treated with (fig. 6A-6D) or without (fig. 6E-6F) Endo-Porter reagent were included as controls.

FIGS. 7A-7B show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) of MBNL1 (exon 5; FIG. 7A) and CLASP1 (exon 19; FIG. 7B) after treatment of DM1 myoblasts with 1. Mu.M of various PMO or PMO-EEV compounds for 48 hours without Endo-Porter transfection reagent. Two controls, namely DM-04 that was not treated with endo-porter and DM-05 that was not treated with endo-porter, were included as controls.

FIGS. 8A-8D show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) of Atp a1 (exon 22; FIG. 8A), nfix (exon 7; FIG. 8B), clcn1 (exon 7a; FIG. 8C), and Mbnl1 (exon 5; FIG. 8D) of gastrocnemius tissue after one week treatment of HSA-LR (DM 1-mouse model) mice with PMO, 20mpk PMO-EEV 221-1106, or 40mpk PMO-EEV 221-1106. FVB/NJ (wild-type inbred mice) and HSA-LR (untreated) mice were included as control groups.

FIGS. 9A-9D show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) of Atp a1 (exon 22; FIG. 9A), nfix (exon 7; FIG. 9B), clcn1 (exon 7a; FIG. 9C), and Mbnl1 (exon 5; FIG. 9D) of quadriceps tissue after one week treatment of HSA-LR (DM 1-mouse model) mice with PMO, 20mpk PMO-EEV 221-1106, or 40mpk PMO-EEV 221-1106. FVB/NJ (wild-type inbred mice) and HSA-LR (untreated) mice were included as control groups.

FIGS. 10A-10D show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) of Atp a1 (exon 22; FIG. 10A), nfix (exon 7; FIG. 10B), clcn1 (exon 7a; FIG. 10C), and Mbnl1 (exon 5; FIG. 10D) in tibial anterior muscle tissue after one week of treatment of HSA-LR (DM 1-mouse model) mice with PMO, 20mpk PMO-EEV 221-1106, or 40mpk PMO-EEV 221-1106. FVB/NJ (wild-type inbred mice) and HSA-LR (untreated) mice were included as control groups.

FIGS. 11A-11F show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) of MBNL1 (exon 5, FIG. 11A), SOS1 (exon 25, FIG. 11B), IR (exon 11, FIG. 11C), DMD (exon 78, FIG. 11D), BIN1 (exon 11, FIG. 11E) and LDB3 (exon 11, FIG. 11F) after treatment of DM1 patient-derived myocytes with different concentrations (10 μm, 3 μm, 1 μm, 0.3 μm) of EEV-PMO (CUG ^exp -777 and CUG ^exp 221-1106) of the targeted DMPKCUG. Alternative RNA splicing events from muscle cells from two groups, healthy people (negative control) and DM1 patients (positive control), were tested as controls. All data were collected from three separate experiments (n=3). T-testing the treated and untreated DM1 myotubes; * p <0.05; * P <0.01; * P <0.001.

FIGS. 12A-12F show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) of MBL1 (exon 5, FIG. 12A), SOS1 (exon 25, FIG. 12B), INSR (exon 11, FIG. 12C), DMD (exon 78, FIG. 12D), BIN1 (exon 11, FIG. 12E) and LDB3 (exon 11, FIG. 12F) after treatment of patient-derived DM1 myoblasts and myotubes with 10 μm, 3 μm or 1 μm of EEV-PMO 197-777 targeted DMPKCUG. Healthy patient cells and DM1 cells were used as controls. All data were collected from three separate experiments (n=3). T-testing the treated and untreated DM1 myotubes; * p <0.05; * P <0.01; * P <0.001.

FIGS. 13A-13B show the relative levels of mRNA after treatment of HSA-LR mice with various concentrations of PMO-EEV 221-1120. Fig. 13A shows the relative mRNA levels of gastrocnemius, triceps, tibialis anterior and diaphragm. Fig. 13B shows relative mRNA levels in the diaphragm.

FIGS. 14A-14C show the relative levels of mRNA in quadriceps (FIG. 14A), gastrocnemius (FIG. 14B), triceps (FIG. 14C) and tibialis anterior (FIG. 14D) tissues after treatment of HSA-LR mice with various concentrations of PMO-EEV 221-1120.

FIGS. 15A-15D show the DM1 splicing index (mDSI) of mice with various genes in quadriceps (FIG. 15A), gastrocnemius (FIG. 15B), triceps (FIG. 15C) and tibialis anterior (FIG. 15D) tissues after treatment of HSA-LR mice with various concentrations of PMO-EEV 221-1120.

FIGS. 16A-16C show the prevalence of RNA foci in tibialis anterior after HSA-LR mice have been untreated or treated with EEV-PMO 221-1120 (EEV-PMO-DM 1-3; DM1-3). Fig. 16A-16B show images of tibialis anterior tissue stained for RNA CUG foci (red) and nuclei (blue). Fig. 16C is a graph quantifying the percentage of nuclei with CUG foci from data associated with the images in fig. 16A-16B.

Fig. 17A-17F are graphs showing dose-dependent responses to drug levels in quadriceps (fig. 17A), triceps (fig. 17B), heart (fig. 17C), gastrocnemius (fig. 17D), tibialis anterior (fig. 17E), diaphragm (fig. 17F), brain (fig. 17H), liver (fig. 17I) and kidney (fig. 17J) tissues after treatment of HSA-LR mice with various concentrations of EEV-PMO-DM 1-3. Fig. 17K shows drug exposure of various tissues at a 60mpk dose level.

FIG. 18 shows dose-dependent myotonic relief in HSA-LR mice after 7 days of treatment with EEV-PMO-DM1-3 at 15, 30, 60 and 90 mpk.

FIGS. 19A-19D are graphs showing the results of principal component analysis comparing gene expression in non-diseased mice (WT), DM1 mice (HSA-LR) and HSA-LR mice treated with PMO-EEV 221-1120. Fig. 19A and 19C are diagrams showing three main components, and fig. 19B and 19D are diagrams showing two main components.

FIGS. 20A-20B show heat maps of differentially expressed genes between non-diseased mice (WT), DM1 mice (HSA-LR) and HSA-LR mice treated with 60mpk PMO-EEV 221-1120. FIG. 20A is a cluster map showing 513 differentially expressed genes. FIG. 20B is a cluster map showing 40 genes known to have CTG CUG repeats.

FIG. 21 is a volcanic chart showing the overall transcriptional changes of untreated HSA-LR mice and mice treated with PMO-EEV 221-1120.

FIGS. 22A to 22E are graphs showing the results of principal component analysis of genes Scube (FIG. 22A), greb1 (FIG. 22B), ttc7 (FIG. 22C), txlnb (CUG) 9 (FIG. 22D) and Ndrg3 (FIG. 22E) from non-diseased mice, HSA-LR mice and HSA-LR mice treated with PMO-EEV 221-1120.

FIGS. 23A-23D show RNA sequencing (RNAseq) data for non-diseased mice (WT-saline), HSA-LR mice (HSA-LR saline), and HSA-LR mice treated with PMO-EEVs 221-1120, atp a1 (FIG. 23A; exon 22 boxed), clcn1 (FIG. 23B; exon 7a boxed), nfix (FIG. 23C; exon 7 boxed), and Mbn1 (FIG. 23D; exon 5 boxed). Each processing group shows two reads.

FIG. 24 shows the Percent Splicing Index (PSI) of individual exons of various genes of interest for non-diseased mice (WT-saline), HSA-LR mice (HSA-LR saline), and HSA-LR mice treated with PMO-EEV 221-1120.

FIGS. 25A-25D show drug levels in tibialis anterior (FIG. 25A), gastrocnemius (FIG. 25B), triceps (FIG. 25C) and quadriceps (FIG. 25D) tissues after 1 to 4 weeks in HSA-LR mice treated with 80mpk (60 mpk oligomer, 80mpk full drug) EEV-PMO-DM 1-3.

FIGS. 26A-26D show drug levels in mice after treatment of HSA-LR mice with a single 80mpk dose of EEV-PMO-DM 1-3. Fig. 26A-26B show drug levels in the liver 1 week to 12 weeks after treatment. Fig. 26C-26D show drug levels in kidneys 1 week to 12 weeks after treatment.

FIGS. 27A-27C are graphs showing the levels of exon inclusion in MBNL1 (exon 5; FIG. 26A), SOS1 (exon 25; FIG. 26B) and NFIX (exon 7; 26C) after treatment of DM 1-derived myocytes with 30. Mu.M EEV-PMO-DM 1-3.

FIGS. 28A-28C show that EEV-PMO-DM1-3 reduces CUG foci (green) in nuclei (blue) of muscle cells from DM1 patients. FIGS. 28A-28B are images of muscle cells from untreated or treated with EEV-PMO-DM1-3 or untreated DM1 patient. Fig. 28C is a quantification of the number of CUG foci per cell nucleus for data related to the image in fig. 28A.

FIGS. 29A-29B show raw data (FIG. 29A) and normalized data (FIG. 29B) of CELLTITER-GLO luminescence activity assays in which RPTEC cells were treated with various concentrations of PMO-DM1 or EEV-PMO-DM 1-3. Melittin was used as a positive control.

FIGS. 30A-30C show images depicting RNA CUG repeat foci in DM1 patient-derived cells (FIG. 30A) and DM1 patient-derived cells treated with EEV-PMO 221-1113 (FIG. 30B). Nuclei (blue; hoechst) and RNA CUG foci (green) of cells were stained. FIG. 30C is a graph of CUG RNA foci per nuclear area for data related to the images of FIGS. 30A-30B.

FIGS. 31A-31B show the prevalence of RNA CUG7 foci in HeLa, untreated HeLa480 cells and HeLa480 cells treated with EEV-PMO 221-1113. FIG. 31A shows images of cells stained for RNA CUG7 foci (green) and nuclei (blue). Fig. 31B is a graph quantifying CUG7 foci per nuclear area for data related to the image in fig. 31A.

FIGS. 32A-32C are graphs showing the percent inclusion of exon 5 in MBNL1 (FIG. 32A), exon 25 in SOS1 (FIG. 32B) and exon 7 in NFIX (FIG. 32C) after treatment of DM1 patient-derived cells with 30. Mu.M EEV-PMO 221-1113.

FIGS. 33A-33E show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion) of MBNL1 (exon 5; FIG. 33A), SOS1 (exon 25; FIG. 33B), CLASP1 (exon 19, FIG. 33C), NFIX (exon 7, FIG. 33D) and INSR (exon 11, FIG. 33E) after treatment of DM1 patient-derived myocytes with various concentrations of PMO-EEV 221-1113. Determining significance using a t-test; * p <0.05; * P <0.01; * P <0.001.

FIGS. 34A-34D show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion) of Atp a1 (exon 22, FIG. 34A), nfix (exon 7, FIG. 34B), clcn1 (exon 7a, FIG. 34C) and Mbnl1 (exon 5, FIG. 34D) in gastrocnemius tissue of mice treated with various concentrations of PMO 221 or EEV-PMO 221-1106.

FIGS. 35A-35C show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion) of Mbnl1 (exon 5, FIG. 35A), nfix (exon 7, FIG. 35B) and Atp a1 (exon 22, FIG. 35C) in tibialis anterior tissue of HSA-LR mice treated with PMO-EEVs 0221-1121 (21-mer) or PMO-EEVs 0325-1121 (24-mer).

FIGS. 36A-36C show RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion) of Mbnl (exon 5, FIG. 36A), nfix (exon 7, FIG. 36B) and Atp a1 (exon 22, FIG. 36C) in gastrocnemius tissue of HSA-LR mice treated with PMO-EEVs 0221-1121 (21-mer) or PMO-EEVs 0325-1121 (24-mer).

FIG. 37 shows the major metabolites of PMO-0221a, PMO-EEV 220-1120 detected in vivo.

FIGS. 38A-38B show the percent exon inclusion of MBNL1 (exon 5) in tibialis anterior (FIG. 38A) and gastrocnemius (FIG. 38B) after treatment of Hela480 cells with various concentrations of EEV-PMO 221-1120.

FIGS. 39A-39B show the percent exon inclusion of NFIX (exon 7) in tibialis anterior (FIG. 39A) and gastrocnemius (FIG. 39B) after treatment of Hela480 cells with various concentrations of EEV-PMO 221-1120.

FIGS. 40A-40B show the percent exon inclusion of Atp a1 (exon 22) in tibialis anterior (FIG. 40A) and gastrocnemius (FIG. 40B) after treatment of Hela480 cells with various concentrations of EEV-PMO 221-1120.

FIG. 41 shows images depicting RNA CUG repeat foci in Hela480 cells after treatment with various concentrations of EEV-PMO 221-1120 (FIG. 41A). FIG. 41B is a graph of RNA foci per nuclear area for data related to the image of FIG. 41A.

FIGS. 42A-42D show relative r (CUG 480) repeat mRNA levels (FIG. 42A), relative DMPK mRNA levels (FIG. 42B), percent exon 5 inclusion of MBNL1 (FIG. 42C) and percent exon 25 inclusion in SOS1 (FIG. 42D) in HeLa480 cells after treatment with various concentrations of EEV-PMO 221-1120.

FIG. 43 is a bar graph showing an example of a gene known to have a CTG CUG repeat sequence expressed in muscle tissue.

FIGS. 44A-44D show reduced phenotypic myotonia in HSA-LR mouse models treated with 20mpk PMO-EEV 221-1106. Fig. 44A and 44C show a relaxation view. Fig. 44B shows an example raw force trace. Fig. 44D shows a representative electromyographic trace.

Detailed Description

Compounds of formula (I)

In embodiments, compounds are provided that modulate the level and/or activity of a gene transcript having an amplified CUG trinucleotide repeat sequence. In embodiments, the compounds of the present disclosure include at least one cyclic cell penetrating peptide (cCPP) and a Therapeutic Moiety (TM). cCPP promote entry of the TM into the cell. In embodiments, the compounds include an Endosomal Escape Vector (EEV) comprising cCPP and an Exocyclic Peptide (EP). cCPP or EEV can allow TM to enter the cytosol or cellular compartment to interact with target transcripts.

Therapeutic section

Generally, TM is the effector moiety that initiates the reaction. In embodiments, the TM initiates the response by modulating the expression, activity, and/or level of the target transcript and/or target protein. In embodiments, the target transcript includes the amplified CUG trinucleotide repeat sequence. In embodiments, the TM modulates the level of a target transcript and/or target protein within the cell. In embodiments, the TM reduces the level of a target transcript and/or target protein within the cell.

In embodiments, the TM modulates the activity of the target transcript by reducing the affinity between the target transcript and one or more proteins that bind to the target transcript. By reducing the affinity between the target transcript and the one or more proteins, the TM can effectively modulate the activity of the one or more proteins that would otherwise associate with the target transcript. For example, if the one or more proteins do not bind to the target transcript, they may perform their function on other molecules. For example, if the one or more proteins are involved in pre-mRNA processing, reducing the affinity of the one or more proteins for transcripts comprising amplified CUG repeats may allow the one or more proteins to process pre-mRNA transcripts that do not comprise amplified CUG repeats. Thus, TM can modulate the activity, expression, and/or level of a downstream gene (a gene that does not contain amplified CTG repeats) that is regulated by the one or more proteins whose interactions with the target transcript are disrupted by TM.

In embodiments, a TM includes oligonucleotides, peptides, antibodies, and/or small molecules. The class and nature of the TM depends on the mechanism used to modulate the level and/or activity of the target transcript, including the amplified CUG trinucleotide repeat sequence.

Antisense compounds

In various embodiments, the compounds disclosed herein comprise a Cell Penetrating Peptide (CPP) conjugated to an Antisense Compound (AC).

The term "antisense compound" refers to an oligonucleotide sequence that is complementary or at least partially complementary to a target nucleotide sequence. AC is an oligonucleotide comprising a natural DNA base, a modified DNA base, a natural RNA base, a modified RNA base, a natural RNA sugar, a modified RNA sugar, a natural DNA sugar, a modified DNA sugar, a natural internucleoside linkage, a modified internucleoside linkage, or any combination thereof. AC includes, but is not limited to, antisense oligonucleotide RNAi, micro RNA, antagomir, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, supermir, miRNA mimics, miRNA inhibitors, U1 adaptors, and combinations thereof.

In embodiments, the AC comprises a nucleotide sequence at least partially complementary to a target transcript having an amplified CUG trinucleotide repeat sequence. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to an amplified CUG trinucleotide repeat sequence in the target mRNA sequence. Several diseases are associated with amplified CUG trinucleotide repeats, such as, for example, type 1 myotonic dystrophy (DM 1), fexofenac corneal endothelial dystrophy (FECD), spinocerebellar ataxia-8 (SCA 8), and huntington-like chorea (HDL 2). Table 1 provides examples of nucleotide repeat disorders, as well as the characteristics of genes having amplified nucleotide repeats associated with such disorders. The following documents describe exemplary oligonucleotides for treating tandem repeat diseases and are incorporated herein by reference in their entirety: zain et al neurotherapeutics.2019;16 (2) 248-262; zarouchlioti et al Am J Hum genet.2018;102 (4) 528-539; fautsch et al Prog Retin Eye res.2021;81:100883.

Table 1: diseases associated with amplified CUG trinucleotide repeats

In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within a target mRNA transcript comprising the amplified ctg.cug trinucleotide repeat sequence. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to the ctg.cug trinucleotide repeat sequence amplified in the target mRNA transcript.

In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the DMPK1 target transcript comprising the amplified ctg.cugtrinucleotide repeat sequence. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within a TCF4 target transcript comprising the amplified ctg.cugtrinucleotide repeat sequence. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the ATXN8OS/ATXN8 target transcript comprising the amplified ctg.cugtrinucleotide repeat sequence. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the JPH3 target transcript comprising the amplified ctg.cugtrinucleotide repeat sequence.

In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a trinucleotide repeat sequence in the 3' utr of the target mRNA transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a ctg.cugtrinucleotide repeat sequence amplified in the 3' utr of the DMPK1 target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to an amplified CTG CUG trinucleotide repeat sequence in the 3' utr of the ATXN8OS/ATXN8 target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to a ctg.cugtrinucleotide repeat sequence amplified in the 3' utr of the JPH3 target transcript.

In embodiments, the AC includes a nucleotide sequence that is at least partially complementary to a trinucleotide repeat sequence, such as a ctg.cug repeat sequence. In embodiments, the target nucleotide sequence comprises at least one amplified trinucleotide repeat sequence (e.g., ctg.cug repeat sequence). In embodiments, the target nucleotide sequence comprises at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, or at least 2000 ctg.cug trinucleotide repeats. In embodiments, the amplified trinucleotide repeat sequence is in the 3' utr of the target nucleotide sequence.

In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of the continuously amplified trinucleotide repeat sequence present in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, and at most 50, at most 100, at most 150, at most 200, at most 300, at most 400, at most 500, at most 600, at most 700, at most 800, at most 900, at most 1000, or at most 2000 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 5 to 10 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 5 to 9 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 5 to 8 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 5 to 7 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 5 to 6 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 5 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 6 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 7 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 8 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizes to 9 trinucleotide repeats in the target transcript. In embodiments, the AC comprises a nucleotide sequence

In embodiments, the AC may comprise a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of a continuously amplified trinucleotide repeat sequence present at any position in the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of the continuously amplified trinucleotide repeat sequence present in the 3' utr of the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of the continuously amplified trinucleotide repeat sequence present in the 3' utr of the DMPK1, SCA8, and/or HDL2 target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of the continuously amplified trinucleotide repeat sequence present in the intron of the target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of the continuously amplified trinucleotide repeat sequence present in intron 3 of the TCF4 target transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of the continuously amplified trinucleotide repeat sequence present in the CTG18.1 locus of the TCF4 transcript. In embodiments, the AC comprises a nucleotide sequence that is at least partially complementary to and hybridizable to at least a portion of the continuously amplified trinucleotide repeat sequence present in the exon of the target transcript.

In embodiments, the AC is 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, or 45 or more nucleic acids in length. In embodiments, the AC is 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, or 10 or less nucleic acids in length. In embodiments, the AC is 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleic acids in length. In embodiments, the AC is 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 nucleic acids in length. In embodiments, the AC is 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, or 15 to 20 nucleic acids in length. In embodiments, the AC is 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, or 20 to 25 nucleic acids in length. In embodiments, the AC is 25 to 50, 25 to 45, 25 to 40, 25 to 35, or 25 to 30 nucleic acids in length. In embodiments, the AC is 30 to 50, 30 to 45, 30 to 40, or 30 to 35 nucleic acids in length. In embodiments, the AC is 35 to 50, 35 to 45, or 35 to 40 nucleic acids in length. In embodiments, the AC is 40 to 50 or 40 to 45 nucleic acids in length. In embodiments, the AC is 45 to 50 nucleic acids in length. In embodiments, the AC is 5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49 or 50 nucleic acids in length.

In embodiments, AC has 100% complementarity to the target nucleotide sequence. In embodiments, AC is not 100% complementary to the target nucleotide sequence. As used herein, the term "percent complementarity" refers to the number of nucleobases (e.g., natural nucleobases or modified nucleobases) in AC that have nucleobase complementarity to the corresponding nucleobase of an oligomeric compound or nucleic acid (e.g., target nucleotide sequence) divided by the total length of AC (the number of nucleobases). One skilled in the art recognizes that it is possible to include mismatches without eliminating the activity of the antisense compound.

In embodiments, AC includes 20% or less, 15% or less, 10% or less, 5% or less, or zero mismatch with the target nucleotide sequence. In some embodiments, the AC comprises 5% or more, 10% or more, or 15% or more mismatches. In embodiments, AC comprises zero to 5%, zero to 10%, zero to 15%, or zero to 20% mismatches with the target nucleotide sequence. In embodiments, AC comprises 5% to 10%, 5% to 15%, or 5% to 20% mismatches with the target nucleotide sequence. In embodiments, AC comprises 10% to 15% or 10% to 20% mismatches with the target nucleotide sequence. In embodiments, AC comprises 10% to 20% mismatches with the target nucleotide sequence.

In embodiments, AC has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more complementarity to the target nucleotide sequence. In embodiments, AC has 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 85% or less complementarity to the target nucleotide sequence. In embodiments, AC has 80% to 100%, 80% to 99%, 80% to 98%, 80% to 97%, 80% to 96%, 80% to 95%, 80% to 90%, or 80% to 85% complementarity to the target nucleotide sequence. In embodiments, AC has 85% to 100%, 85% to 99%, 85% to 98%, 85% to 97%, 85% to 96%, 85% to 95%, or 85% to 90% complementarity to the target nucleotide sequence. In embodiments, AC has 90% to 100%, 90% to 99%, 90% to 98%, 90% to 97%, 90% to 96%, or 90% to 95% complementarity to the target nucleotide sequence. In embodiments, AC has 95% to 100%, 95% to 99%, 95% to 98%, 95% to 97%, or 95% to 96% complementarity to the target nucleotide sequence. In embodiments, AC has 96% to 100%, 96% to 99%, 96% to 98%, or 96% to 97% complementarity to the target nucleotide sequence. In embodiments, AC has 97% to 100%, 97% to 99%, or 97% to 98% complementarity to the target nucleotide sequence. In embodiments, AC has 98% to 100% or 98% to 99% complementarity to the target nucleotide sequence. In embodiments, AC has 99% to 100% complementarity to the target nucleotide sequence.

In embodiments, incorporation of nucleotide affinity modifications allows for a greater number of mismatches than unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than others. One of ordinary skill in the art can determine the appropriate number of mismatches between AC and the target nucleotide sequence, such as by determining the thermal melting temperature (Tm). Tm or Δtm may be calculated by techniques familiar to those of ordinary skill in the art. For example, freier et al (Nucleic ACIDS RESEARCH,1997,25, 22:4429-4443) allow one of ordinary skill in the art to evaluate the ability of nucleotide modifications to increase the melting temperature of RNA to DNA duplex.

In embodiments, the AC includes a nucleotide sequence that is itself a trinucleotide repeat sequence, i.e., a CAG trinucleotide repeat sequence. The reverse complement of 5'-CAG-3' has 100% complementarity and hybridizes to the 5'-CUG-3' trinucleotide repeat sequence. In embodiments, the AC comprises 1 to 50 CAG repeats. In embodiments, the CAG repeat sequence is contiguous. In embodiments, the CAG repeat sequence is not contiguous. In embodiments, the AC comprises a nucleotide sequence comprising an incomplete CAG repeat at the 5 'end or the 3' end. For example, in embodiments, the AC includes a sequence such as AG (CAG) _n、G(CAG)_n、(CAG)_n AG or (CAG) _n a, where n is an integer from 1 to 50. In embodiments, the AC comprises a nucleotide sequence comprising one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, or 50 or more CAG repeats. In embodiments, the AC comprises a nucleotide sequence comprising 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less CAG repeat sequences. In embodiments, the AC comprises a nucleotide sequence comprising 2 to 50, 2 to 20,2 to 10, 4 to 10, 5 to 10, 6 to 9, 6 to 8, or 6 to 7 CAG repeats. In an embodiment, AC comprises any of the nucleotide sequences in Table 2 (SEQ ID NOS: 151-291).

Table 2: CAG repeat AC nucleotide sequence

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In embodiments, an AC having a nucleotide comprising a CAG repeat sequence may comprise additional nucleotide sequences on the 5 'end, 3' end, or both ends of the CAG repeat sequence. In embodiments, the additional nucleotide sequences may have 80% to 100% or 95% to 100% complementarity to the portion of the target transcript to which they hybridize. Additional nucleotide sequences may be added to the CAG repeat nucleotide sequence to increase the selectivity of hybridization of AC to a particular target transcript.

In embodiments, the AC comprises a nucleotide sequence comprising 1 to 50 CAG repeats and is gapmer. gapmer is an oligonucleotide that acts as a DNA/RNA hybrid and induces an rnase attenuation mechanism. For example, a gapmer may have a central DNA or DNA mimetic segment flanked by RNA or RNA mimetic segments at the 5 'and 3' ends of the DNA or DNA mimetic. In embodiments, the AC comprises a gapmer comprising a nucleotide sequence that hybridizes to a target nucleic acid sequence of a target transcript that is separate from an amplified CUG repeat of the target transcript.

In embodiments, the AC of the present disclosure is a gapmer oligonucleotide disclosed in U.S. patent No. 9,550,988, the disclosure of which is incorporated herein by reference.

In embodiments, the disclosed ACs comprise the sequence and/or structure of any of the DMPK-targeting ACs disclosed in U.S. patent publication No. 2017/0260524, the disclosure of which is incorporated herein by reference.

In embodiments, the AC of the present disclosure comprises the sequence and/or structure of any one of the AC or oligonucleotides disclosed in U.S. patent publication US20030235845A1、US20060099616A1、US 2013/0072671 A1、US 2014/0275212 A1、US 2009/0312532 A1、US20100125099A1、US 2010/0125099 A1、US 2009/0269755 A1、US 2011/0294753 A1、US 2012/0022134 A1、US 2011/0263682 A1、US 2014/0128592 A1、US 2015/0073037 A1 and US20120059042A1, the contents of each of which are incorporated herein in their entirety for all purposes.

When using AC to target and/or hybridize to amplified CTG CUG repeats, care must be taken to avoid off-target effects, wherein AC inadvertently binds to off-target transcripts comprising CTG CUG repeats (e.g., transcripts comprising CTG CUG repeats but not amplified CTG CUG repeats). Computer modeling (in-silico) analysis of the human genome revealed that a total of 63 human genes had CTG CUG repeats (Uhlen et al, science 2015 347 (6220): 1260419)). These 63 genes can be rated by the expression of mRNA plus the amount of protein expressed in the total muscles (myocardium, skeletal muscle and smooth muscle). RPM (reads per million) can be used to quantify expression levels. mRNA expression was FPKM (fragments of every kilobase of transcripts per million mapped fragments) and protein expression was pTPM (transcripts per million protein encoding genes), using greater than 10RPM as a cut-off for non-insignificant expression. Fig. 43 shows the results of such computer simulation analysis. Thirty-six genes showed expression levels >10RPM. Of the 36 genes, only three genes (except DMPK) had >10 ctg.cug repeats. Genes with a CUG repeat of 10CTG represent the lowest risk of off-target binding and toxicity. Nevertheless, the number of ctg.cugrepeats in these 3 genes (TCF 4, task, MAP3k 4) (11-24) was significantly lower than that seen in classical and congenital DM1 patients. For example, late onset DM1 patients have 100-600 CTG CUG repeats on DMPK, typical DM1 patients have 250-750 CTG CUG repeats on DMPK, and congenital DM1 patients have 750-1,400 CTG CUG repeats on DMPK. The same computer simulation analysis can be performed on the liver and kidney. CASK is the only important gene in the kidney with >10 ctg.cugrepeats. In the liver, genes with >10 ctg.cug repeats were not apparent.

The ACs described herein may contain one or more asymmetric centers, thus yielding enantiomers, diastereomers, and other stereoisomeric configurations, which may be defined as (R) or (S) depending on absolute stereochemistry; alpha or beta; or (D) or (L). Antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

The efficacy of AC can be assessed by evaluating the antisense activity caused by their administration. As used herein, the term "antisense activity" refers to any detectable and/or measurable activity attributable to hybridization of an antisense compound to its target nucleotide sequence. Such detection and/or measurement may be direct or indirect. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of protein expressed from the transcript of interest. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of a transcript of interest. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of alternatively spliced RNA and/or the amount of protein isoforms translated from the target transcript.

AC modulation mechanism-

In embodiments, AC may modulate the activity and/or level of a target transcript within a cell. FIG. 1 shows an exemplary mechanism of how AC can modulate the level and/or activity of a target transcript.

In embodiments, AC may regulate the level of a target transcript within a cell. For example, in embodiments where AC is gamper, binding of AC to the target transcript induces degradation of the target transcript via the rnase H pathway (fig. 1, arrow a and arrow B). In embodiments, the gapmer hybridizes to a portion of the target transcript that differs from the amplified CUG trinucleotide repeat sequence, thereby inducing degradation of the target transcript via the rnase H pathway (fig. 1, arrow a). In embodiments, the gapmer hybridizes to at least a portion of the CUG repeat sequence amplified within the target transcript, thereby inducing degradation of the target transcript via the rnase H pathway (fig. 1, arrow B).

In embodiments, AC may modulate the activity of a target transcript. Modulation of activity may include increasing or decreasing the ability of the target transcript to bind to the binding partner. In embodiments, AC may modulate the activity of a target transcript by reducing its ability to bind to one or more proteins that may associate with the transcript, particularly to at least a portion of the amplified CUG repeat of the target transcript (fig. 1, arrow C). In embodiments, reducing the ability of the target transcript to bind to one or more proteins comprises reducing the affinity of the target transcript for the one or more proteins. In embodiments, reducing the ability of the target transcript to bind to one or more proteins comprises partially or completely spatially blocking the target transcript from binding to the one or more proteins. For example, AC may occupy at least a portion of a binding site that may be occupied by the one or more proteins if there is no steric hindrance. In embodiments, the binding site of the one or more proteins to the target transcript comprises at least a portion of an amplified trinucleotide repeat sequence of the target transcript. Thus, in embodiments, the AC may occupy at least a portion of the amplified trinucleotide repeat sequence (e.g., the amplified CUG repeat sequence), which may be occupied by the one or more proteins if not blocked spatially. Partial steric blocking of the target transcript may result in a decrease in affinity between the target transcript and the one or more proteins. For example, in embodiments, AC binds to at least a portion of the amplified CUG repeat of the target transcript, thereby spatially blocking and/or reducing the affinity of the target transcript for proteins that may bind to the amplified CUG repeat (fig. 1, arrow C). The following review article describes other applications of sterically blocking antisense oligonucleotides and is incorporated herein by reference in its entirety: roberts et al Nature Reviews Drug Discovery (2020) 19:673-694.

The CUG repeat of the amplified CUG repeat may form a double-stranded hairpin structure. In a disease state, proteins bind to double-stranded hairpin structures and are sequestered and cannot perform other functions. In embodiments, AC binds to at least a portion of the double-stranded hairpin structure, thereby sterically blocking and/or reducing the affinity of the double-stranded hairpin structure for the protein binding partner. In embodiments, AC binds to at least a portion of the single-stranded amplified CUG repeat, thereby inhibiting formation of a double-stranded hairpin structure and, thus, binding of one or more proteins to the double-stranded hairpin structure. In embodiments, hybridization of AC to the double hairpin structure spatially blocks and/or reduces the affinity of one or more proteins to bind to the double hairpin structure. In embodiments, hybridization of AC to at least a portion of the single stranded region of the amplified trinucleotide repeat sequence inhibits formation of a double stranded hairpin structure.

Reducing the ability of a target transcript to bind to the one or more proteins may allow the one or more proteins to perform other functions, such as, for example, regulating splicing of downstream transcripts (transcripts that do not contain amplified CUG repeats). Thus, in embodiments, decreasing the ability of a target transcript to bind to the one or more proteins may increase the level of the one or more proteins available within the cell to provide other functions or to act on other transcripts. In embodiments, decreasing the ability of a target transcript to bind to the one or more proteins may increase the cytoplasmic level of the one or more proteins that may be used within the cell to provide other functions or to act on other transcripts. Thus, in embodiments, binding of AC to a target transcript may result in modulation of the level and/or activity of the one or more proteins interacting with the target transcript.

In embodiments, hybridization of AC to at least a portion of the amplified CUG repeat reduces affinity and/or spatially blocks binding of MNBL1 to the target transcript. MNBL1 is a splicing factor that regulates splicing of downstream gene transcripts. In the DM1 disease phenotype, MNBL1 binds to the amplified CUG repeat of the target transcript. MNBL1 is sequestered in the nucleus when bound to the target transcript and is unable to regulate splicing of downstream gene transcripts (transcripts that do not contain amplified CUG repeats). In embodiments, AC hybridizes to at least a portion of the amplified CUG repeat in the target transcript, spatially blocks and/or reduces affinity of MNBL1 for the target transcript, thereby allowing it to regulate splicing of the downstream gene transcript. In embodiments, AC hybridizes to at least a portion of the amplified CUG repeat in the target transcript, spatially blocks and/or reduces affinity of MNBL1 for the target transcript, thereby increasing the amount of free (e.g., non-binding to a transcript having the CUG repeat) MNBL 1. In embodiments, AC hybridizes to at least a portion of the amplified CUG repeat in the target transcript, spatially blocks and/or reduces affinity of MNBL1 for the target transcript, thereby reducing the amount of MBNL1 bound to and sequestered by the target transcript.

In embodiments where the target transcript is a DMPK, hybridization of AC to at least a portion of the amplified CUG repeat reduces affinity and/or spatially blocks binding of MNBL1 to the target transcript. MNBL1 is a splicing factor that regulates splicing of downstream gene transcripts. In the DM1 disease phenotype, MNBL1 binds to the amplified CUG repeat of DMPK 1. When bound to DMPK1, MNBL1 is sequestered in the nucleus and is unable to regulate splicing of downstream gene transcripts (transcripts that do not contain amplified CUG repeats). In embodiments, AC hybridizes to at least a portion of the CUG repeat amplified in the DMPK, spatially blocks and/or reduces affinity of MNBL1 for DMPK transcripts, thereby allowing it to regulate splicing of downstream gene transcripts. In embodiments, AC hybridizes to at least a portion of the CUG repeat amplified in the DMPK, spatially blocks and/or reduces affinity of MNBL1 for DMPK transcripts, thereby increasing the amount of free (e.g., non-binding to transcripts having CUG repeat) MNBL 1. In embodiments, AC hybridizes to at least a portion of the CUG repeat amplified in the DMPK, spatially blocks and/or reduces affinity of MNBL1 for DMPK transcripts, thereby reducing the amount of MBNL1 that binds to and is sequestered by DMPK1 transcripts.

In embodiments where the target transcript is DMPK, hybridization of AC to at least a portion of the amplified CUG repeat results in a decrease in CUGBP levels. In the DM1 disease state, the level of free MBNL1 (capable of functioning) decreases, while the level of free CUGBP (capable of functioning) increases. An increase in CUGBP1 levels is associated with a disease state. Thus, in embodiments, hybridization of AC to at least a portion of the amplified CUG repeat results in an increase in free (functional) MBNL1 levels and/or a decrease in free (functional) CUGBP1 levels.

Reducing the ability of the target transcript to bind to the one or more proteins may reduce or inhibit CUG repeat foci formation. Transcripts that include amplified nucleotide repeats (e.g., amplified CUG repeats) may be transcribed and then sequestered in the nucleus. Within the nucleus, sequestered transcripts may form aggregates. Proteins that bind to the transcript may then nucleate on the sequestered transcript and/or sequestered transcript aggregates, thereby forming an amplified nucleotide repeat (e.g., CUG repeat) pool. CUG repeat foci can be seen using a microscope. In embodiments, reducing the ability of the target transcript to bind to the one or more proteins may reduce or inhibit the formation of aggregates comprising the target transcript. In embodiments, reducing the ability of the target transcript to bind to the one or more proteins may reduce or inhibit nucleation of the one or more proteins on the target transcript, on a double-stranded hairpin region of the transcript, or on an aggregate of the target transcript. In embodiments where the target transcript is a DMPK, reducing the ability of the DMPK1 target transcript to bind to MNBL1 may reduce or inhibit nucleation of MNBL1 on the DMPK1 target transcript or the DMPK1 target transcript aggregate. In embodiments, hybridization of AC to a target transcript may result in inhibition or reduction of CUG repeat foci formation. In embodiments, hybridization of AC to a target transcript may result in inhibition or reduction of CUG repeat foci formation by DMPK, TCF4, JPH3, and/or ATXN8OS/ATXN8 target transcripts.

In embodiments, hybridization of AC to a target transcript may result in modulation of the level, expression, and/or activity of one or more downstream genes. For example, hybridization of AC to a target transcript may be used to induce degradation of the target transcript or spatially block or reduce affinity of the target transcript for one or more proteins, thereby allowing the one or more proteins sequestered by the target transcript to regulate expression, level and/or activity of a downstream gene. For example, in embodiments, the one or more proteins may include a protein involved in regulating splicing of one or more downstream transcripts (transcripts that do not contain amplified CUG repeats). In embodiments, splicing of downstream transcripts is altered when proteins involved in splicing bind to and sequester on target transcripts. For example, a change in splicing may include the exclusion of one or more exons or the inclusion of one or more introns in a transcript, resulting in the expression of various protein isoforms. In embodiments, the alteration of splicing may result in the inclusion of exons and/or introns containing premature stop codons, thereby producing truncated isoforms that may be inactive or have deleterious activity. Alterations in downstream gene transcript splicing can result in changes in downstream gene product levels, folding, and/or activity that may be correlated with disease phenotypes. When not bound to a target transcript comprising an amplified CUG repeat, the proteins involved in splicing are free to regulate splicing, which can lead to correction (or rescue) of downstream gene transcript splicing, thereby at least partially restoring protein levels, folding, and/or activity of downstream gene products associated with a healthy phenotype.

In embodiments, hybridization of AC to a target transcript may result in modulation of splicing of a downstream gene transcript regulated by a protein sequestered by the target transcript under a disease state associated with an amplified nucleotide repeat (e.g., an amplified trinucleotide repeat). In diseases associated with amplified trinucleotide repeats, downstream gene transcripts are often misprocessed, e.g., mis-spliced. Mis-splicing of downstream gene transcripts can produce gene products that are disrupted prior to translation or translated into proteins with aberrant structure and/or function. For example, isolating a protein that regulates processing of a downstream gene transcript may result in a transcript and/or gene product that includes exons and/or introns with premature stop codons, includes introns, excludes exons, and/or includes selective exons that may be disrupted prior to translation or translated into a gene product with aberrant function. The level change of the downstream gene transcript and/or gene product and/or the abnormal structure and/or function of the downstream gene product is correlated with the amplified trinucleotide disease phenotype. In embodiments, hybridization of AC to a target transcript may result in modulation of exon inclusion, exon exclusion, intron inclusion, and/or intron exclusion in downstream transcripts whose splicing is regulated by proteins sequestered to the target transcript during the disease state. Thus, hybridization of AC to a target transcript may cause up-regulation of downstream protein isoforms and/or transcripts associated with a healthy phenotype. Similarly, hybridization of AC to a target transcript may cause downregulation (e.g., repression) of downstream transcripts and/or protein isoforms associated with the disease phenotype.

In embodiments where the target transcript is a DMPK, hybridization of AC to the target transcript may result in modulation of splicing of downstream gene transcripts regulated by proteins sequestered by the DMPK target transcript during the disease state. In DM1, several downstream gene transcripts are produced by mis-splicing. The mis-spliced gene is associated with a disease phenotype. Thus, modulation of gene splicing may include correcting (e.g., rescuing) splicing of a gene to produce a gene product of a downstream gene associated with a healthy phenotype. In embodiments where the target transcript is a DMPK, hybridization of AC to the target transcript may result in modulation of splicing of the downstream gene transcript regulated by the splicing regulator MNBL1 sequestered by the DMPK target transcript during the disease state. In embodiments where the target transcript is DMPK, hybridization of AC to the target transcript may result in modulation of splicing of the downstream gene transcript regulated by protein CUGBP1 whose activity is affected by the amplified CUG repeat. In embodiments where the target transcript is DMPK, hybridization of AC to the target transcript may cause proper processing (e.g., splicing) of downstream genes regulated by MNBL1 and/or CUGBP 1. In embodiments where the target transcript is DMPK, AC hybridization to the target transcript may result in modulation of splicing of downstream genes including, but not limited to 4833439L19Rik、Abcc9、Atp2a1、Arhgef10、Arhgap28、Armcx6、Angel1、Best3、Bin1、Brd2、Cacna1s、Cacna2d1、Cpd、Cpeb3、Ccpg1、Clasp1、ClC-1、Clcn1、Clk4、Cpeb2、Camk2g、Capzb、Copz2、Coch、cTNT、Ctu2、Cyp2s1、Dctn4、Dnm1l、Eya4、Efna3、Efna2、Fbxo31、Fbxo21、Frem2、Fgd4、Fuca1、Fn1、Gogla4、Gpr37l1、Greb1、Heg1、Insr、Impdh2、IR、Itgav、Jag2、Klc1、Kcan6、Kif13a、Ldb3、Lrrfip2、Mapt、Macf1、Map3k4、Mapkap1、Mbnl1、Mllt3、Mbnl2、Mef2c、Mpdz、Mrpl1、Mxra7、Mybpc1、Myo9a、Ncapd3、Ngfr、Ndrg3、Ndufv3、Neb、Nfix、Numa1、Opa1、Pacsin2、Pcolce、Pdlim3、Pla2g15、Phactr4、Phka1、Phtf2、Ppp1r12b、Ppp3cc、Ppp1cc、Ramp2、Rapgef1、Rur1、Ryr1、Sorcs2、Spsb4、Scube2、Sema6c、Sfc8a3、Slain2、Sorbs1、Spag9、Tmem28、Tacc1、Tacc2、Ttc7、Tnik、Tnfrsf22、Tnfrsf25、Trappc9、Trim55、Ttn、Txnl4a、Txlnb、Ube2d3、Vsp39, or any combination thereof.

Mis-splicing of many of the above downstream gene transcripts resulted in specific DM1 disease phenotypes. For example, MNBL1 is a splicing factor that is disabled in DM1 by the inclusion of exon 5. MNBL1 sequesters and forms RNA foci by DMPK CUG amplification. In addition, SOS1 promotes Ras activation to positively regulate the RAS/MAPK signaling pathway. In DM1, exon 25 of SOS1 was excluded, leading to inhibition of the muscle hypertrophy pathway. In DM1, IR/INSR has an exon 11 exclusion, which results in higher levels of low signaling non-muscle isoforms in DM1 and reduced metabolic response to insulin (insulin resistance). Similarly, the exon 78 exclusion of DMD was observed in DM. Exon 78 excludes out-of-frame transcripts that produce the C-terminal domain. This mutant protein is expressed in DM1 patients and is associated with mechanisms responsible for muscle wasting in patients. BIN1 is required for normal muscle T-tubule formation (EC coupling). Exon 11 is excluded from producing inactive isoforms and is found in DM1 patients. LDB3 interacts with α -actin at the striated muscle Z-disc and maintains muscle structure. Exon 11 of LDB3 was detected in DM1 to contain a sequence that resulted in reduced affinity for Protein Kinase C (PKC). Accordingly, PKC becomes abnormally active in DM 1. In embodiments, modulation of one or more downstream genes results in correction or rescue of transcript splicing associated with a healthy phenotype. Thus, in embodiments, hybridization of AC to a DMPK target transcript results in rescue of the mis-splicing of the downstream gene/transcript, thereby reducing the level of the downstream gene/transcript associated with the disease phenotype. Thus, in embodiments, hybridization of AC to a DMPK target transcript results in rescue of the mis-splicing of the downstream gene/transcript, thereby increasing the level of the downstream gene/transcript associated with a healthy phenotype.

In embodiments, AC inhibits expression of a target transcript. In embodiments, AC inhibits expression of a target transcript by blocking entry into and/or completion of translation by a pre-mRNA processing machinery and/or translation machinery. In embodiments, AC inhibits expression of a target transcript by inducing degradation of the target transcript, e.g., via the rnase H pathway.

AC structure

AC includes oligonucleotides and/or oligonucleotides. Oligonucleotides and/or oligonucleotides are nucleotides or nucleosides linked by internucleoside linkages. Nucleosides include pentoses (e.g., ribose or deoxyribose) and nitrogen-containing bases covalently attached to the sugar. Naturally occurring bases (or conventional bases) found in DNA and/or RNA are adenine (a), guanine (G), thymine (T), cytosine (C) and uracil (U). Naturally occurring sugars (or conventional sugars) found in DNA and/or RNA are Deoxyribose (DNA) and Ribose (RNA). Naturally occurring nucleoside linkages (or traditional internucleoside linkages) are phosphodiester linkages. In embodiments, the AC of the present disclosure may have all natural sugar, base, and internucleoside linkages.

Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for a target RNA. In embodiments, the AC of the present disclosure may have one or more modified nucleosides. In embodiments, the AC of the present disclosure may have one or more modified sugars. In embodiments, the AC of the present disclosure may have one or more modified bases. In embodiments, the ACs of the present disclosure may have one or more modified internucleoside linkages.

In general, a nucleobase is any group containing one or more atoms or groups of atoms capable of hydrogen bonding to the base of another nucleic acid. In addition to "unmodified" or "natural" nucleobases (A, G, T, C and U), many modified nucleobases or nucleobase mimics are known to those of skill in the art and can be affected by the compounds described herein. In general, a modified nucleobase refers to a nucleobase that is very similar in structure to the parent nucleobase, such as 7-deazapurine, 5-methylcytosine, 2-thio-dT (FIG. 2) or G-clamp. Typically, nucleobase mimetics are nucleobases that include more complex structures than modified nucleobases, such as tricyclic phenoxazine nucleobase mimetics. Methods for preparing the modified nucleobases described above are well known to those skilled in the art.

In embodiments, AC may include one or more nucleosides with modified sugar moieties. In embodiments, the furanosyl sugar of the natural nucleoside may have a 2' modification, a modification that forms a limiting nucleoside, or the like (see fig. 2). For example, in embodiments, the furanosyl sugar ring of a natural nucleoside can be modified in a variety of ways, including but not limited to adding substituents, bridging two non-geminal ring atoms to form a Bicyclic Nucleic Acid (BNA) or locked nucleic acid; exchanging the oxygen of the furanosyl ring with C or N; and/or substituted atoms or groups (see figure 2). Modified sugars are well known and can be used to increase or decrease the affinity of AC for its target nucleotide sequence. Modified sugars may also be used to increase resistance of AC to nucleases. The sugar may be replaced with a sugar mimetic group or the like. In embodiments, one or more of the sugars of the nucleoside of AC are replaced with a methylene morpholine ring, as shown at 19 in fig. 2.

In embodiments, AC comprises one or more nucleosides that include a bicyclic modified sugar (BNA; sometimes referred to as a bridging nucleic acid). Examples of BNA include, but are not limited to, LNA (4 '- (CH ₂) -O-2' bridge), 2 '-thio-LNA (4' - (CH ₂) -S-2 'bridge), 2' -amino-LNA (4 '- (CH ₂) -NR-2' bridge), ENA (4 '- (CH ₂)₂ -O-2' bridge), 4'- (CH ₂)₃ -2' bridge BNA, 4'- (CH ₂CH(CH₃)) -2' bridge BNA "cEt (4 '- (CH (CH ₃) -O-2' bridge) and cMOE BNA (4 '- (CH (CH ₂OCH₃) -O-2' bridge). BNA has been prepared and disclosed in the patent literature as well as in the scientific literature (Srivastava et al, J.am.chem. Soc. (2007), ACS newly published online, 10.1021/ja071106y; albaek et al, J.Org. chem. (2006), 71,7731-7740; fluid et al, chembiochem (2005), 6,1104-1109; singh et al, chem. Commun. (1998), 4,455-456; koshkin et al, tetrahedron (1998), 54,3607-3630; wahlestedt et al, proc.Natl. Acad. Sci. U.S. A. (2000), 97,5633-5638; kumar et al, bioorg. Med. Chem. Lett. (1998), 8,2219-2222; WO 94/14226; WO 2005/021570; singh et al, J.Org. chem. (1998), 63,10035-10039, WO 2007/090071; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,748, 6,794,499, 7,034,133 and 8; U.S. UK.S. A. 2000; U.S. publication No. WO 021017,950, 2004-4959, 2003-4959; U.S. A. publication No. 4343,021, 5,1575 2004-0143114 and 20030082807).

In embodiments, the AC comprises one or more nucleosides, including a Locked Nucleic Acid (LNA). In LNA, the 2 '-hydroxyl group of the ribosyl sugar ring is attached to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4' -C-oxymethylene linkage, to form a bicyclic sugar moiety (reviewed in Elayadi et al, curr. Opinion Invens. Drugs (2001), 2,558-561; braasch et al, chem. Biol. (2001), 8 1-7; and Orum et al, curr. Opinion mol. Ther. (2001), 3,239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The bond may be a methylene (-CH ₂ -) group bridging the 2 'oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic moiety; in the case of an ethylene group at this position, the term ENA ^TM (Singh et al, chem. Commun. (1998), 4,455-456; ENA ^TM; morita et al, bioorganic MEDICINAL CHEMISTRY (2003), 11, 2211-2226) is used. LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability (Tm= +3 to +10℃) to complementary DNA and RNA, stability to 3' -exonuclease degradation and good solubility. Effective and nontoxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al, proc. Natl. Acad. Sci. U.S. A. (2000), 97, 5633-5638).

The isomer of LNA that has also been studied is alpha-L-LNA, which has been demonstrated to have excellent stability to 3' -exonucleases. alpha-L-LNA was incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al, nucleic ACIDS RESEARCH (2003), 21, 6365-6372).

The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil, as well as their oligomerization and nucleic acid recognition properties have been described (Koshkin et al Tetrahedron 1998,54,3607-3630). LNA and its preparation are also described in WO 98/39352 and WO 99/14226.

Analogs of LNA, phosphorothioate-LNA and 2' -thio-LNA have also been prepared (Kumar et al, biorg. Med. Chem. Lett.,1998,8,2219-2222). The preparation of LNA analogues containing oligodeoxyribonucleotide duplex as substrates for nucleic acid polymerase has also been described (Wengel et al, WO 99/14226). In addition, synthesis of 2' -amino-LNA, a conformationally constrained high affinity oligonucleotide analogue, has been described (Singh et al, J.Org.chem. (1998), 63, 10035-10039). In addition, 2 '-amino-and 2' -methylamino-LNAs have been prepared and their thermal stability of duplex with complementary RNA and DNA strands has been previously reported.

Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. patent ：4,981,957;5,118,800;5,319,080;5,359,044;5,393,878;5,446,137;5,466,786;5,514,785;5,519,134;5,567,811;5,576,427;5,591,722;5,597,909;5,610,300;5,627,053;5,639,873;5,646,265;5,658,873;5,670,633;5,792,747;5,700,920; and 6,600,032; WO 2005/121371.

Internucleoside linkages

Described herein are internucleoside linking groups that link together nucleosides or other modified nucleoside monomer units to form oligonucleotides and/or oligonucleotide-containing ACs. AC may include naturally occurring internucleoside linkages, non-natural internucleoside linkages, or both.

In naturally occurring DNA and RNA, an internucleoside linkage group is a phosphodiester that covalently links adjacent nucleosides to each other to form a linear polymeric compound. In naturally occurring DNA and RNA, phosphodiester is linked to the 2', 3' or 5' hydroxyl moiety of a sugar. Within an oligonucleotide, phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. In naturally occurring DNA and RNA, the linkage or backbone of RNA and DNA is a3 'to 5' phosphodiester linkage. In embodiments, the internucleoside linking group of AC is a phosphodiester. In embodiments, the internucleoside linkage of AC is a3 'to 5' phosphodiester linkage.

Two main classes of non-natural internucleoside linkages are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Representative phosphorus-free internucleoside linkages include, but are not limited to, methylenemethylamino (-CH ₂-N(CH₃)-O-CH₂ -), thiodiester (-O-C (O) -S-), thiocarbamate (-O-C (O) (NH) -S-); siloxane (-O-Si (H ₂ -O-) and N, N' -dimethylhydrazine (-CH ₂-N(CH₃)-N(CH₃) -). AC with a phosphorus internucleoside linkage is referred to as an oligonucleotide. Antisense compounds having non-phosphorus internucleoside linkages are referred to as oligonucleotides. Modified internucleoside linkages can be used to alter (typically increase) nuclease resistance of antisense compounds as compared to native phosphodiester linkages. Internucleoside linkages having chiral atoms can be prepared as racemic, chiral or mixtures. Representative chiral internucleoside linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free linkages are well known to those skilled in the art.

In embodiments, two or more nucleosides with modified sugars and/or modified nucleobases can be linked using phosphoramidates. In embodiments, two or more nucleosides having a methylene morpholine ring can be linked by phosphoramidate internucleoside linkages.

Antisense compounds comprising nucleobases with a methylene morpholine ring linked by phosphoramidate internucleoside linkages can be referred to as Phosphoramidate Morpholine Oligomers (PMOs).

Conjugation group

In embodiments, AC is modified by covalent attachment of one or more conjugate groups. Typically, the conjugate group modifies one or more properties of the attached AC, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts to connect to a parent compound such as AC either directly or via an optional linking moiety or linking group. Conjugation groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterols, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In embodiments, the conjugation group is polyethylene glycol (PEG), and the PEG is conjugated to AC or CPP (CPP discussed elsewhere herein).

In embodiments, the conjugate group includes a lipid moiety, such as a cholesterol moiety (Letsinger et al, proc.Natl. Acad.Sci.USA (1989), 86,6553); cholic acid (Manoharan et al, biorg. Med. Chem. Lett. (1994), 4,1053); thioethers, for example hexyl-S-tritylthiol (Manoharan et al, ann.N. Y. Acad. Sci. (1992), 660,306; manoharan et al, biorg. Med. Chem. Let. (1993), 3,2765); thiocholesterol (Oberhauser et al, nucleic acids res. (1992), 20,533); aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J. (1991), 10,111; kabanov et al, FEBS Lett. (1990), 259,327; svinarchuk et al, biochimie (1993), 75,49); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium-1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al, tetrahedron lett. (1995), 36,3651; shea et al, nucleic acids res. (1990), 18,3777); polyamine or polyethylene glycol chains (Manoharan et al, nucleic & nucleic acids (1995), 14,969); adamantaneacetic acid (Manoharan et al, tetrahedron lett (1995), 36,3651); palm-based moiety (Mishra et al, biochim. Biophys. Acta. (1995), 1264,229); or octadecylamine or hexylamine-carbonyl-oxy cholesterol moiety (Crooke et al, j. Pharmacol. Exp. Ter. (1996), 277,923).

Type of antisense compound

Various types of ACs may be used, including, for example, antisense oligonucleotides, sirnas, micro RNA, antagomir, aptamers, ribozymes, supermir, miRNA mimics, miRNA inhibitors, or combinations thereof.

Antisense oligonucleotides

In various embodiments, the Antisense Compound (AC) is an antisense oligonucleotide (ASO) complementary to a target nucleotide sequence. The term "antisense oligonucleotide (ASO)" or simply "antisense" is intended to include oligonucleotides complementary to a target nucleotide sequence. The term also encompasses ASOs that may not be fully complementary to the desired target nucleotide sequence. ASOs comprise single strands of DNA and/or RNA complementary to a selected target nucleotide sequence or target gene. ASOs may include one or more modified DNA and/or RNA bases, modified sugars, and/or unnatural internucleoside linkages. In embodiments, the ASO may include one or more phosphoramidate internucleoside linkages. In embodiments, the ASO is a Phosphoroamidate Morpholino Oligomer (PMO). ASOs may have any characteristic, may be of any length, may bind to any target nucleotide sequence and/or sequence element, and may implement any of the mechanisms described in connection with AC.

Antisense oligonucleotides have proven to be effective as targeted inhibitors of protein synthesis and, thus, can be used to specifically inhibit protein synthesis of the targeted gene. The efficacy of ASO in inhibiting protein synthesis is well established. To date, these compounds have shown promise in several in vitro and in vivo models, including models of inflammatory disease, cancer and HIV (Agrawal, trends in Biotech. (1996), 14:376-387). Antisense can also affect cellular activity by specifically hybridizing to chromosomal DNA.

Methods of generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeted to any polynucleotide sequence. The selection of antisense oligonucleotide sequences specific for a given target sequence is based on analysis of the selected target sequence and determination of secondary structure, tm, binding energy and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to target mRNA in a host cell. Target regions of mRNA include those regions at or near AUG translation initiation codon and those sequences that are substantially complementary to the 5' region of mRNA. These secondary structural analysis and target site selection considerations may be performed, for example, using the v.4 (Molecular Biology Insights) and/or BLASTN 2.0.5 algorithm software of the OLIGO primer analysis software (Altschul et al, nucleic Acids Res.1997,25 (17): 3389-402).

RNA interference

In embodiments, the AC includes molecules that mediate RNA interference (RNAi). As used herein, the phrase "mediate RNAi" refers to the ability to silence a target transcript in a sequence-specific manner. While not wishing to be bound by theory, it is believed that silencing uses RNAi machinery or processes and guide RNAs, such as siRNA compounds of about 21 to about 23 nucleotides. In embodiments, the AC targets the target transcript for degradation. Thus, in embodiments, RNAi molecules can be used to disrupt expression of a gene or polynucleotide of interest. In embodiments, the RNAi molecules are used to induce degradation of a target transcript, such as a pre-mRNA or mature mRNA.

In embodiments, the AC comprises small interfering RNAs (sirnas) that elicit an RNAi response.

Small interfering RNAs (sirnas) are nucleic acid duplex, typically about 16 to about 30 nucleotides long, that can associate with cytoplasmic polyprotein complexes known as RNAi-induced silencing complexes (RISC). RISC loaded with siRNA mediates degradation of homologous transcripts, so siRNA can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA functions through a natural mechanism that advances to control gene expression through non-coding RNAs. A variety of RNAi agents, including siRNAs targeting clinically relevant targets, are currently under drug development, for example, as described in Fougerolles, A. Et al, nature Reviews (2007) 6:443-453.

Although the first RNAi molecules described are RNA-to-RNA hybrids comprising an RNA sense strand and an RNA antisense strand, DNA sense-to-RNA antisense hybrids, RNA sense-to-DNA antisense hybrids and DNA-to-DNA hybrids have now been demonstrated to mediate RNAi (Lamberton, J.S. and Christian, A.T., molecular Biotechnology (2003), 24:111-119). In embodiments, RNAi molecules used include any of these different types of double-stranded molecules. In addition, it should be understood that RNAi molecules can be used and introduced into cells in a variety of forms. Thus, as used herein, RNAi molecules encompass any and all molecules capable of mediating RNAi in cells, including, but not limited to: double-stranded oligonucleotides comprising two separate strands, a sense strand and an antisense strand, such as small interfering RNAs (sirnas); a double-stranded oligonucleotide comprising two separate strands joined together by a non-nucleotide linker; an oligonucleotide comprising a hairpin loop that forms a complement of a double stranded region, such as a shRNAi molecule, and an expression vector that expresses one or more polynucleotides capable of forming a double stranded polynucleotide alone or in combination with another polynucleotide.

As used herein, a "single stranded siRNA compound" is an siRNA compound consisting of a single molecule. It may comprise a double stranded region formed by intra-strand pairing, for example, it may be or comprise a hairpin or disc-handle structure. The single stranded siRNA compound may be antisense to the target molecule.

The single stranded siRNA compound may be long enough that it can enter RISC and participate in RISC-mediated cleavage of target mRNA. The single stranded siRNA compound is at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, or at most about 50 nucleotides in length. In certain embodiments, the single stranded siRNA is less than about 200, about 100 or about 60 nucleotides in length.

The hairpin siRNA compound can have a duplex region equal to or at least about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotide pairs. The duplex region may be equal to or less than about 200, about 100, or about 50 nucleotide pairs in length. In certain embodiments, the duplex region ranges in length from about 15 to about 30, from about 17 to about 23, from about 19 to about 23, and from about 19 to about 21 nucleotide pairs. The hairpin may have a single stranded overhang or a terminal unpaired region. In certain embodiments, the length of the overhang is about 2 to about 3 nucleotides. In embodiments, the overhangs are on the same side of the hairpin, and in embodiments on the antisense side of the hairpin.

As used herein, a "double stranded siRNA compound" is an siRNA compound that includes more than one strand, and in some cases, two strands, where strand hybridization can form a region of double stranded structure.

The antisense strand length of the double stranded siRNA compound can be equal to or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, or about 60 nucleotides. It may be equal to or less than about 200, about 100, or about 50 nucleotides in length. The length may range from about 17 to about 25, from about 19 to about 23, and from about 19 to about 21 nucleotides. As used herein, the term "antisense strand" means a strand of an siRNA compound that is sufficiently complementary to a target nucleotide sequence of a target molecule, e.g., a target transcript.

The sense strand length of the double stranded siRNA compound may be equal to or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40 or about 60 nucleotides. It may be equal to or less than about 200, about 100, or about 50 nucleotides in length. The length may range from about 17 to about 25, from about 19 to about 23, and from about 19 to about 21 nucleotides.

The double stranded portion of the double stranded siRNA compound may be equal to or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 40 or about 60 nucleotide pairs in length. It may be equal to or less than about 200, about 100, or about 50 nucleotide pairs in length. The length can range from about 15 to about 30, from about 17 to about 23, from about 19 to about 23, and from about 19 to about 21 nucleotide pairs.

In embodiments, the siRNA compound is sufficiently large that it can be cleaved by an endogenous molecule, such as Dicer, to produce smaller siRNA compounds, such as siRNA agents.

The sense strand and the antisense strand can be selected such that the double stranded siRNA compound comprises a single stranded or unpaired region at one or both ends of the molecule. Thus, a double stranded siRNA compound may contain a sense strand and an antisense strand paired to contain an overhang, such as one or two 5' or 3' overhangs, or a 3' overhang of 1-3 nucleotides. The overhangs may be the result of one strand being longer than the other, or the result of two strands of the same length being interleaved. Some embodiments will have at least one 3' overhang. In embodiments, both ends of the siRNA molecule will have a 3' overhang. In embodiments, the overhang is 2 nucleotides.

In embodiments, the duplex region is about 15 to about 30, or about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides in length, for example in the ssiRNA (cohesive overhang siRNA) compound range discussed above. ssiRNA compounds can be similar in length and structure to the natural Dicer processing products from long dsiRNA. Also included are embodiments wherein both chains of ssiRNA compounds are linked, e.g., covalently linked. In embodiments, hairpins or other single stranded structures that provide a double stranded region and a 3' overhang are included.

The siRNA compounds described herein, including double stranded siRNA compounds and single stranded siRNA compounds, can mediate silencing of target RNA, e.g., mRNA, e.g., transcripts of a gene encoding a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such genes are also referred to as target genes. In general, the RNA to be silenced is an endogenous gene.

In embodiments, the siRNA compound is "sufficiently complementary" to the target transcript such that the siRNA compound silences the production of a protein encoded by the target mRNA. In embodiments, the siRNA compound is "sufficiently complementary" to at least a portion of the target transcript such that the siRNA compound silences the production of a gene product encoded by the target transcript. In another embodiment, the siRNA compound is "exactly complementary" to the target nucleotide sequence (e.g., a portion of the target transcript) such that the target nucleotide sequence and the siRNA compound anneal, e.g., form a hybrid consisting of only watson-crick base pairs in the region of the exact complementarity. "substantially complementary" to a target nucleotide sequence can include an interior region (e.g., at least about 10 nucleotides) that is precisely complementary to the target nucleotide sequence. Moreover, in certain embodiments, the siRNA compounds specifically distinguish single nucleotide differences. In this case, the siRNA compound only mediates RNAi if exact complementarity is found in the region of single nucleotide difference (e.g., within 7 nucleotides).

RNAi has a very wide range of therapeutic applications, as siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against a target protein. To date, siRNA constructs have been shown to specifically down-regulate target proteins in vitro and in vivo models and in clinical studies

MicroRNA

In embodiments, the AC comprises a microrna molecule. Micrornas (mirnas) are a highly conserved class of small RNA molecules that are transcribed from DNA in plant and animal genomes but are not translated into proteins. Processed mirnas are 17-25 nucleotide single stranded RNA molecules that are incorporated into RNA-induced silencing complexes (RISC) and have been identified as key regulatory factors for development, cell proliferation, apoptosis and differentiation. They are believed to play a role in the regulation of gene expression by binding to the 3' -untranslated region of specific mRNAs. RISC mediates down-regulation of gene expression by translational inhibition, transcriptional cleavage, or both. RISC is also involved in transcriptional silencing in a variety of eukaryotic nuclei.

antagomir

In embodiments, AC is antagomir. antagomir is an RNA-like oligonucleotide with various modifications directed against rnase protection and pharmacological properties such as enhanced tissue and cell uptake. They differ from normal RNAs in, for example, the complete 2 '-0-methylation of the sugar, phosphorothioate backbone, and cholesterol moieties, for example, at the 3' -end. antagomir can be used to effectively silence endogenous mirnas by forming a duplex comprising antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. an example of antagomir-mediated miRNA silencing is that of miR-122, described in Krutzfeldt et al, nature (2005), 438:685-689, which is expressly incorporated herein by reference in its entirety. The antagomir RNA can be synthesized using standard solid phase oligonucleotide synthesis protocols (U.S. patent application Ser. Nos. 11/502,158 and 11/657,341; each of which is incorporated herein by reference).

Antagomir may include ligand conjugated monomer subunits and monomers for oligonucleotide synthesis. Monomers are described in U.S. application Ser. No. 10/916,185. antagomir may have a ZXY structure, as described in PCT application No. PCT/US 2004/07070. antagomir may be complexed with an amphiphilic moiety. Amphiphilic moieties for use with oligonucleotide reagents are described in PCT application No. PCT/US 2004/07070.

Aptamer

In embodiments, the AC includes an aptamer. Aptamers are nucleic acid or peptide molecules that bind with high affinity and specificity to a particular target molecule (Tuerk and Gold, science 249:505 (1990); ellington and Szostak, nature 346:818 (1990)). DNA or RNA aptamers have been successfully produced that bind many different entities from large proteins to small organic molecules (Eaton, curr. Opin. Chem. Biol. (1997), 1:10-16; famulok, curr. Opin. Structure. Biol. (1999), 9:324-9; and Hermann and Patel, science (2000), 287:820-5). The aptamer may be RNA or DNA based and may include a riboswitch. Riboswitches are part of an mRNA molecule that can bind directly to a small target molecule, whose binding to the target affects gene activity. Thus, mRNA containing riboswitches is directly involved in regulating its own activity, depending on the presence or absence of the target molecule. In general, aptamers are engineered by multiple rounds of repeated in vitro selection or equivalently SELEX (evolution through an exponentially enriched ligand system) to bind to various molecular targets such as small molecules, proteins, nucleic acids, even cells, tissues and organisms. The aptamer may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other aptamers specific for the same target. In addition, the term "aptamer" also includes "secondary aptamers," which contain consensus sequences obtained by comparing two or more known aptamers to a given target. In embodiments, the aptamer is an "intracellular aptamer", or "intermer", which specifically recognizes an intracellular target (Famulok et al, chem biol. (2001), 8 (10): 931-939; yoon and Rossi, adv. Drug deliv. Rev. (2018), 134:22-35; each of which is incorporated herein by reference).

Ribozyme

In embodiments, AC is a ribozyme. Ribozymes are complexes of RNA molecules with endonuclease activity having specific catalytic domains (Kim and Cech, proc. Natl. Acad. Sci. USA (1987), 84 (24): 8788-92; forster and Symons, cell (1987) 24,49 (2): 211-20). For example, a large number of ribozymes accelerate the phosphotransesterification reaction with a high degree of specificity, typically cleaving only one of several phosphates in an oligonucleotide substrate (Cech et al, cell (1981), 27 (3 Pt 2): 487-96; michel and Westhof, J.mol.biol. (1990), 5,216 (3): 585-610; reinhold-Hurek and Shub, nature (1992), 14,357 (6374): 173-6). This specificity has been attributed to the need for the substrate to bind to the Internal Guide Sequence (IGS) of the ribozyme via specific base pairing interactions prior to the chemical reaction.

At least six basic varieties of naturally occurring enzymatic RNAs are currently known. Under physiological conditions, each ribozyme can catalyze trans-hydrolysis of RNA phosphodiester bonds (and thus can cleave other RNA molecules), and in general, enzymatic nucleic acids act by first binding to the target RNA. Such binding occurs through a target binding moiety of the enzymatic nucleic acid that is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes a target RNA, then binds to the target RNA by complementary base pairing, and once bound to the correct site, enzymatically cleaves the target RNA. Strategic cleavage of such target RNAs would destroy their ability to direct synthesis of the encoded protein. After an enzymatic nucleic acid binds to and cleaves its RNA target, it is released from the RNA to find another target, and can repeatedly bind to and cleave the new target.

For example, the enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, delta hepatitis virus, class I intron, or rnase P RNA (associated with an RNA guide sequence) or a neurospora VS RNA motif. Specific examples of hammerhead motifs are described by Rossi et al Nucleic Acids Res (1992), 20 (17): 4559-65. Examples of hairpin motifs are described by Hampel et al (European patent application publication No. EP 0360257), hampel and Tritz, biochemistry (1989), 28 (12): 4929-33; hampel et al, nucleic Acids Res (1990), 18 (2): 299-304 and U.S. Pat. No.5,631,359. Examples of hepatitis virus motifs are described by Perrotta and ben, biochemistry (1992), 31 (47): 11843-52; examples of RNAse P motifs are described by Guerrier-Takada et al, cell (1983), 35 (3 Pt 2): 849-57; neurospora VS RNA ribozyme motifs are described by Collins (Saville and Collins,Cell(1990),61(4):685-96;Saville and Collins,Proc.Natl.Acad.Sci.USA(1991),88(19):8826-30;Collins and Olive, biochemistry (1993), 32 (l l): 2795-9); and examples of class I introns are described in U.S. Pat. No. 4,987,071. In embodiments, the enzymatic nucleic acid molecules have specific substrate binding sites complementary to one or more target gene DNA or RNA regions, and they have nucleotide sequences within or around the substrate binding sites that confer RNA cleavage activity on the molecule. Thus, the ribozyme construct need not be limited to the specific motifs mentioned herein.

Ribozymes can be designed as described in International patent application publication No. WO 93/23569 and International patent application publication No. WO 94/02595, each expressly incorporated herein by reference, and synthesized as described therein for in vitro and in vivo testing. In embodiments, the ribozyme is targeted to a target nucleotide sequence of a target transcript.

The ribozyme activity may be increased by altering the length of the ribozyme binding arm or chemically synthesizing a ribozyme having modifications that prevent its degradation by serum ribonucleases (see, e.g., international patent application publication No. WO 92/07065; international patent application publication No. WO 93/15187; international patent application publication No. WO 91/03162; european patent application publication No. 92110298.4; U.S. patent 5,334,711; and International patent application publication No. WO 94/13688, which describe various chemical modifications that may be made to the sugar portion of an enzymatic RNA molecule), which modifications enhance their efficacy in cells, and the removal of the stem pi base to shorten RNA synthesis time and reduce chemical requirements.

Supermir

In an embodiment, AC is supermir. supermir refers to single-stranded, double-stranded or partially double-stranded RNA oligomers or polymers, DNA polymers, or both, or modifications thereof having a nucleotide sequence that is substantially identical to a miRNA and antisense to its target, the term including oligonucleotides consisting of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, and which contain at least one functionally similar non-naturally occurring moiety. Such modified or substituted oligonucleotides have desirable properties, e.g., enhanced cellular uptake, enhanced affinity for nucleic acid targets, and enhanced stability in the presence of nucleases. In embodiments, supermir does not include a sense strand, and in another embodiment, supermir does not self-hybridize to a significant extent. supermir may have a secondary structure but is substantially single stranded under physiological conditions. Substantially single-stranded supermir is single-stranded to an extent of less than about 50% (e.g., less than about 40%, about 30%, about 20%, about 10%, or about 5%) of supermir is duplex with itself. supermir can include hairpin segments, e.g., sequences, e.g., duplex regions that can hybridize to themselves and form duplex regions at the 3' end, e.g., duplex regions of at least about 1, about 2, about 3, or about 4 or less than about 8, about 7, about 6, or about 5 nucleotides. The duplex regions may be joined by a linker, such as a nucleotide linker, e.g., about 3, about 4, about 5, or about 6 dT, e.g., modified dT. In another embodiment supermir is duplex with shorter oligomers, e.g., oligomers about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides in length, e.g., at one or both of the 3 'and 5' ends of supermir, or at one end and non-terminal or middle.

MiRNA mimics

In embodiments, AC is a miRNA mimic. miRNA mimics represent a class of molecules that can be used to mimic the gene silencing ability of one or more mirnas. Thus, the term "microrna mimetic" refers to a synthetic non-coding RNA that is capable of entering the RNAi pathway and regulating gene expression (i.e., a miRNA that is not obtained by purification from an endogenous miRNA source). miRNA mimics may be designed as mature molecules (e.g., single-stranded) or as mimetic precursors (e.g., pre-miRNA or precursor miRNA). miRNA mimics may include nucleic acids (modified or modified nucleic acids), including oligonucleotides, including but not limited to RNA, modified RNA, DNA, modified DNA, locked nucleic acids, or 2'-0,4' -C-ethylene bridged nucleic acids (ENA), or any combination of the above (including DNA-RNA hybrids). In addition, miRNA mimics may include conjugates capable of affecting delivery, intracellular compartmentalization, stability, specificity, functionality, strand use, and/or potency. In one design, a miRNA mimic is a double-stranded molecule (e.g., having a duplex region between about 16 and about 31 nucleotides in length) and contains one or more sequences having identity to the mature strand of a given miRNA. Modifications may include 2' modifications (including 2' -0 methyl modifications and 2' f modifications) and internucleoside modifications (e.g., phosphorothioate modifications) that enhance stability and/or specificity of the nucleic acid on one or both strands of the molecule. In addition, the miRNA mimic may include an overhang. The overhang may comprise about 1 to about 6 nucleotides at the 3 'or 5' end of either strand, and may be modified to enhance stability or functionality. In embodiments, the miRNA mimic comprises a duplex region of about 16 to about 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2 '-0-methyl modifications of nucleotide 1 and nucleotide 2 (counted from the 5' end of the sense oligonucleotide) and all C and U; antisense strand modifications can include all C and U2 ' f modifications, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleoside linkages associated with 3' overhangs of 2 nucleotides.

MiRNA inhibitors

In embodiments, AC is a miRNA inhibitor. The terms "antimir", "microrna inhibitor", "miR inhibitor" or "miRNA inhibitor" are synonymous and refer to an oligonucleotide or modified oligonucleotide that interferes with the ability of a particular miRNA. Generally, an inhibitor is essentially a nucleic acid or modified nucleic acid, including an oligonucleotide, including RNA, modified RNA, DNA, modified DNA, locked Nucleic Acid (LNA), or any combination of the above.

Modifications include 2' modifications (including 2' -0 alkyl modifications and 2' f modifications) and internucleoside modifications (e.g., phosphorothioate modifications) that can affect delivery, stability, specificity, intracellular compartmentalization, or potency. In addition, miRNA inhibitors may include conjugates capable of affecting delivery, intracellular compartmentalization, stability, and/or potency. Inhibitors can take a variety of configurations, including single-stranded, double-stranded (RNA/RNA or RNA/DNA duplex) and hairpin designs, and in general, microrna inhibitors comprise one or more sequences or sequence portions that are complementary or partially complementary to the mature strand(s) of the miRNA to be targeted. In addition, the miRNA inhibitor may also include additional sequences 5 'and 3' of the sequence that is the reverse complement of the mature miRNA. The additional sequence may be the reverse complement of the sequence adjacent to the mature miRNA in the pre-miRNA from which the mature miRNA is derived, or the additional sequence may be any sequence (mixture with A, G, C or U). In embodiments, one or both additional sequences are any sequence capable of forming a hairpin. Thus, in embodiments, the sequence that is the reverse complement of a miRNA is flanked on the 5 'and 3' sides by hairpin structures. When double-stranded, microRNA inhibitors can include mismatches between nucleotides on opposite strands. In addition, microrna inhibitors can be linked to the conjugate moiety to facilitate uptake of the inhibitor into the cell. For example, the microrna inhibitor can be linked to cholesteryl 5- (bis (4-methoxyphenyl) (phenyl) methoxy) -3 hydroxypentylcarbamate), which allows passive uptake of the microrna inhibitor into the cell. microRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al RNA 13:723-730 (2007) and WO2007/095387 and WO 2008/036825, the U.S. references are incorporated herein by reference in their entirety. One of ordinary skill in the art can select the sequence of the desired miRNA from a database and design inhibitors useful in the methods disclosed herein.

Linking groups or difunctional linking moieties such as those known in the art are suitable for use in the compounds provided herein. The linking groups can be used to attach chemical functional groups, conjugation groups, reporter groups, and other groups to selective sites in parent compounds such as AC. In general, a difunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One functional group is selected to bind to the parent molecule or compound of interest and the other functional group is selected to bind to essentially any selected group, such as a chemical functional group or a conjugate group. Any of the joints described herein may be used. In embodiments, the linker comprises a chain structure or oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups conventionally used in difunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophiles. In embodiments, the difunctional linking moiety includes amino groups, hydroxyl groups, carboxylic acids, thiols, unsaturations (e.g., double or triple bonds), and the like. Some non-limiting examples of difunctional linking moieties include 8-amino-3, 6-dioxooctanoic Acid (ADO), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), and 6-aminocaproic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl, wherein a non-limiting list of substituents include hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In embodiments, AC includes nucleotide modifications designed to not support rnase H activity. Nucleotide modifications of antisense compounds that do not support rnase H activity are known, including but not limited to 2' -O-methoxyethyl/phosphorothioate (MOE) modifications. Advantageously, the affinity of AC with MOE modification to target RNA is increased and nuclease stability is increased.

Immunostimulatory oligonucleotides

In embodiments, the therapeutic moiety is an immunostimulatory oligonucleotide. Immunostimulatory oligonucleotides (ISS, single-stranded or double-stranded) are capable of inducing an immune response when administered to a patient, which may be a mammal or other patient. ISS include, for example, certain palindromes that create hairpin secondary structures (see Yamamoto s et al (1992) j. Immunol. 148:4072-4076), or CpG motifs, as well as other known ISS features (such as poly G domains, see WO 96/11266).

The immune response may be an innate or adaptive immune response. The immune system is divided into a more innate immune system and an adaptive immune system acquired in vertebrates, the latter being further divided into humoral cellular components. In particular embodiments, the immune response may be mucosal.

Immunostimulatory nucleic acids are considered to be non-sequence specific when it is not desirable for them to specifically bind to a target polynucleotide and reduce its expression in order to elicit an immune response. Thus, certain immunostimulatory nucleic acids may include sequences corresponding to naturally occurring genes or mRNA regions, but they may still be considered non-sequence specific immunostimulatory nucleic acids.

In embodiments, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide having a methylated cytosine. In embodiments, the nucleic acid comprises a single CpG dinucleotide, wherein the cytosine in the CpG dinucleotide is methylated. In a specific embodiment, the nucleic acid comprises the sequence 5'TAACGTTGAGGG'CAT 3' (SEQ ID NO: 369). In another embodiment, the nucleic acid comprises at least two CpG dinucleotides, wherein at least one cytosine in the CpG dinucleotides is methylated. In another embodiment, each cytosine in a CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid comprises a plurality of CpG dinucleotides, wherein at least one of the CpG dinucleotides comprises a methylated cytosine.

Other specific nucleic acid sequences for Oligonucleotides (ODNs) suitable for use in the compositions and methods are described in Raney et al Journal of Pharmacology and Experimental Therapeutics,298:1185-1192 (2001). In certain embodiments, ODNs used in the compositions and methods have a phosphodiester ("PO") backbone or phosphorothioate ("PS") backbone, and/or at least one methylated cytosine residue in a CpG motif.

Decoy oligonucleotides

In embodiments, the therapeutic moiety is a decoy oligonucleotide. Because transcription factors recognize their relatively short binding sequences, short oligonucleotides with consensus binding sequences for specific transcription factors can be used as tools for manipulating gene expression in living cells even in the absence of surrounding genomic DNA. This strategy involves intracellular delivery of such "decoy oligonucleotides" which are then recognized and bound by the target factors. The bait occupies the DNA binding site of the transcription factor so that the transcription factor cannot subsequently bind to the promoter region of the target gene. Decoys may be used as therapeutic agents, to inhibit expression of genes activated by transcription factors, or to up-regulate genes repressed by binding of transcription factors. Examples of the use of decoy oligonucleotides can be found in Mann et al, J.Clin.Invest,2000,106:1071-1075, which is expressly incorporated herein by reference in its entirety.

U1 adapter

In some embodiments, the therapeutic moiety is a U1 adapter. The U1 adapter inhibits the poly A site and is a bifunctional oligonucleotide having a target domain complementary to a site in the terminal exon of the target gene and a "U1 domain" that binds to the U1 smaller nuclear RNA component of U1 snRNP (Goraczniak et al, 2008,Nature Biotechnology,27 (3), 257-263, expressly incorporated herein by reference in its entirety). U1 snRNP is a ribonucleoprotein complex whose main function is to direct early steps of spliceosome formation by binding to pre-mRNA exon-intron boundaries (Brown and Simpson,1998,Annu Rev Plant Physiol Plant Mol Biol49:77-95). Nucleotides 2-11 of the 5 'end of the U1 snRNA base pair bind to the 5' ss of the pre-mRNA. In one embodiment, the oligonucleotide is a U1 adaptor. In one embodiment, the Ul adaptor can be administered in combination with at least one other iRNA agent.

(CRISPR) gene editing machinery

In embodiments, the compounds disclosed herein include one or more CPPs (or cCPP) mechanically conjugated to CRISPR gene editing. As used herein, "CRISPR gene editing machinery" refers to proteins, nucleic acids, or combinations thereof that can be used to edit a genome. Non-limiting examples of gene editing machinery include grnas, nucleases, nuclease inhibitors, combinations and complexes thereof. The following patent documents describe CRISPR gene editing machinery: U.S. Pat. No. 8,697,359, U.S. Pat. No. 8,771,945, U.S. Pat. No. 8,795,965, U.S. Pat. No. 8,865,406, U.S. Pat. No. 8,871,445, U.S. Pat. No. 8,889,356, U.S. Pat. No. 8,895,308, U.S. Pat. No. 8,906,616, U.S. Pat. No. 8,932,814, U.S. Pat. No. 8,945,839, U.S. Pat. No. 8,993,233, U.S. Pat. No. 8,999,641, U.S. patent application Ser. No. 14/704,551, and U.S. patent application Ser. No. 13/842,859. Each of the above patent documents is incorporated by reference in its entirety.

In embodiments, the linker conjugates cCPP to a CRISPR gene editing machinery. Any of the linkers described in this disclosure or known to those skilled in the art may be utilized.

gRNA

In embodiments, the compound comprises a CPP (or cCPP) conjugated to a gRNA. The gRNA targets a genomic locus in a prokaryotic or eukaryotic cell.

In embodiments, the gRNA is a single molecule guide RNA (sgRNA). The sgrnas include spacer sequences and scaffold sequences. The spacer sequence is a short nucleic acid sequence for targeting a nuclease (e.g., cas9 nuclease) to a specific nucleotide region of interest (e.g., genomic DNA sequence to be cleaved). In embodiments, the spacer may be about 17-24 bases in length, such as about 20 bases. In embodiments, the length of the spacer can be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 bases. In embodiments, the length of the spacer can be at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 bases. In embodiments, the length of the spacer can be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 bases. In embodiments, the spacer sequence has a GC content of about 40% to about 80%.

In embodiments, the spacer targets a site immediately preceding the 5' Protospacer Adjacent Motif (PAM). PAM sequences may be selected according to the desired nuclease. For example, the PAM sequence may be any one of the PAM sequences shown in table 3 below, wherein N refers to any nucleic acid, R refers to a or G, Y refers to C or T, W refers to a or T, and V refers to a or C or G.

Table 3: exemplary nuclease and PAM sequences

PAM sequence (5 'to 3')	Nuclease (nuclease)	Separated from
			NGG	SpCas9	Streptococcus pyogenes (Streptococcus pyogenes)
NGRRT or NGRRN	SaCas9	Staphylococcus aureus (Staphylococcus aureus)
			NNNNGATT	NmeCas9	Neisseria meningitidis (NEISSERIA MENINGITIDIS)
NNNNRYAC	CjCas9	Campylobacter jejuni (Campylobacter jejuni)
			NNAGAAW	StCas9	Streptococcus thermophilus (Streptococcus thermophiles)
TTTV	LbCpf1	Bacteria of the family Maotaceae (Lachnospiraceae)
			TTTV	AsCpf1	Species of amino acid coccus (Acidaminococcus sp.)

In embodiments, the spacer may target a sequence of a mammalian gene (such as a human gene). In embodiments, the spacer may target a mutant gene. In embodiments, the spacer may target the coding sequence. In embodiments, the spacer may target an exon sequence. In embodiments, the spacer may target a Polyadenylation Site (PS). In embodiments, the spacer may target a sequence element of PS. In embodiments, the spacer may target polyadenylation signals (PAS), insertion Sequences (IS), cleavage Sites (CS), downstream Elements (DES), or a portion or combination thereof. In embodiments, the spacer may target a Splice Element (SE) or a cis-Splice Regulatory Element (SRE).

The scaffold sequence is the sequence within the sgRNA responsible for nuclease (e.g., cas 9) binding. The scaffold sequence does not include a spacer/targeting sequence. In embodiments, the length of the scaffold may be about 1 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides. In the context of an embodiment of the present invention, the length of the stent may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 50, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 63, about about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, or about 125 nucleotides. In embodiments, the length of the scaffold may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, or at least 125 bases.

In embodiments, the gRNA is a bimolecular guide RNA, such as crRNA and tracrRNA. In embodiments, the gRNA may also include a poly (a) tail.

In embodiments, a compound comprising a CPP is conjugated to a nucleic acid comprising a gRNA. In embodiments, the nucleic acid comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 grnas. In embodiments, the grnas recognize the same target. In embodiments, the gRNA recognizes different targets. In embodiments, the nucleic acid comprising the gRNA comprises a sequence encoding a promoter, wherein the promoter drives expression of the gRNA.

Nuclease (nuclease)

In embodiments, the compounds include a cell penetrating peptide conjugated to a nuclease. In embodiments, the nuclease is Sup>A type II, type V-A, type V-B, type VC, type V-U, type VI-B nuclease. In embodiments, the nuclease is a transcription, activator-like effector nuclease (TALEN), meganuclease, or zinc finger nuclease. In embodiments, the nuclease is a Cas9, cas12a (Cpf 1), cas12B, cas12C, tnp-B like, cas13a (C2), cas13B, or Cas14 nuclease. For example, in some embodiments, the nuclease is a Cas9 nuclease or a Cpf1 nuclease.

In embodiments, the nuclease is a modified form or variant of Cas9, cas12a (Cpf 1), cas12B, cas12C, tnp-B like, cas13a (C2), cas13B, or Cas14 nuclease. In embodiments, the nuclease is a modified form or variant of TAL nuclease, meganuclease or zinc finger nuclease. A "modified" or "variant" nuclease is, for example, a truncated, catalytically inactive nuclease fused to another protein (such as another nuclease). In embodiments, the nuclease may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to a naturally occurring Cas9, cas12a (Cpf 1), cas12B, cas12C, tnp-B like, cas13a (C2), cas13B, cas14 nuclease or TALEN, meganuclease, or zinc finger nuclease. In embodiments, the nuclease is a Cas9 nuclease (SpCas 9) derived from streptococcus pyogenes. In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with a Cas9 nuclease derived from streptococcus pyogenes (SpCas 9). In embodiments, the nuclease is Cas9 (SaCas 9) derived from staphylococcus aureus. In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with Cas9 (SaCas 9) derived from staphylococcus aureus. In embodiments, cpf1 is a Cpf1 enzyme from the genus amino acid coccus (species BV3L6, uniProt accession No. U2UMQ 6). In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the Cpf1 enzyme from the genus amino acid coccus (species BV3L6, uniProt accession No. U2 UMQ).

In embodiments, cpf1 is a Cpf1 enzyme from the family Maspirea (species ND2006, uniProt accession A0A182DWE 3). In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with a Cpf1 enzyme from the family chaetoviridae. In embodiments, the sequence encoding the nuclease is codon optimized for expression in mammalian cells. In embodiments, the sequence encoding the nuclease is codon optimized for expression in a human cell or a mouse cell.

In embodiments, the compound comprising the CPP is conjugated to a nuclease. In embodiments, the nuclease is a soluble protein.

In embodiments, a compound comprising a CPP is conjugated to a nucleic acid encoding a nuclease. In embodiments, the nucleic acid encoding a nuclease comprises a sequence encoding a promoter, wherein the promoter drives expression of the nuclease.

Combinations of gRNA and nucleases

In embodiments, the compounds include one or more CPPs (or cCPP) conjugated to a gRNA and a nuclease. In embodiments, the one or more CPPs (or cCPP) are conjugated to a nucleic acid encoding a gRNA and/or a nuclease. In embodiments, the nucleic acid encoding the nuclease and the gRNA comprises a sequence encoding a promoter, wherein the promoter drives expression of the nuclease and the gRNA. In embodiments, the nucleic acid encoding the nuclease and the gRNA comprises two promoters, wherein a first promoter controls expression of the nuclease and a second promoter controls expression of the gRNA. In embodiments, the nucleic acid encoding the gRNA and nuclease encodes about 1 to about 20 grnas, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19, and up to about 20 grnas. In embodiments, the gRNA recognizes different targets. In embodiments, the grnas recognize the same target.

In embodiments, the compounds include a cell penetrating peptide (or cCPP) conjugated to a Ribonucleoprotein (RNP) that includes gRNA and nucleases.

In embodiments, a composition comprising (a) a CPP conjugated to a gRNA and (b) a nuclease is delivered to a cell. In embodiments, a composition comprising (a) a nuclease conjugated CPP and (b) a gRNA is delivered to a cell.

In embodiments, a composition comprising (a) a first CPP conjugated to a gRNA and (b) a second CPP conjugated to a nuclease is delivered to a cell. In embodiments, the first CPP and the second CPP are the same. In an embodiment, the first CPP and the second CPP are different.

Target genetic element

In embodiments, the compounds disclosed herein include a cell penetrating peptide conjugated to a genetic element of interest. In embodiments, the genetic element of interest replaces a genomic DNA sequence that is cleaved by a nuclease. Non-limiting examples of genetic elements of interest include genes, single nucleotide polymorphisms, promoters or terminators.

Nuclease inhibitors

In embodiments, the compounds disclosed herein include a cell penetrating peptide conjugated to an inhibitor of a nuclease (e.g., cas 9). One limitation of gene editing is potential off-target editing. Delivery of nuclease inhibitors will limit off-target editing. In embodiments, the nuclease inhibitor is a polypeptide, polynucleotide, or small molecule. Exemplary nuclease inhibitors are described in U.S. publication No. 2020/087354, international publication No. 2018/085288, U.S. publication No. 2018/0382741, international publication No. 2019/089761, international publication No. 2020/068304, international publication No. 2020/04384, and international publication No. 2019/076651, each of which is incorporated herein by reference in its entirety.

Therapeutic polypeptides

In embodiments, the therapeutic moiety comprises a polypeptide. In embodiments, the therapeutic moiety comprises a protein or fragment thereof. In embodiments, the therapeutic moiety comprises an RNA binding protein or an RNA binding fragment thereof. In embodiments, the therapeutic moiety comprises an enzyme. In embodiments, the therapeutic moiety comprises an RNA lyase or an active fragment thereof.

Conjugation group

In embodiments, AC is modified by covalent attachment of one or more conjugate groups. Typically, the conjugate group modifies one or more properties of the attached AC, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts to connect to a parent compound such as AC either directly or via an optional linking moiety or linking group. Conjugation groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterols, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In embodiments, the conjugation group is polyethylene glycol (PEG), and the PEG is conjugated to AC or CPP.

The conjugate group includes a lipid moiety such as a cholesterol moiety (Letsinger et al, proc. Natl. Acad. Sci. USA 1989,86,6553); cholic acid (Manoharan et al, biorg. Med. Chem. Lett.,1994,4,1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al, ann.N. Y. Acad. Sci.,1992,660,306; manoharan et al, biorg. Med. Chem. Let.,1993,3,2765); thiocholesterols (Oberhauser et al, nucleic acids res.,1992,20,533); aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J.,1991,10,111; kabanov et al, FEBS Lett.,1990,259,327; svinarchuk et al, biochimie,1993,75,49); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium-1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al, tetrahedron lett.,1995,36,3651; shea et al, nucleic acids res.,1990,18,3777); polyamine or polyethylene glycol chains (Manoharan et al, circulations & circulations, 1995,14,969); adamantaneacetic acid (Manoharan et al, tetrahedron lett.,1995,36,3651); palm-based moieties (Mishra et al, biochim. Biophys. Acta,1995,1264,229); or octadecylamine or hexylamine-carbonyl-oxy cholesterol moiety (Crooke et al, j. Pharmacol. Exp. Ter., 1996,277,923).

In embodiments, AC may be linked to a dipeptide repeat sequence of 10 arginine-serine. AC for artificial recruitment of splice enhancers linked to a dipeptide repeat sequence of 10 arginine-serine has been used in vitro to induce BRCA1 and SMN2 exons containing mutations that would otherwise be skipped. See Cartegni and Krainer 2003, incorporated herein by reference.

Endosome escape carrier (EEV)

Endosomal Escape Vehicles (EEVs) may be used to transport cargo across a cell membrane, for example, to deliver cargo to the cytosol or nucleus of a cell. The cargo may include a Therapeutic Moiety (TM). EEV may include a Cell Penetrating Peptide (CPP), such as a cyclic cell penetrating peptide (cCPP). In an embodiment, the EEV comprises cCPP conjugated to an Exocyclic Peptide (EP). EP may be interchangeably referred to as a regulatory peptide (MP). The EP may include a sequence of Nuclear Localization Signals (NLS). The EP may be coupled to the cargo. The EP may be coupled to cCPP. The EP may be coupled to the cargo and cCPP. The coupling between the EP, cargo, cCPP, or combinations thereof may be non-covalent or covalent. The EP may be attached to the N-terminus of cCPP by a peptide bond. The EP may be attached to the C-terminus of cCPP by a peptide bond. EP can be attached to cCPP via the side chain of the amino acid in cCPP. EP may be attached to cCPP via a side chain of lysine, which may be conjugated to a side chain of glutamine in cCPP. The EP may be conjugated to the 5 'or 3' end of the oligonucleotide cargo. The EP may be coupled to a linker. The exocyclic peptide may be conjugated to the amino group of the linker. The EP may be coupled to the linker via the C-terminal ends of EP and cCPP via the side chains on cCPP and/or EP. For example, an EP may comprise a terminal lysine, which may then be coupled to a glutamine-containing cCPP via an amide linkage. When EP contains a terminal lysine and the side chain of the lysine is available for attachment cCPP, the C-terminus or N-terminus may be attached to a linker on the cargo.

Cyclic exopeptides

The Exocyclic Peptide (EP) may comprise 2 to 10 amino acid residues, for example 2,3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues, including all ranges and values therebetween. An EP may comprise 6 to 9 amino acid residues. An EP may comprise 4 to 8 amino acid residues.

Each amino acid in the exocyclic peptide may be a natural amino acid or a non-natural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homolog of a natural amino acid in that it has a structure similar to that of a natural amino acid such that it mimics the structure and reactivity of a natural amino acid. The unnatural amino acid can be a modified amino acid and/or amino acid analog that is not one of the 20 common naturally occurring amino acids or the rare natural amino acid selenocysteine or pyrrolysine. The unnatural amino acid can also be a D-isomer of the natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allo-isoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids are listed in table 4 along with their abbreviations used herein. For example, the amino acid may be A, G, P, K, R, V, F, H, nal or citrulline.

The EP may comprise at least one positively charged amino acid residue, for example at least one lysine residue and/or at least one amino acid residue comprising a side chain comprising a guanidine group or a protonated form thereof. The EP may comprise 1 or 2 amino acids comprising a side chain comprising a guanidine group or a protonated form thereof. The amino acid residue comprising a guanidine-containing side chain may be an arginine residue. Throughout this disclosure, protonated form may mean a salt thereof.

The EP may comprise at least two, at least three or at least four or more lysine residues. The EP may comprise 2,3 or 4 lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group including, for example, trifluoroacetyl (-COCF ₃), allyloxycarbonyl (Alloc), 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl (Dde) or (4, 4-dimethyl-2, 6-dioxocyclon-1-hexyl-3) -methylbutyl (ivDde). The amino group on the side chain of each lysine residue may be substituted with a trifluoroacetyl group (-COCF ₃). Protecting groups may be included to effect amide conjugation. After conjugation of EP to cCPP, the protecting group may be removed.

An EP may comprise at least 2 amino acid residues with hydrophobic side chains. Amino acid residues having hydrophobic side chains may be selected from valine, proline, alanine, leucine, isoleucine and methionine. The amino acid residue having a hydrophobic side chain may be valine or proline.

The EP may comprise at least one positively charged amino acid residue, e.g. at least one lysine residue and/or at least one arginine residue. An EP may comprise at least two, at least three or at least four or more lysine residues and/or arginine residues.

EP may comprise KK、KR、RR、HH、HK、HR、RH、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKH、KHK、HKK、HRR、HRH、HHR、HBH、HHH、HHHH(SEQ ID NO:1)、KHKK(SEQ ID NO:2)、KKHK(SEQ ID NO:3)、KKKH(SEQ ID NO:4)、KHKH(SEQ IDNO:5)、HKHK(SEQ ID NO:6)、KKKK(SEQ ID NO:7)、KKRK(SEQ ID NO:8)、KRKK(SEQ ID NO:9)、KRRK(SEQ ID NO:10)、RKKR(SEQ ID NO:11)、RRRR(SEQ ID NO:12)、KGKK(SEQ ID NO:13)、KKGK(SEQ ID NO:14)、HBHBH(SEQ ID NO:15)、HBKBH(SEQ ID NO:16)、RRRRR(SEQ ID NO:17)、KKKKK(SEQ ID NO:18)、KKKRK(SEQ ID NO:19)、RKKKK(SEQ ID NO:20)、KRKKK(SEQ ID NO:21)、KKRKK(SEQ ID NO:22)、KKKKR(SEQ ID NO:23)、KBKBK(SEQ ID NO:24)、RKKKKG(SEQ ID NO:25)、KRKKKG(SEQ ID NO:26)、KKRKKG(SEQ ID NO:27)、KKKKRG(SEQ ID NO:28)、RKKKKB(SEQ ID NO:29)、KRKKKB(SEQ ID NO:30)、KKRKKB(SEQ ID NO:31)、KKKKRB(SEQ ID NO:32)、KKKRKV(SEQ ID NO:33)、RRRRRR(SEQ ID NO:34)、HHHHHH(SEQ ID NO:35)、RHRHRH(SEQ ID NO:36)、HRHRHR(SEQ ID NO:37)、KRKRKR(SEQ ID NO:38)、RKRKRK(SEQ ID NO:39)、RBRBRB(SEQ ID NO:40)、KBKBKB(SEQ ID NO:41)、PKKKRKV(SEQ ID NO:42)、PGKKRKV(SEQ ID NO:43)、PKGKRKV(SEQ ID NO:44)、PKKGRKV(SEQ ID NO:45)、PKKKGKV(SEQ ID NO:46)、PKKKRGV(SEQ ID NO:47) or PKKKRKG (SEQ ID NO: 48) where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

EP may comprise KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK(SEQ ID NO:7)、KKRK(SEQ ID NO:8)、KRKK(SEQ ID NO:9)、KRRK(SEQ ID NO:10)、RKKR(SEQ ID NO:11)、RRRR(SEQ ID NO:12)、KGKK(SEQ ID NO:13)、KKGK(SEQ ID NO:14)、KKKKK(SEQ ID NO:18)、KKKRK(SEQ ID NO:19)、KBKBK(SEQ ID NO:24)、KKKRKV(SEQ ID NO:33)、PKKKRKV(SEQ ID NO:42)、PGKKRKV(SEQ ID NO:43)、PKGKRKV(SEQ ID NO:44)、PKKGRKV(SEQ ID NO:45)、PKKKGKV(SEQ ID NO:46)、PKKKRGV(SEQ ID NO:47) or PKKKRKG (SEQ ID NO: 48). EP may comprise PKKKRKV (SEQ ID NO: 42), RR, RRR, RHR, RBR, RBRBR (SEQ ID NO: 49), RBHBR (SEQ ID NO: 50) or HBRBH (SEQ ID NO: 51), wherein B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

EP may consist of KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK(SEQ ID NO:7)、KKRK(SEQ ID NO:8)、KRKK(SEQ ID NO:9)、KRRK(SEQ ID NO:10)、RKKR(SEQ ID NO:11)、RRRR(SEQ ID NO:12)、KGKK(SEQ ID NO:13)、KKGK(SEQ ID NO:14)、KKKKK(SEQ ID NO:18)、KKKRK(SEQ ID NO:19)、KBKBK(SEQ ID NO:24)、KKKRKV(SEQ ID NO:33)、PKKKRKV(SEQ ID NO:42)、PGKKRKV(SEQ ID NO:Z43)、PKGKRKV(SEQ ID NO:Z44)、PKKGRKV(SEQ ID NO:Z45)、PKKKGKV(SEQ ID NO:46)、PKKKRGV(SEQ ID NO:47) or PKKKRKG (SEQ ID NO: 48). EP may consist of PKKKRKV (SEQ ID NO: 42), RR, RRR, RHR, RBR, RBRBR (SEQ ID NO: 49), RBHBR (SEQ ID NO: 50) or HBRBH (SEQ ID NO: 51), wherein B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

An EP may comprise an amino acid sequence identified in the art as a Nuclear Localization Sequence (NLS). An EP may consist of an amino acid sequence identified in the art as a Nuclear Localization Sequence (NLS). EP may comprise an NLS comprising the amino acid sequence PKKKRKV (SEQ ID NO: 42). EP may consist of NLS containing the amino acid sequence PKKKRKV (SEQ ID NO: 42). EP may comprise an NLS comprising amino acid sequences ：NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52)、PAAKRVKLD(SEQ ID NO:53)、RQRRNELKRSF(SEQ ID NO:54)、RMRKFKNKGKDTAELRRRRVEVSVELR(SEQ ID NO:Z55)、KAKKDEQILKRRNV(SEQ ID NO:56)、VSRKRPRP(SEQ ID NO:57)、PPKKARED(SEQ ID NO:58)、PQPKKKPL(SEQ ID NO:59)、SALIKKKKKMAP(SEQ ID NO:60)、DRLRR(SEQ ID NO:61)、PKQKKRK(SEQ ID NO:62)、RKLKKKIKKL(SEQ ID NO:63)、REKKKFLKRR(SEQ ID NO:64)、KRKGDEVDGVDEVAKKKSKK(SEQ ID NO:65) and RKCLQAGMNLEARKTKK (SEQ ID NO: 66) selected from the group consisting of. EP may consist of NLS comprising amino acid sequences ：NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52)、PAAKRVKLD(SEQ ID NO:53)、RQRRNELKRSF(SEQ ID NO:54)、RMRKFKNKGKDTAELRRRRVEVSVELR(SEQ ID NO:55)、KAKKDEQILKRRNV(SEQ ID NO:56)、VSRKRPRP(SEQ ID NO:57)、PPKKARED(SEQ ID NO:58)、PQPKKKPL(SEQ ID NO:59)、SALIKKKKKMAP(SEQ ID NO:60)、DRLRR(SEQ ID NO:61)、PKQKKRK(SEQ ID NO:62)、RKLKKKIKKL(SEQ ID NO:63)、REKKKFLKRR(SEQ ID NO:64)、KRKGDEVDGVDEVAKKKSKK(SEQ ID NO:65) and RKCLQAGMNLEARKTKK (SEQ ID NO: 66) selected from the group consisting of.

All exocyclic sequences may also contain N-terminal acetyl groups. Thus, for example, an EP may have the following structure: ac-PKKKKRKV (SEQ ID NO: 42).

Cell Penetrating Peptide (CPP)

The Cell Penetrating Peptide (CPP) may comprise from 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). cCPP are capable of penetrating cell membranes. The Exocyclic Peptide (EP) may be conjugated to cCPP and the resulting construct may be referred to as an Endosomal Escape Vector (EEV). cCPP can direct cargo (e.g., a Therapeutic Moiety (TM), such as an oligonucleotide, peptide, or small molecule) to penetrate a cell membrane. cCPP can deliver cargo into the cytosol of a cell. cCPP can deliver cargo to a cellular location where a target (e.g., pre-mRNA) is located. To conjugate cCPP to a cargo (e.g., peptide, oligonucleotide, or small molecule), at least one bond or lone pair of electrons on cCPP may be replaced.

The total number of amino acid residues in cCPP is in the range of 6 to 20 amino acid residues, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, including all ranges and subranges therebetween. cCPP may comprise from 6 to 13 amino acid residues. cCPP disclosed herein may comprise from 6 to 10 amino acids. For example, cCPP comprising 6-10 amino acid residues may have a structure according to any of formulas I-a to I-E:

Wherein AA₁、AA₂、AA₃、AA₄、AA₅、AA₆、AA₇、AA₈、AA₉ and AA ₁₀ are amino acid residues.

CCPP may comprise from 6 to 8 amino acids. cCPP may comprise 8 amino acids.

Each amino acid in cCPP may be a natural amino acid or a non-natural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homolog of a natural amino acid in that it has a structure similar to that of a natural amino acid such that it mimics the structure and reactivity of a natural amino acid. The unnatural amino acid can be a modified amino acid and/or amino acid analog that is not one of the 20 common naturally occurring amino acids or the rare natural amino acid selenocysteine or pyrrolysine. The unnatural amino acid can also be a D-isomer of the natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allo-isoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids are listed in table 5 along with their abbreviations used herein.

TABLE 4 amino acid abbreviations

As used herein, "polyethylene glycol" and "PEG" are used interchangeably. "PEGm" and "PEG _m" are or are derived from molecules of the formula HO (CO) - (CH ₂)_n-[OCH₂CH₂)_m-NH₂), where n is any integer from 1 to 5, and m is any integer from 1 to 23.

As used herein, "miniPEGm" or "miniPEG _m" is or derives from a molecule of the formula HO (CO) - (CH ₂)_n-[OCH₂CH₂)_m-NH₂), where n is 1 and m is any integer from 1 to 23, for example, "miniPEG2" or "miniPEG ₂" is or derives from (2- [2- [ 2-aminoethoxy ] ethoxy ] acetic acid), and "miniPEG4" or "miniPEG ₄" is or derives from HO (CO) - (CH ₂)_n-(OCH₂CH₂)_m-NH₂, where n is 1 and m is 4.

CCPP may comprise from 4 to 20 amino acids, wherein (I) at least one amino acid has a side chain comprising a guanidino group or protonated form thereof; (ii) At least one amino acid having no side chains or comprising Or a side chain of a protonated form thereof; and (iii) at least two amino acids independently have side chains comprising aromatic or heteroaromatic groups.

At least two amino acids may have no side chains or have a chain comprising Or a side chain of a protonated form thereof. As used herein, when a side chain is not present, the amino acid has two hydrogen atoms (e.g., -CH ₂ -) on the carbon atoms linking the amine and the carboxylic acid.

The amino acid without a side chain may be glycine or β -alanine.

CCPP may comprise 6 to 20 amino acid residues forming cCPP, wherein (I) at least one amino acid may be a glycine, β -alanine or 4-aminobutyric acid residue; (ii) At least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid has a guanidine group,Or a side chain of a protonated form thereof.

CCPP may comprise 6 to 20 amino acid residues forming cCPP, wherein (I) at least two amino acids may independently be glycine, β -alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid has a guanidine group,Or a side chain of a protonated form thereof.

CCPP may comprise 6 to 20 amino acid residues forming cCPP, wherein (I) at least three amino acids may independently be glycine, β -alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aromatic or heteroaromatic group; and (iii) at least one amino acid may have a guanidine group, Or a side chain of a protonated form thereof.

Glycine and related amino acid residues

CCPP may comprise (i) 1,2, 3, 4, 5, or 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) a 2 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) a3 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) a 4 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) a 5 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) a 6 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) 3, 4, or 5 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 or 4 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof.

CCPP may comprise (i) 1, 2, 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 2 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues. cCPP may comprise (i) 2 or 3 glycine residues. cCPP may comprise (i) 1 or 2 glycine residues.

CCPP may comprise (i) 3, 4, 5, or 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) a 3 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) a 4 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) a 5 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) a 6 glycine, beta-alanine, 4-aminobutyric acid residue, or a combination thereof. cCPP may comprise (i) 3, 4, or 5 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 or 4 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof.

CCPP may comprise at least three glycine residues. cCPP may comprise (i) 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or 5 glycine residues. cCPP can comprise (i) 3 or 4 glycine residues

In embodiments, no glycine, β -alanine, or 4-aminobutyric acid residues in cCPP are consecutive. Two or three glycine, beta-alanine, 4-or aminobutyric acid residues may be consecutive. The two glycine, beta-alanine or 4-aminobutyric acid residues may be consecutive.

In embodiments, no glycine residue in cCPP is continuous. Each glycine residue in cCPP may be separated by an amino acid residue that is not glycine. Two or three glycine residues may be contiguous. The two glycine residues may be contiguous.

Amino acid side chains with aromatic or heteroaromatic groups

CCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues which independently have a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 amino acid residues which independently have a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 3 amino acid residues which independently have a side chain comprising an aromatic or heteroaromatic group. cCPP can comprise (ii) 4 amino acid residues which independently have a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 5 amino acid residues which independently have a side chain comprising an aromatic or heteroaromatic group. cCPP can comprise (ii) 6 amino acid residues that independently have a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2, 3 or 4 amino acid residues which independently have a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues which independently have a side chain comprising an aromatic or heteroaromatic group.

CCPP may comprise (ii) 2, 3, 4,5 or 6 amino acid residues which independently have a side chain comprising an aromatic group. cCPP can comprise (ii) 2 amino acid residues that independently have a side chain comprising an aromatic group. cCPP can comprise (ii) 3 amino acid residues that independently have a side chain comprising an aromatic group. cCPP can comprise (ii) 4 amino acid residues that independently have a side chain comprising an aromatic group. cCPP may comprise (ii) 5 amino acid residues which independently have a side chain comprising an aromatic group. cCPP can comprise (ii) 6 amino acid residues that independently have a side chain comprising an aromatic group. cCPP may comprise (ii) 2, 3 or 4 amino acid residues which independently have a side chain comprising an aromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues which independently have a side chain comprising an aromatic group.

The aromatic group may be a 6 to 14 membered aryl group. Aryl groups may be phenyl, naphthyl or anthracenyl, each of which is optionally substituted. Aryl groups may be phenyl or naphthyl, each of which is optionally substituted. The heteroaromatic group may be a 6 to 14 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S. Heteroaryl may be pyridinyl, quinolinyl or isoquinolinyl.

Amino acid residues having a side chain comprising an aromatic or heteroaromatic group may each independently be bis (homonaphthalanine), homonaphthalanine, naphthalanine, phenylglycine, bis (homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3- (3-benzothienyl) -alanine, 3- (2-quinolinyl) -alanine, O-benzylserine, 3- (4- (benzyloxy) phenyl) -alanine, S- (4-methylbenzyl) cysteine, N- (naphthalen-2-yl) glutamine, 3- (1, 1' -biphenyl-4-yl) -alanine, 3- (3-benzothienyl) -alanine or tryptophan, each of which is optionally substituted with one or more substituents. Amino acids having side chains comprising aromatic or heteroaromatic groups may each be independently selected from:

/>

Wherein H at the N-terminal and/or H at the C-terminal is replaced by a peptide bond.

The amino acid residues having a side chain comprising an aromatic or heteroaromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, homonaphthylalanine, bis (homophenylalanine), bis- (homonaphthylalanine), tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienyl alanine, 4-phenylphenylalanine, 3, 4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5, 6-pentafluorophenylalanine, homophenylalanine, β -homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthryl) -alanine. The amino acid residues having a side chain comprising an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain comprising an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, homophenylalanine, homonaphthylalanine, bis (homonaphthylalanine) or bis (homonaphthylalanine), each of which is optionally substituted with one or more substituents. Amino acid residues having a side chain comprising an aromatic group may each independently be residues of phenylalanine or naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain comprising an aromatic group may be a phenylalanine residue. The at least two amino acid residues having a side chain comprising an aromatic group may be phenylalanine residues. Each amino acid residue having a side chain comprising an aromatic group may be a phenylalanine residue.

In embodiments, no amino acid having a side chain comprising an aromatic or heteroaromatic group is continuous. The two amino acids having side chains comprising aromatic or heteroaromatic groups may be contiguous. Two consecutive amino acids may have opposite stereochemistry. Two consecutive amino acids may have the same stereochemistry. Three amino acids having side chains containing aromatic or heteroaromatic groups may be contiguous. Three consecutive amino acids may have the same stereochemistry. Three consecutive amino acids may have alternating stereochemistry.

The amino acid residue comprising an aromatic or heteroaromatic group may be an L-amino acid. The amino acid residue comprising an aromatic or heteroaromatic group may be a D-amino acid. The amino acid residue comprising an aromatic or heteroaromatic group may be a mixture of D-amino acids and L-amino acids.

An optional substituent may be, for example, any atom or group that does not significantly reduce (e.g., more than 50%) the cytoplasmic delivery efficiency of cCPP as compared to the otherwise identical sequence without the substituent. The optional substituents may be hydrophobic or hydrophilic substituents. The optional substituents may be hydrophobic substituents. The substituents may increase the solvent accessible surface area (as defined herein) of the hydrophobic amino acid. The substituent may be halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamide, alkoxycarbonyl, alkylthio or arylthio. The substituent may be halogen.

While not wishing to be bound by theory, it is believed that amino acids having aromatic or heteroaromatic groups with higher hydrophobicity values (i.e., amino acids having side chains comprising aromatic or heteroaromatic groups) may increase the cytoplasmic delivery efficiency of cCPP relative to amino acids having lower hydrophobicity values. Each hydrophobic amino acid may independently have a greater hydrophobicity value than glycine. Each hydrophobic amino acid may independently be a hydrophobic amino acid having a hydrophobicity value greater than alanine. Each hydrophobic amino acid may independently have a hydrophobicity value greater than or equal to phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 5 lists the hydrophobicity values of various amino acids, as reported by Eisenberg and Weiss (Proc. Natl. Acad. U.S. A.1984;81 (1): 140-144), engleman et al (Ann. Rev. Of Biophys. Chem.1986;1986 (15): 321-53), kyte and Doolittle (J. Mol. Biol.1982;157 (1): 105-132), hoop and Woods (Proc. Natl. Acad. Sci. U.S.A.1981;78 (6): 3824-3828), and Janin (Nature. 1979;277 (5696): 491-492), each of which is incorporated herein by reference in its entirety. Hydrophobicity can be measured using the hydrophobicity scale reported by Engleman et al.

TABLE 5 amino acid hydrophobicity

The size of the aromatic or heteroaromatic groups may be selected to increase the cytoplasmic delivery efficiency of cCPP. While not wishing to be bound by theory, it is believed that larger aromatic or heteroaromatic groups on the amino acid side chains may increase cytoplasmic delivery efficiency compared to otherwise identical sequences with less hydrophobic amino acids. The size of the hydrophobic amino acid may be measured in terms of the molecular weight of the hydrophobic amino acid, the steric effect of the hydrophobic amino acid, the solvent accessible surface area of the side chain (SASA), or a combination thereof. The size of the hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, and the larger hydrophobic amino acid has side chains with a molecular weight of at least about 90 g/mol, or at least about 130 g/mol, or at least about 141 g/mol. The size of an amino acid can be measured in terms of the SASA of the hydrophobic side chain. The hydrophobic amino acid may have a side chain with SASA greater than or equal to alanine, or greater than or equal to glycine. The larger hydrophobic amino acid may have a side chain with a SASA greater than alanine or greater than glycine. The hydrophobic amino acid may have an aromatic or heteroaromatic group with a SASA greater than or equal to about piperidine-2-carboxylic acid, greater than or equal to about tryptophan, greater than or equal to about phenylalanine, or greater than or equal to about naphthylalanine. The first hydrophobic amino acid (AA _H1) may have a SASA of at least aboutAt least about/>At least about/>At least about/>At least aboutAt least about/>At least about/> At least about/>At least about/>At least about/>At least about/>At least about/>Or at least about/>Is a side chain of (c). The second hydrophobic amino acid (AA _H2) may have a SASA of at least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least aboutAt least about/>Or at least about/>Is a side chain of (c). The side chains of AA _H1 and AA _H2 may have a combined SASA of at least about/>At least about/>At least about/>At least about/>At least about/>At least aboutAt least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>Greater than about/>At least aboutAt least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least aboutAt least about/>At least about/>Greater than about/>At least about/>At least about/>At least about/>At least about/>Or at least about/>AA _H2 can be a hydrophobic amino acid residue of SASA with a side chain less than or equal to the hydrophobic side chain of AA _H1. For example, without limitation, cCPP having a Nal-Arg motif may exhibit increased cytoplasmic delivery efficiency compared to cCPP, which is otherwise identical to that having a Phe-Arg motif; cCPP having a Phe-Nal-Arg motif may exhibit increased cytoplasmic delivery efficiency compared to cCPP, which is otherwise identical to that having a Nal-Phe-Arg motif; and the Phe-Nal-Arg motif may exhibit increased cytoplasmic delivery efficiency compared to otherwise identical cCPP with the Nal-Phe-Arg motif.

As used herein, "hydrophobic surface area" or "SASA" refers to the surface area of the solvent accessible amino acid side chains (reported in square angstroms; ). SASA can be calculated using the "rolling ball" algorithm developed by Shrake and Rupley (J Mol biol.79 (2): 351-71), which is incorporated herein by reference in its entirety for all purposes. This algorithm uses solvent "spheres" of specific radius to probe the molecular surface. Typical values for spheres are/> Approaching the radius of water molecules.

The SASA values for some of the side chains are shown in table 6 below. The SASA values described herein are based on the theoretical values listed in Table 6 below, as reported by Tien et al (PLOS ONE (11): e80635, available at doi.org/10.1371/journ.fine.0080635), which is incorporated herein by reference in its entirety for all purposes.

TABLE 6 amino acid SASA values

Residues	Theoretical value	Empirical values	Miller et al (1987)	Rose et al (1985)
					Alanine (Ala)	129.0	121.0	113.0	118.1
Arginine (Arg)	274.0	265.0	241.0	256.0
					Asparagine derivatives	195.0	187.0	158.0	165.5
Aspartic acid	193.0	187.0	151.0	158.7
					Cysteine (S)	167.0	148.0	140.0	146.1
Glutamate salt	223.0	214.0	183.0	186.2
					Glutamine	225.0	214.0	189.0	193.2
Glycine (Gly)	104.0	97.0	85.0	88.1
					Histidine	224.0	216.0	194.0	202.5
Isoleucine (Ile)	197.0	195.0	182.0	181.0
					Leucine (leucine)	201.0	191.0	180.0	193.1
Lysine	236.0	230.0	211.0	225.8
					Methionine	224.0	203.0	204.0	203.4
Phenylalanine (Phe)	240.0	228.0	218.0	222.8
					Proline (proline)	159.0	154.0	143.0	146.8
Serine (serine)	155.0	143.0	122.0	129.8
					Threonine (Thr)	172.0	163.0	146.0	152.5
Tryptophan	285.0	264.0	259.0	266.3
					Tyrosine	263.0	255.0	229.0	236.8
Valine (valine)	174.0	165.0	160.0	164.5

Amino acid residues having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof

Guanidine, as used herein, refers to the following structure:

as used herein, the protonated form of guanidine refers to the following structure:

Guanidine replacement groups refer to functional groups on the side chains of amino acids that will be positively charged at or above physiological pH, or those that are capable of reproducing the hydrogen bond donating and accepting activity of a guanidinium group.

The guanidine replacement group facilitates cellular penetration and delivery of the therapeutic agent while reducing toxicity associated with the guanidine group or protonated form thereof. cCPP can comprise at least one amino acid having a side chain comprising a guanidine or guanidinium replacement group. cCPP can comprise at least two amino acids having side chains comprising guanidine or guanidinium substitution groups. cCPP can comprise at least three amino acids having side chains comprising guanidine or guanidinium substitution groups.

The guanidine or guanidinium group can be an isostere of guanidine or guanidinium. The guanidine or guanidinium replacement group can be less basic than guanidine.

As used herein, guanidine replacement group refers to Or a protonated form thereof.

The present disclosure relates to cCPP comprising 4 to 20 amino acids, wherein: (i) At least one amino acid has a side chain comprising a guanidino group or a protonated form thereof; (ii) At least one amino acid residue has no side chain or has a chain comprising Or a side chain of a protonated form thereof; and (iii) at least two amino acid residues independently have a side chain comprising an aromatic or heteroaromatic group.

At least two amino acid residues may have no side chains or have a chain comprising/>Or a side chain of a protonated form thereof. As used herein, an amino acid residue has two hydrogen atoms (e.g., -CH ₂ -) on the carbon atom connecting the amine and the carboxylic acid when no side chains are present.

CCPP may comprise at least one amino acid whose side chain comprises one of the following moieties: Or a protonated form thereof.

CCPP may comprise at least two amino acids each independently having one of the following moieties: Or a protonated form thereof. At least two amino acids may have side chains comprising the same moiety selected from the group consisting of: Or a protonated form thereof. At least one amino acid may have a nucleotide sequence comprising/> Or a side chain of a protonated form thereof. At least two amino acids may have a nucleotide sequence comprising/>Or a side chain of a protonated form thereof. One, two, three or four amino acids may have a sequence comprising/>Or a side chain of a protonated form thereof. One amino acid may have a nucleotide sequence comprising/>Or a side chain of a protonated form thereof. The two amino acids may have a sequence comprising/>Side chains in their protonated form. Or a protonated form thereof may be attached to the end of the amino acid side chain. /(I)May be attached to the end of the amino acid side chain.

CCPP may comprise (iii) 2, 3,4,5 or 6 amino acid residues which independently have a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. cCPP can comprise (iii) 2 amino acid residues that independently have a side chain comprising a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP can comprise (iii) 3 amino acid residues that independently have a side chain comprising a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP can comprise (iii) 4 amino acid residues that independently have a side chain comprising a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may comprise (iii) 5 amino acid residues which independently have a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. cCPP can comprise (iii) 6 amino acid residues that independently have a side chain comprising a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may comprise (iii) 2, 3,4 or 5 amino acid residues which independently have a side chain comprising a guanidine group, guanidine replacement group or protonated form thereof. cCPP may comprise (iii) 2, 3 or 4 amino acid residues which independently have a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 2 or 3 amino acid residues which independently have a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidino group or protonated form thereof. cCPP may comprise (iii) two amino acid residues having a side chain comprising a guanidino group or protonated form thereof. cCPP may comprise (iii) three amino acid residues having a side chain comprising a guanidino group or protonated form thereof.

The amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, which are discontinuous. The two amino acid residues may independently have a side chain comprising a guanidine group, a guanidine replacement group, or a protonated form thereof, and may be contiguous. The three amino acid residues may independently have a side chain comprising a guanidine group, a guanidine replacement group, or a protonated form thereof, and may be contiguous. The four amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, and may be discontinuous. Consecutive amino acid residues may have the same stereochemistry. Consecutive amino acids may have alternating stereochemistry.

The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be an L-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be a D-amino acid. The amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be a mixture of L-or D-amino acids.

Each amino acid residue having a side chain comprising a guanidino group or a protonated form thereof may independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidino butyric acid, or a protonated form thereof. Each amino acid residue having a side chain comprising a guanidine group or protonated form thereof can independently be an arginine residue or protonated form thereof.

Each amino acid having a side chain comprising a guanidine replacement group or protonated form thereof can independently beOr a protonated form thereof.

Without being bound by theory, it is hypothesized that the guanidine replacement group has a reduced basicity relative to arginine, and in some cases is uncharged at physiological pH (e.g., -N (H) C (O)), and is capable of maintaining bidentate hydrogen bond interactions with phospholipids on plasma membranes, which is believed to promote efficient membrane association and subsequent internalization. It is also believed that removal of the positive charge reduces cCPP toxicity.

Those skilled in the art will appreciate that the N-terminal and/or C-terminal of the above-described unnatural aromatic hydrophobic amino acids, upon incorporation into the peptides disclosed herein, form amide linkages.

CCPP may comprise a first amino acid having a side chain comprising an aromatic or heteroaromatic group and a second amino acid having a side chain comprising an aromatic or heteroaromatic group, wherein the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain comprising an aromatic or heteroaromatic group and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain comprising an aromatic or heteroaromatic group. Although by convention, the term "first amino acid" often refers to the N-terminal amino acid of a peptide sequence, as used herein, the term "first amino acid" is used to distinguish a reference amino acid in cCPP from another amino acid (e.g., "second amino acid"), such that the term "first amino acid" may be or may refer to an amino acid located at the N-terminal end of a peptide sequence.

CCPP can comprise the N-terminus of the second glycine, which forms a peptide bond with an amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the second glycine forms a peptide bond with an amino acid having a side chain comprising a guanidino group or protonated form thereof.

CCPP may comprise a first amino acid having a side chain comprising a guanidino group or a protonated form thereof, and a second amino acid having a side chain comprising a guanidino group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidino group or a protonated form thereof, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain comprising a guanidino group or a protonated form thereof.

CCPP may comprise residues of asparagine, aspartic acid, glutamine, glutamic acid or homoglutamine. cCPP may comprise an asparagine residue. cCPP may comprise a glutamine residue.

CCPP may comprise residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienyl alanine, 4-phenylphenylalanine, 3, 4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5, 6-pentafluorophenylalanine, homophenylalanine, β -homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthracenyl) -alanine.

While not wishing to be bound by theory, it is believed that the chirality of the amino acids in cCPP may affect cytoplasmic uptake efficiency. cCPP may comprise at least one D amino acid. cCPP may comprise one to fifteen D amino acids. cCPP may comprise one to ten D amino acids. cCPP may comprise 1, 2, 3 or 4D amino acids. cCPP may comprise 2, 3,4, 5,6, 7 or 8 consecutive amino acids with alternating D and L chiralities. cCPP may comprise three consecutive amino acids with the same chirality. cCPP may comprise two consecutive amino acids having the same chirality. At least two amino acids may have opposite chiralities. At least two amino acids having opposite chirality may be adjacent to each other. At least three amino acids may have alternating stereochemistry with respect to each other. At least three amino acids having alternating chiralities relative to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry with respect to each other. At least four amino acids having alternating chiralities relative to each other may be adjacent to each other. At least two amino acids may have the same chirality. At least two amino acids having the same chirality may be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids having opposite chiralities may be adjacent to at least two amino acids having the same chirality. Thus, adjacent amino acids in cCPP may have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D. The amino acid residues forming cCPP may all be L-amino acids. The amino acid residues forming cCPP may all be D-amino acids.

At least two amino acids may have different chiralities. At least two amino acids having different chiralities may be adjacent to each other. At least three amino acids may have different chiralities relative to adjacent amino acids. At least four amino acids may have different chiralities relative to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chiralities. One or more of the amino acid residues forming cCPP may be achiral. cCPP may comprise a 3, 4 or 5 amino acid motif, wherein two amino acids having the same chirality may be separated by an achiral amino acid. cCPP may comprise the following sequence: D-X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L, wherein X is an achiral amino acid. The achiral amino acid may be glycine.

Has the following steps:

The amino acid of the side chain, or protonated form thereof, may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Has the following steps: /(I) Or a side chain of a protonated form thereof may be adjacent to at least one amino acid having a side chain comprising guanidine or a protonated form thereof. An amino acid having a side chain comprising guanidine or a protonated form thereof can be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Has the following steps: /(I) Or the two amino acids of the side chain in its protonated form may be adjacent to each other. Two amino acids having side chains comprising guanidine or a protonated form thereof are adjacent to each other. cCPP can comprise at least two consecutive amino acids having side chains that can comprise aromatic or heteroaromatic groups, and at least two amino acids having side chains comprising: or a non-adjacent amino acid of a side chain of a protonated form thereof. cCPP can comprise at least two consecutive amino acids having a side chain comprising an aromatic or heteroaromatic group, and at least two amino acids having a chain comprising/> Or a non-adjacent amino acid of a side chain of a protonated form thereof. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, for example, any of the sequences described in the previous paragraph.

At least two of the devices have a structure comprising:

or a protonated form thereof with at least two amino acids having a side chain comprising a guanidino group or a protonated form thereof.

CCPP may comprise the structure of formula (a):

Or a protonated form thereof,

Wherein:

R ₄、R₅、R₆、R₇ is independently H or an amino acid side chain;

AA _SC is an amino acid side chain; and

Q is 1, 2, 3 or 4.

In an embodiment, compounds are provided comprising a cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids. In embodiments, the at least two charged amino acids of the cyclic peptide are arginine. In embodiments, the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine or naphthylalanine. In embodiments, the at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline or glycine.

In an embodiment, the cyclic peptide of formula (A) is not selected from cyclic peptides having the sequence of SEQ ID NO: 89-117.

In an embodiment, the cyclic peptide of formula (A) is selected from cyclic peptides having the sequence of SEQ ID NO: 89-117.

Φ=l-naphthylalanine; phi = D-naphthylalanine; Ω=l-norleucine

CCPP may comprise the structure of formula (I):

/>

Or a protonated form thereof,

Wherein:

R ₁、R₂ and R ₃ may each independently be H or an amino acid residue having a side chain comprising an aromatic group;

R ₄ and R ₇ are independently H or an amino acid side chain;

AA _SC is an amino acid side chain;

q is 1, 2, 3 or 4; and

Each m is independently an integer of 0, 1,2 or 3.

R ₁、R₂ and R ₃ may each independently be H, -alkylene-aryl or-alkylene-heteroaryl. R ₁、R₂ and R ₃ may each independently be H, -C _1-3 alkylene-aryl or-C _1-3 alkylene-heteroaryl. R ₁、R₂ and R ₃ may each independently be H or-alkylene-aryl. R ₁、R₂ and R ₃ may each independently be H or-C _1-3 alkylene-aryl. The C _1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₁、R₂ and R ₃ may each independently be H, -C _1-3 alkylene-Ph or-C _1-3 alkylene-naphthyl. R ₁、R₂ and R ₃ may each independently be H, -CH ₂ Ph or-CH ₂ naphthyl. R ₁、R₂ and R ₃ may each independently be H or-CH ₂ Ph.

R ₁、R₂ and R ₃ may each independently be a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienyl alanine, 4-phenylphenylalanine, 3, 4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5, 6-pentafluorophenylalanine, homophenylalanine, β -homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthryl) -alanine.

R ₁ can be a side chain of tyrosine. R ₁ can be the side chain of phenylalanine. R ₁ can be the side chain of 1-naphthylalanine. R ₁ can be the side chain of 2-naphthylalanine. R ₁ can be the side chain of tryptophan. R ₁ can be the side chain of 3-benzothiophenylalanine. R ₁ can be the side chain of 4-phenylphenylalanine. R ₁ can be the side chain of 3, 4-difluorophenylalanine. R ₁ can be the side chain of 4-trifluoromethylphenylalanine. R ₁ can be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R ₁ can be the side chain of homophenylalanine. R ₁ can be the side chain of beta-homophenylalanine. R ₁ may be the side chain of 4-tert-butylphenylalanine. R ₁ can be the side chain of 4-pyridylalanine. R ₁ can be the side chain of 3-pyridylalanine. R ₁ can be the side chain of 4-methylphenylalanine. R ₁ can be the side chain of 4-fluorophenylalanine. R ₁ can be the side chain of 4-chlorophenylalanine. R ₁ can be the side chain of 3- (9-anthryl) -alanine.

R ₂ can be a side chain of tyrosine. R ₂ can be the side chain of phenylalanine. R ₂ can be the side chain of 1-naphthylalanine. R ₁ can be the side chain of 2-naphthylalanine. R ₂ can be the side chain of tryptophan. R ₂ can be the side chain of 3-benzothiophenylalanine. R ₂ can be the side chain of 4-phenylphenylalanine. R ₂ can be the side chain of 3, 4-difluorophenylalanine. R ₂ can be the side chain of 4-trifluoromethylphenylalanine. R ₂ can be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R ₂ can be the side chain of homophenylalanine. R ₂ can be the side chain of beta-homophenylalanine. R ₂ may be the side chain of 4-tert-butylphenylalanine. R ₂ can be the side chain of 4-pyridylalanine. R ₂ can be the side chain of 3-pyridylalanine. R ₂ can be the side chain of 4-methylphenylalanine. R ₂ can be the side chain of 4-fluorophenylalanine. R ₂ can be the side chain of 4-chlorophenylalanine. R ₂ can be the side chain of 3- (9-anthryl) -alanine.

R ₃ can be a side chain of tyrosine. R ₃ can be the side chain of phenylalanine. R ₃ can be the side chain of 1-naphthylalanine. R ₃ can be the side chain of 2-naphthylalanine. R ₃ can be the side chain of tryptophan. R ₃ can be the side chain of 3-benzothiophenylalanine. R ₃ can be the side chain of 4-phenylphenylalanine. R ₃ can be the side chain of 3, 4-difluorophenylalanine. R ₃ can be the side chain of 4-trifluoromethylphenylalanine. R ₃ can be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R ₃ can be the side chain of homophenylalanine. R ₃ can be the side chain of beta-homophenylalanine. R ₃ may be the side chain of 4-tert-butylphenylalanine. R ₃ can be the side chain of 4-pyridylalanine. R ₃ can be the side chain of 3-pyridylalanine. R ₃ can be the side chain of 4-methylphenylalanine. R ₃ can be the side chain of 4-fluorophenylalanine. R ₃ can be the side chain of 4-chlorophenylalanine. R ₃ can be the side chain of 3- (9-anthryl) -alanine.

R ₄ can be H, -alkylene-aryl, -alkylene-heteroaryl. R ₄ can be H, -C _1-3 alkylene-aryl, or-C _1-3 alkylene-heteroaryl. R ₄ can be H or-alkylene-aryl. R ₄ can be H or-C _1-3 alkylene-aryl. The C _1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₄ can be H, -C _1-3 alkylene-Ph or-C _1-3 alkylene-naphthyl. R ₄ can be H or the side chain of an amino acid in Table 4 or Table 6. R ₄ may be H or an amino acid residue having a side chain comprising an aromatic group. R ₄ can be H, -CH ₂ Ph or-CH ₂ naphthyl. R ₄ can be H or-CH ₂ Ph.

R ₅ can be H, -alkylene-aryl, -alkylene-heteroaryl. R ₅ can be H, -C _1-3 alkylene-aryl, or-C _1-3 alkylene-heteroaryl. R ₅ can be H or-alkylene-aryl. R ₅ can be H or-C _1-3 alkylene-aryl. The C _1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₅ can be H, -C _1-3 alkylene-Ph or-C _1-3 alkylene-naphthyl. R ₅ can be H or the side chain of an amino acid in Table 4 or Table 6. R ₄ may be H or an amino acid residue having a side chain comprising an aromatic group. R ₅ can be H, -CH ₂ Ph or-CH ₂ naphthyl. R ₄ can be H or-CH ₂ Ph.

R ₆ can be H, -alkylene-aryl, -alkylene-heteroaryl. R ₆ can be H, -C _1-3 alkylene-aryl, or-C _1-3 alkylene-heteroaryl. R ₆ can be H or-alkylene-aryl. R ₆ can be H or-C _1-3 alkylene-aryl. The C _1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₆ can be H, -C _1-3 alkylene-Ph or-C _1-3 alkylene-naphthyl. R ₆ can be H or the side chain of an amino acid in Table 4 or Table 6. R ₆ may be H or an amino acid residue having a side chain comprising an aromatic group. R ₆ can be H, -CH ₂ Ph or-CH ₂ naphthyl. R ₆ can be H or-CH ₂ Ph.

R ₇ can be H, -alkylene-aryl, -alkylene-heteroaryl. R ₇ can be H, -C _1-3 alkylene-aryl, or-C _1-3 alkylene-heteroaryl. R ₇ can be H or-alkylene-aryl. R ₇ can be H or-C _1-3 alkylene-aryl. The C _1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R ₇ can be H, -C _1-3 alkylene-Ph or-C _1-3 alkylene-naphthyl. R ₇ can be H or the side chain of an amino acid in Table 4 or Table 6. R ₇ may be H or an amino acid residue having a side chain comprising an aromatic group. R ₇ can be H, -CH ₂ Ph or-CH ₂ naphthyl. R ₇ can be H or-CH ₂ Ph.

One, two or three of R ₁、R₂、R₃、R₄、R₅、R₆ and R ₇ may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆ and one of R ₇ may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆ and two of R ₇ may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆ and three of R ₇ may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆ and at least one of R ₇ may be-CH ₂Ph.R₁、R₂、R₃、R₄、R₅、R₆ and no more than four of R ₇ may be-CH ₂ Ph.

One, two or three of R ₁、R₂、R₃ and R ₄ are-CH ₂Ph.R₁、R₂、R₃ and one of R ₄ are-CH ₂Ph.R₁、R₂、R₃ and two of R ₄ are-CH ₂Ph.R₁、R₂、R₃ and three of R ₄ are-CH ₂Ph.R₁、R₂、R₃ and at least one of R ₄ are-CH ₂ Ph.

One, two or three of R ₁、R₂、R₃、R₄、R₅、R₆ and R ₇ may be H. One of R ₁、R₂、R₃、R₄、R₅、R₆ and R ₇ may be H. Two of R ₁、R₂、R₃、R₄、R₅、R₆ and R ₇ are H. Three of R ₁、R₂、R₃、R₅、R₆ and R ₇ may be H. At least one of R ₁、R₂、R₃、R₄、R₅、R₆ and R ₇ may be H. No more than three of R ₁、R₂、R₃、R₄、R₅、R₆ and R ₇ may be-CH ₂ Ph.

One, two or three of R ₁、R₂、R₃ and R ₄ are H. One of R ₁、R₂、R₃ and R ₄ is H. Two of R ₁、R₂、R₃ and R ₄ are H. Three of R ₁、R₂、R₃ and R ₄ are H. At least one of R ₁、R₂、R₃ and R ₄ is H.

At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of 4-guanidino-2-aminobutyric acid. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of arginine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of homoarginine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of N-methyl arginine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of N, N-dimethylarginine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of 2, 3-diaminopropionic acid. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of 2, 4-diaminobutyric acid, lysine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of N-methyl lysine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of N, N-dimethyl lysine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of N-ethyl lysine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of N, N-trimethyllysine, 4-guanidinophenylalanine. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of citrulline. At least one of R ₄、R₅、R₆ and R ₇ may be a side chain of N, N-dimethyl lysine, β -homoarginine. At least one of R ₄、R₅、R₆ and R ₇ may be the side chain of 3- (1-piperidinyl) alanine.

At least two of R ₄、R₅、R₆ and R ₇ may be side chains of 3-guanidino-2-aminopropionic acid. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of 4-guanidino-2-aminobutyric acid. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of arginine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of homoarginine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of N-methyl arginine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of N, N-dimethylarginine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of 2, 3-diaminopropionic acid. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of 2, 4-diaminobutyric acid, lysine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of N-methyl lysine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of N, N-dimethyl lysine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of N-ethyl lysine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of N, N, N-trimethyllysine, 4-guanidinophenylalanine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of citrulline. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of N, N-dimethyl lysine, β -homoarginine. At least two of R ₄、R₅、R₆ and R ₇ may be side chains of 3- (1-piperidinyl) alanine.

At least three of R ₄、R₅、R₆ and R ₇ may be side chains of 3-guanidino-2-aminopropionic acid. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of 4-guanidino-2-aminobutyric acid. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of arginine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of homoarginine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of N-methyl arginine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of N, N-dimethylarginine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of 2, 3-diaminopropionic acid. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of 2, 4-diaminobutyric acid and lysine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of N-methyllysine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of N, N-dimethyl lysine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of N-ethyl lysine. At least three of R ₄、R₅、R₆ and R ₇ may be the side chain of N, N, N-trimethyllysine, 4-guanidinophenylalanine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of citrulline. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of N, N-dimethyl lysine, beta-homoarginine. At least three of R ₄、R₅、R₆ and R ₇ may be side chains of 3- (1-piperidinyl) alanine.

AA _SC can be a side chain of an asparagine, glutamine or homoglutamine residue. AA _SC can be a side chain of a glutamine residue. cCPP may also comprise a linker conjugated to AA _SC, such as asparagine, glutamine or homoglutamine residues. Thus cCPP may also comprise linkers conjugated to asparagine, glutamine or homoglutamine residues. cCPP may also comprise a linker conjugated to the glutamine residue.

Q may be 1,2 or 3.q may be 1 or 2.q may be 1.q may be 2.q may be 3.q may be 4.

M may be 1-3.m may be 1 or 2.m may be 0.m may be 1.m may be 2.m may be 3.

CCPP of formula (a) may comprise structure (I) of formula (I) or a protonated form thereof, wherein AA _SC、R₁、R₂、R₃、R₄、R₇, m and q are as defined herein.

CCPP of formula (A) may comprise a structure of formula (I-a) or formula (I-b):

Or a protonated form thereof, wherein AA _SC、R₁、R₂、R₃、R₄ and m are as defined herein.

CCPP of formula (A) may comprise a structure of formula (I-1), formula (I-2), formula (I-3) or formula (I-4):

Or a protonated form thereof, wherein AA _SC and m are as defined herein.

CCPP of formula (A) may comprise a structure of formula (I-5) or formula (I-6):

Or a protonated form thereof, wherein AA _SC is as defined herein.

CCPP of formula (a) may comprise the structure of formula (I-1):

Or a protonated form thereof,

Wherein AA _SC- and m are as defined herein.

CCPP of formula (a) may comprise the structure of formula (I-2):

Or a protonated form thereof, wherein AA _SC- and m are as defined herein.

CCPP of formula (a) may comprise the structure of formula (I-3):

Or a protonated form thereof, wherein AA _SC- and m are as defined herein.

CCPP of formula (a) may comprise the structure of formula (I-4):

Or a protonated form thereof, wherein AA _SC- and m are as defined herein.

CCPP of formula (a) may comprise the structure of formula (I-5):

Or a protonated form thereof,

Wherein AA _SC- and m are as defined herein.

CCPP of formula (a) may comprise the structure of formula (I-6):

Or a protonated form thereof, wherein AA _SC and m are as defined herein.

CCPP may comprise one of the following sequences: FGFGRGR (SEQ ID NO: 68); gfFGrGr (SEQ ID NO: 69); ffPhi GRGR (SEQ ID NO: 70); ffFGRGR (SEQ ID NO: 71); or FfPhi GrGr (SEQ ID NO: 72). cCPP may have one of the following sequences: FGF phi (SEQ ID NO: 73); gfFGrGrQ (SEQ ID NO: 74); ffPhi GRGRQ (SEQ ID NO: 75); ffFGRGRQ (SEQ ID NO: 76); or FfPhi GrGrQ (SEQ ID NO: 77).

The present disclosure also relates to cCPP having the structure of formula (II):

Wherein:

AA _SC is an amino acid side chain;

R ^1a、R^1b and R ^1c are each independently 6 to 14 membered aryl or 6 to 14 membered heteroaryl;

r ^2a、R^2b、R^2c and R ^2d are independently amino acid side chains;

at least one of R ^2a、R^2b、R^2c and R ^2d is Or a protonated form thereof;

at least one of R ^2a、R^2b、R^2c and R ^2d is guanidine or a protonated form thereof;

each n "is independently an integer of 0,1, 2, 3, 4, or 5;

each n' is independently an integer of 0, 1,2 or 3; and

If n' is 0, then R ^2a,R^2b,R^2b or R ^2d are absent.

At least two of R ^2a、R^2b、R^2c and R ^2d may be Or a protonated form thereof. Two or three of R ^2a、R^2b、R^2c and R ^2d may be/> Or a protonated form thereof. One of R ^2a、R^2b、R^2c and R ^2d may be Or a protonated form thereof. At least one of R ^2a、R^2b、R^2c and R ^2d may be/>Or a protonated form thereof, and the remainder of R ^2a、R^2b、R^2c and R ^2d may be guanidine or a protonated form thereof. At least two of R ^2a、R^2b、R^2c and R ^2d may be/>Or a protonated form thereof, the remainder of R ^2a、R^2b、R^2c and R ^2d may be guanidine or a protonated form thereof.

R ^2a、R^2b、R^2c and R ^2d may all be Or a protonated form thereof. At least one of R ^2a、R^2b、R^2c and R ^2d may beOr a protonated form thereof, and the remainder of R ^2a、R^2b、R^2c and R ^2d may be guanidine or a protonated form thereof. At least two R ^2a、R^2b、R^2c and R ^2d groups may be/>Or a protonated form thereof, and the remainder of R ^2a、R^2b、R^2c and R ^2d are guanidine or a protonated form thereof.

Each of R ^2a、R^2b、R^2c and R ^2d may independently be a side chain of 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homolysine, serine, homoserine, threonine, allothreonine, histidine, 1-methylhistidine, 2-aminosuccinic acid, aspartic acid, glutamic acid, or homoglutamic acid.

AA _SC can beWherein t may be an integer from 0 to 5. AA _SC can beWhere t may be an integer from 0 to 5.t may be 1 to 5.t is 2 or 3.t may be 2.t may be 3.

R ^1a、R^1b and R ^1c may each independently be a6 to 14 membered aryl group. R ^1a、R^1b and R ^1c may each independently be a6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O or S. R ^1a、R^1b and R ^1c may each be independently selected from phenyl, naphthyl, anthracenyl, pyridinyl, quinolinyl or isoquinolinyl. R ^1a、R^1b and R ^1c may each be independently selected from phenyl, naphthyl or anthracenyl. R ^1a、R^1b and R ^1c may each independently be phenyl or naphthyl. R ^1a、R^1b and R ^1c may each be independently selected from pyridinyl, quinolinyl or isoquinolinyl.

Each n' may independently be 1 or 2. Each n' may be 1. Each n' may be 2. At least one n' may be 0. At least one n' may be 1. At least one n' may be 2. At least one n' may be 3. At least one n' may be 4. At least one n' may be 5.

Each n "may independently be an integer from 1 to 3. Each n "may independently be 2 or 3. Each n "may be 2. Each n "may be 3. At least one n "may be 0. At least one n "may be 1. At least one n "may be 2. At least one n "may be 3.

Each n "may independently be 1 or 2, and each n' may independently be 2 or 3. Each n "may be 1 and each n' may independently be 2 or 3. Each n "may be 1 and each n' may be 2. Each n "is 1 and each n' is 3.

CCPP of formula (II) may have the structure of formula (II-1):

Wherein R ^1a、R^1b、R^1c、R^2a、R^2b、R^2c、R^2d、AA_SC, n 'and n' are as defined herein.

CCPP of formula (II) may have the structure of formula (IIa):

Wherein R ^1a、R^1b、R^1c、R^2a、R^2b、R^2c、R^2d、AA_SC and n' are as defined herein.

CCPP of formula (II) may have the structure of formula (IIb):

Wherein R ^2a、R^2b、AA_SC and n' are as defined herein.

CCPP can have the structure of formula (IIc):

Or a protonated form thereof,

Wherein:

AA _SC and n' are as defined herein.

CCPP of formula (IIa) has one of the following structures:

/>

Wherein AA _SC and n are as defined herein. cCPP of formula (IIa) has one of the following structures:

/>

Wherein AA _SC and n are as defined herein for formula (IIa) cCPP has one of the following structures:

/>

Wherein AA _SC and n are as defined herein. cCPP of formula (II) may have the following structure: /(I)

CCPP of formula (II) may have the following structure:

cCPP can have the structure of formula (III):

/>

Wherein:

AA _SC is an amino acid side chain;

R ^2a and R ^2c are each independently H, Or a protonated form thereof;

R ^2b and R ^2d are each independently guanidine or a protonated form thereof;

each n "is independently an integer from 1 to 3;

each n' is independently an integer from 1 to 5; and

Each p' is independently an integer from 0 to 5.

CCPP of formula (III) may have the structure of formula (III-1):

Wherein:

AA _SC、R^1a、R^1b、R^1c、R^2a、R^2c、R^2b、R^2d n ', n ", and p' are as defined herein. cCPP of formula (III) may have the structure of formula (IIIa):

Wherein:

AA _SC、R^2a、R^2c、R^2b、R^2d n ', n ", and p' are as defined herein.

In formula (III), formula (III-1) and formula (IIIa), R ^a and R ^c may be H. R ^a and R ^c may be H and R ^b and R ^d may each independently be guanidine or a protonated form thereof. R ^a may be H. R ^b may be H. p' may be 0.R ^a and R ^c may be H and each p' may be 0.

In formulae (III), (III-1) and (IIIa), R ^a and R ^c may be H, R ^b and R ^d may each independently be guanidine or protonated form thereof, n "may be 2 or 3, and each p' may be 0.

P' may be 0.p' may be 1.p' may be 2.p' may be 3.p' may be 4.p' may be 5.

CCPP can have the following structure:

cCPP of formula (a) may be selected from:

CPP sequence	SEQ ID NO:
		FΦRRRRQ	86
fΦRrRrQ	87
		FfΦRrRrQ	78
FfΦCit-r-Cit-rQ	79
		FfΦGrGrQ	80
FfΦRGRGQ	88
		FfFGRGRQ	81
FGFGRGRQ	82
		GfFGrGrQ	83
FGFGRRRQ	84
		FGFRRRRQ	85

In embodiments cCPP is selected from:

wherein Φ=l-naphthylalanine; phi = D-naphthylalanine; Ω=l-norleucine

In embodiments cCPP is not selected from:

Wherein Φ=l-naphthylalanine; phi = D-naphthylalanine; Ω=l-norleucine cCPP may comprise the structure of formula (D):

Or a protonated form thereof, wherein:

R ₄ and R ₆ are independently H or an amino acid side chain;

Y is

AA _SC is an amino acid side chain;

q is 1, 2, 3 or 4;

each m is independently an integer of 0, 1,2 or 3, and

Each n is independently an integer of 0,1, 2 or 3.

CCPP of formula (D) may have the structure of formula (D-I):

Or a protonated form thereof,

Wherein:

R ₄ and R ₆ are independently H or an amino acid side chain;

AA _SC is an amino acid side chain;

q is 1, 2, 3 or 4;

each m is independently an integer of 0, 1,2 or 3, and

Y is

CCPP of formula (D) may have the structure of formula (D-II):

Or a protonated form thereof,

Wherein:

R ₄ and R ₆ are independently H or an amino acid side chain;

AA _SC is an amino acid side chain;

q is 1, 2, 3 or 4;

Each m is independently an integer of 0,1, 2 or 3,

Each m is independently an integer of 0, 1,2 or 3, and

Y is

CCPP of formula (D) may have the structure of formula (D-III):

Or a protonated form thereof,

Wherein:

R ₄ and R ₆ are independently H or an amino acid side chain;

AA _SC is an amino acid side chain;

q is 1, 2, 3 or 4;

Each m is independently an integer of 0,1, 2 or 3,

Each n is independently an integer of 0, 1,2 or 3, and

Y is

CCPP of formula (D) may have the structure of formula (D-IV):

Or a protonated form thereof,

Wherein:

R ₄ and R ₆ are independently H or an amino acid side chain;

AA _SC is an amino acid side chain;

q is 1, 2, 3 or 4;

each m is independently an integer of 0, 1,2 or 3, and

Y is

CCPP of formula (D) may have the structure of formula (D-V):

Or a protonated form thereof,

Wherein:

R ₄ and R ₆ are independently H or an amino acid side chain;

AA _SC is an amino acid side chain;

q is 1, 2, 3 or 4;

each m is independently an integer of 0, 1,2 or 3, and

Y is

AA _SC may be conjugated to a linker.

Joint

CCPP of the present disclosure may be conjugated to a linker. The connector may connect cargo to cCPP. The linker may be attached to the side chain of cCPP amino acids and the cargo may be attached to the appropriate position on the linker.

The linker may be any suitable moiety that may conjugate cCPP to one or more additional moieties, such as a cyclic Exopeptide (EP) and/or cargo. Prior to conjugation to cCPP and one or more additional moieties, the linker has two or more functional groups, each of which is capable of independently forming a covalent bond with cCPP and one or more additional moieties. If the cargo is an oligonucleotide, the linker may be covalently bound to the 5 'end of the cargo or the 3' end of the cargo. The linker may be covalently bound to the 5' end of the cargo. The linker may be covalently bound to the 3' end of the cargo. If the cargo is a peptide, the linker may be covalently bound to the N-terminus or the C-terminus of the cargo. The linker may be covalently bound to the backbone of the oligonucleotide or peptide cargo. The linker may be any suitable moiety that conjugates cCPP described herein to a cargo such as an oligonucleotide, peptide, or small molecule.

The linker may comprise a hydrocarbon linker.

The linker may comprise a cleavage site. The cleavage site may be a disulfide or a caspase cleavage site (e.g., val-Cit-PABC).

The linker may comprise: (i) One or more D or L amino acids, each optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) One or more- (R ^1-J-R²) z "-subunits, wherein each of R ¹ and R ² is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR ³、-NR³ C (O) -, S, and O, wherein R ³ is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; (viii) - (R ^1- J) z "-or- (J-R ¹) z" -, wherein each R ¹ is independently in each occurrence alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, each J is independently C, NR ³、-NR³ C (O) -, S or O, wherein R ³ is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z "is an integer from 1 to 50; or (ix) the linker may comprise one or more of (i) to (x).

The linker may comprise one or more D or L amino acids and/or- (R ^1-J-R²) z "-, wherein each of R ¹ and R ² is each independently alkylene, each J is independently C, NR ³、-NR³ C (O) -, S, and O, wherein R ⁴ is independently selected from H and alkyl, and z" is an integer from 1 to 50; or a combination thereof.

The linker may comprise- (OCH ₂CH₂)_z' - (e.g., as a spacer), where z 'is an integer from 1 to 23, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, "- (OCH ₂CH₂) z' may also be referred to as polyethylene glycol (PEG).

The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise- (OCH ₂CH₂)_z' -and a peptide, wherein z' is an integer from 1 to 23, the peptide may comprise from 2 to 10 amino acids, the linker may further comprise a Functional Group (FG) capable of a click chemistry reaction, FG may be an azide or alkyne, when the cargo binds to the linker, triazole is formed.

The linker may comprise (i) a beta alanine residue and a lysine residue; (ii) - (J-R ¹) z "; or (iii) combinations thereof. Each R ¹ can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J independently is C, NR ³、-NR³ C (O) -, S, or O, wherein R ³ is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z "can be an integer from 1 to 50. Each R ¹ may be alkylene and each J may be O.

The linker may comprise residues of (i) beta-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or a combination thereof; and (ii) - (R ^1- J) z '-or- (J-R ¹) z'. Each R ¹ can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J independently is C, NR ³、-NR³ C (O) -, S, or O, wherein R ³ is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z "can be an integer from 1 to 50. Each R ¹ may be alkylene and each J may be O. The linker may comprise glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid or a combination thereof.

The linker may be a trivalent linker. The joint may have the following structure: Wherein A ₁、B₁ and C ₁ can independently be a hydrocarbon linker (e.g., NRH- (CH ₂)_n -COOH), a PEG linker (e.g., NRH- (CH ₂O)_n -COOH, wherein R is H, methyl, or ethyl), or one or more amino acid residues, and Z independently is a protecting group.

The hydrocarbon may be a glycine or beta-alanine residue.

The linker may be divalent and connects cCPP to the cargo. The linker may be bivalent and connects cCPP to the Exocyclic Peptide (EP).

The linker may be trivalent and connects cCPP to the cargo and EP.

The linker may be a divalent or trivalent C ₁-C₅₀ alkylene group, wherein 1-25 methylene groups are optionally and independently substituted by-N (H) -, -N (C ₁-C₄ alkyl) -, -N (cycloalkyl) -, -O-, -C (O) O-, -S (O) ₂-、-S(O)₂N(C₁-C₄ alkyl) -, -S (O) ₂ N (cycloalkyl) -, -N (H) C (O) -, -N (C ₁-C₄ alkyl) C (O) -, -N (cycloalkyl) C (O) -, -C (O) N (H) -, -C (O) N (C ₁-C₄ alkyl), -C (O) N (cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl or cycloalkenyl. The linker may be a divalent or trivalent C ₁-C₅₀ alkylene group in which 1-25 methylene groups are optionally and independently replaced by-N (H) -, -O-, -C (O) N (H) -or a combination thereof.

The joint may have the following structure:

Wherein: each AA is independently an amino acid residue; * Is an attachment point to AA _SC, and AA _SC is a side chain of the amino acid residue of cCPP; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10. x may be an integer from 1 to 5. x may be an integer from 1 to 3. x may be 1.y may be an integer from 2 to 4. y may be 4.z may be an integer from 1 to 5. z may be an integer from 1 to 3. z may be 1. Each AA may be independently selected from glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid and 6-aminocaproic acid.

CCPP may be attached to the cargo by a joint ("L"). The linker may be conjugated to the cargo through a bonding group ("M").

The joint may have the following structure:

Wherein: x is an integer from 1 to 10; y is an integer from 1 to 5; z is an integer from 1 to 10; each AA is independently an amino acid residue; * Is an attachment point to AA _SC, and AA _SC is a side chain of the amino acid residue of cCPP; and M is a bonding group as defined herein. /(I)

The joint may have the following structure:

wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * Is an attachment point to AA _SC, and AA _SC is a side chain of the amino acid residue of cCPP; and M is a bonding group as defined herein.

The joint may have the following structure:

Wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; and z' is an integer from 1 to 23; * Is an attachment point to AA _SC, and AA _SC is a side chain of the amino acid residue of cCPP.

X may be an integer from 1 to 10, such as 1,2,3,4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.

X' may be an integer from 1 to 23, such as1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. x' may be an integer from 5 to 15. x' may be an integer from 9 to 13. x' may be an integer from 1 to 5. x' may be 1.

Y may be an integer from 1 to 5, such as 1, 2,3, 4 or 5, including all ranges and subranges therebetween. y may be an integer from 2 to 5.y may be an integer from 3 to 5.y may be 3 or 4.y may be 4 or 5.y may be 3.y may be 4.y may be 5.

Z may be an integer from 1 to 10, such as 1,2,3,4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.

Z' may be an integer from 1 to 23, such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. z' may be an integer from 5 to 15. z' may be an integer from 9 to 13. z' may be 11.

As described above, the linker or M (where M is part of the linker) may be covalently bound to the cargo at any suitable location on the cargo. The linker or M (where M is part of the linker) may be covalently bound to the 3 'end of the oligonucleotide cargo or the 5' end of the oligonucleotide cargo. The linker or M (where M is part of the linker) may be covalently bound to the N-terminus or the C-terminus of the peptide cargo. The linker or M (where M is part of the linker) may be covalently bound to the backbone of the oligonucleotide or peptide cargo.

The linker may be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on cCPP, or to a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker may be attached to the side chain of the lysine on cCPP.

The linker may be bound to a side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the peptide cargo, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker may be attached to the side chain of a lysine on the peptide cargo.

The joint may have the following structure:

Wherein the method comprises the steps of

M is a group that conjugates L to cargo such as oligonucleotides;

AA _s is the side chain or terminal to the amino acid on cCPP;

Each AA _x is independently an amino acid residue;

o is an integer from 0 to 10; and

P is an integer from 0 to 5.

The joint may have the following structure:

Wherein the method comprises the steps of

M is a group that conjugates L to cargo such as oligonucleotides;

AA _s is the side chain or terminal to the amino acid on cCPP;

Each AA _x is independently an amino acid residue;

o is an integer from 0 to 10; and

P is an integer from 0 to 5.

M may include alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted. M may be selected from:

wherein R is alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl.

M may be selected from:

wherein: r ¹⁰ is alkylene, cycloalkyl or/> Wherein a is 0 to 10.

M may beR ¹⁰ can be/>And a is 0 to 10.M may be/>

M may be a heterobifunctional crosslinker, e.gIt is disclosed in Williams et al Curr.Protoc Nucleic Acid chem.2010,42,4.41.1-4.41.20, which is incorporated herein by reference in its entirety.

M may be-C (O) -.

AA _s may be the side chain or the end of the amino acid on cCPP. Non-limiting examples of AA _s include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or modified side chains of glutamine or asparagine (e.g., reduced side chains having an amino group). AA _s may be AA _SC as defined herein.

Each AA _x is independently a natural or unnatural amino acid. One or more AA _x may be a natural amino acid. One or more AA _x may be an unnatural amino acid. One or more AA _x may be a β -amino acid. The beta-amino acid may be beta-alanine.

O may be an integer from 0 to 10, such as 0, 1,2, 3, 4, 5, 6, 7, 8, 9, and 10.o may be 0, 1,2 or 3.o may be 0.o may be 1.o may be 2.o may be 3.

P may be 0 to 5, for example 0, 1, 2, 3, 4 or 5.p may be 0.p may be 1.p may be 2.p may be 3.p may be 4.p may be 5.

The joint may have the following structure:

Wherein M, AA _s, each- (R ^1-J-R²) z "-, o, and z" are defined herein; r may be 0 or 1.

R may be 0.r may be 1.

The joint may have the following structure:

Wherein each of M, AA _s, o, p, q, r, and z "may be as defined herein.

Z "may be an integer from 1 to 50, such as 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49 and 50, including all ranges and values therebetween. z "may be an integer from 5 to 20. z "may be an integer from 10 to 15.

The joint may have the following structure:

Wherein:

M, AA _s and o are as defined herein.

Other non-limiting examples of suitable linkers include:

/>

wherein M and AA _s are as defined herein.

Provided herein are compounds comprising cCPP and AC, further comprising L, which is complementary to a target in a precursor mRNA sequence, wherein the linker is conjugated to AC through a linking group (M), wherein M is

Provided herein are compounds comprising cCPP and a cargo comprising an Antisense Compound (AC), such as an antisense oligonucleotide, complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein the linker is conjugated to AC through a linking group (M), wherein M is selected from the group consisting of: wherein: r ¹ is alkylene, cycloalkyl or/> Wherein t' is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, wherein R ¹ is/>And t' is 2.

The joint may have the following structure:

Wherein AA _s is as defined herein, and m' is 0-10.

The linker may have the formula:

the linker may have the formula: wherein the "base" is the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

The linker may have the formula:

Wherein "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.

The linker may have the formula:

wherein "base" is the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.

The linker may have the formula:

The linker may be covalently bound to any suitable location on the cargo. The linker is covalently bound to the 3 'end of the cargo or the 5' end of the oligonucleotide cargo. The linker may be covalently bound to the backbone of the cargo.

CCPP-linker conjugates

CCPP may be conjugated to a linker as defined herein. The linker may be conjugated to AA _SC of cCPP as defined herein.

The linker may comprise a- (OCH ₂CH₂)_z' -subunit (e.g., as a spacer), wherein z' is an integer from 1 to 23, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 "- (OCH ₂CH₂)_z', also known as peg.cpp-linker conjugate, may have a structure selected from table 7:

Table 7: cCPP-linker conjugates and SEQ ID NOs

Ring (FfΦ -4gp-r-4 gp-rQ) -PEG ₄-K-NH₂	Ring (SEQ ID NO: 118) -PEG ₄-K-NH₂
		Ring (FfΦ -Cit-r-Cit-rQ) -PEG ₄-K-NH₂	Ring (SEQ ID NO: 119) -PEG ₄-K-NH₂
Ring (FfPhi-Pia-r-Pia-rQ) -PEG ₄-K-NH₂	Ring (SEQ ID NO: 120) -PEG ₄-K-NH₂
		Ring (FfΦ -Dml-r-Dml-rQ) -PEG ₄-K-NH₂	Ring (SEQ ID NO: 121) -PEG ₄-K-NH₂
Ring (Ffphi-Cit-r-Cit-rQ) -PEG ₁₂ -OH	Ring (SEQ ID NO: 122) -PEG ₁₂ -OH
		Ring (fPhi R-Cit-R-Cit-Q) -PEG ₁₂ -OH	Ring (SEQ ID NO: 123) -PEG ₁₂ -OH

The linker may comprise- (OCH ₂CH₂)_z' -subunit and a peptide subunit, wherein z' is an integer from 1 to 23, the peptide subunit may comprise from 2 to 10 amino acids cCPP-linker conjugate may have a structure selected from table 8:

table 8: cCPP-linker conjugates and SEQ ID NOs

EEVs comprising a cyclic cell penetrating peptide (cCPP), a linker, and an Exocyclic Peptide (EP) are provided. EEV may comprise a structure of formula (B):

Or a protonated form thereof,

Wherein:

R ₄ and R ₇ are independently H or an amino acid side chain;

EP is a cyclic exopeptide as defined herein;

each m is independently an integer from 0 to 3;

n is an integer from 0 to 2;

x' is an integer from 1 to 20;

y is an integer from 1 to 5;

q is 1-4; and

Z' is an integer from 1 to 23.

R ₁、R₂、R₃、R₄、R₇, EP, m, q, y, x ', z' are as defined herein.

N may be 0.n may be 1.n may be 2.

EEVs may comprise structures of formula (B-a) or formula (B-B):

Or a protonated form thereof, wherein EP, R ¹、R²、R³、R⁴, m and z' are as defined in formula (B) above.

EEVs may comprise structures of formula (B-c):

Or a protonated form thereof, wherein EP, R ¹、R²、R³、R⁴ and m are as defined in formula (B) above; AA is an amino acid as defined herein; m is as defined herein; n is an integer from 0 to 2; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10.

The EEV may have a structure of formula (B-1), formula (B-2), formula (B-3) or formula (B-4):

/>

or a protonated form thereof, wherein EP is as defined in formula (B) above.

The EEV may comprise formula (B) and may have the following structure: ac-PKKKRKVAEEA-K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH (Ac-SEQ ID NO:132-K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂ -OH) or Ac-PK-KKR-KV-AEEA-K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH (Ac-SEQ ID NO:133-K (cyclo [ SEQ ID NO:83 ]) -PEG ₁₂ -OH.

The EEV may comprise cCPP of the formula:

EEV may include the formula: ac-PKKKKRKV-miniPEG ₂ -Lys (loop (FfFGRGRQ) -PEG ₂-K(N₃)(Ac-SEQ ID NO:42-miniPEG₂ -Lys (loop (SEQ ID NO: 81) -PEG ₂-K(N₃)).

The EEV may be:

EEVs may be

EEV may be Ac-P-K (Tfa) -K (Tfa) -K (Tfa) -R-K (Tfa) -V-miniPEG ₂ -K (cyclo (Ff-Nal-GrGrQ) -PEG ₁₂-OH(Ac-SEQ ID NO:134-miniPEG₂ -K (cyclo (SEQ ID NO: 135) -PEG ₁₂ -OH).

EEVs may be

EEV may be Ac-P-K-K-K-R-K-V-miniPEG ₂ -K (ring (Ff-Nal-GrGrQ) -PEG ₁₂-OH(Ac-SEQ ID NO:42-miniPEG₂ -K (ring (SEQ ID NO: 135) -PEG ₁₂ -OH).

EEVs may be

The EEV may be:

EEVs may be

EEVs may be selected from

/>

EEV may be selected from:

Ac-PKKKKRKV-Lys (cyclo [ FfΦ GrGrQ ]) -PEG ₁₂-K(N₃)-NH₂

(Ac-SEQ ID NO:42-Lys (cyclo [ SEQ ID NO:80 ]) -PEG ₁₂-K(N₃)-NH₂)Ac-PKKKRKV-miniPEG₂ -Lys (cyclo [ FfPhi GrGrQ ]) miniPEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:80 ]) -miniPEG ₂-K(N₃)-NH₂)

Ac-PKKKKRKV-miniPEG ₂ -Lys (cyclo [ FGFGRGRQ ]) -miniPEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:82 ]) -miniPEG ₂-K(N₃)-NH₂)

Ac-KR-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₂-K(N₃)-NH₂

(Ac-KR-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₂-K(N₃)-NH₂)Ac-PKKKGKV-PEG₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:46-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₂-K(N₃)-NH₂)Ac-PKKKRKG-PEG₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:48-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₂-K(N₃)-NH₂)Ac-KKKRK-PEG₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:19-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₂-K(N₃)-NH₂)Ac-PKKKRKV-miniPEG₂ -Lys (cyclo [ FF. Phi. GRGRQ ]) -miniPEG ₂-K(N₃)-NH₂

Ac-PKKKKRKV-miniPEG ₂ -Lys (cyclo [ beta hFf. Phi. GrGrQ ]) -miniPEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:142 ]) -miniPEG ₂-K(N₃)-NH₂)

Ac-PKKKKRKV-miniPEG ₂ -Lys (cyclo [ FfΦ SrSrQ ]) -miniPEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:143 ]) -miniPEG ₂-K(N₃)-NH₂).

EEV may be selected from:

Ac-PKKKKRKV-miniPEG ₂ -Lys (loop (GfFGrGrQ)) PEG ₁₂ -OH

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo (SEQ ID NO: 133)) -PEG ₁₂-OH)Ac-PKKKRKV-miniPEG₂ -Lys (cyclo [ FGFKRKRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:144 ]) -PEG ₁₂-OH)Ac-PKKKRKV-miniPEG₂ -Lys (cyclo [ FGFRGRGQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:145 ]) -PEG ₁₂-OH)Ac-PKKKRKV-miniPEG₂ -Lys (cyclo [ FGFGRGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:146 ]) -PEG ₁₂-OH)Ac-PKKKRKV-miniPEG₂ -Lys (cyclo [ FGFGRrRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:147 ]) -PEG ₁₂-OH)Ac-PKKKRKV-miniPEG₂ -Lys (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH) and Ac-PKKKKKRKV-miniPEG ₂ -Lys (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys (cyclo [ SEQ ID NO:85 ])) -PEG ₁₂ -OH.

EEV may be selected from:

Ac-K-K-K-R-K-G-miniPEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:148-miniPEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂-OH)Ac-K-K-K-R-K-miniPEG₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:19-miniPEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂-OH)Ac-K-K-R-K-K-PEG₄ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:22-PEG ₄ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂-OH)Ac-K-R-K-K-K-PEG₄ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:21-PEG ₄ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂-OH)Ac-K-K-K-K-R-PEG₄ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:23-PEG ₄ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂-OH)Ac-R-K-K-K-K-PEG₄ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:20-PEG ₄ -K (cyclo [ SEQ ID NO:82 ])) -PEG ₁₂ -OH and Ac-K-K-R-K-PEG ₄ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:19-PEG ₄ -K (cyclo [ SEQ ID NO:82 ])) -PEG ₁₂ -OH.

EEV may be selected from:

Ac-PKKKRKV-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₂-K(N₃)-NH₂)Ac-PKKKRKV-PEG₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂-OH)Ac-PKKKRKV-PEG₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₂-K(N₃)-NH₂

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:133 ]) -PEG ₂-K(N₃)-NH₂) and Ac-PKKKKKKKV-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:133 ])) -PEG ₁₂ -OH.

The cargo may be AC and the EEV may be selected from:

Ac-PKKKRKV-PEG ₂ -K (cyclo [ FfPhi GrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:80 ])) -PEG ₁₂ -OH

Ac-PKKKKRKV-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:79 ])) -PEG ₁₂ -OH

Ac-PKKKRKV-PEG ₂ -K (cyclo [ FfFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-PKKKRKV-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:82 ])) -PEG ₁₂ -OH

Ac-PKKKRKV-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:133 ])) -PEG ₁₂ -OH

Ac-PKKKRKV-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-PKKKRKV-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:42-PEG ₂ -K (cyclo [ SEQ ID NO:85 ])) -PEG ₁₂ -OH

Ac-rr-PEG ₂ -K (cyclo [ FfPhi GrGrQ ]) -PEG ₁₂ -OH

(Ac-rr-PEG ₂ -K (cyclo [ SEQ ID NO:80 ]) -PEG ₁₂ -OH)

Ac-rr-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-rr-PEG ₂ -K (cyclo [ SEQ ID NO:79 ]) -PEG ₁₂ -OH)

Ac-rr-PEG ₂ -K (cyclo [ FfF-GRGRQ ]) -PEG ₁₂ -OH

(Ac-rr-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-rr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-rr-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂ -OH)

Ac-rr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-rr-PEG ₂ -K (cyclo [ SEQ ID NO:133 ]) -PEG ₁₂ -OH)

Ac-rr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-rr-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-rr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-rr-PEG ₂ -K (cyclo [ SEQ ID NO:85 ]) -PEG ₁₂ -OH)

Ac-rrr-PEG ₂ -K (cyclo [ FfPhi GrGrQ ]) -PEG ₁₂ -OH

(Ac-rrr-PEG ₂ -K (cyclo [ SEQ ID NO:80 ]) -PEG ₁₂ -OH)

Ac-rrr-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-rrr-PEG ₂ -K (cyclo [ SEQ ID NO:79 ]) -PEG ₁₂ -OH)

Ac-rrr-PEG ₂ -K (cyclo [ FfFGRGRQ ]) -PEG ₁₂ -OH

(Ac-rrr-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-rrr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-rrr-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂ -OH)

Ac-rrr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-rrr-PEG ₂ -K (cyclo [ SEQ ID NO:133 ]) -PEG ₁₂ -OH)

Ac-rrr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-rrr-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-rrr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-rrr-PEG ₂ -K (cyclo [ SEQ ID NO:85 ]) -PEG ₁₂ -OH)

Ac-rhr-PEG ₂ -K (cyclo [ FfΦ GrGrQ ]) -PEG ₁₂ -OH

(Ac-rhr-PEG ₂ -K (cyclo [ SEQ ID NO:80 ]) -PEG ₁₂ -OH)

Ac-rhr-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-rhr-PEG ₂ -K (cyclo [ SEQ ID NO:79 ]) -PEG ₁₂ -OH)

Ac-rhr-PEG ₂ -K (cyclo [ FfFGRGRQ ]) -PEG ₁₂ -OH

(Ac-rhr-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-rhr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-rhr-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂ -OH)

Ac-rhr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-rhr-PEG ₂ -K (cyclo [ SEQ ID NO:133 ]) -PEG ₁₂ -OH)

Ac-rhr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-rhr-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-rhr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-rhr-PEG ₂ -K (cyclo [ SEQ ID NO:85 ]) -PEG ₁₂ -OH)

Ac-rbr-PEG ₂ -K (cyclo [ FfΦ GrGrQ ]) -PEG ₁₂ -OH

(Ac-rbr-PEG ₂ -K (cyclo [ SEQ ID NO:80 ]) -PEG ₁₂ -OH)

Ac-rbr-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-rbr-PEG ₂ -K (cyclo [ SEQ ID NO:79 ]) -PEG ₁₂ -OH)

Ac-rbr-PEG ₂ -K (cyclo [ FfFGRGRQ ]) -PEG ₁₂ -OH

(Ac-rbr-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-rbr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-rbr-PEG ₂ -K (cyclo [ SEQ ID NO:82 ]) -PEG ₁₂ -OH)

Ac-rbr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-rbr-PEG ₂ -K (cyclo [ SEQ ID NO:133 ]) -PEG ₁₂ -OH)

Ac-rbr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-rbr-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-rbr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-rbr-PEG ₂ -K (cyclo [ SEQ ID NO:85 ]) -PEG ₁₂ -OH)

Ac-rbrbr-PEG ₂ -K (cyclo [ FfΦ GrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:138-PEG ₂ -K (cyclo [ SEQ ID NO:80 ])) -PEG ₁₂ -OH

Ac-rbrbr-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:138-PEG ₂ -K (cyclo [ SEQ ID NO:79 ])) -PEG ₁₂ -OH

Ac-rbrbr-PEG ₂ -K (cyclo [ FfFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:138-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-rbrbr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:138-PEG ₂ -K (cyclo [ SEQ ID NO:82 ])) -PEG ₁₂ -OH

Ac-rbrbr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:138-PEG ₂ -K (cyclo [ SEQ ID NO:133 ])) -PEG ₁₂ -OH

Ac-rbrbr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:138-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-rbrbr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:138-PEG ₂ -K (cyclo [ SEQ ID NO:85 ])) -PEG ₁₂ -OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FfΦ GrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:149-PEG ₂ -K (cyclo [ SEQ ID NO:80 ])) -PEG ₁₂ -OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:149-PEG ₂ -K (cyclo [ SEQ ID NO:79 ])) -PEG ₁₂ -OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FfFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:149-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-rbhbr-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:149-PEG ₂ -K (cyclo [ SEQ ID NO:82 ])) -PEG ₁₂ -OH

Ac-rbhbr-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:149-PEG ₂ -K (cyclo [ SEQ ID NO:133 ])) -PEG ₁₂ -OH

Ac-rbhbr-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:149-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-rbhbr-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:149-PEG ₂ -K (cyclo [ SEQ ID NO:85 ])) -PEG ₁₂ -OH

Ac-hbrbh-PEG ₂ -K (cyclo [ FfΦ GrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:141-PEG ₂ -K (cyclo [ SEQ ID NO:80 ])) -PEG ₁₂ -OH

Ac-hbrbh-PEG ₂ -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:141-PEG ₂ -K (cyclo [ SEQ ID NO:79 ])) -PEG ₁₂ -OH

Ac-hbrbh-PEG ₂ -K (cyclo [ FfFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:141-PEG ₂ -K (cyclo [ SEQ ID NO:81 ]) -PEG ₁₂ -OH)

Ac-hbrbh-PEG ₂ -K (cyclo [ FGFGRGRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:141-PEG ₂ -K (cyclo [ SEQ ID NO:82 ])) -PEG ₁₂ -OH

Ac-hbrbh-PEG ₂ -K (cyclo [ GfFGrGrQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:141-PEG ₂ -K (cyclo [ SEQ ID NO:133 ])) -PEG ₁₂ -OH

Ac-hbrbh-PEG ₂ -K (cyclo [ FGFGRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:141-PEG ₂ -K (cyclo [ SEQ ID NO:84 ]) -PEG ₁₂ -OH)

Ac-hbrbh-PEG ₂ -K (cyclo [ FGFRRRRQ ]) -PEG ₁₂ -OH

(Ac-SEQ ID NO:141-PEG ₂ -K (cyclo [ SEQ ID NO:85 ])) -PEG ₁₂ -OH,

Wherein b is beta-alanine and the exocyclic sequence may be D or L stereochemistry.

Goods (e.g. freight)

A Cell Penetrating Peptide (CPP), such as a cyclic cell penetrating peptide (e.g., cCPP), may be conjugated to the cargo. As used herein, a "cargo" is a compound or moiety that is desired to be delivered into a cell. The cargo may be conjugated to the terminal carbonyl group of the linker. At least one atom of the cyclic peptide may be displaced by the cargo, or at least one lone pair may form a bond with the cargo. The cargo may be conjugated to cCPP via a linker. Cargo may be conjugated to AA _SC via a linker. At least one atom of cCPP may be displaced by cargo, or at least one lone pair of cCPP forms a bond with cargo. The hydroxyl group on the amino acid side chain of cCPP may be replaced with a bond to the cargo. The hydroxyl group on the glutamine side chain of cCPP can be replaced with a bond to the cargo. The cargo may be conjugated to cCPP via a linker. Cargo may be conjugated to AA _SC via a linker.

In embodiments, the amino acid side chain comprises a chemically reactive group conjugated to a linker or cargo. The chemically reactive group may include an amine group, carboxylic acid, amide, hydroxyl group, sulfhydryl group, guanidino group, phenolic group, thioether group, imidazole group, or indole group. In embodiments, the amino acid cCPP conjugated to the cargo includes lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, homoglutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, or tryptophan.

The cargo may include one or more detectable moieties, one or more Therapeutic Moieties (TM), one or more targeting moieties, or any combination thereof. In embodiments, the cargo comprises a TM. In embodiments, the cargo comprises AC.

Cyclic cell penetrating peptides conjugated to cargo moieties (cCPP)

The cyclic cell penetrating peptide (cCPP) may be conjugated to a cargo moiety.

The cargo moiety may be conjugated to the linker at the terminal carbonyl group to provide the following structure:

Wherein:

EP is a cyclic exopeptide and M, AA _SC, cargo, x ', y and z' are as defined above, are attachment points to AA _SC. x' may be 1.y may be 4.z' may be 11.- (OCH ₂CH₂)_x' -and/or- (OCH ₂CH₂)_z' -may independently be replaced by one or more amino acids including, for example, glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or combinations thereof.

An Endosomal Escape Vector (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an Exocyclic Peptide (EP), and a linker, and may be conjugated to cargo to form an EEV conjugate comprising a structure of formula (C):

Or a protonated form thereof,

Wherein:

R ₄ is H or an amino acid side chain;

EP is a cyclic exopeptide as defined herein;

cargo is part as defined herein;

each m is independently an integer from 0 to 3;

n is an integer from 0 to 2;

x' is an integer from 2 to 20;

y is an integer from 1 to 5;

q is an integer from 1 to-4; and

Z' is an integer from 2 to-20.

R ₁、R₂、R₃、R₄, EP, cargo, m, n, x ', y, q, and z' are as defined herein.

EEV may be conjugated to cargo, and EEV conjugates may comprise a structure of formula (C-a) or formula (C-b):

Or a protonated form thereof, wherein EP, m and z are as defined in formula (C) above.

EEV can be conjugated to cargo, and EEV conjugates can comprise a structure of formula (C-C):

Or a protonated form thereof, wherein EP, R ¹、R²、R³、R⁴ and m are as defined in formula (III) above; AA may be an amino acid as defined herein; n may be an integer from 0 to 2; x may be an integer from 1 to 10; y may be an integer from 1 to 5; and z may be an integer from 1 to 10.

EEVs may be conjugated to oligonucleotide cargo, and EEV-oligonucleotide conjugates may comprise structures of formula (C-1), formula (C-2), formula (C-3), or formula (C-4):

/>

EEV can be conjugated to an oligonucleotide cargo, and EEV conjugates can include the following structure:

cytoplasmic delivery efficiency

Modification of the cyclic cell penetrating peptide (cCPP) may increase cytoplasmic delivery efficiency. By comparing the cytoplasmic delivery efficiency of cCPP with the modified sequence to the control sequence, increased cytoplasmic uptake efficiency can be measured. The control sequence does not include specific replacement amino acid residues in the modified sequence (including, but not limited to, arginine, phenylalanine, and/or glycine), but is otherwise identical.

As used herein, cytoplasmic delivery efficiency refers to the ability of cCPP to cross the cell membrane and enter the cytosol of the cell. cCPP are not necessarily dependent on the receptor or cell type. Cytoplasmic delivery efficiency may refer to absolute cytoplasmic delivery efficiency or relative cytoplasmic delivery efficiency.

Absolute cytosolic delivery efficiency is the ratio of the cytosolic concentration of cCPP (or cCPP-cargo conjugate) to the concentration of cCPP (or cCPP-cargo conjugate) in the growth medium. Relative cytosolic delivery efficiency refers to the concentration of cCPP in the cytosol compared to the concentration of control cCPP in the cytosol. Quantification may be achieved by fluorescent labeling cCPP (e.g., with FITC dye) and measuring the fluorescence intensity using techniques well known in the art.

The relative cytoplasmic delivery efficiency is determined by comparing the amount of the invention cCPP that is internalized by (i) a cell type (e.g., a HeLa cell) to the amount of the control cCPP that is internalized by (ii) the same cell type. To measure relative cytoplasmic delivery efficiency, the cell type can be incubated in the presence of cCPP for a specified period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), after which the amount of intracellular cCPP can be quantified using methods known in the art, such as fluorescence microscopy. Separately, the same concentration of control cCPP was incubated in the presence of the cell type for the same period of time and the amount of control cCPP internalized by the cell was quantified.

The relative cytoplasmic delivery efficiency can be determined by measuring cCPP with the modified sequence against IC ₅₀ of the intracellular target and comparing IC ₅₀ (as described herein) with cCPP of the modified sequence to the control sequence.

The relative cytoplasmic delivery efficiency of cCPP compared to loop (Ff Φrrrq, SEQ ID NO: 150) can be in the range of about 50% to about 450%, for example, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, or about 590%. The relative cytoplasmic delivery efficiency of cCPP can be improved by more than about 600% compared to a cyclic peptide comprising a loop (FfΦRrQ, SEQ ID NO: 150).

The absolute cytosolic delivery efficiency is about 40% to about 100%, e.g., about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, including all values and subranges therebetween.

The cCPP of the present invention may increase cytoplasmic delivery efficiency by about 1.1 fold to about 30 fold, for example, all values of about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10, about 10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0, about 19.5, about 20, about 20.5, about 21.0, about 21.5, about 22.22.5, about 23.25.0, about 24.0, about 25.5, about 26.25.0, about 25.5, about 26.0, about 25.0, about 26.0, about 25.0.

Detectable moiety

In embodiments, the compounds disclosed herein include a detectable moiety. In embodiments, the detectable moiety is attached to the cell-penetrating peptide at an amino, carboxylate group, or side chain of any amino acid of the cell-penetrating peptide moiety (e.g., an amino, carboxylate group, or side chain of any amino acid in a CPP). In embodiments, the therapeutic moiety comprises a detectable moiety. The detectable moiety may comprise any detectable label. Examples of suitable detectable labels include, but are not limited to, UV-Vis labels, near infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable moieties, chelate centers, heavy atoms, radioisotopes, isotopically detectable spin resonance labels, paramagnetic moieties, chromophores, or any combination thereof. In embodiments, the label is detectable without the addition of other reagents.

In embodiments, the detectable moiety is a biocompatible detectable moiety, such that the compound may be suitable for use in a variety of biological applications. As used herein, "biocompatible" and "biocompatible" generally refer to compounds that, along with any metabolites or degradation products thereof, are generally non-toxic to cells and tissues and do not cause any significant side effects to cells and tissues when incubated (e.g., cultured) in the presence of them.

The detectable moiety may comprise a luminophore, such as a fluorescent label or a near infrared label. Examples of suitable luminophores include, but are not limited to, metalloporphyrins; benzoporphyrin; azabenzoporphyrins; naphthalene porphyrin; a phthalocyanine; polycyclic aromatic hydrocarbons such as perylene diimide, pyrene; azo dyes; xanthene dyes; boron dipyrromethene, aza boron dipyrromethene, cyanine dyes, metal-ligand complexes such as bipyridine, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine and oxazine derivatives such as benzophenoxazine; aza-rotaene, squaric acid; 8-hydroxyquinoline, polymethine, luminescent nanoparticles such as quantum dots, nanocrystals; a quinolone (carbostyril); terbium complexes; an inorganic phosphor; ionophores, such as crown ether ancillary or derivatized dyes; or a combination thereof. Specific examples of suitable luminophores include, but are not limited to, pd (II) octaethylporphyrin; pt (II) -octaethylporphyrin; pd (II) tetraphenylporphyrin; pt (II) tetraphenylporphyrin; pd (II) meta-tetraphenylporphyrin tetrabenzoporphyrin; pt (II) m-tetraphenylmethyl benzoporphyrin; pd (II) octaethylporphyrin; pt (II) octaethylporphyrin; pd (II) meta-tetrakis (pentafluorophenyl) porphyrin; pt (II) meta-tetrakis (pentafluorophenyl) porphyrin; ru (II) tris (4, 7-diphenyl-1, 10-phenanthroline) (Ru (dpp) ₃); ru (II) tris (1, 10-phenanthroline) (Ru (phen) ₃), tris (2, 2' -bipyridine) ruthenium (II) chloride hexahydrate (Ru (bpy) ₃); erythrosine B; fluorescein; fluorescein Isothiocyanate (FITC); eosin; iridium (III) ((N-methyl-benzoimidazol-2-yl) -7- (diethylamino) -coumarin); 137 benzothiazole) ((benzothiazol-2-yl) -7- (diethylamino) -coumarin)) -2- (acetylacetonate); lumogen dye; macroflex fluorescent red; macrolex fluorescent yellow; texas red; rhodamine B; rhodamine 6G; thiorhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromocresol red; bromothymol blue; cy2; cy3; cy5; cy5.5; cy7; 4-nitrophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; a calcium magnesium indicator (calmagite); bromo-xylenol; phenol red; neutral red; nitrooxazine; 3,4,5, 6-tetrabromophenolphthalein; congo red; fluorescein; eosin; 2',7' -dichlorofluorescein; 5 (6) -carboxy-fluorescein; carboxynaphthalene fluorescein; 8-hydroxypyrene-1, 3, 6-trisulfonic acid; semi-naphthyl rhodamine (semi-naphthorhodafluor); semi-naphthalene fluorescein; tris (4, 7-diphenyl-1, 10-phenanthroline) ruthenium (II) dichloride; (4, 7-diphenyl-1, 10-phenanthroline) ruthenium (II) tetraphenyl boride; platinum (II) octaethylporphyrin; dialkylcarbocyanines; dioctadecyl epoxycarbocyanine; fluorenylmethoxy carbonyl chloride; 7-amino-4-methylcoumarin (Amc); green Fluorescent Protein (GFP); and derivatives or combinations thereof.

In some examples, the detectable moiety may include rhodamine B (Rho), fluorescein Isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green Fluorescent Protein (GFP), or derivatives or combinations thereof.

Preparation method

The compounds described herein may be prepared in various ways known to those skilled in the art of organic synthesis or by modifications thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. The optimal reaction conditions may vary with the particular reactants or solvents used, but such conditions may be determined by one skilled in the art.

Alterations to the compounds described herein include the addition, subtraction, or movement of the various components described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule may vary. In addition, compound synthesis may involve protection and deprotection of various chemical groups. The person skilled in the art can determine the use of protection and deprotection, and the choice of appropriate protecting groups. The chemical structure of the protecting group can be found, for example, in Wuts and Greene, protective Groups in Organic Synthesis, 4 th edition, wiley & Sons,2006, which is incorporated herein by reference in its entirety.

The raw materials and reagents used to prepare the disclosed compounds and compositions are available from commercial suppliers such as Aldrich Chemical Co.,(Milwaukee,WI)、Acros Organics(Morris Plains,NJ)、Fisher Scientific(Pittsburgh,PA)、Sigma(St.Louis,MO)、Pfizer(New York,NY)、GlaxoSmithKline(Raleigh,NC)、Merck(Whitehouse Station,NJ)、Johnson&Johnson(New Brunswick,NJ)、Aventis(Bridgewater,NJ)、AstraZeneca(Wilmington,DE)、Novartis(Basel,Switzerland)、Wyeth(Madison,NJ)、Bristol-Myers-Squibb(New York,NY)、Roche(Basel,Switzerland)、Lilly(Indianapolis,IN)、Abbott(Abbott Park,IL)、Schering Plough(Kenilworth,NJ) or Boehringer Ingelheim (Ingelheim, germany) or are prepared by methods known to those skilled in the art following procedures such as set forth in the following references: FIESER AND FIESER' S REAGENTS for Organic Synthesis, volumes 1-17 (John Wiley and Sons, 1991); rodd' S CHEMISTRY of Carbon Compounds, volumes 1-5 and journals (ELSEVIER SCIENCE Publishers, 1989); organic Reactions, volumes 1-40 (John Wiley and Sons, 1991); march' S ADVANCED Organic Chemistry, (John Wiley and Sons, 4 th edition); and Larock' sComprehensive Organic Transformations (VCH Publishers inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, may be obtained from commercial sources.

The reaction for producing the compounds described herein may be carried out in a solvent, which may be selected by one skilled in the art of organic synthesis. The solvent may be substantially non-reactive with the starting materials (reactants), intermediates or products under the conditions under which the reaction is carried out, i.e., temperature and pressure. The reaction may be carried out in one solvent or a mixture of more than one solvent. The formation of the product or intermediate may be monitored according to any suitable method known in the art. For example, product formation may be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible light), or mass spectrometry, or by chromatography such as High Performance Liquid Chromatography (HPLC) or thin layer chromatography.

The disclosed compounds can be prepared by solid phase peptide synthesis wherein the amino acid α -N-terminus is protected by an acid or base protecting group. Such protecting groups should have properties that are stable to the conditions of peptide bond formation while being easily removed without disrupting racemization of the growing peptide chain or any chiral centers contained therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenyl isopropoxycarbonyl, t-pentyloxycarbonyl, isobornyloxycarbonyl, α -dimethyl-3, 5-dimethoxybenzyloxycarbonyl, o-nitrophenyloxythio, 2-cyano-t-butoxycarbonyl and the like. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. Other preferred side chain protecting groups are 2,5,7, 8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, cbz, boc and adamantyloxy carbonyl for side chain amino groups such as lysine and arginine; for tyrosine are benzyl, o-bromobenzyloxy-carbonyl, 2, 6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopropyl and acetyl (Ac); for serine are tert-butyl, benzyl and tetrahydropyranyl; for histidine are trityl, benzyl, cbz, p-toluenesulfonyl and 2, 4-dinitrophenyl; for tryptophan is formyl; benzyl and t-butyl for aspartic acid and glutamic acid and triphenylmethyl (trityl) for cysteine.

In the solid phase peptide synthesis method, the α -C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful in the above synthesis are those materials which are inert to the reagents and reaction conditions of the progressive condensation-deprotection reaction and insoluble in the medium used. The solid support used for the synthesis of the α -C-terminal carboxy peptide is a 4-hydroxymethylphenoxymethyl-co (styrene-1% divinylbenzene) or 4- (2 ',4' -dimethoxyphenyl-Fmoc-aminomethyl) phenoxyacetamido ethyl resin available from Applied Biosystems (Foster City, calif.). The α -C-terminal amino acid is coupled to the resin by means of N, N ' -Dicyclohexylcarbodiimide (DCC), N ' -Diisopropylcarbodiimide (DIC) or o-benzotriazole-1-yl-N, N ' -tetramethyluronium Hexafluorophosphate (HBTU), with or without 4-Dimethylaminopyridine (DMAP), 1-Hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) or bis (2-oxo-3-oxazolidine) phosphine chloride (BOPCl), at a temperature of 10 ℃ to 50 ℃ for about 1 to about 24 hours in a solvent such as dichloromethane or DMF. When the solid support is 4- (2 ',4' -dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, prior to coupling with the α -C-terminal amino acid as described above. One method of coupling with the deprotected 4- (2 ',4' -dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin is o-benzotriazol-1-yl-N, N ' -tetramethyluronium hexafluorophosphate (HBTU, 1 eq) and 1-hydroxybenzotriazole (HOBT, 1 eq) in DMF. Coupling of the consecutively protected amino acids may be performed in an automated polypeptide synthesizer. In one example, the α -N-terminus in the amino acid of the growing peptide chain is Fmoc protected. Removal of the Fmoc protecting group from the alpha-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Then about a 3-fold molar excess of each protected amino acid is introduced and the coupling is preferably performed in DMF. The coupling agent may be ortho-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (HBTU, 1 eq.) and 1-hydroxybenzotriazole (HOBT, 1 eq.). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either continuously or in a single operation. Removal and deprotection of the polypeptide may be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising anisole (thianisole), water, ethylene dithiol and trifluoroacetic acid. In the case where the alpha-C terminus of the polypeptide is an alkylamide, the resin is cleaved by ammonolysis with an alkylamine. Alternatively, the peptide may be removed by transesterification with methanol, for example, followed by ammonolysis or by direct transamidation. The protected peptide may be purified at this point or directly into the next step. Removal of the side chain protecting groups can be accomplished using the cleavage mixtures described above. The fully deprotected peptide may be purified by a series of chromatographic steps employing any or all of the following types: ion exchange on weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., amberlite XAD); silica gel adsorption chromatography; carboxymethyl cellulose ion exchange chromatography; partition chromatography, e.g. Sephadex G-25, LH-20 or countercurrent partition; high Performance Liquid Chromatography (HPLC), particularly reverse phase HPLC on octyl or octadecylsilyl-silica bonded phase column packing.

The above polymers, such as PEG groups, may be attached to an oligonucleotide, such as AC, under any suitable conditions. The reactive groups (e.g., aldehyde, amino, ester, thiol, a-haloacetyl, maleimide, or hydrazino) on AC can be conjugated through reactive groups (e.g., aldehyde, amino, ester, thiol, a-haloacetyl, maleimide, or hydrazino) on the PEG moiety using any means known in the art, including via acylation, reductive alkylation, michael addition, thiol alkylation, or other chemoselective conjugation/attachment methods. Activating groups useful for attaching the water-soluble polymer to one or more proteins include, but are not limited to, sulfones, maleimides, thiols, triflates (triflates), triflates (tresylates), aziridines (azidirine), oxiranes, 5-pyridinyl, and alpha-halo acyl groups (e.g., alpha-iodoacetic acid, alpha-bromoacetic acid, alpha-chloroacetic acid). If attached to AC by reductive alkylation, the polymer selected should have a single reactive aldehyde in order to control the degree of polymerization. See, e.g., kinstler et al, adv. Drug. Delivery Rev. (2002), 54:477-485; roberts et al, adv. Drug Delivery Rev. (2002), 54:459-476; and Zalipsky et al, adv. Drug Delivery Rev. (1995), 16:157-182.

To covalently attach an AC or linker directly to a CPP, the appropriate amino acid residue of the CPP may be reacted with an organic derivatizing agent capable of reacting with selected side chains or N-or C-termini of the amino acid. Reactive groups on the peptide or conjugate moiety include, for example, aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazine groups. Derivatizing agents include, for example, maleimide benzoyl sulfosuccinimidyl ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, or other agents known in the art.

Methods of preparing and conjugating AC to a linear CPP are generally described in U.S. publication No. 2018/0298383, which is incorporated herein by reference for all purposes. The method may be applied to the cyclic CPPs disclosed herein.

Synthetic schemes are provided in fig. 3A-3D and fig. 4.

Non-limiting examples of compounds including CPPs and reactive groups useful for conjugation to AC are shown in table 9. Exemplary linker groups are also shown. Exemplary reactive groups include tetrafluorophenyl ester (TFP), free carboxylic acid (COOH), and azide (N ₃). In table 9, n is an integer of 0 to 20; pipa6 is AcRXRRBRRXRYQFLIRXRBRXRB, wherein B is β -alanine and X is aminocaproic acid; dap is 2, 3-diaminopropionic acid; NLS is a nuclear localization sequence; βa is βalanine; -ss-is a disulfide; PABC is the C-terminal domain of the poly (A) binding protein; c _x (where x is a number) is an alkyl chain of length x; and BCN is [6.1.0] nonyne.

Table 9: compounds comprising CPP and reactive groups

In embodiments, the CPP has free carboxylic acid groups that are useful for conjugation to AC. In embodiments, EEVs have free carboxylic acid groups available for conjugation to AC.

The following structure is a 3' cyclooctyne modified PMO for click reaction with azide containing compounds:

An example scheme for the conjugation of CPPs and linkers to the 3' end of AC via an amide bond is shown below.

An exemplary scheme for CPP and linker conjugation to 3' -cyclooctyne modified PMO via strain-promoted azide-alkyne cycloaddition is shown below:

Examples of conjugation chemistries for linking AC and CPP to additional linkers containing polyethylene glycol moieties are shown below:

examples of CPP-linkers conjugated to 5' -cyclooctyne modified PMOs via strain-promoted azide-alkyne cycloaddition (click chemistry) are shown below:

Methods of synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited to methods of synthesizing AC. In embodiments, provided herein are compounds having reactive phosphorus groups useful for forming internucleoside linkages, including, for example, phosphodiester and phosphorothioate internucleoside linkages. Methods of preparing and/or purifying the precursor or antisense compound are not limitations of the compositions or methods provided herein. Methods for synthesizing and purifying DNA, RNA, and antisense compounds are well known to those skilled in the art.

Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, agrawal eds. (1993), humana Press) and/or RNA (Scaringe, methods (2001), 23,206-217.Gait et al, applications of Chemically synthesized RNA in RNA: protein Interactions, smith editions (1998), 1-36.Gallo et al, tetrahedron (2001), 57, 5707-5713).

The antisense compounds provided herein can be conveniently and routinely prepared by well known solid phase synthesis techniques. The equipment used for such synthesis is sold by several suppliers including, for example Applied Biosystems (Foster City, calif.). Additionally or alternatively, any other means known in the art for such synthesis may be employed. Similar techniques are well known for preparing oligonucleotides such as phosphorothioates and alkylated derivatives. The invention is not limited by the synthetic method of antisense compounds.

Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analytical methods include Capillary Electrophoresis (CE) and electrospray mass spectrometry. Such synthetic and analytical methods can be performed in multiwell plates. The method of the present invention is not limited by the method of oligomer purification.

In the compounds disclosed herein, AC is coupled to a CPP (e.g., a cyclic peptide). As used herein, "coupled" may refer to covalent or non-covalent association between a CPP and AC, including fusion of a CPP to AC and chemical conjugation of a CPP (e.g., a cyclic peptide) to AC. One non-limiting example of a means of non-covalently attaching a CPP to an AC is through streptavidin/biotin interactions, such as by conjugating biotin to the CPP and fusing the AC to streptavidin. In the resulting compounds, the CPP is coupled to the AC via a non-covalent association between biotin and streptavidin.

In embodiments, a CPP (e.g., a cyclic peptide) is conjugated directly or indirectly to AC, thereby forming a CPP-AC conjugate. Conjugation of AC to CPP can occur at any suitable site on these moieties. For example, in embodiments, the 5 'or 3' end of the AC may be conjugated to the C-terminus, N-terminus, or side chain of an amino acid in the CPP.

In embodiments, the AC is covalently linked to a CPP (e.g., a cyclic peptide). As used herein, covalent linkage refers to a construct in which the CPP moiety is covalently linked to the 5 'and/or 3' end of the AC moiety. Such conjugates may alternatively be described as having a CPP moiety (e.g., a cyclic peptide moiety) and an oligonucleotide moiety. According to certain embodiments, a covalently linked AC-CPP or CPP-AC conjugate includes an AC component and a CPP component that are associated with each other through a linker as described herein.

In embodiments, AC may be conjugated to a CPP (e.g., a cyclic peptide) through a side chain of an amino acid on the CPP. Any amino acid side chain on a CPP that is capable of forming a covalent bond or that can be so modified can be used to attach an AC to a CPP. The amino acid on the CPP may be a natural or unnatural amino acid. In embodiments, the amino acid on the CPP for conjugation to AC is aspartic acid, glutamic acid, glutamine, asparagine, lysine, ornithine, 2, 3-diaminopropionic acid or an analog thereof, wherein the side chain is substituted with a bond to AC or a linker. In embodiments, the amino acid is lysine or an analog thereof. In embodiments, the amino acid is glutamic acid or an analog thereof. In embodiments, the amino acid is aspartic acid or an analog thereof.

In embodiments, the CPP is cyclic. There are many possible configurations of the compounds disclosed herein. In embodiments, compounds of the present disclosure include compounds in which AC is conjugated to a side chain of an amino acid in a cyclic peptide. In embodiments, the compounds disclosed herein have a structure according to formula I-a (i.e., an exocyclic structure):

CPPLAC

(I-A),

Wherein the linker is covalently bound to the side chain of the amino acid on the CPP and the 5 'end of the AC, the backbone of the AC, or the 3' end of the AC.

Disease and target genes

In embodiments, compounds and methods are provided for treating diseases or disorders associated with one or more genes having amplified nucleotide repeats (e.g., amplified trinucleotide repeats, such as amplified trinucleotide repeats). In embodiments, compounds and methods are provided for treating diseases or disorders associated with one or more genes having amplified ctg.cug trinucleotide repeats. In embodiments, compounds and methods are provided for treating diseases or conditions associated with one or more genes having an amplified CTG CUG trinucleotide repeat sequence in the 3' -UTR of the gene. In embodiments, compounds and methods are provided for treating diseases or disorders associated with genes having amplified CTG CUG in the 3' utr, such as DMPK, ATXN8OS ATXN8, and/or JPH 3. In embodiments, compounds and methods are provided for treating diseases or disorders associated with one or more genes having amplified CTG CUG trinucleotide repeats in the introns of the genes. In embodiments, compounds and methods are provided for treating diseases or disorders associated with CTG-CUG trinucleotide repeats amplified in the TCF4 intron. In embodiments, compounds and methods for treating type 1 myotonic dystrophy (DM 1), fechs corneal endothelial dystrophy (FECD), spinocerebellar ataxia-8 (SCA 8), and/or huntington-like chorea (HDL 2) are provided.

Type 1 tonic muscular dystrophy (DM 1)

In embodiments, compounds, compositions, and methods for treating myotonic muscular dystrophy (DM 1 or Steinert disease) are provided. DM1 is a multisystem disease often characterized by muscle degeneration and myotonic or delayed muscle relaxation caused by repeated sequence action potentials in the muscle fibers. Type 1 myotonic muscular dystrophy (DM 1) is the most common form of muscular dystrophy, with 1 in about 8000 people suffering. DM1 is an example of a genetic disorder caused by ctg.cugamplification. DM1 is a neuromuscular disorder caused by amplification of ctg.cug repeats in the 3' -untranslated region (UTR) of the Dystrophic Myotonic Protein Kinase (DMPK) gene. At the RNA level, DMPK transcripts (e.g., amplified CUG repeats) sequester splice regulatory proteins, e.g., myoblindness like (MBNL) proteins, which result in incorrect splicing of many downstream pre-mrnas regulated by MBNL1 (pre-mrnas that do not contain amplified CUG repeats). This functional gain is the reason for DM 1.

Excess CUG repeats confer toxic activity, termed toxic function gain. A number of key proteins are misprocessed, which leads to the multisystemic nature of the disease, including general limb weakness, respiratory muscle damage, heart abnormalities, fatigue, gastrointestinal complications, cataracts, incontinence and excessive daytime sleepiness.

DM1 patients with ctg.cugamplified within the 3' -untranslated region of the DMPK gene are at increased risk for FECD and form CUGexp-MBNL1 foci in the corneal endothelium. (Mootha et al, INVESTIGATIVE OPHTHALMOLOGY & visual science,2017;58, 4579-4585). Association of MBNL1 with mutant RNAs affects the cell pool of free MBNL1 and triggers mis-splicing of some MBNL1 target genes (e.g., regulated by MBNL 1) in the affected brain, muscle, and heart tissue (Jiang et al Hum Mol genet.2004; 13:3079-3088). Gattey et al (Cornea.2014; 33:96-98) report FECD of four DM1 subjects, including a pair of females. Thus, there may be an association between DM1 and the FECD (FECD is described in more detail elsewhere herein).

Without being bound by theory, at least two hypotheses are proposed to explain the pathogenesis of DM 1. One pathogenesis is that the amplified ctg.cug repeat inhibits DMPK mRNA or protein production, resulting in insufficient haploid DMPK. This was supported by studies demonstrating that DMPK mRNA and protein expression was reduced in DM1 muscle (Fu, y.h. et al (1993)Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy.Science 260,235–238). in embodiments, the compounds and methods described herein improve DMPK haploinsufficiency another RNA functional gain hypothesis suggests that mutant RNAs transcribed from amplified alleles are sufficient to induce symptoms of disease, this is suggested by observations that (i) amplified CTG repeats are transcribed into CUG repeats that accumulate in discrete foci, (ii) expression of DMPK 3' -UTRs having only 200 CTG repeats is sufficient to inhibit myogenesis (Davis, b.m. et al (1997)Expansion of a CUG trinucleotide repeat in the 3l untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts.Proc.Natl.Acad.Sci.U.S.A.94,7388–7393;Amack,J.D. et al ,(1999)Cis and trans effects of the myotonic dystrophy(DM)mutation in a cell culture model.Hum.Mol.Genet.8,1975–1984). in embodiments, the compounds and methods described herein reduce transcription of mutant RNAs associated with amplified alleles).

The ctg.cugtrinucleotide repeats amplified in the 3' untranslated region of DMPK mRNA form imperfect stable hairpin structures that accumulate in the nucleus in the form of small ribonucleoplexes or microscopic inclusions and impair the function of proteins involved in transcription, splicing or RNA export. Although the DMPK gene with CUG repeat sequence was transcribed into mRNA, the mutant transcript was sequestered as an aggregate (foci) in the nucleus, which resulted in a decrease in cytoplasmic DMPK mRNA levels. Due to the isolation of the two RNA binding proteins, these aggregates lead to alternative splicing deregulation of many different transcripts: MBNL1 (myoblindness-like protein 1) and CUGBP1 (CUG-binding protein 1), causing loss of MBNL1 function and CUGBP up-regulation (Lee and Cooper. (2009) "Pathogenic mechanisms of myotonic dystrophy," Biochem Soc Trans.37 (06): 1281-1286).

In DM1, the RNA-binding protein MBNL1 is sequestered in a double-stranded hairpin structure formed by the CUG repeat sequence, allowing it to be depleted from the nucleoplasm. The MBNL 1-bound CUG repeat then stimulates Protein Kinase C (PKC) activation through an unknown mechanism, thereby inducing CUGBP a1 hyperphosphorylation and stabilization. Downstream effects include disruption of alternative splicing, mRNA translation, and mRNA attenuation of downstream genes. An important molecular feature of DM1 is the deregulation of alternative splicing, due to the isolation of MBNL1 to CUG repeats with double stranded hairpin structures. Among the twenty or more deregulated splicing events in DM1, aberrant splicing of skeletal muscle-specific ClC-1 (chloride channel 1) is known to be one of the causes of myotonia. In embodiments, the compounds and methods described herein reduce the number of exons containing a premature stop codon, as compared to a subject with DM1 not treated with a compound or method of the disclosure, and thus, in some embodiments, the compounds and methods described herein can reduce the number of deregulated splicing events in one or more downstream genes, such as 4833439L19Rik、Abcc9、Atp2a1、Arhgef10、Arhgap28、Armcx6、Angel1、Best3、Bin1、Brd2、Cacna1s、Cacna2d1、Cpd、Cpeb3、Ccpg1、Clasp1、Clcn1、Clk4、Cpeb2、Camk2g、Capzb、Copz2、Coch、cTNT、Ctu2、Cyp2s1、Dctn4、Dnm1l、Eya4、Efna3、Efna2、Fbxo31、Fbxo21、Frem2、Fgd4、Fuca1、Fn1、Gogla4、Gpr37l1、Greb1、Heg1、Insr、Impdh2、IR、Itgav、Jag2、Klc1、Kcan6、Kif13a、Ldb3、Lrrfip2、Mapt、Macf1、Map3k4、Mapkap1、Mbnl1、Mllt3、Mbnl2、Mef2c、Mpdz、Mrpl1、Mxra7、Mybpc1、Myo9a、Ncapd3、Ngfr、Ndrg3、Ndufv3、Neb、Nfix、Numa1、Opa1、Pacsin2、Pcolce、Pdlim3、Pla2g15、Phactr4、Phka1、Phtf2、Ppp1r12b、Ppp3cc、Ppp1cc、Ramp2、Rapgef1、Rur1、Ryr1、Sorcs2、Spsb4、Scube2、Sema6c、Sfc8a3、Slain2、Sorbs1、Spag9、Tmem28、Tacc1、Tacc2、Ttc7、Tnik、Tnfrsf22、Tnfrsf25、Trappc9、Trim55、Ttn、Txnl4a、Txlnb、Ube2d3 or Vsp39. In embodiments, the compounds and methods described herein reduce the number of exons containing a premature stop codon, as compared to a subject with DM1 not treated with a compound or method of the disclosure, resulting in downregulation of ClC-1.

The levels of MBNL1 and CUGBP1 in the nucleus control a subset of developmentally regulated splicing events that are reversed in DM 1. In embryo stage, MBNL1 nuclear levels were low and CUGBP levels were high. During development, MBNL1 nuclear levels increased, while CUGBP levels decreased, inducing the transition of downstream splicing targets (including IR exon 11, clC-1 exon with stop codon, and cTNT exon 5) from embryos to adults. However, in DM1, MBNL1 sequesters to the CUG repeat, resulting in a decrease in functional MBNL1, while CUGBP levels increase due to phosphorylation and stabilization. This mimics embryo conditions and enhances expression of adult embryo isoforms, resulting in a variety of disease symptoms (Lee and cooper; 2009). In embodiments, the compounds and methods described herein reduce the amount of sequestered MBNL1, increase the amount of functional MBNL1, reduce CUGBP levels compared to a subject with DM1 not treated with a compound or method of the present disclosure.

MBNL1 and CELF (also referred to as "CUGBP 1") are developmental regulators of splicing events during fetal to adult transition, and their altered activity in DM1 results in expression of fetal splicing patterns in adult tissues. Downstream effects of low MBNL1 and high CELF1 include disruption of alternative splicing, mRNA translation, and mRNA attenuation in proteins such as cardiac troponin T (cTNT), insulin receptor (INSR), muscle-specific chloride channel (CLCN 1), and sarcoplasmic/endoplasmic reticulum calcium atpase 1 (ATP 2 A1) transcripts, as well as MBNL1.Konieczny et al (2017)"Myotonic dystrophy:candidate small molecule therapeutics,"Drug Discovery Today.22(11):1740-1748.

Compounds and methods for treating tonic muscular dystrophy using antisense oligomers targeting the poly CUG repeat in the 3' -UTR of the DMPK gene are described in US10106796B2, US10111962B2, US20150080311A1, each of which is incorporated herein by reference in its entirety for all purposes. However, such PMO or PPMO targeting CUG repeats to treat DM1 may have limitations in terms of oligonucleotide delivery to muscle (disease-affected tissue).

In embodiments, the present disclosure teaches the use of different Cell Penetrating Peptides (CPPs) to deliver AC (e.g., PMO or ASO) and degradation sequences described herein, e.g., in tables 2 and 10, to the cytosol of a cell. In embodiments, an AC-conjugated CPP or EEV delivers the AC of interest to the cell location where the target sequence on the pre-mRNA is located.

In embodiments, the disease is a form of myotonic muscular dystrophy (e.g., type 1 myotonic muscular dystrophy or type 2 myotonic muscular dystrophy). In embodiments, the target gene is a DMPK gene encoding myotonic protein kinase. In embodiments, the compounds provided herein comprise an AC (e.g., ASO) that targets a DMPK (e.g., the 3' -untranslated region/polyadenylation of a DMPK gene) to degrade the DMPK gene. Exemplary oligonucleotides targeted for DMPK degradation are provided in table 10. The degradation sequence may be used in combination with an AC sequence comprising 10-40 CAG repeats (including but not limited to the AC provided in table 2).

Table 10. Oligonucleotides (ACs) targeting DMPK for degradation.

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Spinocerebellar ataxia-8 (SCA 8)

In embodiments, compounds, compositions and methods for treating spinocerebellar ataxia-8 (SCA 8) are provided. SCA8 is a hereditary neurodegenerative condition characterized by slow progression of ataxia. Symptoms typically appear from the thirty-th to the fifty-th year of life. Symptoms include abnormal eye movement, sensory neuropathy, dysphagia, cerebellar ataxia, and cognitive disorders.

SCA8 is associated with heterozygous aberrant amplification CTG.CUGrepeat in the 3' UTR of two overlapping genes ATXN8OS and ATXN 8. Healthy individuals typically have 15 to 50 ctg.cugrepeats in the atxn8os and atxn8 genes. In the ATXN8OS and ATXN8 genes, SCA8 patients had more than 50 CTG CUG repeats, sometimes up to 240 CTG CUG repeats.

Huntington-like chorea-2 (HDL 2)

In embodiments, compounds, compositions, and methods for treating huntington-like disease-2 (HDL 2) diseases are provided. HDL2 is an autosomal dominant neurodegenerative disorder phenotypically associated with Huntington's chorea. HDL2 is characterized by symptoms including chorea, dystonia, rigidity, bradykinesia, and psychotic symptoms such as dementia. Symptoms of HDL2 usually appear in middle age and may lead to premature death by about 10-15 years.

HDL2 is related to CTG.CUG amplified in the 3' UTR of the connexin 3 (JPH 3) gene (16q24.3). Healthy individuals typically have 6 to 27 ctg.cugrepeats in the JPH3 gene. In the JPH3 gene, HDL2 patients have more than 40 CTG CUG repeats, sometimes up to 60 or more CTG CUG repeats.

Fukes corneal endothelial dystrophy

In embodiments, compounds, compositions, and methods for treating Fuchs corneal endothelial dystrophy (FECD) are provided. FECD (MIM 136800) is an age-related degenerative disorder of the cornea endothelium. FECD is characterized by progressive loss of corneal endothelial cells, by thickening of the dejection Mei Mo (DESCEMENT's membrane) and by deposition of extracellular matrix in drop form. When the number of endothelial cells becomes extremely low, the cornea swells and causes vision loss (Elhalis et al Ocul surf.2010;8 (4): 173-184).

FECD can be inherited as an autosomal dominant trait with genetic heterogeneity. Rare heterozygous mutations in the collagen VIII type α2 gene (COL 8A2, MIM 120252) can lead to early corneal endothelial dystrophy. Other genes such as solute carrier family 4, sodium borate transporter member 11 (SLC 4a11, MIM 610206), transcription factor 8 (TCF 8, MIM 189909), lipoxygenase homeodomain 1 (LOXHD, MIM 613267) and ATP/GTP-like binding protein 1 (AGBL 1, MIM 615523) are commonly associated with a small fraction of adult onset FECD cases. Whole genome association studies of adult onset FECD have shown that transcription factor 4 (TCF 4, MIM 602272) and the most recent KN motif and ankyrin repeat domain containing protein 4 (KANK 4, MIM 614612), laminin gamma-1 (LAMC 1, MIM 150290), na ⁺/K⁺ transport ATPase and beta-1 polypeptide (ATP 1B1, MIM 182330) have a prominent effect on FECD with the mentioned TCF4 locus (Mootha et al, INVESTIGATIVE OPHTHALMOLOGY & visual science,2017;58, 4579-4585).

The trinucleotide repeat sequence amplified at the CTG18.1 locus in TCF4 intron 2 is related to FECD (Wieben et al, PLoS one.2012;7 (11): e 49083). Each copy of the amplified CTG18.1 allele of more than 40 CTG CUG trinucleotide repeats results in a significant risk of FECD development (Mootha et al, invest Ophthalmol Vis Sci.2014; 55:33-42). RNA foci, i.e. markers of functional RNA toxicity gain, have been reported in neurodegenerative disorders caused by simple repeat amplification. In the corneal endothelium of FECD subjects with CTG18.1 triplet repeat amplification, the amplified CUG repeat RNA accumulated as a foci, whereas it was absent in control samples lacking triplet amplification (Mootha et al, invest Ophthalmol Vis Sci.2015;56 (3): 2003-2011). Amplified CUG repeat RNA was co-localized with mRNA splicing factor, myoblind 1 (MBNL 1) as a molecular marker in endothelial cell nuclear foci. Triplet repeat amplification at the CTG18.1 locus may mediate endothelial dysfunction via aberrant gene splicing due to isolation of MBNL1 by the mutant CUG RNA transcript (Du et al, J biol chem.2015; 290:5979-5990). Thus, two different triplet repeats are concentrated on RNA foci and FECD, and these foci may play a causal role on FECD.

In embodiments, compounds and methods useful for treating Fux corneal endothelial dystrophy (FECD) using antisense oligonucleotides to reduce amplified CUG repeat RNA are described in WO2018165541A1, US10760076B2, each of which is incorporated herein by reference in its entirety for all purposes. However, such Phosphorodiamidate Morpholino Oligomers (PMOs) or peptide-conjugated PMOs (PPMOs) targeting CUG repeats for the treatment of DM1, such as those described in the prior art, may have limitations in the delivery of the oligomers to target tissues (e.g., endothelial cell layers of the cornea), which are tissues affected by disease.

In embodiments, the present disclosure teaches the use of different Cell Penetrating Peptides (CPPs) or Endosomal Escape Vectors (EEVs) to deliver AC (e.g., PMO or ASO) described herein, e.g., in table 6, to the cytosol of a cell. In embodiments, an AC-conjugated CPP or EEV delivers the AC of interest to the cell location where the target sequence on the pre-mRNA is located.

In embodiments, the disease is Fechs Endothelial Corneal Dystrophy (FECD). In embodiments, the target gene is TCF4, which encodes transcription factor 4 (TCF-4), also known as immunoglobulin transcription factor 2 (ITF-2). In embodiments, the compounds provided herein comprise antisense oligonucleotides that target TCF 4. Exemplary oligonucleotides useful for targeting TCF4 are provided in tables 2 and 11.

TABLE 11 exemplary oligonucleotides targeting the amplified triplet repeat sequence of TCF4

PMO: phosphorodiamidite morpholino oligomers

Mootha et al (2017) reported that DM1 and FECD originated from non-coding CTG amplifications, but that both were not the same disease. DMPK amplification in DM1 causes multiple organ disease involving various tissues in the eye including the lens, retina and corneal endothelium. In contrast, TCF4 repeat expansion appears to affect the corneal endothelium without any clinically significant sequelae to other ocular tissues or body organs. Mutant amplification of DMPK and TCF4 has important similarities such as (i) foci containing amplified CUG repeats, (ii) association of foci with MBNL1 protein, and (iii) ability to cause FECD. This suggests that triplet amplification of DMPK and TCF4 may lead to the same FECD corneal endothelial tissue phenotype through a common molecular machinery.

See U.S. patent No. 10760076B2, international application publication No. WO2018165541A1, U.S. patent application publication No. 2016/0355796, and U.S. patent application publication No. 2018/0344817, each of which is incorporated herein by reference, and disclose diseases and corresponding genes that favor formation and/or amplification of tandem nucleotide repeats.

Compositions and methods of administration

The compounds of the present disclosure may be formulated into compositions suitable for in vivo use. These compounds and/or compositions can be administered to a patient suffering from or suspected of suffering from a disease associated with an amplified trinucleotide repeat sequence.

In vivo application of the disclosed compounds and compositions containing these compounds may be accomplished by any suitable methods and techniques currently or contemplated to be known to those of skill in the art. For example, the disclosed compounds may be formulated into physiologically or pharmaceutically acceptable compositions and administered by any suitable route known in the art, including, for example, oral and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal and intrathecal administration, such as by injection. The administration of the disclosed compounds or compositions may be a single administration, or at successive or different intervals, as readily determinable by one of skill in the art.

The compounds disclosed herein and compositions containing these compounds may also be administered using liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide uniform doses over a long period of time. These compounds may also be administered in the form of their salt derivatives or in crystalline form.

The compounds disclosed herein may be formulated into pharmaceutical compositions according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in many sources, which are well known and readily available to those skilled in the art. For example, remington's Pharmaceutical Science (1995) of e.w. martin describes formulations that can be used in conjunction with the disclosed methods. In general, the compounds disclosed herein may be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound. The composition used may also be in various forms. These include, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable solutions and non-infusible solutions and sprays. The form depends on the intended mode of administration and the therapeutic application. These compositions also include conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for administration of such doses for the desired therapeutic treatment, the compositions disclosed herein may advantageously comprise a total of about 0.1 to 100% by weight of one or more subject compounds, based on the weight of the total composition including the carrier or diluent.

Formulations suitable for administration include, for example, sterile injectable aqueous solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only a sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets and the like. It should be understood that the compositions disclosed herein may include other conventional agents in the art in addition to the ingredients specifically mentioned above, taking into account the type of formulation in question.

The compounds disclosed herein and compositions comprising these compounds can be delivered to cells by direct contact with the cells or via carrier means. Carrier means for delivering the compounds and compositions to cells are known in the art and include, for example, encapsulation of the compositions in a liposomal fraction. Another means of delivering the compounds and compositions disclosed herein to cells includes attaching the compounds to proteins or nucleic acids that are targeted for delivery to the target cells. U.S. Pat. No. 6,960,648 and U.S. application publication nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow transport of the composition across a biological membrane. U.S. application publication number 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. The compounds may also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymers for intracranial tumors; poly [ bis (p-carboxyphenoxy) propane: sebacic acid ] (as used in GLIADEL) in a molar ratio of 20:80; chondroitin; chitin; and chitosan.

The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, may be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or salts thereof may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and oils. Under ordinary conditions of storage and use, these formulations may contain preservatives to prevent microbial growth.

Pharmaceutical dosage forms suitable for injection or infusion may comprise sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable solutions or infusions, optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. For example, by forming liposomes, by maintaining the desired particle size in the case of dispersions or by using surfactants, proper fluidity can be maintained. Optionally, prevention of microbial action can be achieved by various other antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In many cases, isotonic agents, for example, sugars, buffers, or sodium chloride, may be included. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents delaying absorption, for example, aluminum monostearate and gelatin.

A sterile injectable solution was prepared by the following procedure: the compounds and/or agents disclosed herein are incorporated in the desired amounts with the various other ingredients enumerated above, as required, into a suitable solvent, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solution.

Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity to in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.

The dosage ranges in which the compositions are administered are those that are large enough to produce the desired effect that affects the symptoms or conditions. The dosage should not be too large to cause adverse side effects such as undesired cross-reactions, allergic reactions, etc. Generally, the dosage will vary with the age, condition, sex and extent of the disease of the patient and can be determined by one skilled in the art. If there are any contraindications, the dosage can be adjusted by the individual physician. The dosage may vary and may be administered in one or more doses per day for one or more days.

Also disclosed are pharmaceutical compositions comprising a compound disclosed herein in combination with a pharmaceutically acceptable carrier. Disclosed herein are pharmaceutical compositions suitable for oral, topical or parenteral administration comprising an amount of a compound. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame without lethal toxicity and without causing side effects or morbidity exceeding acceptable levels. Those skilled in the art will recognize that the dosage will depend on a variety of factors including the condition (health) of the subject, the weight of the subject, the type of concurrent therapy (if any), the frequency of treatment, the rate of treatment, and the severity and stage of the pathological condition.

Also disclosed are kits comprising in one or more containers a compound disclosed herein and/or a pharmaceutical composition containing the compound. The disclosed kits may optionally include a pharmaceutically acceptable carrier and/or diluent. In one embodiment, the kit comprises one or more other components, additives or adjuvants as described herein. In one embodiment, the kit includes instructions or packaging materials describing how to administer the compounds or compositions of the kit. The container of the kit may be of any suitable material, such as glass, plastic, metal, etc., and may be of any suitable size, shape or configuration. In one embodiment, the compounds and/or reagents disclosed herein are provided in a kit in solid form, such as in tablet, pill or powder form. In another embodiment, the compounds and/or reagents disclosed herein are provided in a kit in liquid or solution form. In one embodiment, the kit comprises an ampoule or syringe containing a compound and/or reagent disclosed herein in liquid or solution form.

In embodiments, the compounds and/or compositions of the present disclosure are administered to a patient diagnosed with a disease associated with nucleotide repeat amplification at a dose of between about 0.1mg/kg and about 1000mg/kg, for example, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg, about 33mg/kg, about 34mg/kg, about 35mg/kg, about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg, about 60mg/kg, about 61mg/kg, about 62mg/kg, about 63mg/kg, about 64mg/kg, about 65mg/kg, about 66mg/kg, about 67mg/kg, about 68mg/kg, about 69mg/kg, about 70mg/kg, about 71mg/kg, about 72mg/kg, about 73mg/kg, about 74mg/kg, about 75mg/kg, about 76mg/kg, about 77mg/kg, about 78mg/kg, about 79mg/kg, about 80mg/kg, about 81mg/kg, about 82mg/kg, about 83mg/kg, about 84mg/kg, about 85mg/kg, about 86mg/kg, about 87mg/kg, about 88mg/kg, about 89mg/kg, about 90mg/kg, about 91mg/kg about 92mg/kg, about 93mg/kg, about 94mg/kg, about 95mg/kg, about 96mg/kg, about 97mg/kg, about 98mg/kg, about 99mg/kg, about 100mg/kg, about 110mg/kg, about 120mg/kg, about 130mg/kg, about 140mg/kg, about 150mg/kg, about 160mg/kg, about 170mg/kg, about 180mg/kg, about 190mg/kg, about 200mg/kg, about 210mg/kg, about 220mg/kg, about 230mg/kg, about 240mg/kg, about 250mg/kg, about 260mg/kg, about 270mg/kg, about 280mg/kg, about 290mg/kg, about 300mg/kg, about 310mg/kg, about 320mg/kg, about 330mg/kg, about 340mg/kg, about 350mg/kg, about 360mg/kg, about 370mg/kg, about 380mg/kg, about 390mg/kg, about 400mg/kg, about 410mg/kg, about 420mg/kg, about 430mg/kg, about 440mg/kg, about 450mg/kg, about 460mg/kg, about 470mg/kg, about 480mg/kg, about 490mg/kg, about 500mg/kg, about 510mg/kg, about 520mg/kg, about 530mg/kg, about 540mg/kg, about 550mg/kg, about 560mg/kg, about 570mg/kg, about 580mg/kg, about 590mg/kg, about 600mg/kg, about 610mg/kg, about 620mg/kg, about 630mg/kg, about 640mg/kg, about 650mg/kg, about 660mg/kg, about 670mg/kg, about 680mg/kg, 690mg/kg about 700mg/kg, about 710mg/kg, about 720mg/kg, about 730mg/kg, about 740mg/kg, about 750mg/kg, about 760mg/kg, about 770mg/kg, about 780mg/kg, about 790mg/kg, about 800mg/kg, about 810mg/kg, about 820mg/kg, about 830mg/kg, about 840mg/kg, about 850mg/kg, about 860mg/kg, about 870mg/kg, about 880mg/kg, about 890mg/kg, about 900mg/kg, about 910mg/kg, about 920mg/kg, about 930mg/kg, about 940mg/kg, about 950mg/kg, about 960mg/kg, about 970mg/kg, about 980mg/kg, about 990mg/kg or about 1000mg/kg, including all values and ranges therein and therebetween.

Therapeutic method

The present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering a compound disclosed herein and/or a composition comprising the compound. In embodiments, the disease is any disease provided in the present disclosure. In embodiments, the target gene or gene transcript is any target gene or gene transcript provided in the present disclosure.

In embodiments, the patient is identified as having or at risk of having any of the diseases described herein. In embodiments, methods are provided for treating diseases associated with ctg.cug repeats in the 3' untranslated region of genes/transcripts. In an embodiment, a method for treating myotonic muscular dystrophy is provided. In an embodiment, a method for treating type 1 tonic muscular dystrophy (DM 1) is provided. In an embodiment, a method for treating SCA8 is provided. In an embodiment, a method for treating HDL2 is provided. In an embodiment, a method for treating FECD is provided.

In embodiments, treatment refers to partially or completely alleviating, ameliorating, alleviating, inhibiting, delaying onset, reducing the severity and/or incidence of one or more symptoms in a subject.

Treatment of the disease and/or disease symptoms may be by a variety of molecular mechanisms, such as those described herein.

In embodiments, methods for altering expression and/or activity of a target gene in a subject in need thereof are provided, the methods comprising administering a compound disclosed herein. In embodiments, the treatment results in reduced expression of the target protein from the target transcript. In embodiments, the treatment results in a decrease in the level of the target transcript. In embodiments, the treatment results in modulation of splicing of downstream gene transcripts that are regulated by the target transcript and/or the protein that binds to the target transcript. In embodiments, modulation of downstream gene transcript splicing results in an increase in downstream transcripts and/or downstream protein isoforms associated with a healthy phenotype. In embodiments, alternative splicing results in a decrease in downstream transcripts and/or downstream protein isoforms associated with the disease phenotype.

In embodiments, methods are provided for treating DM1 by reducing the sequestration of at least one RNA binding protein to a pre-mRNA comprising at least one amplified CUG repeat sequence. In embodiments, methods of treating DM1 by reducing accumulation of a precursor mRNA comprising at least one amplified CUG repeat sequence are provided. In embodiments, methods of treating DM1 by correcting splice defects in downstream gene transcripts are provided.

In embodiments, treatment according to the present disclosure results in a reduction in the level of target transcript and/or target transcript (e.g., DMPK, TCF4, JPH3, ATXN8OS, and/or ATXN 8) gene expression by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100% compared to the average protein level of a subject prior to treatment or one or more control subjects having a similar disease but not treated, or compared to treatment with AC not conjugated to a cyclic CPP as disclosed herein. In embodiments, treatment according to the present disclosure results in a reduction in the level of target transcripts (e.g., DMPK, TCF4, JPH3, ATXN8OS, and/or ATXN 8) and/or target transcript expression by about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 50% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 40% to about 95%, about 50% to about 95%, about 70% to about 95%, or about 90% to about 95% as compared to the average level of transcripts and/or proteins in a subject or one or more control subjects having a similar disease but not treated, or as compared to AC treatment with a cyclic CPP not conjugated to the disclosure.

In embodiments, treatment according to the present disclosure results in a reduction in the number of CUG repeat RNA foci of a target gene (e.g., DMPK, TCF4, JPH3, ATXN8OS, and/or ATXN 8) by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average foci level in a subject prior to treatment or one or more control subjects having a similar disease but not treated, or compared to AC treatment not conjugated to a cyclic CPP as disclosed herein. In embodiments, treatment according to the present disclosure results in a reduction in the number of CUG repeat RNA foci of a target gene (e.g., DMPK, TCF4, JPH3, ATXN8OS, and/or ATXN 8) by about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 50% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 40% to about 95%, about 50% to about 95%, about 70% to about 95%, or about 90% to about 95% compared to the average foci level in a subject prior to treatment or one or more control subjects having a similar disease but not treated, or compared to AC treatment with a cyclic CPP not conjugated to the disclosure.

In embodiments, treatment according to the present disclosure results in a reduction in the level of downstream transcript and/or downstream gene product expression associated with a disease phenotype by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average protein level of a subject prior to treatment or one or more control subjects having a similar disease but not treated, or compared to treatment with AC not conjugated to a cyclic CPP disclosed herein. In embodiments, treatment according to the present disclosure results in a reduction in the level of downstream transcript and/or downstream gene product expression associated with a disease phenotype of about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 50% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 40% to about 95%, about 50% to about 95%, about 70% to about 95%, or about 90% to about 95% compared to the average level of protein in a subject prior to treatment or one or more control subjects having a similar disease but not treated, or compared to treatment with AC not conjugated to a cyclic CPP as disclosed herein.

In embodiments, treatment according to the present disclosure results in an increase in the level of downstream transcript and/or downstream gene product expression associated with a healthy phenotype of more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average protein level of a subject prior to treatment or one or more control subjects having a similar disease but not treated, or compared to treatment with AC not conjugated to a cyclic CPP disclosed herein. In embodiments, treatment according to the present disclosure results in an increase in the level of downstream transcript and/or downstream gene product expression associated with a healthy phenotype of about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 50% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 40% to about 95%, about 50% to about 95%, about 70% to about 95%, or about 90% to about 95% compared to the average protein level of a subject prior to treatment or one or more control subjects having a similar disease but not treated, or compared to treatment with AC not conjugated to a cyclic CPP as disclosed herein.

In embodiments, treatment according to the present disclosure results in a reduction in the expression of a protein isoform associated with a disease phenotype in corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or heart of a subject by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average protein level in corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or heart of the subject prior to treatment, or compared to AC treatment with a cyclic CPP not conjugated to the disclosure. In embodiments, treatment according to the present disclosure results in a reduction in the expression of a protein isoform associated with a disease phenotype in corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or heart of a subject by more than about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 50% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 40% to about 95%, about 50% to about 95%, about 70% to about 95%, or about 90% to about 95% in the corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or heart of the subject as compared to the average protein level in the corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or heart of the subject, or as compared to AC treatment not conjugated to the cyclic CPP disclosed herein.

In embodiments, treatment according to the present disclosure results in an increase in expression of an alternatively spliced downstream protein in corneal tissue, muscle tissue, diaphragm tissue, quadriceps, or heart of a subject by more than about 5%, such as about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, or about 1000% or more, compared to the average level of the downstream protein in the corneal tissue, muscle tissue, diaphragm tissue, quadriceps, or heart of the subject prior to treatment, or compared to AC treatment with a cyclic CPP not conjugated thereto.

In embodiments, treatment according to the present disclosure causes an increase or decrease in expression of a wild-type protein isomer in a subject's corneal tissue, muscle tissue, diaphragm tissue, quadriceps, or heart by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the wild-type protein isomer in the subject's corneal tissue, muscle tissue, diaphragm tissue, quadriceps, or heart prior to treatment, or compared to AC treatment with a cyclic CPP not conjugated to the disclosure.

In embodiments, treatment according to the present disclosure results in a reduction in the expression of a protein in a subject's target tissue by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average protein level in the subject's target tissue prior to treatment, or compared to treatment with AC not conjugated to a cyclic CPP disclosed herein.

In embodiments, treatment according to the present disclosure results in an increase in expression of an alternatively spliced downstream protein in a subject's target tissue of more than about 5%, such as about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, or about 1000% or more, compared to the average level of the downstream protein in the target tissue of the subject prior to treatment or compared to treatment with AC treatment not conjugated to a cyclic CPP as disclosed herein.

In embodiments, treatment according to the present disclosure causes an increase or decrease in expression of a wild-type downstream protein isomer in a subject's target tissue by more than about 5%, such as about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the downstream protein in the subject's target tissue prior to treatment, or compared to treatment with AC not conjugated to a cyclic CPP disclosed herein.

In embodiments, the target tissue of the subject is corneal tissue or muscle tissue.

As used herein, the terms "increase", "decrease", and the like indicate values relative to a control. In embodiments, a suitable control is a baseline measurement, such as a measurement of the same individual prior to initiation of the treatment described herein, or a measurement of a control individual (or multiple control individuals) without the treatment described herein. A "control individual" is an individual with the same disease, approximately the same age and/or sex as the individual being treated (to ensure that the disease stage of the treated individual and the control individual are equivalent).

The individual being treated (also referred to as a "patient" or "subject") is an individual (fetus, infant, child, adolescent, or adult) who has a disease or is likely to develop a disease. The individual may have a disorder mediated by aberrant gene expression or aberrant gene splicing. In various embodiments, the level of downstream protein expression or activity in an individual suffering from a disease may be less than about 1% to 99% of the level of normal wild-type protein expression or activity in an individual not suffering from the disease. In embodiments, this range includes, but is not limited to, less than about 80% -99%, less than about 65% -80%, less than about 50% -65%, less than about 30% -50%, less than about 25% -30%, less than about 20% -25%, less than about 15% -20%, less than about 10% -15%, less than about 5% -10%, less than about 1% -5% of the normal wild-type protein expression or activity level. In embodiments, the level of downstream protein expression or activity in the subject is 1% to 500% of the level of normal wild-type target protein expression or activity in a subject not suffering from the disease. In embodiments, this range includes, but is not limited to, from about 1% to 10%, from about 10% to 50%, from about 50% to 100%, from about 100% to 200%, from about 200% to 300%, from about 300% to 400%, from about 400% to 500%, or from about 500% to 1000% greater than the normal wild-type target protein expression or activity level.

In embodiments, the individual is an individual who has been recently diagnosed with the disease. In general, early treatment (starting treatment as soon as possible after diagnosis) is important to minimize the impact of the disease and maximize therapeutic benefit.

Certain definitions

As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a mixture of two or more such compositions, reference to "an agent" includes a mixture of two or more such agents, reference to "the component" includes a mixture of two or more such components, and so forth.

The term "about" when immediately preceding a numerical value means a range (e.g., + or-20%, 10%, or 5% of the value). For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc., unless the context of the present disclosure indicates otherwise or is inconsistent with such interpretation. For example, in a list of values such as "about 49, about 50, about 55, …," about 50 "means extending to a range less than half the interval between the front and back values, e.g., greater than 49.5 to less than 52.5. Furthermore, the phrase "less than about" a value or "greater than about" a value should be understood in accordance with the definition of the term "about" provided herein. Similarly, the term "about" (e.g., "about 10, 20, 30" or "about 10-30") preceding a series of values or ranges of values refers to all values in the series or the endpoints of the range, respectively.

As used herein, "cell penetrating peptide" or "CPP" refers to a peptide that facilitates delivery of cargo, such as a Therapeutic Moiety (TM), into a cell. In an embodiment, the CPP is cyclic and is denoted as "cCPP". In embodiments cCPP are capable of directing the therapeutic moiety to penetrate the cell membrane. In embodiments cCPP delivers the therapeutic moiety to the cytosol of the cell. In embodiments cCPP delivers Antisense Compounds (ACs) to the cellular location where the pre-mRNA is located.

As used herein, the term "endosomal escape vector" (EEV) refers to cCPP conjugated to a linker and/or an Exocyclic Peptide (EP) via a chemical linkage (i.e., covalent bond or non-covalent interaction). The EEV may be an EEV of formula (B).

As used herein, the term "EEV conjugate" refers to an endosomal escape carrier as defined herein that is conjugated to cargo via a chemical linkage (i.e., covalent bond or non-covalent interaction). The cargo may be a therapeutic moiety (e.g., an oligonucleotide, peptide, or small molecule) that can be delivered into the cell by EEV. The EEV conjugate may be an EEV conjugate of formula (C).

As used herein, the terms "exocyclic peptide" (EP) and "modulator peptide" (MP) are used interchangeably to refer to two or more amino acid residues joined by peptide bonds that are conjugated to a cyclic cell penetrating peptide (cCPP) as disclosed herein. When conjugated to the cyclic peptides disclosed herein, EP may alter the tissue distribution and/or retention of the compound. Typically, an EP comprises at least one positively charged amino acid residue, e.g. at least one lysine residue and/or at least one arginine residue. Non-limiting examples of EPs are described herein. An EP may be a peptide that has been identified in the art as a "nuclear localization sequence" (NLS). Non-limiting examples of nuclear localization sequences include the nuclear localization sequence of SV40 viral large T antigen whose smallest functional unit is the seven amino acid sequence PKKKRKV (SEQ ID NO: 42), the nucleoplasmin binary NLS with sequence NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 52), the c-myc nuclear localization sequence with amino acid sequence PAAKRVKLD (SEQ ID NO: 53) or RQRRNELKRSF (SEQ ID NO: 54), the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of the input protein-alpha (SEQ ID NO: 50), the sequence VSRKRPRP of myoma T protein (SEQ ID NO: 57) and PPKKARED (SEQ ID NO: 58), the sequence PQPKKKPL of human p53 (SEQ ID NO: 59), the sequence SALIKKKKKMAP of mouse c-abl IV (SEQ ID NO: 60), the sequence DRLRR of influenza virus NS1 (SEQ ID NO: 61) and PKQKKRK (SEQ ID NO: 62), the sequence RKLKKKIKKL of hepatitis virus delta antigen (SEQ ID NO: 63), the sequence REKKKFLKRR of mouse Mxl protein (SEQ ID NO: 64), the human glucocorticoid sequence of human p53 (SEQ ID NO: 58), and the human glucocorticoid receptor (SEQ ID NO: 9665). Further examples of NLS are described in International publication No. 2001/038547, and incorporated herein by reference in its entirety.

As used herein, "linker" or "L" refers to a moiety that covalently binds one or more moieties (e.g., exocyclic Peptides (EPs) and cargo, such as oligonucleotides, peptides, or small molecules) to a cyclic cell penetrating peptide (cCPP). The linker may comprise a natural or unnatural amino acid or polypeptide. The linker may be a synthetic compound containing two or more suitable functional groups suitable for binding cCPP to the cargo moiety, thereby forming the compounds disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. cCPP may be covalently bound to the cargo via a linker.

The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked to the alpha amino group of one amino acid through the carboxyl group of another amino acid. Two or more amino acid residues may be linked to the alpha amino group by the carboxyl group of one amino acid. Two or more amino acids of a polypeptide may be linked by peptide bonds. A polypeptide may include peptide backbone modifications in which two or more amino acids are covalently attached by bonds other than peptide bonds. A polypeptide may include one or more unnatural amino acids, amino acid analogs, or other synthetic molecules that are capable of being integrated into the polypeptide. The term polypeptide includes naturally occurring and artificially produced amino acids. The term polypeptide includes, for example, peptides comprising from about 2 to about 100 amino acid residues, as well as proteins comprising more than about 100 amino acid residues or more than about 1000 amino acid residues, including but not limited to therapeutic proteins such as antibodies, enzymes, receptors, soluble proteins, and the like.

As used herein, the term "contiguous" refers to two amino acids that are linked by a covalent bond. For example, in a representative cyclic cell penetrating peptide (cCPP) such asIn the case of (a), AA ₁/AA₂、AA₂/AA₃、AA₃/AA₄ and AA ₅/AA₁ illustrate consecutive amino acid pairs.

As used herein, a residue of a chemical refers to a derivative of a chemical present in a particular product. To form a product, at least one atom of the substance is substituted with a bond to another moiety such that the product contains a derivative or residue of the chemical substance. For example, the cyclic cell penetrating peptides (cCPP) described herein have amino acids (e.g., arginine) incorporated therein by formation of one or more peptide bonds. Amino acids incorporated into cCPP may be referred to as residues, or simply amino acids. For example, arginine or arginine residues refer to

The term "protonated form thereof" refers to a protonated form of an amino acid or side chain. For example, the guanidinium group on the arginine side chain may be protonated to form a guanidinium group. The structure of the protonated form of arginine is

As used herein, the term "chiral" refers to molecules having more than one stereoisomer that differ in the three-dimensional arrangement of atoms, wherein one stereoisomer is a non-superimposable mirror image of the other stereoisomer. In addition to glycine, amino acids have a chiral carbon atom adjacent to the carboxyl group. The term "enantiomer" refers to a stereoisomer having chirality. Chiral molecules may be amino acid residues with the "D" and "L" enantiomers. Molecules without chiral centers, such as glycine, may be referred to as "achiral".

As used herein, the term "hydrophobic" refers to a moiety that is insoluble or has minimal solubility in water. Typically, the neutral and/or non-polar moiety, or predominantly neutral and/or non-polar moiety, is hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein.

As used herein, "aromatic" refers to an unsaturated cyclic molecule having 4n+2 pi electrons, wherein n is any integer. "heteroaromatic" as defined below is a subset of aromatic. Examples of aromatic amino acids include phenylalanine and naphthylalanine. The term "non-aromatic" refers to any molecule that does not fall within the definition of aromatic. For example, any linear, branched, or cyclic molecule that is not an aromatic definition is non-aromatic. Examples of non-aromatic amino acids include, but are not limited to, glycine and citrulline.

"Alkyl", "alkyl chain" or "alkyl group" refers to a fully saturated straight or branched hydrocarbon group having 1 to 40 carbon atoms and attached to the remainder of the molecule by a single bond. Including alkyl groups containing any number of carbon atoms from 1 to 40. The alkyl group containing up to 40 carbon atoms is a C ₁-C₄₀ alkyl group, the alkyl group containing up to 10 carbon atoms is a C ₁-C₁₀ alkyl group, the alkyl group containing up to 6 carbon atoms is a C ₁-C₆ alkyl group, and the alkyl group containing up to 5 carbon atoms is a C ₁-C₅ alkyl group. C ₁-C₅ alkyl includes C ₅ alkyl, C ₄ alkyl, C ₃ alkyl, C ₂ alkyl, and C ₁ alkyl (i.e., methyl). C ₁-C₆ alkyl includes all parts described above for C ₁-C₅ alkyl, but also C ₆ alkyl. C ₁-C₁₀ alkyl includes all parts described above for C ₁-C₅ alkyl and C ₁-C₆ alkyl, but also includes C ₇、C₈、C₉ and C ₁₀ alkyl. Similarly, C ₁-C₁₂ alkyl includes all of the foregoing moieties, but also includes C ₁₁ and C ₁₂ alkyl. Non-limiting examples of C ₁-C₁₂ alkyl groups include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless otherwise specifically indicated in the specification, alkyl groups may be optionally substituted.

"Alkylene", "alkylene chain" or "alkylene group" refers to a fully saturated straight or branched divalent hydrocarbon chain group having 1 to 40 carbon atoms. Non-limiting examples of C ₂-C₄₀ alkylene include ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. Unless otherwise specifically indicated in the specification, the alkylene chain may be optionally substituted.

"Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain group having 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the remainder of the molecule by a single bond. Including alkenyl groups containing from 2 to 40 carbon atoms in any number. The alkenyl group containing up to 40 carbon atoms is a C ₂-C₄₀ alkenyl group, the alkenyl group containing up to 10 carbon atoms is a C ₂-C₁₀ alkenyl group, the alkenyl group containing up to 6 carbon atoms is a C ₂-C₆ alkenyl group, and the alkenyl group containing up to 5 carbon atoms is a C ₂-C₅ alkenyl group. C ₂-C₅ alkenyl includes C ₅ alkenyl, C ₄ alkenyl, C ₃ alkenyl and C ₂ alkenyl. C ₂-C₆ alkenyl includes all moieties described above for C ₂-C₅ alkenyl, but also includes C ₆ alkenyl. C ₂-C₁₀ alkenyl includes all of the moieties described above for C ₂-C₅ alkenyl and C ₂-C₆ alkenyl, but also includes C ₇、C₈、C₉ and C ₁₀ alkenyl. Similarly, C ₂-C₁₂ alkenyl includes all of the foregoing moieties, but also includes C ₁₁ and C ₁₂ alkenyl. Non-limiting examples of C ₂-C₁₂ alkenyl include vinyl (vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless otherwise specifically indicated in the specification, alkyl groups may be optionally substituted.

"Alkenylene", "alkenylene" or "alkenylene group" refers to a straight or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. Non-limiting examples of C ₂-C₄₀ alkenylene groups include ethylene, propylene, butene, and the like. Alkenyl ene chains may be optional unless specifically stated otherwise in the specification.

"Alkoxy" OR "alkoxy group" refers to the group-OR, wherein R is alkyl, alkenyl, alkynyl, cycloalkyl, OR heterocyclyl as defined herein. Unless otherwise specifically indicated in the specification, an alkoxy group may be optionally substituted.

"Acyl" or "acyl group" refers to the group-C (O) R, wherein R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl as defined herein. Unless otherwise specifically indicated in the specification, the acyl groups may be optionally substituted.

"Alkylcarbamoyl" or "alkylcarbamoyl group" refers to the group-O-C (O) -NR _aR_b, wherein R _a and R _b are the same or different and are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl as defined herein, or R _aR_b may together form a cycloalkyl group or a heterocyclyl group as defined herein. Unless otherwise specifically indicated in the specification, alkylcarbamoyl groups may be optionally substituted.

"Alkylcarboxamide" or "alkylcarboxamide group" refers to the group-C (O) -NR _aR_b, wherein R _a and R _b are the same or different and are independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl or heterocyclyl group as defined herein, or R _aR_b may together form a cycloalkyl group as defined herein. Unless otherwise specifically indicated in the specification, the alkylcarbamoyl groups may be optionally substituted.

"Aryl" refers to a hydrocarbon ring system group comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems. Aryl groups include, but are not limited to, those derived from acetate, acenaphthylene, acephenanthrene, anthracene, azulene, benzene,Aryl groups of fluoranthene, fluorene, as-dipentane (s-indacene), s-dipentane (s-indacene), indane, indene, naphthalene, phenalene (phenalene), phenanthrene, heptadiene (pleiadiene), pyrene, and benzophenanthrene (TRIPHENYLENE). Unless specifically stated otherwise in the specification, the term "aryl" is intended to include optionally substituted aryl groups.

"Heteroaryl" refers to a 5 to 20 membered ring system group comprising a hydrogen atom, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, heteroaryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples include, but are not limited to, azetidinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxaheptenyl, 1, 4-benzodioxanyl, benzonaphtalenofuranyl, benzoxazolyl, benzodioxo, benzodioxanyl, benzopyranyl, benzopyranonyl (benzopyranonyl), benzofuranyl, benzofuranonyl (benzofuranonyl), benzothienyl (benzothienyl) (benzothienyl (benzothiophenyl)), benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, isothiazolyl, cinnolinyl, benzofuranyl, benzofuranonyl (benzothienyl) imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxocycloheptatrienyl, oxazolyl, oxiranyl, 1-oxopyridinyl, 1-oxopyrimidinyl, 1-oxopyrazinyl, 1-oxopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thienyl (thiophenyl) (i.e., thienyl (thienyl)). Unless otherwise specifically indicated in the specification, heteroaryl groups may be optionally substituted.

The term "substituted" as used herein means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkoxycarbonyl, alkylthio, or arylthio) wherein at least one atom is replaced by a non-hydrogen atom such as, but not limited to: halogen atoms such as F, cl, br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in a group such as a thiol group, a thioalkyl group, a sulfone group, a sulfonyl group, and a sulfoxide group; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylaryl amines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups and triarylsilyl groups; and other heteroatoms in various other groups. "substituted" also means any of the above groups in which one or more atoms are replaced by a higher bond (e.g., double or triple bond) to a heteroatom such as oxo, carbonyl, carboxyl, and oxygen in the ester group; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes any of the above groups in which one or more atoms are replaced with -NR_gR_h、-NR_gC(＝O)R_h、-NR_gC(＝O)NR_gR_h、-NR_gC(＝O)OR_h、-NR_gSO₂R_h、-OC(＝O)NR_gR_h、-OR_g、-SR_g、-SOR_g、-SO₂R_g、-OSO₂R_g、-SO₂OR_g、＝NSO₂R_g and-SO ₂NR_gR_h. "substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by -C(＝O)R_g、-C(＝O)OR_g、-C(＝O)NR_gR_h、-CH₂SO₂R_g、-CH₂SO₂NR_gR_h. In the foregoing, R _g and R _h are the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and/or heteroarylalkyl. "substituted" also means any of the above groups wherein one or more of the atoms is replaced with an amino, cyano, hydroxy, imino, nitro, oxo, thiooxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and/or heteroarylalkyl group. "substituted" may also mean an amino acid in which one or more atoms in the side chain are replaced with an alkyl, alkenyl, alkynyl, acyl, alkylcarboxamide, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl or heteroaryl group. In addition, each of the foregoing substituents may also be optionally substituted with one or more of the foregoing substituents.

As used herein, a symbol(Hereinafter may be referred to as "attachment bond point") means a bond that is an attachment point between two chemical entities, one of which is depicted as attached to the attachment bond point and the other of which is not depicted as attached to the attachment bond point. For example,/>Indicating that chemical entity "XY" is bound to another chemical entity via an attachment bond point. Furthermore, specific points of attachment to a chemical entity not depicted may be specified by inference. For example, compound CH ₃R³, wherein R ³ is H or/>It is inferred that when R ³ is "XY", the attachment bond point is the same bond as the bond depicted as bonding to CH ₃ for R ³.

As used herein, "subject" means an individual. Thus, a "subject" may include domestic animals (e.g., cats, dogs, etc.), farm animals (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "subject" may also include mammals, such as primates or humans. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under treatment by a clinician (e.g., physician).

The terms "inhibit", "inhibit (inhibiting)" or "inhibition" refer to a decrease in an activity, expression, function or other biological parameter and may include, but do not require, complete elimination of the activity, expression, function or other biological parameter. Inhibition may include, for example, at least about a 10% reduction in activity, response, condition, or disease as compared to a control. In embodiments, expression, activity, or function of a gene or protein is reduced by a statistically significant amount. In embodiments, the activity or function is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, and up to about 60%, about 70%, about 80%, about 90%, or about 100%.

"Reduce" or other forms of the word, such as "reduce" or "reduction", means reducing an event or feature (e.g., tumor growth). It will be appreciated that this is typically associated with a certain standard or expected value, in other words, this is relative, but reference to a standard or relative value is not always required. For example, "reducing tumor growth" means reducing the growth rate of a tumor relative to a standard or control (e.g., untreated tumor).

As used herein, "treatment," "treatment," and variants thereof refer to any administration of a disclosed compound that partially or completely alleviates, ameliorates, alleviates, inhibits, delays onset of, reduces the severity of, and/or reduces the incidence of one or more symptoms or features of a disease described herein. In terms of a patient, the term "treatment" refers to the medical management of a patient with the aim of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term includes active treatment, i.e. treatment directed specifically to ameliorating a disease, pathological condition or disorder, as well as causal treatment, i.e. treatment directed to removing the cause of the associated disease, pathological condition or disorder. Furthermore, the term also includes palliative treatment, i.e. treatment designed to alleviate symptoms rather than cure a disease, pathological condition or disorder; prophylactic treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a related disease, pathological condition, or disorder; and supportive treatment, i.e., treatment for supplementing another specific therapy directed to ameliorating a related disease, pathological condition, or disorder.

The term "therapeutically effective" means that the amount of the disclosed compounds and/or compositions used is an amount sufficient to ameliorate one or more causes or symptoms of the disease or disorder. Such improvements need only be reduced or altered and are not necessarily eliminated.

The term "pharmaceutically acceptable" means those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and/or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition of the present disclosure, facilitates or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature for its intended use or purpose, or a combination thereof. For example, the carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier suitable for administration to a patient. The pharmaceutical carrier may be a substance that facilitates or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of a compound or composition of the present disclosure for its intended use or purpose, or a combination thereof. For example, the carrier may be selected to reduce degradation of the compound or to reduce adverse side effects in the patient. In embodiments, the pharmaceutically acceptable carrier may be a sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, as well as a sterile powder for reconstitution into a sterile injectable solution or dispersion prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, sorbitol, and the like. It is also desirable to include isotonic agents, such as sugars, sodium chloride, and the like. The injectable formulations may be sterilized, for example, by filtration through bacterial-retaining filters (bacterial-RETAINING FILTER) or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. Suitable inert carriers may include sugars such as lactose.

The term "pharmaceutically acceptable salts" includes those obtained by reacting an active compound which acts as a base with an inorganic or organic acid to form a salt, for example, salts of hydrochloric, sulfuric, phosphoric, methanesulfonic, camphorsulfonic, oxalic, maleic, succinic, citric, formic, hydrobromic, benzoic, tartaric, fumaric, salicylic, mandelic, carbonic and the like. Those skilled in the art will further recognize that acid addition salts may be prepared by reaction of a compound with an appropriate inorganic or organic acid via any of a number of known methods. The term "pharmaceutically acceptable salts" also includes those salts obtained by reacting an acid-acting active compound with an inorganic or organic base to form salts such as salts of ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, xylylenediamine (ephenamine), dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids and the like. Non-limiting examples of inorganic or metal salts include lithium, sodium, calcium, potassium, magnesium, and the like.

As used herein, the term "parenteral administration" refers to administration by injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous, intravenous, or intramuscular administration.

As used herein, the term "subcutaneous administration" refers to administration just under the skin. By "intravenous administration" is meant administration into a vein.

As used herein, the term "dose" refers to a specified amount of an agent provided in a single administration. In embodiments, one dose may be administered in the form of two or more pills, tablets or injections. In embodiments where subcutaneous administration is desired, the desired dose requires a volume that is not readily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In embodiments, the dose may be administered in two or more injections to reduce the injection site response of the patient.

As used herein, the term "dosage unit" refers to a form that provides a pharmaceutical agent. In embodiments, the dosage unit is a vial comprising a lyophilized compound or composition described herein. In embodiments, the dosage unit is a vial comprising a reconstituted compound or composition described herein.

The term "therapeutic moiety" (TM) refers to a compound that can be used to treat at least one symptom of a disease or disorder, and can include, but is not limited to, therapeutic polypeptides, oligonucleotides, small molecules, and other agents that can be used to treat at least one symptom of a disease or disorder. In embodiments, the TM modulates the activity, expression and/or level of the target transcript. In embodiments, TM reduces the level of target transcripts by an attenuation mechanism. In embodiments, activity is the ability of a target transcript to bind (e.g., sequester) one or more proteins. In embodiments, the TM modulates the activity of the target transcript by reducing the affinity between the target transcript and one or more proteins that bind to the target transcript. The activity of the one or more proteins may be modulated due to the reduced affinity between the target transcript and the one or more proteins. For example, if the one or more proteins do not bind to the target transcript, they may be used to perform their function, e.g., to facilitate splicing, alternative splicing, and/or exon skipping of other transcripts. Due to the function of TM, the activity, expression and/or level of downstream genes regulated by the one or more proteins whose interaction with the target transcript is disrupted by TM can be modulated.

The terms "modulation (modulate)", "modulation" and "modulation" refer to a disturbance of expression, function or activity compared to the level of expression, function or activity prior to modulation. Modulation may include an increase (stimulation or induction) or decrease (inhibition or decrease) in expression, function or activity. In embodiments, the activity of the target transcript is modulated. In embodiments, modulating the activity of the target transcript comprises reducing the ability of the target transcript to bind to one or more proteins. In embodiments, decreasing the affinity between the target transcript and the one or more proteins results in modulating the activity of the one or more proteins interacting with the target transcript. For example, if the one or more proteins do not bind to the target transcript, they may be used to perform their function, such as facilitating splicing, alternative splicing, and/or exon skipping of other transcripts (e.g., downstream transcripts). Thus, modulating the activity of a target transcript may result in modulation of the activity, expression and/or level of a downstream gene regulated by the one or more proteins, the interaction of which with the target transcript may be disrupted.

"Amino acid" means an amino acid comprising an amino group and a carboxylic acid group and having the general formulaWherein R may be any organic group. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid. The amino acid may be a proteinogenic amino acid or a non-proteinogenic amino acid. The amino acid may be an L-amino acid or a D-amino acid. The term "amino acid side chain" or "side chain" refers to a characteristic substituent ("R") that is bound to the α -carbon of a natural or unnatural α -amino acid. Amino acids may be incorporated into polypeptides by peptide bonds.

As used herein, an "uncharged" amino acid is an amino acid having a side chain with a net neutral charge at a pH of 7.35 to 7.45. Examples of uncharged amino acids include, but are not limited to, glycine and citrulline.

As used herein, a "charged" amino acid is an amino acid having a side chain with a net charge at a pH of 7.35 to 7.45. An example of a charged amino acid is arginine.

As used herein, the term "sequence identity" refers to the percentage of nucleic acids or amino acids that are identical and in the same relative position between two oligonucleotide or polypeptide sequences, respectively. Thus, one sequence has a certain percentage of sequence identity compared to another sequence. For sequence comparison, typically one sequence is used as a reference sequence to which the test sequence is compared. One of ordinary skill in the art will appreciate that two sequences are generally considered "substantially identical" if they contain identical residues at the corresponding positions. In embodiments, sequence identity between sequences may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.mol. Biol. 48:443-453) as implemented in the Needle program (EMBOSS:The European Molecular Biology Open Software Suite,Rice et al.,Trends Genet.(2000),16:276-277) of the EMBOSS package by the version of the filing date. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 (the embosm version of BLOSUM 62) substitution matrix. The needle output labeled "longest identity" (obtained using the-nobrief option) was used as percent identity and calculated as follows: (identical residues. Times.100)/(alignment Length-total number of pairs of air)

In other embodiments, sequence identity may be determined using the Smith-Waterman algorithm of the version that exists as of the filing date.

As used herein, "sequence homology" refers to the percentage of amino acids that are homologous and in the same relative position between two polypeptide sequences. Thus, one polypeptide sequence has a certain percentage of sequence homology to another polypeptide sequence. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered "substantially homologous" if they contain homologous residues at the corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be different residues having suitably similar structural and/or functional characteristics. For example, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "nonpolar" side chains, and one amino acid substitution for another amino acid of the same type can be generally considered a "homologous" substitution, as is well known to those of ordinary skill in the art.

Any of a variety of algorithms may be used to compare amino acid sequences, including those available in commercial computer programs, such as BLASTP, vacancy BLAST, and PSI-BLAST, which exist by the date of filing, as is well known in the art. Such procedures are described in Altschul et al, J.mol.biol., (1990), 215 (3): 403-410; altschul et al, nucleic Acids Res (1997), 25:3389-3402; baxevanis et al, bioinformation A PRACTICAL Guide to THE ANALYSIS of Genes and Proteins, wiley,1998; and Misener et al, (editions), bioinformatics Methods and Protocols (Methods in Molecular Biology, volume 132), humana Press, 1999. In addition to identifying homologous sequences, the above-mentioned procedures generally provide an indication of the degree of homology.

As used herein, a "cell targeting moiety" refers to a molecule or macromolecule that specifically binds to a molecule, such as a receptor, on the surface of a target cell. In embodiments, the cell surface molecules are expressed only on the surface of the target cells. In embodiments, the cell surface molecules are also present on the surface of one or more non-target cells, but the amount of cell surface molecule expression is higher on the surface of the target cells. Examples of cell targeting moieties include, but are not limited to, antibodies, peptides, proteins, aptamers, or small molecules.

As used herein, the terms "antisense compound" and "AC" are used interchangeably to refer to a polymeric nucleic acid structure that is at least partially complementary to a target nucleic acid molecule to which it (AC) hybridizes. The AC may be a short (in embodiments, less than 50 bases) polynucleotide or polynucleotide homolog that includes at least a portion of a sequence that is complementary to a target sequence. In embodiments, AC is a polynucleotide or polynucleotide homolog that includes a portion having a sequence that is complementary to a target sequence in a target pre-mRNA strand. AC may be formed from natural nucleic acids, synthetic nucleic acids, nucleic acid homologs, or any combination thereof. In embodiments, the AC comprises an oligonucleotide. In embodiments, the AC comprises an antisense oligonucleotide. In embodiments, AC comprises a conjugate group. Non-limiting examples of ACs include, but are not limited to, primers, probes, antisense oligonucleotides, external Guide Sequence (EGS) oligonucleotides, sirnas, oligonucleotides, oligonucleotide analogs, oligonucleotide mimics, and chimeric combinations of these. Thus, these compounds may be introduced in single-stranded, double-stranded, cyclic, branched or hairpin form, and may contain structural elements such as internal or terminal bulges or loops. The oligomeric double-stranded compound may be two strands that hybridize to form a double-stranded compound, or a single strand that has sufficient self-complementarity to allow hybridization and formation of a complete or partial double-stranded compound. In embodiments, AC modulates (increases, decreases, or alters) expression, levels, and/or activity of a target transcript (e.g., a target nucleic acid). In embodiments, AC reduces the level of target transcripts by inducing an attenuation mechanism. In embodiments, AC modulates the activity of a target transcript. In embodiments, AC modulates the activity of a target transcript by reducing its ability to bind to one or more proteins. In embodiments, decreasing the affinity between the target transcript and the one or more proteins may result in modulating the activity of the one or more proteins. For example, if the one or more proteins do not bind to the target transcript, they may be used to perform their function, such as facilitating splicing, alternative splicing, and/or exon skipping of other transcripts (downstream transcripts). Thus, AC-mediated modulation of the activity of a target transcript may result in modulation of the activity, expression and/or level of a downstream gene regulated by the one or more proteins, the interaction of which with the target transcript may be disrupted.

As used herein, the term "targeting" or "targeting to" refers to the association of a therapeutic moiety (e.g., an antisense compound) with a target nucleic acid molecule or region of a target nucleic acid molecule. In embodiments, the therapeutic moiety comprises an antisense compound capable of hybridizing to the target nucleic acid under physiological conditions. In embodiments, the antisense compound targets a particular portion or site within the target nucleic acid, e.g., a portion of the target nucleic acid having at least one identifiable structure, function, or feature, such as a particular exon or intron, or a selected nucleobase or motif within an exon or intron.

As used herein, the terms "target nucleic acid sequence", "target nucleotide sequence" and "target sequence" refer to a nucleic acid sequence or nucleotide sequence to which a therapeutic moiety (such as an antisense compound) binds or hybridizes. Target nucleic acids include, but are not limited to, a portion of a target transcript, a target RNA (including, but not limited to, pre-mRNA and mRNA or portions thereof), a portion of a target cDNA derived from such RNA, and a portion of a target untranslated RNA (such as miRNA). For example, in embodiments, a target nucleic acid may be part of a target cell gene (or mRNA transcribed from such gene), whose expression or transcription is associated with a particular disorder or disease state. The term "moiety" refers to a defined number of consecutive (i.e., linked) nucleotides in a nucleic acid.

As used herein, the term "transcript" or "gene transcript" refers to an RNA molecule transcribed from DNA, including but not limited to mRNA, pre-mRNA, and partially processed RNA.

The terms "target transcript" and "target RNA" refer to the pre-mRNA or mRNA transcript to which the therapeutic moiety binds. The target transcript may include the target nucleotide sequence. In embodiments, the target transcript comprises a target nucleotide sequence comprising an amplified CUG trinucleotide repeat sequence.

The terms "target gene" and "target gene" refer to genes for which it is desired or expected to regulate expression and/or activity. The target gene may be transcribed into a target transcript comprising the target nucleotide sequence. The target transcript may be translated into the protein of interest.

The term "target protein" refers to a polypeptide or protein encoded by a target transcript (e.g., a target mRNA).

As used herein, the term "mRNA" refers to an RNA molecule encoding a protein, including pre-mRNA and mature mRNA. "Pre-mRNA" refers to a eukaryotic mRNA molecule that is newly synthesized directly after transcription of DNA. In embodiments, the pre-mRNA is capped with a 5 'cap, modified with a 3' poly a tail, and/or spliced to produce a mature mRNA sequence. In embodiments, the pre-mRNA includes one or more introns. In embodiments, the pre-mRNA undergoes a process known as splicing to remove introns and ligate exons. In embodiments, the pre-mRNA comprises one or more splice elements or splice regulatory elements. In embodiments, the pre-mRNA includes a polyadenylation site.

As used herein, the terms "expression," "gene expression," "expression of a gene," and the like refer to all functions and steps in which information encoded in a gene is converted in a cell into a functional gene product, such as a polypeptide or non-coding RNA. Examples of non-coding RNAs include transfer RNAs (trnas) and ribosomal RNAs. Gene expression of a polypeptide includes transcription of the gene to form a pre-mRNA, processing of the pre-mRNA to form a mature mRNA, translocation of the mature mRNA from the nucleus to the cytoplasm, translation of the mature mRNA into the polypeptide, and assembly of the encoded polypeptide. Expression includes partial expression. For example, expression of a gene may be referred to as generation of a gene transcript. Translation of mature mRNA may be referred to as expression of mature mRNA.

As used herein, "modulation of gene expression" and the like refer to modulation of one or more processes associated with gene expression. For example, modification of gene expression may include modification of one or more of gene transcription, RNA processing, translocation of RNA from the nucleus to the cytoplasm, and translation of mRNA into protein.

As used herein, the term "gene" is meant to encompass the 5 'promoter region associated with expression of a gene product, as well as any intronic and exonic regions as well as the 3' untranslated region ("UTR") associated with expression of a gene product.

The term "immune cell" refers to a cell of hematopoietic origin and plays a role in the immune response. Immune cells include, but are not limited to, lymphocytes (e.g., B cells and T cells), natural Killer (NK) cells, and bone marrow cells. The term "bone marrow cells" includes monocytes, macrophages and granulocytes (e.g., basophils, neutrophils, eosinophils and mast cells). Monocytes are lymphocytes that circulate through the blood for 1-3 days, after which they migrate into the tissue and differentiate into macrophages or inflammatory dendritic cells, or die. The term "macrophage" as used herein includes macrophages of fetal origin (also may be referred to as resident tissue macrophages) and macrophages derived from monocytes that have migrated from the blood stream to tissue in the body (also may be referred to as monocyte-derived macrophages). Depending on the tissue in which the macrophages are located, it is called Coulopfer cells (liver), mesangial cells (kidney), alveolar macrophages (lung), sinus tissue cells (lymph node), huo Fubao's cells (placenta), microglial cells (brain and spinal cord), langerhans cells (skin) or the like.

As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides of the oligonucleotide may be modified. The oligonucleotides may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides may consist of natural and/or modified nucleobases, sugars, and covalent internucleoside linkages, and may also include non-nucleic acid conjugates.

As used herein, the term "nucleoside" refers to a glycosylamine that includes a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having simulated bases and/or sugar groups. "Natural nucleoside" or "unmodified nucleoside" is a nucleoside that includes a natural nucleobase and a natural sugar. Natural nucleosides include RNA and DNA nucleosides.

As used herein, the term "natural sugar" refers to a sugar of a nucleoside that has not been modified from its naturally occurring form in RNA (2 '-OH) or DNA (2' -H).

As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group covalently linked to a sugar. The nucleotide may be modified with any of a variety of substituents.

As used herein, the term "nucleobase" refers to a nucleoside or a base portion of a nucleotide. A nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid. A natural nucleobase is a nucleobase that has not been modified from its naturally occurring form in RNA or DNA.

As used herein, the term "heterocyclic base moiety" refers to a nucleobase that includes a heterocycle.

As used herein, "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides.

As used herein, "natural internucleoside linkage" refers to a 3 'to 5' phosphodiester linkage.

As used herein, the term "modified internucleoside linkage" refers to a nucleoside or any linkage between nucleotides other than a naturally occurring internucleoside linkage.

As used herein, "oligonucleotide" refers to an oligonucleotide in which the internucleoside linkage does not contain a phosphorus atom.

As used herein, the term "chimeric antisense compound" refers to antisense compounds having at least one sugar, nucleobase, and/or internucleoside linkage that is differentially modified compared to other sugar, nucleobase, and internucleoside linkages within the same oligomeric compound. The remaining sugar, nucleobase and internucleoside linkages may be independently modified or unmodified. In general, chimeric oligomeric compounds will have modified nucleosides that can be in separate positions or clustered together in regions that will define a particular motif. Any combination of modifying and/or mimicking groups may include chimeric oligomeric compounds as described herein.

As used herein, the term "mixed-backbone antisense oligonucleotide" refers to an antisense oligonucleotide in which at least one internucleoside linkage of the antisense oligonucleotide is different from at least one other internucleoside linkage of the antisense oligonucleotide.

As used herein, the term "nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (a) is complementary to thymine (T). For example, in RNA adenine (A) is complementary to uracil (U). In embodiments, complementary nucleobases refer to nucleobases in an antisense compound that are capable of base pairing with nucleobases of their target nucleic acids. For example, if a nucleobase at a position of an antisense compound is capable of hydrogen bonding with a nucleobase at a position of a target nucleic acid, the hydrogen bonded position between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds with each other or do not support hybridization.

As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid by nucleobase complementarity. In embodiments, an antisense compound is complementary to its target when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond to each other to allow stable bonding between the antisense compound and the target. Those skilled in the art recognize that it is possible to include mismatches without eliminating the ability of the oligomeric compounds to remain associated. Thus, antisense compounds described herein can include up to about 20% mismatched nucleotides (i.e., nucleobases that are not complementary to the corresponding nucleotides of the target). In embodiments, the antisense compound contains no more than about 15%, such as no more than about 10%, such as no more than 5%, or no mismatches. The remaining nucleotides are complementary nucleobases or do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art will recognize that the compounds provided herein have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleobase complementarity to the target nucleic acid.

As used herein, "hybridization" means pairing of complementary oligomeric compounds (e.g., antisense compounds and their target nucleic acids). Although not limited to a particular mechanism, the most common pairing mechanism involves hydrogen bonding between complementary nucleosides or nucleotide bases (nucleobases), which may be Watson-Crick, hoogsteen or reverse Hoogsteen hydrogen bonding. For example, the natural base adenine is a nucleobase that is complementary to the natural nucleobases thymine and uracil that pair by forming hydrogen bonds. The natural base guanine is a nucleobase complementary to the natural bases cytosine and 5-methylcytosine. Hybridization may occur in different environments.

As used herein, the term "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In embodiments, the antisense oligonucleotide hybridizes specifically to more than one target site. In embodiments, the oligomeric compounds specifically hybridize to their targets under stringent hybridization conditions.

"Stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent in the case of nucleic acid hybridization and are different under different environmental parameters. Extensive guidelines for nucleic acid hybridization are found in chapter 2, I"Overview of principles of hybridization and the strategy of nucleic acid probe assays"Elsevier,New York(1993) of Tijssen,Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes. Generally, highly stringent hybridization and wash conditions are selected to be about 5 ℃ lower than the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen to be equal to the Tm of the particular probe. In Southern or Northern blots, an example of stringent hybridization conditions for hybridization of complementary nucleotide sequences having 100 or more complementary residues on a filter is hybridization of 50% formamide with 1mg heparin at 42℃overnight. An example of highly stringent wash conditions is 0.15M NaCl wash at 72℃for about 15 minutes. An example of stringent wash conditions is a 0.2 XSSC wash at 65℃for 15 minutes (see Sambrook and Russel, molecular Cloning: A laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press,2001 for a description of SSC buffers). Typically, the high stringency wash is preceded by a low stringency wash to remove background probe signal. For duplex of, for example, more than 100 nucleotides, an example of a moderate stringency wash is a 1 XSSC wash at 45℃for 15 minutes. For duplex of, for example, more than 100 nucleotides, an example of a low stringency wash is 4-6 XSSC wash at 40℃for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0M Na ion, typically about 0.01 to 1.0M Na ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is typically at least about 30 ℃. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

As used herein, the term "2' -modified" or "2' -substituted" means a sugar that includes substituents other than H or OH at the 2' position. 2' -modified monomers include, but are not limited to, BNA and monomers (e.g., nucleosides and nucleotides) having a 2' -substituent such as allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, -OCF3, O- (CH 2) 2-O-CH3, 2' -O (CH 2) 2SCH3, O- (CH 2) 2-O-N (Rm) (Rn) or O-CH2-C (=o) -N (Rm) (Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C ₁-C₁₀ alkyl.

As used herein, the term "MOE" refers to a 2' -O-methoxyethyl substituent.

As used herein, the term "high affinity modified nucleotide" refers to a nucleotide having at least one modified nucleobase, internucleoside linkage, or sugar moiety such that the modification increases the affinity of an antisense compound comprising the modified nucleotide for a target nucleic acid. High affinity modifications include, but are not limited to, BNA, LNA, and 2' -MOE.

As used herein, the term "mimetic" refers to a group that replaces a sugar, nucleobase, and/or internucleoside linkage in AC. Typically, a mimetic is used in place of a sugar or sugar-internucleoside linkage combination and retains nucleobases to hybridize to a selected target. Representative examples of glycomimetics include, but are not limited to, cyclohexenyl or morpholinyl. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide Nucleic Acids (PNAs) and morpholino groups linked by uncharged achiral linkages. In some cases, the nucleobase is replaced with a mimetic. Representative nucleobase mimics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al, nuc Acid res.2000,28:2911-14, which is incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.

As used herein, the term "bicyclic nucleoside" or "BNA" refers to a nucleoside in which the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring, thereby forming a bicyclic ring system. BNA includes, but is not limited to, alpha-L-LNA, beta-D-LNA, ENA, oxyBNA (2 '-O-N (CH 3) -CH 2-4') and aminooxyBNA (2 '-N (CH 3) -O-CH 2-4').

As used herein, the term "4 'to 2' bicyclic nucleoside" refers to BNA in which a bridge connecting two atoms of a furanose ring bridges the 4 'carbon atom and the 2' carbon atom of the furanose ring, thereby forming a bicyclic ring system.

As used herein, "locked nucleic acid" or "LNA" refers to a nucleotide that is modified such that the 2 '-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring via a methylene group, thereby forming a 2'-C,4' -C-oxymethylene linkage. LNAs include, but are not limited to, alpha-L-LNA and beta-D-LNA.

As used herein, the term "cap structure" or "terminal cap portion" refers to a chemical modification that has been incorporated into either end of an AC. The term "therapeutic polypeptide" refers to a naturally occurring or recombinantly produced macromolecule that includes two or more amino acids and that has therapeutic, prophylactic, or other biological activity.

The term "small molecule" refers to an organic compound that has pharmacological activity and a molecular weight of less than about 2000 daltons, or less than about 1000 daltons, or less than about 500 daltons. Small molecule therapeutics are typically manufactured by chemical synthesis.

"Wild-type target protein" refers to a natural, functional protein isoform produced from a wild-type, normal or unmutated version of a target gene. Wild-type target protein also refers to a protein produced from a target pre-mRNA that has been alternatively spliced.

As used herein, "alternatively spliced target protein" refers to a protein encoded by mRNA resulting from splicing of AC hybridized target pre-mRNA. The alternatively spliced target protein may be identical to the wild-type target protein, may be homologous to the wild-type target protein, may be a functional variant of the wild-type target protein, may be an isoform of the wild-type target protein, or may be an active fragment of the wild-type target protein.

As used herein, "amplified trinucleotide repeats," such as "amplified" CUG or "amplified" CTG repeats, means that the gene containing or encoding the trinucleotide repeats contains a greater number of repeated consecutive trinucleotides than are present in the wild-type gene. Amplified nucleotide repeats may be written as XXX. NNN or (XXX. NNN), where XXX refers to DNA repeats and NNN refers to RNA repeats transcribed from DNA repeats. For example, a ctg.cug repeat refers to a gene having a CTG DNA repeat sequence from which RNA having a CUG repeat sequence is transcribed. In embodiments, the number of repeats in the amplified trinucleotide repeats is 5 or more, 10 or more, 15 or more, or 20 or more greater than the wild type gene. In embodiments, amplified trinucleotide repeats include 2x, 3x, 4x, 5x, 10x, 20x, 50x or more times that of the wild-type gene. Amplified trinucleotide repeats can cause disease in subjects having a gene containing amplified trinucleotide repeats. For example, a subject having an amplified CTG repeat in a gene may have DM1 or FECD. In DM1, the DPMK gene contains an amplified CTG repeat. A subject with DM1 may have 50 or more CTG repeats in the 3 'untranslated region (UTR) of the DPMK gene, whereas a non-diseased subject typically has 5 to 34 CTG repeats in the 3' UTR of the DPMK gene. In FECD, the TCF4 gene contains amplified CTG repeats. Subjects with FECD may have 40 or more CTG repeats in the CTG18.1 locus of the TCF4 gene, whereas non-diseased subjects typically have 30 or less CTG repeats in the CTG18.1 locus of the TCF4 gene. mRNA transcribed from a gene with amplified CTG repeats will have amplified CUG repeats.

The term "downstream" in this disclosure, when it relates to a gene, mRNA, or protein, refers to a gene, mRNA, or protein that is affected by AC binding to a target nucleotide (e.g., target transcript), but not the gene, mRNA, or protein corresponding to the target nucleotide. Binding of AC to a target nucleotide can reduce aggregation or sequestration of RNA binding proteins such as MBNL1 or CUGBP1 on accumulated mRNA with CUG repeats, which can allow for proper transcription, RNA processing, and/or expression of such RNA binding proteins for downstream gene products.

As used herein, "functional fragment" or "active fragment" refers to a portion of a eukaryotic wild-type target protein that exhibits activity, such as one or more activities of a full-length wild-type target protein, or has another activity. In embodiments, an alternatively spliced target protein sharing at least one biological activity of a wild-type target protein is considered an active fragment of the wild-type target protein. The activity may be any percentage (i.e., more or less) of the activity of the full-length wild-type target protein, including but not limited to about 1% of the activity compared to the wild-type target protein, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 200%, about 300%, about 400%, about 500% or more (including all values and ranges between these values). Thus, in embodiments, the active fragment may retain at least a portion of one or more biological activities of the wild-type target protein. In embodiments, the active fragment may enhance one or more biological activities of the wild-type target protein.

"Wild-type target protein" refers to a natural, functional protein isoform produced from a wild-type, normal or unmutated version of a target gene. Wild-type target protein also refers to a protein produced from a target pre-mRNA that has been spliced correctly.

As used herein, the terms "splicing" and "processing" refer to the modification of post-transcriptional pre-mRNA in which introns are removed and exons are linked. Splicing occurs in a series of reactions that are catalyzed by large RNA-protein complexes consisting of five small nuclear ribonucleoproteins (snrnps), called spliceosomes. Within the intron, splicing requires a 3 'splice site, a 5' splice site, and a branching site. The RNA component of snRNP interacts with introns and may be involved in catalysis.

As used herein, alternative splicing refers to splicing of different combinations of exons present in a gene, resulting in the production of different mRNA transcripts from a single gene.

As used herein, "alternatively spliced target protein" refers to a protein encoded by mRNA resulting from splicing of AC hybridized target pre-mRNA. The alternatively spliced target protein may be identical to the wild-type target protein, may be homologous to the wild-type target protein, may be a functional variant of the wild-type target protein, or may be an active fragment of the wild-type target protein.

All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Examples

Example 1 evaluation of the Effect of PMO and PMO-EEV Compounds on RNA foci formation and splice rescue in DM 1-associated cell lines

The effect of (CUG) ₇ repeat PMO and (CUG) ₇ repeat PMO-EEV compounds (a and D in table 12) on RNA foci formation and splice rescue in DM 1-associated cell lines was evaluated. PMO-EEV compounds with mismatched PMO sequences and PMOs with chaotic sequences are also included (B and C in Table 12).

Experiment

Cells PMO and PMO-EEV were evaluated in DM1 HeLa cell model (HeLa-480), which is a stable cell line with high CUG repeat load and downstream splice defects, and DM1 myoblasts derived from DM1 patients with about 2600 CTG repeats and downstream splice defects. HeLa-480 reproduced the pathogenic features of DM1, including missplicing of pre-mRNA targets for CUG ribonucleophiles and Myoblindness (MBNL) alternative splicing factors. Note that DM1 myoblasts grew very slowly (doubling time of about 7 days) and did not transfect well. Control HeLa and HeLa-480 cells were treated with ENDOPORTER without other compounds. HeLa-480 cells exhibited disease states and HeLa cells exhibited non-disease states.

RNA foci analysis. HeLa-480 cells or DM1 myoblasts were treated with 1. Mu.M, 3. Mu.M or 10. Mu.M of compounds A-D (Table 12). All compounds were transfected without transfection reagent or with ENDOPORTER (available from Philomath, oregon, GENETOOLS LLC) transfection reagent designed to deliver naturally charged PMO into cells. Cells were incubated for 24 hours and then fixed for qualitative RNA focus analysis via microscopy. Compound a (PMO-EEV) showed minimal RNA foci following qualitative visual inspection of the treated Hela-480 cells. No conclusion was drawn from DM1 myoblasts.

Splice analysis and RT-PCR. HeLa-480 cells treated as described above were harvested after 24 hours or 48 hours of incubation, and total RNA was extracted. RT-PCR is performed to measure and/or quantify the splice pattern of the exons affected by DM in target RNAs such as MBNL1 and CLASP 1. The percentage of target exon inclusion was assessed.

Table 12: PMO and PMO-EEV tested in example 1.

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As a result. FIGS. 6A-6D show the RT-PCR results of alternative splicing events for MBNL1 (6A and 6C, exon 5 comprising) and CLASP1 (6B and 6D, exon 19 comprising) 24 hours (6A and 6B) and 48 hours (6C and 6D) after treatment of HeLa-480 cells with compounds A-D and ENDOPORTER transfection agents. A decrease in exon 5 inclusion in MBNL1 was observed at the 24 hour and 48 hour time points for cells treated with each of compounds a-D (fig. 6A and 6C). Cells treated with compound a showed the greatest rescue (exon 5 inclusion reduced). An increase in exon 19 inclusion in CLASP1 was observed at 24 hour and 48 hour time points for cells treated with each of compounds a-D (fig. 6B and 6D). Cells treated with compound a showed the greatest rescue (exon 19 contained increased). In contrast, when HeLa-480 cells were treated with A-D in the absence of ENDOPORTER transfection reagent, no change in splicing events of MBNL1 (FIG. 6E) or CLASP1 (FIG. 6F) was observed other than compound A.

FIGS. 7A-7B show RT-PCR results of alternative splicing events for MBNL1 and CLASP1 after treatment of DM1 myoblasts with compound A-D, negative control DM-04 or positive control DM-05. Treatment with all compounds caused rescue of splicing events of MBNL1 (fig. 7A) and CLASP1 (fig. 7B).

Example 2 evaluation of the Effect of EEV-PMO 221-1106 on splice rescue in DM 1-mouse model

The effect of PMO and PMO-EEV alone on splice rescue was evaluated in vivo using the HSA-LR DM 1-mouse model (Table 13).

And (5) experiment.

A mouse model. HSA-LR is a transgenic mouse model that has an amplified long CUG repeat (LR) in the 3' -UTR of a Human Skeletal Actin (HSA) transgene and expresses CUGexp RNA (e.g., amplified CUG RNA) at high levels in skeletal muscle (Mankodi et al, science 2000,289 (5485):1769-1773). HSA-LR mice showed myotonic phenotype and splice deficiency. The Friend virus B NIH Jackson (FVB/NJ) mouse model was used as a control and for the production of HSA-LR transgenic mice.

Experiment design. Compound a-D was administered to mice via retroorbital injection or Intravenous (IV) injection in a single dose of 100 μl of compound solution per 20 grams of body weight. The scale is proportional to the body weight of each mouse (e.g., 150 μl per 30g body weight).

Table 13: PMO and PMO-EEV tested in example 2.

Animals were age matched and assigned to six treatment groups. Two control groups were used; group 1: FVB/NJ mice (FVB/NJ; non-diseased control) and group 2: HSA-LR (disease control) mice, each injected with saline. Four treatment groups (groups a-D) were used; HSA-LR mice were injected with compound A, B, C or D. FVB/NJ mice were 5 weeks zero 4 days old at injection, while HSA-LR mice were 6 weeks zero 1 day or 2 days old at injection. Four mice per group (two males and two females) were used for this experiment. Mice were sacrificed 1 week after treatment. Tissues (gastrocnemius, quadriceps, tibialis Anterior (TA)) were harvested and flash frozen in liquid nitrogen and stored at-80 ℃ for further evaluation of splice rescue analysis.

Total RNA was extracted from tissue samples and analyzed by RT-PCR to evaluate the AC-induced alternative RNA splice rescue events on (i) Atp a1 exon 22, (ii) Nfix exon 7, (iii) Clcn exon 7a, and (iv) Mbnl exon 5. The percentage of target exon inclusion was assessed.

As a result. All HSA-LR mice had myotonia prior to treatment. After injection of the compound, all mice were left undirected. Mice injected with compound a and compound B (groups a and B) recovered within 15 minutes, while mice injected with compound C and compound D (groups C and D) took several hours to recover. All treated mice recovered completely by the next day. When the animal is sacrificed, the muscle rigidity of the A group and the B group is achieved; however, groups C and D clearly did not have myotonia.

Figures 8A-10D show RNA splice measurements of Atp a1 (included for exon 22; figures 8A, 9A and 10A), nfix (included for exon 7; figures 8B, 9B and 10B), clcn1 (included for exon 7 a; figures 8C, 9C and 10C) and Mbnl1 (included for exon 5; figures 8D, 9D and 10D) in gastrocnemius (figures 8A-8D), quadriceps (figures 9A-9D) and tibialis anterior (figures 10A-10D) musculature of treated mice. Mice treated with compound C and compound D (PMO-EEV) showed rescue of Atp a1 and Nfix splicing events in gastrocnemius, quadriceps and anterior tibial tissues, whereas PMO and saline groups did not show rescue of splicing events (fig. 8A, 8B, 9A, 9B, 10A and 10B). Similarly, mice treated with compound C and compound D showed rescue of Clcn (fig. 8C and 9C) and Mbnl1 (fig. 8D and 9D) splicing events in gastrocnemius and quadriceps tissues, whereas PMO and saline groups did not show rescue of splicing events. Regarding splicing of Clcn and Mbnl1 in tibial tissues (fig. 10C-10D), no alternative splicing defect was detected in the control mouse line (FVB/NJ) and DM1 mouse model (HSA-LR), and therefore treatment with compounds a-D did not lead to splice rescue of the Clcn and Mbnl1 genes in tibial tissues.

These results demonstrate the positive impact of PMO-EEV treatment on splice rescue in vivo studies using a DM1 mouse model, as well as the potential use of PMO-EEV compounds to treat myotonic muscular Dystrophies (DM).

Example 3 evaluation of various PMO-EEV Compounds for correction of missplice events in immortalized myoblasts from DM1 patients

The effect of two PMO-EEVs targeting DMPK CUG (197-777 and 221-1106; table 14) on splice rescue of DM 1-related genes was evaluated in vitro using myoblasts and myotubes derived from DM1 patients.

And (5) experiment.

And (5) culturing the cells. Immortalized myoblasts from DM1 patient (ASA 308DM 1) and unaffected individuals (KM 1421; AB 1190) were obtained. Myoblasts from DM1 patients carry 2600 CTG repeats in the 3' -UTR of DMPK. Myoblasts were cultured in skeletal muscle cell growth medium (available from PromoCell in Heidelberg, germany), 2% horse serum (available from Gibco of Bristol, RI), 1% chicken embryo extract (available from USB company of Cleveland, ohio) and 0.5mg/mL penicillin/streptomycin (Gibco) growth medium. For myogenic differentiation, confluent cultures were switched to DMEM differentiation medium supplemented with 2% horse serum and cultured for 4 days.

Table 14: compounds tested in example 3

And (5) processing. The myoblasts of DM1 patients were treated with 10 μm, 3 μm, 1 μm or 0.3 μm compounds using two different treatment conditions. Under the first condition, myoblasts were plated at 75% -80% confluence, the compounds were serially diluted in growth medium, and the cells were soaked for 24 hours to allow free uptake of the compounds. The medium containing the compounds was removed, myoblasts were washed with 1X DPBS (Gibco) and differentiated for 4 days before harvest. For the second condition, which runs in parallel, myoblasts differentiated three days prior to treatment, compounds were serially diluted in differentiation medium and myotubes were harvested after 24 hours for analysis.

RNA isolation and PCR. Total RNA was isolated using RNEASY MINI kit (available from Qiagen of Germanown, MD) according to the manufacturer's instructions. For exon inclusion, 100ng RNA was reverse transcribed and used for PCR (OneStep RT-PCR kit, qiagen). Samples were analyzed by LabChip (available from Perkinelmer of Waltham, MD) using the HT DNA high sensitivity assay kit.

As a result. PMO targeting DMPK CUG (not conjugated to EEV) improved the mis-splicing of MBNL1 exon 5 (data not shown). FIGS. 11A-11F show the mixed rescue of splice defects of MBNL1 (FIG. 11A) and its targets (SOS 1, IR, DMD, BIN1, LDB3; FIGS. 11B-11F) in DM1 patient-derived myocytes treated with various concentrations of EEV-PMO (CUG ^exp 197-777 and CUG ^exp 221-1106) targeting DMPK CUG. EEV-PMO 197-777 causes a moderate correction of the missplice event in the muscle cells of DM1 patients. MBNL1 and SOS1 showed the best response to the correction of mis-splicing. EEV-PMO 197-777 was selected as the tool compound for the following experiments.

Myoblasts and myotubes from DM1 patients were treated with EEV PMO 197-777 targeting DMPK CUG at 10 μm, 3 μm and 1 μm using methods similar to those described above. Rescue of MNBL1 and MNBL1 target alternative RNA splicing events was evaluated. After treatment of myoblasts and myotubes with EEV-PMO, different degrees of splice correction were observed for MNBL1 (exon 5 exclusion; fig. 12A), SOS1 (exon 25 inclusion; fig. 12B), INSR (exon 11 inclusion; fig. 12C), DMD (exon 78 inclusion, fig. 12D), BIN1 (exon 11 inclusion; fig. 12E) and LDB3 (exon 11 exclusion; fig. 12F).

FIGS. 44A-44D show reversal of myotonic phenotype in HSA-LR mice treated with 20mpk 221-1106 quantified by muscle relaxation. Fig. 44A and 44C show graphs of relaxation times for equal length to 80% peak, while fig. 44B shows force trace raw data. FIG. 44D shows the reversal of myotonic phenotype in HSA-LR mice treated with 20mpk 221-1106 quantified by representative Electromyography (EMG) traces.

EXAMPLE 4 evaluation of PMO-EEV 221-1120 in the DM1 mouse model

The effect of EEV-PMO 221-1120 (also known as EEV-PMO-DM1-3 or DM1-3; PMO221= 5'-CAG CAG CAG CAG CAG CAG CAG-3' (SEQ ID NO:154, all PMO monomers); EEV1120=Ac-PKKKRKV-AEEA-Lys (ring [ FGFGRGRQ ] -PEG12-OH (Ac- (SEQ ID NO: 42) -AEEA-Lys (SEQ ID NO: 82) -PEG 12-OH)) on splicing and mRNA levels of downstream genes in the same HSA-LR transgenic mouse model as described in example 2. PMO and EEV were conjugated using amide chemical structures.

And (5) experiment. There are two conventional treatment groups: 1) Wild-type mice; and 2) HSA-LR mice (DM 1 disease model). In the HSA-LR treatment group, there are two sub-treatment groups: 1) HSA-LR treated with saline (control); and 2) HSA-LR+EEV-PMO 221-1120. Mice were treated with 15mpk, 30mpk, 60mpk or 90mpk (PMO-based) PMO-EEV or saline via tail vein injection. After 7 days of treatment, mice were sacrificed and tissues were collected for analysis.

RT-PCR assay (correction of splicing). Tissues were homogenized with an OMNI bead mill homogenizer and RNA was extracted with QIACUBEQ. RT-PCR analysis was performed using a one-step RT-PCT kit (Qiagen) following the manufacturer's protocol, and 35 PCR cycles were performed: 94℃for 30 seconds; 60℃for 30 seconds and 72℃for 30 seconds. The gene-specific primer sequences were as follows: clcn1 exon 7a comprises forward primer= 5'-TTCACATCGCCAGCATCTGTGC-3' (SEQ ID NO: 319), reverse primer= 5'-CACGGAACACAAAGGCACTGAATGT-3' (SEQ ID NO: 320); mbnl1 exon 5 comprises forward primer= 5'-GCTGCCCAATACCAGGTCAAC-3' (SEQ ID NO: 321), reverse primer= 5'-TGGTGGGAGAAATGCTGTATGC-3' (SEQ ID NO: 322);

Atp2 exon 2a1 comprises forward primer= 5'-GCTCATGGTCCTCAAGATCTCAC-3' (SEQ ID NO: 323), reverse primer: 5'-GGGTCAGTGCCTCAGCTTTG-3' (SEQ ID NO: 324);

Nfix exon 7 comprises forward primer= 5'-TCGACGACAGTGAGATGGAG-3' (SEQ ID NO: 325) and reverse primer 5 'CAAACTCTTCAGCGAGTCC-3' (SEQ ID NO: 326). Clcn1, mbnl1 and Atp a1 from Klein et al, the Journal of Clinical investigation.2019,129 (11), page 4739; and Nfix primers from Chen et al Scientific reports.2016,6 (1), page 1. The cDNA product was isolated on a 2% agarose E-gel using SYBR SAFE dye. The exon inclusion percentage was calculated by the ratio of non-skipped bands/(non-skipped band + skipped band).

Calculation of the mouse DM1 splicing index (mDSI). mDSI (Nucleic acids research.2021,49 (4), pages 2240-54) was calculated according to the literature protocol from Tanner et al. For each sample i, a normalized splice value (PSI _i,j–PSI_{Wild type ,j})/(PSI_HSALR,j–PSI_{Wild type ,j}) was calculated for each splice event j, where PSI _{Wild type ,j} is the average PSI for event j in wild-type mice and PSI _HSALR,j is the average PSI for event j in HSALR mice. mDSI was then calculated as the average of all normalized splice values, atp a1, nfix, mbnl1 and Clcn1 in the study.

QRT-PCR assay (HSA mRNA knockdown). Reverse transcription was performed using the high-capacity cDNA reverse transcription kit from Life Technologies Corporation according to the manufacturer's protocol. Quantitative real-time PCR with gene specific primers was performed using Bio-Rad SyBr Green Supermix and QuantStudio qPCR machine: HSA mRNA forward primer = 5'-TTCCATCGTCCACCGCAAAT-3' (SEQ ID NO: 327), reverse primer = 5'-AGTTTACGATGGCAGCAACG-3' (SEQ ID NO: 328), both primers from Klein et al The Journal of Clinical investment.2019, 129 (11), page 4739; and mouse GAPDH forward primer = 5'-AGGTCGGTGTGAACGGATTTG-3' (SEQ ID NO: 329), reverse primer = 5'-TGTAGACCATGTAGTTGAGGTCA-3' (SEQ ID NO: 330).

RNAseq. PolyA RNAseq was used for transcriptome profiling using next generation sequencing. The Z-score for each gene was calculated as (sample value-average)/(standard deviation). Differential splicing analysis was performed on the RNAseq data to calculate the Percent Splicing (PSI) of individual exons for each gene. PSI is the ratio of the normalized read count indicating the inclusion of a transcriptional element to the total normalized reads (both inclusion and exclusion reads) of the event. For example, if exons are contained 100% of the time in the reads, then PSI is 1. In addition, PSI is 0 if exons are all excluded from reads 100% of the time.

Analysis of 22 target genes known to predict DM 1. In addition, the genes studied by Wagner et al (PLOS Gen 2016 (47)) and Tanner et al (NAR 2021 (48), 4, 2240-2254) were analyzed. Mouse exons map to positions in humans. In some cases, the boundaries of exons in the mouse and/or human genome are not completely known. Thus, different boundaries are used to analyze the data. Correct boundaries are verified using RNAseq data.

RNA CUG foci analysis. Tibialis anterior sections were stained for CUG foci (FISH, red) and nuclei (Hoechst, blue). TA muscle sections were imaged and the number of nuclei with CUG RNA foci were quantified.

Results

HSA mRNA knockdown. In contrast to quadriceps, tibialis anterior, and triceps, diaphragmatis expresses only 5% -10% of HSA mRNA levels (fig. 13A). EEV-PMO treatment did not appear to alter HSA mRNA levels in the diaphragm (fig. 13B). For the mis-spliced phenotype in DM1 diaphragm, the expression level of the HSA 220CUG repeat may be insufficient.

EEV-PMO knockdown HSA mRNA in a dose-dependent manner, confirming target engagement in quadriceps (fig. 14A), gastrocnemius (fig. 14B), triceps (fig. 14C) and tibialis anterior (fig. 14D) tissues. In addition, the Ct (circulation threshold) value of HSA mRNA was similar to the level of mouse GAPDH (-15), indicating high expression of HSA transgene in the quadriceps of HSA-LR mice.

MDSI (correction of splicing). The mouse DM1 splicing index of quadriceps, gastrocnemius, triceps and tibialis anterior is shown in FIGS. 15A-15D (mDSI). Treatment with EEV-PMO in quadriceps (fig. 15A), gastrocnemius (fig. 15B), triceps (fig. 15C) and tibialis anterior (fig. 15D) 1 weeks after injection, DM 1-related splice defects (Atp a1 exon 22, nfix exon 7, clcn1 exon 7a, mbnl1 exon 5) were corrected in a dose-dependent manner, with higher doses approaching or equivalent to wild-type (complete correction). Approximately 50% -60% of human skeletal actin RNA knockdown in HSA-LR mice was achieved at drug concentrations that achieved near-complete splice correction.

Fig. 16A-B show images of pre-tibial tissues stained for CUG foci (red) and nuclei (blue) in HSA-LR mice (fig. 16A) and HSA-LR mice treated with EEV-PMO (fig. 16B). Qualitative and quantitative evaluation (FIG. 16C) showed that EEV-PMO treatment reduced the number of nuclei with CUG foci.

Drug exposure. Drug exposure was studied using LC-MS. Figures 17A-17D show the dose-dependent response of PMO-EEV exposure in quadriceps (figure 17A), triceps (figure 17B), heart (figure 17C), gastrocnemius (figure 17D), tibialis anterior (TA; figure 17F), liver (figure 17I) and kidney (figure 17J). No dose-dependent response was observed in the diaphragm (fig. 17G). No EEV-PMO was detected in the brain except at 60mpk and 90mpk dose levels. Fig. 17K shows drug exposure of various tissues at a 60mpk dose level.

Myotonic response: dose-dependent myotonic reduction was observed in HSA-LR mice 7 days after treatment with EEV-PMO-DM1-3 at 15mpk, 30mpk, 60mpk and 90mpk (FIG. 18A). Myotonia may be improved one week after treatment with EEV-PMO-DM 1-3. HSA-LR mice treated with a single dose of 90mpk EEV-PMO-DM1-3 showed no evidence of hindlimb myotonia after induction.

RNAseq data analysis. Fig. 19A to 19D show the results of principal component analysis. Principal component analysis can be used to reveal similarities between samples based on distance matrices. This type of graph can be used to visualize the overall and batch effects of experimental covariates. The x-axis is the direction that accounts for the greatest variance and the y-axis is the second greatest. The percentage of the total variance of each direction is shown as PCA. Wild type and HSA-LR mice are different groups. The gene expression in the gastrocnemius muscle of HSA-LR mice treated with PMO-EEV was shifted to that of wild-type mice.

FIG. 20A is a heat map showing differentially expressed genes (by Z-score) from three treatment groups: 1) WT mice; 2) HSA-LR mice; and 3) HSA-LR+EEV-PMO 221-1120 (60 mpk). There were a total of 956 (p < 0.5) gene differential expressions between the treatment groups, knowledge of the differences between wild-type mice (WT) and disease model mice (HSA-LR). Treatment with EEV-PMO (HSA-LR (++)) resulted in correction of overall gene expression, shift from disease profile (red, HSA-LR (-, -)) to wild-type mice (WT above).

FIG. 20B is a heat map for visualizing the expression profile of 40 genes among 43 genes found to have 7 or more CTG repeats from BLAST analysis. The three CTG repeat genes found in Blast analysis (Crb 2, hsd b6 and Inhbe) were not included due to the low number of reads. Such analysis can be used to identify genes co-regulated in the treatment conditions.

FIG. 21 shows volcanic diagrams of global transcriptional changes in EEV-PMO treated and HSA-LA groups. Each data point in the scatter plot represents a gene. Fold change for each gene is shown on the x-axis, which adjusts log10 of p-values on the y-axis. Genes whose adjusted p-value was less than 0.05 and fold change was greater than 2 were indicated by red dots. These represent up-regulated genes. Genes with adjusted p-values less than 0.05 and fold change less than-2 are indicated by blue dots. These represent down-regulated genes. Three genes were found to be significantly down-regulated (Txlnb, scube2 and Greb 1) and one gene was significantly up-regulated (Txlnb). Most transcripts containing at least (CUG) ₇ were not significantly affected. PCA analysis of these genes showed that Scube2 (FIG. 22A), greb1 (FIG. 22B), ttc7 (FIG. 22C), txlnb (CUG) 9 and Ndrg3 (FIG. 22E) showed correction when treated with EEV-PMO. Txlnb overcorrection by handling (fig. 22D).

FIGS. 23A-23D show transcriptome data for various genes and various treatment groups. The HSA-lr+eev-PMO 221-1120 treatment group showed correction of inclusion of exon 22 of Atp a1 (fig. 23A), exclusion of exon 7 of Clcn1 (fig. 23B), exclusion of exon 7 of Nfix (fig. 23C) and exclusion of exon 7 of Mbnl1 (fig. 23D).

FIG. 24 shows the Percent Splicing (PSI) of individual exons of various genes. These genes are MBNL-1 reactive splicing biomarkers (e.g., downstream genes). The selection of MBNL-1-dependent biomarkers was selected based on dynamic range between wild-type and disease groups as described in the literature. The HSA-LR+EEV-PMO 221-1120 treatment group showed correction of exon inclusion/exclusion for all 20 genes of interest, including Mbnl1、Nfix、Atp2a1、Ldb3、Camk2g、Trim55、Fbox31、Slc8a3、Map3k4、Dctn4、Cacna1s、Ryr1、Slain2、Phka1、Ppp3cc、Ttn、Neb、lrrfip2、Rapgef1 and Vsp39.

EXAMPLE 5 evaluation of PMO-EEV 221-1120 in the DM1 mouse model, the second DM1 mouse model

PMO-EEV DM1-3 was evaluated in a second DM1 mouse model using methods similar to those described in example 4 (221-1120; see sequence of example 4).

And (5) experiment. Seven week old HSA-LR mice were intravenously administered a single dose of 80mpk EEV-PMO-DM1-3 or 20mpk dose of EEV-PMO-DM1-3 every other week for six weeks (4 doses total 80 mpk) and tissues were harvested 1 week to 12 weeks after the single dose or two weeks after the final dose. Alternative splicing of specific genes (Atp a1, clcn1, nfix, MBNL 1) was determined using RT-PCR. Reduction of actin-HSA mRNA levels after treatment was determined using Q-PCR. Drug levels in quadriceps, gastrocnemius, tibialis anterior, triceps, diaphragm, heart, kidney, liver, brain and plasma were determined using LC-mass. After 7 days of treatment with EEV-PMO-DM1-3 compound, a decrease in myotonia was noted.

As a result. For rescue of Atp a1, clcn1, nfix and MBNL1 splicing in tibialis anterior, gastrocnemius, triceps and quadriceps tissues, a trend similar to that observed in example 4 was observed (data not shown). In addition, for the tibialis anterior, gastrocnemius, triceps and quadriceps tissues 1 week and 4 weeks after treatment, a trend similar to that observed in example 4 was observed (data not shown).

Figures 25A-25D are graphs showing the decrease in drug levels after 1 week to 8 weeks with 80mpk EEV-PMO-DM1-3 in tibialis anterior (figure 25A), gastrocnemius (figure 25B), triceps (figure 25C) and quadriceps (figure 25D) tissues. EEV-PMO-DM1-3 (60 mpk oligomer, 80mpk total) was completely corrected for mis-splicing in gastrocnemius, triceps, tibialis anterior and quadriceps after 1 week of treatment. FIGS. 26A-26B are graphs showing the decrease in drug levels observed in the liver after 1 week to 4 weeks, to 8 weeks, and to 12 weeks with a single 80mpk dose of EEV-PMO-DM1-3. A relatively higher amount of EEV-PMO-DM1-3 was observed in the liver 2 weeks after the last dose of the 6 week dosing regimen compared to 4 weeks after the single dose regimen.

FIGS. 26C-26D show the decrease in drug levels observed in the kidneys after 1 week to 4 weeks, to 8 weeks, and to 12 weeks with a single 80mpk dose of EEV-PMO-DM1-3. Relatively low amounts of EEV-PMO-DM1-3 were observed in the kidneys starting 2 weeks after the last dose of the 6-week dosing regimen compared to 4 weeks after the single dose regimen. The drug was still present in the kidney but not in the liver 12 weeks after a single dose of EEV-PMO-DM1-3.

Subjective myotonic observations were made and are shown in table 15. The multi-dose regimen (Q2W) showed no sign of rescue of myotonia two weeks after treatment. In mice treated with a single 80mpk dose, there was a mixed effect on myotonia, which disappeared 12 weeks after treatment.

TABLE 15 myotonic observations

Similar experiments were performed to evaluate the longer duration and higher dose intravenous administration of EEV-PMO-DM 1-3. Eight weeks old HSA-LR mice were treated intravenously with EEV-PMO-DM1-3 at 40mpk, 60mpk, 80mpk or 120mpk and tissues harvested after 4 to 12 weeks. Alternative splicing of specific genes (Atp a1, clcn1, nfix, MBNL 1) was determined using RT-PCR. After 7 days of treatment with EEV-PMO-DM1-3, a decrease in myotonia was noted. For rescue of Atp a1, clcn1, nfix and MBNL1 splicing in tibialis anterior, gastrocnemius tissue, a trend similar to that observed in example 4 was observed (data not shown).

Subjective myotonic observations were made and are shown in table 16. Females exhibit more myotonia than males. At 8 weeks post treatment, neither male nor female mice dosed at 120mpk had signs of myotonia.

TABLE 16 myotonic observations

Example 6 treatment of patient-derived DM1 cells with EEV-PMO-DM1-3

PMO-EEV DM1-3 was evaluated in myoblasts derived from DM1 patients (221-1120; see example 4 for sequence).

And (5) experiment. Patient myoblasts were treated with 30 micromolar DM1-3 during four days of differentiation. Splice correction was assessed by one-step RT-PCR and Labpchip (plot mean ± SD; n=4). RNA CUG foci were detected using HCR-FISH and isolated MBNL1 protein detection assays. Results: EEV-PMO-DM1-3 promotes significant biomarker splice correction and reduction of foci in DM1 patient-derived myocytes.

As a result. FIGS. 27A-27C are graphs showing that EEV-PMO-DM1-3 promotes significant biomarker splice correction (MBLN 1, SOS1, and NFIX) in DM1 patient-derived myocytes. In addition, treatment with DM1-3 caused a reduction in nuclear foci in the DM1 patient-derived myocytes (FIGS. 28A-28C).

Example 7 cytotoxicity screening of EEV-PMO-DM1-3 in renal cells

PMO-EEV DM1-3 was evaluated in human kidney cells (221-1120; see example 4 for sequence).

And (5) experiment. Human primary kidney proximal tubule epithelial cells (RPTEC) were exposed to varying concentrations of PMO-DM1 and EEV-PMO-DM1-3 (serial dilutions 1:2 in saline, final dilution factor of 4x, from about 6. Mu.M to about 800. Mu.M) for 24 hours and screened for viability using a CELLTITER-GLO luminometric assay. Melittin was used as a positive control at 16.6. Mu.M.

As a result. FIGS. 29A-29B show that PMO-DM1 or its conjugated EEV-PMO-DM1-3 does not show any toxicity even at the highest concentrations of 817. Mu.M or 797. Mu.M, respectively.

Example 8 evaluation of PMO-EEV 221-1113 ability to correct mis-splicing events and downstream splicing in immortalized cell DM1 patients and HeLa-480 cells

Immortalized DM1 patient-derived (2,600 CUG repeats) muscle cells and HeLa-480 (DM 1 model cell line, see example 1) were treated with EEV-PMO constructs 221-1113 and analyzed for correction of aberrant splicing and focal quantification. EEV 1113 is Ac-PKKKRKV-miniPEG-K(cyclo(Ff-Nal-GrGrQ)-PEG12-OH(Ac-(SEQ ID NO:42)-miniPEG-K(cyclo(SEQ ID NO:80)-PEG12-OH).EEV-PMO 221-1113 is EEV 1113 conjugated to PMO sequence 221 via an amide bond chemical structure (5'-CAG CAG CAG CAG CAG CAG CAG-3' (SEQ ID NO:154; all PMO monomers).

And (5) experiment. Methods similar to those described in example 1 and example 3 were used.

As a result.

RNA CUG foci analysis. Nuclei (Hoeschet, blue) and RNA CUG repeat foci (green) of the cells were stained and imaged. A decrease in RNA CUG foci was observed between untreated DM1 patient cells and EEV-PMO treated DM1 cells (FIGS. 30A-30C). Similarly, a decrease in RNA CUG-foci was observed between untreated HeLa-480 cells and EEV-PMO HeLa-480 cells (FIGS. 31A-31B).

Correction of downstream splicing. The PMO-EEV treated DM1 patient-derived cells and HeLa-480 cells were analyzed for percent exon 5 inclusion of MBLN, percent exon 25 inclusion of SOS1 and percent exon 7 inclusion of NFIX. Treatment with EEV-PMO caused rescue of splice events of Mbnl1 (fig. 32A), sos1 (fig. 32B) and NFIX (fig. 32C).

In addition, EEV-PMO treated HeLa-480 cells showed dose-dependent correction of MBNL1 (fig. 33A) splicing and downstream mis-splicing of SOS1 (fig. 33B), CLASP1 (fig. 33C), NFIX (fig. 33D) and INSR (fig. 33E) in a dose-dependent manner.

Example 9 evaluation of EEV-PMO 221-1106 in the second DM1 mouse model

The effect of EEV-PMO 221-1106 on downstream gene splicing and mRNA levels was studied using the DM1 mouse model.

And (5) experiment. Human skeletal actin long repeat (HSA-LR) transgenic mice were used as a model for DM1 disease. Methods similar to those described in example 5 were used.

As a result. Fig. 34A-34D show the dose-dependent correction of inclusion of exon 22 in Atp a1 (fig. 34A), exon 7 in Nfix (fig. 34B), exon 7A in Clcn1 (fig. 34C) and Mbnl1 (fig. 34D) in gastrocnemius muscles of mice treated with various concentrations of EEV-PMOs 221-1106. Treatment with PMO 221 alone did not cause correction of splicing.

Example 10 DM1 mouse model for studying the influence of different lengths of the CUG repeat in PMO

The effect of PMO-EEV 221-1121 (PMO has 7 CAG repeats, 21-mer) and PMO-EEV 0325-1121 (PMO has 8 CAG repeats, 24-mer) on downstream gene splicing and mRNA levels was studied using the DM1 mouse model. PMO-EEV 221-1121 is PMO 221 (5'-CAG CAG CAG CAG CAG CAG CAG-3'; SEQ ID NO:154; all PMO monomers) conjugated to EEV 1121 (Ac-PKKKRKV-miniPEG 2-Lys (cyclo [ GfFGrGrQ ]) -PEG12-OH; ac- (SEQ ID NO: 42) -miniPEG2-Lys (SEQ ID NO: 74) -PEG 12-OH) via an amide chemical structure. PMO-EEV 0325-1121 is PMO 0325 conjugated to EEV 1121 via an amide chemical structure (5 '-CAG CAG CAG CAG CAG CAG CAG-CAG-3'; SEQ ID NO:155; all PMO monomers).

And (5) experiment. Human skeletal actin long repeat (HSA-LR) transgenic mice were used as a model for DM1 disease. Briefly, HSA-LR mice were administered intravenously via the tail vein at 0221-1121 or 0325-1121 of 20mpk, 40mpk or 60 mpk. One week after injection, mice were sacrificed and tissues were collected. Other experimental methods similar to those described in example 5 were used.

As a result. 0221-1121 (21-mer) was more effective at correcting exon splicing in Mbnl (FIG. 35A), nfix (FIG. 35B) and Atp a1 (FIG. 35C) in tibialis anterior tissue than 0325-1121 (24-mer). This result is unexpected. It is expected that 24-mer will be more efficient because it will have higher hybridization efficiency and higher thermal melting temperature. In gastrocnemius tissue, as shown in fig. 36A-36C, the difference is less pronounced. Subjective myotonic observations were performed using male mice (table 17). Mixed myotonia was observed 1 week after 21-mer treatment at 40mpk, similar to the results in Table 11.

Table 17: myotonic observations

Grouping	Before administration of the drug	1 Week after administration	4 Weeks after administration
				Sex (sex)	M	M	M
FVB	0	0	NA
				HSA-LR	++	++	NA
221-112 20mpk	++	++	NA
				221-1120 40mpk	++	+
325-1120 20mpk	++	++	NA
				325-1120 40mpk	++	++

M = male; 0 = no mice show myotonia; ++ = mixed myotonia, some mice showed

The myotonia is relieved; ++ = all mice all show myotonia

Example 11 pharmacokinetic study of EEV-PMO 221-1120 in CD1 mice

Drug exposure (AUC) of plasma, kidney and tibialis anterior to EEV-PMO constructs 221-1120 (see sequence example 4) and PMO-0221a was studied using the CD1 mouse model, as well as the major metabolites of 221-1120 (see fig. 37).

And (5) experiment. CD1 mice 5 to 7 weeks old were treated with 80mpk EEV-PMO construct 221-1120 via intravenous injection. Mice were bled and/or sacrificed at different time points.

As a result. Tables 18, 19 and 20 show the pharmacokinetic properties observed in plasma, kidney and tibialis anterior, respectively. For the table: AUC _Finally = area under the curve from zero to the last quantifiable concentration; d = dose; c _max = maximum serum or plasma concentration; t _max = time to reach C _max; CL = total plasma, serum or blood clearance; t _1/2 = elimination half-life; v _ss = apparent distribution volume at equilibrium; q _h = liver blood flow (ml/min/kg).

The AUC values for metabolites in tibialis anterior muscle were lower by a factor of 1000 compared to kidney. The Mean Residence Time (MRT) value of the metabolite in the plasma may be directly related to the MRT value of the tissue due to transfer from the tissue to the plasma prior to urinary excretion.

Table 18: pharmacokinetic properties of plasma

	221-1120	PMO-0221a
			AUC_{Finally (}nM*hr)	9290	953
AUC_Finally/D	1121	115
			C_max(nM)	16217	12
C_max/D	1956	1.4
			T _max (hours)	0.1	24
CL(mL/min/kg)	15	-
			Q_h(％)	16	-
T _1/2 (hours)	19	68
			MRT _Finally (hours)	1.2	60.0
V_ss(mL/kg)	1325	-

Table 19: renal pharmacokinetic profile

Table 20: tibialis anterior pharmacokinetic properties

EXAMPLE 12 evaluation of PMO-EEV 221-1120 in the third DM1 mouse model

A DM1 mouse model study similar to example 5 and example 9 was performed to evaluate the effect of different doses of PMO-EEV 221-220 (see sequence example 4) in HSA-LR mice.

And (5) experiment. Eight weeks old HSA-LR mice were intravenously administered 40mpk, 60mpk, 80mpk or 120mpk PMO-EEV 221-1120 and tissues were harvested after 4 to 12 weeks. Alternative splicing of specific genes (Atp a1, clcn1, nfix, MBNL 1) was determined using RT-PCR. Drug levels in quadriceps (Quad), gastrocnemius (gastro), tibialis Anterior (TA), triceps, diaphragm, heart, kidney, liver, brain, and plasma were determined using LC-mass. The RNA-seq was used to determine transcriptional level changes between the treated disease model, the untreated disease model and the wild type. Reduction of actin-HSA mRNA levels after treatment was determined using Q-PCR.

As a result. The reduction of RNA foci after treatment with EEV-oligomeric compounds was determined using fluorescence imaging (data not shown). After 7 days of treatment with EEV-oligomeric compounds, myotonic decrease was recorded (data not shown). The results of these experiments showed similar trends to the mice treated with PMO-EEV 221-1120 in example 5 and example 14.

MBNL1, NFIX and ATP2A1 splice correction were observed in tibialis anterior (fig. 38A, 39A, 40A) and gastrocnemius (fig. 38B, 39B, 40B) at different doses of EEV-PMO. MBNL1, NFIX and ATP2A1 splice correction were observed in both tibialis anterior and gastrocnemius after 12 weeks of treatment with 120mpk EEV-PMO.

EXAMPLE 13 evaluation of EEV-PMO 221-1120 in HeLa480 cells

HeLa480 cells were treated with different concentrations of EEV-PMO 221-1120 (see sequence example 4) and analyzed for CUG repeat foci, selective r (CUG) reduction, and downstream splicing correction of MBNL1 and SOS 1.

And (5) experiment. Hela480 cells were constructed as described in the previous examples. RT-PCR and foci staining were performed similarly to the other examples described herein.

As a result. FIGS. 41A-41B show exemplary images of control cells (untreated) and cells treated with EEV-PMO 221-1120 at 5. Mu.M, 10. Mu.M, 20. Mu.M, 50. Mu.M, and 100. Mu.M. CUG foci (green) were reduced in the treated HeLa480 group compared to untreated HeLa480 cells. FIG. 41B is a graph of foci quantifying area per nucleus. FIGS. 41A-41B show that EEV-PMO 221-1120 can reduce nuclear CUG RNA foci. Almost complete reduction was observed in the 5 μm dose.

FIGS. 42A-42B indicate that EEV-PMO 221-1120 treatment selectively knockdown of amplified DMPK transcripts containing repeat sequences in the HeLa480 cell line.

FIGS. 42C-42D show correction of MBNL1 (FIG. 42C) and SOCS1 (FIG. 42D) splicing in a dose dependent manner with EEV-PMO 221-1120 treatments.

Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A compound, the compound comprising:

A cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids; and

An Antisense Compound (AC) complementary to at least a portion of an amplified CUG repeat in a target mRNA sequence, wherein the AC comprises Phosphorodiamidate Morpholino (PMO) nucleotides.

2. The compound of claim 1, wherein at least two charged amino acids of the cyclic peptide are arginine.

3. The compound of claim 1 or 2, wherein the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine, or a combination thereof.

4. The compound of any one of claims 1 to 3, wherein at least two uncharged non-aromatic amino acids are citrulline, glycine, or a combination thereof.

5. The compound of claim 1, wherein the cyclic peptide has one of the following structures:

In its protonated form, the precursor of the precursor,

Wherein:

R ₄ is H or an amino acid side chain;

Each m is independently an integer of 0, 1,2 or 3.

6. The compound of claim 5, wherein the cyclic peptide has one of the following structures:

Or (b)

In its protonated form.

7. The compound of claim 5 or 6, wherein AA _SC is a side chain of an asparagine residue, an aspartic acid residue, a glutamic acid residue, a homoglutamic acid residue, or a homoglutamate residue.

8. The compound according to claim 5 or 6, wherein

R ₄ is H or an amino acid side chain;

m is 2, and

AA _SC is the side chain of a glutamic acid residue.

9. The compound of claim 5 or 6, wherein AA _SC is: Wherein t is an integer from 0 to 5.

10. The compound of any one of claims 1 to 9, further comprising a linker, wherein the linker conjugates the antisense compound to the AA _SC.

11. The compound of claim 10, wherein the linker comprises a- (OCH ₂CH₂)_z' -subunit, wherein z' is an integer from 1 to 23.

12. The compound of claim 10, wherein the linker comprises:

(i) - (OCH ₂CH₂)_z -subunit wherein z' is an integer from 1 to 23;

(ii) One or more amino acid residues such as residues of glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminocaproic acid or combinations thereof; or alternatively

(Iii) A combination of (i) and (ii).

13. The compound of claim 10, wherein the linker comprises:

(i) - (OCH ₂CH₂)_z -subunit wherein z is an integer from 2 to 20;

(ii) One or more residues of glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or a combination thereof; or alternatively

(Iii) A combination of (i) and (ii).

14. A compound, the compound comprising:

An endosomal escape vector comprising a cyclic peptide and an exocyclic peptide, wherein the cyclic peptide comprises 6 to 12 amino acids and the exocyclic peptide comprises 2 to 10 amino acids; and

15. The compound of claim 14, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids.

16. The compound of claim 15, wherein at least two charged amino acids of the cyclic peptide are arginine, at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine, or a combination thereof, and at least two uncharged non-aromatic amino acids are citrulline, glycine, or a combination thereof.

17. The compound of claim 14, wherein the cyclic peptide has one of the following structures:

Or (b)

In its protonated form, the precursor of the precursor,

Wherein:

R ₄ is H or an amino acid side chain;

AA _SC is the amino acid side chain to which the antisense compound and the exocyclic peptide are conjugated; and

Each m is independently an integer of 0, 1,2 or 3.

18. The compound of claim 17, wherein the cyclic peptide has one of the following structures:

Or (b)

In its protonated form.

19. The compound of claim 17 or 18, wherein AA _SC is a side chain of an asparagine residue, an aspartic acid residue, a glutamic acid residue, a homoglutamic acid residue, or a homoglutamate residue.

20. The compound of claim 17 or 18, wherein AA _SC is a side chain of a glutamic acid residue.

21. The compound of claim 17 or 18, wherein

R ₄ is H or an amino acid side chain;

m is 2, and

AA _SC is the side chain of a glutamic acid residue.

22. The compound of any one of claims 17 to 21, wherein AA _SC is: Wherein t is an integer from 0 to 5.

23. The compound of any one of claims 14 to 22, wherein the exocyclic peptide comprises 4 to 8 amino acid residues.

24. The compound of any one of claims 14 to 23, wherein the exocyclic peptide comprises 1 or 2 amino acid residues comprising a side chain comprising a guanidino group, or a protonated form or salt thereof.

25. The compound of any one of claims 14 to 24, wherein the exocyclic peptide comprises 2,3 or 4 lysine residues.

26. The compound of claim 25, wherein the amino group on the side chain of each lysine residue is substituted with trifluoroacetyl (-COCF ₃), allyloxycarbonyl (Alloc), 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl (Dde) or (4, 4-dimethyl-2, 6-dioxocyclon-1-hexyl-3) -methylbutyl (ivDde).

27. The compound of any one of claims 14 to 24, wherein the exocyclic peptide comprises at least 2 amino acid residues having a hydrophobic side chain.

28. The compound of claim 27, wherein the amino acid residue having a hydrophobic side chain is selected from valine, proline, alanine, leucine, isoleucine and methionine.

29. The compound of any one of claims 14 to 24, wherein the exocyclic peptide comprises one of the following sequences ：KK、KR、RR、HH、HK、HR、RH、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKH、KHK、HKK、HRR、HRH、HHR、HBH、HHH、HHHH、KHKK、KKHK、KKKH、KHKH、HKHK、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、HBHBH、HBKBH、RRRRR、KKKKK、KKKRK、RKKKK、KRKKK、KKRKK、KKKKR、KBKBK、RKKKKG、KRKKKG、KKRKKG、KKKKRG、RKKKKB、KRKKKB、KKRKKB、KKKKRB、KKKRKV、RRRRRR、HHHHHH、RHRHRH、HRHRHR、KRKRKR、RKRKRK、RBRBRB、KBKBKB、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV or PKKKRKG, wherein B is β -alanine.

30. The compound of any one of claims 14 to 24, wherein the exocyclic peptide comprises one of the following sequences: PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, wherein B is β -alanine.

31. The compound of any one of claims 14 to 24, wherein the exocyclic peptide comprises one of the following sequences ：KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、KKKKK、KKKRK、KBKBK、KKKRKV、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV or PKKKRKG.

32. The compound of any one of claims 14 to 24, wherein the exocyclic peptide comprises PKKKRKV.

33. The compound of any one of claims 14 to 24, wherein the exocyclic peptide comprises one of the following sequences ：NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK or RKCLQAGMNLEARKTKK.

34. The compound of any one of claims 14 to 33, further comprising a linker that conjugates the antisense compound and the exocyclic peptide to the AA _SC.

35. The compound of claim 34, wherein the linker comprises:

(i) - (OCH ₂CH₂)_z' -subunit wherein z' is an integer from 1 to 23;

(Iii) A combination of (i) and (ii).

36. The compound of claim 34, wherein the linker comprises:

(i) - (OCH ₂CH₂)_z -subunit wherein z is an integer from 2 to 20;

(Iii) A combination of (i) and (ii).

37. The compound of claim 34, wherein the linker comprises a divalent or trivalent C ₁-C₅₀ alkylene group, wherein 1-25 methylene groups are optionally and independently replaced by-N (H) -, -N (C ₁-C₄ alkyl) -, -N (cycloalkyl) -, -O-, -C (O) O-, -S (O) ₂-、-S(O)₂N(C₁-C₄ alkyl) -, -S (O) ₂ N (cycloalkyl) -, -N (H) C (O) -, -N (C ₁-C₄ alkyl) C (O) -, -N (cycloalkyl) C (O) -, -C (O) N (H) -, -C (O) N (C ₁-C₄ alkyl), -C (O) N (cycloalkyl), aryl, heteroaryl, cycloalkyl, or cycloalkenyl.

38. The compound of claim 34, wherein the linker has the structure:

Wherein:

x' is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * Is the attachment point of the amino acid residue of the cyclic peptide to the amino acid side chain; and M is a bonding group.

39. The compound of claim 38, wherein z' is 11.

40. The compound of claim 38 or 39, wherein x' is 1.

41. The compound of any one of claims 38 to 40, wherein the exocyclic peptide is conjugated to the linker at the amino terminus of the linker.

42. The compound of any one of claims 38 to 41, wherein the antisense compound is conjugated to M.

43. The compound of claim 17, wherein the compound has formula (C):

Or (b)

In its protonated form or in the form of a salt,

Wherein:

R ₄ and R ₇ are independently H or an amino acid side chain;

EP is the cyclic exopeptide;

each m is independently an integer from 0 to 3;

n is an integer from 0 to 2;

x' is an integer from 1 to 23;

y is an integer from 1 to 5;

q is an integer from 1 to 4;

z' is an integer from 1 to 23, and

The cargo is said antisense compound.

44. The compound of claim 43, wherein R ₁、R₂ and R ₃ are H or side chains comprising aryl.

45. The compound of claim 44, wherein said side chain comprising aryl is a side chain of phenylalanine.

46. The compound of claim 45, wherein two of R ₁、R₂ and R ₃ are side chains of phenylalanine.

47. The compound of any one of claims 44 to 46, wherein two of R ₁、R₂、R₃ and R ₄ are H.

48. The compound of any one of claims 44 to 47, wherein z' is 11.

49. The compound of any one of claims 44 to 48, wherein x' is 1.

50. The compound of claim 17, comprising a structure of formula (C-1), (C-2), (C-3), or (C-4):

/>

Or a protonated form or salt thereof,

Wherein EP is said cyclic exopeptide, and

Oligonucleotides are the antisense compounds.

51. The compound of claim 50, wherein the oligonucleotide comprises the sequence: 5'-CAG CAG CAG CAG CAG CAG CAG-3'.

52. The compound of claim 50 or 51, wherein the EP comprises the sequence: PKKKRKV.

53. The compound of any one of the preceding claims, wherein the amplified trinucleotide repeat sequence is located in the 3' utr of the target mRNA sequence.

54. The compound of claim 53, wherein the target mRNA sequence is a DMPK mRNA sequence.

55. The compound of claim 53, wherein the target mRNA sequence is an ATXN8OS/ATXN8 mRNA sequence.

56. The compound of claim 53, wherein the target mRNA sequence is a JPH3mRNA sequence.

57. The compound of any one of the preceding claims, wherein the AC comprises 5-10 CAG repeats.

58. The compound of any one of the preceding claims, wherein the mRNA is a pre-mRNA or a mature mRNA.

59. The compound of any one of the preceding claims, wherein the mRNA is a pre-mRNA.

60. A pharmaceutical composition comprising an effective amount of a compound of any one of claims 1-59.

61. A cell comprising a compound of any one of claims 1-59.

62. A method of treating myotonic muscular Dystrophy (DM) in a subject in need thereof, the method comprising administering to the subject the compound of any one of claims 1-59 or the composition of claim 60.

63. The method of claim 62, wherein the administration results in increased expression of a wild-type protein in muscle tissue.

64. The method of claim 63, wherein the administration causes increased expression of the wild-type protein in diaphragmatic tissue, quadriceps tissue, and/or cardiac tissue.

65. The method of claim 63 or 64, wherein the wild-type protein is a protein expressed from a gene that does not have an amplified CUG repeat.

66. The method of any one of claims 62-65, wherein the administration prevents or reduces lesion formation.

67. The method of any one of claims 62-66, wherein the nucleotide repeat amplification is located in the 3' utr of DMPK mRNA.

68. A method of treating a disease associated with mRNA having an amplified CUG repeat in the 3' untranslated region (UTR), the method comprising administering to a subject in need thereof a compound comprising:

An Antisense Compound (AC) complementary to at least a portion of the amplified CUG repeat in the mRNA; and

A cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids.

69. The method of claim 68, wherein the at least two charged amino acids of the cyclic peptide are arginine, the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine, or a combination thereof, and the at least two non-charged non-aromatic amino acids of the cyclic peptide are citrulline, glycine, or a combination thereof.

70. The method of claim 68, wherein the cyclic peptide has the structure:

Or (b)

In its protonated form, the precursor of the precursor,

Wherein:

R ₄、R₅、R₆、R₇ is independently H or an amino acid side chain;

at least one of R ₄、R₅、R₆、R₇ is H or the side chain of citrulline;

Q is 1, 2, 3 or 4.

71. The method of claim 70, wherein at least one of R ₄、R₅、R₆、R₇ is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N-dimethylarginine, 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, lysine, N-methyllysine, N-dimethyllysine, N-ethyllysine, N-trimethyllysine, 4-guanidinophenylalanine, N-dimethyllysine, β -homoarginine, or 3- (1-piperidinyl) alanine.

72. The method of claim 68, wherein the cyclic peptide has the structure:

Or a protonated form thereof,

Wherein:

R ₄ and R ₇ are independently H or an amino acid side chain;

q is 1, 2, 3 or 4; and

Each m is independently an integer of 0, 1,2 or 3.

73. The method of claim 72, wherein the cyclic peptide has one of the following structures:

/>

Or (b)

In its protonated form.

74. The method of claim 72, wherein the cyclic peptide has one of the following structures:

/>

Or (b)

In its protonated form.

75. The method of any one of claims 68-74, wherein the antisense compound comprises a Phosphorodiamidate Morpholino (PMO) nucleotide.

76. The method of any one of claims 68-75, further comprising conjugating the antisense compound to a linker of AA _SC.

77. The method of any one of claims 68-76, comprising an endosomal escape vector, wherein the endosomal escape vector comprises a cyclic peptide and an exocyclic peptide.

78. The method of any one of claims 68-77, wherein the disease is tonic muscular Dystrophy (DM).

79. The method of any one of claims 68 to 77, wherein the disease is spinocerebellar ataxia type 8 (SCA 8).

80. The method of any one of claims 68-77, wherein the disease is huntington's disease-like 2 (HDL 2).

81. The compound, composition or method of any one of the preceding claims, wherein the antisense compound comprises a nucleotide sequence set forth in table 2.