KR20240038967A - Antisense compounds and methods for targeting CUG repeats - Google Patents

Abstract

Compounds including cyclic peptides, such as cyclic cell penetrating peptides, and antisense compounds are provided. Antisense compounds bind to genes with expanded CTG repeats or to gene transcripts with expanded CUG repeats. The compounds can be delivered to a subject to treat diseases associated with expanded CTG·CUG repeats, such as myotonic dystrophy type 1 (DM1), spinocerebellar ataxia type 8 (SCA8), and Huntington's disease-like-2 (HDL2). .

Description

Antisense compounds and methods for targeting CUG repeats

Cross-reference to related applications

This application is related to U.S. Provisional Patent Application No. 63/213,900, filed on June 23, 2021; U.S. Provisional Patent Application No. 63/239,847, filed September 1, 2021; U.S. Provisional Patent Application No. 63/290,892, filed December 17, 2021; U.S. Provisional Patent Application No. 63/305,071, filed January 31, 2022; U.S. Provisional Patent Application No. 63/314,369, filed February 26, 2022; U.S. Provisional Patent Application No. 63/316,634, filed March 4, 2022; U.S. Provisional Patent Application No. 63/317,856, filed March 8, 2022; U.S. Provisional Patent Application No. 63/326,201, filed March 31, 2022; U.S. Provisional Patent Application No. 63/327,179, filed April 4, 2022; U.S. Provisional Patent Application No. 63/339,250, filed May 6, 2022; U.S. Provisional Patent Application No. 63/362,295, filed March 31, 2022; U.S. Provisional Patent Application No. 63/239,671, filed September 1, 2021; U.S. Provisional Patent Application No. 63/290,960, filed December 17, 2021; U.S. Provisional Patent Application No. 63/298,565, filed January 11, 2022; and U.S. Provisional Patent Application No. 63/268,577, filed February 25, 2022.

Technology field

The present disclosure relates to compounds, compositions, and methods for modulating the activity and/or level of genes comprising expanded nucleotide repeats, particularly expanded CTG · CUG repeats. The compounds and compositions containing them can be used to treat diseases associated with genes containing expanded nucleotide repeats, especially expanded CTG · CUG repeats.

Some diseases are associated with genes with expanded nucleotide repeats, that is, genes with a greater number of nucleotide repeats than observed in the healthy phenotype. Expanded repeats may cause aggregation and/or nucleation of expanded repeat-containing transcripts and/or may cause nucleation of proteins that bind to expanded repeat-containing transcripts. Expanded repeats sequester some proteins, such as pre-mRNA processing proteins, on the repeat, preventing these proteins from performing their normal functions, such as processing pre-mRNA transcripts from other genes that do not contain expanded repeats. It can be suppressed.

There are several diseases associated with genes with expanded CTG · CUG trinucleotide repeats (CTG refers to a DNA repeat and CUG refers to the corresponding RNA repeat that occurs after transcription). Diseases associated with genes with expanded CTG · CUG trinucleotide repeats include myotonic dystrophy type 1 (DM1), spinocerebellar ataxia type 8 (SCA8), Huntington's disease-like-type 2 (HDL2), and Fuchs' corneal endothelial dystrophy (FECD). Including, but not limited to.

Myotonic dystrophy type 1 (DM1), which affects 1 in 8500 people worldwide and is the most common cause of muscular dystrophy in adults, is associated with genes with expanded trinucleotide repeats (Lee and Cooper (2009 ) (“Pathogenic mechanisms of myotonic dystrophy,” Biochem Soc Trans. 37(06): 10.1042/BST0371281]). DM1 is a disorder that affects not only skeletal and smooth muscles, but also the eyes, heart, endocrine system, and central nervous system. DM1 is caused by abnormal expansion of CTG-trinucleotide repeats in the non-coding region of the gene encoding dystrophic myotonic dystrophy protein kinase (DMPK). The CTG extension is located within a region corresponding to the 3' untranslated region (3'-UTR) of DMPK mRNA. The DMPK gene in healthy individuals contains 5 to 40 CTG trinucleotide repeats, whereas DM1 patients have 50 to up to several thousand CTG trinucleotide repeats. CTG-trinucleotide repeat expansions result in a global loss of regulation of gene expression in diseased individuals due to nucleation of some regulatory RNA-binding proteins within the CUG-expansion in the 3' untranslated region (3'-UTR). , RNA-binding proteins such as muscleblind-like protein (MBNL1-3) prevent normal cellular functions. Nuclear RNA binding proteins are not available to bind to other mRNA transcripts and affect their translation. These CUG-extended mRNA-protein aggregates form distinct nuclear foci. The activity of additional splicing factors, such as CUGBP Elav-like family member 1 (CELF1), is also disrupted, leading to mis-splicing of multiple downstream gene transcripts that are associated with symptoms of DM1. As the number of repetitions increases, disease severity increases and the age of onset decreases (Pettersson et al. [(2015) “Molecular mechanisms in DM1 - a focus on foci.” Nucleic Acids Res. 43(4):2433-2441]) .

CUG-trinucleotide repeats in the 3' untranslated region of DMPK mRNA form incomplete stable hairpin structures, which accumulate in the cell nucleus as small ribonuclear complexes or microscopic inclusions and are not involved in transcription, splicing, or RNA export. Damages the function of proteins involved in Although the DMPK gene with CUG repeats is transcribed into mRNA, the mutant transcripts are sequestered as aggregates (foci) in the nucleus, resulting in decreased cytoplasmic DMPK mRNA levels. This aggregation leads to loss of regulation of alternative splicing of many different transcripts due to sequestration of two RNA binding proteins: muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1), resulting in loss of function of MBNL1. and causes upregulation 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 (CELF1) are developmental regulators of splicing events during fetal transition to adulthood, and in DM1, their active modifications result in expression of fetal splicing patterns in adult tissues. The downstream effects of decreased levels of MBNL1 and increased levels of CELF1 include, in addition to MBNL1, cardiac troponin T (cTNT), insulin receptor (INSR), muscle-specific chloride ion channel (CLCN1), and sarcoplasmic reticulum/endoplasmic reticulum calcium ATPase 1. (ATP2A1) involves disruption of alternative splicing, mRNA translation, and mRNA decay in protein-like transcripts (Konieczny et al. (2017) “Myotonic dystrophy: candidate small molecule therapeutics.” Drug Discov Today. 22 (11):1740-174]).

Possible therapeutic approaches for treating DM1, or other diseases associated with expanded CTG · CUG repeats, include the use of therapeutic oligonucleotide-containing compounds. However, a major problem associated with the use of oligonucleotide compounds in therapeutics is their limited ability to access intracellular compartments when these compounds are administered systemically. Intracellular delivery of oligonucleotide compounds can 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. Improved delivery systems are still needed to increase the efficacy of these compounds.

There is an unmet need for effective compositions for delivering therapeutic oligonucleotide compounds to intracellular compartments to treat diseases such as DM1 caused by expanded CTG · CUG repeats.

Described herein are compounds, compositions, and methods for treating diseases associated with expanded CTG · CUG repeats. In embodiments, the present disclosure relates to compounds comprising antisense compounds (AC) and cyclic peptides, such as cyclic cell penetrating peptide (cCPP). In an embodiment, the AC binds to a gene or gene transcript comprising expanded CUG repeats. In embodiments, the cyclic peptide facilitates intracellular localization of AC. The compound may include an endosomal escape vehicle (EEV). EEV may include 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 the target nucleotide. In an embodiment, the target nucleotide comprises at least one extended CUG or CTG repeat. In an embodiment, the target nucleotide is a gene comprising at least one expanded CTG repeat. In an embodiment, the target nucleotide is RNA comprising at least one expanded CUG repeat. In an embodiment, the RNA comprising at least one expanded CUG repeat is a pre-mRNA sequence. In an embodiment, the expanded CUG repeat corresponds to an expanded CTG repeat in the gene from which the pre-mRNA is transcribed. In an embodiment, the antisense compound binds to an expanded CTG repeat or an expanded CUG repeat. In embodiments, the AC has 5 to 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 an embodiment, the AC comprises a sequence of nucleotides listed in Table 2, Table 10 , or Table 11 . In an embodiment, the AC comprises a sequence of nucleotides listed in Table 2 .

In an embodiment, the AC comprises at least one modified nucleotide or nucleic acid selected from: phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino (PMO) nucleotides, locked nucleic acids (LNA), peptides. Nucleic acid (PNA), nucleotides containing a 2'-O-methyl (2'-OMe) modified backbone, 2'O-methoxy-ethyl (2'-MOE) nucleotides, 2',4' tethered ethyl ( cEt) nucleotides, 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA), and combinations thereof. In an embodiment, AC comprises PMO nucleotides.

In an embodiment, provided is a compound comprising a cyclic peptide having 6 to 12 amino acids, wherein at least 2 amino acids of the cyclic peptide are charged amino acids, at least 2 amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and wherein the cyclic peptide At least two amino acids are uncharged, non-aromatic amino acids. In an embodiment, the antisense compound (AC) is complementary to at least a portion of the expanded CUG repeat in the target mRNA sequence. In an embodiment, AC is a phosphorodiamidate morpholino (PMO) nucleotide.

In an embodiment, at least two charged amino acids of the cyclic peptide are arginine. In an embodiment, the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine (3-naphth-2-yl-alanine), or a combination thereof. In an embodiment, the at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, glycine, or a combination thereof. In an embodiment, the compound is a cyclic peptide having 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; The at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine, and combinations thereof.

In an embodiment, the compound comprises an endosomal escape vehicle comprising a cyclic peptide and an extracyclic peptide (EP). In an embodiment, the EP is conjugated to the linker at the amino group. The linker may be a linker such as those described herein. In an embodiment, the EP is conjugated to the cyclic peptide via a linker. In an embodiment, EP is conjugated to AC via a linker. In an embodiment, 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, EP comprises 4 to 8 amino acid residues. In an embodiment, the EP comprises one or two amino acids comprising a side chain comprising a guanidine group or a protonated form thereof. In embodiments, EP comprises 1, 2, 3, or 4 lysine residues. In an embodiment, the amino group on the side chain of each lysine residue is a trifluoroacetyl (-COCF ₃ ) group, allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexyly) It is substituted with den)ethyl (Dde), or (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) group. In an embodiment, the EP comprises at least two amino acid residues with hydrophobic side chains. In an embodiment, the amino acid residue having a hydrophobic side chain is selected from valine, proline, alanine, leucine, isoleucine, and methionine. In an embodiment, the exophytic 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; PKGRKV; PKKKGKV; PKKKRGV; Or PKKKRKG. In an embodiment, the exophytic 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; PKGRKV; PKKKGKV; PKKKRGV; Or PKKKRKG. In an embodiment, the exophytic peptide has the following structure: Ac-PKKKRKV-.

In an embodiment, the cyclic pentide contains 4 to 12 amino acids. In an embodiment, the cyclic pentide contains 6 to 12 amino acids. In an embodiment, 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 an embodiment, 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, and combinations thereof, and the at least two uncharged non-aromatic amino acids are citrulline. , glycine, and combinations thereof.

In an embodiment, the cyclic peptide has 4 to 12 amino acids, at least two amino acids of the cyclic peptide are arginine and at least two amino acids comprise hydrophobic side chains, but the cyclic peptide has a cyclic peptide having the sequence of SEQ ID NOs: 89-117. It's not a peptide. In an embodiment, the cyclic peptide is not a cyclic peptide having the sequence of SEQ ID NOs: 89-117.

where F is L-phenylalanine, f is D-phenylalanine, Φ is L-3-(2-naphthyl)-alanine, Φ is D-3-(2-naphthyl)-alanine, and R is 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 an embodiment, the cyclic peptide has the following structure:

(A), or

It has a protonated form,

During the ceremony:

R ₁ , R ₂ , and R ₃ are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

R ₄ , R ₅ , R ₆ , and R ₇ are independently H or an amino acid side chain;

At least one of R ₄ , R ₅ , R ₆ , and R ₇ is 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N, N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethylysine, N,N,N-trimethyllysine, It is the side chain of 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, and 3-(1-piperidinyl)alanine;

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

q is 1, 2, 3, or 4.

In embodiments, at least one of R ₄ , R ₅ , R ₆ , and R ₇ is independently an uncharged, non-aromatic side chain of an amino acid. In embodiments, at least one of R ₄ , R ₅ , R ₆ , and R ₇ is independently H or a side chain of citrulline.

In an embodiment, the cyclic peptide has the structure of Formula I:

(I)

or a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ are each independently H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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;

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

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

(Ia),

(Ib), or

It has its protonated form.

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

(I-1),

(I-2),

(I-3),

(I-4),

(I-5),

(I-6), or

or a protonated form thereof.

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

(I-1),

(I-2), or

or a protonated form thereof.

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

(I-1), or

or a protonated form thereof.

In an embodiment, the compound has the structure of Formula C:

(C), or

has a protonated form or salt thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ are each independently H or a side chain containing an aryl or heteroaryl group, and at least one of R ₁ , R ₂ , and R ₃ is a side chain containing an aryl or heteroaryl group;

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

EP is an exophytic peptide;

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;

The cargo is an antisense compound.

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

(C-1),

(C-2),

(C-3),

(C-4)

or a protonated form or salt thereof,

In the formula, EP is an exophytic peptide,

Oligonucleotides are antisense compounds.

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

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

Brief description of the drawing
Figure 1 is a schematic showing multiple strategies for targeting CUG repeats in mRNA.
Figure 2 shows modified nucleotides used in the antisense oligonucleotides described herein. Structures 1 to 3 (1 = phosphorothioate; 2 = (S _C5 -R _p )-α,β-CAN; 3 = PMO) are phosphate backbone modifications; 4(2-thio-dT) is a base modification; 5 to 8 (5 = 2'-OMe-RNA; 6 = 2'O-MOE-RNA; 7 = 2'F-RNA; 8 = 2'F-ANA) are 2' sugar modifications; 9-11 are bound nucleotides; 12-14 (9 = LNA; 10 = ( S )-cET; 11 = tcDNA; 12 = FHNA; 13 = (S)5'-C-methyl; 14 = UNA) are additional sugar modifications; 15-18 (15 = E -VP; 16 = methyl phosphonate; 17 = 5'phosphorothioate; 18 = (S)-5'-C-methyl with phosphate) are the 5' phosphate stabilizing modifications; ; 19 is a morpholino sugar. Khvorova, A., et al. [Nat. Biotechnology. 2017 Mar; Format reconstructed from [35(3): 238-248].
Figures 3A-3D depict the conjugation chemistry for linking AC to a cyclic cell penetrating peptide. Figure 3a shows the formation of an amide bond between a peptide with a carboxylic acid group or a peptide with a TFP activated ester and the primary amine residue at the 5' end of AC. Figure 3b shows the conjugation of peptide-TFP ester with secondary amine modified AC or primary amine modified AC at the 3' through amide bond formation. Figure 3C shows peptide-azide conjugation to 5' cyclooctyne modified AC via copper-free azide-alkyne cycloaddition. Figure 3D shows linker-azide with 3' modified cyclooctyne AC or 3' modified azide AC via copper-free azide-alkyne cycloaddition or copper catalyzed azide-alkyne cycloaddition (click reaction). Alternatively, another example is shown in which a CPP containing a linker-alkyne/cyclooctyne moiety is conjugated.
Figure 4 depicts the conjugation chemistry for linking AC and CPP with additional linker modalities containing polyethylene glycol (PEG) moieties.
Figures 5A-5D show adesine ( 5a ), cytosine ( 5b ), guanine ( 5c ), and thymine ( 5d ) morpholinos that can be used to synthesize phosphorodiamidate-linked morpholino oligomers (PMOs). The structure of the subunit monomer is provided.
Figures 6a-6f show various PMO or PMO-EEV at 1 μM, 3 μM, or 10 μM with ( 6a-6c ) or without ( 6e-6f ) Endo-Porter transfection agent. 24 h ( 6a–6b ) and 48 h ( 6c–6d ) after treatment of HeLa-48 cells with the compounds, MBNL1 (exon 5; Figures 6a, 6b, 6e ) and CLASP1 (exon 19; Figures 6b, 6d; 6f ) shows RT-PCR analysis of alternative RNA splicing events (e.g. exon inclusion or exclusion). Parental HeLa cell lines and HeLa-480 cell lines treated with endo-porter agents ( 6a-6d ) or without endo-porter agents ( 6e-6f ) were included as controls.
Figures 7A-7B show MBNL1 (exon 5; Figure 7A ) and CLASP1 (exon 19; Figure 7B ) 48 hours after treatment of DM1 myoblasts with 1 μM of various PMO or PMO-EEV compounds without endo-porter transfectant. RT-PCR analysis of alternative splicing events (e.g., exon inclusion or exclusion) is shown. Two controls were included as controls: DM-04 treated without Endo-Porter and DM-05 treated without Endo-Porter.
Figures 8A-8D show gastrocnemius tissue samples from HSA-LR (DM1-mouse model) mice 1 week after treatment with PMO, 20 mpk PMO-EEV 221-1106, or 40 mpk PMO-EEV 221-1106. Alternative RNA splicing events in Atp2a1 (exon 22; Fig. 8A ), Nfix (exon 7; Fig. 8B ), Clcn1 (exon 7a; Fig. 8C ), and Mbnl1 (exon 5; Fig. 8D ), e.g. or exclusion). FVB/NJ (wild-type crossbred mice) and HSA-LR (no treatment) mice were included as controls.
Figures 9A-9D show quadriceps tissue samples from HSA-LR (DM1-mouse model) mice 1 week after treatment with PMO, 20 mpk PMO-EEV 221-1106, or 40 mpk PMO-EEV 221-1106. Alternative RNA splicing events in Atp2a1 (exon 22; Fig. 9A ), Nfix (exon 7; Fig. 9B ), Clcn1 (exon 7a; Fig. 9C ), and Mbnl1 (exon 5; Fig. 9D ), e.g. or exclusion). FVB/NJ (wild-type crossbred mice) and HSA-LR (no treatment) mice were included as controls.
Figures 10A-10D show tibialis anterior tissue from HSA-LR (DM1-mouse model) mice 1 week after treatment with PMO, 20 mpk PMO-EEV 221-1106, or 40 mpk PMO-EEV 221-1106. Alternative RNA splicing events (e.g. exon 5; Figure 10d ) of Atp2a1 (exon 22; Figure 10a ), Nfix (exon 7; Figure 10b ), Clcn1 (exon 7a; Figure 10c ), and Mbnl1 (exon 5; Figure 10d ) were harvested. RT-PCR analysis for inclusion or exclusion) is shown. FVB/NJ (wild-type crossbred mice) and HSA-LR (no treatment) mice were included as controls.
Figures 11A-11F show muscle cells from DM1 patients after treatment with different concentrations (10 μm, 3 μm, 1 μm, 0.3 μm) of DMPK CUG-targeted EEV-PMO (CUG ^exp 197-777 and CUG ^exp 221-1106). MBNL1 (exon 5, Figure 11A ), SOS1 (exon 25, Figure 11B ), IR (exon 11, Figure 11C ), DMD (exon 78, Figure 11D ), BIN1 (exon 11, Figure 11E ), and LDB3 (exon 11) , Figure 11f ) shows RT-PCR analysis of alternative RNA splicing events (e.g., exon inclusion or exclusion). As controls, two groups of muscle cells from healthy individuals (negative control) and DM1 patients (positive control) were tested for alternative RNA splicing events. All data were collected from three individual experiments (n=3). A T-test of treated versus untreated DM1 root canals was performed; *p <0.05; **p <0.01; ***p < 0.001.
Figures 12A-12F show MBL1 (exon 5, Figure 12A ), SOS1 (exon 25, Figure 12b ), INSR (exon 11, Figure 12c ), DMD (exon 78, Figure 12d ), BIN1 (exon 11, Figure 12e ), and LDB3 (exon 11, Figure 12f ) alternative RNA splicing events (examples) : shows RT-PCR analysis for exon inclusion or exclusion). Healthy patient cells and DM1 cells were used as controls. All data were collected from three individual experiments (n=3). A T-test of treated versus untreated DM1 root canals was performed; *p <0.05; **p <0.01; ***p < 0.001.
Figures 13A-13B show relative levels of mRNA after treatment of HSA-LR mice with various concentrations of PMO-EEV 221-1120. Figure 13A shows relative mRNA levels for gastrocnemius, triceps, tibialis anterior, and diaphragm. Figure 13B shows relative mRNA levels in the diaphragm.
Figures 14A-14C show relative mRNA expression in quadriceps ( 14a ), gastrocnemius ( 14b ), triceps ( 14c ), and tibialis anterior ( 14d ) tissues after treatment of HSA-LR mice with various concentrations of PMO-EEV 221-1120. Shows the level.
Figures 15A-15D show the quadriceps ( Figure 15A ), gastrocnemius ( Figure 15B ), triceps ( Figure 15C ), and tibialis anterior ( Figure 15D ) after treatment of HSA-LR mice with various concentrations of PMO-EEV 221-1120. Mouse DM1 splicing index (mDSI) for various genes is shown.
Figures 16A-16C show the prevalence of RNA foci in the tibialis anterior muscle of HSA-LR mice untreated or after treatment with EEV-PMO 221-1120 (EEV-PMO-DM1-3; DM1-3). Figures 16A-16B show images of tibialis anterior tissue stained for RNA CUG foci (red) and nuclei (blue). Figure 16C is a chart quantifying the percentage of nuclei with CUG foci from data associated with the images in Figures 16A-16B .
Figures 17a-17f show the quadriceps ( 17a ), triceps ( 17b ), heart ( 17c ), gastrocnemius ( 17d ), and tibialis anterior ( 17e ) muscles after treatment of HSA-LR mice with various concentrations of EEV-PMO-DM1-3. , a diagram showing the dose-dependent response to drug levels in diaphragm ( 17f ), brain ( 17h ), liver ( 17i ), and kidney ( 17j ) tissues. Figure 17K shows drug exposure of various tissues at the 60 mpk dose level.
Figure 18 shows dose-dependent reduction in myotonia in HSA-LR mice 7 days after treatment with 15, 30, 60, and 90 mpk of EEV-PMO-DM1-3.
19A-19D show 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. This is a diagram showing: Figures 19a and 19c are charts showing three main components, and Figures 19b and 19d are charts showing two main components.
Figures 20A-20B show heatmaps of differentially expressed genes between non-diseased mice (WT), DM1 mice (HSA-LR), and HSA-LR mice treated with 60 mpk PMO-EEV 221-1120. Figure 20A is a clustered heatmap showing 513 differentially expressed genes. Figure 20b is a clustered heatmap showing 40 genes known to have CTG·CUG repeats.
Figure 21 is a volcano plot showing overall transcriptional changes across untreated HSA-LR mice and mice treated with PMO-EEV 221-1120.
Figures 22A-22E show Scube2 ( 22a ), Greb1 ( 22b ), Ttc7 ( 22c ), and Txlnb(CUG)9 from non-disease mice, HSA-LR mice, and HSA-LR mice treated with PMO-EEV 221-1120. This is a chart showing the results of principal component analysis for ( 22d ), and Ndrg3 ( 22e ) genes.
23A-23D show Atp2a1 ( 23a ; exon 22 is boxed) in non-diseased mice (WT-saline), HSA-LR mice (HSA-LR saline), and HSA-LR mice treated with PMO-EEV 221-1120. ), Clcn1 ( 23b ; exon 7a is boxed), Nfix ( 23c ; exon 7 is boxed), and Mbn1 ( 23d ; exon 5 is boxed). Two readings are shown for each treatment group.
Figure 24 Splicing index of individual exons for various genes of interest in non-diseased mice (WT-saline), HSA-LR mice (HSA-LR saline), and HSA-LR mice treated with PMO-EEV 221-1120. Shows percentage (PSI).
Figures 25A-25D show tibialis anterior ( 25a ), gastrocnemius ( 25b ), 1-4 weeks after treatment of HSA-LR mice with 80 mpk (60 mpk oligo, 80 mpk full drug) EEV-PMO-DM1-3. triceps ( 25c ), and Shows drug levels in quadriceps ( 25d ) tissue.
Figures 26A-26D show drug levels in mice after treating HSA-LR mice with a single dose of 80 mpk of EEV-PMO-DM1-3. Figures 26A-26B show drug levels in the liver from 1 to 12 weeks after treatment. Figures 26C-26D show drug levels in the kidneys from 1 to 12 weeks after treatment.
Figures 27A-27C show MBNL1 (exon 5; 26a ), SOS1 (exon 25; 26b ), and NFIX (exon 7; 26c ) levels after treatment of muscle cells from DM1 patients with 30 μM EEV-PMO-DM1-3. This is a chart showing the level of exon inclusion for .
Figures 28A-28C show that EEV-PMO-DM1-3 reduces CUG nuclear foci (green) in the nuclei (blue) of muscle cells from DM1 patients. Figures 28A-28B are images of muscle cells from DM1 patients untreated or treated with EEV-PMO-DM1-3. Figure 28C quantifies the number of CUG foci per nucleus for data associated with the images in Figure 28A .
Figures 29A-29B show raw data ( 29a ) and normalized data ( 29b ) from the CELLTITER-GLO luminescence viability assay in which RPTEC cells were treated with various concentrations of PMO-DM1 or EEV-PMO-DM1-3. Melittin was used as a positive control.
Figures 30A-30C show images depicting RNA CUG repeat foci in DM1 patient-derived cells ( 30A ) and DM1 patient-derived cells treated with EEV-PMO 221-1113 ( 30B ). Cells were stained for nuclei (blue; Hoechst) and RNA CUG foci (green). Figure 30C is a plot of CUG RNA foci per nuclear area for data associated with the images of Figures 30A-30B .
Figures 31A-31B show the prevalence of RNA CUG7 foci in HeLa, untreated HeLa480, and HeLa480 cells treated with EEV-PMO 221-1113. Figure 31A shows cells stained for RNA CUG7 foci (green) and nuclei (blue). Shows the image. Figure 31B is a plot quantifying CUG7 foci per nuclear area for the data associated with the image in Figure 31A .
Figures 32A-32C show percent exon 5 inclusion in MBNL1 ( 32a ), percent exon 25 inclusion in SOS1 ( 32b ), and exon 7 inclusion in NFIX after treatment of DM1 patient-derived cells with 30 μM EEV-PMO 221-1113. This is a chart showing the percentage ( 32c ).
Figures 33a-33e show the expression of MBNL1 (exon 5; 33a ), SOS1 (exon 25; 33b ), CLASP1 (exons 19, 33c ), and NFIX after treating muscle cells from DM1 patients with various concentrations of PMO-EEV 221-1113. (exon 7, 33d ), and INSR (exon 11, 33e ). Significance was determined using the T-test; *p <0.05; **p <0.01; ***p < 0.001.
Figures 34A-34D show Atp2a1 (exon 22, 34a ), Nfix (exon 7, 34b ), Clcn1 (exon 7a, 34c ), and Shown is RT-PCR analysis of alternative RNA splicing events (including exon 5, 34d ) of Mbnl1 (exon 5, 34d).
Figures 35a-35c show Mbnl1 (exon 5, 35a ) and Nfix (exon 7) in tibialis anterior tissue of HSA-LR mice treated with PMO-EEV 0221-1121 (21-mer) or PMO-EEV 0325-1121 (24-mer). , 35b ), and RT-PCR analysis of alternative RNA splicing events (i.e., exon inclusion or exclusion) of Atp2a1 (exons 22, 35c ).
Figures 36a-36c show Mbnl1 (exon 5, 36a ), Nfix (exon 7, 36b ), and RT-PCR analysis of alternative RNA splicing events (including exons 22 and 36c ) of Atp2a1 (exons 22, 36c).
Figure 37 shows PMO-0221a, the main metabolite of PMO-EEV 220-1120 detected in vivo.
Figures 38A-38B show the percentage exon coverage for MBNL1 (exon 5) in tibialis anterior ( 38a ) and gastrocnemius ( 38b ) after treatment of Hela480 cells with various concentrations of EEV-PMO 221-1120.
Figures 39A-39B show the percentage exon coverage for NFIX (exon 7) in tibialis anterior ( 39a ) and gastrocnemius ( 39b ) after treatment of Hela480 cells with various concentrations of EEV-PMO 221-1120.
Figures 40A-40B show the percentage of exon coverage for Atp2a1 (exon 22) in tibialis anterior ( 40a ) and gastrocnemius ( 40b ) after treatment of Hela480 cells with various concentrations of EEV-PMO 221-1120.
Figure 41 shows an image depicting RNA CUG repeat foci in Hela480 cells after treatment with various concentrations of EEV-PMO 221-1120 ( 41a ). Figure 41B is a plot of RNA foci per nuclear area for the data associated with the image of Figure 41A .
Figures 42A-42D show relative r(CUG480) repeat mRNA levels ( 42a ), relative DMPK mRNA levels ( 42b ), and percent exon 5 coverage of MBNL1 ( 42c ) in HeLa480 cells after treatment with various concentrations of EEV-PMO 221-1120. , and percent inclusion of exon 25 in SOS1 ( 42d ).
Figure 43 is a bar chart showing examples of genes expressed in muscle tissue known to have CTG·CUG repeats.
Figures 44A-44D show reduction of myotonia phenotype in HSA-LR mouse model treated with 20 mpk of PMO-EEV 221-1106. Figures 44a and 44c show diagrams of muscle relaxation. Figure 44B shows raw data of an example muscle strength trace. Figure 44D shows a representative electromyography trace.

Specific details for carrying out the invention

compound

In embodiments, compounds are provided that modulate the level and/or activity of gene transcripts with expanded CUG trinucleotide repeats. In an embodiment, the compounds of the present disclosure comprise at least one cyclic cell penetrating peptide (cCPP) and a therapeutic moiety (TM). cCPP facilitates TM entry into cells. In an embodiment, the compound comprises an endosomal escape vehicle (EEV) comprising cCPP and an exophytic peptide (EP). cCPP or EEV can allow TM to enter cytosolic or cellular compartments and interact with target transcripts.

therapeutic moiety

Generally, TM is the actuator moiety that drives the reaction. In embodiments, the TM induces a response by modulating the expression, activity, and/or level of the target transcript and/or target protein. In an embodiment, the target transcript comprises an expanded CUG trinucleotide repeat. In embodiments, the TM modulates the level of the target transcript and/or target protein within the cell. In embodiments, the TM reduces the level of the 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. TM can effectively modulate the activity of one or more proteins that may be associated with a target transcript by reducing the affinity between the target transcript and one or more proteins. For example, if one or more proteins do not bind to the target transcript, they can target other molecules to perform their function. For example, if one or more proteins are involved in pre-mRNA processing, reducing the affinity of one or more proteins for transcripts containing expanded CUG repeats will cause these one or more proteins to contain expanded CUG repeats. It is possible to process unused pre-mRNA transcripts. As such, a TM can modulate the activity, expression, and/or levels of downstream genes (genes that do not contain expanded CTG repeats) that are regulated by one or more proteins whose interaction with the target transcript is disrupted by the TM. there is.

In embodiments, TMs include oligonucleotides, peptides, antibodies, and/or small molecules. The class and identity of the TM depends on the mechanism used to regulate the level and/or activity of the target transcript containing the extended CUG trinucleotide repeat.

antisense compounds

In various embodiments, the compounds disclosed herein include 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 stands for natural DNA bases, modified DNA bases, natural RNA bases, modified RNA bases, natural RNA sugars, modified RNA sugars, natural DNA sugars, modified DNA sugars, natural internucleoside linkages, modified nucleosides. It is an oligonucleotide containing interlinkages, or any combination thereof. AC includes antisense oligonucleotides RNAi, microRNA, antagomir, aptamer, ribozyme, immunostimulatory oligonucleotide, decoy oligonucleotide, supermir, miRNA mimic, miRNA inhibitor, U1 adapter, and these. Including, but not limited to, combinations of.

In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to the target transcript with expanded CUG trinucleotide repeats. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to an expanded CUG trinucleotide repeat in the target mRNA sequence. Several diseases, such as myotonic dystrophy type 1 (DM1), Fuchs' corneal endothelial dystrophy (FECD), spinocerebellar ataxia type 8 (SCA8), and Huntington's disease-like-type 2 (HDL2), are associated with expanded CUG trinucleotide repeats. . Table 1 provides examples of nucleotide repeat disorders and characteristics of genes with expanded nucleotide repeats associated with these disorders. The following documents describe exemplary oligonucleotides for treating tandem repeat diseases, which 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].

Diseases Associated with Expanded CUG Trinucleotide Repeats Disease (abbreviation) gene normal repeat length extended repeat length gene product repeat sequence repeat position DM1 DMPK 5-35 > 50 Myotonic muscular dystrophy protein kinase CTG·CAG 3’UTR FECD TCF4 < 30 >40 transcription factor 4 CTG·CAG intron 3 SCA8 ATXN8OS and/or ATXN8 15-50 > 50 Ataxin 8 and Ataxin 8 opposing strands CTG·CAG 3’UTR HDL2 JPH3 6-27 >40 Junktophyllin 3 CTG·CAG 3’UTR

In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the target mRNA transcript comprising an expanded CTG·CUG trinucleotide repeat. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to an expanded CTG·CUG trinucleotide repeat in the target mRNA transcript.

In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the DMPK1 target transcript comprising an expanded CTG·CUG trinucleotide repeat. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the TCF4 target transcript comprising an expanded CTG·CUG trinucleotide repeat. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the ATXN8OS/ATXN8 target transcript comprising an expanded CTG·CUG trinucleotide repeat. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence within the JPH3 target transcript comprising an expanded CTG·CUG trinucleotide repeat.

In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to a trinucleotide repeat in the 3'UTR of the target mRNA transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to an extended CTG·CUG trinucleotide repeat in the 3'UTR of the DMPK1 target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to an extended CTG·CUG trinucleotide repeat in the 3'UTR of the ATXN8OS/ATXN8 target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to an extended CTG·CUG trinucleotide repeat in the 3'UTR of the JPH3 target transcript.

In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to a trinucleotide repeat, such as a CTG·CUG repeat. In an embodiment, the target nucleotide sequence comprises at least one nucleotide repeat (e.g., CTG·CUG repeat). In embodiments, the target nucleotide sequence is 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, It contains at least 700, at least 800, at least 900, at least 1000, or at least 2000 CTG·CUG trinucleotide repeats. In an embodiment, the expanded trinucleotide repeat is in the 3'UTR of the target nucleotide sequence.

In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of the contiguous expanded trinucleotide repeats present in the target transcript. In embodiments, the AC is 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 up to 50, up to 100, up to 150, up to 200, up to 300, up to 400, up to 500, up to 600, up to 700, up to 800, up to and a nucleotide sequence that is at least partially complementary to and capable of hybridizing to 900, up to 1000, or up to 2000 trinucleotide repeats. In embodiments, the AC is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 trees within the target transcript. It comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to the nucleotide repeat. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to 5 to 10 trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to 5 to 9 trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to 5 to 8 trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to 5 to 7 trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to 5 to 6 trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to five trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to six trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to a 7 trinucleotide repeat in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to eight trinucleotide repeats in the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to nine trinucleotide repeats in the target transcript. In embodiments, AC comprises a nucleotide sequence

In embodiments, the AC may comprise a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of a contiguous expanded trinucleotide repeat present at any position within the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of the contiguous expanded trinucleotide repeats present in the 3' UTR of the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of the contiguous extended trinucleotide repeats present in the 3' UTR of the DMPK1, SCA8, and/or HDL2 target transcript. . In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of the contiguous expanded trinucleotide repeats present in the intron of the target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of the contiguous expanded trinucleotide repeats present in intron 3 of the TCF4 target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of the contiguous expanded trinucleotide repeats present in the CTG18.1 locus of the TCF4 target transcript. In an embodiment, the AC comprises a nucleotide sequence that is at least partially complementary to and capable of hybridizing to at least a portion of the contiguous expanded trinucleotide repeats present in the exon of the target transcript.

In embodiments, AC is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 nucleic acids in length. In embodiments, AC is no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, or no more than 10 nucleic acids in length. In embodiments, 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, 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, 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, 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, AC is 25 to 50, 25 to 45, 25 to 40, 25 to 35, or 25 to 30 nucleic acids in length. In embodiments, AC is 30 to 50, 30 to 45, 30 to 40, or 30 to 35 nucleic acids in length. In embodiments, AC is 35 to 50, 35 to 45, or 35 to 40 nucleic acids in length. In embodiments, AC is 40 to 50 or 40 to 45 nucleic acids in length. In embodiments, AC is 45 to 50 nucleic acids in length. In embodiments, 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 is the length of

In an embodiment, the AC has 100% complementarity to the target nucleotide sequence. In embodiments, the AC does not have 100% complementarity to the target nucleotide sequence. As used herein, the term “percent complementarity” refers to the number of nucleobases of an AC (e.g., a native nucleobase or a modified nucleobase) that has nucleobase homology to the corresponding nucleobase of an oligomeric compound or nucleic acid (e.g., a target nucleotide sequence). It refers to the number of bases) divided by the total length of AC (number of nucleobases). Those skilled in the art recognize that it is possible to include mismatches without eliminating the activity of the antisense compound.

In embodiments, the AC contains no more than 20%, no more than 15%, no more than 10%, no more than 5% mismatch to the target nucleotide sequence, or no mismatch. In some embodiments, the AC includes a mismatch of at least 5%, at least 10%, or at least 15%. In embodiments, the AC comprises 0 to 5%, 0 to 10%, 0 to 15%, or 0 to 20% mismatch to the target nucleotide sequence. In embodiments, the AC comprises a mismatch of 5% to 10%, 5% to 15%, or 5% to 20% relative to the target nucleotide sequence. In embodiments, the AC comprises a mismatch of 10% to 15% or 10% to 20% relative to the target nucleotide sequence. In an embodiment, the AC comprises a mismatch of 10% to 20% relative to the target nucleotide sequence.

In embodiments, the AC has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementarity to the target nucleotide sequence. In embodiments, the AC has no more than 100%, no more than 99%, no more than 98%, no more than 97%, no more than 96%, no more than 95%, no more than 90%, no more than 85% complementarity to the target nucleotide sequence. In embodiments, AC is 80% to 100%, 80% to 99%, 80% to 98%, 80% to 97%, 80% to 96%, 80% to 95%, 80% to 80% relative to the target nucleotide sequence. It has a complementarity of 90%, or 80% to 85%. In embodiments, the AC is 85% to 100%, 85% to 99%, 85% to 98%, 85% to 97%, 85% to 96%, 85% to 95%, or 85% relative to the target nucleotide sequence. It has a complementarity of up to 90%. In embodiments, the 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. have In embodiments, the 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, the AC has 96% to 100%, 96% to 99%, 96% to 98%, or 96% to 97% complementarity to the target nucleotide sequence. In embodiments, the AC has 97% to 100%, 97% to 99%, or 97% to 98% complementarity to the target nucleotide sequence. In embodiments, the AC has 98% to 100% or 98% to 99% complementarity to the target nucleotide sequence. In embodiments, the 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 compared to unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences. A person skilled in the art can determine the appropriate number of mismatches between the AC and the target nucleotide sequence, such as by determining the thermal melting temperature (Tm). Tm or ΔTm can be calculated by techniques familiar to those skilled in the art. For example, the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allows those skilled in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes. let it be

In an embodiment, the AC comprises a nucleotide sequence that is itself a trinucleotide repeat, i.e., a CAG trinucleotide repeat. The reverse complement of 5'-CAG-3' has 100% complementarity and can hybridize with the 5'-CUG-3' trinucleotide repeat. In embodiments, AC comprises 1 to 50 CAG repeats. In an embodiment, the CAG repeats are consecutive. In an embodiment, CAG repeats are not consecutive. In an embodiment, the AC comprises a nucleotide sequence comprising an incomplete CAG repeat on the 5' or 3' end. For example, in embodiments, AC comprises 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 is 1 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, It contains a nucleotide sequence containing at least 30, or at least 50 CAG repeats. In embodiments, the AC is 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, It contains a nucleotide sequence containing no more than 3, or no more than 2 CAG repeats. In embodiments, AC is a nucleotide sequence comprising 2 to 50, 2 to 20, 2 to 10, 4 to 10, 5 to 10, 6 to 10, 6 to 9, 6 to 8, or 6 to 7 CAG repeats. Includes. In an embodiment, the AC comprises any one of the nucleotide sequences in Table 2 (SEQ ID NOs: 151-291) .

CAG repeat AC nucleotide sequence AC sequence (5’ to 3’ direction) sequence number C.A.G. N.A. CAG-CAG N.A. CAG-CAG-CAG N.A. CAG-CAG-CAG-CAG 151 CAG-CAG-CAG-CAG-CAG 152 CAG-CAG-CAG-CAG-CAG-CAG 153 CAG-CAG-CAG-CAG-CAG-CAG-CAG 154 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 155 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 156 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 157 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 158 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 159 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 160 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 161 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 162 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 163 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 164 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 165 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 166 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 167 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 168 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 169 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 170 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 171 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 172 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- C.A.G. 173 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG 174 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG 175 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG 176 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG 177 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG 178 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG 179 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 180 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 181 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 182 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 183 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 184 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 185 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 186 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 187 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 188 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 189 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 190 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 191 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 192 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 193 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 194 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 195 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 196 CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG- CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG-CAG 197 AGC N.A. AGCAGC N.A. AGCAGCAGC N.A. AGCAGCAGCAGC 198 AGCAGCAGCAGC 199 AGCAGCAGCAGCAGCAGC 200 AGCAGCAGCAGCAGCAGC 201 AGCAGCAGCAGCAGCAGCAGCAGC 202 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 203 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 204 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 205 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 206 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 207 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 208 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 209 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 210 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 211 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 212 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 213 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 214 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 215 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 216 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 217 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 218 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 219 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 220 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 221 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 222 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 223 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 224 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 225 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 226 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 227 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 228 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 229 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 230 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 231 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 232 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 233 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 234 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 235 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 236 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 237 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 238 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 239 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 240 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 241 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 242 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 243 AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC 244 GCA N.A. GCAGCA N.A. GCAGCAGCA N.A. GCAGCAGCAGCA 245 GCAGCAGCAGCAGCA 246 GCAGCAGCAGCAGCAGCA 247 GCAGCAGCAGCAGCAGCAGCA 248 GCAGCAGCAGCAGCAGCAGCAGCA 249 GCAGCAGCAGCAGCAGCAGCAGCAGCA 250 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 251 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 252 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 253 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 254 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 255 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 256 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 257 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 258 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 259 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 260 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 261 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 262 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 263 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 264 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 265 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 266 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 267 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 268 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 269 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 270 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 271 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 272 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 273 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 274 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 275 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 276 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 277 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 278 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 279 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 280 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 281 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 282 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 283 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 284 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 285 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 286 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 287 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 288 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 289 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 290 GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA 291

In embodiments, an AC with nucleotides comprising a CAG repeat may include additional nucleotide sequences at the 5' end, the 3' end, or both of the CAG repeat. In embodiments, the additional nucleotide sequences can have 80% to 100% or 95% to 100% complementarity to the portion of the target transcript to which they hybridize. Additional nucleotide sequences can be added to the CAG repeat nucleotide sequence to increase the selectivity of the AC to hybridize to a specific target transcript.

In an embodiment, the AC comprises a nucleotide sequence that is a gapmer containing 1 to 50 CAG repeats. Gapmers are oligonucleotides that are DNA/RNA hybrids and induce an RNase decay mechanism. For example, a gapmer may have a central DNA or DNA mimic segment flanked by RNA or RNA mimic segments at both the 5' and 3' ends of the DNA or DNA mimic segment. In an embodiment, the AC comprises a gapmer comprising a nucleotide sequence that hybridizes to a target nucleic acid sequence of the target transcript that is distinct from the expanded CUG repeat of the target transcript.

In an embodiment, the AC of the present disclosure is a gapmer oligonucleotide such as that disclosed in U.S. Pat. No. 9,550,988, the disclosure of which is incorporated herein by reference.

In an embodiment, the AC of the present disclosure comprises the sequence and/or structure of any one of the ACs targeting DMPK disclosed in US Patent Publication No. 2017/0260524, the disclosure of which is incorporated herein by reference.

In embodiments, the AC of the present disclosure is disclosed in US 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, including the sequence and/or structure of any of the AC or oligonucleotides disclosed in the above The contents of each patent disclosure are incorporated herein in its entirety for all purposes.

When using AC to target and/or hybridize to expanded CTG·CUG repeats, the AC may be used to detect off-target transcripts that contain CTG·CUG repeats (e.g., contain CTG·CUG repeats that are not expanded CTG·CUG repeats). Care must be taken to avoid off-target effects due to unintended binding to transcripts. In silico analysis of the human genome shows that a total of 63 human genes have CTG·CUG repeats (Uhlen et al., Science 2015 347(6220):1260419). The 63 genes can be ranked by expression of mRNA + amount of protein expressed in total muscle (cardiac, skeletal, and smooth muscle). Expression levels can be quantified using RPM (reads per million), mRNA expression can be quantified using FPKM (fragments per kilobase of transcript per million mapped fragments), and protein expression can be quantified using pTPM (fragments per kilo base of transcript per million mapped fragments). Transcripts per protein-coding gene), where 10 RPM can be used as a cutoff for non-significant expression to exclude expression above this. Figure 43 shows the results of this in silico analysis. 36 genes show expression levels >10 RPM. Among the 36 genes, only 3 genes (other than DMPK) had > 10 CTG·CUG repeats. Genes with 10 or fewer CTG·CUG repeats present the lowest risk for off-target binding and toxicity. Nevertheless, the number of CTG·CUG repeats (11–24) in these three genes (TCF4, CASK, MAP3k4) is significantly lower than that observed in patients with classical and congenital DM1. For example, patients with late-onset DM1 have 100 to 600 CTG·CUG repeats on the DMPK, patients with classic DM1 have 250 to 750 CTG·CUG repeats on the DMPK, and patients with congenital DM1 have 750 to 1,400 repeats on the DMPK. It has CTG·CUG repetitions. The same in silico analysis can be performed on liver and kidney. CASK is the only significant gene in the kidney with more than 10 CTG·CUG repeats. There were no significant genes in the liver with more than 10 CTG·CUG repeats.

The ACs described herein contain one or more asymmetric centers, thereby, in terms of absolute stereochemistry, either (R) or (S); α or β; or (D) or (L). Antisense compounds provided herein include all of these possible isomers, as well as their racemic and optically pure forms.

The efficacy of ACs can be assessed by assessing the antisense activity exerted by their administration. As used herein, the term “antisense activity” refers to any detectable and/or measurable activity resulting from 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 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.

Regulating Mechanism of AC

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

In embodiments, AC can modulate the level of a target transcript within a cell. For example, in embodiments where the AC is a gapmer, binding of the AC to the target transcript induces degradation of the target transcript via the RNase H pathway ( Figure 1 , arrows A and B). In an embodiment, the gapmer hybridizes to a portion of the target transcript that is distinct from the expanded CUG trinucleotide repeat, leading to degradation of the target transcript via the RNase H pathway ( Figure 1 , arrow A). In an embodiment, the gapmer hybridizes to at least a portion of the expanded CUG repeat in the target transcript, thereby inducing degradation of the target transcript via the RNase H pathway ( Figure 1 , arrow B).

In embodiments, AC can modulate the activity of a target transcript. Modulating activity may involve increasing or decreasing the ability of a target transcript to bind a binding partner. In embodiments, the AC modifies the activity of the target transcript by reducing the ability of the target transcript to bind one or more proteins capable of binding the transcript, particularly a protein that binds at least a portion of the expanded CUG repeat of the target transcript. can be adjusted ( Figure 1 , arrow C). In embodiments, reducing the ability of the target transcript to bind one or more proteins includes reducing the affinity of the target transcript for the one or more proteins. In embodiments, reducing the ability of a target transcript to bind one or more proteins includes partially or completely sterically blocking the target transcript from binding to one or more proteins. For example, AC may occupy at least a portion of the binding site, a portion that could be occupied by one or more proteins if they were not sterically blocked. In an embodiment, the binding site of the one or more proteins for the target transcript comprises at least a portion of an extended trinucleotide repeat of the target transcript. As such, in embodiments, the AC may occupy at least a portion of an extended trinucleotide repeat (e.g., an extended CUG repeat), which portion may be occupied by one or more proteins if they were not sterically blocked. It's part. Partial steric blocking of the target transcript may reduce the affinity between the target transcript and one or more proteins. For example, in an embodiment, the AC binds to at least a portion of the expanded CUG repeat of the target transcript, thereby sterically blocking the target transcript and/or targeting the target transcript for a protein capable of binding to the expanded CUG repeat. decreases the affinity ( Figure 1 , arrow C). Additional applications for sterically blocking antisense oligonucleotides are described in the following review paper, which is incorporated herein by reference in its entirety: Roberts et al., Nature Reviews Drug Discovery (2020) 19: 673 -694].

The CUG repeats of the expanded CUG repeats can form double-stranded hairpin structures. In a diseased state, the protein binds to the double-stranded hairpin structure and becomes sequestered, making it unable to perform other functions. In embodiments, the AC binds to at least a portion of the double-stranded hairpin structure to sterically block the double-stranded hairpin structure and/or reduce the affinity of the double-stranded hairpin structure for a protein binding partner. In an embodiment, the AC inhibits the formation of the double-stranded hairpin structure by binding to at least a portion of the single-stranded extended CUG repeat, thereby inhibiting the binding of one or more proteins to the double-stranded hairpin structure. In embodiments, hybridization of the AC to the double hairpin structure sterically blocks one or more proteins from binding to the double hairpin structure and/or reduces the affinity of the one or more proteins to bind to the double hairpin structure. In an embodiment, hybridization of AC to at least a portion of the single-stranded region of the extended trinucleotide repeat inhibits the formation of double-stranded hairpin structures.

Reducing the ability of a target transcript to bind one or more proteins prevents one or more proteins from performing other functions, such as regulating splicing of downstream transcripts (transcripts that do not contain expanded CUG repeats). It can be done. As such, in embodiments, reducing the ability of a target transcript to bind one or more proteins increases the level of one or more proteins that may serve another function within the cell or may serve a function for another transcript. You can do it. In embodiments, reducing the ability of a target transcript to bind one or more proteins may increase cytosolic levels of one or more proteins that may serve a different function within the cell or may serve a function for another transcript. there is. Accordingly, in embodiments, binding AC to a target transcript can modulate the level and/or activity of one or more proteins that interact with the target transcript.

In embodiments, hybridization of AC to at least a portion of the expanded CUG repeat reduces the affinity of MNBL1 for the target transcript or sterically 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 expanded CUG repeats of target transcripts. While bound to the target transcript, MNBL1 is sequestered in the nucleus and is unable to regulate splicing of downstream gene transcripts (transcripts that do not contain expanded CUG repeats). In embodiments, hybridization of the AC to at least a portion of the expanded CUG repeat within the target transcript sterically blocks and/or reduces the affinity of MNBL1 for the target transcript, such that the AC is capable of splicing the downstream gene transcript. Allows you to control the sound. In embodiments, hybridization of AC to at least a portion of the expanded CUG repeat within the target transcript sterically blocks and/or reduces the affinity of MNBL1 for the target transcript, thereby freeing (e.g., having the CUG repeat) Increases the amount of MNBL1 (not bound to the transcript). In embodiments, hybridization of AC to at least a portion of the expanded CUG repeat within the target transcript sterically blocks and/or reduces the affinity of MNBL1 for the target transcript, such that it binds to the target transcript and binds to the target transcript. Reduces the amount of MNBL1 sequestered by .

In embodiments where the target transcript is DMPK, hybridization of AC to at least a portion of the expanded CUG repeat reduces the affinity of MNBL1 for the target transcript and sterically 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 expanded CUG repeat of DMPK1. While bound to DMPK1, MNBL1 is sequestered in the nucleus and is unable to regulate splicing of downstream gene transcripts (transcripts that do not contain expanded CUG repeats). In embodiments, hybridization of AC to at least a portion of the expanded CUG repeat in DMPK sterically blocks and/or reduces the affinity of MNBL1 for DMPK transcripts, such that AC inhibits splicing of downstream gene transcripts. Allows you to control it. In embodiments, hybridization of AC to at least a portion of the expanded CUG repeats in DMPK sterically blocks and/or reduces the affinity of MNBL1 for DMPK transcripts, thereby releasing (e.g., transcripts with CUG repeats) increases the amount of MNBL1 (unbound). In embodiments, hybridization of AC to at least a portion of the expanded CUG repeats in DMPK sterically blocks and/or reduces the affinity of MNBL1 for DMPK transcripts, thereby binding to DMPK1 transcripts and thereby sequestering MBNL1. reduces the amount of

In embodiments where the target transcript is DMPK, hybridization of AC to at least a portion of the expanded CUG repeat reduces CUGBP1 levels. In the DM1 disease state, levels of free MBNL1 (functional MBNL1) are decreased, while levels of free CUGBP1 (functional CUGBP1) are increased. Increased CUGBP1 levels are associated with disease states. As such, in an embodiment, hybridization of AC to at least a portion of the expanded CUG repeat increases the level of free MBNL1 (functional MBNL1) and/or decreases the level of free CUGBP1 (functional CUGBP1) I order it.

Reducing the ability of a target transcript to bind to one or more proteins can reduce or inhibit the formation of CUG repeat foci. Transcripts containing extended nucleotide repeats (e.g. expanded CUG repeats) can be transcribed and then sequestered in the nucleus. Within the nucleus, sequestered transcripts can form aggregates. Proteins that bind to the transcript may then nucleate on the isolated transcript and/or sequester the transcript aggregates, forming foci of extended nucleotide repeats (e.g., CUG repeats). CUG repeat lesions can be seen using a microscope. In embodiments, reducing the ability of a target transcript to bind one or more proteins can reduce or inhibit the formation of aggregates comprising the target transcript. In embodiments, reducing the ability of the target transcript to bind one or more proteins reduces or inhibits nucleation of the one or more proteins on the target transcript, in the double-stranded hairpin region of the transcript, or on aggregates of the target transcript. can do. In embodiments where the target transcript is DMPK, reducing the ability of the DMPK1 target transcript to bind MNBL1 may reduce or inhibit MNBL1 nucleation on the DMPK1 target transcript or on DMPK1 target transcript aggregates. In embodiments, hybridizing AC to the target transcript can inhibit or reduce the formation of CUG repeat nuclear foci. In embodiments, hybridizing AC to a target transcript can inhibit or reduce the formation of CUG repeat nuclear foci from DMPK, TCF4, JPH3, and/or ATXN8OS/ATXN8 target transcripts.

In embodiments, hybridizing AC to a target transcript can modulate 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 to sterically block or reduce the affinity of the target transcript for one or more proteins, thereby binding one or more proteins sequestered by the target transcript. Proteins can regulate the expression, level, and/or activity of downstream genes. For example, in an embodiment, the one or more proteins may include proteins involved in regulating splicing of one or more downstream transcripts (transcripts that do not contain expanded CUG repeats). In embodiments, splicing of a downstream transcript is altered when a protein involved in splicing binds to and sequesters the target transcript. For example, alterations in splicing may include excluding one or more exons or including one or more introns in the transcript, which may lead to expression of different protein isoforms. In embodiments, alterations in splicing may result in the inclusion of exons and/or introns containing premature stop codons, which may form truncated isoforms that may be inactive or have deleterious activity. Alterations in downstream gene transcript splicing may lead to changes in the level, folding, and/or activity of the downstream gene product, which may be associated with the disease phenotype. When not bound to a target transcript containing expanded CUG repeats, proteins involved in splicing are free to regulate splicing, which may result in correction (or rescue) of splicing of downstream gene transcripts. Induction can at least partially restore protein levels, folding, and/or activity of downstream gene products associated with a healthy phenotype.

In embodiments, hybridization of the AC to the target transcript results in the formation of a downstream gene transcript that is regulated by the protein sequestered by the target transcript in a disease state associated with an extended nucleotide repeat (e.g., an extended trinucleotide repeat). Splicing can be controlled. In diseases associated with expanded trinucleotide repeats, downstream gene transcripts are often misprocessed and, for example, mis-spliced. Incorrect splicing of a downstream gene transcript can destroy the gene product before it is translated or translate the gene product into a protein with abnormal structure and/or function. For example, sequestering proteins that regulate the processing of downstream gene transcripts can result in inclusion of exons and/or introns with premature stop codons, inclusion of introns, exclusion of exons, and/or inclusion of alternative exons; , which can destroy transcripts and/or gene products before they are translated, or translate transcripts and/or gene products into gene products with abnormal functions. Changes in the levels of downstream gene transcripts and/or gene products and/or abnormal structure and/or function of downstream gene products are associated with an extended trinucleotide disease phenotype. In embodiments, hybridizing AC to a target transcript results in 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 disease states. It can be adjusted. As such, hybridization of AC to a target transcript may lead to upregulation of downstream protein isomers and/or transcripts associated with a healthy phenotype. Similarly, hybridization of AC to a target transcript can downregulate (e.g., suppress) downstream transcripts and/or protein isomers associated with the disease phenotype.

In embodiments where the target transcript is DMPK, hybridization of AC to the target transcript can modulate splicing of downstream gene transcripts regulated by proteins sequestered by the DMPK target transcript during disease states. In DM1, several downstream gene transcripts are mis-spliced reads. Misspliced genes are associated with disease phenotypes. As such, regulation of gene splicing may include correcting (e.g., rescuing) the splicing of a gene to generate the gene product of the downstream gene associated with a healthy phenotype. In embodiments where the target transcript is DMPK, hybridization of AC to the target transcript may modulate splicing of downstream gene transcripts regulated by MNBL1, a splicing regulator that is sequestered by the DMPK target transcript during disease states. there is. In embodiments where the target transcript is DMPK, hybridization of AC to the target transcript can modulate splicing of the downstream gene transcript regulated by CUGBP1, a protein whose activity is affected by expanded CUG repeats. In embodiments where the target transcript is DMPK, hybridization of AC to the target transcript allows correct processing (e.g., splicing) of downstream genes regulated by MNBL1 and/or CUGBP1. In embodiments where the target transcript is DMPK, hybridizing AC to the target transcript can modulate 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.

Missplicing of many of the aforementioned downstream gene transcripts results in specific DM1 disease phenotypes. For example, MNBL1 is a splicing factor that has lost its function in DM1 due to exon 5 inclusion. MNBL1 is sequestered by DMPK CUG expansion and forms RNA nuclear foci. Additionally, SOS1 positively regulates the RAS/MAPK signaling pathway by promoting Ras activation. In DM1, exon 25 of SOS1 is excluded, which leads to inhibition of the muscle hypertrophy pathway. In DM1, IR/INSR has exon 11 exclusion, which increases levels of low signaling non-muscle isoforms and reduces the metabolic response to insulin in DM1 (insulin resistance). Similarly, exon 78 exclusion of DMD is observed in DM. Exon 78 exclusion results in an out-of-frame transcript in the C-terminal domain. These mutated proteins are expressed in DM1 patients and are involved in the mechanisms responsible for their muscle wasting. BIN1 is required for proper muscle T-tube formation (EC binding). Exon 11 exclusion generates an inactive isoform and is found in DM1 patients. LDB3 interacts with α-actinin at the Z-plate within striated muscle and maintains muscle structure. Inclusion of exon 11 of LDB3 detected in DM1 reduces its affinity for protein kinase C (PKC). As a result, PKC becomes hyperactive in DM1. In embodiments, regulating one or more downstream genes corrects or rescues transcription splicing associated with a healthy phenotype. As such, in embodiments, hybridization of AC to a DMPK target transcript rescues splicing errors in the downstream gene/transcript, thereby reducing the level of the downstream gene/transcript associated with the disease phenotype. As such, in embodiments, hybridization of AC to a DMPK target transcript rescues splicing errors in the downstream gene/transcript, thereby increasing the level of the downstream gene/transcript associated with a healthy phenotype.

In an embodiment, the AC inhibits expression of the target transcript. In embodiments, the AC inhibits expression of the target transcript by blocking the pre-mRNA processing machinery and/or the translation machinery from accessing and/or completing translation and/or pre-mRNA processing. In embodiments, the AC inhibits expression of the target transcript by inducing degradation of the target transcript, e.g., via the RNase H pathway.

AC structure

AC includes oligonucleotides and/or oligonucleosides. Oligonucleotides and/or oligonucleotides are nucleotides or nucleosides linked through internucleoside linkages. Nucleosides include a pentose sugar (e.g., ribose or deoxyribose) and a nitrogen base covalently attached to the sugar. The naturally occurring bases (or traditional bases) found in DNA and/or RNA are adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The naturally occurring sugars (or traditional sugars) found in DNA and/or RNA are deoxyribose (DNA) and ribose (RNA). The naturally occurring nucleoside linkage (or traditional internucleoside linkage) is a phosphodiester linkage. In embodiments, the ACs of the present disclosure can all have natural sugars, bases, and internucleoside linkages.

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

Generally, 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 the “unmodified” or “natural” nucleobases (A, G, T, C, and U), many modified nucleobases or nucleobase mimetics are known to those skilled in the art and suitable for use with the compounds described herein. do. Generally, a modified nucleobase refers to a nucleobase that is substantially similar in structure to the parent nucleobase, such as 7-deaza purine, 5-methyl cytosine, 2-thio-dT ( Figure 2 ), or G-clamp. In general, nucleobase mimetics are nucleobases that contain a more complex structure 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, the AC may comprise one or more nucleosides with modified sugar moieties. In embodiments, the furanosyl sugar of a natural nucleoside can have 2' modifications, modifications to produce tethered nucleosides, and other modifications (see Figure 2 ). For example, in embodiments, the furanosyl sugar ring of a natural nucleoside can be modified by addition of a substituent, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA) or locked nucleic acid, oxygen of the furanosyl ring. exchanging for C or N; and/or substitution of such 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 can also be used to increase the resistance of AC to nucleases. Sugars may in particular be substituted with sugar mimetic groups. In an embodiment, one or more sugars of the nucleoside of AC are substituted with a methylenemorpholine ring, shown as 19 in Figure 2 .

In an embodiment, the AC comprises one or more nucleosides comprising a bicyclic modified sugar (BNA; also referred to as a bridged nucleic acid). Examples of BNAs include 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' cross-linked BNA, 4' -(CH ₂ CH(CH ₃ )-2' cross-linked BNA" cEt (4'-(CH(CH ₃ )-O-2' bridge), and cMOE BNA (4'-(CH(CH ₂ OCH ₃ ) -O-2' bridge) BNA has been prepared and described in the scientific literature as well as in the patent literature, such as: Srivastava et al., J. Am. Chem. Soc. (2007), ACS Advanced online publication, 10.1021/ja071106y; Albaek et al., J. Org. Chem. (2006), 71, 7731-7740; Fluiter et al., Chembiochem (2005), 6, 1104-1109; Singh Chem. Commun. (1998), 4, 455-456; Koshkin et al. (Tetrahedron (1998), 54, 3607-3630); Wahlestedt et al. (Proc. Natl. Acad. Sci. USA) (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; US Patent No. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and 6,525,191; US Application Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; No. 2004-0143114; and No. 20030082807.

In an embodiment, the AC comprises one or more nucleosides comprising a locked nucleic acid (LNA). In LNA, the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring to form a 2'-C,4'-C-oxymethylene linkage, forming a bicyclic sugar moiety (Elayadi Curr. (2001), 3, 239-243; see also US Pat. Nos. 6,268,490 and 6,670,461). The linkage may be a methylene (-CH ₂ -) group bridging the 2' oxygen atom and the 4' carbon atom, where the term LNA is used for the bicyclic moiety; If there is an ethylene group at this position, the term ENA™ is used (Singh et al., Chem. Commun. (1998), 4, 455-456; ENA™; Morita et al., Bioorganic Medicinal Chemistry (2003), 11, 2211-2226]). LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability (Tm = +3 to +10° C.) with complementary DNA and RNA, stability against 3'-exonucleolytic degradation, and good dissolution properties. Potent and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA (2000), 97, 5633-5638).

The isomer of LNA also studied is alpha-L-LNA, which has been shown to have excellent stability against 3'-exonucleases. Alpha-L-LNA was incorporated into antisense gapmers and chimeras that showed strong antisense activity (Frieden et al., Nucleic Acids Research (2003), 21, 6365-6372).

The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, along with their oligomerization and nucleic acid recognition properties, have been described by 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. [Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222]). The preparation of LNA analogues containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Additionally, the synthesis of 2'-amino-LNA, a conformationally restricted high-affinity oligonucleotide analog, has been described (Singh et al., J. Org. Chem. (1998), 63, 10035-10039). Additionally, 2'-amino- and 2'-methylamino-LNAs have been prepared, and the thermal stability of their duplexes 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 teaching the preparation of these modified sugars include U.S. Pat. No. 4,981,957; No. 5,118,800; No. 5,319,080; No. 5,359,044; No. 5,393,878; No. 5,446,137; No. 5,466,786; No. 5,514,785; No. 5,519,134; No. 5,567,811; No. 5,576,427; No. 5,591,722; No. 5,597,909; No. 5,610,300; No. 5,627,053; No. 5,639,873; No. 5,646,265; No. 5,658,873; No. 5,670,633; No. 5,792,747; No. 5,700,920; and 6,600,032; and WO 2005/121371.

Internucleoside linkages

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

In naturally occurring DNA and RNA, internucleoside linkers are phosphodiesters that covalently link adjacent nucleosides to each other to form linear polymer compounds. In naturally occurring DNA and RNA, the phosphodiester is linked to the 2', 3', or 5' hydroxyl moiety of the sugar. Within oligonucleotides, the phosphate group is generally 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 a phosphodiester linkage in the 3' to 5' direction. In an embodiment, the internucleoside linker of AC is a phosphodiester. In an embodiment, the internucleoside linkage of AC is a phosphodiester linkage in the 3' to 5' direction.

The two main classes of non-natural internucleoside linkers are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Representative non-phosphorus containing internucleoside linkages include methylenemethylimino (-CH ₂ -N(CH ₃ )-O-CH ₂ -), thiodiester (-OC(O)-S-), ti. Onocarbamate (-OC(O)(NH)-S-); Including, but not limited to, siloxanes (-O-Si(H ₂ -O-); and N,N'-dimethylhydrazine (-CH ₂ -N(CH ₃ )-N(CH ₃ )-). ACs with internucleoside linkages are referred to as oligonucleotides. Antisense compounds with non-phosphorus internucleoside linkages are referred to as oligonucleosides. Compared to natural phosphodiester linkages, modified nucleosides Interseed linkages can be used to modify and typically increase the nuclease resistance of antisense compounds.Internucleoside linkages with chiral atoms can be prepared as racemic, chiral, or mixtures. Representative chiral nucleosides Intercleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for making phosphorus containing and non-phosphorus containing linkages are well known to those skilled in the art.

In embodiments, two or more nucleosides having modified sugars and/or modified nucleobases can be linked using phosphoramidates. In an embodiment, two or more nucleosides having a methylenemorpholine ring can be linked via a phosphoramidate internucleoside linkage.

Antisense compounds comprising a nucleobase with methylenemorpholine rings linked through phosphoramidate internucleoside linkages may be referred to as phosphoramidate morpholino oligomers (PMOs).

Conjugate Groups

In embodiments, the AC is modified by covalent attachment of one or more adapters. Typically, the conjugator modifies one or more properties of the attached AC, including but not limited to pharmacodynamics, pharmacokinetics, binding, uptake, cellular distribution, cellular uptake, charge, and clearance. Conjugators are routinely used in the field of chemistry, linking either directly to the parent compound, such as AC, or through an optional linking moiety or linker. Adapters include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, Includes, but is not limited to, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In an embodiment, the conjugator is polyethylene glycol (PEG), and PEG is conjugated to AC or CPP (CPP discussed elsewhere herein).

In embodiments, the adapter is a lipid moiety, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA (1989), 86, 6553); Cholic acid (Manoharan et al., Bioorg. 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., Bioorg. Med. Chem. Let. ( 1993), 3, 2765]); thiocholesterol (Oberhauser et al., Nucl. Acids Res. (1992), 20, 533); Aliphatic chains, such as dodecanediol or undecyl moieties (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-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. (1995), 36, 3651; Shea et al. (Nucl. Acids Res. (1990), 18, 3777); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides (1995), 14, 969); Adamantane acetic acid (Manoharan et al., Tetrahedron Lett. (1995), 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta. (1995), 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. (1996), 277,923).

Types of Antisense Compounds

Various types of ACs can be used, including for example antisense oligonucleotides, siRNAs, microRNAs, entagomers, aptamers, ribozymes, supermers, miRNA mimics, miRNA inhibitors, or combinations thereof.

Antisense oligonucleotide

In various embodiments, the antisense compound (AC) is an antisense oligonucleotide (ASO) complementary to the target nucleotide sequence. The term “antisense oligonucleotide (ASO)” or simply “antisense” refers to comprising an oligonucleotide that is complementary to a target nucleotide sequence. The term also includes ASOs that may not be completely complementary to the desired target nucleotide sequence. ASOs contain a single strand of DNA and/or RNA that is complementary to a selected target nucleotide sequence or target gene. ASOs may comprise one or more modified DNA and/or RNA bases, modified sugars, and/or non-natural internucleoside linkages. In embodiments, the ASO may comprise one or more phosphoramidate internucleoside linkages. In an embodiment, the ASO is phosphoramidate morpholino oligomer (PMO). ASOs can have any properties, can be any length, can bind any target nucleotide sequence and/or sequence element, and can exert any mechanism as described for AC.

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

Methods for producing antisense oligonucleotides are known in the art and can be readily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. The selection of an antisense oligonucleotide sequence 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 may be selected based on their relative ability to form dimers, hairpins, or other secondary structures that reduce or inhibit specific binding to the target mRNA in the host cell. The target region of the mRNA includes a region at or near the AUG translation initiation codon, and a sequence substantially complementary to the 5' region of the mRNA. Considerations for such secondary structure analysis and target site selection can be considered, for example, in OLIGO primer analysis software v.4 (Molecular Biology Insights) and/or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402]).

RNA interference

In an embodiment, the AC comprises a molecule that mediates RNA interference (RNAi). As used herein, the phrase “mediating RNAi” refers to the ability to silence a target transcript in a sequence-specific manner. Without wishing to be bound by theory, silencing is believed to use the RNAi machinery or process and a guide RNA, for example, an siRNA compound of about 21 to about 23 nucleotides. In embodiments, the AC targets a target transcript for degradation. As such, in embodiments, RNAi molecules can be used to disrupt the expression of a gene or polynucleotide of interest. In embodiments, RNAi molecules are used to induce degradation of target transcripts, such as pre-mRNA or mature mRNA.

In an embodiment, the AC comprises small interfering RNA (siRNA) that induces an RNAi response.

Small interfering RNAs (siRNAs) are nucleic acid duplexes, typically about 16 to about 30 nucleotides long, that can associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Because siRNA-loaded RISCs mediate degradation of homologous transcripts, siRNAs can be designed to knockdown protein expression with high specificity. Unlike other antisense technologies, siRNA functions through natural mechanisms that have evolved to control gene expression through non-coding RNA. A variety of RNAi reagents, including siRNAs targeting clinically relevant targets, are currently in pharmaceutical development, as described, for example, in de Fougerolles, A. et al., Nature Reviews (2007) 6:443-453. It's in progress.

The first described RNAi molecules were RNA:RNA hybrids containing both RNA sense and RNA antisense strands, but now DNA sense:RNA antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrids mediate RNAi. It has been proven that it can be done (Lamberton, J.S. and Christian, A.T. [Molecular Biotechnology (2003), 24:111-119]). In embodiments, RNAi molecules comprising any of these different types of double-stranded molecules are used. It will also be appreciated that RNAi molecules can be used and introduced into cells in a variety of forms. Accordingly, as used herein, RNAi molecule includes any and all molecules capable of mediating RNAi in a cell, including double-stranded oligos comprising two separate strands (sense strand and antisense strand) Nucleotides, such as small interfering RNA (siRNA); A double-stranded oligonucleotide comprising two separate strands joined together by a non-nucleotidyl linker; Expressing oligonucleotides comprising hairpin loops of complementary sequence, e.g., shRNAi molecules, forming a double-stranded region, and one or more polynucleotides capable of forming double-stranded polynucleotides alone or together with other polynucleotides. Includes, but is not limited to, expression vectors.

As used herein, a “single-stranded siRNA compound” is an siRNA compound consisting of a single molecule. It may comprise a duplexed region formed by intra-strand pairing, for example it may be or include a hairpin or fan-handle structure. Single-stranded siRNA compounds may be antisense to the target molecule.

Single-stranded siRNA compounds can be long enough to 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 up to 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 may have a duplex region of at least about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotide pairs or an equivalent length. The duplex region may be no more than about 200, about 100, or about 50 nucleotide pairs in length. In certain embodiments, the range for the duplex region is about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21 nucleotide pairs in length. Hairpins may have single-stranded overhangs or terminal unpaired regions. In certain embodiments, the overhang is about 2 to about 3 nucleotides long. In embodiments, the overhang is on the same side as the hairpin, and in embodiments, it is on the antisense side of the hairpin.

As used herein, a “double-stranded siRNA compound” is an siRNA compound comprising two or more, and in some cases two, chains in which interchain hybridization can form a region of duplex structure.

The antisense strand of a double-stranded siRNA compound may be 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 long. It may be less than about 200, about 100, or about 50 nucleotides in length. Ranges can be from about 17 to about 25, about 19 to about 23, and about 19 to about 21 nucleotides in length. As used herein, the term “antisense strand” refers to a strand of an siRNA compound that is sufficiently complementary to the target nucleotide sequence of a target molecule, e.g., a target transcript.

The sense strand of a double-stranded siRNA compound may be 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 long. It may be up to about 200, about 100, or about 50 nucleotides in length. Ranges can be from about 17 to about 25, about 19 to about 23, and about 19 to about 21 nucleotides in length.

The double-stranded portion of a double-stranded siRNA compound has 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 It may be 40, or about 60 nucleotide pairs in length. It may be no more than about 200, about 100, or about 50 nucleotide pairs in length. Ranges can be about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21 nucleotide pairs in length.

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

The sense and antisense strands can be selected so that the double-stranded siRNA compound contains single-stranded regions or unpaired regions at one or both ends of the molecule. Accordingly, a double-stranded siRNA compound may contain sense and antisense strands paired to contain overhangs, e.g., one or two 5' or 3' overhangs, or a 3' overhang of one to three nucleotides. You can. An overhang may be caused by one strand being longer than the other, or by two strands of the same length crossing each other. Some embodiments will have at least one 3' overhang. In an embodiment, both ends of the siRNA molecule will have a 3' overhang. In an embodiment, the overhang is 2 nucleotides.

In embodiments, the length of the duplex region is from about 15 to about 30, or about 18, about 19, about 20, about 21, e.g., in the range of ssiRNA (siRNA with sticky overhangs) compounds described above. It is about 22, or about 23 nucleotides long. ssiRNA compounds may be similar in length and structure to natural Dicer-processed products from long dsiRNAs. Embodiments in which the two strands of the ssiRNA compound are linked, for example covalently linked, are also included. In embodiments, hairpins, or other single stranded structures that provide double stranded regions and 3' overhangs 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., transcript of a gene encoding a protein. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. These genes are also referred to as target genes. Typically, 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 production of the 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 production of the genetic product encoded by the target transcript. In other embodiments, the siRNA compound is a target nucleotide sequence (e.g., a target nucleotide sequence) such that the target nucleotide sequence and the siRNA compound anneal to form a hybrid made only of Watson-Crick base pairs, e.g., in regions of exact complementarity. It is “exactly complementary” to the part of the corpse). “Sufficiently complementary” to a target nucleotide sequence may include an internal region (e.g., at least about 10 nucleotides) that is exactly complementary to the target nucleotide sequence. Additionally, in certain embodiments, siRNA compounds specifically distinguish single nucleotide differences. In this case, the siRNA compound mediates RNAi only if exact complementarity is found in the region of a single nucleotide difference (e.g., within 7 nucleotides).

The therapeutic applications of RNAi are very broad, because siRNA and miRNA constructs can be synthesized with arbitrary nucleotide sequences directed against the target protein. To date, siRNA constructs have shown the ability to specifically downregulate target proteins in both in vitro and in vivo models as well as in clinical studies.

microRNA

In an embodiment, the AC comprises a microRNA molecule. MicroRNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals but are not translated into proteins. Engineered miRNAs are single-stranded RNA molecules of 17-25 nucleotides that are incorporated into the RNA-induced silencing complex (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis, and differentiation. They are believed to play a role in regulating gene expression by binding to the 3'-untranslated region of specific mRNAs. RISC mediates downregulation of gene expression through translational repression, transcript cleavage, or both. RISC is also involved in transcriptional silencing in a wide range of eukaryotic nuclei.

Antagomers

In an embodiment, AC is an antagomer. Antagomers are RNA-like oligonucleotides that possess various modifications for RNAse protection and pharmacological properties, such as enhancing tissue and cellular uptake. They differ from normal RNA by, for example, the sugar, the phosphorothioate backbone, and the complete 2'-0-methylation of the cholesterol moiety, for example, at the 3'-end. Antagomers can be used to efficiently silence endogenous miRNAs by forming a duplex containing an antagomer and an endogenous miRNA to prevent miRNA-induced gene silencing. An example of antagomer-mediated miRNA silencing is the silencing of miR-122 described in Krutzfeldt et al. (Krutzfeldt et al., Nature (2005), 438: 685-, expressly incorporated herein by reference in its entirety). 689]). Antagomeric RNA can be synthesized using standard solid-phase oligonucleotide synthesis protocols (U.S. Patent Application Nos. 11/502,158 and 11/657,341; the disclosures of each of these are incorporated herein by reference).

Antagomers can include monomers and ligand-conjugated monomeric subunits for oligonucleotide synthesis. The monomer is described in US patent application Ser. No. 10/916,185. Antagomers may have the ZXY structure as described in PCT Application No. PCT/US2004/07070. Antagomers can form complexes with amphipathic moieties. Amphipathic moieties for use with oligonucleotide preparations are described in PCT Application No. PCT/US2004/07070.

Aptamers

In an embodiment, the AC comprises an aptamer. Aptamers are nucleic acid or peptide molecules that bind to a specific molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). DNA or RNA aptamers that bind to many different entities, from large proteins to small organic molecules, have been successfully produced (Eaton, Curr. Opin. Chem. Biol. (1997), 1: 10-16; Famulok, [Curr. Opin. Struct. Biol. (1999), 9:324-9]; and Hermann and Patel, Science (2000), 287:820-5. Aptamers may be of the RNA or DNA family and may contain riboswitches. A riboswitch is a part of an mRNA molecule that can bind directly to a small target molecule, and whose target binding affects the activity of the gene. Therefore, mRNA containing a riboswitch is directly involved in regulating its activity depending on the presence or absence of its target molecule. Typically, aptamers are engineered through repeated rounds of in vitro selection to bind to a variety of molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organs, or, equivalently, by SELEX (systemic sequencing of ligands by exponential enrichment). is manipulated through evolution. Aptamers can be prepared by any known method, including synthetic, recombinant, and purification methods, and can be used alone or in combination with other aptamers specific for the same target. The term “aptamer” also includes “secondary aptamers” containing a consensus sequence derived by comparing two or more known aptamers for a given target. In embodiments, the aptamer is an “intracellular aptamer” or “intramer” that 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 incorporated herein by reference).

Ribozyme

In an embodiment, AC is a ribozyme. Ribozymes are complexes of RNA molecules with specific catalytic domains with endonuclease activity (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, many ribozymes accelerate phosphoester transfer reactions with high specificity, often cleaving only one of several phosphoesters 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 was due to the requirement that the substrate bind to the internal guide sequence (IGS) of the ribozyme through specific base pairing interactions prior to the chemical reaction.

At least six basic variants of naturally occurring enzyme RNA are currently known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus cleave other RNA molecules) under physiological conditions. Generally, enzyme nucleic acids act by first binding to target RNA. This binding occurs through the target binding portion of the enzymatic nucleic acid being held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Therefore, the enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA through complementary base pairing, and after binding to the correct site, acts enzymatically to cleave the target RNA. This strategic cleavage of the target RNA will destroy its ability to induce synthesis of the encoded protein. After the enzyme nucleic acid binds to and cleaves the RNA target, it is released from the RNA and can find another target and repeatedly bind to and cleave new targets.

The enzymatic nucleic acid molecule may be formed of a hammerhead, hairpin, hepatitis δ virus, group I intron, or RNaseP RNA (associated with an RNA guide sequence), or, for example, a Neurospora VS RNA motif. Specific examples of hammerhead motifs include Rossi et al., Nucleic Acids Res. (1992), 20(17):4559-65. Examples of hairpin motifs include European Patent Application Publication No. EP 0360257 by Hampel et al., Hampel and Tritz, Biochemistry (1989), 28(12):4929-33; Hampel et al. [Nucleic Acids Res. (1990), 18(2):299-304] and U.S. Patent No. 5,631,359. Examples of hepatitis virus motifs are described in Perrotta and Been (Biochemistry (1992), 31(47): 11843-52; Examples of RNaseP motifs are described in Guerrier-Takada et al., Cell (1983), 35(3 Pt 2):849-57; The Neurospora VS RNA ribozyme motif was 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); Examples of group I introns are described in U.S. Pat. No. 4,987,071. In an embodiment, the enzymatic nucleic acid molecule has a specific substrate binding site complementary to one or more regions of the target gene DNA or RNA, and has a nucleotide sequence within or surrounding the substrate site, which nucleotide sequence has RNA cleavage activity. is given to the molecule. Accordingly, ribozyme constructs 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 of which is specifically incorporated herein by reference) and can be grown in vitro as described herein. It can be synthesized for testing in vitro and in vivo. In an embodiment, the ribozyme is targeted to the target nucleotide sequence of the target transcript.

Ribozyme activity can be achieved by altering the length of the ribozyme binding arm, by chemically synthesizing the ribozyme with modifications that prevent degradation by serum ribonucleases (for example, by sugar modification of the enzyme RNA molecule). 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 No. 5,334,711, and International Patent Application Publication No. 92110298.4, which describe chemical modifications. See Patent Application Publication No. WO 94/13688), modifications to improve their efficacy in cells, and removal of stem Π bases to shorten RNA synthesis time and reduce chemical requirements.

Supermir

In an embodiment, AC is a supermer. Supermer refers to a single-stranded, double-stranded, or partially double-stranded oligomer, or a polymer of RNA, a polymer of DNA, or both, or a modification thereof, wherein the supermer is substantially identical to a miRNA and is antisense to its target. It has a nucleotide sequence. The term includes oligonucleotides consisting of naturally occurring nucleobases, sugars, and shared internucleoside (backbone) linkages and containing at least one non-naturally occurring moiety that functions similarly. These modified or substituted oligonucleotides have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases. In embodiments, the supermer does not include a sense strand, and in other embodiments, the supermer does not self-hybridize to a significant degree. Supermers may have secondary structures, but are substantially single stranded under physiological conditions. A substantially single-stranded supermer is such that less than about 50% (e.g., less than about 40%, about 30%, about 20%, about 10%, or about 5%) of the supermer is duplexed with itself. It is a single strand. A supermer is a hairpin segment, e.g., a sequence at the 3' end that self-hybridizes and has a duplex region, e.g., at least about 1, about 2, about 3, or about 4, or about 8, about 7, or about 6. , or less than about 5 nucleotides, or may form a duplex region of about 5 nucleotides. The duplex regions may be connected by a linker, for example a nucleotide linker, for example about 3, about 4, about 5, or about 6 dT, for example a modified dT. In another embodiment, the supermer is a shorter oligo, e.g., about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides long, e.g., 3' and 5 nucleotides of the supermer. ' Duplexed at one or both of the ends, or at one end and at the non-terminus or middle of the supermer.

miRNA mimic

In an embodiment, AC is a miRNA mimetic. MiRNA mimics represent a class of molecules that can be used to mimic the gene silencing ability of one or more miRNAs. Accordingly, the term “microRNA mimetic” refers to synthetic non-coding RNA (i.e., a miRNA not obtained by purification from a source of endogenous miRNA) that can enter the RNAi pathway and regulate gene expression. MiRNA mimics can be designed as mature molecules (e.g., single-stranded) or mimic precursors (e.g., pri- or pre-miRNA). A miRNA mimetic may include a nucleic acid (modified or modified nucleic acid) containing oligonucleotides, including but not limited to: RNA, modified RNA, DNA, modified DNA, locked nucleic acid, or 2'- 0,4'-C-ethylene-crosslinked nucleic acid (ENA), or any combination thereof (including DNA-RNA hybrids). Additionally, miRNA mimetics can include conjugates that can affect delivery, intracellular compartmentalization, stability, specificity, functionality, strand usage, and/or efficacy. In one design, a miRNA mimetic is a double-stranded molecule (e.g., having a duplex region of about 16 to about 31 nucleotides in length) and contains one or more sequences that have identity to the mature strand of a given miRNA. Modifications include 2' modifications to one or both strands of the molecule (including 2'-0 methyl modifications and 2' F modifications) and internucleoside modifications that improve nucleic acid stability and/or specificity (e.g. phosphorothioate modification). Additionally, the miRNA mimic may contain overhangs. The overhang may comprise from about 1 to about 6 nucleotides on the 3' or 5' end of either strand and may be modified to improve stability or functionality. In an embodiment, the miRNA mimetic comprises a duplex region of about 16 to about 31 nucleotides and one or more of the following chemical modification patterns: nucleotides 1 and 2, the sense strand (counted from the 5' end of the sense oligonucleotide) ) and 2'-0-methyl modifications of both C and U; Antisense strand modifications may include 2' F modifications of both C and U, phosphorylation of the 5' end of the oligonucleotide, and a stabilized internucleoside linkage associated with a 2 nucleotide 3' overhang.

miRNA inhibitors

In an embodiment, 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 specific miRNA. Typically, the inhibitor is a nucleic acid or modified nucleic acid in nature, including an oligonucleotide comprising RNA, modified RNA, DNA, modified DNA, locked nucleic acid (LNA), or any combination thereof.

Modifications can include 2' modifications (including 2'-0 alkyl modifications and 2' F modifications) and internucleoside modifications (e.g., phosphoro- thioate modification). Additionally, miRNA inhibitors may include conjugates that may affect delivery, intracellular compartmentalization, stability, and/or efficacy. Inhibitors can adopt a variety of configurations, including single-stranded, double-stranded (RNA/RNA or RNA/DNA duplex), and hairpin designs, and generally microRNA inhibitors combine the mature strands (strands) of the miRNA to be targeted. Contains one or more complementary or partially complementary sequences or portions of sequences. Additionally, the miRNA inhibitor may contain additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA. The additional sequence may be the reverse complement of a sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequence may be any sequence (with a mixture of A, G, C, or U). In an embodiment, one or both of the additional sequences are any sequence capable of forming a hairpin. Accordingly, in an embodiment, the sequence that is the reverse complement of the miRNA has hairpin structures located on the 5' and 3' sides. When a micro-RNA inhibitor is double stranded, it may contain mismatches between nucleotides on opposite strands. Additionally, micro-RNA inhibitors can be linked to a conjugate moiety to facilitate uptake of the inhibitor into the cell. For example, micro-RNA inhibitors include cholesteryl 5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate, which can passively uptake micro-RNA inhibitors into cells. ) can be connected to. Micro-RNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al. (RNA 13: 723-730 (2007)) and in WO2007/095387 and WO 2008/036825 (each of which is referenced in its entirety). (incorporated herein). One skilled in the art can select a sequence for a desired miRNA from a database and design inhibitors useful in the methods disclosed herein.

Linking groups or bifunctional linking moieties, such as those known in the art, may be suitable for the compounds provided herein. Linkers are useful for attaching chemical functional groups, adapter groups, reporter groups, and other groups to selective sites of the parent compound, such as AC. Typically, bifunctional linking moieties include a hydrocarbyl moiety with two functional groups. One of the functional groups is selected to bind to the parent molecule or compound of interest, and the other is selected to bind essentially any selected functional group, such as a chemical functional group or conjugation group. Any of the linkers described herein may be used. In embodiments, the linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups routinely used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In embodiments, the bifunctional linking moiety includes amino, hydroxyl, carboxylic acid, thiol, unsaturated (e.g., double or triple bond), etc. Some non-limiting examples of bifunctional binding moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) , and 6-aminohexanoic 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 includes hydroxyl, Includes amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In an embodiment, the AC comprises nucleotide modifications designed not to support RNase H activity. Nucleotide modifications of antisense compounds that do not support RNase H activity are known and include, but are not limited to, the 2'-O-methoxy ethyl/phosphorothioate (MOE) modification. Advantageously, ACs with MOE modifications have increased affinity for target RNA and increase nuclease stability.

Immunostimulatory oligonucleotides

In an embodiment, the therapeutic moiety is an immunostimulatory oligonucleotide. Immunostimulatory oligonucleotides (ISS; single or double stranded) can induce an immune response when administered to a patient, which may be a mammal or other patient. ISSs can, for example, contain specific palindromes leading to hairpin secondary structures (Yamamoto S. et al. (1992) J. Immunol. 148: 4072-4076), or CpG motifs as well as other known ISS features (e.g. multi-G domain, see WO 96/11266).

The immune response may be an innate or adaptive immune response. The immune system is divided into the more innate immune system and the adaptive immune system of vertebrates, the latter of which is further divided into humoral cellular components. In certain embodiments, the immune response may be mucosal.

Immunostimulatory nucleic acids are considered non-sequence specific if these nucleic acids do not need to specifically bind to and reduce the expression of the target polynucleotide to elicit an immune response. Accordingly, although certain immunostimulatory nucleic acids may contain sequences that correspond to regions of naturally occurring genes or mRNAs, they may still be considered non-sequence specific immunostimulatory nucleic acids.

In an embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. Oligonucleotides or CpG dinucleotides may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide with a methylated cytosine. In an embodiment, the nucleic acid comprises a single CpG dinucleotide, wherein the cytosine within the CpG dinucleotide is methylated. In certain embodiments, the nucleic acid comprises sequence 5' TAACGTTGAGGG'CAT 3' (SEQ ID NO: 369). In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides, wherein at least one cytosine within the CpG dinucleotide is methylated. In a further embodiment, each cytosine within 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.

Additional specific nucleic acid sequences of 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, the ODN used in the compositions and methods have a phosphodiester (“PO”) Xbone or a phosphorothioate (“PS”) backbone, and/or at least one methylated cytosine residue within a CpG motif. has

decoy oligonucleotide

In an embodiment, the therapeutic moiety is a decoy oligonucleotide. Because transcription factors recognize their relatively short binding sequences even in the absence of surrounding genomic DNA, short oligonucleotides with consensus binding sequences of specific transcription factors can be used as tools for manipulating gene expression in living cells. This strategy involves intracellular delivery of such “decoy oligonucleotides,” which are recognized and bound by targeting factors. After the transcription factor occupies the DNA binding site by attraction, the transcription factor is unable to bind to the promoter region of the target gene. Attractants can be used as a therapeutic agent to suppress the expression of genes activated by transcription factors or to upregulate genes suppressed by binding of transcription factors. An example of the use of decoy oligonucleotides is 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 suppresses a polyA site, a targeting domain complementary to a site within the terminal exon of the target gene; and a 'U1 domain' that binds to the U1 smaller nuclear RNA component of the U1 snRNP (Goraczniak et al., 2008, Nature Biotechnology, 27(3), 257-263, by reference in its entirety. expressly incorporated herein). U1 snRNP is a ribonucleoprotein complex that acts primarily to induce the initial steps of spliceosome formation by binding to the pre-mRNA exon-intron boundary (Brown and Simpson, 1998, Annu Rev Plant Physiol Plant Mol Biol 49:77 -95]). Nucleotides 2 to 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 adapter. In one embodiment, the Ul adapter can be administered in combination with at least one other iRNA agent.

(CRISPR) gene editing machine

In an embodiment, the compounds disclosed herein comprise one or more CPPs (or cCPPs) conjugated to the CRISPR gene editing machine. As used herein, “CRISPR gene editing machine” refers to a protein, nucleic acid, or combination thereof that can be used to edit a genome. Non-limiting examples of gene editing machinery include gRNA, nucleases, nuclease inhibitors, and combinations and complexes thereof. The following patent documents describe CRISPR gene editing machines: U.S. Patent No. 8,697,359, U.S. Patent No. 8,771,945, U.S. Patent No. 8,795,965, U.S. Patent No. 8,865,406, U.S. Patent No. 8,871,445, U.S. Patent No. 8,889,356, U.S. Patent No. Patent Nos. 8,895,308, US Patent 8,906,616, US Patent 8,932,814, US Patent 8,945,839, US Patent 8,993,233, US Patent 8,999,641, US Patent 14/704,551, and US Patent 13/842,859. like. Each of the foregoing patent documents is incorporated herein by reference in its entirety.

In an embodiment, the linker conjugates cCPP to the CRISPR gene editing machine. Any linker described in this disclosure or known to those skilled in the art may be used.

gRNA

In an embodiment, the compound comprises CPP (or cCPP) conjugated to a gRNA. gRNA targets genomic loci in prokaryotic or eukaryotic cells.

In an embodiment, the gRNA is a single molecule guide RNA (sgRNA). sgRNA includes a spacer sequence and a scaffold sequence. A spacer sequence is a short nucleic acid sequence used to target a nuclease (e.g., Cas9 nuclease) to a specific nucleotide region of interest (e.g., the genomic DNA sequence to be cleaved). In embodiments, the spacer can be about 17-24 bases in length, such as about 20 bases in length. In embodiments, the spacer is 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 may be about 30 bases long. In embodiments, the spacer is 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 may be at least 30 bases in length. In embodiments, the spacer is 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 may be about 30 bases long. In embodiments, the spacer sequence has a GC content of about 40% to about 80%.

In an embodiment, the spacer targets the region immediately preceding the 5' protospacer adjacent motif (PAM). PAM sequences can be selected based on the desired nuclease. For example, the PAM sequence can be any of the PAM sequences shown in Table 3 below, where N refers to any nucleic acid, R refers to A or G, Y refers to C or T, and W refers to A or T, and V refers to A or C or G.

Exemplary nuclease and PAM sequences PAM sequence (5’ to 3’ direction) nuclease Isolated from NGG SpCas9 Streptococcus pyogenes NGRRT or NGRRN SaCas9 Staphylococcus aureus NNNNGATT NmeCas9 Meningococci NNNNRYAC CjCas9 Campylobacter jejuni NNAGAAW StCas9 Streptococcus thermophilus TTTV LbCpf1 Lachnospiraceae bacterium TTTV AsCpf1 Acidaminococcus spp.

In embodiments, the spacer may target the sequence of a mammalian gene, such as a human gene. In embodiments, the spacer can target a mutant gene. In embodiments, a spacer can target a coding sequence. In embodiments, the spacer can target an exon sequence. In embodiments, the spacer can target a polyadenylation site (PS). In embodiments, the spacer can target a sequence element of PS. In embodiments, the spacer may target a polyadenylation signal (PAS), an intervening sequence (IS), a cleavage site (CS), a downstream element (DES), or a portion or combination thereof. In embodiments, the spacer may target a splicing element (SE) or cis-splicing regulatory element (SRE).

The scaffold sequence is the sequence within the sgRNA that is responsible for nuclease (e.g. Cas9) binding. The scaffold sequence does not include a spacer/targeting sequence. In embodiments, the scaffold has 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 It may be 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 length. In embodiments, the scaffold has 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 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 60, about 61, about 62, about 63, 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 It may be 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 length. In embodiments, the scaffolds have 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. , may be at least 120 nucleotides, or at least 125 nucleotides in length.

In an embodiment, the gRNA is a dual molecular guide RNA, such as crRNA and tracrRNA. In embodiments, the gRNA may further comprise a poly(A) tail.

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

nuclease

In an embodiment, the compound comprises a cell penetrating peptide conjugated to a nuclease. In an embodiment, the nuclease is a type II, type V-A, type V-B, type VC, type V-U, or 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 Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. For example, in some embodiments, the nuclease is Cas9 nuclease or Cpf1 nuclease.

In an embodiment, the nuclease is a modified form or variant of a Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. In embodiments, the nuclease is a modified form or variant of a TAL nuclease, meganuclease, or zinc-finger nuclease. “Modified” or “variant” nucleases are, for example, truncated, fused to another protein (e.g., another nuclease), catalytically inactivated, etc. In an embodiment, the nuclease is a naturally occurring Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, Cas14 nuclease, or TALEN, meganuclease, or zinc-finger. It 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 with the nuclease. In an embodiment, the nuclease is Cas9 nuclease (SpCas9) from Streptococcus pyogenes ( S. pyogenes ). In embodiments, the nuclease is 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% different from the Cas9 nuclease from Streptococcus pyogenes (SpCas9). % sequence identity. In an embodiment, the nuclease is Cas9 (SaCas9) from Staphylococcus aureus ( S. aureus ). In embodiments, the nuclease has a sequence that is 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% identical to Cas9 (SaCas9) from Staphylococcus aureus. have sameness. In an embodiment, Cpf1 is the Cpf1 enzyme from Acidaminococcus (species BV3L6, UniProt accession number U2UMQ6). In an embodiment, the nuclease is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% identical to the Cpf1 enzyme from Asidaminococcus (strain BV3L6, UniProt Accession No. U2UMQ6). , or has a sequence identity of at least about 99%.

In an embodiment, Cpf1 is the Cpf1 enzyme from Lachnospiraceae (species ND2006, UniProt accession number A0A182DWE3). In embodiments, the nuclease has 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 Lachnospiraceae. has In an embodiment, the sequence encoding the nuclease is codon optimized for expression in mammalian cells. In an embodiment, the sequence encoding the nuclease is codon optimized for expression in human cells or mouse cells.

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

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

gRNA and nuclease combination

In an embodiment, the compound comprises one or more CPPs (or cCPPs) conjugated to a gRNA and a nuclease. In an embodiment, one or more CPPs (or cCPPs) are conjugated to a nucleic acid encoding a gRNA and/or nuclease. In an embodiment, 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 an embodiment, the nucleic acid encoding the nuclease and the gRNA comprises two promoters, wherein the first promoter regulates expression of the nuclease and the second promoter regulates expression of the gRNA. In embodiments, the nucleic acids encoding gRNAs and nucleases comprise from 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, encodes 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 an embodiment, the compound comprises a cell penetrating peptide (or cCPP) conjugated to a ribonucleoprotein (RNP) containing a gRNA and a nuclease.

In an embodiment, a composition comprising: (a) CPP conjugated to gRNA is delivered to the cell; and (b) nuclease. In an embodiment, a composition comprising: (a) CPP conjugated to a nuclease; and (b) gRNA.

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

Genetic element of interest

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

nuclease inhibitor

In an embodiment, the compounds disclosed herein include a cell penetrating peptide conjugated to a nuclease inhibitor (e.g., Cas9). A limitation of gene editing is potential off-target editing. Delivery of nuclease inhibitors limits off-target editing. In embodiments, the nuclease inhibitor is a polypeptide, polynucleotide, or small molecule. Exemplary nuclease inhibitors include US Publication No. 2020/087354, International Publication No. 2018/085288, US Publication No. 2018/0382741, International Publication No. 2019/089761, International Publication No. 2020/068304, International Publication No. 2020/041384, and International Publication No. 2019/076651, each of which is incorporated herein by reference in its entirety.

therapeutic polypeptide

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

Conjugate Groups

In embodiments, the AC is modified by covalent attachment of one or more adapters. Typically, the conjugator modifies one or more properties of the attached AC, including but not limited to pharmacodynamics, pharmacokinetics, binding, uptake, cellular distribution, cellular uptake, charge, and clearance. Conjugators are routinely used in the field of chemistry, linking either directly to the parent compound, such as AC, or through an optional linking moiety or linker. Adapters include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, Includes, but is not limited to, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In an embodiment, the conjugating group is polyethylene glycol (PEG), and PEG is conjugated to AC or CPP.

The conjugator may be a lipid moiety, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. 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., Bioorg. Med. Chem. Let., 1993, 3, 2765]); thiocholesterol (Oberhauser et al. [Nucl. 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-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. , 1995, 36, 3651]; Shea et al. [Nucl. Acids Res., 1990, 18, 3777]; polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); Adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996,277,923).

Linking groups or bifunctional linking moieties, such as those known in the art, may be suitable for the compounds provided herein. Linkers are useful for attaching chemical functional groups, adapter groups, reporter groups, and other groups to selective sites of the parent compound, such as AC. Typically, bifunctional linking moieties include a hydrocarbyl moiety with two functional groups. One of the functional groups is selected to bind to the parent molecule or compound of interest, and the other is selected to bind essentially any selected functional group, such as a chemical functional group or conjugation group. Any of the linkers described herein may be used. In embodiments, the linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups routinely used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In embodiments, the bifunctional linking moiety includes amino, hydroxyl, carboxylic acid, thiol, unsaturated (e.g., double or triple bond), etc. Some non-limiting examples of bifunctional binding moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) , and 6-aminohexanoic 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 includes hydroxyl, Includes amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In an embodiment, the AC may be linked to 10 arginine-serine dipeptide repeats. AC linked to 10 arginine-serine dipeptide repeats was applied in vitro for artificial recruitment of splicing enhancer factors, leading to inclusion of mutated BRCA1 and SMN2 exons that may be skipped. See Cartegni and Krainer, 2003, incorporated herein by reference.

Endosomal escape vehicle (EEV)

Endosomal escape vehicles (EEVs) can be used to transport cargo across the cell membrane, for example, to deliver the cargo to the cytosol or nucleus of the cell. The cargo may include a therapeutic moiety (TM). EEV may contain cell penetrating peptides (CPP), such as cyclic cell penetrating peptide (cCPP). In an embodiment, the EEV comprises cCPP conjugated to an exophytic peptide (EP). EPs may be referred to interchangeably as regulatory peptides (MPs). The EP may contain the sequence of a nuclear localization signal (NLS). EP can be combined with cargo. EP can be bound to cCPP. EP can be bound to cargo and cCPP. The bond between EP, cargo, cCPP, or combinations thereof may be non-covalent or covalent. EP can be attached to the N-terminus of cCPP via a peptide bond. EP can be attached to the C-terminus of cCPP via a peptide bond. EP can be attached to cCPP through the side chains of amino acids within cCPP. EP can be attached to cCPP through the side chain of lysine, which can be conjugated to the side chain of glutamine in cCPP. EP can be conjugated to the 5' or 3' end of the oligonucleotide cargo. EP can be linked to a linker. The extracyclic peptide may be conjugated to the amino group of the linker. The EP may be bound to a linker through the C-terminus of the EP and to cCPP through the side chains on cCPP and/or EP. For example, EP may contain a terminal lysine, and EP may then be linked to cCPP containing glutamine via an amide bond. If the EP contains a terminal lysine, and the side chain of the lysine can be used to attach cCPP, either the C-terminus or the N-terminus can be attached to a linker on the cargo.

Exocyclic Peptides

The exophytic peptide (EP) may comprise from 2 to 10 amino acid residues, for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, and all ranges and values in between. Includes. EP may contain 6 to 9 amino acid residues. EP may contain 4 to 8 amino acid residues.

Each amino acid in the exophytic peptide may be a natural or non-natural amino acid. The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid to mimic the structure and reactivity of the natural amino acid. Non-natural amino acids may be modified amino acids and/or amino acid analogs that are not one of the 20 common naturally occurring amino acids and are not the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids are alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, and serine. , threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids are listed in Table 4 along with their abbreviations as used herein. For example, the amino acid can be A, G, P, K, R, V, F, H, Nal, or citrulline.

The EP may comprise at least one positively charged amino acid residue, such as 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. EP may contain one or two amino acid residues containing a side chain containing a guanidine group or a protonated form thereof. The amino acid residue containing the side chain containing the guanidine group may be an arginine residue. Protonated forms may refer to salts thereof throughout this disclosure.

EP may contain at least 2, at least 3, or at least 4 or more lysine residues. EP may contain 2, 3, or 4 lysine residues. The amino group on the side chain of each lysine residue can be, for example, trifluoroacetyl (-COCF ₃ ), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexylidene) ) It may be substituted with a protecting group containing ethyl (Dde), or (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) group. The amino group on the side chain of each lysine residue may be substituted with a trifluoroacetyl (-COCF ₃ ) group. Protecting groups may be included to enable amide conjugation. The protecting group can be removed after EP is conjugated to cCPP.

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

The EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one arginine residue. The EP may comprise at least 2, at least 3, or at least 4 or more lysine residues and/or arginine residues.

EP is 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 ID NO: 5), HKHK (SEQ ID NO: 6), KKKK (SEQ ID NO: 7), KKRK (SEQ ID NO: Number 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. Amino acids in EP can have D or L stereochemistry.

EP is 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 include 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), where B is beta-alanine am. Amino acids in EP can have D or L stereochemistry.

EP is 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), where B is beta-alanine . Amino acids in EP can have D or L stereochemistry.

EPs may include amino acid sequences identified in the art as nuclear localization sequences (NLS). EPs may consist of amino acid sequences identified in the art as nuclear localization sequences (NLS). The EP may contain an NLS containing the amino acid sequence PKKKRKV (SEQ ID NO: 42). EP may consist of an NLS containing the amino acid sequence PKKKRKV (SEQ ID NO: 42). The EP may comprise an NLS comprising an amino acid sequence selected from: 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), SALIKKKKKKMAP (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). The EP may consist of an NLS comprising an amino acid sequence selected from: 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), SALIKKKKKKMAP (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).

All extracyclic sequences may contain an N-terminal acetyl group. Thus, for example, EP may have the following structure: Ac-PKKKRKV (SEQ ID NO: 42).

Cell penetrating peptide (CPP)

Cell penetrating peptides (CPPs) may contain 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). cCPP can penetrate cell membranes. An exophytic peptide (EP) can be conjugated to cCPP, and the resulting construct can be referred to as an endosomal escape vehicle (EEV). cCPP can induce cargo (e.g. therapeutic moieties (TM) such as oligonucleotides, peptides, or small molecules) to permeate cell membranes. cCPP can deliver cargo to the cytosol of cells. cCPP can deliver cargo to cellular locations where targets (e.g. pre-mRNA) are located. To conjugate cCPP to a cargo (e.g., a peptide, oligonucleotide, or small molecule), at least one bond or pair of electrons on cCPP can be replaced.

The total number of amino acid residues in cCPP ranges from 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 between them. cCPP may contain 6 to 13 amino acid residues. cCPP disclosed herein may contain 6 to 10 amino acids. By way of example, cCPP comprising 6-10 amino acid residues may have a structure according to any one of Formulas I-A to I-E:

or , where AA ₁ , AA ₂ , AA ₃ , AA ₄ , AA ₅ , AA ₆ , AA ₇ , AA ₈ , AA ₉ , and AA ₁₀ are amino acid residues.

cCPP may contain 6 to 8 amino acids. cCPP may contain 8 amino acids.

Each amino acid in cCPP can be a natural or non-natural amino acid. The term “non-natural amino acid” refers to an organic compound that is a class of natural amino acids in that it has a similar structure to a natural amino acid so as to mimic the structure and reactivity of the natural amino acid. Non-natural amino acids may be modified amino acids and/or amino acid analogs that are not one of the 20 common naturally occurring amino acids and are not the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids are alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, and serine. , threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids are listed in Table 5 along with their abbreviations as used herein.

Amino Acid Abbreviations amino acid abbreviation*
L-amino acid abbreviation*
D-amino acid 2-[2-[2-aminoethoxy]ethoxy]acetic acid AEEA, miniPEG, PEG2 N.A. alanine Ala (A) ala (a) Allo-isoleucine Aile Aile arginine Arg (R) arg (r) Asparagine Asn(N) asn (n) aspartic acid Asp (D) asp (d) cysteine Cys (C) cys (c) Citrulline Cit Cit Cyclohexylalanine Cha cha 2,3-Diaminopropionic acid Dap daps 4-Fluorophenylalanine Fpa (Σ) pfa glutamic acid Glu (E) glu (e) glutamine Gln (Q) gln(q) glycine Gly (G) gly (g) histidine His (H) his (h) Homoproline (also known as pipecolic acid) Pip (Θ) pip (*?*) isoleucine Ile (I) ile (i) leucine Leu (L) leu (l) Lee Sin Lys (K) lys (k) methionine Met (M) meet (m) 3-(2-naphthyl)-alanine Nal (Φ) nal (*?*) 3-(1-naphthyl)-alanine 1-Nal 1-nal Norleucine Nle (Ω) nle Phenylalanine Phe (F) phe (f) phenylglycine Phg (Ψ) phg 4-(phosphonodifluoromethyl)phenylalanine F ₂ Pmp (Λ) _f2pmp proline Pro (P) pro (p) Sarcosine Sar (Ξ) sar Selenocysteine Sec (U) sec (u) serine Ser(S) ser(s) threonine Thr (T) thr(y) tyrosine Tyr (Y) tyr (y) tryptophan Trp(W) trp(w) Valine Val (V) val (v) tert-butyl-alanine Tle tle penicillamine Pen Pen Homoarginine HomoArg homoarg Nicotine-lysine Lys(NIC) lys(NIC) Trifluoroacetyl-lysine Lys(TFA) lys(TFA) Methyl-leucine MeLeu meLeu 3-(3-benzothienyl)-alanine Bta bta * One-letter abbreviation: uppercase letters indicate the L-amino acid form, lowercase letters indicate the D-amino acid form.

As used herein, “polyethylene glycol” and “PEG” are used interchangeably. “PEGm” and “PEG _m ” are molecules of the formula HO(CO)-(CH ₂ ) _n -(OCH2CH ₂ ) _m -NH ₂ , where n is any integer from 1 to 5; m is any integer from 1 to 23. In embodiments, n is 1 or 2. In embodiments, n is 1. In an embodiment, n is 2. In an embodiment, n is 1 and m is 2. In an embodiment, n is 2 and m is 2. In an embodiment, n is 1 and m is 4. In an embodiment, n is 2 and m is 4. In an embodiment, n is 1 and m is 12. In an embodiment, n is 2 and m is 12.

As used herein, “miniPEGm” and “miniPEG _m ” are or are derived from a molecule of the formula HO(CO)-(CH ₂ ) _n -(OCHCH ₂ ) _m -NH ₂ , where n is 1 and , m is any integer from 1 to 23. For example, “miniPEG2” or “miniPEG ₂ ” is or is derived from (2-[2-[2-aminoethoxy]ethoxy]acetic acid, and “miniPEG4” or “miniPEG ₄ ” is HO(CO)- (CH ₂ ) _n -(OCH ₂ CH ₂ ) _m -NH ₂ or derived therefrom, where n is 1 and m is 4.

cCPP may comprise 4 to 20 amino acids, where: (i) at least one amino acid has a side chain comprising a guanidine group, or a protonated form thereof; (ii) at least one amino acid has no side chain or or has a side chain comprising a protonated form thereof; (iii) at least two amino acids independently have side chains containing aromatic or heteroaromatic groups.

At least two amino acids have no side chains, or

or a side chain comprising a protonated form thereof. As used herein, when no side chain is present, an amino acid has two hydrogen atoms (eg, -CH ₂ -) on the carbon atom(s) linking the amine and the carboxylic acid.

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

A cCPP may comprise 6 to 20 amino acid residues forming a cCPP, where: (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; (iii) at least one amino acid is a guanidine group, or a protonated form thereof.

A cCPP may comprise 6 to 20 amino acid residues forming a cCPP, where: (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; (iii) at least one amino acid is a guanidine group, or a protonated form thereof.

A cCPP may comprise 6 to 20 amino acid residues forming a cCPP, where: (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; (iii) at least one amino acid is a guanidine group, or a side chain comprising 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 combinations thereof. cCPP may comprise (i) two glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) three glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) four glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) five glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) six glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) 3, 4, or 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof.

cCPP may include (i) 1, 2, 3, 4, 5, or 6 glycine residues. cCPP may include (i) two glycine residues. cCPP may include (i) three glycine residues. cCPP may include (i) four glycine residues. cCPP may include (i) five glycine residues. cCPP may contain (i) six glycine residues. cCPP may include (i) 3, 4, or 5 glycine residues. cCPP may include (i) 3 or 4 glycine residues. cCPP may include (i) 2 or 3 glycine residues. cCPP may include (i) 1 or 2 glycine residues.

cCPP may comprise (i) 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) three glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) four glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) five glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) six glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) 3, 4, or 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may comprise (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof.

cCPP may contain at least 3 glycine residues. cCPP may include (i) 3, 4, 5, or 6 glycine residues. cCPP may include (i) three glycine residues. cCPP may include (i) four glycine residues. cCPP may include (i) five glycine residues. cCPP may contain (i) six glycine residues. cCPP may include (i) 3, 4, or 5 glycine residues. cCPP may include (i) 3 or 4 glycine residues.

In an embodiment, none of the glycine, β-alanine, or 4-aminobutyric acid residues in cCPP are continuous. Two or three glycine, β-alanine, 4- or aminobutyric acid residues may be consecutive. The two glycine, β-alanine, and 4-aminobutyric acid residues may be consecutive.

In an embodiment, none of the glycine residues in cCPP are consecutive. Each glycine residue in cCPP may be separated by an amino acid residue, where the amino acid residue cannot be glycine. Two or three glycine residues may be consecutive. Two glycine residues may be consecutive.

Amino acid side chains with aromatic or heteroaromatic groups

cCPP may comprise 2, 3, 4, 5, or 6 amino acid residues that independently have (ii) side chains comprising aromatic or heteroaromatic groups. cCPP may comprise (ii) two amino acid residues that independently have side chains comprising aromatic or heteroaromatic groups. cCPP may comprise three amino acid residues that independently have (ii) side chains comprising aromatic or heteroaromatic groups. cCPP may comprise four amino acid residues that independently have (ii) side chains comprising aromatic or heteroaromatic groups. cCPP may comprise five amino acid residues that independently have (ii) side chains comprising aromatic or heteroaromatic groups. cCPP may comprise six amino acid residues that independently have (ii) side chains comprising aromatic or heteroaromatic groups. cCPP may comprise 2, 3, or 4 amino acid residues that independently have (ii) side chains comprising aromatic or heteroaromatic groups. cCPP may comprise 2 or 3 amino acid residues that independently have (ii) side chains comprising aromatic or heteroaromatic groups.

cCPP may comprise 2, 3, 4, 5, or 6 amino acid residues that independently have (ii) a side chain containing an aromatic group. cCPP may comprise (ii) two amino acid residues that independently have side chains containing an aromatic group. cCPP may comprise (ii) three amino acid residues that independently have side chains containing aromatic groups. cCPP may comprise four amino acid residues that independently have (ii) side chains containing aromatic groups. cCPP may comprise five amino acid residues that independently have (ii) side chains containing aromatic groups. cCPP may comprise six amino acid residues that independently have (ii) side chains containing aromatic groups. cCPP may comprise 2, 3, or 4 amino acid residues that independently have (ii) a side chain containing an aromatic group. cCPP may comprise 2 or 3 amino acid residues that independently have (ii) side chains containing aromatic groups.

The aromatic group may be 6-membered to 14-membered aryl. Aryl can be phenyl, naphthyl, or anthracenyl, each of which is optionally substituted. Aryl can be phenyl or naphthyl, each of which is optionally substituted. Heteroaromatic groups can be 6-14 membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S. Heteroaryl can be pyridyl, quinolyl, or isoquinolyl.

The amino acid residues having a side chain containing an aromatic or heteroaromatic group are each independently bis(homonaphthylalanine), homonaphthylalanine, naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3 -(3-benzothienyl)-alanine, 3-(2-quinolyl)-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 tyrosine, each of these is optionally substituted with one or more substituents. Amino acids having side chains containing aromatic or heteroaromatic groups may each be independently selected from:

and , wherein H on the N-terminus and/or H on the C-terminus are replaced by a peptide bond.

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

In an embodiment, none of the amino acids having side chains containing aromatic or heteroaromatic groups are continuous. Two amino acids with side chains containing aromatic or heteroaromatic groups may be consecutive. Two consecutive amino acids can have opposite stereochemistry. Two consecutive amino acids can have the same stereochemistry. Three amino acids with side chains containing aromatic or heteroaromatic groups may be consecutive. Three consecutive amino acids can have the same stereochemistry. Three consecutive amino acids can have alternating stereochemistry.

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

The optional substituent may be any atom or group that does not significantly (e.g., by more than 50%) reduce the cytosolic transport efficiency of cCPP, e.g., compared to the same sequence but without the substituent. Optional substituents may be hydrophobic or hydrophilic substituents. The optional substituent may be a hydrophobic substituent. Substituents can increase the solvent accessible surface area (as defined herein) of a hydrophobic amino acid. Substituents include halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, It may be alkoxycarbonyl, alkylthio, or arylthio. The substituent may be halogen.

Without wishing to be bound by theory, amino acids with aromatic or heteroaromatic groups with higher hydrophobicity values (i.e., amino acids with side chains containing aromatic or heteroaromatic groups) have a greater cytosolic transport of cCPP than amino acids with lower hydrophobicity values. It is believed that efficiency can be improved. Each hydrophobic amino acid can independently have a hydrophobicity value greater than that of glycine. Each hydrophobic amino acid may independently be a hydrophobic amino acid having a hydrophobicity value greater than that of alanine. Each hydrophobic amino acid can independently have a hydrophobicity value greater than or equal to phenylalanine. Hydrophobicity can be measured using hydrophobicity scales known in the art. Table 5 shows Eisenberg and Weiss [Proc. Natl. Acad. Sci. USA 1984;81(1):140-144], Engleman et al. [Ann. Rev. of Biophys. 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. USA 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.

amino acid hydrophobicity amino acid energy Eisenberg and Weiss Engleman et al. Kyrie and Doolittle Hoop and Woods Janine Ile nonpolar 0.73 3.1 4.5 -1.8 0.7 Phe nonpolar 0.61 3.7 2.8 -2.5 0.5 Val nonpolar 0.54 2.6 4.2 -1.5 0.6 Leu nonpolar 0.53 2.8 3.8 -1.8 0.5 Trp nonpolar 0.37 1.9 -0.9 -3.4 0.3 Met nonpolar 0.26 3.4 1.9 -1.3 0.4 Ala nonpolar 0.25 1.6 1.8 -0.5 0.3 Gly nonpolar 0.16 1.0 -0.4 0.0 0.3 Cys uncharged/polarized 0.04 2.0 2.5 -1.0 0.9 Tyr Uncharged/polar 0.02 -0.7 -1.3 -2.3 -0.4 Pro nonpolar -0.07 -0.2 -1.6 0.0 -0.3 Thr uncharged/polarized -0.18 1.2 -0.7 -0.4 -0.2 Ser Uncharged/polar -0.26 0.6 -0.8 0.3 -0.1 His charged -0.40 -3.0 -3.2 -0.5 -0.1 Glu charged -0.62 -8.2 -3.5 3.0 -0.7 Asn uncharged/polarized -0.64 -4.8 -3.5 0.2 -0.5 Gln uncharged/polarized -0.69 -4.1 -3.5 0.2 -0.7 Asp charged -0.72 -9.2 -3.5 3.0 -0.6 Lys charged -1.10 -8.8 -3.9 3.0 -1.8 Arg charged -1.80 -12.3 -4.5 3.0 -1.4

The size of the aromatic or heteroaromatic group can be selected to improve the cytosolic transport efficiency of cCPP. Without wishing to be bound by theory, it is believed that larger aromatic or heteroaromatic groups on the side chains of amino acids can improve cytosolic transport efficiency compared to otherwise identical sequences with smaller hydrophobic amino acids. The size of a hydrophobic amino acid can 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 (SASA) of the side chain, or a combination thereof. The size of a hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, with the more hydrophobic amino acid having a side chain with a molecular weight of at least about 90 g/mole, or at least about 130 g/mole, or at least about 141 g/mole. . The size of an amino acid can be measured in terms of the SASA of the hydrophobic side chain. Hydrophobic amino acids can have side chains with a SASA that is greater than or equal to the SASA of alanine, or greater than or equal to the SASA of glycine. A more hydrophobic amino acid may have a side chain with a SASA greater than that of alanine or greater than that of glycine. The hydrophobic amino acid may have an aromatic or heteroaromatic group with a SASA that is approximately equal to or greater than the SASA of piperidine-2-carboxylic acid, approximately equal to or greater than the SASA of tryptophan, approximately equal to or greater than the SASA of phenylalanine, or approximately equal to or greater than the SASA of naphthylalanine. The first hydrophobic amino acid (AA _H1 ) has a length of at least about 200 Å ² , at least about 210 Å ² , at least about 220 Å ² , at least about 240 Å ² , at least about 250 Å ² , at least about 260 Å ² , at least about 270 Å ² , at least about 280 Å ² , at least about 290 Å ² , at least about 300 Å ² , at least about 310 Å ² , at least about 320 Å ² , or at least about 330 Å ² . The second hydrophobic amino acid (AA _H2 ) is at least about 200 Å ² , at least about 210 Å ² , at least about 220 Å ² , at least about 240 Å ² , at least about 250 Å ² , at least about 260 Å ² , at least about 270 Å ² , at least about 280 Å ² , at least about 290 Å ² , at least about 300 Å ² , at least about 310 Å ² , at least about 320 Å ² , or at least about 330 Å ² . The side chains of AA _H1 and AA _H2 are at least about 350 Å ² , at least about 360 Å ² , at least about 370 Å ² , at least about 380 Å ² , at least about 390 Å ² , at least about 400 Å ² , at least about 410 Å ² , at least about 420 Å ² , at least about 430 Å ² , At least about 440 Å ² , at least about 450 Å ² , at least about 460 Å ² , at least about 470 Å ² , at least about 480 Å ² , At least about 490 Å ² , Greater than about 500 Å ² but at least about 510 Å ² , at least about 520 Å ² , at least about 530 Å ² , at least about 540 Å ² , at least about 550 Å ² , at least about 560 Å ² , at least about 570 Å ² , at least about 580 Å ² , at least about 590 Å ² , at least about 600 Å ² , at least about 610 Å ² , at least about 620 Å ² , at least about 630 Å ² , at least about 640 Å ² , greater than about 650 Å ² , and can have a combined SASA of at least about 660 Å ² , at least about 670 Å ² , at least about 680 Å ² , at least about 690 Å ² , or at least about 700 Å ² . AA _H2 may be a hydrophobic amino acid residue having a side chain with a SASA that is less than or equal to the SASA of the hydrophobic side chain of AA _H1 . For example, but not by way of limitation, a cCPP with a Nal-Arg motif may exhibit improved cytosolic transport efficiency compared to an otherwise identical cCPP with a Phe-Arg motif; cCPP with a Phe-Nal-Arg motif can exhibit improved cytosolic transport efficiency compared to an otherwise identical cCPP with a Nal-Phe-Arg motif; The phe-Nal-Arg motif may exhibit improved cytosolic transport efficiency compared to an otherwise identical cCPP with a nal-Phe-Arg motif.

As used herein, “hydrophobic surface area” or “SASA” refers to the surface area (angstrom squared; reported as Å ² ) of amino acid side chains accessible to solvents, SASA being the ‘rolling ball’ developed by rolling ball' algorithm (Shrake and Rupley, J Mol Biol . 79 (2): 351-71, incorporated herein by reference in its entirety for all purposes). This algorithm uses a “sphere” of solvent with a specific radius to probe the surface of the molecule. A typical value for a sphere is 1.4 Å, which approximates the radius of a water molecule.

SASA values for specific 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. (Tien et al. [PLOS ONE 8(11): e80635], available at doi.org/10.1371/journal.pone.0080635, incorporated herein by reference in its entirety for all purposes).

Amino Acid SASA Value residue theoretical empirical Miller et al. (1987) Rose et al. (1985) alanine 129.0 121.0 113.0 118.1 arginine 274.0 265.0 241.0 256.0 Asparagine 195.0 187.0 158.0 165.5 Aspartate 193.0 187.0 151.0 158.7 cysteine 167.0 148.0 140.0 146.1 glutamate 223.0 214.0 183.0 186.2 glutamine 225.0 214.0 189.0 193.2 glycine 104.0 97.0 85.0 88.1 histidine 224.0 216.0 194.0 202.5 isoleucine 197.0 195.0 182.0 181.0 leucine 201.0 191.0 180.0 193.1 Lee Sin 236.0 230.0 211.0 225.8 methionine 224.0 203.0 204.0 203.4 Phenylalanine 240.0 228.0 218.0 222.8 proline 159.0 154.0 143.0 146.8 serine 155.0 143.0 122.0 129.8 threonine 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 174.0 165.0 160.0 164.5

Amino acid residues with side chains containing a guanidine group, a guanidine substituent, or a protonated form thereof

As used herein, guanidine refers to the following structure:

.

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

.

A guanidine substituent refers to a functional group on the side chain of an amino acid that is positively charged at or above physiological pH, or that can reproduce hydrogen bonds that donate and accept the activity of the guanidinium group.

The guanidine substituent facilitates cellular penetration and delivery of the therapeutic agent while reducing the toxicity associated with the guanidine group or its protonated form. cCPP may include at least one amino acid with a side chain containing a guanidine or guanidinium substituent. cCPP may comprise at least two amino acids with side chains containing guanidine or guanidinium substituents. cCPP may comprise at least three amino acids with side chains containing guanidine or guanidinium substituents.

The guanidine or guanidinium group may be an isostere of guanidine or guanidinium. The guanidine or guanidinium substituent may be less basic than guanidine.

As used herein, a guanidine substituent is , , or its protonated form.

The present disclosure relates to cCPPs comprising 4 to 20 amino acid residues, where: (i) at least one amino acid has a side chain comprising a guanidine group, or a protonated form thereof; (ii) at least one amino acid residue has no side chain, or , , or has a side chain comprising a protonated form thereof; (iii) at least two amino acid residues independently have side chains containing aromatic or heteroaromatic groups.

At least two amino acid residues do not have side chains, or , , or a side chain comprising a protonated form thereof. As used herein, when no side chain is present, the amino acid residue has two hydrogen atoms (eg, -CH ₂ -) on the carbon atom(s) linking the amine and the carboxylic acid.

cCPP may comprise at least one amino acid with a side chain comprising 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. The at least two amino acids may have side chains comprising the same moiety selected from: , , or a protonated form thereof. at least one or a side chain comprising a protonated form thereof. at least 2 amino acids or a side chain comprising a protonated form thereof. 1, 2, 3, or 4 amino acids or a side chain comprising a protonated form thereof. one amino acid or a side chain comprising a protonated form thereof. 2 amino acids or a side chain comprising a protonated form thereof. , , or its protonated form may be attached to the terminus of an amino acid side chain. Can be attached to the end of the amino acid side chain.

cCPP may comprise 2, 3, 4, 5, or 6 amino acid residues that independently have (iii) a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise (iii) two amino acid residues that independently have side chains comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise three amino acid residues that independently have (iii) side chains comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise four amino acid residues that independently have (iii) a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise five amino acid residues that independently have (iii) a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise six amino acid residues that independently have (iii) side chains comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise 2, 3, 4, or 5 amino acid residues that independently have (iii) a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise 2, 3, or 4 amino acid residues that independently have (iii) a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise two or three amino acid residues that independently have (iii) a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof. cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidine group or a protonated form thereof. cCPP may comprise (iii) two amino acid residues with side chains comprising a guanidine group or a protonated form thereof. cCPP may comprise (iii) three amino acid residues with side chains comprising a guanidine group or a protonated form thereof.

The amino acid residues may independently have a side chain comprising a non-contiguous guanidine group, a guanidine substituent, or a protonated form thereof. The two amino acid residues may independently have side chains containing a guanidine group, a guanidine substituent, or a protonated form thereof, which may be consecutive. The three amino acid residues may independently have side chains containing guanidine groups, guanidine substituents, or protonated forms thereof, which may be consecutive. The four amino acid residues may independently have side chains containing guanidine groups, guanidine substituents, or protonated forms thereof, which may be consecutive. Consecutive amino acid residues may have the same stereochemistry. Sequential amino acids can have alternating stereochemistry.

An amino acid residue that independently has a side chain comprising a guanidine group, a guanidine substituent, or a protonated form thereof may be an L-amino acid. An amino acid residue that independently has a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be a D-amino acid. The amino acid residues that independently have a side chain containing a guanidine group, a guanidine substituent, or a protonated form thereof may be an L-amino acid or a mixture of D-amino acids.

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

Each amino acid having a side chain containing a guanidine substituent or a protonated form thereof is independently , , or it may be a protonated form thereof.

Without being bound by theory, the guanidine substituent has reduced basicity compared to arginine and, in some cases, is uncharged at physiological pH (e.g. -N(H)C(O)), facilitating effective membrane binding and subsequent internalization. It is assumed that it can maintain bidentate hydrogen bonding interactions with phospholipids on the plasma membrane, which are believed to allow it to do so. Removing the positive charge is also believed to reduce the toxicity of cCPP.

Those skilled in the art will understand that after the non-natural aromatic hydrophobic amino acids are incorporated into the peptides disclosed herein, their N-termini and/or C-termini form amide bonds.

cCPP is a first amino acid having a side chain comprising an aromatic or heteroaromatic group; and a second amino acid having a side chain containing 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 containing an aromatic or heteroaromatic group, and 1 The C-terminus of glycine forms a peptide bond with a second amino acid having a side chain containing an aromatic or heteroaromatic group. By convention, the term “first amino acid” often refers to the N-terminal amino acid of a peptide sequence, but as used herein, a “first amino acid” refers to a reference amino acid within a cCPP that is replaced by another amino acid (e.g., “second amino acid”). As used to distinguish between "amino acids"), the term "first amino acid" may or may refer to an amino acid located at the N-terminus of a peptide sequence.

cCPP is the N-terminus of a second glycine that forms a peptide bond with an amino acid having a side chain containing an aromatic or heteroaromatic group; and the C-terminus of the second glycine that forms a peptide bond with an amino acid having a side chain containing a guanidine group or a protonated form thereof.

cCPP comprises a first amino acid having a side chain comprising a guanidine group or a protonated form thereof, and a second amino acid having a side chain comprising a guanidine group or a protonated form thereof, wherein the N-terminus of the third glycine is Forms a peptide bond with a first amino acid having a side chain containing a guanidine group or a protonated form thereof, and the C-terminus of the third glycine is linked to a second amino acid having a side chain containing a guanidine group or a protonated form thereof. Forms peptide bonds.

cCPP may include the residues of asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine. cCPP may contain a residue of asparagine. cCPP may contain residues of glutamine.

cCPP contains tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2 ,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluoro It may include residues of lophenylalanine, 4-chlorophenylalanine, and 3-(9-anthryl)-alanine.

Without wishing to be bound by theory, it is believed that the chirality of the amino acids in cCPP may affect cytosolic uptake efficiency. cCPP may contain at least one D amino acid. cCPP may contain 1 to 15 D amino acids. cCPP may contain 1 to 10 D amino acids. cCPP may contain 1, 2, 3, or 4 D amino acids. cCPP may comprise 2, 3, 4, 5, 6, 7, or 8 consecutive amino acids of alternating D and L chirality. cCPP may contain three consecutive amino acids with the same chirality. cCPP may contain two consecutive amino acids with the same chirality. At least two of the amino acids may have opposite chirality. At least two amino acids with opposite chirality may be adjacent to each other. At least three amino acids can have alternating stereochemistry with respect to each other. At least three amino acids with alternating chirality with respect 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 with alternating chirality with respect to each other may be adjacent to each other. At least two of the amino acids may have the same chirality. At least two amino acids with the same chirality may be adjacent to each other. At least two amino acids have identical chirality and at least two amino acids have opposite chirality. At least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Accordingly, contiguous amino acids within cCPP can 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 that form cCPP may all be L-amino acids. The amino acid residues that form cCPP may all be D-amino acids.

At least two of the amino acids may have different chiralities. At least two amino acids with different chirality may be adjacent to each other. At least three amino acids may have different chirality compared to adjacent amino acids. At least four amino acids may have different chirality compared to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chirality. One or more amino acid residues forming cCPP may be achiral. cCPP may contain a motif of 3, 4, or 5 amino acids, where two amino acids with the same chirality can be separated by an achiral amino acid. cCPP may comprise the following sequences: 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, where X is an achiral amino acid. The achiral amino acid may be glycine.

, , Alternatively, an amino acid with a side chain comprising a protonated form thereof may be adjacent to an amino acid with a side chain comprising an aromatic or heteroaromatic group. , , Alternatively, the amino acid having a side chain comprising guanidine or 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 with a side chain comprising guanidine or a protonated form thereof may be adjacent to an amino acid with a side chain comprising an aromatic or heteroaromatic group. , , Alternatively, two amino acids with side chains comprising protonated forms thereof may be adjacent to each other. Two amino acids with side chains comprising guanidine or a protonated form thereof are adjacent to each other. cCPP is at least two consecutive amino acids with side chains that may include aromatic or heteroaromatic groups; and , , or at least two non-contiguous amino acids with side chains comprising a protonated form thereof. cCPP is at least two consecutive amino acids with side chains containing aromatic or heteroaromatic groups; and or at least two non-contiguous amino acids with side chains comprising a protonated form thereof. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chiralities. Other combinations of amino acids can have any arrangement of D and L amino acids, for example any of the sequences described in the previous paragraph.

, , or at least two amino acids having side chains comprising a guanidine group or a protonated form thereof alternate with at least two amino acids having side chains comprising a guanidine group or a protonated form thereof.

cCPP has the structure of equation (A):

(A)

or a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

R ₄ , R ₅ , R ₆ , and R ₇ are independently H or an amino acid side chain;

At least one of R ₄ , R ₅ , R ₆ , R ₇ is 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N, N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethylysine, N,N,N-trimethyllysine, It is the side chain of 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, and 3-(1-piperidinyl)alanine;

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4.

In embodiments, at least one of R ₄ , R ₅ , R ₆ , and R ₇ is independently an uncharged, non-aromatic side chain of an amino acid. In embodiments, at least one of R ₄ , R ₅ , R ₆ , and R ₇ is independently H or a side chain of citrulline.

In an embodiment, provided is a compound comprising a cyclic peptide having 6 to 12 amino acids, wherein at least 2 amino acids of the cyclic peptide are charged amino acids, at least 2 amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and wherein the cyclic peptide At least two amino acids are uncharged, non-aromatic amino acids. In an embodiment, at least two charged amino acids of the cyclic peptide are arginine. In an embodiment, the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine or naphthylalanine. In an embodiment, 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 NOs: 89-117.

In an embodiment, the cyclic peptide of formula (A) is selected from cyclic peptides having the sequence of SEQ ID NOs: 89-117.

cCPP has the structure of formula (I):

(I)

or a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4;

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

R ₁ , R ₂ , and R ₃ may each independently be H, -alkylene-aryl, or -alkylene-heteroaryl. R ₁ , R ₂ , and R ₃ may each independently be H, -C _1-3 alkylene-aryl, or -C _1-3 alkylene-heteroaryl. R ₁ , R ₂ , and R ₃ may each independently be H or -alkylene-aryl. R ₁ , R ₂ , and R ₃ may each independently be H or -C _1-3 alkylene-aryl. C _1-3 alkylene may be methylene. Aryl may be a 6-membered to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl may be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R ₁ , R ₂ , and R ₃ may each independently be H, -C _1-3 alkylene-Ph, or -C _1-3 alkylene-naphthyl. R ₁ , R ₂ , and R ₃ may each independently be H, -CH ₂ Ph, or -CH ₂ naphthyl. R ₁ , R ₂ , and R ₃ may each independently be H or -CH ₂ Ph.

R ₁ , R ₂ , and R ₃ are each independently tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluoro Phenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridinylalanine, 3-pyridine. It may be the side chain of dinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 3-(9-anthryl)-alanine.

R ₁ may be the side chain of tyrosine. R ₁ may be the side chain of phenylalanine. R ₁ may be the side chain of 1-naphthylalanine. R ₁ may be the side chain of 2-naphthylalanine. R ₁ may be a side chain of tryptophan. R ₁ may be the side chain of 3-benzothienylalanine. R ₁ may be the side chain of 4-phenylphenylalanine. R ₁ may be the side chain of 3,4-difluorophenylalanine. R ₁ may be the side chain of 4-trifluoromethylphenylalanine. R ₁ may be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R ₁ may be the side chain of homophenylalanine. R ₁ may be the side chain of β-homophenylalanine. R ₁ may be the side chain of 4-tert-butyl-phenylalanine. R ₁ may be the side chain of 4-pyridinylalanine. R ₁ may be the side chain of 3-pyridinylalanine. R ₁ may be the side chain of 4-methylphenylalanine. R ₁ may be the side chain of 4-fluorophenylalanine. R ₁ may be the side chain of 4-chlorophenylalanine. R ₁ may be the side chain of 3-(9-anthryl)-alanine.

R ₂ may be the side chain of tyrosine. R ₂ may be the side chain of phenylalanine. R ₂ may be the side chain of 1-naphthylalanine. R ₁ may be the side chain of 2-naphthylalanine. R ₂ may be a side chain of tryptophan. R ₂ may be the side chain of 3-benzothienylalanine. R ₂ may be the side chain of 4-phenylphenylalanine. R ₂ may be the side chain of 3,4-difluorophenylalanine. R ₂ may be the side chain of 4-trifluoromethylphenylalanine. R ₂ may be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R ₂ may be the side chain of homophenylalanine. R ₂ may be the side chain of β-homophenylalanine. R ₂ may be the side chain of 4-tert-butyl-phenylalanine. R ₂ may be the side chain of 4-pyridinylalanine. R ₂ may be the side chain of 3-pyridinylalanine. R ₂ may be the side chain of 4-methylphenylalanine. R ₂ may be the side chain of 4-fluorophenylalanine. R ₂ may be the side chain of 4-chlorophenylalanine. R ₂ may be the side chain of 3-(9-anthryl)-alanine.

R ₃ may be the side chain of tyrosine. R ₃ may be the side chain of phenylalanine. R ₃ may be the side chain of 1-naphthylalanine. R ₃ may be the side chain of 2-naphthylalanine. R ₃ may be a side chain of tryptophan. R ₃ may be the side chain of 3-benzothienylalanine. R ₃ may be the side chain of 4-phenylphenylalanine. R ₃ may be the side chain of 3,4-difluorophenylalanine. R ₃ may be the side chain of 4-trifluoromethylphenylalanine. R ₃ may be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R ₃ may be the side chain of homophenylalanine. R ₃ may be the side chain of β-homophenylalanine. R ₃ may be the side chain of 4-tert-butyl-phenylalanine. R ₃ may be the side chain of 4-pyridinylalanine. R ₃ may be the side chain of 3-pyridinylalanine. R ₃ may be the side chain of 4-methylphenylalanine. R ₃ may be the side chain of 4-fluorophenylalanine. R ₃ may be the side chain of 4-chlorophenylalanine. R ₃ may be the side chain of 3-(9-anthryl)-alanine.

R ₄ may be H, -alkylene-aryl, -alkylene-heteroaryl. R ₄ may be H, -C _1-3 alkylene-aryl, or -C _1-3 alkylene-heteroaryl. R ₄ may be H or -alkylene-aryl. R ₄ may be H or -C _1-3 alkylene-aryl. C _1-3 alkylene may be methylene. Aryl may be a 6-membered to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl may be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R ₄ may be H, -C _1-3 alkylene-Ph, or -C _1-3 alkylene-naphthyl. R ₄ may 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 containing an aromatic group. R ₄ may be H, -CH ₂ Ph, or -CH ₂ naphthyl. R ₄ may be H or -CH ₂ Ph.

R ₅ may be H, -alkylene-aryl, -alkylene-heteroaryl. R ₅ may be H, -C _1-3 alkylene-aryl, or -C _1-3 alkylene-heteroaryl. R ₅ may be H or -alkylene-aryl. R ₅ may be H or -C _1-3 alkylene-aryl. C _1-3 alkylene may be methylene. Aryl may be a 6-membered to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl may be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R ₅ may be H, -C _1-3 alkylene-Ph, or -C _1-3 alkylene-naphthyl. R ₅ may 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 containing an aromatic group. R ₅ may be H, -CH ₂ Ph, or -CH ₂ naphthyl. R ₄ may be H or -CH ₂ Ph.

R ₆ may be H, -alkylene-aryl, -alkylene-heteroaryl. R ₆ may be H, -C _1-3 alkylene-aryl, or -C _1-3 alkylene-heteroaryl. R ₆ may be H or -alkylene-aryl. R ₆ may be H or -C _1-3 alkylene-aryl. C _1-3 alkylene may be methylene. Aryl may be a 6-membered to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl may be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R ₆ may be H, -C _1-3 alkylene-Ph, or -C _1-3 alkylene-naphthyl. R ₆ may 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 containing an aromatic group. R ₆ may be H, -CH ₂ Ph, or -CH ₂ naphthyl. R ₆ may be H or -CH ₂ Ph.

R ₇ may be H, -alkylene-aryl, -alkylene-heteroaryl. R ₇ may be H, -C _1-3 alkylene-aryl, or -C _1-3 alkylene-heteroaryl. R ₇ may be H or -alkylene-aryl. R ₇ may be H or -C _1-3 alkylene-aryl. C _1-3 alkylene may be methylene. Aryl may be a 6-membered to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl may be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R ₇ may be H, -C _1-3 alkylene-Ph, or -C _1-3 alkylene-naphthyl. R ₇ may 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 containing an aromatic group. R ₇ may be H, -CH ₂ Ph, or -CH ₂ naphthyl. R ₇ may be H or -CH ₂ Ph.

₁ , 2, or 3 of R 1 , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be -CH ₂ Ph. One of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be -CH ₂ Ph. Two of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be -CH ₂ Ph. Three of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be -CH ₂ Ph. At least one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be -CH ₂ Ph. Four or less of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be -CH ₂ Ph.

1, ₂ , or 3 of R1, R 2 , R ₃ , and R ₄ are -CH ₂ Ph. One of R ₁ , R ₂ , R ₃ , and R ₄ is -CH ₂ Ph. Two of R1, R ₂ , R ₃ , and R ₄ are -CH ₂ Ph. Three of R1, R ₂ , R ₃ , and R ₄ are -CH ₂ Ph. At least one of R ₁ , R ₂ , R ₃ , and R ₄ is -CH ₂ Ph.

₁ , ₂ , or 3 of R 1 , R 2 , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be H. One of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be H. Two of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ are H. Three of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be H. At least one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be H. Three or less of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and R ₇ may be -CH ₂ Ph.

₁ , ₂ , or ₃ of R 1 , R 2 , R 3 , and R ₄ are H. One of R ₁ , R ₂ , R ₃ , and R ₄ is H. Two of R ₁ , R ₂ , R ₃ , and R ₄ are H. Three of R ₁ , R ₂ , R ₃ , and R ₄ are H. At least one of R ₁ , R ₂ , R ₃ , and R ₄ is H.

At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of 4-guanidino-2-aminobutanoic acid. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be the side chain of arginine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of homoarginine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of N-methylarginine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of N,N-dimethylarginine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of 2,3-diaminopropionic acid. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be the side chain of 2,4-diaminobutanoic acid or lysine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of N-methyllysine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of N,N-dimethyllysine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of N-ethylysine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be the side chain of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of citrulline. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be the side chain of N,N-dimethyllysine or β-homoarginine. At least one of R ₄ , R ₅ , R ₆ , and R ₇ may be a side chain of 3-(1-piperidinyl)alanine.

At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 3-guanidino-2-aminopropionic acid. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 4-guanidino-2-aminobutanoic acid. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of arginine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of homoarginine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N-methylarginine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N-dimethylarginine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 2,3-diaminopropionic acid. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be the side chain of 2,4-diaminobutanoic acid or lysine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N-methyllysine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N-dimethyllysine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N-ethylysine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of citrulline. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N-dimethyllysine or β-homoarginine. At least two of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 3-(1-piperidinyl)alanine.

At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 3-guanidino-2-aminopropionic acid. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 4-guanidino-2-aminobutanoic acid. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of arginine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of homoarginine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N-methylarginine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N-dimethylarginine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 2,3-diaminopropionic acid. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be the side chain of 2,4-diaminobutanoic acid or lysine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N-methyllysine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N-dimethyllysine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N-ethylysine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of citrulline. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of N,N-dimethyllysine, β-homoarginine. At least three of R ₄ , R ₅ , R ₆ , and R ₇ may be side chains of 3-(1-piperidinyl)alanine.

AA _SC may be the side chain of a residue of asparagine, glutamine, or homoglutamine. AA _SC may be the side chain of a glutamine residue. cCPP may further comprise a linker conjugated to AA _SC , such as the residue of asparagine, glutamine, or homoglutamine. Accordingly, cCPP may further comprise a linker conjugated to an asparagine, glutamine, or homoglutamine residue. cCPP may further comprise a linker conjugated to a glutamine residue.

q can 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 to 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) has the structure of formula (I) (I) or a protonated form thereof, wherein AA _SC , R ₁ , R ₂ , R ₃ , R ₄ , R _{7 ,} m, and q is as defined herein.

cCPP of formula (A) has the structure of formula (I-a) or formula (I-b):

(Ia), (Ib),

or protonated forms thereof, wherein AA _SC , R ₁ , R ₂ , R ₃ , R ₄ , and m is as defined herein.

cCPP of formula (A) has the structure of formula (I-1), (I-2), (I-3), or (I-4):

(I-1), (I-2),

(I-3), (I-4)

or protonated forms thereof, wherein AA _SC and m are as defined herein.

cCPP of formula (A) has the structure of formula (I-5) or (I-6):

(I-5), or

(I-6) or protonated forms thereof, wherein AA _SC is as defined herein.

cCPP of formula (A) has the structure of formula (I-1):

(I-1), or a protonated form thereof,

where AA _SC and m are as defined herein.

cCPP of formula (A) has the structure of formula (I-2):

(I-2), or a protonated form thereof,

where AA _SC and m are as defined herein.

cCPP of formula (A) has the structure of formula (I-3):

(I-3), or a protonated form thereof,

where AA _SC and m are as defined herein.

cCPP of formula (A) has the structure of formula (I-4):

(I-4), or a protonated form thereof,

where AA _SC and m are as defined herein.

cCPP of formula (A) has the structure of formula (I-5):

(I-5), or a protonated form thereof,

where AA _SC and m are as defined herein.

cCPP of formula (A) has the structure of formula (I-6):

(I-6), or protonated forms thereof, where 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), FfΦGRGR (SEQ ID NO: 70); FfFGRGR (SEQ ID NO: 71); or FfΦGrGr (SEQ ID NO: 72). cCPP may have one of the following sequences: FGFΦ (SEQ ID NO: 73); GfFGrGrQ (SEQ ID NO: 74), FfΦGRGRQ (SEQ ID NO: 75); FfFGRGRQ (SEQ ID NO: 76); or FfΦGrGrQ (SEQ ID NO: 77).

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

(II)

During the ceremony:

AA _SC is the 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 , , 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 the integer 0, 1, 2, 3, 4, or 5;

Each n' is independently an integer 0, 1, 2, or 3;

When n' is 0, R ^2a , R ^2b , R ^2b , or R ^2d is absent.

At least two of R ^2a , R ^2b , R ^2c , and R ^2d , , or it may be a protonated form thereof. 2 or 3 of R ^2a , R ^2b , R ^2c , and R ^2d , , or it may be a protonated form thereof. One of R ^2a , R ^2b , R ^2c , and R ^2d , , or it may be a protonated form thereof. At least one of R ^2a , R ^2b , R ^2c , and R ^2d , 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 , 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.

R ^2a , R ^2b , R ^2c , and R ^2d are all , , or it may be a protonated form thereof. At least R ^2a , R ^2b , R ^2c , and R ^2d , 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 groups , 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.

R ^2a , R ^2b , R ^2c , and R ^2d each independently selected from 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, methyl lysine, dimethyl lysine, trimethyl lysine, homo-lysine, It may be serine, homo-serine, threonine, allo-threonine, histidine, 1-methylhistidine, 2-aminobutanedioic acid, aspartic acid, glutamic acid, or homo-glutamic acid.

AA _SC is or may be, where t may be an integer from 0 to 5. AA _SC is may be, where 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 a 6-membered to 14-membered aryl. R ^1a , R ^1b , and R ^1c may each independently be a 6- to 14-membered heteroaryl 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, pyridyl, quinolyl, or isoquinolyl. 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 pyridyl, quinolyl, or isoquinolyl.

Each n’ can independently be 1 or 2. Each n’ can be 1. Each n’ can be 2. At least one n’ can be 0. At least one n’ can be 1. At least one n’ can be 2. At least one n’ can be 3. At least one n’ can be 4. At least one n’ can be 5.

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

Each n” can independently be 1 or 2, and each n’ can independently be 2 or 3. Each n” can be 1, and each n’ can independently be 2 or 3. Each n” can be 1, and each n’ can be 2. Each n” is 1 and each n’ is 3.

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

(II-1),

In the formula, 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):

(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):

(IIb),

where R ^2a , R ^2b , AA _SC , and n' are as defined herein.

cCPP has the structure of formula (IIc):

(IIc), or a protonated form thereof,

During the ceremony:

AA _SC and n' are as defined herein.

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

, , , or , where AA _SC , and n are as defined herein.

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

or , where AA _SC , and n are as defined herein.

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

, or

, where AA _SC , and n are as defined herein.

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

.

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

cCPP may have the following structure in formula (III):

(III),

During the ceremony:

AA _SC is the 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 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;

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

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

(III-1),

During the ceremony:

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):

(IIIa),

During the ceremony:

AA _SC , R ^2a , R ^2c , R ^2b , R ^2d , n’, n”, and p’ are as defined herein.

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

p’ can be 0. p’ may be 1. p’ may be 2. p’ may be 3. p’ could be 4. p’ may be 5.

cCPP may have the following structure:

.

cCPP in equation (A) can be selected from:

cCPP in equation (A) can be selected from:

In an embodiment, cCPP is selected from:

In an embodiment, the cCPP is not selected from:

cCPP is the structure of formula (D) (D)

or a protonated form thereof, wherein:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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

Y is , or ego;

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4;

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

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

cCPP of formula (D) has the structure of formula (D-I):

(DI)

or may have a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4;

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

Y is am.

cCPP of formula (D) has the structure of formula (D-II):

(D-II)

or may have a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4;

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

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

Y is am.

cCPP of formula (D) has the structure of formula (D-III):

(D-III)

or may have a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4;

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

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

Y is am.

cCPP of formula (D) has the structure of formula (D-IV):

(D-IV)

or may have a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4;

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

Y is am.

cCPP of formula (D) has the structure of formula (D-V):

(DV)

or may have a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;

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

AA _SC is the amino acid side chain;

q is 1, 2, 3, or 4;

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

Y is am.

AA _SC can be conjugated to a linker.

linker

The cCPP of the present disclosure can be conjugated to a linker. A linker can connect the cargo to cCPP. The linker can be attached to the side chain of an amino acid of cCPP, and the cargo can be attached to the appropriate position on the linker.

The linker may be any suitable moiety capable of conjugating cCPP to one or more additional moieties, such as an exophytic peptide (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 can independently form a covalent bond to cCPP and one or more additional moieties. If the cargo is an oligonucleotide, the linker may be covalently attached to the 5' end of the cargo or to the 3' end of the cargo. The linker may be covalently attached to the 5' end of the cargo. The linker may be covalently attached to the 3' end of the cargo. If the cargo is a peptide, the linker may be covalently attached to the N-terminus or C-terminus of the cargo. The linker may be covalently attached to the backbone of the oligonucleotide or peptide cargo. The linker may be any suitable moiety that conjugates the cCPP described herein to a cargo such as an oligonucleotide, peptide, or small molecule.

Linkers may include hydrocarbon linkers.

The linker may contain a cleavage site. The cleavage site may be a disulfide site, or a caspase-cleavage site (eg, 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- JR ² )z”-subunits, wherein each of R ¹ and R ² is, at each occurrence, selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl. is independently selected; each J is independently C, NR ³ , -NR ³ C(O)-, S, and O; wherein R ³ is each optionally substituted H, alkyl, alkenyl, alkynyl, independently selected from carbocyclyl, and heterocyclyl; z” is an integer from 1 to 50; (viii) -(R ^1- J)z”- or -(JR ¹ )z”- (wherein each R ¹ is independently at each occurrence alkylene, alkenylene, alkynylene, carbocyclyl, or hetero cyclyl; each J is independently C, NR ³ , -NR ³ C(O)-, S, or O, where R ³ is H, alkyl, alkenyl, alkynyl, carbocyclyl, or hetero cyclyl, or each of these is optionally substituted; z” is an integer from 1 to 50; or (ix) a linker that may include one or more of (i) to (x).

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

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

The linker may contain one or more amino acids. Linkers may include peptides. The linker may include -(OCH ₂ CH ₂ ) _z' - (z' is an integer from 1 to 23) and a peptide. Peptides may contain 2 to 10 amino acids. The linker may additionally contain functional groups (FGs) capable of reacting through click chemistry. FG can be an azide or an alkyne, and a triazole is formed when the cargo is conjugated to the linker.

The linker consists of (i) a β alanine residue and a lysine residue; (ii) -(JR ¹ )z”; or (iii) a combination thereof. Each R ¹ may independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J may independently be C, NR ³ , -NR ³ C(O)-, S, or O, where 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 include (i) the residues of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof; and (ii) -(R ^1- J)z”- or -(JR ¹ )z”. Each R ¹ may independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J may independently be C, NR ³ , -NR ³ C(O)-, S, or O, where 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 include glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

The linker may be a trivalent linker. A linker may have the following structure: , In the formula, A ₁ , B ₁ , and C ₁ are independently a hydrocarbon linker (e.g. NRH-(CH ₂ ) _n -COOH), a PEG linker (e.g. NRH-(CH ₂ O) _n -COOH, where R is H , methyl, or ethyl), or one or more amino acid residues, and Z is independently a protecting group. The linker may incorporate a cleavage site containing the disulfide [NH ₂ -(CH 2 O) _n -SS-(CH ₂ O)n-COOH], or a caspase-cleavage site (Val-Cit-PABC).

The hydrocarbon may be the residue of glycine or beta-alanine.

The linker can be bivalent and connect cCPP to the cargo. The linker can be bivalent and connect cCPP to an exophytic peptide (EP).

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

The linker may be a divalent or trivalent C ₁ -C ₅₀ alkylene, where 1 to 25 methylene groups are -N(H)-, -N(C ₁ -C ₄ alkyl)-, -N(cycloalkyl)- , -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ N(C ₁ - C ₄ alkyl)-, -S(O) ₂ N(cycloalkyl)-, -N(H)C(O)-, -N(C ₁ -C ₄ alkyl)C(O)-, -N(cyclo Alkyl)C(O)-, -C(O)N(H)-, -C(O)N(C ₁ -C ₄ alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl , optionally and independently substituted by heteroaryl, cycloalkyl, or cycloalkenyl. The linker may be a divalent or trivalent C ₁ -C ₅₀ alkylene, where 1 to 25 methylene groups are -N(H)-, -O-, -C(O)N(H)-, or combinations thereof. is optionally and independently substituted by.

A linker may have the following structure:

, where: each AA is independently an amino acid residue; * is the point of attachment to AA _SC ; AA _SC is the side chain of the amino acid residues of cCPP; x is an integer from 1 to 10; y is an integer from 1 to 5; 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 between 2 and 4. z can 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 can be independently selected from glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminohexanoic acid.

cCPP can be attached to the cargo via a linker (“L”). The linker can be conjugated to the cargo via a linker (“M”).

A linker may have the following structure:

, where: 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 the point of attachment to AA _SC , where AA _SC is the side chain of the amino acid residue of cCPP; M is a linking group as defined herein.

A linker may have the following structure:

,

In the formula: 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 point of attachment to AA _SC , where AA _SC is the side chain of the amino acid residue of cCPP; M is a linking group as defined herein.

A linker may have the following structure:

In the formula: 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 point of attachment to AA _SC , and AA _SC is the side chain of the amino acid residue of cCPP.

x may be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (including all ranges and subranges therebetween).

x' is an integer from 1 to 23, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, It can be 20, 21, 22, or 23 (inclusive of all ranges and subranges in between). x’ can be an integer between 5 and 15. x’ can be an integer between 9 and 13. x’ can be an integer from 1 to 5. x’ may be 1.

y may be an integer from 1 to 5, for example, 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 between 3 and 5. y can be 3 or 4. y can be 4 or 5. y can be 3. y can be 4. y can be 5.

z may be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (including all ranges and subranges therebetween).

z' is an integer from 1 to 23, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, It can be 20, 21, 22, or 23 (inclusive of all ranges and subranges in between). z’ can be an integer between 5 and 15. z’ can be an integer from 9 to 13. z’ could be 11.

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

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

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

A linker may have the following structure:

,

During the ceremony

M is a group that conjugates L to a cargo (e.g., oligonucleotide);

AA _s is the side chain or terminus of an amino acid on cCPP;

Each AA _x is independently an amino acid residue;

o is an integer from 0 to 10;

y is an integer from 0 to 5.

A linker may have the following structure:

,

During the ceremony

M is a group that conjugates L to a cargo (e.g., oligonucleotide);

AA _s is the side chain or terminus of an amino acid on cCPP;

Each AA _x is independently an amino acid residue;

o is an integer from 0 to 10;

y is an integer from 0 to 5.

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

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

M can be selected from:

where: R ¹⁰ is alkylene, cycloalkyl, or , where a is 0 to 10.

M is And R ¹⁰ is and a is 0 to 10. M is It can be.

M is a heterobifunctional crosslinker, e.g. It may be, which is described in Williams et al. [ Curr. Protoc Nucleic Acid Chem. 2010 , 42 , 4.41.1-4.41.20, which is hereby incorporated by reference in its entirety.

M may be -C(O)-.

AA _s can be a side chain or terminal of an 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 with amino groups). AA _s may be AA _SC as defined herein.

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

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

p can 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.

A linker may have the following structure:

or ,

wherein M, AA _s , each of -(R ^1- JR ² )z”-, o, and z” are defined herein; r can be 0 or 1.

r may be 0. r may be 1.

A linker may have the following structure:

or ,

In the formula, each of M, AA _s , o, p, q, r, and z” may be as defined herein.

z” is 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, It can be 45, 46, 47, 48, 49, and 50 (inclusive of all ranges and values in between). z” can be an integer from 5 to 20. z” may be an integer from 10 to 15.

A linker may have the following structure:

,

During the ceremony:

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, wherein AC is complementary to the target in a pre-mRNA sequence further comprising L, the linker is conjugated to AC through a linker (M), and M is am.

Provided herein are compounds comprising cCPP and a cargo, wherein the cargo is an antisense compound (AC). For example, comprising an antisense oligonucleotide complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, the linker is conjugated to AC through a linker (M), and M is selected from:

, ; where: R ¹ is alkylene, cycloalkyl, or and t' is 0 to 10, each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, and R ¹ is and t' is 2.

A linker may have the following structure:

,

In the formula, AA _s is as defined herein, and m' is 0 to 10.

The linker may be a linker of the formula:

.

The linker may be a linker of the formula: , where “base” is the nucleobase at the 3’ end of the cargo phosphorodiamidate morpholino oligomer.

The linker may be a linker of the formula:

, where “base” corresponds to the nucleobase at the 3’ end of the cargo phosphorodiamidate morpholino oligomer.

The linker may be a linker of the formula:

, where “base” is the nucleobase at the 3’ end of the cargo phosphorodiamidate morpholino oligomer.

The linker may be a linker of the formula: , where “base” is the nucleobase at the 3’ end of the cargo phosphorodiamidate morpholino oligomer.

The linker may be a linker of the formula:

.

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

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

cCPP-linker conjugate

cCPP can be conjugated to linkers defined herein. The linker may be conjugated to the AA _SC of cCPP as defined herein.

The linker may comprise (e.g. as a spacer) -(OCH ₂ CH ₂ ) _z' -subunit, where z' is an integer from 1 to 23, for example 1, 2, 3, 4, 5, It is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. “-(OCH ₂ CH ₂ ) _z’ is also referred to as PEG. The cCPP-linker conjugate may have a structure selected from Table 7 :

cCPP-linker conjugate and SEQ ID NO: Cyclo (FfФ-4gp-r-4gp-rQ)-PEG ₄ -K-NH ₂ Cyclo (SEQ ID NO: 118)-PEG ₄ -K-NH ₂ Cyclo (FfФ-Cit-r-Cit-rQ)-PEG ₄ -K-NH ₂ Cyclo (SEQ ID NO: 119)-PEG ₄ -K-NH ₂ Cyclo (FfФ-Pia-r-Pia-rQ)-PEG ₄ -K-NH ₂ Cyclo (SEQ ID NO: 120)-PEG ₄ -K-NH ₂ Cyclo (FfФ-Dml-r-Dml -rQ)-PEG ₄ -K-NH ₂ Cyclo (SEQ ID NO: 121)-PEG ₄ -K-NH ₂ Cyclo (FfФ-Cit-r-Cit-rQ)-PEG ₁₂ -OH Cyclo (SEQ ID NO: 122)-PEG ₁₂ -OH Cyclo (fФR-Cit-R-Cit-Q)-PEG ₁₂ -OH Cyclo (SEQ ID NO: 123)-PEG ₁₂ -OH

The linker may include a -(OCH ₂ CH ₂ ) _z' -subunit, where z' is an integer from 1 to 23, and a peptide subunit. A peptide subunit may contain 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table 8 :

cCPP-linker conjugate and SEQ ID NO: Ac-PKKKRKV-Lys(cyclo[FfФ-Rr-Cit-rQ])-PEG ₁₂ -K(N ₃ )-NH ₂ Ac-SEQ ID NO: 42-Lys (cyclo[SEQ ID NO: 127])-PEG ₁₂ -K(N ₃ )-NH ₂ Ac-PKKKRKV-Lys(cyclo[FfФ-Cit-rR-rQ])-PEG ₁₂ -K(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-Lys (cyclo[SEQ ID NO: 128])-PEG ₁₂ -K(N ₃ )-NH ₂ Ac-PKKKRKV-K(cyclo(FfФR-cit-R-cit-Q))-PEG ₁₂ -K(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-K (Cyclo (SEQ ID NO: 129)) -PEG ₁₂ -K(N ₃ )-NH ₂ Ac-PKKKRKV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-Bk(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-PEG2-Lys(cyclo[SEQ ID NO: 130])-Bk(N ₃ )-NH ₂ Ac-PKKKRKV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG2-k(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-PEG2-Lys (cyclo[SEQ ID NO: 130])-PEG2-k(N ₃ )-NH ₂ Ac-PKKKRKV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG4-k(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-PEG2-Lys (cyclo[SEQ ID NO: 130])-PEG4-k(N ₃ )-NH ₂ Ac-PKKKRKV-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-k(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-Lys (cyclo[SEQ ID NO: 130])-PEG12-k(N ₃ )-NH ₂ Ac-pkkkrkv-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-k(N ₃ )-NH ₂ Ac- SEQ ID NO: 131-PEG2-Lys (cyclo[SEQ ID NO: 130])-PEG12-k(N ₃ )-NH ₂ Ac-rrv-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-OH Ac-rrv-PEG2-Lys (cyclo [SEQ ID NO: 130]) -PEG12-OH Ac-PKKKRKV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-k(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-PEG2-Lys (cyclo[SEQ ID NO: 130])-PEG12-k(N ₃ )-NH ₂ Ac-PKKK-Cit-KV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-k(N ₃ )-NH ₂ Ac- SEQ ID NO: 126-PEG2-Lys (cyclo[SEQ ID NO: 130])-PEG12-k(N ₃ )-NH ₂ Ac-PKKKRKV-PEG2-Lys( cyclo [FfФ-Cit-r-Cit-rQ] - PEG12-K(N ₃ )-NH ₂ Ac- SEQ ID NO: 42-PEG2-Lys ( cyclo [SEQ ID NO: 130] - PEG12-K(N ₃ )-NH ₂

EEVs comprising a circular cell penetrating peptide (cCPP), a linker, and an exocyclic peptide (EP) are provided. EEV has the structure of equation (B):

(B), or a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

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

EP is an exophytic peptide 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 to 4;

z’ is an integer from 1 to 23.

R ₁ , R ₂ , R ₃ , R ₄ , R ₇ , EP, m, q, y, x', z' are as described herein.

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

EEV has the structure of formula (B-a) or (B-b):

(Ba),

(Bb), or a protonated form thereof, wherein EP, R ¹ , R ² , R ³ , R ⁴ , m, and z’ are as defined in formula (B).

EEV has the structure of equation (B-c):

(BC),

or protonated forms thereof, wherein EP, R ¹ , R ² , R ³ , R ⁴ , and m are as defined above in formula (B); 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; z is an integer from 1 to 10.

EEV has the structure of formula (B-1), (B-2), (B-3), or (B-4):

(B-1),

(B-2),

(B-3),

(B-4), or a protonated form thereof, wherein EP is as defined above in formula (B).

EEV may comprise a structure of 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).

EEV may contain cCPP of the formula:

EEV may comprise the following formula: Ac-PKKKRKV-miniPEG ₂ -Lys(cyclo(FfFGRGRQ)-PEG ₂ -K(N ₃ ) (Ac-SEQ ID NO: 42-miniPEG ₂ -Lys(cyclo(SEQ ID NO: 81) -PEG ₂ -K(N ₃ )).

EEV can be:

EEV can be:

.

EEV is Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-miniPEG ₂ -K( cyclo (Ff-Nal-GrGrQ)-PEG ₁₂ -OH (Ac- SEQ ID NO: 134 -miniPEG ₂ -K ( cyclo (SEQ ID NO: 135) -PEG ₁₂ -OH).

EEV can be:

.

EEV can be Ac-PKKKRKV-miniPEG ₂ -K( Cyclo (Ff-Nal-GrGrQ)-PEG ₁₂ -OH (Ac-SEQ ID NO:42-miniPEG ₂ -K( Cyclo (SEQ ID NO:135)-PEG ₁₂ -OH) there is.

EEV can be:

.

EEV can be:

.

EEV can be:

EEV can be:

EEV can be:

.

EEV can be:

EEV can be:

EEV can be:

EEV can be:

.

EEV can be:

.

EEV can be:

.

EEV can be selected from:

EEV can be selected from:

Ac-PKKKRKV-Lys( cyclo [FfΦGrGrQ])-PEG ₁₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 42-Lys ( cyclo [SEQ ID NO: 80])-PEG ₁₂ -K(N ₃ )-NH ₂ )

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FfΦGrGrQ])-miniPEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 42-miniPEG ₂ -Lys( cyclo [SEQ ID NO: 80])-miniPEG ₂ -K(N ₃ )-NH ₂ )

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FGFGRGRQ])-miniPEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 42-miniPEG ₂ -Lys( cyclo [SEQ ID NO: 82])-miniPEG ₂ -K(N ₃ )-NH ₂ )

Ac-KR-PEG ₂ -K( cyclo [FGFGRGRQ])-PEG ₂ -K(N ₃ )-NH ₂

(Ac-KR-PEG ₂ -K( cyclo [SEQ ID NO: 82])-PEG ₂ -K(N ₃ )-NH ₂ )

Ac-PKKKGKV-PEG ₂ -K( cyclo [FGFGRGRQ])-PEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 46-PEG ₂ -K( cyclo [SEQ ID NO: 82])-PEG ₂ -K(N ₃ )-NH ₂ )

Ac-PKKKRKG-PEG ₂ -K( cyclo [FGFGRGRQ])-PEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 48-PEG ₂ -K( cyclo [SEQ ID NO: 82])-PEG ₂ -K(N ₃ )-NH ₂ )

Ac-KKKRK-PEG ₂ -K( cyclo [FGFGRGRQ])-PEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 19-PEG ₂ -K( cyclo [SEQ ID NO: 82])-PEG ₂ -K(N ₃ )-NH ₂ )

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FFΦGRGRQ])-miniPEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 42-miniPEG ₂ -Lys( cyclo [SEQ ID NO: 80])-miniPEG ₂ -K(N ₃ )-NH ₂ )

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [βhFfΦGrGrQ])-miniPEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 42-miniPEG ₂ -Lys( cyclo [SEQ ID NO: 142])-miniPEG ₂ -K(N ₃ )-NH ₂ )

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FfΦSrSrQ])-miniPEG ₂ -K(N ₃ )-NH ₂

(Ac-SEQ ID NO:42-miniPEG ₂ -Lys( cyclo [SEQ ID NO:143])-miniPEG ₂ -K(N ₃ )-NH ₂ ).

EEV can be selected from:

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo (GfFGrGrQ])-PEG ₁₂ -OH

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

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FGFKRKRQ])-PEG ₁₂ -OH

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

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FGFRGRGQ])-PEG ₁₂ -OH

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

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FGFGRGRGRQ])-PEG ₁₂ -OH

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

Ac-PKKKRKV-miniPEG ₂ -Lys( cyclo [FGFGRrRQ])-PEG ₁₂ -OH

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

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

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

Ac-PKKKRKV-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 can be selected from:

Ac-KKKRKG-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-KKKRK-miniPEG ₂ -K( cyclo [FGFGRGRQ])-PEG ₁₂ -OH

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

Ac-KRKRKK-PEG ₄ -K( cyclo [FGFGRGRQ])-PEG ₁₂ -OH

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

Ac-KRKKK-PEG ₄ -K( cyclo [FGFGRGRQ])-PEG ₁₂ -OH

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

Ac-KKKKR-PEG ₄ -K( cyclo [FGFGRGRQ])-PEG ₁₂ -OH

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

Ac-RKKKK-PEG ₄ -K( cyclo [FGFGRGRQ])-PEG ₁₂ -OH

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

Ac-KKKRK-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 can be selected from:

Ac-PKKKRKV-PEG ₂ -K( cyclo [FGFGRGRQ])-PEG ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 42-PEG ₂ -K( cyclo [SEQ ID NO: 82])-PEG ₂ -K(N ₃ )-NH ₂ )

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 ₂ -K(N ₃ )-NH ₂

(Ac- SEQ ID NO: 42-PEG ₂ -K( cyclo [SEQ ID NO: 133])-PEG ₂ -K(N ₃ )-NH ₂ ) and

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).

Cargo can be AC and EEV can be selected from:

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

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

Ac-PKKKRKV-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 [FfΦGrGrQ])-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 [FfΦGrGrQ])-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 [FfΦGrGrQ])-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 [FfΦGrGrQ])-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 [FfΦGrGrQ])-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 [FfΦGrGrQ])-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 [FfΦGrGrQ])-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),

where b is beta-alanine, and the extracircular sequence may be D or L stereochemistry.

Cargo

Cell-penetrating peptides (CPPs), such as cyclic cell-penetrating peptides (e.g., cCPP), can be conjugated to the cargo. As used herein, “cargo” is a compound or moiety that requires delivery into a cell. The cargo may be conjugated to the terminal carbonyl group of the linker. At least one atom of the cyclic peptide can be substituted for the cargo, or at least one single pair can form a bond to the cargo. Cargo can be conjugated to cCPP by a linker. The cargo can be conjugated to AA _SC by a linker. At least one atom of cCPP is substituted by the cargo, or at least one lone pair of cCPP forms a bond to the cargo. The hydroxyl group on the amino acid side chain of cCPP can be replaced by binding to the cargo. The hydroxyl group on the glutamine side chain of cCPP can be replaced by binding to the cargo. Cargo can be conjugated to cCPP by a linker. The cargo can be conjugated to AA _SC by a linker.

In embodiments, the amino acid side chain comprises a chemically reactive group to which a linker or cargo is conjugated. Chemically reactive groups may include amine groups, carboxylic acids, amides, hydroxyl groups, sulfhydryl groups, guanidinyl groups, phenol groups, thioether groups, imidazolyl groups, or indolyl groups. In an embodiment, the amino acids of cCPP to which the cargo is conjugated include lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, homoglutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, or tryptophan.

The cargo may comprise one or more detectable moieties, one or more therapeutic moieties (TM), one or more targeting moieties, or any combination thereof. In an embodiment, the cargo includes TM. In embodiments, the cargo includes AC.

Cyclic cell penetrating peptide (cCPP) conjugated to cargo moiety

Circular cell penetrating peptide (cCPP) can be conjugated to the cargo moiety.

The cargo moiety can be conjugated to a linker at the terminal carbonyl group to give the following structure:

, in the formula:

EP is the exophytic peptide, M, AA _SC , cargo, x', y, and z' are as defined above, * is the point of attachment to AA _SC , and x' may be 1. y can be 4. z' may be 11. -(OCH ₂ CH ₂ ) _x' - and/or -(OCH ₂ CH ₂ ) _z' - may be independently substituted with one or more amino acids, including for example: glycine, beta-alanine, 4- Aminobutyric acid, 5-aminopentanic acid, 6-aminohexanoic acid, or combinations thereof.

The endosomal escape vehicle (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an extracyclic peptide (EP), and a linker, which are conjugated to the cargo to form the structure of formula (C):

(C)

or form an EEV-conjugate comprising a protonated form thereof,

During the ceremony:

R ₁ , R ₂ , and R ₃ may each independently be H or an amino acid residue having a side chain containing an aromatic group;

R ₄ is H or an amino acid side chain;

EP is an exophytic peptide as defined herein;

Cargo is a moiety 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;

z’ is an integer from 2 to 20.

R ₁ , R ₂ , R _{3 ,} R ₄ , EP, cargo, m, n, x', y, q, and z' are as defined herein.

EEV can be conjugated to a cargo and the EEV-conjugate has the structure of formula (C-a) or (C-b):

(Ca),

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

EEV can be conjugated to a cargo, and the EEV-conjugate has the structure of formula (C-c):

(Cc),

or a protonated form thereof, wherein EP, R ¹ , R ² , R ³ , R ⁴ , 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 can be an integer from 1 to 10; y may be an integer from 1 to 5; z can be an integer from 1 to 10.

EEV can be conjugated to an oligonucleotide cargo, and the EEV-oligonucleotide conjugate can comprise a structure of formula (C-1), (C-2), (C-3), or (C-4):

(C-1),

(C-2),

(C-3),

(C-4)

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

.

Cell fluid delivery efficiency

Modifications to cyclic cell penetrating peptide (cCPP) can improve cytosolic transport efficiency. Improved cytosolic uptake efficiency can be measured by comparing the cytosolic transport efficiency of cCPPs with modified sequences to control sequences. The control sequence does not contain specific substituted amino acid residues (including but not limited to arginine, phenylalanine, and/or glycine) in the modified sequence, but is otherwise identical.

As used herein, cytosolic transport efficiency refers to the ability of cCPP to cross the cell membrane and enter the cytosol of a cell. The cytosolic transport efficiency of cCPP does not necessarily vary depending on the receptor or cell type. Cytosolic transfer efficiency may refer to absolute cytosolic transfer efficiency or relative cytosolic transfer 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 can be accomplished by fluorescently labeling cCPP (e.g. with FITC dye) and measuring the fluorescence intensity using techniques well known in the art.

Relative cytosolic transfer efficiency is determined by comparing (i) the amount of cCPP of the invention internalized by a cell type (e.g., HeLa cells) to (ii) the amount of control cCPP internalized by the same cell type. To measure relative cytosolic transport efficiency, cell types can be incubated in the presence of cCPP for a specified period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), and then the amount of cCPP internalized by the cells is measured as described in the art. Quantification is performed using known methods, for example fluorescence microscopy. Separately, equal concentrations of control cCPP are incubated in the presence of cell types over the same period of time, and the amount of control cCPP internalized by the cells is quantified.

Relative cytosolic delivery efficiency can be determined by measuring the IC ₅₀ of cCPP with modified sequences against an intracellular target and comparing the IC ₅₀ of cCPP with modified sequences to a control sequence (as described herein).

The relative cytosolic transport efficiency of cCPP ranges from about 50% to about 450%, for example about 60%, about 70%, about 80%, about 90%, about 100% compared to cyclo (FfФRrRrQ, SEQ ID NO: 150). , 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 It may be 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, or about 590%. (Includes all values and subranges between them). The relative cytosolic transport efficiency of cCPP can be improved by more than about 600% compared to a cyclic peptide comprising cyclo (FfФRrRrQ, SEQ ID NO: 150).

The absolute cytosolic transfer efficacy is about 40% to about 100%, for example 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% (any value in between) and subranges).

The cCPP of the present disclosure increases the cytosolic transfer efficiency by about 1.1 times to about 30 times, for example, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9 compared to the same sequence. , 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 22.0, about 22.5, about 23.0, about 23.5, about 24.0, about 24.5, about 25.0, about 25.5, about 26.0, about 26.5, about 27.0 , about 27.5, about 28.0, about 28.5, about 29.0, or about 29.5 times (including all values and subranges therebetween).

Detectable Moieties

In embodiments, compounds disclosed herein include a detectable moiety. In an embodiment, the detectable moiety is in the amino group, carboxylate group, or side chain of any of the amino acids of the cell-penetrating peptide moiety (e.g., in the amino group, carboxylate group, or side chain of any of the amino acids in CPP). ) is attached to a cell-penetrating peptide. In embodiments, the therapeutic moiety comprises a detectable moiety. A detectable moiety may include any detectable label. Examples of suitable detectable labels include UV-Vis labels, near-infrared labels, luminescent groups, phosphor groups, magnetic spin resonance labels, photosensitizers, photocleavable moieties, chelation centers, heavy atoms, radioactive isotopes, and isotopically detectable spin resonance labels. Including, but not limited to, labels, paramagnetic moieties, chromophores, or any combination thereof. In embodiments, the label is detectable without addition of additional 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 “biologically compatible” generally refer to compounds that are generally non-toxic to cells and tissues, including any of their metabolites or degradation products. It is a compound that does not cause any significant side effects to cells and tissues when cells and tissues are incubated (e.g., cultured) in its presence.

The detectable moiety may contain a luminophore, such as a fluorescent label or a near-infrared label. Examples of suitable luminophores include metal porphyrins; benzoporphyrin; Azabenzoporphyrin; naphtoporphyrin; phthalocyanine; polycyclic aromatic hydrocarbons such as perylene diimine, pyrene; azo dye; xanthene dye; boron dipioromethene, aza-boron dipioromethene, cyanine dyes, metal-ligand complexes such as bipyridine, bipyridyl, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; Oxazine derivatives such as acridine and benzophenoxazine; aza-annulene, squarine; Luminescence-producing nanoparticles and nanocrystals such as 8-hydroxyquinoline, polymethine, and quantum dots; Carbostyryl; Terbium complex; inorganic phosphor; Ionic carriers such as crown ether-related or derivatized dyes; or combinations thereof, but are not limited thereto. Specific examples of suitable luminophores include Pd (II) octaethylporphyrin; Pt (II)-octaethylporphyrin; Pd (II) tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II) meso-tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetraphenyl methylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II) octaethylporphyrin ketone; Pd (II) meso-tetra(pentafluorophenyl)porphyrin; Pt (II) meso-tetra (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-benzimidazol-2-yl)-7-(diethylamino)-coumarin));enzothiazole) ((benzothiazol-2-yl)-7-(di Ethylamino)-coumarin))-2-(acetylacetonate); lumogen dye; Macroflex Fluorescent Red; Macrolex Fluorescent Yellow; Texas Red; rhodamine B; Rhodamine 6G; rhodamine sulfur; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; Bromocresol Red; bromothymol blue; Cy2; Cy3; Cy5; Cy5.5; Cy7; 4-nitirophenol; alizarin; phenolphthalein; o -cresolphthalein; Chlorophenol Red; Kalmagit; bromo-xylenol; phenol red; neutral red; nitrazine; 3,4,5,6-tetrabromophenolphthalein; Congo Red; fluorescein; Eosin; 2',7'-dichlorofluorescein;5(6)-carboxy-fluorescein;carboxynaphthofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid; semi-naphthorodafluoro; semi-naphthofluorescein; Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride; (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron; Platinum(II) octaethylporphyine; dialkylcarbocyanine; dioctadecylcyclooxacarbocyanine; fluorenylmethyloxycarbonyl chloride; 7-amino-4-methylcoumarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof.

In some embodiments, the detectable moiety is rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), or It may include derivatives or combinations of.

Manufacturing method

The compounds described herein can be prepared in a variety of ways known to those skilled in the art of organic synthesis, or with modifications understood thereto by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.

Modifications to the compounds described herein include adding, subtracting, or moving various components as described for each compound. Similarly, when more than one chiral center is present in a molecule, the chirality of the molecule can vary. Additionally, compound synthesis may involve protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups, can be determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene (Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006), which is incorporated herein by reference in its entirety.

Starting materials and reagents used to prepare the disclosed compounds and compositions are from Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), and 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 from commercial suppliers such as Boehringer Ingelheim (Ingelheim, Germany), or prepared by methods known to those skilled in the art following the procedures set forth therein with reference to the following literature: 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 Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as pharmaceutical carriers disclosed herein, may be obtained from commercial sources.

The reactions producing the compounds described herein can be carried out in solvents, which can be selected by those skilled in the art of organic synthesis. The solvent may not substantially react with the starting materials (reactants), intermediates, or products under the conditions at which the reaction is conducted, i.e., temperature and pressure. The reaction can be carried out in one solvent or a mixture of two or more solvents. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation is monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., ¹ H or ¹³ C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry; It can be monitored 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. These protecting groups must be easily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein, while being stable against the conditions of peptide bond formation. Suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, iso Bornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, etc. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. Other preferred side chain protecting groups are: for side chain amino groups such as lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; For tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclophenyl, and acetyl (Ac); For serine, t-butyl, benzyl, and tetrahydropyranyl; For histidine, trityl, benzyl, Cbz, p-toluenesulfonyl, and 2,4-dinitrophenyl; For tryptophan, formyl; for aspartic acid and glutamic acid, benzyl and t-butyl; and for cysteine, triphenylmethyl (trityl).

In solid-phase peptide synthesis methods, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for this synthesis are materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction as well as insoluble in the medium used. Solid supports for the synthesis of α-C-terminal carboxy peptides were 4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or 4-(, available from Applied Biosystems (Foster City, Calif.). It is 2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin. The α-C-terminal amino acids are 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), and benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). , or N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide, with or without bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl). bound to a resin by med (DIC), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU), such as dichloromethane or DMF. The coupling is mediated in a solvent at a temperature of 10° C. to 50° C. for about 1 to about 24 hours. When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, prior to coupling with the α-C-terminal amino acid as described above, Fmoc The group is cleaved with a secondary amine, preferably piperidine. One method for coupling to deprotected 4(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N in DMF. ,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). Coupling of sequential protected amino acids can be performed in an automated polypeptide synthesizer. In one embodiment, the α-N-terminus of the amino acids of the growing peptide chain is protected with Fmoc. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is achieved by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in approximately 3-fold molar excess and coupling is preferably performed in DMF. The coupling agent is O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) ) can be. After solid phase synthesis is complete, the polypeptide is removed from the resin and deprotected, which can be done sequentially or in a single operation. Removal and deprotection of the polypeptide can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thianisole, water, ethanedithiol, and trifluoroacetic acid. When the α-C-terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, peptides can be removed by transesterification (e.g. using methanol) followed by aminolysis or direct transamidation. Protected peptides can be purified at this point or proceed directly to the next step. Removal of side chain protecting groups can be achieved using the cleavage cocktail described above. Fully deprotected peptides can be purified by a sequence of chromatographic steps using any or all of the following types: ion exchange using a mildly basic resin (acetate form); Hydrophobic adsorption chromatography using nonderivatized polystyrene-divinylbenzene (e.g., Amberlite XAD); silica gel adsorption chromatography; Ion exchange chromatography using carboxymethylcellulose; Partition chromatography using, for example, Sephadex G-25, LH-20, or countercurrent partitioning; High-performance liquid chromatography (HPLC), especially reversed-phase HPLC using octyl- or octadesilyl-silica bonded phase column packing.

Such polymers, such as PEG groups, can be attached to oligonucleotides such as AC under any suitable conditions. Any means known in the art may be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation, or conjugation of reactive groups on the PEG moiety (e.g., aldehyde, amino, ester, thiol, α). -Chemiselectively to a reactive group on the AC (e.g., an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazino group) via a haloacetyl, maleimido, or hydrazino group. Other methods of joining/ligating are included. Activating groups that can be used to link water-soluble polymers to one or more proteins include sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidiline, oxirane, 5-pyridyl, and alpha-halogenated azide. Includes, but is not limited to, real groups (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). When attached to AC by reductive alkylation, the polymer selected should have a single reactive aldehyde so that the extent of polymerization is controlled. For example, 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 link an AC or linker directly to a CPP, the appropriate amino acid residue of the CPP can be reacted with an organic derivative capable of reacting with the side chain or N-terminus or C-terminus of the selected amino acid. Reactive groups on the peptide or conjugate moiety include, for example, aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazino groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation via a cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, or others known in the art. Includes preparations.

Methods for making AC and joining AC to linear CPP are generally described in US Patent Publication No. 2018/0298383, which is incorporated herein by reference for all purposes. The method can be applied to the cyclic CPP disclosed herein.

Synthetic schemes are provided in Figures 3a-3d and Figure 4 .

Non-limiting examples of compounds containing CPP and reactive groups useful for conjugation to AC are shown in Table 9 . An exemplary linkage is also shown. Exemplary reactive groups include tetrafluorophenyl ester (TFP), free carboxylic acid (COOH), and azide (N ₃ ). In Table 9 , n is an integer from 0 to 20; Pipa6 is AcRXRRBRRXRYQFLIRXRBRXRB, where B is β-alanine and X is aminohexanoic acid; Dap is 2,3-diaminopropionic acid; NLS is a nuclear localization sequence; βA is beta alanine; -ss- is disulfide; PABC is the poly(A) binding protein C-terminal domain; C _x is an alkyl chain of length x (x is a number); BCN is bicyclo[6.1.0]nonine.

Compounds containing CPP and reactive groups TFP- _PEGn -K(CPP) TFP-PEG _n -K(CPP)-PEG _n -Dap(palmitoyl) TFP-PEG _n -K(CPP)-PEG _n -Dap(CPP) TFP-Pip6a CPP- _PEGn -TFP CPP- _PEGn -K(CPP) _-PEGn -TFP CPP-PEG _n -Lys(N ₃ ) CPP-K(CPP)-PEG _n -K(N ₃ ) CPP-PEG _n -K(PEG _n -CPP)-PEG _n -K(N ₃ ) CPP-PEG _n -K(PEG _n -CPP)-PEG _n -K(N ₃ ) CPP-K(CPP)-K(CPP)-PEG _n -K(N ₃ ) CPP-PEG _n -K(PEG _n -CPP)-K(PEG _n -CPP)-PEG _n -K(N ₃ ) CPP-PEG _n -K(PEG _n -CPP)-K(PEG _n -CPP)-PEG _n -K(N ₃ ) Ac-NLS-Lys(CPP)-PEG _n -K(N ₃ ) K(N ₃ )-PEG _n -NLS-ss-PEG _n -CPP BCN-NLS-ss-CPP CPP- _PEGn -Val-Cit-PABC-K(N ₃ ) CPP-PEG _n -Cys-ss-Cys-K(N ₃ ) CPP-PEG _n -Cys-ss-Cys-K(N ₃ ) CPP- _PEGn -TFP CPP-PEG _n -Lys(N ₃ ) CPP-PEG _n -Cys-prodisulfide-K(N ₃ ) CPP-PEG _n -K(N ₃ ) CPP-K(CPP)-PEG _n -K(N ₃ ) CPP- _PEGn -K(CPP) _-PEGn -TFP CPP- _C6 -TFP CPP-PEG _n -K(PEG _n -CPP)PEG _n -K(N ₃ ) Ac-T9-PEG _n -Lys(CPP-PEG _n )-K(N ₃ ) Ac-MSP-PEG _n -K(CPP-PEG _n )-K(N ₃ ) CPP- _PEGn -TFP (ENTRD 802) CPP-C ₆ -TFP (ENTRD 696) CPP-PEG _n -K(CPP)-PEG _n -TFP (ENTRD-344) CPP-PEG _n -COOH CPP-C ₁₂ -TFP (ENTD-695) Palmitoyl-PEG _n -K(CPP)-PEG _n -TFP (ENTD-343) CPP-PEG _n -K(N ₃ ) (ENTRD-617) Ac-T9-PEG _n -K(CPP)-K(N ₃ ) (ENTRD 673) Ac-MSP-PEG _n -K(CPP-PEG _n )-K(N ₃ ) (ENTRD 675) Ac-NLS-K(CPP)-PEG _n -K(N ₃ ) (ENTRD 684) K(N3)-PEG _n -NLS-ss-PEG _n -CPP (ETRD-681) K(N3)-PEG _n -NLS-K-βA-βA-CPP (ETRD-682)

In an embodiment, CPP has a free carboxylic acid group that can be used to conjugate to AC. In embodiments, EEV has a free carboxylic acid group that can be used to conjugate to AC.

The structure below is a 3' cyclooctyne modified PMO used for click reactions with compounds containing azides:

An exemplary scheme for conjugating CPP and a linker to the 3' end of AC via an amide bond is shown below.

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

Examples of conjugation chemistries used to link AC and CPP with additional linkers containing polyethylene glycol moieties are shown below:

An example of conjugation of a CPP-linker to a 5'-cyclooctyne modified PMO via lineage-promoted azide-alkyne cycloaddition (click chemistry) is shown below:

Methods for synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited by the method of synthesizing AC. In embodiments, provided herein are compounds with reactive properties that are useful for forming internucleoside linkages, including, for example, linkages between phosphodiester and phosphorothioate nucleosides. Methods for making and/or purifying precursor or antisense compounds are not limited to 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 performed using DNA (Protocols for Oligonucleotides and Analogs, (1993), Humana Press) and/or RNA (Scaringe, Methods). (2001), 23, 206-217]; Gait et al. [Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36]; Gallo et al. [Tetrahedron (2001), 57 , 5707-5713]) can be routinely performed according to literature procedures.

Antisense compounds provided herein can be conveniently and routinely prepared through well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be used. It is well known to use similar techniques to prepare oligonucleotides such as phosphorothioates and alkylated derivatives. The present invention is not limited by the method of synthesizing antisense compounds.

Methods for oligonucleotide purification and analysis are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. These synthesis and analysis methods can be performed in multi-well plates. The method of the present invention is not limited by the oligomer purification method.

In the compounds disclosed herein, AC is linked to CPP (eg, a cyclic peptide). As used herein, “bound” may refer to a covalent or non-covalent bond between a CPP and an AC, including but not limited to fusing a CPP to an AC; and chemically conjugating CPP (e.g., cyclic peptide) to AC. A non-limiting example of a means to non-covalently attach CPP to AC is through streptavidin/biotin interactions, for example, by conjugating biotin to CPP and fusing AC to streptavidin. In the resulting compound, CPP is bound to AC through a non-covalent bond between biotin and streptavidin.

In embodiments, a CPP (e.g., a cyclic peptide) is conjugated directly or indirectly to AC to form 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 AC may be conjugated to the C-terminus, N-terminus, or side chain of an amino acid in CPP.

In an embodiment, 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' ends of the AC moiety. These conjugates may alternatively be described as having a CPP moiety (eg, a cyclic peptide moiety) and an oligonucleotide moiety. According to certain embodiments, a covalently linked AC-CPP or CPP-AC conjugate comprises an AC component and a CPP component linked to each other by a linker described herein.

In embodiments, AC may be conjugated to a CPP (e.g., a cyclic peptide) through the side chain of an amino acid on the CPP. Any amino acid side chain on the CPP that can form a covalent bond or be so modified can be used to link the AC to the CPP. Amino acids on CPP may be natural or non-natural amino acids. In an embodiment, the amino acid on the CPP used to conjugate the AC is aspartic acid, glutamic acid, glutamine, asparagine, lysine, ornithine, 2,3-diaminopropionic acid, or an analog thereof, wherein the side chain is for the AC or linker. replaced by bonding. In an embodiment, the amino acid is lysine or an analog thereof. In an embodiment, the amino acid is glutamic acid or an analog thereof. In an embodiment, the amino acid is aspartic acid or an analog thereof.

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

,

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

Diseases and target genes

In embodiments, compounds and methods are provided for treating diseases or disorders associated with one or more genes having extended nucleotide repeats (e.g., extended trinucleotide repeats, such as extended trinucleotide repeats). In embodiments, compounds and methods are provided for treating diseases or disorders associated with one or more genes having expanded CTG · CUG trinucleotide repeats. In embodiments, compounds and methods are provided for treating diseases or disorders associated with one or more genes having expanded CTG·CUG trinucleotide repeats in the 3'-UTR of the gene. In embodiments, compounds and methods are provided for treating diseases or disorders associated with genes with extended CTG·CUG in the 3' UTR, such as DMPK, ATXN8OS ATXN8, and/or JPH3. In embodiments, compounds and methods are provided for treating diseases or disorders associated with one or more genes having expanded CTG·CUG trinucleotide repeats in the introns of the genes. In embodiments, compounds and methods are provided for treating diseases or disorders associated with expanded CTG·CUG trinucleotide repeats in the intron of TCF4. In embodiments, compounds and methods are provided for treating myotonic dystrophy type 1 (DM1), Fuchs' corneal endothelial dystrophy (FECD), spinocerebellar ataxia type 8 (SCA8), and/or Huntington's disease pseudo-type 2 (HDL2). do.

Myotonic dystrophy type 1 (DM1)

In embodiments, compounds, compositions, and methods for treating myotonic dystrophy (DM1 or Steinert disease) are provided. DM1 is a multisystem disorder characterized by muscle degeneration and myotonia, or delayed muscle relaxation, often due to repetitive action potentials in muscle fibers. Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy, affecting approximately 1 in 8,000 people. DM1 is a paradigm for genetic disorders caused by CTG·CUG expansion. DM1 is a neuromuscular disorder caused by CTG·CUG repeat expansions in the 3’-untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK) gene. At the RNA level, DMPK transcripts (e.g., expanded CUG repeats) sequester splicing regulatory proteins, such as muscleblind-like (MBNL) proteins, which regulate a number of downstream pre- It results in incorrect splicing of mRNA (pre-mRNA that does not contain expanded CUG repeats). This gain of function is the cause of DM1.

Excessive numbers of CUG repeats confer toxic activity, referred to as toxic gain of function. A number of key proteins are incorrectly processed, contributing to the multisystemic nature of the disease, which includes generalized limb weakness, respiratory muscle damage, cardiac abnormalities, fatigue, gastrointestinal complications, cataracts, incontinence, and excessive daytime sleepiness.

DM1 patients with CTG·CUG expansions 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]). Binding of MBNL1 to mutant RNA affects the cellular pool of free MBNL1 and causes mis-splicing of some MBNL1 target genes (e.g., genes regulated by MBNL1) in infected brain, muscle, and heart tissues. (Jiang et al. [Hum Mol Genet. 2004; 13: 3079-3088]). Gattey et al. (Cornea. 2014; 33: 96-98) reported FECD in four DM1 subjects, including mother-daughter pairs. Therefore, it is likely that an association exists between DM1 and FECD (FECD is described in more detail elsewhere herein).

Without wishing to be bound by theory, at least two hypotheses have been proposed to explain the pathogenesis of DM1. One is that expanded CTG·CUG repeats suppress DMPK mRNA or protein production, resulting in DMPK haploid dysfunction. This was supported by a study demonstrating decreased expression of DMPK mRNA and protein in DM1 muscle (Fu, Y.H. et al. [(1993) Reduced 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 haplodysfunction. Another RNA gain-of-function hypothesis proposes that mutant RNA transcribed from the expanded allele is sufficient to induce disease symptoms. This is suggested by the observations: (i) expanded CTG repeats are transcribed into CUG repeats and accumulate in discrete nuclear foci; (ii) Expression of DMPK 3'-UTR with 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 RNA associated with an expanded allele.

Expanded CTG·CUG trinucleotide repeats in the 3' untranslated region of DMPK mRNA form incomplete stable hairpin structures, which accumulate in the cell nucleus as small ribonuclear complexes or microscopic inclusions, and are involved in transcription, splicing, and transcription processes. Alternatively, it impairs the function of proteins involved in RNA export. Although the DMPK gene with CUG repeats is transcribed into mRNA, the mutant transcripts are sequestered as aggregates (foci) in the nucleus, resulting in decreased cytoplasmic DMPK mRNA levels. This aggregation leads to loss of regulation of alternative splicing of many different transcripts due to sequestration of two RNA binding proteins: muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1), resulting in loss of function of MBNL1. and causes upregulation of CUGBP1 (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 CUG repeats and is depleted from the nucleoplasm. MBNL1-bound CUG repeats then stimulate protein kinase C (PKC) activity through an unknown mechanism, which leads to CUGBP1 hyperphosphorylation and stabilization. Downstream effects include disruption of alternative splicing, mRNA translation, and mRNA decay of downstream genes. An important molecular feature of DM1 is the misregulation of alternative splicing due to the sequestration of MBNL1 to the CUG repeats by a double-stranded hairpin structure. Among the more than 24 splicing events misregulated in DM1, aberrant splicing of skeletal muscle-specific chloride channel 1 (ClC-1) is known to be one of the causes of myotonia. Increased inclusion of exons containing premature stop codons results in downregulation of ClC-1 mRNA and protein, which is sufficient to cause myotonia (Charlet-B et al. (2002) Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol. Cell 10, 45-53]; Mankodi, A. et al. [(2002) Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol. Cell 10, 35-44]). In embodiments, the compounds and methods described herein improve downstream effects, including disruption of alternative splicing, mRNA translation, and mRNA decay of downstream genes. In embodiments, the compounds and methods described herein reduce the number of misregulated splicing events in DM1 compared to subjects with DM1 who were not treated with a compound or method of the present disclosure. For example, in some embodiments, the compounds and methods described herein can reduce the number of misregulated 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 premature stop codons that result in downregulation of ClC-1 mRNA compared to subjects with DM1 not treated with a compound or method of the present disclosure. I order it.

The levels of MBNL1 and CUGBP1 in the nucleus regulate a subset of developmentally regulated splicing events that are reversed in DM1. At embryonic stages, MBNL1 nuclear levels are low and CUGBP1 levels are high. During development, MBNL1 nuclear levels increase, while CUGBP1 levels decrease, resulting in increased inhibition of downstream splicing targets (including IR exon 11, ClC-1 exon containing a stop codon, and cTNT exon 5) from embryo to adult. induce metastasis. However, in DM1, MBNL1 is sequestered to CUG repeats, reducing functional MBNL1, while CUGBP1 levels increase due to phosphorylation and stabilization. This simulates embryonic conditions and enhances the expression of embryonic isoforms in the adult, resulting in multiple 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, and reduce CUGBP1 levels compared to subjects with DM1 not treated with a compound or method of the present disclosure. I order it.

MBNL1 and CELF1 (also referred to as “CUGBP1”) are developmental regulators of splicing events during fetal transition to adulthood, and in DM1, their active modifications result in expression of fetal splicing patterns in adult tissues. Downstream effects of low MBNL1 and high CELF1 include, in addition to MBNL1, cardiac troponin T (cTNT), insulin receptor (INSR), muscle-specific chloride ion channel (CLCN1), and sarcoplasmic/endoplasmic reticular calcium ATPase. These include disruption of alternative splicing, mRNA translation, and mRNA decay in proteins such as 1(ATP2A1) transcripts. Konieczny et al. (2017) “Myotonic dystrophy: candidate small molecule therapeutics,” Drug Discovery Today. 22(11):1740-1748].

Compounds and methods for treating myotonic dystrophy using antisense oligomers targeting polyCUG repeats in the 3'-UTR of the DMPK gene are described in US10106796B2, US10111962B2, US20150080311A1, each of which is incorporated in its entirety for all purposes. Incorporated herein by reference. However, these PMOs or PPMOs targeting CUG repeats to treat DM1 may have limitations in uptake and delivery to muscle, the diseased tissue.

In embodiments, the present disclosure provides for the use of various cell penetrating peptides (CPPs) to deliver ACs (e.g., PMOs or ASOs) and cleavage sequences described herein (e.g., in Tables 2 and 10 ) to the cytosol of cells. I teach. In an embodiment, CPP or EEV conjugated with an AC delivers the AC of interest to the cellular location where the target sequence on the pre-mRNA is located.

In an embodiment, the condition is a form of myotonic dystrophy (e.g., myotonic dystrophy type 1 or myotonic dystrophy type 2). In an embodiment, the target gene is the DMPK gene encoding myotonic-protein kinase. In embodiments, compounds provided herein include ACs (e.g., ASOs) that target DMPK (e.g., 3'-untranslated region/polyadenylation of the DMPK gene) to degrade the DMPK gene. Exemplary oligonucleotides targeting DMPK for degradation are provided in Table 10 . Degradation sequences can be used in combination with AC sequences containing 10-40 CAG repeats, including but not limited to the ACs provided in Table 2 .

Oligonucleotides (AC) targeting DMPK for degradation. Oligo(AC) ID Sequence (5' to 3' direction) sequence number target 5'-CAG CAG CAG CAG CAG CAG CAG-3'-Click-K-PEG12-Lys(CPP12)-NLS-AC
(all PMO monomers) DMPK DMPK-A-17 GGGCCTTTTATTCGCGAGGGTCGGG 151 DMPK DMPK-A-18 GAGGGCCTTTATTCGCGAGGGTCG 152 DMPK DMPK-A-19 TGGAGGGCCTTTTATTCGGCGAGGGT 153 DMPK DMPK-A-20 GATGGAGGCCTTTTATTCGGCGAGG 154 DMPK DMPK-A-21 CAGATGGAGGGCCTTTTATTCGCGA 155 DMPK DMPK-A-22 GGCAGATGGAGGGGCCTTTTATTCGC 156 DMPK DMPK-A-23 TGGGCAGATGGAGGGCCTTTTATTC 157 DMPK DMPK-A-24 TTGGGCAGATGGAGGGCCTTTTAT 158 DMPK DMPK-A-25 GCTTTGGGCAGATGGAGGGCCTTTT 159 DMPK DMPK-A-26 GAGCTTTGGGCAGATGGAGGGCCTT 160 DMPK DMPK-A-27 CAGAGCTTTGGGCAGATGGAGGGCC 161 DMPK DMPK-A-28 TCCAGAGCTTTGGGCAGATGGAGGG 162 DMPK DMPK-A-29 AGTCCAGAGCTTTGGGCAGATGGAG 163 DMPK DMPK-A-30 GGAGTCCAGAGCTTTGGGCAGATGG 164 DMPK DMPK-A-31 GTGGAGTCCAGAGCTTTGGCAGAT 165 DMPK DMPK-A-32 CTGTGGAGTCCAGAGCTTTGGGCAG 166 DMPK DMPK-A-33 CACTGTGGAGTCCAGAGCTTTTGGGC 177 DMPK DMPK-A-34 GACACTGTGGAGTCCAGAGCTTTGG 178 DMPK DMPK-A-35 CGGACACTGTGGAGTCCAGAGCTTT 179 DMPK DMPK-A-36 CGCGGACACTGTGGAGTCCAGAGCT 180 DMPK DMPK-A-37 ACCGCGGACACTGTGGAGTCCAGAG 181 DMPK DMPK-A-38 AAACCGCGGACACTGTGGAGTCCAG 182 DMPK DMPK-A-39 GCAAACCGCGGACACTGTGGAGGTCC 183 DMPK DMPK-A-40 ACGCAAACCGCGGACACTGTGGAGT 184 DMPK DMPK-A-41 CAACGCAAACCGCGGACACTGTGGA 185 DMPK

Spinocerebellar ataxia type 8 (SCA8)

In embodiments, compounds, compositions, and methods for treating spinocerebellar ataxia type 8 (SCA8) are provided. SCA8 is an inherited neurodegenerative condition characterized by slowly progressive ataxia. Symptoms generally appear in the 30s to 50s. Symptoms include oculomotor abnormalities, sensory neuropathy, dysphagia, cerebellar ataxia, and cognitive impairment.

SCA8 is associated with heterozygous abnormal expanded CTG·CUG repeats in the 3’ UTR of two overlapping genes, ATXN8OS and ATXN8. Healthy individuals typically have 15 to 50 CTG·CUG repeats in the ATXN8OS and ATXN8 genes. SCA8 patients have more than 50 CTG·CUG repeats in the ATXN8OS and ATXN8 genes, and may have as many as 240 CTG·CUG repeats.

Huntington's disease pseudo-type 2 (HDL2)

In embodiments, compounds, compositions, and methods for treating Huntington's disease-like-2 (HDL2) disease are provided. HDL2 is an autosomal dominant neurodegenerative disorder phenotypically related to Huntington's disease. HDL2 is characterized by symptoms including chorea, dystonia, rigidity, bradykinesia, and psychiatric symptoms such as dementia. Symptoms of HDL2 typically occur in middle age and can cause premature death by about 10 to 15 years.

HDL2 is associated with an expanded CTG·CUG in the 3' UTR of the junktophilin 3 (JPH3) gene (16q24.3). Healthy individuals typically have 6 to 27 CTG·CUG repeats in the JPH3 gene. HDL2 patients have more than 40 CTG·CUG repeats in the JPH3 gene, and sometimes have more than 60 CTG·CUG repeats.

Fuchs' Endothelial Corneal 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 corneal endothelium. FECD is characterized by progressive loss of corneal endothelial cells, thickening of Descemet's membrane, and deposition of extracellular matrix in the form of guttae. When the number of endothelial cells becomes very low, the cornea swells, causing 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, type VIII, alpha 2 gene (COL8A2, MIM 120252) can cause early onset corneal endothelial dystrophy. Solute carrier family 4, sodium borate transporter, member 11 (SLC4A11, MIM 610206), transcription factor 8 (TCF8, MIM 189909), lipoxygenase homology domain 1 (LOXHD1, MIM 613267), and ATP/GTP binding protein- Other genes, such as pseudo-1 (AGBL1, MIM 615523), are collectively associated with a small proportion of adult-onset FECD cases. Genome-wide association studies of adult-onset FECD have implicated transcription factor 4 (TCF4, MIM 602272) and, more recently, KN motif- and ankyrin repeat domain-containing protein 4 (KANK4, MIM 614612) and laminin gamma-1 (LAMC1). , MIM150290), Na ⁺ / K ⁺ transport ATPase, and beta-1 polypeptide (ATP1B1, MIM 182330), and the mentioned TCF4 locus has a dominant effect on FECD (Mootha et al. [Investigative ophthalmology & visual science, 2017;58, 4579-4585]).

Expanded trinucleotide repeats at the CTG18.1 locus in intron 2 of TCF4 are associated with FECD (Wieben et al., PLoS One. 2012; 7(11):e49083). Each copy of the CTG18.1 allele with an expansion of more than 40 CTG·CUG trinucleotide repeats carries a significant risk for the development of FECD (Mootha et al. [Invest Ophthalmol Vis Sci. 2014; 55: 33-42] ). RNA nuclear foci, a hallmark of toxic gain-of-function RNA, have been reported in neurodegenerative disorders caused by simple repeat expansions. Expanded CUG repeat RNA accumulates as nuclear foci in the corneal endothelium of FECD subjects with the CTG18.1 triplet expansion, whereas it is absent in control samples lacking the triplet expansion (Mootha et al., Invest Ophthalmol Vis Sci. 2015;56(3):2003-2011]). Expanded CUG repeat RNA colocalizes in nuclear foci of the endothelium with the mRNA-splicing factor muscleblind-like 1 (MBNL1) as a molecular signature. Triplet repeat expansion at the CTG18.1 locus may mediate endothelial dysfunction through aberrant gene splicing due to mutant CUG RNA transcripts sequestering MBNL1 (Du et al., J Biol Chem. 2015; 290: 5979-5990]). Therefore, two distinct triplet repeats converge on the RNA foci and FECD, with the foci likely playing a causal role for FECD.

In embodiments, compounds and methods useful for the treatment of Fuchs endothelial corneal dystrophy (FECD), which reduce expanded CUG repeat RNA with antisense oligonucleotides, are described in WO2018165541A1, US10760076B2, each of which is incorporated in its entirety for all purposes. Incorporated herein by reference. However, such phosphorodiamidate morpholino oligomers (PMOs) or peptide-conjugated PMOs (PPMOs) that target the CUG repeat to treat DM1, such as those described in the prior art, do not target the diseased tissue, e.g. : There may be limitations in delivering oligos to the endothelial layer within the cornea.

In embodiments, the present disclosure provides various cell penetrating peptides (CPP) or endosomal escape vehicles (EEV) for delivering ACs (e.g., PMOs or ASOs) described herein (e.g., in Table 6) to the cytosol of cells. Explain its use. In an embodiment, CPP or EEV conjugated to an AC delivers the AC of interest to the cellular location where the target sequence on the pre-mRNA is located.

In an embodiment, the disease is Fuchs corneal endothelial dystrophy (FECD). In an embodiment, the target gene is TCF4 , which encodes transcription factor 4 (TCF-4), also known as immunoglobulin transcription factor 2 (ITF-2). In embodiments, compounds provided herein include antisense oligonucleotides that target TCF4 . Exemplary oligonucleotides that can be used to target TCF4 are provided in Table 2 and Table 11 .

Exemplary Oligonucleotides Targeting the Expanded Triplet Repeat of TCF4 oligo chemistry design target 21-mer PMO 5'-CAG CAG CAG CAG CAG CAG CAG -3'
(All PMO monomers; SEQ ID NO: 146) TCF4(CUG)n 25-mer PMO 5'-CAG CAG CAG CAG CAG CAG CAG CAG C-3'
(All PMO monomers; SEQ ID NO: 147) TCF4(CUG)n 21-mer EEV-NLS-PMO EEV-NLS_PMO_CAG 21-mer
(Conjugation from SEQ ID NO: 146) TCF4(CUG)n 30-mer PMO 5'-CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG-3'
(All PMO monomers; SEQ ID NO: 148) TCF4(CUG)n 30-mer PMO 5'-AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC-3'
(All PMO monomers; SEQ ID NO: 149) TCF4(CUG)n 30-mer PMO 5'-GCA GCA GCA GCA GCA GCA GCA GCA GCA GCA-3'
(All PMO monomers; SEQ ID NO: 150) TCF4(CUG)n

PMO: phosphorodiamidate morpholino oligomerMootha et al. reported that DM1 and FECD are not all the same disease but result from non-coding CTG expansions (Mootha et al. (2017)). DMPK expansion in DM1 results in a multisystem disease involving various tissues of the eye, including the lens, retina, and corneal endothelium. In contrast, TCF4 repeat expansions appear to affect the corneal endothelium without clinically apparent sequelae on other ocular tissues or body organs. Mutant expansions in DMPK and TCF4 share important similarities, including (i) nuclear foci containing proliferated CUG repeats, (ii) association of foci with the MBNL1 protein, and (iii) the ability to cause FECD. It is suggested that triplet expansions in both DMPK and TCF4 may cause the same corneal endothelial tissue phenotype of FECD through shared molecular mechanisms.

See US Patent No. 10760076B2, International Application Publication No. WO2018165541A1, US Patent Application Publication No. 2016/0355796, and US Patent Application Publication No. 2018/0344817, each of which is incorporated herein by reference; Disclosed are diseases and corresponding genes prone to forming and/or expanding tandem nucleotide repeats.

Compositions and Administration Methods

Compounds of the present disclosure can be formulated into compositions suitable for in vivo application. Compounds and/or compositions may be administered to patients who have or are suspected of having a disease associated with expanded trinucleotide repeats.

In vivo application of the disclosed compounds and compositions containing them can be accomplished by any suitable method and technique now known or hereinafter known to those skilled in the art. For example, the disclosed compounds can be formulated in physiologically or pharmaceutically acceptable compositions and administered by any suitable route known in the art, including, for example, oral and parenteral routes of administration. there is. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal, and intrathecal administration, such as by injection. Administration of the disclosed compounds or compositions may be single administration, sequential administration, or administration at sharply spaced intervals as can be readily determined by those skilled in the art.

The compounds disclosed herein and compositions containing them may be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide a uniform dosage over an extended period of time. The compounds may be administered in salt derivative form or in crystalline form.

The compounds disclosed herein can be formulated into pharmaceutical compositions according to known methods for preparing pharmaceutically acceptable compositions. Formulations are well known to those skilled in the art and are described in detail in a number of readily available literature sources. For example, EW Martin in Remington's Pharmaceutical Science (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with an appropriate carrier to facilitate effective administration of the compound. The compositions used may also be in various forms. This includes solid, semisolid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectables and insoluble solutions, and sprays. The form depends on the intended mode of administration and therapeutic application. The composition also includes 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 dosages for the desired therapeutic treatment, the compositions disclosed herein advantageously contain from about 0.1% to about 0.1% by weight of one or more of the compounds of interest, based on the weight of the total composition including the carrier or diluent. It can be included at 100% by weight.

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

The compounds disclosed herein and compositions comprising them can be delivered to cells through direct contact with the cells or via carrier means. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the compositions in liposomal moieties. Other means for delivering compounds and compositions disclosed herein to cells include attaching the compound to a targeted protein or nucleic acid for delivery to the target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can bind to other compositions and allow the compositions to translocate across biological membranes. US Published Application No. 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, for intracranial tumors, poly(D-L lactide-co-glycolide) polymer; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); Chondroitin; chitin; and chitosan.

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

Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles may be solvents or liquid dispersions including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. It could be a beating. Adequate fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using surfactants. Optionally, prevention of the action of microorganisms can be achieved by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents may be included, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the compounds and/or agents disclosed herein in the required amount in an appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, manufacturing methods include vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient plus any additional desired ingredient present in a previously sterile-filtered solution. .

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

The dosage range for administration of the composition is a sufficiently large range to produce the desired effect affecting the symptom or disorder. The dosage should not be large enough to cause side effects such as unwanted cross-reactions, anaphylactic reactions, etc. In general, the dosage will vary depending on the patient's age, condition, sex, and extent of the disease, and can be determined by one skilled in the art. If any contraindications appear, the individual physician may adjust the dosage. Dosages may vary and may be administered in one or more doses per day for one or several days.

Also disclosed are pharmaceutical compositions comprising the compounds disclosed herein together with a pharmaceutically acceptable carrier. Disclosed herein are pharmaceutical compositions suitable for oral, topical, or parenteral administration and comprising an amount of a compound. The dosage administered to a patient (particularly a human) should be sufficient to achieve a therapeutic response in the patient for a reasonable period of time, without lethal toxicity, and should not cause an acceptable level of side effects or death. 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 subject's weight, the type of concurrent treatment, if present, the frequency of treatment, the cure rate, as well as the severity and stage of the pathological condition. will be.

Also disclosed are kits comprising a compound disclosed herein and/or a pharmaceutical composition containing the same in one or more containers. The disclosed kit may optionally include a pharmaceutically acceptable carrier and/or diluent. In one embodiment, the kit includes one or more other ingredients, concomitants, or adjuvants as described herein. In one embodiment, the kit includes instructions or packaging that describe how to administer the compound or composition 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 agents disclosed herein are provided in a kit as a solid form, such as in tablet, pill, or powder form. In another embodiment, the compounds and/or agents disclosed herein are provided in a kit as a liquid or solution. In one embodiment, the kit includes an ampoule or syringe containing a compound and/or agent disclosed herein in liquid or solution form.

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

Treatment method

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

In embodiments, the patient is identified as having or at risk of having any of the conditions described herein. In embodiments, methods are provided for treating diseases associated with CTG·CUG repeats in the 3' untranslated region of a gene/transcript. In embodiments, methods for treating myotonic dystrophy are provided. In embodiments, methods for treating myotonic dystrophy type 1 (DM1) are provided. In embodiments, methods for treating SCA8 are provided. In embodiments, methods for treating HDL2 are provided. In embodiments, methods for treating FECD are provided.

In embodiments, treatment refers to partial or complete relief, amelioration, alleviation, inhibition, delaying the onset, reducing the severity and/or occurrence of one or more symptoms in a subject.

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

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

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

In embodiments, treatment according to the present disclosure is performed when compared to the average protein level in the subject prior to treatment, or compared to the average protein level without treatment in one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the level of target transcripts and/or the expression of target transcript (e.g., DMPK, TCF4, JPH3, ATXN8OS, and/or ATXN8) genes was reduced by approximately 5%. % greater than 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%. In embodiments, treatment according to the present disclosure is effective when compared to the average level of the transcripts and/or proteins in the subject prior to treatment, or when not treated in one or more control subjects with similar disease. The level of target transcripts (e.g., DMPK, TCF4, JPH3, ATXN8OS, and/or ATXN8) when compared to the average level of, or compared to treatment with AC not conjugated to the cyclic CPP disclosed herein, and /or increase the expression of the target transcript from 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% Reduces by about 95%.

In embodiments, treatment according to the present disclosure is compared to the average lesion level in the subject prior to treatment, or compared to the average lesion level without treatment in one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the number of CUG repeat RNA nuclear foci of target genes (e.g. DMPK, TCF4, JPH3, ATXN8OS and/or ATXN8) was increased by approximately 5%, e.g. For 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 Reduce by 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%. In embodiments, treatment according to the present disclosure is compared to the average lesion level in the subject prior to treatment, or compared to the average lesion level without treatment in one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the reduction of CUG repeat RNA nuclear foci in target genes (e.g., DMPK, TCF4, JPH3, ATXN8OS, and/or ATXN8) was reduced by approximately 5% to approximately 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 Reduce by 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 embodiments, treatment according to the present disclosure is performed when compared to the average protein level in the subject prior to treatment, or compared to the average protein level without treatment in one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the level of downstream transcripts and/or expression of downstream gene products associated with the disease phenotype is reduced 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 Reduce by 75%, about 80%, about 85%, about 90%, about 95%, and about 100%. In embodiments, treatment according to the present disclosure is performed when compared to the average protein level in the subject prior to treatment, or compared to the average protein level without treatment in one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the levels of downstream transcripts and/or expression of downstream gene products associated with the disease phenotype are reduced by about 5% to 100%, about 10% to 100%, and about 20%. % to 100%, about 50% to 100%, about 70% to 100%, about 80% to 100%, about 90% to 100%, about 95% to 100%, about 40% to 95%, about 50% to 95%, about 70% to 95%, or about 90% to 95%.

In embodiments, treatment according to the present disclosure is performed when compared to the average protein level in the subject prior to treatment, or compared to the average protein level without treatment in one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the levels of downstream transcripts and/or expression of downstream gene products associated with a healthy phenotype are reduced 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 Increase by 75%, about 80%, about 85%, about 90%, about 95%, and about 100%. In embodiments, treatment according to the present disclosure is performed when compared to the average protein level in the subject prior to treatment, or compared to the average protein level without treatment in one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the levels of downstream transcripts and/or expression of downstream gene products associated with a healthy phenotype were reduced by about 5% to 100%, about 10% to 100%, and about 20%. % to 100%, about 50% to 100%, about 70% to 100%, about 80% to 100%, about 90% to 100%, about 95% to 100%, about 40% to 95%, about 50% to 95%, about 70% to 95%, or about 90% to 95%.

In embodiments, treatment according to the present disclosure is performed on subjects with similar disease when compared to the average protein expression level in the corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or heart of the subject prior to treatment. When the above control subjects were compared to untreated or treated with AC not conjugated to the annular CPP disclosed herein, the subjects' corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior. , gastrocnemius, or heart by greater 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 decrease by about 100%. In embodiments, treatment according to the present disclosure is performed on subjects with similar disease when compared to the average protein expression level in the corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior, gastrocnemius, or heart of the subject prior to treatment. When the above control subjects were compared to untreated or treated with AC not conjugated to the annular CPP disclosed herein, the subjects' corneal tissue, muscle tissue, diaphragm tissue, quadriceps, triceps, tibialis anterior. , gastrocnemius, or heart, 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 Reduces by about 95%, about 90% to about 95%.

In embodiments, treatment according to the present disclosure does not treat one or more control subjects with similar disease when compared to the average level of the downstream protein in the subject's corneal tissue, muscle tissue, diaphragmatic tissue, quadriceps muscle, or heart prior to treatment. alternatively spliced cells in the subject's corneal tissue, muscle tissue, diaphragm tissue, quadriceps muscle, or heart when compared to treatment with AC that is not conjugated to the annular CPP disclosed herein. Reduce the expression of the downstream protein by greater 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%, 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%, Increase by about 850%, about 900%, about 950%, or about 1000% or more.

In embodiments, treatment according to the present disclosure results in one or more control subjects with similar disease as compared to the average expression level of the wild-type protein isomer in the subject's corneal tissue, muscle tissue, diaphragm tissue, quadriceps muscle, or heart prior to treatment. Expression of the wild-type protein isomer in the subject's corneal tissue, muscle tissue, diaphragm tissue, quadriceps muscle, or heart compared to untreated or compared to treatment with AC not conjugated to the cyclic CPP disclosed herein By more than about 5%, for example about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, Increase or decrease by about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%.

In embodiments, treatment according to the present disclosure is performed when compared to the average level of the protein in the subject's tissue of interest prior to treatment, or when compared to no treatment in one or more control subjects with similar disease, or when compared to an annular protein as disclosed herein. Compared to treatment with AC not conjugated to CPP, the expression of the protein in the subject's tissue of interest is reduced by more than about 5%, for example 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% , reduces by about 90%, about 95%, and about 100%.

In embodiments, treatment according to the present disclosure is performed when compared to the average level of the downstream protein in the subject's tissue of interest prior to treatment, compared to no treatment of one or more control subjects with similar disease, or Compared to treatment with AC not conjugated to circular CPP, the expression of alternatively spliced downstream proteins in the subject's tissue of interest is reduced 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%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, Increase by 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. .

In embodiments, treatment according to the present disclosure is performed when compared to the average level of the downstream protein in the subject's tissue of interest prior to treatment, or compared to no treatment in one or more control subjects with similar disease, or herein. Compared to treatment with AC not conjugated to the disclosed cyclic CPP, the expression of the wild-type downstream protein isomer in the subject's tissue of interest is increased by greater 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 %, increase or decrease by about 85%, about 90%, about 95%, and about 100%.

In an embodiment, the tissue of interest in the subject is corneal tissue or muscle tissue.

As used herein, the terms “improve,” “increase,” “reduce, decrease,” etc. refer to values relative to a control group. In embodiments, an appropriate control is a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of a treatment described herein. A “control subject” is an individual suffering from the same disease, of similar age, and/or of the same sex as the subject being treated (this is to ensure that the stage of the treated and control subject(s) is similar).

The individual being treated (also referred to as a “patient” or “subject”) is an individual (fetal, infant, child, adolescent, or adult human) who has a disease or is likely to develop a disease. An individual may have a disease mediated by abnormal gene expression or abnormal gene splicing. In various embodiments, an individual with a disease may have a level of downstream protein expression or activity that is less than about 1-99% of the normal wild-type protein expression or activity level in an individual without the disease. In embodiments, the range is 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%, about Including, but not limited to, 20 to less than 25%, about 15 to less than 20%, about 10 to less than 15%, about 5 to less than 10%, and about 1 to less than 5%. In embodiments, the individual may have a level of downstream protein expression or activity that is 1-500% higher than the normal wild-type target protein expression or activity level in a non-diseased individual. In embodiments, the range is about 1-10%, about 10-50%, about 50-100%, about 100-200%, about 200-300%, about 300-400% of normal wild-type target protein expression or activity level. , including, but not limited to, about 400-500%, or about 500-1000%.

In embodiments, the individual is an individual who has recently been diagnosed with a disease. In general, early treatment (initiating treatment as soon as possible after diagnosis) is important to minimize the effects of the disease and maximize the benefits of treatment.

specific definition

As used in the description and appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. do. Thus, for example, a reference to a “composition” includes a mixture of two or more such compositions, a reference to an “agent” includes a mixture of two or more such agents, and a reference to an “ingredient” includes two or more such compositions. Contains mixtures of more than one of these ingredients.

When the term “about” appears immediately before a numeric value, it refers to a range (e.g. ±20%, 10%, or 5% of that value). For example, “about 50” can mean 45 to 55, and “about 25,000” can mean 22,500 to 27,500, unless the context of the disclosure indicates otherwise or the context of the disclosure contradicts such interpretation. can do. For example, in a list of numeric values such as “about 49, about 50, about 55, ...”, “about 50” means a range that extends less than half the interval(s) between the previous and subsequent values; For example, it means greater than 49.5 and less than 52.5. Additionally, the expressions “less than about” or “more than about” should be understood in light of the definition of the term “about” provided herein. Similarly, when the term “about” is placed before a consecutive numeric value or range of values (e.g., “about 10, 20, 30” or “about 10 to 30”), the term “about” is used for all consecutive numeric values or ranges of values. Refers to both endpoints of a value or range, respectively.

As used herein, “cell penetrating peptide” or “CPP” refers to a peptide that facilitates the delivery of a cargo, e.g., a therapeutic moiety (TM), into a cell. In an embodiment, CPP is cyclic and is denoted as “cCPP”. In embodiments, cCPP can induce 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 an antisense compound (AC) to the cellular location where the pre-mRNA is located.

As used herein, the term “endosomal escape vehicle” (EEV) refers to cCPP that is conjugated to a linker and/or an exophytic peptide (EP) by a chemical linkage (i.e., covalent or non-covalent interaction). EEV may be the EEV of equation (B).

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

As used herein, the terms “extracyclic peptide” (EP) and “modulatory peptide” (MP) refer to two or more amino acids linked by a peptide bond that can be conjugated to a cyclic cell penetrating peptide (cCPP) disclosed herein. Can be used interchangeably to refer to residues. When EP is conjugated to a cyclic peptide disclosed herein, the EP can alter the tissue distribution and/or retention of the compound. Typically, the EP comprises at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one arginine residue. Non-limiting examples of EP are described herein. EP may be a peptide identified in the art as a “nuclear localization sequence” (NLS). Non-limiting examples of nuclear localization sequences include the nuclear localization sequence of the SV40 virus large T-antigen with the minimal functional unit consisting of the seven amino acid sequence PKKKRKV (SEQ ID NO: 42); Nucleoplasmin two-part NLS with sequence NLSKRPAAIKAGQAKKKK (SEQ ID NO: 52); c-myc nuclear localization sequence with amino acid sequence PAAKRVKLD (SEQ ID NO: 53) or RQRRNELKRSF (SEQ ID NO: 54); The sequence of the IBB domain from importin-alpha RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 50); The sequences VSRKRPRP (SEQ ID NO: 57) and PPKKARED (SEQ ID NO: 58) of the myoma T protein; Sequence PQPKKKPL (SEQ ID NO: 59) of human p53; Sequence SALIKKKKKKMAP (SEQ ID NO: 60) of mouse c-abl IV; Sequences DRLRR (SEQ ID NO: 61) and PKQKKRK (SEQ ID NO: 62) of influenza virus NS1; Sequence RKLKKKIKKL (SEQ ID NO: 63) of hepatitis virus delta antigen; Sequence REKKKFLKRR (SEQ ID NO: 64) of mouse Mxl protein; Sequence of human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 65); and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 66) of the steroid hormone receptor (human) glucocorticoid. International Publication No. 2001/038547 describes additional examples of NLS, which is hereby incorporated by reference in its entirety.

As used herein, “linker” or “L” refers to linking one or more moieties (e.g., an exocyclic peptide (EP) and a cargo, e.g., an oligonucleotide, peptide, or small molecule) to a cyclic cell penetrating peptide (cCPP). It refers to a moiety that is covalently bound to. Linkers may include natural or non-natural amino acids or polypeptides. The linker may be a synthetic compound containing two or more suitable functional groups suitable for linking cCPP to a cargo moiety to form a compound disclosed herein. The linker may include a polyethylene glycol (PEG) moiety. The linker may contain one or more amino acids. cCPP can be covalently linked to the cargo via a linker.

The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule containing two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino 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 in a polypeptide may be joined by peptide bonds. A polypeptide may contain a peptide backbone modification in which two or more amino acids are covalently attached by a bond other than a peptide bond. A polypeptide may include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that may be incorporated into the polypeptide. The term polypeptide includes naturally occurring amino acids and artificially occurring amino acids. The term polypeptide includes, for example, peptides containing from about 2 to about 100 amino acid residues, as well as proteins containing more than about 100 amino acid residues or more than about 1000 amino acid residues, including antibodies, enzymes, receptors, etc. , soluble proteins, etc. include, but are not limited to, therapeutic proteins.

As used herein, the term “contiguous” refers to two amino acids joined by a covalent bond. For example, in the context of representative cyclic cell penetrating peptides (cCPPs), e.g. , AA ₁ /AA _2, AA ₂ /AA _3, AA ₃ /AA _4, and AA ₅ /AA ₁ exemplify pairs of consecutive amino acids.

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

The term “protonated form thereof” refers to the protonated form of an amino acid or side chain. For example, a guanidine group on the side chain of arginine can be protonated to form a guanidinium group. The structure of the protonated form of arginine is: .

As used herein, the term “chirality” refers to a molecule having two or more stereoisomers that differ in the three-dimensional spatial arrangement of the atoms, where one stereoisomer is a non-superimposable mirror image of the other stereoisomer. . Amino acids except glycine have a chiral carbon atom adjacent to the carboxyl group. The term “enantiomer” refers to a stereoisomer that is chiral. Chiral molecules can be amino acid residues that have “D” and “L” enantiomers. Molecules without a chiral center, such as glycine, may be referred to as “achiral.”

As used herein, the term “hydrophobic” refers to a moiety that is not soluble in water or has minimal solubility in water. Generally, neutral moieties and/or nonpolar moieties, or moieties that are predominantly neutral and/or nonpolar, are 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 π electrons, where n is any integer. “Heteroaromatics,” as defined below, are a subset of aromatics. Examples of aromatic amino acids include phenylalanine and naphthylalinine. 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 does not fall within the definition of aromatic 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 chain radical having 1 to 40 carbon atoms and attached to the remainder of the molecule by single bonds. Alkyls containing any number of carbon atoms from 1 to 40 are included. Alkyl containing up to 40 carbon atoms is C ₁ -C ₄₀ alkyl, alkyl containing up to 10 carbon atoms is C ₁ -C ₁₀ alkyl, and alkyl containing up to 6 carbon atoms is C ₁ -C ₆ alkyl, and alkyl containing up to 5 carbon atoms is C ₁ -C ₅ alkyl. C ₁ -C ₅ alkyl includes C ₅ alkyl, C ₄ alkyl, C ₃ alkyl, C ₂ alkyl, and C ₁ alkyl (ie, methyl). C ₁ -C ₆ alkyl includes all moieties previously described for C ₁ -C ₅ alkyl, but also includes C ₆ alkyl. C ₁ -C ₁₀ alkyl includes all moieties previously described for C 1 -C ₅ alkyl and C ₁ -C ₆ alkyl, but also includes C ₇ , C ₈ , C ₉ , and C ₁₀ alkyl. Similarly, C ₁ -C ₁₂ alkyl includes all moieties described above, but also includes C ₁₁ and C ₁₂ alkyl. Non-limiting examples of C ₁ -C ₁₂ alkyl include methyl, ethyl, n -propyl, i -propyl, sec- propyl, n -butyl, i -butyl, sec -butyl, t -butyl, n -pentyl, t - Includes amyl, n -hexyl, n -heptyl, n -octyl, n -nonyl, n -decyl, n -undecyl, and n -dodecyl. Unless specifically stated otherwise in the specification, an alkyl group may be optionally substituted.

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

“Alkenyl,” “alkenyl chain,” or “alkenyl group” refers to a straight or branched hydrocarbon chain radical having 2 to 40 carbon atoms and one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl containing any number of carbon atoms from 2 to 40 is included. An alkenyl group containing up to 40 carbon atoms is C ₂ -C ₄₀ alkenyl, an alkenyl group containing up to 10 carbon atoms is C ₂ -C ₁₀ alkenyl, and an alkenyl group containing up to 6 carbon atoms. The group is C ₂ -C ₆ alkenyl, and alkenyl containing up to 5 carbon atoms is C ₂ -C ₅ alkenyl. C ₂ -C ₅ alkenyl includes C ₅ alkenyl, C ₄ alkenyl, C ₃ alkenyl, and C ₂ alkenyl. C ₂ -C ₆ alkenyl includes all moieties previously described for C ₂ -C ₅ alkenyl, but also includes C ₆ alkenyl. C ₂ -C ₁₀ alkenyl includes all moieties previously described for C ₂ -C ₅ alkenyl and C ₂ -C ₆ alkenyl, but also includes C ₇ , C ₈ , C ₉ , and C ₁₀ alkenyl. do. Similarly, C ₂ -C ₁₂ alkenyl includes all moieties described above, but also includes C ₁₁ and C ₁₂ alkenyl. Non-limiting examples of C ₂ -C ₁₂ alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 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-undecenyl, 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 specifically stated otherwise in the specification, an alkyl group may be optionally substituted.

“Alkenylene,” “alkenylene chain,” or “alkenylene group” refers to a straight or branched divalent hydrocarbon chain radical having 2 to 40 carbon atoms and one or more carbon-carbon double bonds. Non-limiting examples of C ₂ -C ₄₀ alkenylene include ethene, propene, butene, and the like. Unless specifically stated otherwise in the specification, the alkenylene chain may be arbitrary.

“Alkoxy” or “alkoxy group” refers to the group -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl as defined herein. Unless specifically stated otherwise in the specification, an alkoxy group may be optionally substituted.

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

“Alkylcarbamoyl” or “alkylcarbamoyl group” refers to the group -OC(O)-NR _a R _b , where R _a and R _b is the same or different and is independently alkyl, alkenyl, alkynyl, aryl, heteroaryl as defined herein, or R _a R _b taken together to form a cycloalkyl group or heterocyclyl group as defined herein can do. Unless specifically stated otherwise in the specification, an alkylcarbamoyl group may be optionally substituted.

“Alkylcarboxamidyl” or “alkylboxamidyl group” refers to the group -C(O)-NR _a R _b , where R _a and R _b is the same or different and, independently, is an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, or heterocyclyl group as defined herein, or R _a R _b may be combined to form a cycloalkyl group as defined herein. Unless specifically stated otherwise in the specification, an alkylcarboxamidyl group may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system radical containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl radicals may be mono-, bi-, tri-, or tetra-cyclic ring systems and may include fused or bridged ring systems. Aryl radicals include aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as -indacene, s -indacene, indane, indene, naphthalene, and phenalene. , phenanthrene, pliaden, pyrene, and triphenylene. Unless specifically stated otherwise in this specification, the term “aryl” is meant to include optionally substituted aryl radicals.

“Heteroaryl” refers to a 5- to 20-membered ring system radical 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, a heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; The nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized; The nitrogen atom may be arbitrarily quaternized. Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[ b ][1 ,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxynyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofura Nonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl , furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridi Nyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1- Phenyl-1 H -pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, Quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thiophenyl) including, but not limited to, Neal). Unless specifically stated otherwise in the specification, heteroaryl groups may be optionally substituted.

As used herein, the term “substituted” refers to any of the following groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, means that at least one atom of any one of alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio) is replaced by a non-hydrogen atom such as, but not limited to: F halogen atoms such as , Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; Silicon atoms in groups such as trialkylsilyl group, dialkylarylsilyl group, alkyldiarylsilyl group, and trialylsilyl group; and other heteroatoms within various other groups. “Substituted” also means that one or more atoms of any of the above groups are replaced by a higher bond (e.g. a 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” means that one or more atoms of any of the above groups are -NR _g R _h , -NR _g C(=O)R _h , -NR _g C(=O)NR _g R _h , -NR _g C(=O)OR _h , -NR _g SO ₂ R _h , -OC(=O)NR _g R _h , -OR _g , -SR _g , -SOR _g , -SO ₂ R _g , -OSO ₂ R _g , -SO ₂ OR _g , =NSO ₂ R _g , and -SO ₂ NR _g R _h . “Substituted” also means that one or more hydrogen atoms of any of the above groups are -C(=O)R _g , -C(=O)OR _g , -C(=O)NR _g R _h , -CH ₂ SO ₂ R _g , -CH ₂ SO ₂ NR _g R _h means replaced. Among the foregoing, R _g and R _h are the same or different and 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. Additionally, “substituted” means that one or more atoms of any of the above groups are amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, Thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N -heterocyclyl, heterocyclylalkyl, heteroaryl , N -heteroaryl, and/or heteroarylalkyl group. “Substituted” means that one or more atoms on the side chain of an amino acid are replaced with alkyl, alkenyl, alkynyl, acyl, alkylcarboxamidyl, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl. You may. Additionally, each of the above-described substituents may be optionally substituted with one or more of the above substituents.

As used herein, the symbol “ ” (which may hereinafter be referred to as a “point of attachment bond”) refers to a bond that is the point of attachment between two chemical entities, where one of the two chemical entities is shown attached to the point of attachment bond and the other is shown as being attached to the point of attachment bond. It is not shown as being attached to. for example, " ” indicates that the chemical entity “XY” is bonded to another chemical entity through a point of attachment bond. Additionally, specific points of attachment to chemical entities not shown may be specified by inference. For example, if R ³ is H or “ "For the compound CH ³ -R ³ , when R ³ is "XY", it is inferred that the point of attachment bond is the same bond as the bond where R ³ is shown bonded to CH ₃ .

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

The term “inhibit, inhibiting, or inhibition” refers to a decrease in activity, expression, function, or other biological parameter, which may include, but is not required to completely eliminate, the activity, expression, function, or other biological parameter. Do not do so. Inhibition may include a reduction in activity, response, pathology, or disease, e.g., a reduction of at least about 10%, as compared to a control. In embodiments, the expression, activity, or function of a gene or protein is reduced by a statistically significant amount. In embodiments, the activity or function is 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%. It is reduced by %.

“Reduce, reduce, or reduce” means to slow down an event or characteristic (e.g., tumor growth). This usually relates to some standard or expected value, in other words it is relative, but it will be understood that it is not always necessary to refer to a standard or relative value. For example, “reducing tumor growth” means reducing the growth rate of a tumor compared to a standard or control group (e.g., an untreated tumor).

As used herein, “treat, treating, treatment” and other expressions thereof mean partially or completely alleviating, ameliorating, alleviating, suppressing, delaying the onset of, or delaying the onset of a condition as described herein; Refers to any administration of a disclosed compound that reduces the severity thereof and/or reduces the occurrence of one or more symptoms or characteristics thereof. With respect to a patient, the term “treatment” refers to the medical management of a patient with the intent to cure, improve, stabilize, or prevent a disease, pathological condition, or disorder. The term includes active treatment, i.e. treatment specifically directed at improving the disease, pathological condition, or disorder, and causal treatment, i.e. treatment directed at eliminating the cause of the associated disease, pathological condition, or disorder. Also includes. Additionally, the term refers to palliative care, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, or disorder; Preventive treatment, i.e. treatment aimed at minimizing or partially or completely suppressing the occurrence of an associated disease, pathological condition, or disorder; and adjuvant treatment, i.e., treatment used to supplement another specific therapy aimed at improving an associated disease, pathological condition, or disorder.

The term “therapeutically effective” refers to an amount of a disclosed compound and/or composition used in an amount sufficient to alleviate one or more causes or symptoms of a disease or disorder. These improvements again require reduction or modification, not necessarily elimination.

The term “pharmaceutically acceptable” means that these compounds, substances, compositions, and/or dosage forms are suitable for human use and use without undue toxicity, irritation, allergic reaction or other problems or complications, within the scope of sound medical judgment. /or suitable for use in contact with animal tissue and commensurate with a reasonable benefit/risk ratio.

The term “carrier” means that, when combined with a compound or composition of the present disclosure, it aids in the manufacture, storage, administration, delivery, efficiency, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. It refers to a compound, composition, material, or structure that facilitates this, or a combination thereof. For example, the carrier can be selected to minimize any degradation of the active ingredient and minimize any side effects in the subject.

As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier suitable for administration to a patient. A pharmaceutical carrier may be a substance that aids or facilitates the manufacture, storage, administration, delivery, efficiency, selectivity, or any other characteristic of the compound or composition of the present disclosure for its intended use or purpose. For example, the carrier may be selected to reduce degradation of the compound in the patient or reduce side effects. In embodiments, pharmaceutically acceptable carriers may be sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and Injectable organic esters such as ethyl oleate. Adequate fluidity can be maintained by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may be desirable to include isotonic agents such as sugars, sodium chloride, etc. Injectable formulations may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. there is. Suitable inert carriers may include sugars such as lactose.

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

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

As used herein, the term “subcutaneous administration” refers to administration just beneath the skin. “Intravenous administration” means administration within a vein.

As used herein, the term “dose” refers to a specified amount of pharmaceutical agent given in one administration. In embodiments, the dosage may be administered in two or more boluses, tablets, or injections. In embodiments where subcutaneous administration is preferred, the preferred dosage requires a volume that is not readily accommodated by a single injection. In this embodiment, two or more injections may be used to achieve the desired dosage. In embodiments, the dosage may be administered in two or more injections to reduce injection site reactions in the patient.

As used herein, the term “dosage unit” refers to the form in which the pharmaceutical formulation is provided. In an embodiment, the dosage unit is a vial containing a lyophilized compound or composition described herein. In an embodiment, the dosage unit is a vial containing the 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, including a therapeutic polypeptide, oligonucleotide, or oligonucleotide that can be used to treat at least one symptom of a disease or disorder. , small molecules, and other agents. In embodiments, the TM modulates the activity, expression, and/or level of the target transcript. In embodiments, the TM reduces the level of the target transcript through a decay 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 one or more proteins may be modulated as a result of reducing the affinity between the target transcript and one or more proteins. For example, if one or more proteins do not bind to a target transcript, they may alter their function, for example, to facilitate splicing of other transcripts, alternative splicing, and/or exon skipping. can be performed. As a result of the function of the TM, the activity, expression, and/or level of downstream genes whose interaction with the target transcript is regulated by one or more proteins disrupted by the TM may be modulated.

The terms “modulate, modulating, and modulation” refer to perturbing expression, function, or activity compared to the level of expression, function, or activity prior to modulation. Modulation may include increasing (stimulating or inducing) or decreasing (inhibiting or reducing) expression, function, or activity. In embodiments, the activity of the target transcript is modulated. In an embodiment, modulating the activity of a target transcript includes reducing the ability of the target transcript to bind one or more proteins. In embodiments, reducing the affinity between a target transcript and one or more proteins modulates the activity of one or more proteins that interact with the target transcript. For example, if one or more proteins do not bind to a target transcript, they facilitate, for example, splicing of other transcripts (e.g., downstream transcripts), alternative splicing, and/or exon skipping. They can perform their functions such as: As such, modulating the activity of a target transcript can modulate the activity, expression, and/or level of downstream genes regulated by one or more proteins whose interaction with the target transcript can be disrupted.

“Amino acid” includes an amino group and a carboxylic acid group and has the general formula (wherein R may be any organic group). Amino acids may be naturally occurring amino acids or non-naturally occurring amino acids. Amino acids may be proteinogenic amino acids or non-proteinogenic amino acids. 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 characterizing substituent (“R”) attached to the α-carbon of a natural or non-natural α-amino acid. Amino acids can be incorporated into polypeptides through peptide bonds.

As used herein, an “uncharged” amino acid is an amino acid that has a side chain that has a net neutral charge at pH 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 that has a side chain that has a net charge at pH 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, respectively, between two oligonucleotide sequences or polypeptide sequences that are identical and in the same relative position. As such, one sequence has a certain percentage of sequence identity compared to another sequence. For sequence comparison, generally one sequence against which test sequences are compared serves as a reference sequence. Those skilled in the art will understand that two sequences are generally considered “substantially identical” if they contain identical residues at corresponding positions. In embodiments, sequence identity between sequences is determined using the EMBOXX package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet. (2000), 16: 276-277) can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 , J. Mol. Biol. 48: 443-453), in the version existing as of the filing date. there is. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The result of the Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity, which is calculated as follows: (identical residues × 100)/(alignment length - total number of gaps in the alignment) number)

In other embodiments, sequence identity can be determined using the Smith-Waterman algorithm in its existing version as of the filing date.

As used herein, “sequence homology” refers to the percentage of amino acids between two polypeptide sequences that are homologous and in the same relative position. As such, one polypeptide sequence has a certain percentage of sequence homology compared to another polypeptide sequence. Those skilled in the art will understand that two sequences are generally considered “substantially homologous” if they contain homologous residues at corresponding positions. Homology residues may be identical residues. Alternatively, homologous residues may be non-identical residues that have suitably similar structural and/or functional properties. For example, as is well known to those skilled in the art, certain amino acids are typically classified as either “hydrophobic” or “hydrophilic” amino acids and/or as having “polar” or “non-polar” side chains, and may be classified as having an amino acid of the same type. Substitutions of amino acids with other amino acids can often be considered “homologous” substitutions.

As is well known in the art, amino acid sequences can be compared using any of a variety of algorithms existing as of the filing date, including those available in commercial computer programs such as BLASTP, gapped BLAST, and PSI-BLAST. Such a program is 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., Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener (eds.), et al., Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs described above generally provide an indication of the degree of homology.

As used herein, “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 molecule is expressed only on the surface of the target cell. In embodiments, the cell surface molecule is 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 cell. 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 polymeric nucleic acid structures that are at least partially complementary to the target nucleic acid molecule (AC) to which they hybridize. The AC may be a short (in embodiments, less than 50 bases) polynucleotide or polynucleotide homolog where at least a portion of the sequence is complementary to the target sequence. In an embodiment, the AC is a polynucleotide or polynucleotide homolog, some of which has a sequence complementary to the target sequence within the target pre-mRNA strand. ACs may be formed from natural nucleic acids, synthetic nucleic acids, nucleic acid homologs, or any combination thereof. In an embodiment, AC comprises an oligonucleoside. In an embodiment, the AC comprises an antisense oligonucleoside. In an embodiment, the AC includes an adapter. Non-limiting examples of ACs include primers, probes, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, siRNA, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations thereof. However, it is not limited to this. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular, branched, or hairpins, and may contain structural elements such as internal or terminal bulges or loops. An oligomeric double-stranded compound may be two strands that hybridize to form a double-stranded compound, or may be a single strand with sufficient self-complementarity to allow hybridization and formation of a complete or partial double-stranded compound. In embodiments, the AC modulates (increases, decreases, or changes) the expression, level, and/or activity of a target transcript (e.g., a target nucleic acid). In embodiments, AC reduces the level of the target transcript by inducing a decay mechanism. In embodiments, the AC modulates the activity of a target transcript. In embodiments, the AC modulates the activity of a target transcript by reducing the ability of the target transcript to bind to one or more proteins. In embodiments, reducing the affinity between a target transcript and one or more proteins may modulate the activity of one or more proteins. For example, if one or more proteins do not bind to a target transcript, they may, for example, facilitate splicing of other transcripts (downstream transcripts), alternative splicing, and/or exon skipping. They can perform the same functions as: As such, AC-mediated regulation of the activity of a target transcript can lead to modulation of the activity, expression, and/or levels of downstream genes regulated by one or more proteins whose interaction with the target transcript can be disrupted. .

As used herein, the term “targeting or targeted to” refers to linking a therapeutic moiety, such as an antisense compound, to a target nucleic acid molecule or region of a target nucleic acid molecule. In embodiments, the therapeutic moiety comprises an antisense compound that is capable of hybridizing to a target nucleic acid under physiological conditions. In embodiments, the antisense compound is a specific portion or region within a target nucleic acid, e.g., having at least one identifiable structure, function, or characteristic (e.g., a specific exon or intron, or a selected nucleobase or motif within an exon or intron). Targets part of the target nucleic acid.

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 portions of target transcripts, target RNAs (including, but not limited to, pre-mRNA and mRNA or portions thereof), portions of target cDNA derived from such RNAs, as well as portions of target untranslated RNAs such as miRNAs. Including, but not limited to this. For example, in embodiments, the target nucleic acid may be a portion of a target cell gene (or an mRNA transcribed from such a gene) whose expression or transcription is associated with a particular disorder or disease state. The term “portion” refers to a defined number of consecutive (i.e. linked) nucleotides of a nucleic acid.

As used herein, the term “transcript” or “gene transcript” refers to an RNA molecule transcribed from DNA and includes, but is 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 bound by the therapeutic moiety. The target transcript may include a target nucleotide sequence. In an embodiment, the target transcript comprises a target nucleotide sequence comprising an expanded CUG trinucleotide repeat.

The terms “target gene” and “gene of interest” refer to a gene whose expression and/or activity is desired or intended. The target gene can be transcribed into a target transcript containing the target nucleotide sequence. The target transcript can be translated into the protein of interest.

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

As used herein, the term “mRNA” refers to an RNA molecule that encodes a protein and includes pre-mRNA and mature mRNA. “Pre-mRNA” refers to newly synthesized eukaryotic mRNA molecules immediately after DNA transcription. In an embodiment, the pre-mRNA is capped with a 5' cap, modified with a 3' poly-A tail, and/or spliced to generate the mature mRNA sequence. In embodiments, the pre-mRNA includes one or more introns. In embodiments, pre-mRNA undergoes a process known as splicing, which removes introns and joins exons. In an embodiment, the pre-mRNA comprises one or more splicing elements or splicing regulatory elements. In an embodiment, the pre-mRNA includes a polyadenylation site.

As used herein, the terms “expression,” “gene expression,” “expression of a gene,” etc. refer to all functions and steps by which information encoded in a gene is converted in a cell to a functional gene product, such as a polypeptide or non-coding RNA. refers to Examples of non-coding RNA include transfer RNA (tRNA) and ribosomal RNA. Gene expression of a polypeptide involves transcribing the gene to form pre-mRNA, processing the pre-mRNA to form mature mRNA, translocating the mature mRNA from the nucleus to the cytoplasm, and translating the mature mRNA into a polypeptide. and assembling the encoded polypeptide. Expression includes partial expression. For example, expression of a gene may be referred to as producing a gene transcript. Translation of mature mRNA may be referred to as expression of mature mRNA.

As used herein, “regulation of gene expression” or similar expressions refers to regulating one or more of the processes associated with gene expression. For example, modification of gene expression may include one or more of gene transcription, RNA processing, RNA translocation from the nucleus to the cytoplasm, and translation of mRNA into protein.

As used herein, the term “gene” includes a 5' promoter region associated with expression of the gene product, and any intron and exon regions and 3' untranslated regions (“UTRs”) associated with expression of the gene product. refers to a nucleic acid sequence.

The term “immune cell” refers to cells of hematopoietic origin that play a role in immune responses. Immune cells include, but are not limited to, lymphocytes (e.g., B cells and T cells), natural killer (NK) cells, and myeloid cells. The term “myeloid cells” includes monocytes, macrophages, and granulocytes (e.g., basophils, neutrophils, eosinophils, and mast cells). Monocytes are lymphocytes that circulate through the blood for 1 to 3 days and then migrate into tissues and differentiate into macrophages or inflammatory dendritic cells or die. As used herein, the term “macrophage” refers to fetal-derived macrophages (which may also be referred to as resident tissue macrophages), and macrophages derived from monocytes that have migrated from the bloodstream into the tissues of the body (which may also be referred to as monocyte-derived macrophages). includes). Depending on the tissue in which they are located, macrophages are specifically Kupffer cells (liver), intraglomerular mesangial cells (kidney), alveolar macrophages (lung), sinus histiocytes (lymph nodes), Hofbauer cells (placenta), and microglia (brain). and spinal cord), or Langerhans (skin).

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. Oligonucleotides may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides may be composed of natural and/or modified nucleobases, sugars, and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.

As used herein, the term “nucleoside” refers to a glycosylamine containing a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups. “Natural nucleosides” or “unmodified nucleosides” are nucleosides that contain natural nucleobases and natural sugars. 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. Nucleotides may be modified with any of a variety of substituents.

As used herein, the term “nucleobase” refers to the base portion of a nucleoside or nucleotide. A nucleobase can include any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid. A native 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 containing a heterocycle.

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

As used herein, “native internucleoside linkage” refers to a phosphodiester linkage in the 3’ to 5’ direction.

As used herein, the term “modified internucleoside linkage” refers to any linkage between nucleosides or between nucleotides that is not a naturally occurring internucleoside linkage.

As used herein, “oligonucleoside” refers to an oligonucleotide in which the internucleoside linkages do not contain phosphorus atoms.

As used herein, the term “chimeric antisense compound” refers to a compound containing at least one sugar, nucleobase, and/or nucleoside that is differently modified compared to the linkages between other sugars, nucleobases, and nucleosides in the same oligomeric compound. Refers to an antisense compound with an intercleoside linkage. The remainder of the sugar, nucleobase, and internucleoside linkages may or may not be independently modified. Typically, chimeric oligomeric compounds will have modified nucleosides, which may be in isolated positions or grouped together in regions defining specific motifs. Any combination of modifications and/or mimetic groups may be included in the chimeric oligomeric compounds 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. do.

As used herein, the term “nucleobase complementarity” refers to the ability of a nucleobase to form base pairs 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 an embodiment, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of forming base pairs with a nucleobase of a target nucleic acid. For example, if a nucleobase at a specific position in an antisense compound can hydrogen bond with a nucleobase at a specific position in a target nucleic acid, the hydrogen bond site between the oligonucleotide and the target nucleic acid is considered complementary for that nucleobase pair. It is considered.

As used herein, the term “non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with each other or otherwise 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 through nucleobase complementarity. In an embodiment, an antisense compound and its target can be used when a sufficient number of corresponding positions in each molecule are occupied by a nucleobase, which nucleobase can form a bond that can stably bind the antisense compound and the target. are complementary to each other. Those skilled in the art recognize that it is possible to include mismatches in an antisense compound without eliminating its ability to maintain binding. Accordingly, antisense compounds are described herein that can contain up to 20% mismatched nucleobases (i.e., nucleobases that are not complementary to the corresponding nucleotide of the target). In embodiments, the antisense compound contains no more than about 15% mismatch, such as no more than about 10%, such as no more than 5% mismatch, or no mismatch. The remaining nucleotides are complementary nucleobases or nucleobases that do not otherwise destroy hybridization (e.g., universal bases). Those skilled in the art will determine that the compounds provided herein have nucleobases that are 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% complementary to the target nucleic acid. will recognize

As used herein, “hybridization” refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not wishing to be limited to a particular mechanism, the most common mechanisms of pairing involve hydrogen bonding between complementary nucleoside or nucleotide bases (nucleobases), such as Watson-Crick hydrogen bonding, Hoogsteen hydrogen bonding, or inverted hydrogen bonding. Could be a Hoogsteen hydrogen bond. For example, the natural base adenine is a nucleobase complementary to the natural nucleobases thymine and uracil that pair together through the formation of hydrogen bonds. Guanine, a natural base, is a nucleobase complementary to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur in a variety of situations.

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 to another nucleic acid site. In embodiments, the antisense oligonucleotide specifically hybridizes to two or more target sites. In embodiments, the oligomeric compound specifically hybridizes to its target under stringent hybridization conditions.

“Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization are sequence dependent and are different under different environmental parameters. An extensive guide to hybridization of nucleic acids is given by Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York (1993). )]. Typically, very stringent hybridization and washing conditions are chosen to be about 5°C 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 matching probe. Very stringent conditions are chosen to be equivalent to the Tm for a particular probe. An example of stringent hybridization conditions for hybridizing complementary nucleotide sequences with more than 100 complementary residues using filters in Southern or Northern blots is 50% formamide at 42°C with 1 mg of heparin, and hybridization is allowed overnight. It is carried out. An example of very stringent washing conditions is washing with 0.15M NaCl at 72°C for approximately 15 minutes. An example of stringent wash conditions is washing with 0.2x SSC for 15 minutes at 65° C. (For a description of SSC buffer, see Sambrook and Russel, Molecular Cloning: A laboratory Manual, 3 ^rd ed., Cold Spring Harbor Laboratory Press, 2001]). A low-stringency wash is often preceded by a high-stringency wash to remove background probe signal. For example, an example of a moderate stringency wash for duplexes of more than 100 nucleotides is washing with 1x SSC for 15 min at 45°C. An example of a low stringency wash for duplexes of more than 100 nucleotides would be washing with 4-6x SSC for 15 min at 40°C. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically include a salt concentration of Na ions of less than about 1.0 M at pH 7.0 to 8.3, typically a Na ion concentration of about 0.01 to 1.0 M (or different salt concentrations), and the temperature is typically at least about 30°C. Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide.

As used herein, the term "2'-modified" or "2'-substituted" means that the sugar contains a substituent other than H or OH at the 2' position. 2'-Modified monomers include BNA and monomers (e.g., nucleosides and nucleotides) with 2'-substituents, such as allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(Rm)(Rn), or O-CH2-C (=O)-N(Rm) (Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

As used herein, the term “MOE” refers to the 2′-O-methoxyethyl substituent.

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

As used herein, the term “mimetic” refers to a group within AC that substitutes for a sugar, nucleobase, and/or internucleoside linkage. Typically, a mimetic is used in place of a sugar or sugar-internucleoside linkage combination, and the nucleobase is kept hybridized to the selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics for 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, mimetics are used in place of nucleobases. Representative nucleobase mimetics 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, incorporated herein by reference). integrated). Methods for synthesizing sugars, nucleosides, 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, wherein the furanose portion of the nucleoside connects two atoms on the furanose ring to form a bicyclic It contains bridges that form a ring system. BNAs include α-L-LNA, β-D-LNA, ENA, oxyamino BNA (2'-O-N(CH3)-CH2-4'), and aminooxy BNA (2'-N(CH3)-O-CH2 -4'), but is not limited thereto.

As used herein, the term "4'-2' bicyclic nucleoside" means that a bridge connecting two atoms of the furanose ring bridges the 4' and 2' carbon atoms of the furanose ring. Refers to BNA that forms a bicyclic ring system.

As used herein, “locked nucleic acid” or “LNA” means that the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring through a methylene group to form a 2'-C,4'-C -refers to a nucleotide that has been modified to form an oxymethylene linkage. LNAs include, but are not limited to, α-L-LNA and β-D-LNA.

As used herein, the term “cap structure” or “terminal cap moiety” refers to a chemical modification incorporated into either terminus of AC. The term “therapeutic polypeptide” refers to a naturally occurring or recombinantly produced macromolecule that contains two or more amino acids and has therapeutic, prophylactic, or other biological activity.

The term “small molecule” refers to an organic compound that has pharmacological activity and has 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 generally manufactured by chemical synthesis.

“Wild-type target protein” refers to the unique functional protein isomer produced by the wild-type, normal, or non-mutated version of the target gene. Wild-type target protein also refers to a protein produced from re-spliced target pre-mRNA.

As used herein, “re-spliced target protein” refers to a protein encoded by an mRNA resulting from splicing of a target pre-mRNA to which AC hybridizes. The re-spliced target protein may be identical to the wild-type target protein, homologous to the wild-type target protein, a functional variant of the wild-type target protein, an isoform of the wild-type target protein, or an active fragment of the wild-type target protein.

As used herein, “expanded trinucleotide repeats,” such as “expanded” CUG and/or “expanded” CTG repeats, refer to genes containing or encoding trinucleotide repeats in greater numbers than are present in the wild-type gene. means containing repeated consecutive trinucleotides. Extended nucleotide repeats can be written as XXX · NNN or (XXX · NNN), where XXX refers to the DNA repeat and NNN refers to the RNA repeat transcribed from the DNA repeat. For example, CTG · CUG repeats refer to genes with CTG DNA repeats from which RNA with CUG repeats is transcribed. In embodiments, the number of repeats in the expanded trinucleotide repeat is at least 5, at least 10, at least 15, or at least 20 more than the wild type gene. In an embodiment, the expanded trinucleotide repeat comprises 2x, 3x, 4x, 5x, 10x, 20x, 50x or more trinucleotide repeats than the wild type gene. Expanded trinucleotide repeats can cause disease in subjects whose genes contain the expanded trinucleotide repeats. For example, a subject with an expanded CTG repeat in a gene may have DM1 or FECD. In DM1, the DPMK gene contains expanded CTG repeats. Subjects suffering from DM1 may have more than 50 CTG repeats in the 3' untranslated region (UTR) of the DPMK gene, whereas non-disease subjects typically have 5 to 34 CTG repeats in the 3' UTR of the DPMK gene. . In FECD, the TCF4 gene contains expanded CTG repeats. Subjects suffering from FECD may have 40 or more CTG repeats at the CTG18.1 locus of the TCF4 gene, whereas non-disease subjects typically have 30 or fewer CTG repeats at the CTG18.1 locus of the TCF4 gene. mRNA transcribed from a gene with expanded CTG repeats will have expanded CUG repeats.

When the term “downstream” in the present disclosure refers to a gene, mRNA, or protein, the term refers to a gene affected by the binding of an AC to a target nucleotide (e.g., a target transcript) but corresponding to the target nucleotide; Refers to a gene, mRNA, or protein other than mRNA or protein. Binding of AC to target nucleotides may reduce the aggregation or sequestration of RNA-binding proteins such as MBNL1 or CUGBP1 on accumulated mRNAs with CUG repeats, which may enable these RNA-binding proteins to participate in the proper transcription of downstream gene products, RNA processing. , and/or can be made available for expression.

As used herein, “functional fragment” or “active fragment” refers to a portion of a eukaryotic wild-type target protein that exhibits an activity equal to or different from one or more activities of the full-length wild-type target protein. In an embodiment, a re-spliced target protein that shares at least one biological activity of the wild-type target protein is considered to be an active fragment of the wild-type target protein. The activity may be any (approximate) percentage of the activity of the full-length wild-type target protein, such as about 1%, 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 activity (including all values and ranges between these values). Accordingly, 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 can enhance one or more biological activities of a wild-type target protein.

“Wild-type target protein” refers to the unique functional protein isomer produced by the wild-type, normal, or non-mutated version of the target gene. Wild-type target protein also refers to a protein produced from properly spliced target pre-mRNA.

As used herein, the terms “splicing” and “processing” refer to the post-transcriptional modification of pre-mRNA in which introns are removed and exons are joined. Splicing occurs in a series of reactions catalyzed by a large RNA-protein complex made up of five small nuclear ribonucleoproteins (snRNPs) called the spliceosome. Within the intron, a 3' splice site, 5' splice site, and branch site are required for splicing. The RNA component of snRNP can interact with introns and participate in catalysis.

As used herein, alternative splicing refers to the splicing of different combinations of exons present in a gene, which results in the production of different mRNA transcripts from a single gene.

As used herein, “re-spliced target protein” refers to a protein encoded by an mRNA resulting from splicing of a target pre-mRNA to which AC hybridizes. The re-spliced target protein may be identical to the wild-type target protein, homologous to the wild-type target protein, a functional variant of the wild-type target protein, or an active fragment of the wild-type target protein.

All publications, patents, and patent applications mentioned herein 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 incorporated by reference.

Example

Example 1. Evaluation of PMO and PMO-EEV compounds for their effects on RNA foci formation and splicing rescue in DM1-related 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 splicing rescue was evaluated in DM1-related cell lines. PMO-EEV compounds with mismatched PMO sequences and PMOs with scrambled sequences were also included ( Table 12 , B and C).

Experiment

cell. PMO and PMO-EEV were combined in the DM1 HeLa cell model (HeLa-480), a stable cell line with a high CUG repeat load and a downstream splicing defect, and DM1 patient-derived DM1 with approximately 2600 CTG repeats and a downstream splicing defect. Evaluated in myoblasts. HeLa-480 reproduces the pathogenic features of DM1, including CUG ribonuclear foci and splicing errors in the pre-mRNA target of the muscle blind (MBNL) alternative splicing factor. DM1 myoblasts grew quite slowly (doubling time approximately 7 days) and appeared to be poorly transfected. Control HeLa and HeLa-480 cells were treated with ENDOPORTER without additional compounds. HeLa-480 cells represent a diseased state and HeLa cells represent a non-disease state.

RNA foci analysis . HeLa-480 cells or DM1 myoblasts were treated with 1 μM, 3 μM, or 10 μM of Compounds A-D ( Table 12 ). All compounds were transfected without transfection reagent or using the ENDOPORTER (available from GENETOOLS LLC, Philomath, Oregon) transfection agent, which is designed to deliver naturally charged PMOs into cells. Cells were incubated for 24 hours and then fixed for qualitative RNA foci analysis by microscopy. After qualitative visual inspection of treated Hela-480 cells, Compound A (PMO-EEV) showed minimal amounts of RNA foci. No conclusions were drawn from DM1 myoblasts.

Splicing analysis and RT-PCR. HeLa-480 cells treated as described above were incubated and harvested after 24 or 48 hours, and total RNA was extracted. RT-PCR was performed to measure and/or quantify the splicing patterns of DM-infected exons in target RNAs such as MBNL1 and CLASP1. Percentage of exon coverage of interest was assessed.

result. Figures 6A-6D show MBNL1 ( 6a and 6c, including exon 5) and CLASP1 24 h ( 6a and 6c ) and 48 h ( 6c and 6d ) after treatment of HeLa-480 cells with compounds A-D and ENDOPORTER transfection agent. Shows RT-PCR results for alternative splicing events ( 6b and 6d , involving exon 19). For cells treated with compounds A to D, respectively, a decrease in exon 5 inclusion in MBNL1 was observed at both 24 and 48 h time points ( Figures 6A and 6C ). Cells treated with Compound A showed the greatest rescue (reduced exon 5 inclusion). For cells treated with compounds A to D, respectively, an increase in exon 19 inclusion in CLASP1 was observed at both 24 and 48 h time points ( Figures 6B and 6D ). Cells treated with Compound A showed the greatest rescue (increased inclusion of exon 19). In contrast, when HeLa-480 cells were treated with A to D without ENDOPORTER transfection reagent, no changes in the splicing events of MBNL1 ( Figure 6E ) or CLASP1 ( Figure 6F ) were observed with compound A. Except, it was not observed.

Figures 7A-7B show RT-PCR results for alternative splicing events of MBNL1 and CLASP1 after treating DM1 myoblasts with compounds A-D, negative control DM-04, or positive control DM-05. Treatment with all compounds showed rescue of splicing events of MBNL1 ( Figure 7A ) and CLASP1 ( Figure 7B ).

Example 2. Evaluation of the effect of EEV-PMO 221-1106 on splicing rescue in the DM1-mouse model

The effects of PMO alone and PMO-EEV ( Table 13 ) on splicing rescue were evaluated in vivo using the HSA-LR DM1-mouse model.

Experiment.

Mouse model. HSA-LR is a transgenic mouse model carrying an expanded long CUG repeat (LR) in the 3′-UTR of the human skeletal actin (HSA) transgene, which produces high levels of CUGexp RNA (i.e. expanded CUG RNA) in skeletal muscle. expressed (Mankodi et al., Science 2000, 289(5485):1769-1773]). HSA-LR mice display a myotonic phenotype with splicing defects. The Friend virus B NIH Jackson (FVB/NJ) mouse model was used as a control, and HSA-LR transgenic mice were created.

Experimental Design. Compounds A-D were administered to mice via retroorbital injection or intravenous (IV) injection at a single dose of 100 μL compound solution per 20 g of body weight. The dosage was proportional to the body weight of each mouse (e.g., 150 μL per 30 g body weight).

Age-matched animals were assigned to six treatment groups. Two control groups were used, each of which was injected with saline; Group 1: FVB/NJ mice (FVB/NJ; disease-free control) and Group 2: HSA-LR (disease control) mice. Four treatment groups (groups A-D) were used; HSA-LR mice were injected with compound A, B, C, or D. At the time of injection, FVB/NJ mice were 5 weeks and 4 days old, and HSA-LR mice were 6 weeks and 1 or 2 days old. Four mice (2 males and 2 females) per group were used in this experiment. Mice were sacrificed 1 week after treatment. Tissues (gastrocnemius, quadriceps, tibialis anterior (TA)) were harvested, flash frozen in liquid nitrogen, and stored at -80°C for further evaluation of splicing structure analysis.

Total RNA was extracted from tissue samples and analyzed by RT-PCR to evaluate AC-induced alternative RNA splicing rescue events for: (i) Atp2a1 exon 22, (ii) Nfix exon 7, and (iii) ) Clcn1 exon 7a, and (iv) Mbnl1 exon 5. The percentage inclusion of exons of interest was assessed.

result. Before treatment, all HSA-LR mice had myotonia. After injection of the compound, all mice became disoriented. Mice injected with compounds A and B (groups A and B) recovered within 15 minutes, whereas mice injected with compounds C and D (groups C and D) recovered after several hours. All treated mice fully recovered by the next day. At the time of sacrifice, Groups A and B had myotonia; Groups C and D clearly did not have myotonia.

Figures 8A-10D show Atp2a1 (for exon 22 inclusions; Figures 8A-9A , and 10a ), Nfix (for inclusion of exon 7; Figures 8B, 9B, and 10B ), Clcn1 (for inclusion of exon 7a; Figures 8C, 9C, and 10C ), and Mbnl1 (for inclusion of exon 5; Figures 8D, 9D Shows RNA splicing measurements for , and 10d ). Mice treated with compounds C and D (PMO-EEV) showed rescue of Atp2a1 and Nfix splicing events in gastrocnemius, quadriceps, and tibialis anterior tissues, whereas PMO and saline treatment groups did not show rescue of splicing events. ( Figures 8a, 8b, 9a, 9b, 10a, and 10b ). Similarly, in gastrocnemius and quadriceps tissue, mice treated with compounds C and D expressed Clcn1 ( Figures 8C and 9C ). and Mbnl1 ( Figures 8D and 9D ). showed rescue of splicing events, whereas PMO and saline treatment groups did not show rescue of splicing events. Regarding the splicing of Clcn1 and Mbnl1 in tibialis anterior tissue ( Figures 10C-10D ), no alternative splicing defects were detected in the control mouse strain (FVB/NJ) and DM1 mouse model (HSA-LR). Likewise, treatment with compounds A to D did not result in splicing rescue of Clcn1 and Mbnl1 genes in tibial tissue.

These results not only demonstrate the positive impact of PMO-EEV treatment in in vivo studies of splicing rescue using the DM1 mouse model, but also demonstrate the potential of PMO-EEV compounds to treat myotonic dystrophy (DM). It also proves the intended use.

Example 3. Evaluation of various PMO-EEV compounds for correcting splicing error events in immortalized myoblasts from DM1 patients.

Using DM1 patient-derived muscle myoblasts and myotubes, the effects of two DMPK CUG-targeting PMO-EEVs (197-777 and 221-1106; Table 14 ) on splicing rescue of DM1-related genes were examined in vitro. evaluated.

Experiment.

Cell Culture Immortalized myoblasts from DM1 patients (ASA308DM1) and unaffected individuals (KM1421; AB1190) were obtained. DM1 patient myoblasts possess 2600 CTG repeats in the 3′-UTR of DMPK. Skeletal muscle cell growth medium (available from PromoCell (Heidelberg, Germany)), 2% horse serum (available from Gibco, Bristol, RI), 1% forehead embryo extract (available from Gibco (Bristol, RI)), and Myoblasts were cultured in growth medium consisting of 0.5 mg/mL penicillin/streptomycin (Gibco). For myoblast differentiation, confluent cultures were switched to differentiation medium of DMEM supplemented with 2% horse serum and cultured for 4 days.

therapy. DM1 patient muscle cells were treated with 10 μM, 3 μM, 1 μM, or 0.3 μM compounds using two different treatment conditions. In the first condition, myoblasts were plated at 75-80% confluence, compounds were serially diluted in growth medium, and cells were soaked in bath for 24 hours to allow free uptake of compounds. Compound-containing medium was removed, and myoblasts were washed with 1X DPBS (Gibco) and differentiated for 4 days before harvest. For a second condition run in parallel, myoblasts were differentiated 3 days prior to treatment, compounds were serially diluted in differentiation medium, and myotubes were harvested for analysis 24 hours later.

RNA isolation and PCR . Total RNA was isolated using the RNEASY Mini Kit (available from Qiagen (Germantown, MD) according to the manufacturer's instructions. For exon inclusion, 100 ng RNA was reverse transcribed and used for PCR (OneStep RT-PCR kit, Qiagen). Samples were analyzed by LabChip (available from PerkinElmer (Waltham, MD)) with the HT DNA High Sensitivity Assay Kit.

result. DMPK CUG targeting PMO (not conjugated with EEV) improved splicing errors in MBNL1 exon 5 (data not shown). Figures 11A-11F show MBNL1 ( Figure 11A ) and its targets (SOS1, IR) in muscle cells from DM1 patients treated with various concentrations of DMPK CUG-targeted EEV-PMO (CUG ^exp 197-777 and CUG ^exp 221-1106). , DMD, BIN1, and LDB3; Figures 11b-11f ) show mixed rescue of splicing defects. EEV-PMO 197-777 induced moderate correction of splicing error events in DM1 patient muscle cells. MBNL1 and SOS1 showed the best response of splicing error correction. EEV-PMO 197-777 was selected as the instrumental compound for the follow-up experiments described below.

Myoblasts and myotubes from DM1 patients were treated with 10 μm, 3 μm, and 1 μm of DMPK CUG-targeted EEV-PMO 197-777 using methods similar to those described above. Rescue of alternative RNA splicing events of MNBL1 and MNBL1 targets was assessed. After treatment of myoblasts and myotubes with EEV-PMO, MNBL1 (excluding exon 5; Figure 12A ), SOS1 (including exon 25; Figure 12B ), INSR (including exon 11; Figure 12C ), and DMD (including exon 78; Figure 12C ) Various degrees of splicing correction were observed for BIN1 ( including exon 11; Figure 12e ), and LDB3 (excluding exon 11; Figure 12f ).

Figures 44A-44D show reversal of the myotonia phenotype in HSA-LR mice treated with 20 mpk of 221-1106, quantified by muscle relaxation assay. Figures 44A and 44C show plots of relaxation time to 80% of peak isometric strength, and Figure 44B shows the raw data of the strength trace. Figure 44D shows reversal of myotonia phenotype in HSA-LR mice treated with 20 mpk of 221-1106, quantified by representative electromyographic (EMG) traces.

Example 4. Evaluation of PMO-EEV 221-1120 in DM1 mouse model

EEV-PMO 221-1120 (also referred to as EEV-PMO-DM1-3 or DM1-3; PMO) affected splicing and mRNA levels of downstream genes in the same HSA-LR transgenic mouse model as described in Example 2. 221 = 5'-CAG CAG CAG CAG CAG CAG CAG CAG-3' (SEQ ID NO: 154; all are PMO monomers); EEV 1120 = Ac-PKKKRKV-AEEA-Lys (cyclo[FGFGRGRQ]-PEG12-OH (Ac-(sequence To study the effect of No. 42)-AEEA-Lys (SEQ ID No. 82)-PEG12-OH)), a DM1 mouse model was performed. PMO and EEV were conjugated using amide chemistry.

Experiment. There were two general treatment groups: 1) wild-type mice; and 2) HSA-LR mice (DM1 disease model). Within the HSA-LR treatment group, there were two sub-treatment groups: 1) HSA-LR treated with saline (control); and 2) HSA-LR + EEV-PMO 221-1120. Mice were treated by injecting 15 mpk, 30 mpk, 60 mpk, or 90 mpk (based on PMO) of PMO-EEV or saline via the tail vein. Seven days after treatment, mice were sacrificed and tissues were collected for analysis.

RT-PCR assay (splicing correction). Tissue was homogenized with an OMNI bead mill homogenizer, and RNA was extracted with QIACUBEQ. RT-PCR assays were performed using a one-step RT-PCT kit (Qiagen) according to the manufacturer's protocol (35 cycles of PCR, 30 s at 94°C; 30 s/cycle at 60°C; and 30 s/cycle at 72°C). . The sequences of the gene-specific primers are as follows: forward primer containing Clcn1 exon 7a = 5'-TTCACATCGCCAGCATCTGTGC-3' (SEQ ID NO: 319), reverse primer = 5'-CACGGAACAAAAGGCACTGAATGT-3' (SEQ ID NO: 320); Forward primer containing Mbnl1 exon 5 = 5'-GCTGCCCAATACCAGGTCAAC-3' (SEQ ID NO: 321), reverse primer = 5'-TGGTGGGAGAAATGCTGTATGC-3' (SEQ ID NO: 322);

Forward primer containing Atp2a1 exon 22 = 5'-GCTCATGGTCCTCAAGATCTCAC-3' (SEQ ID NO: 323), reverse primer: 5'-GGGTCAGTGCCTCAGCTTTG-3' (SEQ ID NO: 324);

Forward primer containing Nfix exon 7 = 5'-TCGACGACAGTGAGATGGAG-3' (SEQ ID NO: 325), reverse primer 5' CAAACTCCTTCAGCGAGTCC-3' (SEQ ID NO: 326). Primers for Clcn1, Mbnl1, and Atp2a1 are described in Klein et al., The Journal of Clinical Investigation. 2019, 129 (11), pg. 4739]; Primers for Nfix are described in Chen et al. [Scientific Reports. 2016, 6(1), pg. 1]. cDNA products were separated on a 2% agarose E-gel containing SYBR SAFE dye. The percentage of exon inclusion was calculated as the ratio of unskipped band/(unskipped band + skipped band).

Mouse DM1 splicing index (mDSI) calculation. mDSI was calculated according to the protocol of Tanner et al. (Tanner et al. [Nucleic acids research. 2021, 49 (4), pg. 2240-54]). For each sample (i), the normalized splicing value for each splicing event (j) is given as (PSI _i,j - PSI _wild-type,j )/(PSI _HSALR,j -PSI _wild-type,j ). It was calculated, where PSI _{wild type,j} is the average PSI for events (j) in all wild type mice, and PSI _HSALR,j is the average PSI for events (j) in all HSALR mice. The mDSI of Atp2a1, Nfix, Mbnl1, and Clcn1 in clinical trials is then calculated as the average of all normalized splicing values.

qRT-PCR assay (HSA mRNA knockdown). Reverse transcription was performed using a high-capacity cDNA reverse transcription kit from Life Technologies Corporation according to the manufacturer's protocol. Quantitative real-time PCR was performed using a Bio-Rad SyBr Green Supermix and QuantStudio3 qPCR machine, and the gene-specific primers were as follows: HSA mRNA forward primer = 5'-TTCCATCGTCCACCGCAAAT-3' (SEQ ID NO: 327), reverse primer = 5. '-AGTTTACGATGGCAGCAACG-3' (SEQ ID NO: 328) (both from Klein et al. [The Journal of Clinical Investigation. 2019, 129 (11), pg. 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 using next-generation sequencing was performed for transcriptome profiling. Z-score for each gene was calculated as (sample value - mean)/(standard deviation). Differential splicing analysis was performed on RNAseq data to calculate percent splicing (PSI) of individual exons for each gene. PSI is the ratio of normalized read counts indicating inclusion of a transcript element relative to the total normalized reads (included and excluded reads) for that event. For example, if an exon is included in the read 100% of the time, the PSI is 1. Additionally, if an exon is excluded from reads 100% of the time, the PSI is 0.

Twenty-two genes of interest known to predict DM1 were analyzed. Additionally, genes studied in Wagner et al. [PLOS Gen 2016 (47)] and Tanner et al. [NAR 2021 (48), 4, 2240-2254] were analyzed. Mouse exons were mapped to human positions. In some cases, the boundaries of exons in the mouse and/or human genome are not completely known. As such, the data were analyzed using different boundaries. The exact boundaries were confirmed using RNAseq data.

RNA CUG lesion analysis Tibialis anterior sections were stained for CUG lesions (FISH, red) and nuclei (Hoechst, blue). TA muscle sections were imaged, and the number of nuclei with CUG RNA foci was quantified.

result

HSA mRNA knockdown. Compared to the quadriceps, tibialis anterior, and triceps, only the diaphragm showed 5-10% higher HSA mRNA levels (Figure 13A). EEV-PMO treatment did not appear to change HSA mRNA levels in the diaphragm ( Figure 13B ). Expression levels of the HSA 220 CUG repeat may not be sufficient for the splicing error phenotype of DM1 in the diaphragm.

EEV-PMO knocked down HSA mRNA in a dose-dependent manner, thereby downregulating quadriceps ( Figure 14A ), gastrocnemius ( Figure 14B ), triceps ( Figure 14C ), and tibialis anterior ( Figure 14D ). Target binding was confirmed in tissue. Additionally, the Ct (cycle threshold) value of HSA mRNA is similar to the level of mouse GAPDH (approximately 15), suggesting high expression of the HSA transgene in the quadriceps muscle of HSA-LR mice.

mDSI (correction of splicing). Mouse DM1 splicing index (mDSI) for quadriceps, gastrocnemius, triceps, and tibialis anterior are shown in Figures 15A-15D . EEV - PMO treatment resulted in DM1-related splicing defects (Atp2a1 exon 22 , Nfix exon 7, Clcn1 exon 7a, and Mbnl1 exon 5) were corrected in a dose-dependent manner, with increasing doses resulting in corrections approaching or equivalent to wild type (complete correction). In HSA-LR mice, complete splicing was observed. Approximately 50% to 60% human skeletal actin RNA knockdown was achieved at concentrations that achieved correction.

Figures 16a-b show tibialis anterior tissue from HSA-LR mice ( Figure 16a ) and tibialis anterior muscle tissue from HSA-LR mice treated with EEV-PMO ( Figure 16b ) staining for CUG foci (red) and nuclei (blue). Shows one image. Qualitative and quantitative evaluation ( Figure 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 quadriceps ( Figure 17A ), triceps ( Figure 17B ), heart ( Figure 17C ), gastrocnemius ( Figure 17D ), tibialis anterior (TA; Figure 17F ), liver ( Figure 17I ), and kidney ( Figure 17J ). shows a dose-dependent response to PMO-EEV exposure. No dose-dependent response was observed in the diaphragm ( Figure 17g ). EEV-PMO was not detected in the brain except at the 60 mpk and 90 mpk dose levels. Figure 17K shows drug exposure of various tissues at the 60 mpk dose level.

Myotonia response: After 7 days of treatment with 15, 30, 60, and 90 mpk of EEV-PMO-DM1-3, a dose-dependent reduction in myotonia was observed in HSA-LR mice ( Figure 18A ). Myotonia is likely to improve after 1 week of treatment with EEV-PMO-DM1-3. HSA-LR mice treated with a single dose of 90 mpk of EEV-PMO-DM1-3 showed no obvious signs of hindlimb myotonia after induction.

RNAseq data analysis. Figures 19a-19d show the results of principal component analysis. Principal component analysis can be used to reveal similarities between samples based on a distance matrix. This type of plot is useful for visualizing the overall effect of experimental covariates and the effect of placement. The x-axis is the direction that explains the largest variance, and the y-axis is the direction that explains the second largest variance. The percentage of total variance per direction is plotted as PCA. Wild-type and HSA-LR mice belong to distinct groups. Gene expression in the gastrocnemius muscle of HSA-LR mice treated with PMO-EEV shifted toward that of wild-type mice.

Figure 20A is a heatmap 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). A total of 956 genes (p < 0.5) were differentially expressed between treatment groups, indicating differences between wild-type mice (WT) and disease model mice (HSA-LR). Treatment with EEV-PMO (HSA-LR(+,+)) resulted in an overall correction of gene expression, and the disease profile (red, HSA-LR(-,-)) compared to that of wild-type mice (WT above). moved towards.

Figure 20B is a heatmap used to visualize the expression profile of 40 genes found to have more than 7 CTG repeats out of 43 genes from BLAST analysis. Three of the CTG repeat genes discovered in BLAST analysis (Crb2, Hsd3b6, and Inhbe) were not included due to low number of reads. This analysis is useful for identifying genes that are co-regulated across treatment conditions.

Figure 21 shows a volcano plot of overall transcriptional changes across EEV-PMO treatment groups and HSA-LA treatment groups. Each data point within the scatterplot represents a gene. The fold change of each gene is shown on the x-axis and the log10 of its adjusted p-value is shown on the y-axis. Genes with adjusted p-values less than 0.05 and fold changes greater than 2 are marked with red dots. These represent upregulated genes. Genes with an adjusted p-value less than 0.05 and a fold change less than -2 are marked with blue dots. These represent down-regulated genes. Three genes were found to be significantly down-regulated (Txlnb, Scube2, and Greb1), and one gene (Txlnb) was found to be significantly up-regulated. Most transcripts containing at least (CUG) ₇ were not significantly affected. PCA analysis of these genes showed that Scube2 ( Figure 22A ), Greb1 ( Figure 22B ), Ttc7 ( Figure 22C ), Txlnb(CUG)9, and Ndrg3 ( Figure 22E ) showed correction when treated with EEV-PMO. gave. Txlnb is overcorrected by treatment ( Figure 22D ).

Figures 23A-23D show transcriptome data for various genes and various treatment groups. The HSA-LR+EEV-PMO 221-1120 treatment group contained exon 22 of Atp2a1 ( Figure 23A ), excluded exon 7 of Clcn1 ( Figure 23B ), excluded exon 7 of Nfix ( Figure 23C ), and excluded exon 7 of Mbnl1 ( Figure 23D ) was corrected.

Figure 24 shows percent splicing (PSI) of individual exons for various genes. The gene is an MBNL-1 reactive splicing biomarker (e.g., downstream gene). The selection of MBNL-1 dependent biomarkers was based on the dynamic range between wild type and disease groups as described in the literature. The HSA-LR+EEV-PMO 221-1120 treatment group had Mbnl1, Nfix, Atp2a1, Ldb3, Camk2g, Trim55, Fbox31, Slc8a3, Map3k4, Dctn4, Cacna1s, Ryr1, Slain2, Phka1, Ppp3cc, Ttn, Neb, lrrfip2, Rapgef1, and corrections for exon inclusion/exclusion were shown for all 20 genes of interest, including Vsp39.

Example 5. Evaluation of PMO-EEV 221-1120 in the DM1 mouse model, a second DM1 mouse model

PMO-EEV DM1-3 (221-1120; see Example 4 for sequence) was evaluated in a second DM1 mouse model using methods similar to those described in Example 4.

Experiment. Seven-week-old HSA-LR mice were administered a single dose of 80 mpk of EEV-PMO-DM1-3 or intravenous administration of 20 mpk of EEV-PMO-DM1-3 every other week for 6 weeks (4 times for a total of 80 mpk). administered), tissues were collected 1 to 12 weeks after the first administration or 2 weeks after the final administration. Alternative splicing for specific genes (Atp2a1, Clcn1, Nfix, MBNL1) was determined using RT-PCR. Using Q-PCR, the decrease in mRNA levels of actin-HSA after treatment was determined. LC-mass was used to determine drug levels in the quadriceps, gastrocnemius, tibialis anterior, triceps, diaphragm, heart, kidney, liver, brain, and plasma. A reduction in myotonia was recorded after 7 days of treatment with EEV-PMO-DM1-3.

result. A similar trend to that observed in Example 4 was observed for rescue of splicing for Atp2a1, Clcn1, Nfix, and MBNL1 in tibialis anterior, gastrocnemius, triceps, and quadriceps tissues (data not shown). Additionally, a trend similar to that observed in Example 4 was observed for HSA mRNA knockdown tibialis anterior, gastrocnemius, triceps, and quadriceps tissues both 1 and 4 weeks after treatment (data not shown).

Figures 25A-25D show tibialis anterior ( Figure 25A ), gastrocnemius ( Figure 25B ) , triceps ( Figure 25C ), and triceps muscles 1 to 8 weeks after treatment with 80 mpk of EEV-PMO-DM1-3. Diagram showing decreased drug levels in quadriceps ( FIG. 25D ) tissue. EEV-PMO-DM1-3 (60 mpk oligo, 80 mpk full drug) completely corrects splicing errors in the gastrocnemius, triceps, tibialis anterior, and quadriceps after 1 week of treatment. Figures 26A-26B are charts showing that a decrease in drug levels in the liver was observed after 1 to 4 weeks, 4 to 8 weeks, and 8 to 12 weeks after administration of 80 mpk of EEV-PMO-DM1-3. Relatively higher amounts of EEV-PMO-DM1-3 were observed in the liver 2 weeks after treatment with the last dose of the 6-week dosing regimen compared to that observed 4 weeks after treatment with the single-dose regimen.

Figures 26C-26D show that a decrease in drug levels in the kidney was observed after 1 to 4 weeks, 4 to 8 weeks, and 8 to 12 weeks after administration of 80 mpk of EEV-PMO-DM1-3. Relatively small amounts of EEV-PMO-DM1-3 were also observed in the kidneys 2 weeks after treatment with the last dose of the 6-week dosing regimen, compared to what was observed 4 weeks after treatment with the single-dose regimen. At 12 weeks after a single dose of EEV-PMO-DM1-3, drug was still present in the kidneys but not in the liver.

Subjective myotonia observations were made and are shown in Table 15 . The multiple dose regimen (Q2W) showed no signs of relief of myotonia after 2 weeks of treatment. There was a mixed effect on myotonia in mice treated with a single dose of 80 mpk, but this disappeared before 12 weeks after treatment.

A similar experiment was performed to evaluate intravenous administration of EEV-PMO-DM1-3 at higher doses for longer periods of time. Eight-week-old HSA-LR mice were treated with intravenous administration of 40 mpk, 60 mpk, 80 mpk, or 120 mpk of EEV-PMO-DM1-3, and tissues were collected 4 to 12 weeks later. Alternative splicing for specific genes (Atp2a1, Clcn1, Nfix, MBNL1) was determined using RT-PCR. A reduction in myotonia was recorded after 7 days of treatment with EEV-PMO-DM1-3. A similar trend to that observed in Example 4 was observed for rescue of splicing for Atp2a1, Clcn1, Nfix, and MBNL1 in tibialis anterior and gastrocnemius tissues (data not shown).

Subjective myotonia observations were made and are shown in Table 16 . Myotonia was more common in females than in males. There are no signs of myotonia at 8 weeks after treatment in both male and female mice administered 120 mpk.

Example 6. Treating Patient-Derived DM1 Cells with EEV-PMO-DM1-3 PMO-EEV DM1-3 (221-1120; see Example 4 for sequence) was evaluated in DM1 patient-derived myoblasts.

Experiment. Patient's myoblasts were treated with 30 μM DM1-3 for all 4 days of differentiation. Splicing correction was assessed by one-step RT-PCR and Labpchip (mean ± SD plotted; n=4). RNA CUG foci were detected using HCR-FISH and isolated MBNL1 protein detection assays. Results: EEV-PMO-DM1-3 promotes splicing correction and reduction of nuclear foci, significant biomarkers, in muscle cells derived from DM1 patients.

result. Figures 27A-27C are diagrams showing that EEV-PMO-DM1-3 promotes splicing correction (MBLN1, SOS1, and NFIX), a significant biomarker in muscle cells derived from DM1 patients. Additionally, treatment with DM1-3 resulted in a decrease in nuclear sickness in muscle cells derived from DM1 patients ( Figures 28A-28C ).

Example 7. Cytotoxicity screening of EEV-PMO-DM1-3 in kidney cells

PMO-EEV DM1-3 (221-1120; see Example 4 for sequence) was evaluated in human kidney cells.

Experiment. Human Primary Renal Proximal Tubular Epithelial Cells (RPTEC) were incubated with PMO-DM1 and EEV at various concentrations (1:2 serial dilutions in saline, final dilution factor 4x, from about 6 μM to about 800 μM). -PMO-DM1-3 for 24 hours and screened for viability using the CELLTITER-GLO luminescence viability assay. 16.6 μM melittin was used as a positive control.

result. Figures 29A-29B show that PMO-DM1 or conjugated EEV-PMO-DM1-3 showed no toxicity even at the highest concentration of 817 μM or 797 μM, respectively.

Example 8. Evaluation of PMO-EEV 221-1113 for its ability to correct splicing error events and downstream splicing in immortalized cells from DM1 patients and HeLa-480 cells.

Immortalized muscle cells from DM1 patients (2,600 CUG repeats) and HeLa-480 (DM1 model cell line, see Example 1) were treated with EEV-PMO construct 221-1113 and used for correction of aberrant splicing and quantification of lesions. analyzed. 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 (5'-CAG CAG CAG CAG CAG CAG CAG-3' (SEQ ID NO: 154; all are PMO monomers) via amide linkage chemistry.

Experiment. Methods similar to those described in Examples 1 and 3 were used.

result.

RNA CUG foci analysis. Cells were stained for nuclei (Hoechst, blue) and RNA CUG repeat foci (green). A reduction in RNA CUG foci was observed between untreated DM1 patient cells and DM1 cells treated with EEV-PMO ( FIGS. 30A-30C ). Similarly, a reduction in RNA CUG foci was observed between untreated HeLa-480 cells and EEV-PMO HeLa-480 cells ( FIGS. 31A-31B ).

Correction of downstream splicing. Treated with PMO-EEV DM1 patient-derived cells and HeLa-480 cells were analyzed for percent exon 5 coverage for MBLN1, percent exon 25 coverage for SOS1, and percent exon 7 coverage for NFIX. Treatment with EEV-PMO rescued splicing events for Mbnl1 ( Figure 32A ), Sos1 ( Figure 32B ), and NFIX ( Figure 32C ).

Additionally, HeLa-480 cells treated with EEV-PMO showed dose-dependent correction of MBNL1 splicing ( Figure 33A ), SOS1 ( Figure 33B ), CLASP1 ( Figure 33C ), NFIX ( Figure 33D ), and INSR. ( Figure 33E ) showed dose-dependent correction of downstream splicing errors.

Example 9. Evaluation of EEV-PMO 221-1106 in a second DM1 mouse model

A DM1 mouse model was performed to study the effects of EEV-PMO 221-1106 on splicing and mRNA levels of downstream genes.

Experiment. Human skeletal actin long repeat (HSA-LR) transgenic mice were used as a DM1 disease model . A method similar to that described in Example 5 was used.

result. Figures 34a-34d show inclusion of exon 22 for Atp2a1 ( Figure 34a ), inclusion of exon 7 for Nfix ( Figure 34b ), and Clcn1 in the gastrocnemius muscle of mice treated with various concentrations of EEV-PMO 221-1106. It is shown that inclusion of exon 7A ( Figure 34C ), and Mbnl1 (Figure 34D ) are corrected in a dose-dependent manner. Treatment with PMO 221 alone did not correct splicing.

Example 10. DM1 mouse model to study the effect of different lengths of CUG repeats on PMO

PMO-EEV 221-1121 (PMO has 7 CAG repeats, 21-mer) and PMO-EEV 0325-1121 (PMO has 8 CAG repeats, 24-mer) on the splicing and mRNA levels of downstream genes. To study the effect, a DM1 mouse model was performed. PMO-EEV 221-1121 is PMO 221 (5'-CAG CAG CAG CAG CAG CAG CAG-3'; SEQ ID NO: 154; all are PMO monomers) via amide chemistry to EEV 1121 (Ac-PKKKRKV-miniPEG2-Lys (cyclo [GfFGrGrQ])-PEG12-OH; Ac-(SEQ ID NO: 42)-miniPEG2-Lys (SEQ ID NO: 74)-PEG12-OH). PMO-EEV 0325-1121 is PMO 0325 (5'-CAG CAG CAG CAG CAG CAG CAG-CAG-3'; SEQ ID NO: 155; all are PMO monomers) conjugated to EEV 1121 via amide chemistry.

Experiment. Human skeletal actin long repeat (HSA-LR) transgenic mice were used as a DM1 disease model . Briefly, HSA-LR mice were administered 20 mpk, 40 mpk, or 60 mpk of 0221-1121 or 0325-1121 via intravenous injection into the tail vein. One week after injection, mice were sacrificed and tissues were collected. Another experimental method similar to that described in Example 5 was used.

result. 0221-1121 (21-mer) was more effective than 0325-1121 (24-mer) in correcting exon splicing in Mbnl1 ( Figure 35A ) , Nfix ( Figure 35B ), and Atp2a1 ( Figure 35C ) in tibialis anterior tissue. . This result was unexpected. It was expected that the 24-mer would be more effective, as it would have higher hybridization efficiency and higher thermal melt temperature. As shown in Figures 36A-36C , differences in gastrocnemius tissue were less pronounced. Subjective myotonia observations were made using male mice ( Table 17 ). For the 21-mer at 40 mpk, mixed myotonia was observed 1 week after treatment, similar to the results in Table 11.

Example 11. Pharmacokinetic Study of EEV-PMO 221-1120 in CD1 Mice Plasma, Kidney, and Transplantation for EEV-PMO Constructs 221-1120 (see Example 4 for sequence) and PMO-0221a using the CD1 mouse model. Tibialis muscle drug exposure (AUC) was studied and the major metabolite 221-1120 (see Figure 37 ) was also measured.

Experiment. Five to seven week old CD1 mice were treated via intravenous injection of 80 mpk of EEV-PMO construct 221-1120. Mice were bled and/or sacrificed at various time points.

result. Table 18 , Table 19 , and Table 20 show the pharmacokinetic properties observed in plasma, kidney, and anterior tibia, respectively. Description of the table: AUC _last = area under the curve from 0 to the final quantifiable concentration; D = dose; C _max = maximum serum or plasma concentration; T _max = time to C _max : CL = total plasma, serum, or blood removal; t _1/2 = elimination half-life; V _ss = apparent volume of distribution at equilibrium; Q _h = liver blood flow (ml/min/kg).

AUC values for metabolites are approximately 1000-fold lower in the anterior tibia compared to the kidney. The mean retention time (MRT) value of a metabolite in plasma can be directly related to the tissue MRT value as a result of the movement of metabolites from tissue to plasma prior to urination.

Example 12. Evaluation of PMO-EEV 221-1120 in a third DM1 mouse model

A DM1 mouse model study similar to Examples 5 and 9 was performed to evaluate the effect of various doses of PMO-EEV 221-220 (see Example 4 for sequence) in HSA-LR mice.

Experiment. 8-week-old HSA-LR mice were intravenously administered 40, 60, 80, or 120 mpk of PMO-EEV 221-1120, and tissues were collected 4 to 12 weeks later. Alternative splicing for specific genes (Atp2a1, Clcn1, Nfix, MBNL1) was determined using RT-PCR. LC-mass was used to determine drug levels in the quadriceps, gastrocnemius, TA, triceps, diaphragm, heart, kidney, liver, brain, and plasma. Transcript level changes between treated disease model, untreated disease model, and wild type were determined using RNA-seq. Using Q-PCR, the decrease in mRNA levels of actin-HSA after treatment was determined.

result. Fluorescence imaging was used to determine RNA foci reduction following treatment with EEV-oligo compounds (data not shown). Reduction in myotonia was recorded 7 days after treatment with EEV-oligo compounds (data not shown). The results of these experiments show a similar trend to the mice treated with PMO-EEV 221-1120 in Examples 5 and 14.

After treatment with various concentrations of EEV-PMO, correction of MBNL1, NFIX, and ATP2A1 splicing was observed in the tibialis anterior ( Figures 38A, 39A, 40A ) and gastrocnemius ( Figures 38B, 39B, 40B ). After 12 weeks of treatment with 120 mpk EEV-PMO, correction of MBNL1, NFIX, and ATP2A1 splicing was observed in both tibialis anterior and gastrocnemius muscles.

Example 13. Evaluation of EEV-PMO 221-1120 in HeLa480 cells

HeLa480 cells were treated with various concentrations of EEV-PMO 221-1120 (see Example 4 for sequences) and analyzed for CUG repeat foci, selective r(CUG) reduction, and downstream splicing correction of MBNL1 and SOS1. .

Experiment. Hela480 cells were constructed as described in previous examples. RT-PCR and lesion staining were performed similarly to other examples described herein.

result. Figures 41A-41B show example images of control cells (untreated) and cells treated with 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM of EEV-PMO 221-1120. Compared to untreated HeLa480 cells, CUG foci (green) are reduced in the treated HeLa480 group. Figure 41B is a chart quantifying lesions per nuclear area. Figures 41A-41B show that EEV-PMO 221-1120 can reduce nuclear CUG RNA foci. Almost complete reduction is observed at 5 μM dose.

Figures 42A-42B show that treatment with EEV-PMO 221-1120 can selectively knock down repeat expansion-containing DMPK transcripts in the HeLa480 cell line.

Figures 42C-42D show that treatment with EEV-PMO 221-1120 corrected MBNL1 ( Figure 42C ) and SOCS1 ( Figure 42D ) splicing in a dose-dependent manner.

A number of embodiments of the 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 implementations are within the scope of the following claims.

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His Lys Lys 1 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Lys Lys His Lys 1 <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide < 400> 4 Lys Lys Lys His 1 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Lys His Lys His 1 <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 His Lys His Lys 1 <210> 7 <211> 4 <212> PRT <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Lys Lys Lys Lys 1 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Lys Lys Arg Lys 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Lys Arg Lys Lys 1 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Lys Arg Arg Lys 1 <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Arg Lys Lys Arg 1 <210> 12 <211> 4 <212> PRT <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Arg Arg Arg Arg 1 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Lys Gly Lys Lys 1 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Lys Lys Gly Lys 1 <210 > 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Beta -alanine <220> <221> MOD_RES <222> (4)..(4) <223> Beta-alanine <400> 15 His Ala His Ala His 1 5 <210> 16 <211> 5 <212> PRT < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Beta-alanine <220> <221> MOD_RES <222 > (4)..(4) <223> Beta-alanine <400> 16 His Ala Lys Ala His 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Arg Arg Arg Arg Arg 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Lys Lys Lys Lys Lys Lys 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Lys Lys Lys Arg Lys 1 5 < 210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Arg Lys Lys Lys Lys 1 5 <210> 21 <211> 5 <212 > PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Lys Arg Lys Lys Lys 1 5 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Lys Lys Arg Lys Lys 1 5 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Lys Lys Lys Lys Arg 1 5 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Beta-alanine <220> <221> MOD_RES <222> (4)..(4) <223> Beta-alanine <400> 24 Lys Ala Lys Ala Lys 1 5 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Arg Lys Lys Lys Lys Gly 1 5 <210> 26 <211 > 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Lys Arg Lys Lys Lys Gly 1 5 <210> 27 <211> 6 <212> PRT <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Lys Lys Arg Lys Lys Gly 1 5 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic peptide <400> 28 Lys Lys Lys Lys Lys Arg Gly 1 5 <210> 29 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (6)..(6) <223> Beta-alanine <400> 29 Arg Lys Lys Lys Lys Ala 1 5 <210> 30 <211> 6 <212> PRT <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (6)..(6) <223> Beta-alanine <400> 30 Lys Arg Lys Lys Lys Ala 1 5 <210> 31 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (6)..(6) <223> Beta-alanine <400> 31 Lys Lys Arg Lys Lys Ala 1 5 <210> 32 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220 > <221> MOD_RES <222> (6)..(6) <223> Beta-alanine <400> 32 Lys Lys Lys Lys Arg Ala 1 5 <210> 33 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Lys Lys Lys Arg Lys Val 1 5 <210> 34 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 Arg Arg Arg Arg Arg Arg 1 5 <210> 35 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400 > 35 His His His His His His His 1 5 <210> 36 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 Arg His Arg His Arg His 1 5 <210> 37 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 His Arg His Arg His Arg 1 5 <210> 38 <211 > 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 Lys Arg Lys Arg Lys Arg 1 5 <210> 39 <211> 6 <212> PRT <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 Arg Lys Arg Lys Arg Lys 1 5 <210> 40 <211> 6 <212> PRT <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Beta-alanine <220> <221> MOD_RES <222> (4)..(4) <223> Beta-alanine <220> <221> MOD_RES <222> (6)..(6) <223> Beta-alanine <400> 40 Arg Ala Arg Ala Arg Ala 1 5 <210> 41 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Beta-alanine <220> < 221> MOD_RES <222> (4)..(4) <223> Beta-alanine <220> <221> MOD_RES <222> (6)..(6) <223> Beta-alanine <400> 41 Lys Ala Lys Ala Lys Ala 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Pro Lys Lys Lys Arg Lys Val 1 5 < 210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Pro Gly Lys Lys Arg Lys Val 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 Pro Lys Gly Lys Arg Lys Val 1 5 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Pro Lys Lys Gly Arg Lys Val 1 5 <210> 46 <211> 7 <212> PRT <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic peptide <400> 46 Pro Lys Lys Lys Gly Lys Val 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Pro Lys Lys Lys Arg Gly Val 1 5 <210> 48 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Pro Lys Lys Lys Arg Lys Gly 1 5 <210> 49 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2) ..(2) <223> Beta-alanine <220> <221> MOD_RES <222> (4)..(4) <223> Beta-alanine <400> 49 Arg Ala Arg Ala Arg 1 5 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Beta-alanine <220> <221> MOD_RES <222> (4)..(4) <223> Beta-alanine <400> 50 Arg Ala His Ala Arg 1 5 <210> 51 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> Beta-alanine <220> <221> MOD_RES <222> ( 4)..(4) <223> Beta-alanine <400> 51 His Ala Arg Ala His 1 5 <210> 52 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Asn Leu Ser Lys Arg Pro Ala Ala Ile Lys Lys Ala Gly Gln Ala Lys 1 5 10 15 Lys Lys Lys <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Phe 1 5 10 <210> 55 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Arg Met Arg Lys Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu Arg 1 5 10 15 Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg 20 25 <210> 56 <211> 14 < 212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 56 Lys Ala Lys Lys Asp Glu Gln Ile Leu Lys Arg Arg Asn Val 1 5 10 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Val Ser Arg Lys Arg Pro Arg Pro 1 5 <210> 58 <211> 8 <212> PRT <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Pro Pro Lys Lys Ala Arg Glu Asp 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Pro Gln Pro Lys Lys Lys Pro Leu 1 5 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 <210> 61 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic peptide <400> 61 Asp Arg Leu Arg Arg 1 5 <210> 62 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 62 Pro Lys Gln Lys Lys Arg Lys 1 5 <210> 63 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu 1 5 10 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 <210> 65 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10 15 Lys Ser Lys Lys 20 <210> 66 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys <210> 67 <400> 67 000 <210> 68 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Phe Gly Phe Gly Arg Gly Arg 1 5 <210> 69 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220 > <221> MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 69 Gly Phe Phe Gly Arg Gly Arg 1 5 <210> 70 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> ( 2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <400> 70 Phe Phe Ala Gly Arg Gly Arg 1 5 <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223 > D-phenylalanine <400> 71 Phe Phe Phe Gly Arg Gly Arg 1 5 <210> 72 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <220> <221 > MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 72 Phe Phe Ala Gly Arg Gly Arg 1 5 <210> 73 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (4). .(4) <223> L-naphthylalanine <400> 73 Phe Gly Phe Ala 1 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide < 220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 74 Gly Phe Phe Gly Arg Gly Arg Gln 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> ( 3)..(3) <223> L-naphthylalanine <400> 75 Phe Phe Ala Gly Arg Gly Arg Gln 1 5 <210> 76 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <400> 76 Phe Phe Phe Gly Arg Gly Arg Gln 1 5 <210> 77 < 211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine < 220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <220> <221> MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 77 Phe Phe Ala Gly Arg Gly Arg Gln 1 5 <210> 78 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <220> <221> MOD_RES <222> (5)..(5) <223> D-arginine <220 > <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 78 Phe Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 79 <211> 8 <212> PRT <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <220> <221> MOD_RES <222> (4)..(4) <223> Citrulline <220> <221> MOD_RES <222> (5). .(5) <223> D-arginine <220> <221> MOD_RES <222> (6)..(6) <223> Citrulline <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 79 Phe Phe Ala <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <220 > <221> MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 80 Phe Phe Ala Gly Arg Gly Arg Gln 1 5 <210> 81 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <400> 81 Phe Phe Phe Gly Arg Gly Arg Gln 1 5 <210> 82 <211> 8 <212> PRT <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic peptide <400> 82 Phe Gly Phe Gly Arg Gly Arg Gln 1 5 <210> 83 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 83 Gly Phe Phe Gly Arg Gly Arg Gln 1 5 <210> 84 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Phe Gly Phe Gly Arg Arg Arg Gln 1 5 <210> 85 <211> 8 <212> PRT <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Phe Gly Phe Arg Arg Arg Arg Gln 1 5 <210> 86 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <400> 86 Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 87 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> D-phenylalanine <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <220> <221> MOD_RES <222> (4)..(4) <223> D-arginine <220> <221 > MOD_RES <222> (6)..(6) <223> D-arginine <400> 87 Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 88 <211> 8 <212> PRT <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES <222> (3). .(3) <223> L-naphthylalanine <400> 88 Phe Phe Ala Arg Gly Arg Gly Gln 1 5 <210> 89 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <400> 89 Phe Ala Arg Arg Arg Gln 1 5 <210> 90 <211> 6 <212 > PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <400> 90 Phe Ala Arg Arg Arg Cys 1 5 <210> 91 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2). .(2) <223> L-naphthylalanine <220> <221> MOD_RES <222> (6)..(6) <223> Selenocysteine <400> 91 Phe Ala Arg Arg Arg Xaa 1 5 <210> 92 <211 > 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (4)..(4) <223> L-naphthylalanine <400 > 92 Arg Arg Arg Ala Phe Gln 1 5 <210> 93 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (5)..(5) <223> L-naphthylalanine <400> 93 Arg Arg Arg Arg Ala Phe 1 5 <210> 94 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <400> 94 Phe Ala Arg Arg Arg Arg 1 5 <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-naphthylalanine <220> < 221> MOD_RES <222> (3)..(3) <223> D-arginine <220> <221> MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-glutamine <400> 95 Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 96 <211> 7 <212> PRT <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-naphthylalanine <220> <221> MOD_RES <222> (3).. (3) <223> D-arginine <220> <221> MOD_RES <222> (5)..(5) <223> D-arginine <400> 96 Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 97 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <400> 97 Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 98 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> D-phenylalanine <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <220> <221> MOD_RES <222 > (4)..(4) <223> D-arginine <220> <221> MOD_RES <222> (6)..(6) <223> D-arginine <400> 98 Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 99 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (5)..(5) <223> L-naphthylalanine <400> 99 Arg Arg Phe Arg Ala Arg Gln 1 5 <210> 100 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide < 220> <221> MOD_RES <222> (6)..(6) <223> L-naphthylalanine <400> 100 Phe Arg Arg Arg Arg Ala Gln 1 5 <210> 101 <211> 7 <212> PRT <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> D-arginine <220> <221> MOD_RES <222> (5)..(5) <223> L-naphthylalanine <400> 101 Arg Arg Phe Arg Ala Arg Gln 1 5 <210> 102 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <400> 102 Arg Arg Ala Phe Arg Arg Gln 1 5 <210> 103 <211 > 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 103 Cys Arg Arg Arg Arg Phe Trp Gln 1 5 <210> 104 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> D-phenylalanine <220> <221> MOD_RES < 222> (3)..(3) <223> L-naphthylalanine <220> <221> MOD_RES <222> (5)..(5) <223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 104 Phe Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 105 <211> 8 <212> PRT <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <400> 105 Phe Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 106 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (6)..(6) <223> L-naphthylalanine <400> 106 Arg Phe Arg Phe Arg Ala Arg Gln 1 5 <210> 107 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> Selenocysteine <400> 107 <223> Description of Artificial Sequence: Synthetic peptide <400> 108 Cys Arg Arg Arg Arg Phe Trp Gln 1 5 <210> 109 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <400> 109 Phe Ala Arg Arg Arg Arg Gln Lys 1 5 <210> 110 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <400> 110 Phe Ala Arg Arg Arg Arg Gln Cys 1 5 <210> 111 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> D-phenylalanine <220> <221> MOD_RES <222> (2)..(2) <223> L-naphthylalanine <220> <221> MOD_RES <222> (4 )..(4) <223> D-arginine <220> <221> MOD_RES <222> (6)..(6) <223> D-arginine <400> 111 Phe Ala Arg Arg Arg Arg Arg Gln 1 5 <210> 112 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223 > L-naphthylalanine <400> 112 Phe Ala Arg Arg Arg Arg Arg Gln 1 5 <210> 113 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220 > <221> MOD_RES <222> (5)..(5) <223> L-naphthylalanine <220> <221> MOD_RES <222> (8)..(8) <223> Norleucine <400> 113 Arg Arg Arg Arg Ala Phe Asp 2)..(2) <223> L-naphthylalanine <400> 114 Phe Ala Arg Arg Arg 1 5 <210> 115 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 115 Phe Trp Arg Arg Arg 1 5 <210> 116 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221 > MOD_RES <222> (4)..(4) <223> L-naphthylalanine <400> 116 Arg Arg Arg Ala Phe 1 5 <210> 117 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 117 Arg Arg Arg Trp Phe 1 5 <210> 118 <400> 118 000 <210> 119 <400> 119 000 <210> 120 <400> 120 000 <210> 121 <400> 121 000 <210> 122 <400> 122 000 <210> 123 <400> 123 000 <210> 124 <400> 124 000 <210> 125 <400> 125 000 <210> 126 <400> 126 000 <210> 127 <400> 127 000 <210> 128 <400> 128 000 <210> 129 <400> 129 000 <210> 130 <400> 130 000 <210> 131 <400> 131 000 <210> 132 <400> 132 000 <210> 133 <400> 133 000 <210> 134 <400> 134 000 <210> 135 <400> 135 000 <210> 136 <400> 136 000 <210> 137 <400> 137 000 <210> 138 <400> 138 000 <210> 139 <400> 139 000 <210> 140 <400> 140 000 <210> 141 <400> 141 000 <210> 142 <400> 142 000 <210> 143 <400> 143 000 <210> 144 <400> 144 000 <210> 145 <400> 145 000 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 146 cagcagcagc agcagcagca g 21 <210> 147 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 147 cagcagcagc agcagcagca gcagc 25 <210> 148 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 148 cagcagcagc agcagcagca gcagcagcag 30 <210> 149 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 149 agcagcag ca gcagcagcag cagcagcagc 30 <210> 150 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (2)..(2) ) <223> D-phenylalanine <220> <221> MOD_RES <222> (3)..(3) <223> L-naphthylalanine <220> <221> MOD_RES <222> (5)..(5) < 223> D-arginine <220> <221> MOD_RES <222> (7)..(7) <223> D-arginine <400> 150 Phe Phe Ala Arg Arg Arg Arg Gln 1 5 <210> 151 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 151 cagcagcagc ag 12 <210> 152 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 152 cagcagcagc agcag 15 <210> 153 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 153 Cagcagcagc AGCAGCAG 18 <210> 154 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence > 154 cagcagcAgcAgca G 21 <210> 155 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 155 cagcagcagc agcagcagca gcag 24 <210> 156 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 156 cagcagcagc agcagcagca gcagcag 27 <210> 157 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400 > 157 cagcagcagc agcagcagca gcagcagcag 30 <210> 158 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 158 cagcagcagc agcagcagca gcagcagcag cag 33 <210> 159 < 211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 159 cagcagcagc agcagcagca gcagcagcag cagcag 36 <210> 160 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 160 cagcagcagc agcagcagca gcagcagcag cagcagcag 39 <210> 161 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic oligonucleotide <400> 161 cagcagcagc agcagcagca gcagcagcag cagcagcagc ag 42 <210> 162 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 162 cagcagca gc agcagcagca gcagcagcag cagcagcagc agcag 45 <210> 163 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 163 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcag 48 <210 > 164 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 164 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca g 51 <210> 165 <211> 54 <212> DNA <213> Artificial ial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 165 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcag 54 <210> 166 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 166 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag 57 <210> 167 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oli gonucleotide <400> 167 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 <210> 168 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 168 cagcagcagc agca gcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cag 63 <210> 169 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 169 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcag 66 <21 0> 170 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 170 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcag 69 <210> 171 <211> 72 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 171 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc ag 72 <210> 172 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oli gonucleotide <400> 172 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcag 75 <210> 173 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 173 cagcagcagc agca gcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcag 78 <210> 174 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 174 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 ca gcagcagc agcagcagca g 81 <210> 175 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 175 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca g cag 84 <210> 176 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 176 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcag 87 <210> 177 <211> 90 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 177 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag 90 <210> 178 <211> 93 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 178 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cag 93 <210> 179 <211> 96 < 212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic oligonucleotide <400> 179 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcag 96 <210> 180 <211> 99 <212> DNA <2 13> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 180 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcag 99 <210> 181 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 181 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc ag 102 <210> 182 <211> 105 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 182 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcag 105 <210> 183 <211> 108 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 183 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcag 108 <210> 184 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Syn thetic polynucleotide <400> 184 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca g 111 <210> 185 <211> 114 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 185 cagcagcag c agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcag 114 <210> 186 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 186 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag 117 <210> 187 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 187 cagcagcagc agcagcagca gcagcagcag cagcagca gc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 <210> 188 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 188 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcag cagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cag 123 <210> 189 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 189 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 ca gcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcag 126 <210> 190 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 190 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 6 0 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcag 129 <210> 191 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 191 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc ag 132 <210> 192 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 192 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagca gcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcag 135 <210> 193 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 193 cagcagcagc agcagcagca gcagcagcag cagcagcagc ag cagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcag 138 <210> 194 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 194 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca g 141 <210> 195 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 195 cagcagcagc agcagca gca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcag 144 <210> 196 <211> 147 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 196 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcag 147 <210> 197 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequ ence: Synthetic polynucleotide <400> 197 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag 150 <210> 198 <211> 12 <212> DNA <213> Artificial Sequence <2 20> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 198 agcagcagca gc 12 <210> 199 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 199 agcagcagca gcagc 15 <210> 200 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 200 agcagcagca gcagcagc 18 <210> 201 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 201 agcagcagca gcagcagcag c 21 <210> 202 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 202 agcagcagca gcagcagcag cagc 24 <210> 203 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 203 agcagcagca gcagcagcag cagcagc 27 <210> 204 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 204 agcagcagca gcagcagcag cagcagcagc 30 <210> 205 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 205 agcagcagca gcagcagcag cagcagcagc agc 33 <210> 206 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 206 agcagcagca gcagcagcag cagcagcagc agcagc 36 <210> 207 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 207 agcagcagca gcagcagcag cagcagcagc agcagca gc 39 <210> 208 <211 > 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 208 agcagcagca gcagcagcag cagcagcagc agcagcagca gc 42 <210> 209 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 209 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagc 45 <210> 210 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 210 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagc 48 <210> 211 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 11 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag c 51 <210> 212 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 212 agcagcagca gcagcagcag cagcagcagc agcagcagca gca gcagcag cagc 54 <210> 213 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 213 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagc 57 <210> 214 <211> 60 <212 > DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 214 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 <210> 215 <211> 63 <212> DNA <213> Artificial Sequ ence <220> <223 > Description of Artificial Sequence: Synthetic oligonucleotide <400> 215 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agc 63 <210> 216 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Art ificial Sequence: Synthetic oligonucleotide <400> 216 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagc 66 <210> 217 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oli gonucleotide <400> 217 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagc 69 <210> 218 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 218 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gc 72 <210> 219 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 219 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagc 75 <210> 220 < 211> 78 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 220 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagc 78 <21 0> 221 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 221 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag c 81 <210> 222 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 222 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagc 84 <210> 223 <211> 87 <212 > DNA <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 223 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagc 87 <210> 224 <211> 90 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 224 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc 90 <210> 225 <211> 93 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic oligonucleotide < 400> 225 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agc 93 <210> 226 <211> 96 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic oligonucleotide <400> 226 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagc 96 <210> 227 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 227 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagc 99 <210> 228 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 228 agcagcagca gcagcagcag ca gcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gc 102 <210> 229 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 229 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag ca gcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagc 105 <210> 230 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 230 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagc 108 <210 > 231 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 231 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag ca gcagcagc agcagcagca gcagcagcag c 111 <210> 232 <211> 114 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 232 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcag c agcagcagca gcagcagcag cagc 114 <210> 233 <211 > 117 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 233 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agca gcagca gcagcagcag cagcagc 117 <210> 234 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 234 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca g cagcagcag cagcagcagc 120 <210> 235 <211> 123 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 235 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag cag cagcagcagc 120 agc 123 <210> 236 <211> 126 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 236 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag cag cagcagcagc 120 agcagc 126 <210> 237 <211> 129 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 237 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag cag cagcagcagc 120 agcagcagc 129 <210> 238 <211> 132 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 238 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gc 132 <210> 239 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 239 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gcagc 135 <210> 240 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide < 400> 240 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gcagcagc 138 <210> 241 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial S equence: Synthetic polynucleotide <400> 241 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gcagcagcag c 141 <210> 242 <211> 144 <212> DNA <213> Artificial Sequence <220> <2 23> Description of Artificial Sequence: Synthetic polynucleotide <400> 242 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gcagcagcag cagc 144 <210> 243 <211> 147 <212> DNA <2 13> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 243 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gcagcagcag cagcagc 147 <210> 244 < 211> 150 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400 > 244 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gcagcagcag cagcagcagc 150 <21 0> 245 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 245 gcagcagcag ca 12 <210> 246 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 246 gcagcagcag cagca 15 <210> 247 <211 > 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 247 gcagcagcag cagcagca 18 <210> 248 <211> 21 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 248 gcagcagcag cagcagcagc a 21 <210> 249 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide < 400>249 gcagcagcag cagcagcagc agca 24 <210> 250 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 250 gcagcagcag cagcagcagc agcagca 27 <210> 251 <211 >30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 251 gcagcagcag cagcagcagc agcagcagca 30 <210> 252 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 252 gcagcagcag cagcagcagc agcagcagca gca 33 <210> 253 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide < 400> 253 gcagcagcag cagcagcagc agcagcagca gcagca 36 <210> 254 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 254 gcagcagcag cagcagcagc agcag cagca gcagcagca 39 <210> 255 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 255 gcagcagcag cagcagcagc agcagcagca gcagcagcag ca 42 <210> 256 <211> 45 <212> DNA <212> 213> Artificial sequence <220> <223> Description of Artificial sequence: Synthetic Oligonucleotide G cagca 45 <210> 257 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 257 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagca 48 <210> 258 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <40 0> 258 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc a 51 <210> 259 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 259 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agca 54 <210> 260 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 260 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagca 57 <210 > 261 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 261 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 <210> 262 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 262 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gca 63 <210> 263 <211> 66 <212> DNA <213 > Artificial Sequence < 220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 263 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagca 66 <210> 264 <211> 69 <212> DNA <213> Artificial Sequence <22 0> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 264 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagca 69 <210> 265 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Description of Art ificial Sequence: Synthetic oligonucleotide <400 > 265 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag ca 72 <210> 266 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <40 0> 266 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagca 75 <210> 267 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 267 gcagcagcag cagcagcagc agcagcagca gcagca gcag cagcagcagc agcagcagca 60 gcagcagcag cagcagca 78 <210> 268 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 268 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagca gcagc a 81 <210> 269 < 211> 84 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 269 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agca 84 <210> 270 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligo nucleotide <400> 270 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagca 87 <210> 271 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 271 gcagcagcag cagcagcagc agcagcagca gca gcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca 90 <210> 272 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 272 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gca 93 <210> 273 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 273 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc a gcagcagca gcagca 96 <210> 274 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 274 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcag cagca 99 <210> 275 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 275 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag ca 102 < 210> 276 <211> 105 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 276 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagca 105 <210 > 277 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 277 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagca 108 <210> 278 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 278 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc a 111 <210> 279 <2 11> 114 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic polynucleotide <400> 279 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agca 114 <210> 280 <211> 117 <212> DNA <213> Artificial Sequence <2 20> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 280 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagca 117 <210> 281 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial ial Sequence: Synthetic polynucleotide <400> 281 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 <210> 282 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <4 00> 282 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gca 123 <210> 283 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 283 g cagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagca 126 <210> 284 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 28 4 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagca 129 <210> 285 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 285 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagcag ca 132 <210> 286 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400 > 286 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagcag cagca 135 <210> 287 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 287 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagcag cagcagca 138 <210> 288 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 288 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagcag cagcagcagc a 141 <210> 289 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 289 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagcag cagcagcagc agca 144 <210> 290 <21 1> 147 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400 > 290 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagcag cagcagcagc agcagca 147 <210> 291 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 291 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 120 gcagcagcag cagcagcagc agcagcagca 15 0 <210> 292 <400> 292 000 <210> 293 <400> 293 000 <210> 294 <400> 294 000 <210> 295 <400> 295 000 <210> 296 <400> 296 000 <210> 297 <400> 297 000 <210> 298 <400> 298 000 <210> 299 <400> 299 000 <210> 300 <400> 300 000 <210> 301 <400> 301 000 <210> 302 <400> 302 000 <210> 303 <400> 303 000 <210> 304 <400> 304 000 <210> 305 <400> 305 000 <210> 306 <400> 306 000 <210> 307 <400> 307 000 <210> 308 <400> 308 000 <210> 309 <400> 309 000 <210> 310 <400> 310 000 <210> 311 <400> 311 000 <210> 312 <400> 312 000 <210> 313 <400> 313 000 <210> 314 <400> 314 000 <210> 315 <400> 315 000 <210> 316 <400> 316 000 <210> 317 <400> 317 000 <210> 318 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 318 agccagagca ccgcaaccgg acgag 25 <210> 319 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 319 ttcacatcgc cagcatctgt gc 22 <210> 320 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 320 cacggaacac aaaggcactg aatgt 25 <210> 321 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 321 gctgcccaat accaggtcaa c 21 <210> 322 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 322 tggtggggaga aatgctgtat gc 22 <210> 323 <211> 23 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 323 gctcatggtc ctcaagatct cac 23 <210> 324 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 324 gggtcagtgc ctcagctttg 20 <210> 325 <211> 20 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 325 tcgacgacag tgagatggag 20 <210> 326 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthetic oligonucleotide <400> 326 caaactcctt cagcgagtcc 20 <210> 327 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 327 ttccatcgtc caccgcaaat 20 <210> 328 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 328 agtttacgat ggcagcaacg 20 <210> 329 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 329 aggtcggtgt gaacggattt g 21 <210> 330 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 330 tgtagaccat gtagttgagg tca 23 <210> 331 <400> 331 000 <210> 332 <400> 332 000 <210> 333 <400> 333 000 <210> 334 <400> 334 000 <210> 335 <400> 335 000 <210> 336 <400> 336 000 <210> 337 <400> 337 000 <210> 338 <400> 338 000 <210> 339 <400> 339 000 <210> 340 <400> 340 000 <210> 341 <400> 341 000 <210> 342 <400> 342 000 <210> 343 <400> 343 000 <210> 344 <400> 344 000 <210> 345 <400> 345 000 <210> 346 <400> 346 000 <210> 347 <400> 347 000 <210> 348 <400> 348 000 <210> 349 <400> 349 000 <210> 350 <400> 350 000 <210> 351 <400> 351 000 <210> 352 <400> 352 000 <210> 353 <400> 353 000 <210> 354 <400> 354 000 <210> 355 <400> 355 000 <210> 356 <400> 356 000 <210> 357 <400> 357 000 <210> 358 <400> 358 000 <210> 359 <400> 359 000 <210> 360 <400> 360 000 <210> 361 <400> 361 000 <210> 362 <400> 362 000 <210> 363 <400> 363 000 <210> 364 <400> 364 000 <210> 365 <400> 365 000 <210> 366 <400> 366 000 <210> 367 <400> 367 000 <210> 368 <400> 368 000 <210> 369 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 369 taacgttgag ggcat 15 <210> 370 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 370 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 371 <211> 41 <212> PRT <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic polypeptide <400> 371 Arg Met Arg Lys Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu Arg 1 5 10 15 Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys Asp 20 25 30 Glu Gln Ile Leu Lys Arg Arg Asn Val 35 40 <210> 372 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 372 gcagcagcag cagcagcagc agcagcagca 30 <210> 373 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 373 gggcctttta ttcgcgaggg tcggg 25 <210> 374 <211> 25 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 374 gagggccttt tattcgcgag ggtcg 25 <210> 375 <211> 25 <212> DNA <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 375 tggagggcct tttattcgcg agggt 25 <210> 376 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 376 gatggagggc cttttatattcg cgagg 25 <210> 377 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 377 cagatggagg gccttttatt cgcga 25 <210> 378 <2 11> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 378 ggcagatgga gggcctttta ttcgc 25 <210> 379 <211> 25 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 379 tgggcagatg gagggccttt tattc 25 <210> 380 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide < 400> 380 tttgggcaga tggagggcct tttat 25 <210> 381 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 381 gctttgggca gatggagggc ctttt 25 <210> 382 < 211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 382 gagctttggg cagatggagg gcctt 25 <210> 383 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 383 cagagctttg ggcagatgga gggcc 25 <210> 384 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 384 tccagagctt tgggcagatg gaggg 25 <210> 385 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 385 agtccagagc tttgggcaga tggag 25 <210 > 386 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 386 ggagtccaga gctttgggca gatgg 25 <210> 387 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 387 gtggagtcca gagctttggg cagat 25 <210> 388 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic oligonucleotide <400> 388 ctgtggagtc cagagctttg ggcag 25 <210> 389 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 389 cactgtggag tccagagctt tgggc 25 < 210> 390 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 390 gacactgtgg agtccagagc tttgg 25 <210> 391 <211> 25 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 391 cggacactgt ggagtccaga gcttt 25 <210> 392 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 392 cgcggacact gtggagtcca gagct 25 <210> 393 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 393 accgcggaca ctgtggagt c cagag 25 <210> 394 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 394 aaaccgcgga cactgtggag tccag 25 <210> 395 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 395 gcaaaccgcg gacactgtgg agtcc 25 <210> 396 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 396 acgcaaaccg cggacactgt ggagt 25 <210> 397 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 397 caacgcaaac cgcggacact gtgga 25 <210> 398 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(120) ) <223> This sequence may encompass 5-40 "cag" repeating units <400> 398 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 <2 10> 399 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(150) <223> This sequence may encompass 1-50 "cag" repeating units <400> 399 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag 150 <210 > 400 <211> 152 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide < 220> <221> misc_feature <222> (3)..(152) <223> This region may encompass 1-50 "cag" repeating units <400> 400 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc 60 agcagcagca gcagcagcag cagcagca gc agcagcagca gcagcagcag cagcagcagc 120 agcagcagca gcagcagcag cagcagcagc ag 152 <210> 401 <211> 151 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (2).. (151) <223> This region may encompass 1-50 "cag" repeating units <400> 401 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 60 gcagcagcag cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca 1 20 gcagcagcag cagcagcagc agcagcagca g 151 <210> 402 <211> 152 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(150) <223> This region may encompass 1-50 " cag" repeating units <400> 402 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagca gcag ag 152 <210> 403 <211> 151 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(150) <223> This region may encompass 1-50 "cag" repeating units <400> 403 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag a 151 <210> 404 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial ial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(150) <223> This sequence may encompass 2-50 "cag" repeating units <400> 404 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcag ca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag 150 <210 > 405 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(60) <223> This sequence may encompass 2-20 "cag" repeating units <400> 405 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 <210> 406 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(30) <223> This sequence may encompass 2-10 "cag" repeating units <400> 406 cagcagcagc agcagcagca gcagcagcag 30 <210> 407 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(30) <223> This sequence may encompass 4- 10 "cag" repeating units <400> 407 cagcagcagc agcagcagca gcagcagcag 30 <210> 408 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(30) <223> This sequence may encompass 5-10 "cag" repeating units <400> 408 cagcagcagc agcagcagca gcagcagcag 30 <210> 409 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1)..(30) <223> This sequence may encompass 6-10 "cag" repeating units <400 > 409 cagcagcagc agcagcagca gcagcagcag 30 <210> 410 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> misc_feature <222> (1). .(27) <223> This sequence may encompass 6-9 "cag" repeating units <400> 410 cagcagcagc agcagcagca gcagcag 27 <210> 411 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> misc_feature <222> (1)..(24) <223> This sequence may encompass 6-8 "cag" repeating units <400> 411 cagcagcagc agcagcagca gcag 24 <210 > 412 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> misc_feature <222> (1)..(21) <223> This sequence may encompass 6-7 "cag" repeating units <400> 412 cagcagcagc agcagcagca g 21 <210> 413 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide < 400> 413 cugcugcugc ugcugcugcu g 21 <210> 414 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> misc_feature <222> (1) ..(120) <223> This sequence may encompass 10-40 "cag" repeating units <400> 414 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag cag 120

Claims (81)

Compounds containing:
a cyclic peptide having 6 to 12 amino acids, wherein at least 2 amino acids are charged amino acids, at least 2 amino acids are aromatic hydrophobic amino acids, and at least 2 amino acids are uncharged non-aromatic amino acids; and
An antisense compound (AC) complementary to at least a portion of an expanded CUG repeat in a target mRNA sequence, the AC comprising a phosphorodiamidate morpholino (PMO) nucleotide. 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 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 1-3, wherein the at least two uncharged non-aromatic amino acids are citrulline, glycine, or combinations thereof. 2. The method of claim 1, wherein the cyclic peptide has one of the following structures:
(Ia);
(Ib); or
It has a protonated form,
During the ceremony:
R ₁ , R ₂ , and R ₃ are each independently H or an amino acid residue having a side chain containing an aromatic group;
At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;
R ₄ is H or an amino acid side chain;
AA _SC is the amino acid side chain to which the antisense compound is conjugated;
A compound wherein each m is independently an integer of 0, 1, 2, or 3. 6. The method of claim 5, wherein the cyclic peptide has one of the following structures:
(I-1);
(I-2);
(I-3);
(I-4);
(I-5);
(I-6); or
A compound having 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. According to claim 5 or 6,
R ₁ , R ₂ , and R ₃ are each independently H or an amino acid residue having a side chain containing an aromatic group;
At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;
R ₄ is H or an amino acid side chain;
m is 2;
AA _SC is the side chain of a glutamic acid residue. The method of claim 5 or 6, wherein AA _SC : or , where 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 AA _SC . 11. The compound of claim 10, wherein the linker comprises a -(OCH ₂ CH ₂ ) _z'- subunit, wherein z' is an integer from 1 to 23. 11. The compound of claim 10, wherein the linker comprises:
(i) -(OCH ₂ CH ₂ ) _z -subunit (where z' is an integer from 1 to 23);
(ii) one or more amino acid residues, such as those of glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, or 6-aminohexanoic acid, or combinations thereof; or
(iii) A combination of (i) and (ii). 11. The compound of claim 10, wherein the linker comprises:
(i) -(OCH ₂ CH ₂ ) _z -subunit, where z is an integer from 2 to 20;
(ii) the residues of glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof; or
(iii) A combination of (i) and (ii). Compounds containing:
An endosomal escape vehicle comprising a cyclic peptide and an extracyclic peptide, wherein the cyclic peptide comprises 6 to 12 amino acids and the extracyclic peptide comprises 2 to 10 amino acids; and
An antisense compound (AC) complementary to at least a portion of an expanded CUG repeat in a target mRNA sequence, the AC comprising a phosphorodiamidate morpholino (PMO) nucleotide. 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 method 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, and combinations thereof, and at least two uncharged non-aromatic amino acids. A compound that is citrulline, glycine, and combinations thereof. 15. The method of claim 14, wherein the cyclic peptide has one of the following structures:
(Ia);
(Ib); or
It has a protonated form,
During the ceremony:
R ₁ , R ₂ , and R ₃ are each independently H or an amino acid residue having a side chain containing an aromatic group;
At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;
R ₄ is H or an amino acid side chain;
AA _SC is the amino acid side chain to which the antisense compound and exophytic peptide are conjugated;
A compound wherein each m is independently an integer of 0, 1, 2, or 3. 18. The method of claim 17, wherein the cyclic peptide has one of the following structures:
(I-1);
(I-2);
(I-3);
(I-4);
(I-5);
(I-6); or
A compound having 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. 19. The compound according to claim 17 or 18, wherein AA _SC is the side chain of a glutamic acid residue. According to claim 17 or 18,
R ₁ , R ₂ , and R ₃ are each independently H or an amino acid residue having a side chain containing an aromatic group;
At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;
R ₄ is H or an amino acid side chain;
m is 2;
AA _SC is the side chain of a glutamic acid residue. 22. The method according to any one of claims 17 to 21, wherein AA _SC is: or , where t is an integer from 0 to 5. 23. The compound of any one of claims 14 to 22, wherein the extracyclic peptide comprises 4 to 8 amino acid residues. 24. A compound according to any one of claims 14 to 23, wherein the extracyclic peptide comprises one or two amino acid residues comprising a side chain comprising a guanidine group, or a protonated form or salt thereof. 25. The compound of any one of claims 14 to 24, wherein the extracyclic peptide comprises 2, 3, or 4 lysine residues. 26. The method of claim 25, wherein the amino group on the side chain of each lysine residue is trifluoroacetyl (-COCF ₃ ), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexane) A compound substituted with silidene)ethyl (Dde), or (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) group. 25. A compound according to any one of claims 14 to 24, wherein the exocyclic peptide comprises at least two amino acid residues with hydrophobic side chains. 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. 25. 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 (B is beta-alanine). 25. The compound of any one of claims 14 to 24, wherein the extracyclic peptide comprises one of the following sequences: PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH (B is beta-alanine) ). 25. 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. 25. The compound of any one of claims 14-24, wherein the extracyclic peptide comprises PKKKRKV. 25. 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, SALIKKKKKKMAP, DRLRR, PKQKKRK, RKLKKK IKKL , REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, or RKCLQAGMNLEARKTKK. 34. The compound of any one of claims 14 to 33, further comprising a linker conjugating the antisense compound and the extracyclic peptide to AA _SC . 35. The compound of claim 34, wherein the linker comprises:
(i) -(OCH ₂ CH ₂ ) _z' -subunit (where z' is an integer from 1 to 23);
(ii) one or more amino acid residues, such as those of glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, or 6-aminohexanoic acid, or combinations thereof; or
(iii) A combination of (i) and (ii). 35. The compound of claim 34, wherein the linker comprises:
(i) -(OCH ₂ CH ₂ ) _z -subunit, where z is an integer from 2 to 20;
(ii) the residues of glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof; or
(iii) A combination of (i) and (ii). The method of claim 34, wherein the linker comprises a divalent or trivalent C ₁ -C ₅₀ alkylene, and 1 to 25 methylene groups are -N(H)-, -N(C ₁ -C ₄ alkyl)-, - N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ N(C ₁ -C ₄ alkyl)-, -S(O) ₂ N(cycloalkyl)-, -N(H)C(O)-, -N(C ₁ -C ₄ alkyl)C(O) -, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C ₁ -C ₄ alkyl), -C(O)N(cycloalkyl) , aryl, heteroaryl, cycloalkyl, or cycloalkenyl. 35. The method of claim 34, wherein the linker has the following structure:
,
During the ceremony:
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 point of attachment to the amino acid side chain of the amino acid residue of the cyclic peptide; M is a linking group. 39. The compound of claim 38, wherein z' is 11. 40. The compound of claim 38 or 39, wherein x' is 1. 41. A compound according to 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 38-41, wherein the antisense compound is conjugated to M. 18. The method of claim 17, wherein the compound is a compound of formula (C):
(C),
or a protonated form or salt thereof,
During the ceremony:
R ₁ , R ₂ , and R ₃ are each independently H or a side chain containing an aryl or heteroaryl group, and at least one of R ₁ , R ₂ , and R ₃ is a side chain containing an aryl or heteroaryl group;
R ₄ and R ₇ are independently H or an amino acid side chain;
EP is an exophytic peptide;
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;
Compound, wherein the cargo is an antisense compound. 44. The compound of claim 43, wherein R ₁ , R ₂ , and R ₃ are H or a side chain comprising an aryl group. 45. The compound of claim 44, wherein the side chain comprising the aryl group is the side chain of phenylalanine. 46. The compound of claim 45, wherein two of R ₁ , R ₂ , and R ₃ are side chains of phenylalanine. 47. The compound of any one of claims 44-46, wherein two of R ₁ , R ₂ , R ₃ , 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. 18. The structure of claim 17, wherein the structure of formula (C-1), (C-2), (C-3), or (C-4):
(C-1),
(C-2),
(C-3),
(C-4)
or protonated forms or salts thereof,
In the formula, EP is an exophytic peptide,
The oligonucleotide is an antisense compound. 51. The compound of claim 50, wherein the oligonucleotide comprises the following sequence:
5'-CAG CAG CAG CAG CAG CAG CAG-3'. 52. The compound of claim 50 or 51, wherein EP comprises the following sequence: PKKKRKV. 53. The compound of any one of claims 1-52, wherein the expanded trinucleotide repeat is 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. 54. The compound of claim 53, wherein the target mRNA sequence is the ATXN8OS/ATXN8 mRNA sequence. 54. The compound of claim 53, wherein the target mRNA sequence is the JPH3 mRNA sequence. 57. The compound of any one of claims 1-56, wherein AC comprises 5-10 CAG repeats. 58. The compound of any one of claims 1 to 57, wherein the mRNA is pre-mRNA or mature mRNA. 59. The compound of any one of claims 1-58, wherein the mRNA is pre-mRNA. A pharmaceutical composition comprising an effective amount of the compound of any one of claims 1 to 59. A cell containing the compound of any one of claims 1 to 59. 1. A method of treating myotonic dystrophy (DM) in a subject in need thereof, comprising administering to the subject a compound of any one of claims 1 to 59 or the composition of claim 60. 63. The method of claim 62, wherein said administering increases expression of wild-type protein in muscle tissue. 64. The method of claim 63, wherein said administering increases expression of the wild-type protein in diaphragm tissue, quadriceps muscle 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 expanded CUG repeats. 66. The method of any one of claims 62-65, wherein said administering prevents or reduces lesion formation. 67. The method of any one of claims 62-66, wherein the nucleotide repeat expansion is located in the 3'UTR of DMPK mRNA. 1. A method for treating a disease associated with an mRNA having an expanded CUG repeat in the 3' untranslated region (UTR), comprising administering to a subject in need thereof a compound comprising:
An antisense compound (AC) complementary to at least a portion of the expanded CUG repeat in the mRNA; and
A cyclic peptide having 6 to 12 amino acids, wherein at least 2 amino acids are charged amino acids, at least 2 amino acids are aromatic hydrophobic amino acids, and at least 2 amino acids 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, and combinations thereof, and the at least two uncharged amino acids of the cyclic peptide are arginine. The method of claim 1, wherein the non-aromatic amino acid is citrulline, glycine, or a combination thereof. 69. The method of claim 68, wherein the cyclic peptide has the following structure:
(A), or
It has a protonated form,
During the ceremony:
R ₁ , R ₂ , and R ₃ are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;
R ₄ , R ₅ , R ₆ , and R ₇ are independently H or an amino acid side chain;
At least one of R ₄ , R ₅ , R ₆ , and R ₇ is the side chain of H citrulline;
AA _SC is the amino acid side chain to which the antisense compound is conjugated;
q is 1, 2, 3, or 4, method. The method of claim 70, wherein at least one of R ₄ , R ₅ , R ₆ , R ₇ is 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N -Methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethylysine, N,N , a side chain of N-trimethyllysine, 4-guanidinophenylalanine, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidinyl)alanine. 69. The method of claim 68, wherein the cyclic peptide has the following structure:
(I)
or a protonated form thereof,
During the ceremony:
R ₁ , R ₂ , and R ₃ are each independently H or an amino acid residue having a side chain containing an aromatic group;
At least one of R ₁ , R ₂ , and R ₃ is an aromatic or heteroaromatic side chain of an amino acid;
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;
wherein 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:
(Ia),
(Ib), or
A method having a protonated form thereof. 73. The method of claim 72, wherein the cyclic peptide has one of the following structures:
(I-1),
(I-2),
(I-3),
(I-4),
(I-5),
(I-6), or
A method having a protonated form thereof. 75. The method of any one of claims 68-74, wherein the antisense compound comprises phosphorodiamidate morpholino (PMO) nucleotides. 76. The method of any one of claims 68-75, further comprising a linker conjugating the antisense compound to AA _SC . 77. The method of any one of claims 68-76, comprising an endosomal escape vehicle, wherein the endosomal escape vehicle comprises a cyclic peptide and an extracyclic peptide. 78. The method of any one of claims 68-77, wherein the disease is myotonic dystrophy (DM). 78. The method of any one of claims 68-77, wherein the disease is spinocerebellar ataxia type 8 (SCA8). 78. The method of any one of claims 68-77, wherein the disease is Huntington's disease-like 2 (HDL2). 81. The compound, composition, or method of any one of claims 1-80, wherein the antisense compound comprises the sequence of nucleotides listed in Table 2.