CN118215504A - Compounds and methods for skipping exon 44 in duchenne muscular dystrophy - Google Patents

Compounds and methods for skipping exon 44 in duchenne muscular dystrophy Download PDF

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CN118215504A
CN118215504A CN202280066121.9A CN202280066121A CN118215504A CN 118215504 A CN118215504 A CN 118215504A CN 202280066121 A CN202280066121 A CN 202280066121A CN 118215504 A CN118215504 A CN 118215504A
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compound
amino acid
side chain
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ccpp
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李翔
钱自清
马赫布贝·海拉瓦迪
马克·怀斯克
纳塔拉詹·塞瑟拉曼
连文龙
马斯韦塔·吉尔根拉特
内尔萨·埃斯特雷拉
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Ant Rada Therapeutics Ltd By Share Ltd
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Ant Rada Therapeutics Ltd By Share Ltd
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Priority claimed from PCT/US2022/075691 external-priority patent/WO2023034817A1/en
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Abstract

Described herein in various embodiments are compositions comprising (a) a cyclic peptide; and (b) an antisense compound, wherein the antisense compound targets exon 44 of the DMD gene in the pre-mRNA sequence.

Description

Compounds and methods for skipping exon 44 in duchenne muscular dystrophy
The priority of U.S. provisional application Ser. No. 63/239,645 submitted at month 1 of 2021, U.S. provisional application Ser. No. 63/292,685 submitted at month 22 of 2022, U.S. provisional application Ser. No. 63/268,580 submitted at month 2 of 2022, U.S. provisional application Ser. No. 63/362,294 submitted at month 31 of 2022, U.S. provisional application Ser. No. 63/362,423 submitted at month 4 of 2022, U.S. provisional application Ser. No. 63/337,560 submitted at month 2 of 2022, U.S. provisional application Ser. No. 63/354,456 submitted at month 22 of 2022, U.S. provisional application Ser. No. 63/239,671 submitted at month 9 of 2021, U.S. provisional application Ser. No. 63/290,960 submitted at month 12 of 2021, U.S. provisional application Ser. No. 63/298,565 of 2022 and U.S. provisional application Ser. 63/268,577 submitted at month 2 of 2022.
Background
Duchenne muscular dystrophy (Duchenne Muscular Dystrophy, DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness due to alterations in the protein dystrophin. Genetic modification in DMD, the gene encoding dystrophin, results in DMD. These genetic modifications shift the reading frame of DMD resulting in a non-functionally truncated DMD protein. One method of treating DMD patients entails delivering to the patient a compound that restores the reading frame of the DMD. Antisense compounds can restore the reading frame of DMD by skipping internal exons associated with the shift in the reading frame of DMD, which results in a non-functional truncated DMD protein. Exon skipping produces dystrophin, which retains lost function in the disease state.
One significant problem with antisense oligonucleotide therapeutics is their limited ability to enter intracellular compartments when administered systemically. Intracellular delivery of antisense compounds can be facilitated by the use of carrier systems such as polymers, cationic liposomes, or by chemical modification of the construct, e.g., by covalent attachment of cholesterol molecules. However, intracellular delivery efficiency remains low, and improved delivery systems are still needed to increase the efficacy of these antisense compounds.
The need for effective compositions for delivering antisense compounds to intracellular compartments to treat diseases caused by, for example, aberrant gene transcription, splicing and/or translation, has not been met.
Disclosure of Invention
Described herein are compounds for delivering nucleic acids. In embodiments, the nucleic acid is an Antisense Compound (AC). In embodiments, the antisense compound targets exon 44 in a subject with Duchenne Muscular Dystrophy (DMD).
The present disclosure relates to compounds comprising:
(a) A Cell Penetrating Peptide (CPP) sequence (e.g., a cyclic peptide); and
(B) An Antisense Compound (AC) complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence. In embodiments, the AC is complementary to a target sequence comprising: at least a portion of the exons 44 of the DMD genes in the pre-mRNA sequence, at least a portion of the intronic sequences flanking the exons 44 of the DMD genes in the pre-mRNA sequence, or both. In embodiments, hybridization of AC to the target sequence alters the splicing pattern of DMD pre-mRNA to restore the reading frame and enable the production of functional dystrophin.
In embodiments, the AC comprises at least one modified nucleotide or nucleic acid selected from Phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino (phosphorodiamidate morpholino, PMO) nucleotides, locked Nucleic Acids (LNA), peptide Nucleic Acids (PNA), nucleotides comprising a 2' -O-methyl (2 ' -OMe) modified backbone, 2' O-methoxy-ethyl (2 ' -MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides, and 2' -deoxy-2 ' -fluoro- β -D-arabinonucleic acid (2 ' f-ANA). In embodiments, the AC comprises at least one PMO (e.g., ,1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49 or 50 PMOs, including all ranges therein). In embodiments, each nucleotide in AC is PMO.
In embodiments, the AC comprises the following sequence:
5’-AAA CGC CGC CAT TTC TCA ACA GAT C-3’。
In embodiments, the cyclic peptide is FGFGRGRQ. In embodiments, the cyclic peptide is GfFGrGrQ. In embodiments, the cyclic peptide is Ff Φ GRGRQ.
In embodiments, the EEV is: ac-PKKKRKV-AEEA-Lys- (cyclo [ FGFGRGRQ ]) -PEG12-OH.
The present disclosure relates to pharmaceutical compositions comprising the compounds described herein.
The present disclosure relates to cells comprising a compound described herein.
The present disclosure relates to a method of treating DMD, comprising administering to a patient a compound described herein.
Drawings
FIGS. 1A and 1B illustrate conjugation chemistry for linking a therapeutic moiety, such as an Antisense Compound (AC), to a Cell Penetrating Peptide (CPP). CPP can be conjugated to the 5 'end, 3' end, or backbone of AC.
FIGS. 2A and 2B illustrate methods for linking Cell Penetrating Peptides (CPPs) (e.g.)Shown) with an Antisense Compound (AC), wherein the CPP comprises a PEG4 linker and shows an AC without a linker containing a polyethylene glycol (PEG 2 or miniPeG) moiety (fig. 2A) and an AC with a linker containing a polyethylene glycol (PEG 2 or miniPeG) moiety (fig. 2B). In the figure, "R" represents palmitoyl.
FIG. 3 shows an example of an Endosomal Escape Vector (EEV) design using a representative CPP. It should be understood that a CPP may include any of the CPPs disclosed herein.
FIG. 4A shows a schematic of the preparation of EEV-PMO-MDX-23-1. FIG. 4B is a RT-PCR analysis showing that mice treated with EEV-PMO-MDX-23-1 produced dystrophin lacking the internal exon (exon 23) as compared to mice treated with PMO-MDX-23-1. FIG. 4C shows the exon skipping products of dystrophin in different treated muscle groups after administration of PMO-MDX-23-1 and EEV-PMO-MDX-23-1.
Fig. 5A-5D show the percentage of exon skipping in quadriceps (fig. 5A), tibialis Anterior (TA) (fig. 5B), diaphragmatia (fig. 5C), and heart (fig. 5D) of MDX mice after delivery of PMO-MDX-23-1 or EEV-PMO-MDX-23-1.
Figures 6A-6D show the percentage of exon 23 splicing in the Tibialis Anterior (TA) of the MDX mice (figure 6A), quadriceps (figure 6B), diaphragm (figure 6C) and heart (figure 6D) after delivery of EEV-PMO-MDX-23-1.
Figures 7A-7D show the corrected dystrophin amounts of exon 23 in quadriceps (figure 7A), tibialis Anterior (TA) (figure 7B), diaphragm (figure 7C) and heart (figure 7D) detected by western blotting after delivery of PMO-MDX-23-1 or EEV-PMO-MDX-23-1.
Figures 8A-8D show western blots of corrected dystrophin and α -actin by exon 23 in the diaphragm (figure 8A), heart (figure 8B), quadriceps (figure 8C) and tibialis anterior (figure 8D) following intravenous delivery of 10mpk or 30 mpkEEV-PMO-MDX-23-1.
FIGS. 9A-9B show dystrophin levels in two weeks (FIG. 9A) and four weeks (FIG. 9B) of MDX mice after treatment with 30mpk EEV-PMO-MDX-23-1 or 30 mpkPMO-MDX-23-1.
FIGS. 10A-10D show the percentage of exon 23 correction in the tibialis anterior (FIG. 10A), quadriceps (FIG. 10B), diaphragm (FIG. 10C) and heart (FIG. 10D) of MDX mice administered 30mpk PMO-MDX-23-1 or 30 mpkEEV-PMO-MDX-23-1. Mice administered EEV-PMO-MDX-23-1 exhibited enhanced splice correction compared to mice administered PMO-MDX-23-1 alone.
FIGS. 11A-11C show exon 23 skipping and dystrophin correction in heart (FIG. 11A), tibialis anterior (FIG. 11B) and diaphragm (FIG. 11C) observed in MDX mice up to 8 weeks after a single IV dose (40 mg/kg) of EEV-PMO-MDX-23-1.
FIGS. 12A-12D show exon 23 skipping in the D2-MDX model following repeated doses (20 mg/kg) of EEV-PMO-MDX-23-2. FIG. 12A (heart); fig. 12B (diaphragm); fig. 12C (tibialis anterior); FIG. 12D (triceps)
Figures 13A-13C show that D2-MDX mice showed significantly improved normalized serum Creatine Kinase (CK) levels (figure 12A) and muscle function (figure 12B-12C) after treatment with 20mg/kgEEV-PMO-MDX-23-2 per month compared to PMO-MDX-23 alone. (ns is not significant, <0.05, < 0.01, < 0.001, < 0.0001, < p).
FIGS. 14A-14D show dose-dependent exon skipping 1 week after EEV-PMO-MDX-23-2 injection assessed by two-step RT-PCR. Fig. 14A (triceps); fig. 14B (tibialis anterior); fig. 14C (separator); fig. 15D (heart).
FIGS. 15A-15D show the duration of effect after administration of 80mpk EEV-PMO-MDX-23-2. Fig. 15A (triceps); fig. 15B (tibialis anterior); fig. 15C (diaphragm); fig. 15D (heart).
Figure 16 shows cumulative exon skipping in all 4 tissues (triceps, tibialis anterior, diaphragmatia and heart).
Fig. 17 shows d2.Mdx line hanging data (WIRE HANG DATA). After 12 weeks of treatment, the line-up time of animals treated with EEV-PMO-MDX23-1 80mpk Q2W was statistically indistinguishable from WT animals. DBA WT vehicle (saline); d2.mdx vehicle (saline); d2.mdxEEV-PMO-MDX 23-1; d2.mdxEEV-PMO-MDX 23-2; D2.mdxPMO-MDX 23 (5'-GGCCAAACCTCGGCTTACCTGAAAT-3')
Fig. 18A to 18D show creatine kinase activity in D2MDX mice before (fig. 18A) and 4 weeks after (fig. 18B), 8 weeks (fig. 18C) and 12 weeks (fig. 18D).
Fig. 19A to 19B show grip strength of D2MDX mice before administration (fig. 19A) and 12 weeks after administration (fig. 19B).
FIGS. 20A-20D show the synthesis of EEV-PMO-DMD44-1 (FIG. 5A), EEV-PMO-DMD44-2 (FIG. 5B), EEV-PMO-DMD44-3 (FIG. 5C) and EEV-PMO-DMD44-4 (FIG. 5D).
FIG. 21 shows the use of 1, 3 or 10. Mu.M EEV-PMO-DMD44-1; dystrophin recovery in DMD delta 45 myocytes after EEV-PMO-DMD44-2 or EEV-PMO-DMD-3 treatment.
FIGS. 22A and 22B show exon skipping and drug concentration in hDMD mouse tissue treated with EEV-PMO-DMD44-1 (FIG. 7A) and EEV-PMO-DMD44-2 (FIG. 7B) via IV injection.
FIGS. 23A-23B depict exon skipping (FIG. 23A) and drug exposure (FIG. 23B) of EEV-PMO-DMD44-1 in the NHP model.
FIGS. 24A-24B depict exon skipping (FIG. 24A) and drug exposure (FIG. 24B) of EEV-PMO-DMD44-2 in the NHP model.
FIGS. 25A-25B show exon skipping (FIG. 25A) and recovery of dystrophin (FIG. 25B) in DMD patient-derived myocytes treated with EEV-PMO-DMD 44-1.
FIGS. 26A-26C observed dose-dependent tissue exposure and exon skipping in heart (FIG. 26A) and skeletal muscle (FIGS. 26B and 26C) of hDMD transgenic mice following Intravenous (IV) administration of EEV-PMO-DMD44-1 at 10, 20, 40 and 80 mg/kg.
Fig. 27 shows that EEV-PMO-DMD44-1 has an extended circulatory half-life after administration to a non-human primate (NHP).
FIG. 28 shows that a single dose of EEV-PMO-DMD44-1 produced meaningful levels of exon skipping in skeletal muscle and heart of NHP 7 days after infusion at 30mg/kg IV for 1 hour.
Fig. 29A-29C depict exon skipping in hDystrophin mouse hearts (fig. 29A), diaphragms (fig. 29B) and triceps (fig. 29C) after single IV dose (15 mg/kg) EEV-PMO-DMD44-1 or R6 (polyarginine) conjugated exon 44 skipping PMO.
Fig. 30A-30E depict exon skipping in the hmdmd mouse heart (fig. 30A), diaphragm (fig. 30B), tibialis anterior (fig. 30C), gastrocnemius (fig. 30D), and triceps (fig. 30E) detected by 1-STEP RT-PCR for up to 12 weeks.
Figure 31 shows exon skipping in NHPs up to 12 weeks after a single IV dose.
FIGS. 32A-32C show localization of PMO and EEV-NLS-PMO in THP cells as determined by LC-MS/MS: whole cell uptake (fig. 32A); subcellular localization (fig. 32B); and nuclear uptake (fig. 32C).
Detailed Description
Compounds of formula (I)
Disclosed herein are compounds for use in the treatment of Duchenne Muscular Dystrophy (DMD). In embodiments, DMD is caused by a mutation in exon 44. In embodiments, the compound is designed to deliver an Antisense Compound (AC) complementary to a target sequence of a DMD gene in a pre-mRNA sequence, wherein the target sequence comprises at least a portion of a 5 'flanking intron of exon 44, at least a portion of a 3' flanking intron of exon 44, or a combination thereof. In embodiments, the compounds are designed to deliver the Antisense Compound (AC) intracellular to a subject in need thereof.
In embodiments, the compound alters the splicing pattern of the target pre-mRNA that binds to AC, resulting in the formation of a re-spliced target protein. In one embodiment, the re-spliced target protein is more functional than a target protein produced by splicing target pre-mRNA in the absence of AC. In embodiments, re-splicing the target protein increases the function of the target protein by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500% or more as compared to the function of the target protein produced by splicing. In embodiments, re-splicing the target protein increases the function of the target protein by about 1%, 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 99%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500% or more, including all values and ranges therebetween, as compared to the function of the target protein produced by splicing. In embodiments, the re-spliced target protein restores function to at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%, and at most about 100% of the function of the wild-type target protein. In embodiments, the re-spliced target protein restores function to about 1%, 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 99%, or about 100% of the function of the wild-type target protein, including all values and ranges therebetween.
In various embodiments, the compounds disclosed herein have an AC moiety and a Cell Penetrating Peptide (CPP) moiety. In some embodiments, the CPP moiety is cyclic (referred to herein as a cyclic peptide). In embodiments, the compound is capable of penetrating the cell membrane and binding to the target pre-mRNA in vivo. In embodiments, the compound comprises: a) At least one CPP portion; and b) at least one AC, wherein the CPP is coupled to the AC directly or indirectly (e.g., via a linker). In embodiments, the compound comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AC moieties. In embodiments, a compound comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more CPP moieties. In embodiments, the compound comprises one AC moiety. In embodiments, the compound comprises two AC moieties. As used herein, "coupled" may refer to covalent or non-covalent association between a CPP and AC, including fusion of a CPP to AC and chemical conjugation of a CPP to AC. A non-limiting example of a method of non-covalently attaching a CPP to an AC is through a streptavidin/biotin interaction, for example, by conjugating biotin to the CPP and fusing the AC to streptavidin. In the resulting compounds, the CPP is coupled to the AC via non-covalent association between biotin and streptavidin.
In embodiments, the CPP is conjugated directly or indirectly via a linker to AC, thereby forming a CPP-AC conjugate. Conjugation of AC to CPP may occur at any suitable site on these moieties. In embodiments, the 5 'or 3' end of the AC may be conjugated to the C-terminus, N-terminus, or side chain of an amino acid in the CPP. In embodiments, the CPP is a cyclic peptide.
In embodiments, the AC may be chemically conjugated to the CPP through a moiety on the 5 'or 3' end of the AC. In embodiments, AC may be conjugated to CPP through the side chain of an amino acid on the CPP. Any amino acid side chain on a CPP capable of forming a covalent bond or that can be so modified can be used to link an AC to a CPP. The amino acid on the CPP may be a natural or unnatural amino acid. In embodiments, the amino acid on the CPP for conjugation to AC is aspartic acid, glutamic acid, glutamine, asparagine, lysine, ornithine, 2, 3-diaminopropionic acid or an analog thereof. In embodiments, the side chain is substituted with a bond attached to the AC or linker. In embodiments, the amino acid is lysine or an analog thereof. In embodiments, the amino acid is glutamic acid or an analog thereof. In embodiments, the amino acid is aspartic acid or an analog thereof. In embodiments, the CPP is a cyclic peptide.
Endosome escape carrier (EEV)
Provided herein are Endosomal Escape Vectors (EEVs) that can be used to transport AC across a cell membrane, e.g., to deliver AC to the cytoplasm or nucleus of a cell. EEV may comprise a Cell Penetrating Peptide (CPP), for example, a cyclic cell penetrating peptide (cCPP) conjugated to an Exocyclic Peptide (EP). EP is interchangeably referred to as a regulatory peptide (MP). The EP may comprise a sequence of Nuclear Localization Signals (NLS). The EP may be coupled with AC. EP's may be coupled to cCPP. EP's can be coupled to AC's and cCPP's. EP, AC, cCPP or combinations thereof may be non-covalent or covalent. The EP may be attached to the N-terminus of cCPP by a peptide bond. The EP may be attached to the C-terminal end of cCPP by a peptide bond. EP may be attached to cCPP by a side chain of an amino acid in cCPP. EP may be attached to cCPP through the side chain of lysine, which may be conjugated to the side chain of glutamine in cCPP. The EP may be conjugated to the 5 'or 3' end of the AC. The EP may be coupled to a linker. The exocyclic peptide may be conjugated to the amino group of the linker. The EP may be coupled to the linker via the C-terminal ends of EP and cCPP via cCPP and/or a side chain on the EP. For example, an EP may comprise a terminal lysine, which may then be coupled to a glutamine-containing cCPP via an amide linkage. When EP contains a terminal lysine and the side chain of lysine is available for attachment cCPP, the C-terminus or N-terminus may be attached to a linker on AC.
Cyclic exopeptides
The Exocyclic Peptide (EP) may comprise 2 to 10 amino acid residues, for example 2,3,4,5,6, 7, 8, 9 or 10 amino acid residues, including all ranges and values therebetween. An EP may comprise 6 to 9 amino acid residues. An EP may comprise 4 to 8 amino acid residues.
Each amino acid in the exocyclic peptide may be a natural or unnatural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homolog of a natural amino acid in that it has a structure similar to that of a natural amino acid, thereby mimicking the structure and reactivity of the natural amino acid. The unnatural amino acid can be a modified amino acid and/or amino acid analog that is not one of the 20 common naturally occurring amino acids, nor the rare natural amino acid selenocysteine or pyrrolysine. The unnatural amino acid can also be a D-isomer of the natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine (allosoleucine), arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids are listed in table 1 along with their abbreviations used herein. For example, the amino acid may be A, G, P, K, R, V, F, H, nal or citrulline.
The EP may comprise at least one positively charged amino acid residue, e.g. 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. An EP may comprise 1 or 2 amino acid residues comprising a side chain comprising a guanidine group or a protonated form thereof. The amino acid residue comprising a guanidine-containing side chain may be an arginine residue. Protonated form may mean salts thereof throughout the disclosure.
The EP may comprise at least two, at least three or at least four or more lysine residues. An EP may comprise 2, 3 or 4 lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group including, for example, a trifluoroacetyl (-COCF 3), allyloxycarbonyl (Alloc), 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) 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 group (-COCF 3). Protecting groups may be included to effect amide conjugation. The protecting group may be removed after conjugation of EP to cCPP.
An EP may comprise at least 2 amino acid residues with hydrophobic side chains. Amino acid residues having hydrophobic side chains may be selected from valine, proline, alanine, leucine, isoleucine and methionine. The amino acid residue having a hydrophobic side chain may be valine or proline.
The EP may comprise at least one positively charged amino acid residue, e.g. at least one lysine residue and/or at least one arginine residue. The EP may comprise at least two, at least three or at least four or more lysine residues and/or arginine residues.
EP's may comprise KK、KR、RR、HH、HK、HR、RH、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKH、KHK、HKK、HRR、HRH、HHR、HBH、HHH、HHHH、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, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.
The EP may comprise 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. EP's may comprise PKKKRKV, PR, RRR, RHR, RBR, RBRBR, RBHBR or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.
The EP may consist of 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. EP may consist of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.
An EP may comprise an amino acid sequence identified in the art as a Nuclear Localization Sequence (NLS). An EP may consist of an amino acid sequence identified in the art as a Nuclear Localization Sequence (NLS). The EP may comprise an NLS comprising the amino acid sequence PKKKRKV. EP may consist of NLS comprising the amino acid sequence PKKKRKV. The EP may comprise an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK. EP may consist of NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK、PAAKRVKLD、RQRRNELKRSF、RMRKFKNKGKDTAELRRRRVEVSVELR、KAKKDEQILKRRNV、VSRKRPRP、PPKKARED、PQPKKKPL、SALIKKKKKMAP、DRLRR、PKQKKRK、RKLKKKIKKL、REKKKFLKRR、KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK
All exocyclic sequences may also contain N-terminal acetyl groups. Thus, for example, an EP may have the following structure: ac-PKKKRKV.
Cell Penetrating Peptide (CPP)
The Cell Penetrating Peptide (CPP) may comprise from 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). cCPP are capable of penetrating cell membranes. The Exocyclic Peptide (EP) may be conjugated to cCPP and the resulting construct may be referred to as an Endosomal Escape Vector (EEV). cCPP can direct AC to penetrate the cell membrane. cCPP can deliver AC to the cytoplasm of the cell. cCPP can deliver AC to a cell site where a target (e.g., pre-mRNA) is located. To conjugate cCPP to AC, at least one bond or lone pair of electrons on cCPP may be replaced.
The total number of amino acid residues in cCPP is in the range of 6 to 20 amino acid residues, e.g., 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, including all ranges and subranges therebetween. cCPP may comprise from 6 to 13 amino acid residues. cCPP disclosed herein may comprise from 6 to 10 amino acids. 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:
Wherein AA1、AA2、AA3、AA4、AA5、AA6、AA7、AA8、AA9 and AA 10 are amino acid residues.
CCPP may comprise from 6 to 8 amino acids. cCPP may comprise 8 amino acids.
CCPP may be natural or unnatural amino acids. The term "unnatural amino acid" refers to an organic compound that is a homolog of a natural amino acid in that it has a structure similar to that of a natural amino acid, thereby mimicking the structure and reactivity of the natural amino acid. The unnatural amino acid can be a modified amino acid and/or amino acid analog that is not one of the 20 common naturally occurring amino acids, nor the rare natural amino acid selenocysteine or pyrrolysine. The unnatural amino acid can also be a D-isomer of the natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids are listed in table 1 along with their abbreviations used herein.
TABLE 1 amino acid abbreviations
* Single letter abbreviations: when shown in uppercase letters herein, it means an L-amino acid form, and when shown in lowercase letters herein, it means a D-amino acid form.
CCPP may comprise from 4 to 20 amino acids, wherein: (i) At least one amino acid has a side chain comprising a guanidino group or a protonated form thereof; (ii) At least one amino acid having no side chain or having a chain comprising Or a side chain of a protonated form thereof; and (iii) at least two amino acids independently have side chains comprising aromatic or heteroaromatic groups.
At least two amino acids may have no side chains or have a chain comprising Or a side chain of a protonated form thereof. As used herein, when a side chain is not present, the amino acid has two hydrogen atoms (e.g., -CH 2 -) on the carbon atoms linking the amine and the carboxylic acid.
The amino acid without a side chain may be glycine or β -alanine.
CCPP may comprise 6 to 20 amino acid residues that form cCPP, wherein: (i) At least one amino acid may be glycine, beta-alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid has a guanidine group, Or a side chain of a protonated form thereof.
CCPP may comprise 6 to 20 amino acid residues that form cCPP, wherein: (i) At least two amino acids may independently be glycine, beta-alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid has a guanidine group, Or a side chain of a protonated form thereof.
CCPP may comprise 6 to 20 amino acid residues that form cCPP, wherein: (i) At least three amino acids may independently be glycine, beta-alanine or 4-aminobutyric acid residues; (ii) At least one amino acid may have a side chain comprising an aromatic or heteroaromatic group; and (iii) at least one amino acid may have a guanidine group, Or a side chain of a protonated form thereof.
Glycine and related amino acid residues
CCPP may comprise (i) 1, 2, 3, 4, 5, or 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 2 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 4 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 5 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3, 4, or 5 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 or 4 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof.
CCPP may comprise (i) 1,2,3,4,5 or 6 glycine residues. cCPP may comprise (i) 2 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3,4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues. cCPP may comprise (i) 2 or 3 glycine residues. cCPP may comprise (i) 1 or 2 glycine residues.
CCPP may comprise (i) 3, 4, 5, or 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 4 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 5 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 6 glycine, β -alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3, 4, or 5 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof. cCPP may comprise (i) 3 or 4 glycine, beta-alanine, 4-aminobutyric acid residues, or a combination thereof.
CCPP may comprise at least three glycine residues. cCPP may comprise (i) 3,4, 5 or 6 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3,4 or 5 glycine residues. cCPP can comprise (i) 3 or 4 glycine residues
In embodiments, none of the glycine, β -alanine, or 4-aminobutyric acid residues in cCPP are contiguous. Two or three glycine, beta-alanine or 4-aminobutyric acid residues may be contiguous. The two glycine, β -alanine or 4-aminobutyric acid residues may be contiguous.
In embodiments, none of the glycine residues cCPP are contiguous. Each glycine residue in cCPP may be separated by an amino acid residue that is not glycine. Two or three glycine residues may be contiguous. The two glycine residues may be contiguous.
Amino acid side chains with aromatic or heteroaromatic groups
CCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2, 3 or 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group.
CCPP may comprise (ii) 2,3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2,3 or 4 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic group.
The aromatic group may be a 6 to 14 membered aryl group. Aryl groups may be phenyl, naphthyl or anthracenyl, each of which is optionally substituted. Aryl groups may be phenyl or naphthyl, each of which is optionally substituted. The heteroaromatic group may be a 6 to 14 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S. Heteroaryl may be pyridinyl, quinolinyl or isoquinolinyl.
Amino acid residues having a side chain comprising an aromatic or heteroaromatic group may each independently be bis (Gao Naiji alanine), gao Naiji alanine, naphthylalanine, phenylglycine, bis (homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3- (3-benzothienyl) -alanine, 3- (2-quinolinyl) -alanine, O-benzylserine, 3- (4- (benzyloxy) phenyl) -alanine, S- (4-methylbenzyl) cysteine, N- (naphthalen-2-yl) glutamine, 3- (1, 1' -biphenyl-4-yl) -alanine, 3- (3-benzothienyl) -alanine or tyrosine, each of which is optionally substituted with one or more substituents. Amino acids having side chains comprising aromatic or heteroaromatic groups may each be independently selected from:
Wherein the H at the N-terminal and/or H at the C-terminal is replaced by a peptide bond.
The amino acid residues having a side chain comprising an aromatic or heteroaromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, gao Naiji alanine, bis (homophenylalanine), bis- (Gao Naiji alanine), tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienyl alanine, 4-phenylphenylalanine, 3, 4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5, 6-pentafluorophenylalanine, homophenylalanine, β -homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthryl) -alanine. The amino acid residues having a side chain comprising an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain comprising an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, homophenylalanine, gao Naiji alanine, bis (Gao Naiji alanine) or bis (Gao Naiji alanine), each of which is optionally substituted with one or more substituents. Amino acid residues having a side chain comprising an aromatic group may each independently be residues of phenylalanine or naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain comprising an aromatic group may be a residue of phenylalanine. The at least two amino acid residues having a side chain comprising an aromatic group may be residues of phenylalanine. Each amino acid residue having a side chain comprising an aromatic group may be a residue of phenylalanine.
In embodiments, none of the amino acids having side chains comprising aromatic or heteroaromatic groups are contiguous. The two amino acids having side chains comprising aromatic or heteroaromatic groups may be contiguous. The two contiguous amino acids may have opposite stereochemistry. Two contiguous amino acids may have the same stereochemistry. Three amino acids having side chains containing aromatic or heteroaromatic groups may be contiguous. The three contiguous amino acids may have the same stereochemistry. The three contiguous amino acids may have alternating stereochemistry.
The amino acid residue comprising an aromatic or heteroaromatic group may be an L-amino acid. The amino acid residue comprising an aromatic or heteroaromatic group may be a D-amino acid. The amino acid residue comprising an aromatic or heteroaromatic group may be a mixture of D-amino acids and L-amino acids.
The optional substituents may be any atom or group that does not significantly reduce (e.g., more than 50%) the cytoplasmic delivery efficiency of cCPP, e.g., as compared to an otherwise identical sequence without the substituents. The optional substituents may be hydrophobic or hydrophilic substituents. The optional substituents may be hydrophobic substituents. Substituents may increase the solvent accessible surface area (as defined herein) of the hydrophobic amino acid. The substituents may be halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamido (alkylcarboxamidyl), alkoxycarbonyl, alkylthio or arylthio. The substituent may be halogen.
While not wishing to be bound by theory, it is believed that amino acids having aromatic or heteroaromatic groups with higher hydrophobicity values (i.e., amino acids having side chains comprising aromatic or heteroaromatic groups) can improve the cytoplasmic delivery efficiency of cCPP relative to amino acids having lower hydrophobicity values. Each hydrophobic amino acid may independently have a hydrophobicity value that is greater than glycine. Each hydrophobic amino acid can independently be a hydrophobic amino acid having a hydrophobicity value greater than alanine. Each hydrophobic amino acid independently can have a hydrophobicity value greater than or equal to phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 2 lists the hydrophobicity values of the various amino acids reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S. A.1984;81 (1): 140-144), engleman et al (Ann. Rev. OfBiophys. Chem.1986;1986 (15): 321-53), kyte and Doolittle (J. Mol. Biol.1982;157 (1): 105-132), hoop and Woods (Proc. Natl. Acad. Sci. U.S. A.1981;78 (6): 3824-3828), and Janin (Nature. 1979;277 (5696): 491 4-492), each of which is incorporated herein by reference in its entirety. Hydrophobicity can be measured using the hydrophobicity scale reported by Engleman et al.
TABLE 2 amino acid hydrophobicity
The size of the aromatic or heteroaromatic groups may be selected to improve the cytoplasmic delivery efficiency of cCPP. While not wishing to be bound by theory, it is believed that larger aromatic or heteroaromatic groups on the amino acid side chains may improve cytoplasmic delivery efficiency compared to otherwise identical sequences with smaller hydrophobic amino acids. The size of the hydrophobic amino acid may be measured in terms of the molecular weight of the hydrophobic amino acid, the steric effect of the hydrophobic amino acid, the solvent accessible surface area of the side chain (SASA), or a combination thereof. The size of the hydrophobic amino acid can be measured in terms of the molecular weight of the hydrophobic amino acid, and the larger hydrophobic amino acid has side chains with a molecular weight of at least about 90g/mol, or at least about 130g/mol, or at least about 141 g/mol. The size of an amino acid can be measured in terms of the SASA of the hydrophobic side chain. The hydrophobic amino acid may have a side chain with SASA greater than or equal to alanine, or greater than or equal to glycine. The larger hydrophobic amino acid may have a side chain with a SASA greater than alanine or greater than glycine. The hydrophobic amino acid may have an aromatic or heteroaromatic group with a SASA greater than or equal to about piperidine-2-carboxylic acid, greater than or equal to about tryptophan, greater than or equal to about phenylalanine, or greater than or equal to about naphthylalanine. The first hydrophobic amino acid (AA H1) may have a SASA of at least aboutAt least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least aboutAt least about/>At least about/>At least about/>At least about/>Or at least about/>Is a side chain of (c). The second hydrophobic amino acid (AA H2) may have a SASA of at least about/>At least about/>At least aboutAt least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>Or at least about/>Is a side chain of (c). The side chains of AA H1 and AA H2 may have at least about/>At least about/>At least about/>At least about/> At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>Greater than about/>At least about/>At least about/>At least about/>At least about/>At least aboutAt least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>At least about/>Greater than about/>At least aboutAt least about/>At least about/>At least about/>Or at least about/>Is a combination of SASA. AA H2 can be a hydrophobic amino acid residue whose side chain SASA is less than or equal to the SASA of the hydrophobic side chain of AA H1. By way of example and not limitation, cCPP having a Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to cCPP which is otherwise identical to that having a Phe-Arg motif; cCPP having a Phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to otherwise identical cCPP having a Nal-Phe-Arg motif; and the Phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to otherwise identical cCPP with the Nal-Phe-Arg motif.
As used herein, "hydrophobic surface area" or "SASA" refers to the solvent accessible surface area of an amino acid side chain (reported in square angstroms; ). SASA can be calculated using the 'rolling ball' algorithm developed by Shrake & Rupley (J Mol biol.79 (2): 351-71), which is incorporated herein by reference in its entirety for all purposes. This algorithm uses solvent "spheres" of specific radius to probe the molecular surface. Typical values for spheres are/> Which approximates the radius of a water molecule.
The SASA values for some of the side chains are shown in table 3 below. The SASA values described herein are based on the theoretical values set forth in Table 3 below, as reported by Tien et al (PLOS ONE (11): e80635.https:// doi.org/10.1371/journ.fine.fine.0080635), which is incorporated herein by reference in its entirety for all purposes.
TABLE 3 amino acid SASA values
Residues Theoretical value Empirical values Miller et al (1987) Rose et al (1985)
Alanine (Ala) 129.0 121.0 113.0 118.1
Arginine (Arg) 274.0 265.0 241.0 256.0
Asparagine derivatives 195.0 187.0 158.0 165.5
Aspartic acid 193.0 187.0 151.0 158.7
Cysteine (S) 167.0 148.0 140.0 146.1
Glutamic acid 223.0 214.0 183.0 186.2
Glutamine 225.0 214.0 189.0 193.2
Glycine (Gly) 104.0 97.0 85.0 88.1
Histidine 224.0 216.0 194.0 202.5
Isoleucine (Ile) 197.0 195.0 182.0 181.0
Leucine (leucine) 201.0 191.0 180.0 193.1
Lysine 236.0 230.0 211.0 225.8
Methionine 224.0 203.0 204.0 203.4
Phenylalanine (Phe) 240.0 228.0 218.0 222.8
Proline (proline) 159.0 154.0 143.0 146.8
Serine (serine) 155.0 143.0 122.0 129.8
Threonine (Thr) 172.0 163.0 146.0 152.5
Tryptophan 285.0 264.0 259.0 266.3
Tyrosine 263.0 255.0 229.0 236.8
Valine (valine) 174.0 165.0 160.0 164.5
Amino acid residues having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof
Guanidine, as used herein, refers to the following structure:
As used herein, the protonated form of guanidine refers to the following structure:
guanidine replacement group refers to a functional group on the side chain of an amino acid that will be positively charged at or above physiological pH, or that reproduces the hydrogen bonding donating and accepting activity of a guanidinium (guanidinium) group.
The guanidine replacement group facilitates cell permeation and delivery of therapeutic agents while reducing toxicity associated with the guanidine group or protonated form thereof. cCPP may comprise at least one amino acid having a side chain comprising a guanidine or guanidinium substitution group. cCPP may comprise at least two amino acids having side chains comprising guanidine or guanidinium substitution groups. cCPP can comprise at least three amino acids having side chains comprising guanidine or guanidinium substitution groups
The guanidine or guanidinium group can be an isostere of guanidine or guanidinium. The guanidine or guanidinium replacement group can be less basic than guanidine.
As used herein, guanidine replacement group refers to Or a protonated form thereof.
The present disclosure relates to cCPP comprising 4 to 20 amino acid residues, wherein: (i) At least one amino acid has a side chain comprising a guanidino group or a protonated form thereof; (ii) At least one amino acid residue having no side chain or having a chain comprising Or a side chain of a protonated form thereof; and (iii) at least two amino acid residues independently have a side chain comprising an aromatic or heteroaromatic group.
At least two amino acid residues may have no side chains or have a chain comprising Or a side chain of a protonated form thereof. As used herein, an amino acid residue has two hydrogen atoms (e.g., -CH 2 -) on the carbon atom connecting the amine and the carboxylic acid when no side chains are present.
CCPP may comprise at least one amino acid having 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. At least two amino acids may have side chains comprising the same moiety selected from the group consisting of: Or a protonated form thereof. At least one amino acid may have a nucleotide sequence comprising/> Or a side chain of a protonated form thereof. At least two amino acids may have a nucleotide sequence comprising/>Or a side chain of a protonated form thereof. One, two, three or four amino acids may have a sequence comprisingOr a side chain of a protonated form thereof. One amino acid may have a nucleotide sequence comprising/>Or a side chain of a protonated form thereof. Two amino acids may have a nucleotide sequence comprising/>Or a side chain of a protonated form thereof. Or a protonated form thereof may be attached to the end of the amino acid side chain. /(I)May be attached to the end of the amino acid side chain.
CCPP may comprise (iii) 2,3, 4,5 or 6 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 2,3, 4, or 5 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group, or a protonated form thereof. cCPP may comprise (iii) 2,3 or 4 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof. cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidino group or a protonated form thereof. cCPP may comprise (iii) two amino acid residues having a side chain comprising a guanidino group or a protonated form thereof. cCPP may comprise (iii) three amino acid residues having a side chain comprising a guanidino group or a protonated form thereof.
The amino acid residues may independently have side chains comprising non-contiguous guanidine groups, guanidine replacement groups, or protonated forms thereof. The two amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, which may be contiguous. The three amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, which may be contiguous. The four amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups, or protonated forms thereof, which may be contiguous. Contiguous amino acid residues may have the same stereochemistry. Contiguous amino acids may have alternating stereochemistry.
The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be an L-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be a D-amino acid. The amino acid residues independently having a side chain comprising a guanidino group, a guanidine replacement group or a protonated form thereof may be an L-amino acid or a mixture of D-amino acids.
Each amino acid residue having a side chain comprising a guanidino group or a protonated form thereof may independently be an arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidino butyric acid, or residue of a protonated form thereof. Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof can independently be an arginine residue or a residue of a protonated form thereof.
Each amino acid having a side chain comprising a guanidine replacement group or protonated form thereof can independently be Or a protonated form thereof.
Without being bound by theory, it is hypothesized that the guanidine replacement group has a reduced basicity relative to arginine, and in some cases is uncharged at physiological pH (e.g., -N (H) C (O)), and is capable of sustaining a bidentate hydrogen bonding interaction with phospholipids on the plasma membrane, which is believed to promote efficient membrane binding and subsequent internalization. Removal of the positive charge is also believed to reduce cCPP's toxicity.
Those skilled in the art will appreciate that the N-terminus and/or C-terminus of the above-described unnatural aromatic hydrophobic amino acids form amide linkages upon incorporation into the peptides disclosed herein.
CCPP may comprise a first amino acid having a side chain comprising an aromatic or heteroaromatic group and a second amino acid having a side chain comprising an aromatic or heteroaromatic group, wherein the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain comprising an aromatic or heteroaromatic group and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain comprising an aromatic or heteroaromatic group. Although the term "first amino acid" generally refers to the N-terminal amino acid of a peptide sequence by convention, as used herein, a "first amino acid" is used to distinguish the referred amino acid from another amino acid (e.g., "second amino acid") in cCPP, such that the term "first amino acid" may refer to or may refer to an amino acid located at the N-terminal end of a peptide sequence.
CCPP may comprise: the N-terminus of the second glycine forms a peptide bond with an amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the second glycine forms a peptide bond with an amino acid having a side chain comprising a guanidino group or protonated form thereof.
CCPP may comprise a first amino acid having a side chain comprising a guanidino group or a protonated form thereof, and a second amino acid having a side chain comprising a guanidino group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidino group or a protonated form thereof, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain comprising a guanidino group or a protonated form thereof.
CCPP may comprise residues of asparagine, aspartic acid, glutamine, glutamic acid or homoglutamine. cCPP may comprise residues of asparagine. cCPP may comprise residues of glutamine.
CCPP may comprise residues of 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-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthracenyl) -alanine.
While not wishing to be bound by theory, it is believed that the chirality of the amino acids in cCPP can affect cytoplasmic uptake efficiency. cCPP may comprise at least one D amino acid. cCPP may comprise one to fifteen D amino acids. cCPP may comprise one to ten D amino acids. cCPP may comprise 1,2, 3 or 4D amino acids. cCPP may comprise 2,3,4, 5, 6, 7 or 8 contiguous amino acids with alternating D and L chiralities. cCPP may comprise three contiguous amino acids with the same chirality. cCPP may comprise two contiguous amino acids having the same chirality. At least two amino acids may have opposite chiralities. At least two amino acids having opposite chiralities may be adjacent to each other. At least three amino acids may have alternating stereochemistry with respect to each other. At least three amino acids having alternating chiralities relative to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry with respect to each other. At least four amino acids having alternating chiralities relative to each other may be adjacent to each other. At least two amino acids may have the same chirality. At least two amino acids having the same chirality may be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chiralities. At least two amino acids having opposite chiralities may be adjacent to at least two amino acids having the same chirality. Thus, adjacent amino acids in cCPP may have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D. The amino acid residues forming cCPP may all be L-amino acids. The amino acid residues forming cCPP may all be D-amino acids.
At least two amino acids may have different chiralities. At least two amino acids having different chiralities may be adjacent to each other. At least three amino acids may have different chiralities relative to adjacent amino acids. At least four amino acids may have different chiralities relative to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chiralities. One or more of the amino acid residues forming cCPP may be achiral. cCPP may comprise a 3,4 or 5 amino acid motif, wherein two amino acids having the same chirality may be separated by an achiral amino acid. cCPP may comprise the following sequence: D-X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L, wherein X is an achiral amino acid. The achiral amino acid may be glycine.
An amino acid having a side chain comprising:
or a protonated form thereof, may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. An amino acid having a side chain comprising: 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 having a side chain comprising guanidine or a protonated form thereof can be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Two amino acids having side chains comprising: /(I) Or protonated forms thereof, may be adjacent to each other. Two amino acids having side chains comprising guanidine or a protonated form thereof are adjacent to each other. cCPP may comprise at least two contiguous amino acids having a side chain which may comprise an aromatic or heteroaromatic group, and at least two non-contiguous amino acids having a side chain comprising: Or a protonated form thereof. cCPP can comprise at least two contiguous amino acids having a side chain comprising an aromatic or heteroaromatic group and at least two amino acids having a chain comprising/> Or a side chain of a protonated form thereof. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, e.g., any of the sequences described in the preceding paragraphs.
At least two amino acids having side chains comprising:
Or a protonated form thereof, with at least two amino acids having side chains comprising a guanidino group or a protonated form thereof.
CCPP may comprise the structure of formula (a):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
At least one of R 1、R2 and R 3 is an aromatic or heteroaromatic side chain of an amino acid;
r 4、R5、R6、R7 is independently H or an amino acid side chain;
At least one of R 4、R5、R6、R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N, N-dimethylarginine, 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, lysine, N-methyllysine, N, N-dimethyllysine, N-ethyllysine, N, N, N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N, N-dimethyllysine, β -homoarginine, 3- (1-piperidinyl) alanine;
AA SC is an amino acid side chain; and
Q is 1, 2, 3 or 4.
In embodiments, at least one of R 4、R5、R6、R7 is independently an uncharged non-aromatic side chain of an amino acid. In an embodiment, at least one of R 4、R5、R6、R7 is independently H or a side chain of citrulline.
In an embodiment, compounds are provided comprising a cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids. In embodiments, the at least two charged amino acids of the cyclic peptide are arginine. In embodiments, the at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthalanine (3-naphthalen-2-yl-alanine), or a combination thereof. In embodiments, the at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, glycine, or a combination thereof. In embodiments, the compound is a cyclic peptide having from 6 to 12 amino acids, wherein two amino acids of the cyclic peptide are arginine, at least two amino acids are aromatic hydrophobic amino acids selected from phenylalanine, naphthylalanine, and combinations thereof, and at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine, and combinations thereof.
In embodiments, the cyclic peptide of formula (a) is not a cyclic peptide having the sequence:
Wherein F is L-phenylalanine, F is D-phenylalanine, Φ is L-3- (2-naphthyl) -alanine, Φ is D-3- (2-naphthyl) -alanine, R is L-arginine, R is D-arginine, Q is L-glutamine, Q is D-glutamine, C is L-cysteine, U is L-selenocysteine, W is L-tryptophan, K is L-lysine, D is L-aspartic acid, and Ω is L-norleucine.
CCPP may comprise the structure of formula (I):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1,2, 3 or 4; and
Each m is independently an integer of 0,1, 2 or 3.
R 1、R2 and R 3 may each independently be H, -alkylene-aryl or-alkylene-heteroaryl. R 1、R2 and R 3 may each independently be H, -C 1-3 alkylene-aryl or-C 1-3 alkylene-heteroaryl. R 1、R2 and R 3 may each independently be H or-alkylene-aryl. R 1、R2 and R 3 may each independently be H or-C 1-3 alkylene-aryl. The C 1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R 1、R2 and R 3 may each independently be H, -C 1-3 alkylene-Ph or-C 1-3 alkylene-naphthyl. R 1、R2 and R 3 may each independently be H, -CH 2 Ph or-CH 2 naphthyl. R 1、R2 and R 3 may each independently be H or-CH 2 Ph.
R 1、R2 and R 3 may each independently be a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3, 4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5, 6-pentafluorophenylalanine, homophenylalanine, β -homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3- (9-anthracenyl) -alanine.
R 1 can be a side chain of tyrosine. R 1 can be the side chain of phenylalanine. R 1 can be the side chain of 1-naphthylalanine. R 1 can be the side chain of 2-naphthylalanine. R 1 can be the side chain of tryptophan. R 1 can be the side chain of 3-benzothiophenylalanine. R 1 can be the side chain of 4-phenylphenylalanine. R 1 can be the side chain of 3, 4-difluorophenylalanine. R 1 can be the side chain of 4-trifluoromethylphenylalanine. R 1 can be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R 1 can be the side chain of homophenylalanine. R 1 can be the side chain of beta-homophenylalanine. R 1 may be the side chain of 4-tert-butyl-phenylalanine. R 1 can be the side chain of 4-pyridylalanine. R 1 can be the side chain of 3-pyridylalanine. R 1 can be the side chain of 4-methylphenylalanine. R 1 can be the side chain of 4-fluorophenylalanine. R 1 can be the side chain of 4-chlorophenylalanine. R 1 can be the side chain of 3- (9-anthryl) -alanine.
R 2 can be a side chain of tyrosine. R 2 can be the side chain of phenylalanine. R 2 can be the side chain of 1-naphthylalanine. R 1 can be the side chain of 2-naphthylalanine. R 2 can be the side chain of tryptophan. R 2 can be the side chain of 3-benzothiophenylalanine. R 2 can be the side chain of 4-phenylphenylalanine. R 2 can be the side chain of 3, 4-difluorophenylalanine. R 2 can be the side chain of 4-trifluoromethylphenylalanine. R 2 can be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R 2 can be the side chain of homophenylalanine. R 2 can be the side chain of beta-homophenylalanine. R 2 may be the side chain of 4-tert-butyl-phenylalanine. R 2 can be the side chain of 4-pyridylalanine. R 2 can be the side chain of 3-pyridylalanine. R 2 can be the side chain of 4-methylphenylalanine. R 2 can be the side chain of 4-fluorophenylalanine. R 2 can be the side chain of 4-chlorophenylalanine. R 2 can be the side chain of 3- (9-anthryl) -alanine.
R 3 can be a side chain of tyrosine. R 3 can be the side chain of phenylalanine. R 3 can be the side chain of 1-naphthylalanine. R 3 can be the side chain of 2-naphthylalanine. R 3 can be the side chain of tryptophan. R 3 can be the side chain of 3-benzothiophenylalanine. R 3 can be the side chain of 4-phenylphenylalanine. R 3 can be the side chain of 3, 4-difluorophenylalanine. R 3 can be the side chain of 4-trifluoromethylphenylalanine. R 3 can be the side chain of 2,3,4,5, 6-pentafluorophenylalanine. R 3 can be the side chain of homophenylalanine. R 3 can be the side chain of beta-homophenylalanine. R 3 may be the side chain of 4-tert-butyl-phenylalanine. R 3 can be the side chain of 4-pyridylalanine. R 3 can be the side chain of 3-pyridylalanine. R 3 can be the side chain of 4-methylphenylalanine. R 3 can be the side chain of 4-fluorophenylalanine. R 3 can be the side chain of 4-chlorophenylalanine. R 3 can be the side chain of 3- (9-anthryl) -alanine.
R 4 can be H, -alkylene-aryl, -alkylene-heteroaryl. R 4 can be H, -C 1-3 alkylene-aryl, or-C 1-3 alkylene-heteroaryl. R 4 can be H or-alkylene-aryl. R 4 can be H or-C 1-3 alkylene-aryl. The C 1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R 4 can be H, -C 1-3 alkylene-Ph or-C 1-3 alkylene-naphthyl. R 4 can be H or the side chain of an amino acid in Table 1 or Table 3. R 4 can be H or an amino acid residue having a side chain comprising an aromatic group. R 4 can be H, -CH 2 Ph or-CH 2 naphthyl. R 4 can be H or-CH 2 Ph.
R 5 can be H, -alkylene-aryl, -alkylene-heteroaryl. R 5 can be H, -C 1-3 alkylene-aryl, or-C 1-3 alkylene-heteroaryl. R 5 can be H or-alkylene-aryl. R 5 can be H or-C 1-3 alkylene-aryl. The C 1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R 5 can be H, -C 1-3 alkylene-Ph or-C 1-3 alkylene-naphthyl. R 5 can be H or the side chain of an amino acid in Table 1 or Table 3. R 4 can be H or an amino acid residue having a side chain comprising an aromatic group. R 5 can be H, -CH 2 Ph or-CH 2 naphthyl. R 4 can be H or-CH 2 Ph.
R 6 can be H, -alkylene-aryl, -alkylene-heteroaryl. R 6 can be H, -C 1-3 alkylene-aryl, or-C 1-3 alkylene-heteroaryl. R 6 can be H or-alkylene-aryl. R 6 can be H or-C 1-3 alkylene-aryl. The C 1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R 6 can be H, -C 1-3 alkylene-Ph or-C 1-3 alkylene-naphthyl. R 6 can be H or the side chain of an amino acid in Table 1 or Table 3. R 6 can be H or an amino acid residue having a side chain comprising an aromatic group. R 6 can be H, -CH 2 Ph or-CH 2 naphthyl. R 6 can be H or-CH 2 Ph.
R 7 can be H, -alkylene-aryl, -alkylene-heteroaryl. R 7 can be H, -C 1-3 alkylene-aryl, or-C 1-3 alkylene-heteroaryl. R 7 can be H or-alkylene-aryl. R 7 can be H or-C 1-3 alkylene-aryl. The C 1-3 alkylene group may be methylene. The aryl group may be a 6 to 14 membered aryl group. The heteroaryl group may be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. The aryl group may be phenyl. Heteroaryl groups may be pyridinyl, quinolinyl and isoquinolinyl. R 7 can be H, -C 1-3 alkylene-Ph or-C 1-3 alkylene-naphthyl. R 7 can be H or the side chain of an amino acid in Table 1 or Table 3. R 7 can be H or an amino acid residue having a side chain comprising an aromatic group. R 7 can be H, -CH 2 Ph or-CH 2 naphthyl. R 7 can be H or-CH 2 Ph.
One, two or three of R 1、R2、R3、R4、R5、R6 and R 7 may be-CH 2Ph.R1、R2、R3、R4、R5、R6 and one of R 7 may be-CH 2Ph.R1、R2、R3、R4、R5、R6 and two of R 7 may be-CH 2Ph.R1、R2、R3、R4、R5、R6 and three of R 7 may be-CH 2Ph.R1、R2、R3、R4、R5、R6 and at least one of R 7 may be-CH 2Ph.R1、R2、R3、R4、R5、R6 and no more than four of R 7 may be-CH 2 Ph.
One, two or three of R 1、R2、R3 and R 4 are-CH 2Ph.R1、R2、R3 and one of R 4 are-CH 2Ph.R1、R2、R3 and two of R 4 are-CH 2Ph.R1、R2、R3 and three of R 4 are-CH 2Ph.R1、R2、R3 and at least one of R 4 are-CH 2 Ph.
One, two or three of R 1、R2、R3、R4、R5、R6 and R 7 may be H. One of R 1、R2、R3、R4、R5、R6 and R 7 may be H. Two of R 1、R2、R3、R4、R5、R6 and R 7 may be H. Three of R 1、R2、R3、R4、R5、R6 and R 7 may be H. At least one of R 1、R2、R3、R4、R5、R6 and R 7 may be H. No more than three of R 1、R2、R3、R4、R5、R6 and R 7 may be-CH 2 Ph.
One, two or three of R 1、R2、R3 and R 4 are H. One of R 1、R2、R3 and R 4 is H. Two of R 1、R2、R3 and R 4 are H. Three of R 1、R2、R3 and R 4 are H. At least one of R 1、R2、R3 and R 4 is H.
At least one of R 4、R5、R6 and R 7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R 4、R5、R6 and R 7 may be a side chain of 4-guanidino-2-aminopropionic acid. At least one of R 4、R5、R6 and R 7 may be a side chain of arginine. At least one of R 4、R5、R6 and R 7 may be a side chain of homoarginine. At least one of R 4、R5、R6 and R 7 may be a side chain of N-methyl arginine. At least one of R 4、R5、R6 and R 7 may be a side chain of N, N-dimethylarginine. At least one of R 4、R5、R6 and R 7 may be a side chain of 2, 3-diaminopropionic acid. At least one of R 4、R5、R6 and R 7 may be a side chain of 2, 4-diaminobutyric acid, lysine. At least one of R 4、R5、R6 and R 7 may be a side chain of N-methyl lysine. At least one of R 4、R5、R6 and R 7 may be a side chain of N, N-dimethyl lysine. At least one of R 4、R5、R6 and R 7 may be a side chain of N-ethyl lysine. At least one of R 4、R5、R6 and R 7 may be a side chain of N, N-trimethyllysine, 4-guanidinophenylalanine. At least one of R 4、R5、R6 and R 7 may be a side chain of citrulline. At least one of R 4、R5、R6 and R 7 may be a side chain of N, N-dimethyl lysine, β -homoarginine. At least one of R 4、R5、R6 and R 7 may be the side chain of 3- (1-piperidinyl) alanine.
At least two of R 4、R5、R6 and R 7 may be side chains of 3-guanidino-2-aminopropionic acid. At least two of R 4、R5、R6 and R 7 may be side chains of 4-guanidino-2-aminobutyric acid. At least two of R 4、R5、R6 and R 7 may be side chains of arginine. At least two of R 4、R5、R6 and R 7 may be side chains of homoarginine. At least two of R 4、R5、R6 and R 7 may be side chains of N-methyl arginine. At least two of R 4、R5、R6 and R 7 may be side chains of N, N-dimethylarginine. At least two of R 4、R5、R6 and R 7 may be side chains of 2, 3-diaminopropionic acid. At least two of R 4、R5、R6 and R 7 may be side chains of 2, 4-diaminobutyric acid, lysine. At least two of R 4、R5、R6 and R 7 may be side chains of N-methyl lysine. At least two of R 4、R5、R6 and R 7 may be side chains of N, N-dimethyl lysine. At least two of R 4、R5、R6 and R 7 may be side chains of N-ethyl lysine. At least two of R 4、R5、R6 and R 7 may be side chains of N, N, N-trimethyllysine, 4-guanidinophenylalanine. At least two of R 4、R5、R6 and R 7 may be side chains of citrulline. At least two of R 4、R5、R6 and R 7 may be side chains of N, N-dimethyl lysine, β -homoarginine. At least two of R 4、R5、R6 and R 7 may be side chains of 3- (1-piperidinyl) alanine.
At least three of R 4、R5、R6 and R 7 may be side chains of 3-guanidino-2-aminopropionic acid. At least three of R 4、R5、R6 and R 7 may be side chains of 4-guanidino-2-aminobutyric acid. At least three of R 4、R5、R6 and R 7 may be side chains of arginine. At least three of R 4、R5、R6 and R 7 may be side chains of homoarginine. At least three of R 4、R5、R6 and R 7 may be side chains of N-methyl arginine. At least three of R 4、R5、R6 and R 7 may be side chains of N, N-dimethylarginine. At least three of R 4、R5、R6 and R 7 may be side chains of 2, 3-diaminopropionic acid. At least three of R 4、R5、R6 and R 7 may be side chains of 2, 4-diaminobutyric acid and lysine. At least three of R 4、R5、R6 and R 7 may be side chains of N-methyllysine. At least three of R 4、R5、R6 and R 7 may be side chains of N, N-dimethyl lysine. At least three of R 4、R5、R6 and R 7 may be side chains of N-ethyl lysine. At least three of R 4、R5、R6 and R 7 may be the side chain of N, N, N-trimethyllysine, 4-guanidinophenylalanine. At least three of R 4、R5、R6 and R 7 may be side chains of citrulline. At least three of R 4、R5、R6 and R 7 may be side chains of N, N-dimethyl lysine, beta-homoarginine. At least three of R 4、R5、R6 and R 7 may be side chains of 3- (1-piperidinyl) alanine.
AA SC can be a side chain of an asparagine, glutamine or homoglutamine residue. AA SC can be a side chain of a glutamine residue. cCPP can also comprise a linker conjugated to AA SC (e.g., residues of asparagine, glutamine, or homoglutamine). Thus cCPP may also comprise linkers conjugated to asparagine, glutamine or homoglutamine residues. cCPP may also comprise a linker conjugated to the glutamine residue.
Q may be 1,2 or 3.q may be 1 or 2.q may be 1.q may be 2.q may be 3.q may be 4.
M may be 1-3.m may be 1 or 2.m may be 0 and m may be 1.m may be 2.m may be 3.
CCPP of formula (a) may comprise the structure of formula (I): Or a protonated form thereof, wherein AA SC、R1、R2、R3、R4、R7, m and q are as defined herein
CCPP of formula (A) may comprise a structure of formula (I-a) or formula (I-b):
Or a protonated form thereof, wherein AA SC、R1、R2、R3、R4 and m are as defined herein.
CCPP of formula (A) may comprise the structure of formula (I-1), (I-2), (I-3) or (I-4):
Or a protonated form thereof, wherein AA SC and m are as defined herein.
CCPP of formula (A) may comprise the structure of formula (I-5) or (I-6):
Or a protonated form thereof, wherein AA SC is as defined herein.
CCPP of formula (A) may comprise the structure of formula (I-1):
Or a protonated form thereof, wherein AA SC and m are as defined herein.
CCPP of formula (A) may comprise the structure of formula (I-2):
Or a protonated form thereof, wherein AA SC and m are as defined herein.
CCPP of formula (A) may comprise the structure of formula (I-3):
Or a protonated form thereof, wherein AA SC and m are as defined herein.
CCPP of formula (A) may comprise the structure of formula (I-4):
Or a protonated form thereof, wherein AA SC and m are as defined herein.
CCPP of formula (a) may comprise the structure of formula (I-5):
Or a protonated form thereof, wherein AA SC and m are as defined herein.
CCPP of formula (A) may comprise the structure of formula (I-6):
Or a protonated form thereof, wherein AA SC and m are as defined herein.
CCPP may comprise one of the following sequences: FGFGRGR; gfFGrGr; ffPhi GRGR; ffFGRGR; or FfΦ GrGr. cCPP may have one of the following sequences: FGFGRGRQ; gfFGrGrQ; ffPhi GRGRQ; ffFGRGRQ; or FfΦ GrGrQ.
The present disclosure also relates to cCPP having the structure of formula (II):
Wherein:
AA SC is an amino acid side chain;
R 1a、R1b and R 1c are each independently 6 to 14 membered aryl or 6 to 14 membered heteroaryl;
r 2a、R2b、R2c and R 2d are independently amino acid side chains;
At least one of R 2a、R2b、R2c and R 2d is Or a protonated form thereof;
At least one of R 2a、R2b、R2c and R 2d is guanidine or a protonated form thereof;
each n "is independently an integer of 0,1, 2,3, 4, or 5;
each n' is independently an integer of 0,1, 2 or 3; and
If n' is 0, then R 2a、R2b、R2b or R 2d are absent.
At least two of R 2a、R2b、R2c and R 2d may be Or a protonated form thereof. Two or three of R 2a、R2b、R2c and R 2d may be/> Or a protonated form thereof. One of R 2a、R2b、R2c and R 2d may be Or a protonated form thereof. At least one of R 2a、R2b、R2c and R 2d may be/>Or a protonated form thereof, and the remainder of each of R 2a、R2b、R2c and R 2d may be guanidine or a protonated form thereof. At least two of R 2a、R2b、R2c and R 2d may beOr a protonated form thereof, and the remainder of each of R 2a、R2b、R2c and R 2d may be guanidine or a protonated form thereof.
All R 2a、R2b、R2c and R 2d may be Or a protonated form thereof. At least one of R 2a、R2b、R2c and R 2d may be/>Or a protonated form thereof, and the remainder of each of R 2a、R2b、R2c and R 2d may be guanidine or a protonated form thereof. At least two of R 2a、R2b、R2c and R 2d may be/>Or a protonated form thereof, and the remainder of each of R 2a、R2b、R2c and R 2d may be guanidine or a protonated form thereof.
Each of R 2a、R2b、R2c and R 2d may independently be a side chain of 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid, the following acids: ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homolysine, serine, homoserine, threonine, allothreonine, histidine, 1-methylhistidine, 2-aminobutyric acid, aspartic acid, glutamic acid or homoglutamic acid.
AA SC can beWherein t may be an integer from 0 to 5. AA SC can be/>Wherein 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.
The AC described herein may be coupled to AA SC. In embodiments, the linker (L) couples AC to AA SC. In embodiments, the linker (L) is covalently bound to the backbone of the AC.
AA SC can be a side chain of an asparagine, glutamine or homoglutamine residue. AA SC can be a side chain of a glutamine residue. The cyclic peptide may comprise a linker conjugated to AA SC (e.g., residues of asparagine, glutamine, or homoglutamine).
R 1a、R1b and R 1c may each independently be a 6 to 14 membered aryl group. R 1a、R1b and R 1c may each independently be a 6 to 14 membered heteroaryl group having one or more heteroatoms selected from N, O or S. R 1a、R1b and R 1c may each be independently selected from phenyl, naphthyl, anthracenyl, pyridinyl, quinolinyl or isoquinolinyl. R 1a、R1b and R 1c may each be independently selected from phenyl, naphthyl or anthracenyl. R 1a、R1b and R 1c may each independently be phenyl or naphthyl. R 1a、R1b and R 1c may each be independently selected pyridinyl, quinolinyl or isoquinolinyl.
Each n' may independently be 1 or 2. Each n' may be 1. Each n' may be 2. At least one n' may be 0. At least one n' may be 1. At least one n' may be 2. At least one n' may be 3. At least one n' may be 4. At least one n' may be 5.
Each n "may independently be an integer from 1 to 3. Each n "may independently be2 or 3. Each n "may be 2. Each n "may be 3. At least one n "may be 0. At least one n "may be 1. At least one n "may be 2. At least one n "may be 3.
Each n "may independently be 1 or 2, and each n' may independently be 2 or 3. Each n "may be 1 and each n' may independently be 2 or 3. Each n "may be 1 and each n' may be 2. Each n "is 1 and each n' is 3.
CCPP of formula (II) may have the structure of formula (II-1):
Wherein R 1a、R1b、R1c、R2a、R2b、R2c、R2d、AASC, n 'and n' are as defined herein.
CCPP of formula (II) may have the structure of formula (IIa):
Wherein R 1a、R1b、R1c、R2a、R2b、R2c、R2d、AASC and n' are as defined herein.
CCPP of formula (II) may have the structure of formula (IIb):
Wherein R 2a、R2b、AASC and n' are as defined herein.
CCPP can have the structure of formula (IIb):
or a protonated form thereof,
Wherein:
AA SC and n' are as defined herein.
CCPP of formula (IIa) has one of the following structures:
wherein AA SC and n are as defined herein.
CCPP of formula (IIa) has one of the following structures:
/> Wherein AA SC and n are as defined herein
CCPP of formula (IIa) has one of the following structures:
wherein 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 can have the structure of formula (III):
Wherein:
AA SC is an amino acid side chain;
R 1a、R1b 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; and
Each p' is independently an integer from 0 to 5.
The AC described herein may be coupled to AA SC. The linker may couple AC to AA SC. The linker may be covalently bound to the backbone of AC, the 5 'end of AC or the 3' end of AC.
CCPP of formula (III) may have the structure of formula (III-1):
Wherein:
AA SC、R1a、R1b、R1c、R2a、R2c、R2b、R2d, n ', n ", and p' are as defined herein.
CCPP of formula (III) may have the structure of formula (IIIa):
Wherein:
AA SC、R2a、R2c、R2b、R2d, 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 protonated form thereof, n "may be 2 or 3, and each p' may be 0.
P' may be 0.p' may be 1.p' may be 2.p' may be 3.p' may be 4.p' may be 5
CCPP can have the following structure:
cCPP of formula (a) may be selected from:
CPP sequence
(FfФRrRrQ)
(FfФCit-r-Cit-rQ)
(FfФGrGrQ)
(FfFGRGRQ)
(FGFGRGRQ)
(GfFGrGrQ)
(FGFGRRRQ)
(FGFRRRRQ)
CCPP of formula (a) may be selected from:
CPP sequence
FФRRRRQ
fФRrRrQ
FfФRrRrQ
FfФCit-r-Cit-rQ
FfФGrGrQ
FfФRGRGQ
FfFGRGRQ
FGFGRGRQ
GfFGrGrQ
FGFGRRRQ
FGFRRRRQ
In embodiments cCPP is selected from:
CPP sequence CPP sequence CPP sequence
FФRRRQ RRFRФRQ FФRRRRQK
FФRRRC FRRRRФQ FФRRRRQC
FФRRRU rRFRФRQ fФRrRrRQ
RRRФFQ RRФFRRQ FФRRRRRQ
RRRRФF CRRRRFWQ RRRRФFDΩC
FФRRRR FfФRrRrQ FФRRR
FφrRrRq FFФRRRRQ FWRRR
FφrRrRQ RFRFRФRQ RRRФF
FФRRRRQ URRRRFWQ RRRWF
fФRrRrQ CRRRRFWQ
Φ=l-naphthylalanine; Φ=d-naphthylalanine; Ω=l-norleucine
In embodiments, cCPP is not selected from:
CPP sequence CPP sequence CPP sequence
FФRRRQ RRFRФRQ FФRRRRQK
FФRRRC FRRRRФQ FФRRRRQC
FФRRRU rRFRФRQ fФRrRrRQ
RRRФFQ RRФFRRQ FФRRRRRQ
RRRRФF CRRRRFWQ RRRRФFDΩC
FФRRRR FfФRrRrQ FФRRR
FφrRrRq FFФRRRRQ FWRRR
FφrRrRQ RFRFRФRQ RRRФF
FФRRRRQ URRRRFWQ RRRWF
fФRrRrQ CRRRRFWQ
Φ=l-naphthylalanine; phi = D-naphthylalanine; Ω=l-norleucine
CCPP may comprise the structure of formula (D):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
Y is
Q is 1, 2, 3 or 4;
each m is independently an integer of 0, 1,2 or 3, and
Each n is independently an integer of 0,1, 2 or 3.
CCPP of formula (D) may have the structure of formula (D-I):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer of 0, 1,2 or 3, and
Y is
CCPP of formula (D) may have the structure of formula (D-II):
/>
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer of 0,1, 2 or 3,
Each n is independently an integer of 0, 1,2 or 3, and
Y is
CCPP of formula (D) may have the structure of formula (D-III):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer of 0,1, 2 or 3,
Each n is independently an integer of 0, 1,2 or 3, and
Y is/>
CCPP of formula (D) may have the structure of formula (D-IV):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer of 0, 1,2 or 3, and
Y is
CCPP of formula (D) may have the structure of formula (D-V):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1, 2, 3 or 4;
each m is independently an integer of 0, 1,2 or 3, and
Y is
AA SC may be conjugated to a linker.
Joint
CCPP of the present disclosure may be conjugated to a linker. The connector may connect the AC to cCPP. The linker may be attached to the side chain of the amino acid of cCPP and the AC may be attached at a suitable position on the linker.
The linker may be any suitable moiety that can conjugate cCPP to one or more additional moieties, such as an Exocyclic Peptide (EP) and/or AC. Prior to conjugation with cCPP and one or more additional moieties, the linker has two or more functional groups, each of which is capable of independently forming a covalent bond with cCPP and one or more additional moieties. The linker may be covalently bound to the 5 'end of the AC or the 3' end of the AC. The linker may be covalently bound to the 5' end of the AC. The linker may be covalently bound to the 3' end of the AC. The linker may be any suitable moiety that conjugates cCPP described herein with AC.
The linker may comprise a hydrocarbon linker.
The linker may comprise a cleavage site. The cleavage site may be a disulfide or caspase cleavage site (e.g., val-Cit-PABC).
The joint may comprise: (i) One or more D or L amino acids, each of which is optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) One or more- (R 1-J-R2) z "-subunits, wherein R 1 and R 2 are each independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S, and O, wherein R 3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; (viii) - (R 1- J) z "-or- (J-R 1) z" -, wherein R 1 is each independently alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S or O, wherein R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z "is an integer from 1 to 50; or (ix) the linker may comprise one or more of (i) to (x).
The linker may comprise one or more D or L amino acids and/or- (R 1-J-R2) z "-, wherein R 1 and R 2 are each independently alkylene, each J is independently C, NR 3、-NR3 C (O) -, S and O, wherein R 4 is independently selected from H and alkyl, and z" is an integer from 1 to 50; or a combination thereof.
The linker may comprise- (OCH 2CH2)z - (e.g., as a spacer), wherein z 'is an integer from 1 to 23, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, "- (OCH 2CH2) z'" may also be referred to as polyethylene glycol (PEG).
The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise- (OCH 2CH2)z · -and a peptide, wherein z' is an integer from 1 to 23, the peptide may comprise from 2 to 10 amino acids, the linker may further comprise a Functional Group (FG) capable of a click chemistry reaction, FG may be an azide or alkyne, and triazole is formed when AC is conjugated to the linker.
The linker may comprise (i) a beta alanine residue and a lysine residue; (ii) - (J-R 1) z "; or (iii) combinations thereof. Each R 1 can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S, or O, wherein R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z "can be an integer from 1 to 50. Each R 1 can be alkylene and each J can be O.
The linker may comprise residues of (i) beta-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or a combination thereof; and (ii) - (R 1- J) z '-or- (J-R 1) z'. Each R 1 can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S, or O, wherein R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z "can be an integer from 1 to 50. Each R 1 can be alkylene and each J can be O. The linker may comprise glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or a combination thereof.
The linker may be a trivalent linker. The joint may have the following structure: Wherein A 1、B1 and C 1 can independently be a hydrocarbon linker (e.g., NRH- (CH 2)n -COOH), a PEG linker (e.g., NRH- (CH 2O)n -COOH, wherein R is H, methyl, or ethyl), or one or more amino acid residues, and Z independently is a protecting group.
The hydrocarbon may be a glycine or beta-alanine residue.
The linker may be divalent and connects cCPP to AC. The linker may be bivalent and connects cCPP to the Exocyclic Peptide (EP).
The linker may be trivalent and connects cCPP to AC and EP.
The linker may be a divalent or trivalent C 1-C50 alkylene group, wherein 1-25 methylene groups are optionally and independently replaced by-N (H) -, -N (C 1-C4 alkyl) -, -N (cycloalkyl) -, -O-, -C (O) O-, -S (O) 2-、-S(O)2N(C1-C4 alkyl) -, -S (O) 2 N (cycloalkyl) -, -N (H) C (O) -, -N (C 1-C4 alkyl) C (O) -, -N (cycloalkyl) C (O) -, -C (O) N (H) -, -C (O) N (C 1-C4 alkyl), -C (O) N (cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl or cycloalkenyl. The linker may be a divalent or trivalent C 1-C50 alkylene group in which 1-25 methylene groups are optionally and independently replaced by-N (H) -, -O-, -C (O) N (H) -or a combination thereof.
AC may be coupled to the glutamic acid of the cyclic peptide, which converts the glutamic acid to glutamine. The linker (L) may couple AC to glutamine/glutamate of the cyclic peptide. In embodiments, the linker (L) is covalently bound to the backbone of the AC.
The joint may have the following structure:
Wherein: each AA is independently an amino acid residue; * Is an attachment point to AA SC, and AA SC is a side chain of the amino acid residue of cCPP; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10. x may be an integer from 1 to 5. x may be an integer from 1 to 3. x may be 1.y may be an integer from 2 to 4. y may be 4.z may be an integer from 1 to 5. z may be an integer from 1 to 3. z may be 1. Each AA may be independently selected from glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminocaproic acid.
CCPP may be attached to the AC by a joint ("L"). The linker may be conjugated to AC through a bonding group ("M").
The joint may have the following structure:
Wherein: x is an integer from 1 to 10; y is an integer from 1 to 5; z is an integer from 1 to 10; each AA is independently an amino acid residue; * Is an attachment point to AA SC, and AA SC is a side chain of the amino acid residue of cCPP; and M is a bonding group as defined herein.
The joint may have the following structure:
Wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * Is an attachment point to AA SC, and AA SC is a side chain of the amino acid residue of cCPP; and M is a bonding group as defined herein.
The joint may have the following structure:
Wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; and z' is an integer from 1 to 23; * Is an attachment point to AA SC, and AA SC is a side chain of the amino acid residue of cCPP.
X may be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.
X' may be an integer from 1 to 23, such as1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. x' may be an integer from 5 to 15. x' may be an integer from 9 to 13. x' may be an integer from 1 to 5. x' may be 1.
Y may be an integer from 1 to 5, such as 1,2, 3, 4 or 5, including all ranges and subranges therebetween. y may be an integer from 2 to 5.y may be an integer from 3 to 5.y may be 3 or 4.y may be 4 or 5.y may be 3.y may be 4.y may be 5.
Z may be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.
Z' may be an integer from 1 to 23, such as 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. z' may be an integer from 5 to 15. z' may be an integer from 9 to 13. z' may be 11.
As discussed above, the linker or M (where M is part of the linker) may be covalently bound to AC (any suitable position on AC). The linker or M (where M is part of the linker) may be covalently bound to the 3 'end of the AC or the 5' end of the AC. The linker or M (where M is part of the linker) may be covalently bound to the backbone of the AC.
The linker may be bound to a side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker may be attached to the side chain of the lysine on cCPP.
The joint may have the following structure:
Wherein the method comprises the steps of
M is a group that conjugates L with AC;
AA s is the side chain or terminal of the amino acid on cCPP;
Each AA x is independently an amino acid residue;
o is an integer from 0 to 10; and
P is an integer from 0 to 5.
The joint may have the following structure:
Wherein the method comprises the steps of
M is a group that conjugates L with AC;
AA s is the side chain or terminal of the amino acid on cCPP;
Each AA x is independently an amino acid residue;
o is an integer from 0 to 10; and
P is an integer from 0 to 5.
M may include alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted. M may be selected from:
wherein R is alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl.
M may be selected from:
Wherein: r 10 is alkylene, cycloalkyl or Wherein a is 0 to 10.
M may beR 10 can be/>And a is 0to 10.M may be/>
M may be a heterobifunctional crosslinker, e.gIt is disclosed in Williams et al Curr. Protoc Nucleic Acid chem.2010, 42,4.41.1-4.41.20, which is incorporated herein by reference in its entirety.
M may be-C (O) -.
AA s may be a side chain or a terminal end 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 having an amino group). AA s may be AA SC as defined herein.
Each AA X is independently a natural or unnatural amino acid. One or more AA X may be a natural amino acid. One or more AA X may be an unnatural amino acid. One or more AA X may be a β -amino acid. The beta-amino acid may be beta-alanine.
O may be an integer from 0 to 10, such as 0, 1,2, 3,4, 5, 6, 7, 8, 9, and 10.o may be 0, 1,2 or 3.o may be 0.o may be 1.o may be 2.o may be 3.
P may be 0 to 5, for example 0, 1, 2, 3,4 or 5.p may be 0.p may be 1.p may be 2.p may be 3.p may be 4.p may be 5.
The joint may have the following structure:
Wherein M, AA s, each- (R 1-J-R2) z "-, o, and z" are as defined herein; r may be 0 or 1.
R may be 0.r may be 1.
The joint may have the following structure:
Wherein M, AA s, o, p, q, r, and z "each may be as defined herein.
Z "may be an integer from 1 to 50, such as 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49 and 50, including all ranges and values therebetween. z "may be an integer from 5 to 20. z "may be an integer from 10 to 15.
The joint may have the following structure:
Wherein:
M, AA s and o are as defined herein.
Other non-limiting examples of suitable linkers include:
/>
wherein M and AA s are as defined herein.
Provided herein are compounds comprising cCPP and AC complementary to a target in a pre-mRNA sequence, the compounds further comprising L, wherein the linker is conjugated to AC through a linking group (M), wherein M is
Provided herein are compounds comprising cCPP and an Antisense Compound (AC), such as an antisense oligonucleotide, which is complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein the linker is conjugated to AC through a linking group (M), wherein M is selected from the group consisting of:
Wherein: r 1 is alkylene, cycloalkyl or/> Wherein t' is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, wherein R 1 is/>And t' is 2.
The joint may have the following structure:
Wherein AA s is as defined herein, and m' is 0-10.
The linker may have the formula:
/>
the linker may have the formula: wherein "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.
The linker may have the formula:
wherein "base" corresponds to the nucleobase at the 3' end of the phosphorodiamidate morpholino oligomer.
The linker may have the formula:
Wherein the "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
The linker may have the formula: Wherein the "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
The linker may have the formula:
the linker may be covalently bound to the cargo at any suitable position on the AC. The linker may be covalently bound to the 3 'end of the cargo or the 5' end of the AC. The linker may be covalently bound to the backbone of the AC.
The linker may be bound to a side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker may be attached to the side chain of the lysine on cCPP.
CCPP-linker conjugates
CCPP may be conjugated to a linker as defined herein. The linker may be conjugated to AA SC of cCPP as defined herein.
The linker may comprise a- (OCH 2CH2)z' -subunit (e.g., as a spacer), wherein z' is an integer from 1 to 23, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 "- (OCH 2CH2)z'", also known as peg.cpp-linker conjugate) may have a structure selected from table 4:
Table 4: cCPP-linker conjugates
Ring (FfΦ -4gp-r-4 gp-rQ) -PEG 4-K-NH2
Ring (FfΦ -Cit-r-Cit-rQ) -PEG 4-K-NH2
Ring (FfPhi-Pia-r-Pia-rQ) -PEG 4-K-NH2
Ring (FfΦ -Dml-r-Dml-rQ) -PEG 4-K-NH2
Ring (Ffphi-Cit-r-Cit-rQ) -PEG 12 -OH
Ring (fPhi R-Cit-R-Cit-Q) -PEG 12 -OH
The linker may comprise- (OCH 2CH2)z' -subunit and a peptide subunit, wherein z' is an integer from 1 to 23, the peptide subunit may comprise from 2 to 10 amino acids cCPP-linker conjugate may have a structure selected from table 5:
table 5: endosomal escape vehicle (cCPP-linker conjugate)
Ac-PKKKKRKV-Lys (cyclo [ FfΦ -R-R-Cit-rQ ]) -PEG 12-K(N3)-NH2
Ac-PKKKKRKV-Lys (cyclo [ FfΦ -Cit-R-R-rQ ]) -PEG 12-K(N3)-NH2
Ac-PKKKKRKV-K (Ring (FfΦR-cit-R-cit-Q)) -PEG 12-K(N3)-NH2
Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -B-k (N) 3)-NH2
Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG2-k (N) 3)-NH2
Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG4-k (N) 3)-NH2
Ac-PKKKKRKV-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG12-k (N 3)-NH2
Ac-pkkkrkv-PEG2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG12-k (N 3)-NH2
Ac-rrv-PEG2-Lys (cyclo [ FfΦ -Cit-r-Cit-rQ ]) -PEG12-OH
Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-r-Q ]) -PEG12-k (N) 3)-NH2
Ac-PKKK-Cit-KV-PEG2-Lys (cyclo [ FfΦ -Cit-r-Cit-r-Q ]) -PEG12-k (N 3)-NH2
Ac-PKKKKRKV-PEG 2-Lys (cyclo [ FfΦ -Cit-r-Cit-r-Q ] -PEG12-K (N) 3)-NH2
CCPP-linker conjugates may be Ac-PKKKRKV-K (cyclo [ Ff GrGrQ ])-PEG 12-K (N 3)-NH2).
EEVs comprising a cyclic cell penetrating peptide (cCPP), a linker, and an Exocyclic Peptide (EP) are provided. EEV may comprise a structure of formula (B):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
R 4 and R 6 are independently H or an amino acid side chain;
EP is a cyclic exopeptide as defined herein;
Each m is independently an integer from 0 to 3;
n is an integer from 0 to 2;
x' is an integer from 1 to 20;
y is an integer from 1 to 5;
q is 1-4; and
Z' is an integer from 1 to 23.
R 1、R2、R3、R4、R6, EP, m, q, y, x ', z' are as described herein.
N may be 0.n may be 1.n may be 2.
EEVs may comprise structures of formula (B-a) or (B-B):
Or a protonated form thereof, wherein EP, R 1、R2、R3、R4, m and z' are as defined above in formula (B).
EEVs may comprise structures of formula (B-c):
or a protonated form thereof, wherein EP, R 1、R2、R3、R4 and m are as defined 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; and z is an integer from 1 to 10.
EEVs may have the structure of formula (B-1), (B-2), (B-3) or (B-4):
/>
Or a protonated form thereof, wherein EP is as defined above in formula (B).
The EEV may comprise formula (B) and may have the following structure: ac-PKKKRKV-AEEA-K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH or Ac-PKKRKV-AEEA-K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH.
The EEV may comprise cCPP of the formula:
The EEV may comprise the formula: ac-PKKKRKV-miniPEG-Lys (loop (FfFGRGRQ) -miniPEG2-K (N3).
The EEV may be:
The EEV may be: ac-PKKKKRKV-K (cyclo (Ff-Nal-GrGrQ) -PEG 12-K(N3)-NH2.
EEVs may be
EEVs may be Ac-P-K (Tfa) -K (Tfa) -K (Tfa) -R-K (Tfa) -V-AEEA-K (cyclo (Ff-Nal-GrGrQ) -PEG 12 -OH or Ac-P-K (Tfa) -K (Tfa) -K (Tfa) -R-K (Tfa) -V-AEEA-K (cyclo (FGFGRGRQ) -PEG 12 -OH).
EEVs may be
EEV may be Ac-PKKKRKV-miniPEG-K (ring (Ff-Nal-GrGrQ) -PEG12-OH.
EEVs may be
EEVs may be
EEVs may be
EEVs may be
EEVs may be
EEVs may be
The EEV may be:
EEVs may be
EEVs may be
EEVs may be
EEVs may be
EEVs may be selected from
Ac-rr-miniPEG-Dap [ ring (FfΦ -Cit-r-Cit-rQ) ] -PEG12-OH
Ac-frr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-rfr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-rbfbr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-rrr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-rbr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-rbrbr-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-hh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-hbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-hbhbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-rbhbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-hbrbh-PEG2-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -PEG12-OH
Ac-rr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-frr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-rfr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-rbfbr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-rrr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-rbr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-rbrbr-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-hh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-hbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-hbhbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-rbhbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-hbrbh-Dap (Ring (FfΦ -Cit-r-Cit-rQ)) -b-OH
Ac-KKKK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KGKK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KKGK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KKK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG2-K (N3) -NH2
Ac-KGK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KBK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KBKBK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KR-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KBR-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PKKKRKV-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PKKKRKV-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PGKKRKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PKGKRKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PKKGRKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PKKKGKV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PKKKRGV-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-PKKKRKG-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KKKRK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2
Ac-KKRK-miniPEG-Lys (cyclo (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2 and
Ac-KRK-miniPEG-Lys (Ring (Ff-Nal-GrGrQ)) -miniPEG-K (N3) -NH2.
EEV may be selected from:
Ac-PKKKRKV-Lys (cyclo [ FfΦ GrGrQ ]) -PEG 12-K(N3)-NH2
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ Ff. Phi. GrGrQ ]) -miniPEG 2-K(N3)-NH2
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ FGFGRGRQ ]) -miniPEG 2-K(N3)-NH2
Ac-KR-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 2-K(N3)-NH2
Ac-PKKKGKV-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 2-K(N3)-NH2
Ac-PKKKRKG-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 2-K(N3)-NH2
Ac-KKKRK-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 2-K(N3)-NH2
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ FF. Phi. GRGRQ ]) -miniPEG 2-K(N3)-NH2
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ beta hFf. Phi. GrGrQ ]) -miniPEG 2-K(N3)-NH2 and
Ac-PKKKRKV-miniPEG 2 -Lys-cyclo [ FfPhi SrSrQ ]) -miniPEG 2-K(N3)-NH2.
EEV may be selected from:
Ac-PKKKKRKV-miniPEG 2 -Lys (loop (GfFGrGrQ)) PEG 12 -OH
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ FGFKRKRQ ]) -PEG 12 -OH
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ FGFRGRGQ ]) -PEG 12 -OH
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ FGFGRGRGRQ ]) -PEG 12 -OH
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ FGFGRrRQ ]) -PEG 12 -OH
Ac-PKKKKRKV-miniPEG 2 -Lys (cyclo [ FGFGRRRQ ]) -PEG 12 -OH and
Ac-PKKKRKV-miniPEG 2 -Lys (cyclo [ FGFRRRRQ ]) -PEG 12 -OH.
EEV may be selected from:
Ac-K-K-K-R-K-G-miniPEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-K-K-K-R-K-miniPEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-K-K-R-K-K-PEG 4 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-K-R-K-K-K-PEG 4 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-K-K-K-K-R-PEG 4 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-R-K-K-K-K-PEG 4 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH and
Ac-K-K-K-R-K-PEG 4 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH.
EEV may be selected from:
Ac-PKKKRKV-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 2-K(N3)-NH2
Ac-PKKKRKV-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-PKKKKRKV-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 2-K(N3)-NH2 and
Ac-PKKKRKV-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH.
The cargo may be AC and the EEV may be selected from:
Ac-PKKKRKV-PEG 2 -K (cyclo [ FfPhi GrGrQ ]) -PEG 12 -OH
Ac-PKKKKRKV-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-PKKKRKV-PEG 2 -K (cyclo [ FfFGRGRQ ]) -PEG 12 -OH
Ac-PKKKRKV-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-PKKKRKV-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-PKKKRKV-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH
Ac-PKKKRKV-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH
Ac-rr-PEG 2 -K (cyclo [ FfPhi GrGrQ ]) -PEG 12 -OH
Ac-rr-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-rr-PEG 2 -K (cyclo [ FfF-GRGRQ ]) -PEG 12 -OH
Ac-rr-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-rr-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-rr-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH
Ac-rr-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH
Ac-rrr-PEG 2 -K (cyclo [ FfPhi GrGrQ ]) -PEG 12 -OH
Ac-rrr-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-rrr-PEG 2 -K (cyclo [ FfFGRGRQ ]) -PEG 12 -OH
Ac-rrr-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-rrr-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-rrr-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH
Ac-rrr-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH
Ac-rhr-PEG 2 -K (cyclo [ FfΦ GrGrQ ]) -PEG 12 -OH
Ac-rhr-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-rhr-PEG 2 -K (cyclo [ FfFGRGRQ ]) -PEG 12 -OH
Ac-rhr-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-rhr-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-rhr-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH
Ac-rhr-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH
Ac-rbr-PEG 2 -K (cyclo [ FfΦ GrGrQ ]) -PEG 12 -OH
Ac-rbr-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-rbr-PEG 2 -K (cyclo [ FfFGRGRQ ]) -PEG 12 -OH
Ac-rbr-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-rbr-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-rbr-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH
Ac-rbr-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH
Ac-rbrbr-PEG 2 -K (cyclo [ FfΦ GrGrQ ]) -PEG 12 -OH
Ac-rbrbr-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-rbrbr-PEG 2 -K (cyclo [ FfFGRGRQ ]) -PEG 12 -OH
Ac-rbrbr-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-rbrbr-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-rbrbr-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH
Ac-rbrbr-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH
Ac-rbhbr-PEG 2 -K (cyclo [ FfΦ GrGrQ ]) -PEG 12 -OH
Ac-rbhbr-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-rbhbr-PEG 2 -K (cyclo [ FfFGRGRQ ]) -PEG 12 -OH
Ac-rbhbr-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH
Ac-rbhbr-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-rbhbr-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH
Ac-rbhbr-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH
Ac-hbrbh-PEG 2 -K (cyclo [ FfΦ GrGrQ ]) -PEG 12 -OH
Ac-hbrbh-PEG 2 -K (cyclo [ FfΦCit-r-Cit-rQ ]) -PEG 12 -OH
Ac-hbrbh-PEG 2 -K (cyclo [ FfFGRGRQ ]) -PEG 12 -OH
Ac-hbrbh-PEG 2 -K (cyclo ] FGFGRGRQ) -PEG 12 -OH
Ac-hbrbh-PEG 2 -K (cyclo [ GfFGrGrQ ]) -PEG 12 -OH
Ac-hbrbh-PEG 2 -K (cyclo [ FGFGRRRQ ]) -PEG 12 -OH and
Ac-hbrbh-PEG 2 -K (cyclo [ FGFRRRRQ ]) -PEG 12 -OH,
Wherein b is beta-alanine and the exocyclic sequence may be D or L stereochemistry.
In embodiments, the compound comprising the cyclic peptide and AC has improved cytoplasmic uptake efficiency compared to the compound comprising AC alone. Cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of a compound comprising a cyclic peptide and AC with the cytoplasmic delivery efficiency of AC alone.
Antisense compounds
In various embodiments, the compounds disclosed herein comprise a CPP (e.g., a cyclic peptide) conjugated to an Antisense Compound (AC). In embodiments, the AC comprises an antisense oligonucleotide directed against the target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" is intended to include oligonucleotides complementary to a target polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA complementary to a selected sequence (e.g., target gene mRNA).
Antisense oligonucleotides can modulate one or more aspects of protein transcription, translation, and expression. In embodiments, the antisense oligonucleotide is directed against a target sequence within a target pre-mRNA to modulate one or more aspects of pre-mRNA splicing. As used herein, modulation of splicing refers to altering the processing of a pre-mRNA transcript such that a spliced mRNA molecule contains a combination of different exons due to exon skipping or exon inclusion, deletion of one or more exons, or deletion or addition of sequences (e.g., intron sequences) that are not normally present in a spliced mRNA. In embodiments, hybridization of the antisense oligonucleotide to a target sequence in a pre-mRNA molecule restores native splicing to the mutated pre-mRNA sequence. In embodiments, antisense oligonucleotide hybridization results in alternative splicing of the target pre-mRNA. In embodiments, antisense oligonucleotide hybridization results in exon inclusion or exon skipping of one or more exons. In embodiments, the skipped exon sequence comprises a frameshift mutation, a nonsense mutation, or a missense mutation. In embodiments, the skipped exon sequence comprises a nucleic acid deletion, substitution, or insertion. In embodiments, the skipped exons do not themselves contain sequence mutations, but adjacent exons contain mutations that result in frame shift mutations or nonsense mutations. In embodiments, hybridization of the antisense oligonucleotide to a target sequence within the target pre-mRNA prevents inclusion of an exon sequence in the mature mRNA molecule. In embodiments, hybridization of the antisense oligonucleotide to a target sequence within the target pre-mRNA results in preferential expression of the wild-type target protein isoform. In embodiments, hybridization of the antisense oligonucleotide to a target sequence within the target pre-mRNA results in expression of a re-spliced target protein comprising an active fragment of the wild-type target protein.
The antisense mechanism functions via hybridization of the antisense oligonucleotide compound to the target nucleic acid. In embodiments, hybridization of an antisense oligonucleotide to its target sequence inhibits expression of a target protein. In embodiments, hybridization of an antisense oligonucleotide to its target sequence inhibits expression of one or more wild-type target protein isoforms. In embodiments, hybridization of an antisense oligonucleotide to its target sequence upregulates expression of the target protein. In embodiments, hybridization of an antisense oligonucleotide to its target sequence increases expression of one or more wild-type target protein isoforms.
In embodiments, antisense compounds can inhibit gene expression by binding to complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression by preventing translation of the complementary mRNA strand (by sterically blocking RNA-binding proteins involved in translation) or by causing degradation of the target mRNA. Antisense DNA can be used to target specific complementary (coding or non-coding) RNAs. If binding occurs, the DNA/RNA hybrid can be degraded by RNase H. In embodiments, the antisense oligonucleotide contains from about 10 to about 50 nucleotides or from about 15 to about 30 nucleotides. In embodiments, the antisense oligonucleotide may not be fully complementary to the target nucleotide sequence.
Antisense oligonucleotides have proven to be effective targeted inhibitors of protein synthesis and are therefore useful for specifically inhibiting protein synthesis by targeting genes. The efficacy of antisense oligonucleotides in inhibiting protein synthesis has been well established. For example, synthesis of polygalacturonase (polygalactauronase) and muscarinic type 2 acetylcholine receptors is inhibited by antisense oligonucleotides directed against their corresponding mRNA sequences (U.S. patent 5,739,119 and U.S. patent 5,759,829). In addition, examples of antisense inhibition have been demonstrated with nucleoprotein cyclin, multiple drug resistance genes (MDG 1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor and human EGF (Jaskulski et al, science 6, 10, 1988; 240 (4858): 1544-6; vasanthakumar and Ahmed, cancer Commun.1989;1 (4): 225-32; peris et al, brain Res Mol Brain Res.1998, 15, 57 (2): 310-20; U.S. patent 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). In addition, antisense constructs have also been described to inhibit and are useful in the treatment of various abnormal cell proliferation, such as cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683).
Methods of generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeted to any polynucleotide sequence. The selection of antisense oligonucleotide sequences specific for a given target sequence is based on analysis of the selected target sequence and determination of secondary structure, tm, binding energy and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that will reduce or inhibit specific binding to target mRNA in a host cell. Target regions of the mRNA may include those at or near the AUG translation initiation codon and those sequences that are substantially complementary to the 5' region of the mRNA. These secondary structural analysis and target site selection considerations may be performed, for example, using the 4 th edition of OLIGO primer analysis software (Molecular Biology Insights) and/or BLASTN 2.0.5 algorithm software (Altschul et al, nucleic Acids res.1997, 25 (17): 3389-402).
According to the present disclosure, antisense Compounds (ACs) alter one or more aspects of splicing, translation, or expression of a target gene, for example, by altering splicing of eukaryotic target pre-mRNA. An AC according to the present disclosure comprises a nucleic acid sequence that is complementary to a sequence found within a target pre-mRNA sequence (e.g., at a sequence that includes at least a portion of an exon, at least a portion of an intron, or both). The use of these ACs provides a straightforward genetic approach that can regulate splicing of specific pathogenic genes. The principle behind antisense technology is that antisense compounds hybridized to a target nucleic acid regulate gene expression events such as splicing or translation by one of a variety of antisense mechanisms. The sequence specificity of AC makes the technology attractive as a therapeutic approach to selectively modulate splicing of pre-mRNA involved in the pathogenesis of any of a variety of diseases. Antisense technology is an effective means for altering the expression of one or more specific gene products and may therefore prove useful in many therapeutic, diagnostic and research applications.
The compounds described herein may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which may be defined as (R) or (S), α or β, or (D) or (L) depending on the absolute stereochemistry. Antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.
Antisense compound hybridization sites
The antisense mechanism relies on hybridization of an antisense compound to a target nucleic acid. In embodiments, the present disclosure provides antisense compounds complementary to a target nucleic acid. In embodiments, the target nucleic acid sequence is present in a pre-mRNA molecule. In embodiments, the target nucleic acid sequence is present in an exon of a pre-mRNA molecule. In embodiments, the target nucleic acid sequence is present in an intron of a pre-mRNA molecule.
The pre-mRNA molecules are produced in the nucleus and processed before or during transport to the cytoplasm for translation. The processing of pre-mRNA involves the addition of a 5 'methylated cap and a poly (A) tail of about 200-250 bases at the 3' end of the transcript. The next step in mRNA processing is splicing of pre-mRNA, which occurs during the maturation of 90-95% of mammalian mRNA. Introns (or intervening sequences) are regions of the primary transcript (or DNA encoding it) that are not included in the coding sequence of the mature mRNA. Exons are regions of the primary transcript in which mature mRNAs remain when they reach the cytoplasm. Exons splice together to form the mature mRNA sequence. The splice junction is also referred to as a splice site, wherein the 5 'side of the junction is commonly referred to as the "5' splice site" or "splice donor site" and the 3 'side is referred to as the "3' splice site" or "splice acceptor site". In splicing, the 3 'end of the upstream exon is joined to the 5' end of the downstream exon. Thus, the non-spliced RNA (pre-mRNA) has an exon/intron junction at the 5 'end of the intron and an intron/exon junction at the 3' end of the intron. After introns are removed, exons are contiguous in the mature mRNA at what is sometimes referred to as an exon/exon junction or boundary. Cryptic splice sites are those splice sites that are not frequently used but can be used when the common splice site is blocked or otherwise unavailable. Alternative splicing, defined as splicing together different combinations of exons, typically results in the production of multiple mRNA transcripts from a single gene.
In embodiments, AC hybridizes to a sequence in a splice site. In embodiments, AC hybridizes to a sequence comprising a portion of a splice site. In embodiments, AC hybridizes to a sequence comprising part or all of the splice site. In embodiments, AC hybridizes to a sequence comprising part or all of the splice donor site. In embodiments, AC hybridizes to a sequence comprising part or all of a splice acceptor site. In embodiments, AC hybridizes to a sequence comprising a portion or all of a cryptic splice site. In embodiments, the AC hybridizes to a sequence comprising an exon/intron junction.
Pre-mRNA splicing involves two consecutive biochemical reactions. Both reactions involve a spliceosome transesterification reaction between RNA nucleotides. In the first reaction, the 2'-OH of a particular branch point nucleotide (defined during assembly of the spliceosome) within the intron nucleophilic attack the first nucleotide of the intron at the 5' splice site, thereby forming a lasso intermediate (LARIAT INTERMEDIATE). In the second reaction, the 3' -OH of the released 5' exon nucleophilic attack on the last nucleotide of the intron at the 3' splice site, thereby splicing the exon and releasing the intronic lasso. pre-mRNA splicing is regulated by Intronic Silencing Sequences (ISS) and Terminal Stem Loop (TSL) sequences. As used herein, the terms "Intronic Silencing Sequence (ISS)" and "Terminal Stem Loop (TSL)" refer to sequence elements within introns and exons, respectively, that control alternative splicing through the binding of trans-acting protein factors within pre-mRNA, resulting in the different use of splice sites. Typically, intronic silencing sequences are between 8 and 16 nucleotides and are less conserved than splice sites at exon-intron junctions. Terminal stem-loop sequences are typically between 12 and 24 nucleotides and form secondary loop structures due to complementarity and thus binding within the 12-24 nucleotide sequences.
In embodiments, the AC hybridizes to a sequence comprising a portion or all of an intron silencing sequence. In embodiments, AC hybridizes to a sequence comprising part or all of the terminal stem loop.
Up to 50% of human genetic diseases caused by point mutations are caused by aberrant splicing. Such point mutations may disrupt the current splice site or create new splice sites, resulting in mRNA transcripts containing different combinations of exons or exon deletions. Point mutations may also result in activation of cryptic splice sites or disruption of cis-regulatory elements (i.e., splice enhancers or silencers).
In embodiments, AC hybridizes to a sequence comprising part or all of the aberrant splice site created by a mutation in the target gene. In embodiments, the AC hybridizes to a sequence comprising part or all of the regulatory elements. Antisense compounds targeting cis regulatory elements are also provided. In embodiments, the regulatory element is in an exon. In embodiments, the regulatory element is in an intron.
In embodiments, the AC can specifically hybridize to sequences in the translational initiator region, the 5' cap region, the intron/exon junction, the coding sequence, the translational stop codon region, or the 5' -untranslated region or the 3' -untranslated region. In embodiments, AC may hybridize to part or all of the pre-mRNA splice site, exon-exon junction or intron-exon junction. In embodiments, the AC may hybridize to an aberrant fusion junction due to rearrangement or deletion. In embodiments, AC may hybridize to a particular exon in an alternatively spliced mRNA.
In embodiments, AC hybridizes to a sequence between 5 and 50 nucleotides in length, which may also be referred to as the length of AC. In embodiments, the AC is between 5 and 50 nucleotides in length, such as between 5 and 10, 10 and 15, 15 and 20, 20 and 25, 25 and 30, 30 and 35, 35 and 40, 40 and 45, or 45 and 50 nucleotides in length. In embodiments, the AC is about 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 nucleotides in length. In embodiments, the length of the AC is at least 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, and at most about 21, about 22, about 23, about 24, or about 25, and at most 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, or about 40, and at most about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 nucleotides. In some embodiments, the AC is about 10 nucleotides in length. In some embodiments, the AC is about 15 nucleotides in length. In some embodiments, the AC is about 16 nucleotides in length. In some embodiments, the AC is about 17 nucleotides in length. In some embodiments, the AC is about 18 nucleotides in length. In some embodiments, the AC is about 19 nucleotides in length. In some embodiments, the AC is about 20 nucleotides in length. In some embodiments, the AC is about 21 nucleotides in length. In some embodiments, the AC is about 22 nucleotides in length. In some embodiments, the AC is about 23 nucleotides in length. In some embodiments, the AC is about 24 nucleotides in length. In some embodiments, the AC is about 25 nucleotides in length. In some embodiments, the AC is about 26 nucleotides in length. In some embodiments, the AC is about 27 nucleotides in length. In some embodiments, the AC is about 28 nucleotides in length. In some embodiments, the AC is about 29 nucleotides in length. In some embodiments, the AC is about 30 nucleotides in length.
In embodiments, AC can be less than 100% complementary to the target nucleic acid sequence. As used herein, the term "percent complementarity" refers to the number of nucleobases of AC that have nucleobase complementarity to the corresponding nucleobase of an oligomeric compound or nucleic acid divided by the total length of AC (the number of nucleobases). One skilled in the art recognizes that it is possible to include mismatches without eliminating the activity of the antisense compound. In embodiments, the AC may contain up to about 20% nucleotides that disrupt base pairing of the AC with the target nucleic acid. In embodiments, AC contains no more than about 15%, no more than about 10%, no more than 5% mismatch or no mismatch. In embodiments, the AC contains no more than 1, 2, 3, 4, or 5 mismatches. In embodiments, AC is 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. The percent complementarity of an oligonucleotide is calculated by dividing the number of complementary nucleobases by the total number of nucleobases of the oligonucleotide. The percent complementarity of an oligonucleotide region is calculated by dividing the number of complementary nucleobases in the region by the total number of nucleobase bases regions.
In embodiments, incorporating nucleotide affinity modifications allows for a greater number of mismatches than unmodified compounds. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than other oligonucleotide sequences. One of ordinary skill in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, such as by determining the melting temperature (Tm). Tm or ATm can be calculated by techniques familiar to those of ordinary skill in the art. For example, freier et al (Nucleic ACIDS RESEARCH,1997, 25, 22:4429-4443) describe techniques that allow one of ordinary skill in the art to evaluate nucleotide modification-enhancing RNAs: ability of DNA duplex melting temperature.
Antisense mechanism
AC according to the present disclosure may regulate one or more aspects of protein transcription, translation, and expression. In embodiments, AC hybridized to a target sequence within a target pre-mRNA modulates one or more aspects of pre-mRNA splicing. As used herein, modulation of splicing refers to altering the processing of a pre-mRNA transcript such that a spliced mRNA molecule contains a combination of different exons due to exon skipping or exon inclusion, deletion of one or more exons, or deletion or addition of sequences (e.g., intron sequences) that are not normally present in a spliced mRNA. In embodiments, AC hybridization to a target sequence within a pre-mRNA molecule restores native splicing to the mutated pre-mRNA sequence. In embodiments, AC hybridization results in alternative splicing of the target pre-mRNA. In embodiments, AC hybridization results in exon inclusion or exon skipping of one or more exons. In embodiments, the skipped exon sequence comprises a frameshift mutation, a nonsense mutation, or a missense mutation. In embodiments, the skipped exon sequence comprises a nucleic acid deletion, substitution, or insertion. In embodiments, the skipped exons do not themselves contain sequence mutations, but adjacent exons contain mutations that result in frame shift mutations or nonsense mutations. In embodiments, the deletion of an exon that does not contain a sequence mutation restores the reading frame of the mature mRNA. In embodiments, hybridization of AC to a target sequence within a target pre-mRNA results in preferential expression of a wild-type target protein isoform. In embodiments, hybridization of AC to a target sequence within a target pre-mRNA results in expression of a re-spliced target protein comprising an active fragment of a wild-type target protein.
The antisense mechanism functions via hybridization of the antisense compound to the target nucleic acid. In embodiments, hybridization of AC to its target sequence inhibits expression of the target protein. In embodiments, hybridization of AC to its target sequence inhibits expression of one or more wild-type target protein isoforms. In embodiments, hybridization of AC with its target sequence up-regulates expression of the target protein. In embodiments, hybridization of AC to its target sequence increases expression of one or more wild-type target protein isoforms.
The efficacy of the AC of the present disclosure can be assessed by evaluating the antisense activity affected by its administration. As used herein, the term "antisense activity" refers to any detectable and/or measurable activity attributable to hybridization of an antisense compound to its target nucleic acid. Such detection and/or measurement may be direct or indirect. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of a target protein. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of the re-spliced target protein. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of target nucleic acid and/or cleaved target nucleic acid and/or alternatively spliced target nucleic acid.
Antisense compound design
The design of an AC according to the present disclosure will depend on the sequence targeted. Targeting AC to a specific target nucleic acid molecule can be a multi-step process. The process typically begins with the identification of a target nucleic acid whose expression is to be modulated. As used herein, the terms "target nucleic acid" and "nucleic acid encoding a target gene" encompass DNA encoding a selected target gene, RNA transcribed from such DNA (including pre-mRNA and mRNA), and cDNA derived from such RNA. For example, the target nucleic acid may be a nucleic acid molecule that expresses a cellular gene (or mRNA transcribed from the gene) associated with a particular disorder or disease condition, or an infectious agent.
Those skilled in the art will be able to design, synthesize and screen antisense compounds of different nucleobase sequences to identify sequences that produce antisense activity. For example, antisense compounds can be designed that alter splicing of target pre-mRNA or inhibit expression of target protein. Methods for designing, synthesizing, and screening antisense compounds for antisense activity against a preselected target nucleic acid can be found, for example, in "ANTISENSE DRUG TECHNOLOGY, principles, strategies, and Applications," CRC Press, boca Raton, florida, edited by Stanley T.Crooke, which is incorporated by reference in its entirety for any purpose.
In embodiments, antisense compounds comprise modified nucleosides, modified internucleoside linkages, and/or conjugate groups.
In embodiments, the antisense compound is a "tricyclo-DNA (tc-DNA)", which refers to a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane loop to limit conformational flexibility of the backbone and optimize backbone geometry at torsion angle γ. tc-DNA containing the homobases adenine and thymine forms very stable A-T base pairs with complementary RNA.
Nucleoside
In embodiments, antisense compounds comprising linked nucleosides are provided. In embodiments, some or all of the nucleosides are modified nucleosides. In embodiments, one or more nucleosides comprise a modified nucleobase. In embodiments, one or more nucleosides comprise a modified sugar. Chemically modified nucleosides are typically used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for a target RNA.
In general, a nucleobase is any group containing one or more atoms or groups of atoms capable of hydrogen bonding with the base of another nucleic acid. In addition to "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (a) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are also suitable for use in the compounds described herein. The terms modified nucleobase and nucleobase mimetic may overlap, but typically modified nucleobases refer to nucleobases that are similar in structure to the parent nucleobase, such as, for example, 7-deazapurine, 5-methylcytosine, or G-clamp, whereas nucleobase mimetics will include more complex structures, such as, for example, 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 provided herein comprises one or more nucleosides having a modified sugar moiety. In embodiments, the furanosyl sugar ring of a natural nucleoside can be modified in a variety of ways, including but not limited to adding substituents, bridging two non-geminal ring atoms to form a Bicyclic Nucleic Acid (BNA), and substituting the epoxy at the 4' -position with an atom or group such as-S-, -N (R) -C (R 1)(R2). Modified sugar moieties are well known and can be used to alter (typically increase) the affinity of antisense compounds for their targets and/or increase nuclease resistance. Representative lists of modified sugars include, but are not limited to, non-bicyclic substituted sugars, particularly non-bicyclic 2' -substituted sugars having a 2' -F, 2' -OCH 3, or 2' -O (CH 2)2-OCH3 substituent; and 4' -thio modified sugars, sugar moieties may also be substituted with sugar mimetic groups, and the like, e.g., furanose rings may be substituted with morpholino rings.
In embodiments, nucleosides comprise bicyclic modified sugars (BNA), including 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' bridged BNA, 4'- (CH 2CH(CH3)) -2' bridged BNA "cEt (4 '- (CH 3) -O-2' bridge), and cMOE BNA (4 '- (CH 2OCH3) -O-2' bridge). Some such BNA has been prepared and disclosed in the patent literature as well as in the scientific literature (see e.g. 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, fluidier et al Chembiochem 2005,6, 1104-1109, singh et al, chem. Commun., 1998,4, 455-456; koshkin et al Tetrahedron,1998, 54, 3607-3630; wahlstedt et al, proc.Natl.Acad.Sci.U.S.A.,2000, 97, 5633-5638; kumar et al, biorg. 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; examples of issued U.S. patents and published applications disclosing BNA include, for example, U.S. patent No. 7,053,207;6,268,490;6,770,748;6,794,499;7,034,133; and 6,525,191; U.S. pre-grant publication No. 2004-0171570;2004-0219565;2004-0014959;2003-0207841;2004-0143114; and 20030082807.
Also provided herein is a "locked nucleic acid" (LNA) in which the 2 '-hydroxyl group of the ribosyl sugar ring is attached to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4' -C-oxymethylene linkage to form a bicyclic sugar moiety (reviewed in Elayadi et al, curr. Opinion Invens. Drugs,2001,2, 558-561; braasch et al, chem. Biol.,2001, 81-7, and Orum et al, curr. Opinion mol. Ther.,2001,3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage may be a methylene (-CH 2 -) group bridging the 2 'oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic moiety; in the case of ethylene in said position, the term ENA TM (Singh et al, chem. Commun.,1998,4, 455-456; ENA TM: morita et al, bioorganic MEDICINAL CHEMISTRY,2003, 11, 2211-2226) is used. LNAs and other bicyclic sugar analogs exhibit very high duplex thermal stability (tm= +3 to +10 ℃) to complementary DNA and RNA, stability to 3' -exonucleolytic degradation, and good solubility. Effective and nontoxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al, proc. Natl. Acad. Sci. U.S.A.,2000, 97, 5633-5638).
The LNA isomer that has also been investigated is α -L-LNA, which has been shown to have improved stability to 3' -exonucleases. alpha-L-LNA is incorporated into antisense spacers and chimeras that exhibit potent antisense activity (Frieden et al, nucleic ACIDS RESEARCH,2003, 21, 6365-6372).
The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil and their oligomerization and nucleic acid recognition properties have been described (Koshkin et al, tetrahedron,1998, 54, 3607-3630). LNA and its preparation are also described in WO 98/39352 and WO 99/14226.
Analogs of LNA, phosphorothioate-LNA and 2' -thio-LNA (Kumar et al, biorg. Med. Chem. Lett.,1998,8, 2219-2222) were also prepared. The preparation of locked nucleoside analogues containing oligodeoxyribonucleotide duplex as substrates for nucleic acid polymerase has also been described (Wengel et al, WO 99/14226). The synthesis of a novel conformationally constrained high affinity oligonucleotide analogue, 2' -amino-LNA, has been described in the art (Singh et al, j. Org. Chem.,1998, 63, 10035-10039). In addition, 2 '-amino-LNAs and 2' -methylamino-LNAs have been prepared and their thermal stability with duplex of complementary RNA and DNA strands has been previously reported.
Internucleoside linkage
Described herein are internucleoside linking groups that link together nucleosides or otherwise modified monomer units to form antisense compounds. Two main classes of internucleoside linkages are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate (including phosphorodiamidate), and phosphorothioate. Representative phosphorus-free internucleoside linkages include, but are not limited to, methyleneimino (-CH 2-N(CH3)-O-CH2 -), thiodiester (-O-C (O) -S-), thiocarbamate (-O-C (O) (NH) -S-); siloxane (-O-Si (H) 2 -O-); and N, N' -dimethylhydrazine (-CH 2-N(CH3)-N(CH3) -). Antisense compounds having non-phosphorus internucleoside linkages are referred to as oligonucleotides. Modified internucleoside linkages can be used to alter (typically increase) nuclease resistance of antisense compounds compared to native phosphodiester linkages. Internucleoside linkages having chiral atoms can be prepared as racemic, chiral or as mixtures. Representative chiral internucleoside linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free linkages are well known to those skilled in the art.
In embodiments, the phosphate group may be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming the oligonucleotide, phosphate groups covalently link nucleosides adjacent to one another to form a linear polymeric compound. Within an oligonucleotide, phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3 'to 5' phosphodiester linkage.
Conjugation group
In embodiments, AC is modified by covalent attachment of one or more conjugate groups. Typically, the conjugate group modifies one or more properties of the attached AC, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance. The conjugate groups are conventionally used in the chemical arts and are attached to the parent compound, such as AC, either directly or via an optional linking moiety or linking group. Conjugation groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterols, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes. In embodiments, the conjugation group is polyethylene glycol (PEG), and the PEG is conjugated to AC or a cyclic peptide.
The conjugate group includes a lipid moiety such as a cholesterol moiety (Letsinger et al, proc. Natl. Acad. Sci. USA,1989, 86, 6553); cholic acid (Manoharan et al, biorg. Med. Chem. Lett.,1994,4, 1053); thioethers, for example hexyl-S-tritylthiol (Manoharan et al, ann.N. Y. Acad. Sci.,1992, 660, 306; manoharan et al, biorg. Med. Chem. Let.,1993,3, 2765); sulphur cholesterol (Oberhauser et al, nucleic acids res.,1992, 20, 533); aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J.,1991, 10, 111; kabanov et al, FEBS Lett.,1990, 259, 327; svinarchuk et al, biochimie,1993, 75, 49); phospholipids, such as di-hexadecyl racemic glycerol or triethylammonium-1, 2-di-O-hexadecyl racemic glycerol-3-H-phosphonate (Manoharan et al, tetrahedron lett.,1995, 36, 3651; shea et al, nucleic acids res.,1990, 18, 3777); polyamine or polyethylene glycol chains (Manoharan et al, nucleic & nucleic acids, 1995, 14, 969); adamantaneacetic acid (Manoharan et al, tetrahedron lett.,1995, 36, 3651); palm-based moieties (Mishra et al, biochim. Biophys. Acta,1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al, j. Pharmacol. Exp. Ter., 1996, 277, 923).
Linking groups or difunctional linking moieties such as those known in the art may be included in the compounds provided herein. The linking groups can be used to attach chemical functional groups, conjugation groups, reporter groups, and other groups to selective sites of parent compounds (such as AC, for example). In embodiments, the difunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. In embodiments, one of the functional groups is selected to bind to a parent molecule or compound of interest, and the other is selected to bind to substantially any selected group, such as a chemical functional group or a conjugate group. Any of the linkers described herein may be used. In embodiments, the linker comprises a chain structure or oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups used in the difunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In embodiments, the difunctional linking moiety may include amino groups, hydroxyl groups, carboxylic acids, thiols, unsaturation (e.g., double or triple bonds), and the like. Some non-limiting examples of difunctional linking moieties include 8-amino-3, 6-dioxaoctanoic Acid (ADO), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), and 6-aminocaproic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C 1-C10 alkyl, substituted or unsubstituted C 2-C10 alkenyl, or substituted or unsubstituted C 2-C10 alkynyl, wherein a non-limiting list of substituent groups includes hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.
In embodiments, AC may be linked to a 10 arginine-serine dipeptide repeat sequence. AC linked to a 10 arginine-serine dipeptide repeat for artificial recruitment of splice enhancers has been used in vitro to induce BRCA1 and SMN2 exons containing mutations that would otherwise be skipped. See Cartegni and Krainer 2003, incorporated herein by reference.
In embodiments, the length of AC may be 5 to 50 nucleotides (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、40、41、42、43、44、45、46、47、48、49 or 50, including all values and ranges therein). In embodiments, the length of AC may be 5-10 nucleotides. In embodiments, the length of AC may be 10-15 nucleotides. In embodiments, the length of AC may be 15-20 nucleotides. In embodiments, the length of AC may be 20-25 nucleotides. In embodiments, the length of AC may be 25-30 nucleotides. In embodiments, the length of AC may be 30-35 nucleotides. In embodiments, the length of AC may be 35-40 nucleotides. In embodiments, the length of AC may be 40-45 nucleotides. In embodiments, the length of AC may be 45-50 nucleotides.
In embodiments, AC binds to the human DMD gene encoding dystrophin. In an embodiment, AC binds to at least a portion of exon 44 of DMD. In an embodiment, AC binds to at least a portion of the 3' flank of exon 44 of DMD. In embodiments, AC binds to at least a portion of the 5' flanking intron of exon 44 of DMD. In embodiments, the AC bound to exon 44 of DMD is about 18 to about 30 nucleic acids in length, e.g., about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleic acids in length.
In embodiments, the nucleic acid sequence of exon 44 of DMD from 5 'to 3' is:
In embodiments, the nucleic acid sequence of exon 44 of DMD from 5 'to 3' is ：GGCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTITCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAG(SEQ ID NO：2). in embodiments, the sequence of exon 44 is set forth in SEQ ID NO: the 5' end of 1 comprises 1, 2,3, 4 or 5 nucleotides or more. In embodiments, the sequence of exon 44 is set forth in SEQ ID NO: the 5' end of 2 comprises 1, 2,3, 4 or 5 nucleotides or more. In embodiments, the sequence of exon 44 is set forth in SEQ ID NO: the 3' end of 1 comprises 1, 2,3, 4 or 5 nucleotides or more. In embodiments, the sequence of exon 44 is set forth in SEQ ID NO: the 3' end of 2 comprises 1, 2,3, 4 or 5 nucleotides or more.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is an 18-mer), wherein the first nucleotide of the 18-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130 or 131. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is an 18-mer), wherein the first nucleotide of the 18-mer starts at SEQ ID NO:2, at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131 or 132.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 19-mer), wherein the first nucleotide of the 19-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129 or 130. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 19-mer), wherein the first nucleotide of the 19-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130 or 131.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 20-mer), wherein the first nucleotide of the 20-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128 or 129. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 20-mer), wherein the first nucleotide of the 20-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129 or 130.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is 21-mer), wherein the first nucleotide of the 21-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127 or 128. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is 21-mer), wherein the first nucleotide of the 21-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128 or 129.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 22-mer), wherein the first nucleotide of the 22-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126 or 127. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 22-mer), wherein the first nucleotide of the 22-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127 or 128.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 23-mer), wherein the first nucleotide of the 23-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125 or 126. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 23-mer), wherein the first nucleotide of the 23-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126 or 127.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 24-mer), wherein the first nucleotide of the 24-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124 or 125. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 24-mer), wherein the first nucleotide of the 24-mer starts at SEQ ID NO:2, at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125 or 126.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 25-mer), wherein the first nucleotide of the 25-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123 or 124. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 25-mer), wherein the first nucleotide of the 25-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124 or 125.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 26-mer), wherein the first nucleotide of the 26-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122 or 123. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 26-mer), wherein the first nucleotide of the 26-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123 or 124.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 27-mer), wherein the first nucleotide of the 27-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121 or 122. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 27-mer), wherein the first nucleotide of the 27-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122 or 123.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 28-mer), wherein the first nucleotide of the 28-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120 or 121. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 28-mer), wherein the first nucleotide of the 28-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121 or 122.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is 29-mer), wherein the first nucleotide of the 29-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119 or 120. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is 29-mer), wherein the first nucleotide of the 29-mer starts at SEQ ID NO:2 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120 or 121.
In embodiments, AC comprises SEQ ID NO:1 (e.g., AC is a 30-mer), wherein the first nucleotide of the 30-mer starts at SEQ ID NO:1 at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118 or 119. In embodiments, AC comprises SEQ ID NO:2 (e.g., AC is a 30-mer), wherein the first nucleotide of the 30-mer starts at SEQ ID NO:2, at position 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119 or 120.
In embodiments, the AC that binds to exon 44 of DMD is selected from any one of the nucleic acid sequences shown in tables 6A-6M, the reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto.
In an embodiment, the AC that binds to the sequence of exon 44 of DMD is selected from any one of the nucleic acid sequences shown in tables 6A-6M. In embodiments, the AC that binds to exon 44 of DMD is selected from any one of the nucleic acid sequences within tables 6A-6M, the reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto. In embodiments, the AC that binds to exon 44 of DMD comprises one or more modified nucleic acids, one or more modified nucleotide linkages, or a combination thereof. In embodiments, the AC that binds to exon 44 comprises one or more morpholine rings, one or more phosphorodiamidate linkages, or a combination thereof. In embodiments, the AC that binds to exon 44 of DMD is an antisense Phosphorodiamidate Morpholino Oligomer (PMO) having a sequence selected from any one of the nucleic acid sequences within tables 6A-6M, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity thereto.
In an embodiment, the AC bound to exon 44 of DMD is 5'-TGAAAACGCCGCCATTTCTCAACAG-3'. In an embodiment, the AC bound to exon 44 of DMD is 5'-ACTGTTCAGCTTCTGTTAGCCACTG-3'. In embodiments, the AC that binds to exon 44 of DMD comprises one or more modified nucleic acids, one or more modified nucleotide linkages, or a combination thereof. In embodiments, the AC that binds to exon 44 comprises one or more morpholine rings, one or more phosphorodiamidate linkages, or a combination thereof. In an embodiment, the AC bound to exon 44 of DMD is an antisense Phosphorodiamidate Morpholino Oligomer (PMO) having the sequence 5 '-TGAAAACGCCGCCATITCTCAACAG-3'. In an embodiment, the AC bound to exon 44 of DMD is an antisense Phosphorodiamidate Morpholino Oligomer (PMO) having sequence 5'-ACTGTTCAGCTTCTGTTAGCCACTG-3'.
TABLE 6A 18-merAC binding to exon 44 of DMD
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TABLE 6B 19-mer AC binding to exon 44 of DMD
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TABLE 6C 20-mer AC binding to exon 44 of DMD
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TABLE 6D 21-mer AC binding to exon 44 of DMD
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TABLE 6E 22-mer AC binding to exon 44 of DMD
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TABLE 6F 23 mer AC binding to exon 44 of DMD
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TABLE 6G 24-mer AC binding to exon 44 of DMD
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TABLE 6H 25-mer AC binding to exon 44 of DMD
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TABLE 6I 26-mer AC binding to exon 44 of DMD
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TABLE 6J 27 mer AC binding to exon 44 of DMD
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TABLE 6K 28-mer AC binding to exon 44 of DMD
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TABLE 6L 29 mer dAC binding to exon 44 of DMD
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TABLE 6M 30-mer AC binding to exon 44 of DMD
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In embodiments, any AC described herein, including an AC in tables 6A-6M, a reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto, comprises at least one modified nucleotide or nucleic acid selected from the group consisting of: phosphorothioate (PS) nucleotides, phosphorodiamidate Morpholino (PMO) nucleotides, locked Nucleic Acids (LNAs), peptide Nucleic Acids (PNAs), nucleotides comprising a 2' -O-methyl (2 ' -OMe) modified backbone, 2' O-methoxy-ethyl (2 ' -MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides and 2' -deoxy-2 ' -fluoro- β -D-arabinonucleic acid (2 ' f-ANA). In embodiments, hybridization of AC to a target sequence promotes or induces splicing of exon 44. In embodiments, the AC comprises at least one Phosphorodiamidate Morpholino (PMO) nucleotide. In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1,4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2,7, 28, 29, 30 or more nucleotides are modified. In embodiments, each nucleotide in AC is a Phosphorodiamidate Morpholino (PMO) nucleotide.
In embodiments, the compounds have the following structure:
Wherein:
CPPs are cyclic peptides (also referred to as cell penetrating peptides) described herein;
L is a linker;
b are each independently nucleobases complementary to bases in the target sequence; and
N is an integer from 1 to 50. In embodiments, the sum of B and n corresponds to the sequences shown in tables 6A-6M, their reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto.
Cyclic cell penetrating peptide conjugated to AC (cCPP)
The cyclic cell penetrating peptide (cCPP) may be conjugated to AC.
AC may be conjugated to cCPP via a linker. The cargo moiety may be conjugated to a linker at the terminal carbonyl group to provide the following structure:
Wherein:
EP is a cyclic exopeptide and M, AA SC, AC, x ', y and z' are as defined above, are attachment points to AA SC. x' may be 1.y may be 4.z' may be 11.- (OCH 2CH-2)x' -and/or- (OCH 2CH-2)z' -may independently be replaced by one or more amino acids including, for example, glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminocaproic acid, or combinations thereof.
An Endosomal Escape Vector (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an Exocyclic Peptide (EP), and a linker, and may be conjugated with AC to form an EEV-conjugate comprising a structure of formula (C):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
R 4 and R 6 are independently H or an amino acid side chain;
EP is a cyclic exopeptide as defined herein;
AC is as defined herein;
Each m is independently an integer from 0 to 3;
n is an integer from 0 to 2;
x' is an integer from 2 to 20;
y is an integer from 1 to 5;
q is an integer from 1 to 4; and
Z' is an integer from 2 to 20.
R 1、R2、R3、R4, EP, AC, m, n, x ', y, q and z' are as defined herein.
EEV can be conjugated to AC, and EEV-conjugates can comprise a structure of formula (C-a) or (C-b):
or a protonated form thereof, wherein EP, m and z are as defined above in formula (C).
EEV can be conjugated to AC, and EEV-conjugates can comprise a structure of formula (C-C):
Or a protonated form thereof, wherein EP, R 1、R2、R3、R4 and m are as defined above in formula (III); AA may be an amino acid as defined herein; n may be an integer from 0 to 2; x may be an integer from 1 to 10; y may be an integer from 1 to 5; and z may be an integer from 1 to 10.
EEV may be conjugated to oligonucleotide AC, and EEV-oligonucleotide conjugates may comprise structures of formula (C-1), (C-2), (C-3), or (C-4):
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In the above formula, EP is a cyclic exopeptide and AC may have a sequence of 15-30 nucleotides that is complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence. In embodiments, AC may be selected from the oligonucleotides shown in tables 6A-6M, reverse complement thereof, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto.
In embodiments, the compounds described herein form multimers. In embodiments, multimerization occurs via non-covalent interactions, such as by hydrophobic interactions, ionic interactions, hydrogen bonding, or dipole-dipole interactions. In embodiments, the compounds form dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, or nonamers. In embodiments, a compound comprises 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 cyclic peptides. In embodiments, the compound comprises 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 ACs. In embodiments, the compound comprises 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 EPs. In embodiments, the compound comprises 1 to 10 cyclic peptides and 1 to 10 ACs. In embodiments, the compound comprises 1 to 10 cyclic peptides, 1 to 10 ACs, or 1 to 10 EPs.
In embodiments, the compounds of the present disclosure comprise any one of the following structures. The following compounds are merely illustrative, and any of the cyclic peptides, linkers, and AC in any of the following structures may be replaced with any of the cyclic peptides, linkers, or AC described herein.
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Cytoplasmic delivery efficiency
Modification of the cyclic cell penetrating peptide (cCPP) may increase cytoplasmic delivery efficiency. By comparing the cytoplasmic delivery efficiency of cCPP with the modified sequence to a control sequence, improved cytoplasmic uptake efficiency can be measured. The control sequence does not include specific replacement amino acid residues in the modified sequence (including, but not limited to, arginine, phenylalanine, and/or glycine), but is otherwise identical.
In embodiments, the compound comprising the cyclic peptide and AC has improved cytoplasmic uptake efficiency compared to the compound comprising AC alone. Cytoplasmic uptake efficiency can be measured by comparing the cytoplasmic delivery efficiency of a compound comprising a cyclic peptide and AC with the cytoplasmic delivery efficiency of AC alone.
As used herein, cytoplasmic delivery efficiency refers to the ability of cCPP to cross the cell membrane and enter the cytoplasm of the cell. cCPP are not necessarily dependent on the receptor or cell type. Cytoplasmic delivery efficiency may refer to absolute cytoplasmic delivery efficiency or relative cytoplasmic delivery efficiency.
Absolute cytosolic delivery efficiency is the ratio of the cytosolic concentration of cCPP (or cCPP-AC conjugate) to the concentration of cCPP (or cCPP-AC conjugate) in the growth medium. Relative cytoplasmic delivery efficiency refers to the concentration of cCPP in the cytoplasm as compared to the concentration of control cCPP in the cytoplasm. Quantification may be achieved by fluorescent labeling cCPP (e.g., with FITC dye) and measuring the fluorescence intensity using techniques well known in the art.
The relative cytoplasmic delivery efficiency is determined by comparing the amount of the invention cCPP that is internalized by a cell type (e.g., a HeLa cell) to the amount of the control cCPP that is internalized by the same cell type. To measure relative cytoplasmic delivery efficiency, the cell type can be incubated in the presence of cCPP for a specified period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), after which the amount of cCPP internalized by the cell can be quantified using methods known in the art, such as fluorescence microscopy. Separately, the same concentration of control cCPP was incubated in the presence of the cell type for the same period of time and the amount of control cCPP internalized by the cells was quantified.
Relative cytoplasmic delivery efficiency can be determined by measuring cCPP with modified sequence versus IC 50 of the intracellular target and comparing cCPP with modified sequence versus IC 50 of the control sequence (as described herein).
The relative cytosolic delivery efficiency of cCPP may be in the range of about 50% to about 450%, such as about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, or about 590%, as compared to ring (Ff Φ RrRrQ), including all values and subranges therebetween. The relative cytoplasmic delivery efficiency of cCPP can be improved by greater than about 600% as compared to a cyclic peptide comprising a loop (Ff Φ RrRrQ).
The absolute cytoplasmic delivery potency 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%, including all values and subranges therebetween.
The cCPP of the present disclosure can increase cytoplasmic delivery efficiency by a factor of about 1.1 to about 30 times, e.g., about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10, about 10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0, about 19.5, about 21.5, about 20.5, about 25.0, about 22.5, about 21.5, about 25.0, about 25.5, about 22.0, about 25.5, about 21.5, about 26.0, about 25.5, about 26.0, about 26.5, about 25.0, about 26.0, about 26.5, about 0.
Resplicing target proteins
A "target protein" is an amino acid sequence that results from transcription and translation of a target gene. As used herein, "re-spliced target protein" refers to a protein encoded by AC binding to target pre-mRNA transcribed from a target gene. "wild-type target protein" refers to a naturally occurring, correctly translated protein isoform resulting from the correct splicing of a target pre-mRNA encoded by a wild-type target gene. The compounds and methods of the invention can produce an alternative splicing target protein that contains one or more amino acid substitutions, deletions, and/or insertions compared to the wild-type target protein. In embodiments, the re-spliced target protein retains some wild-type target protein activity. In embodiments, the re-spliced target protein produced by administration of a compound of the invention is homologous to a wild-type target protein. In embodiments, the re-spliced target protein has an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% and up to 100% identical to the wild-type target protein. In embodiments, the re-spliced target protein is substantially identical to the wild-type target protein. In embodiments, the amino acid sequence of the re-spliced target protein is at least 50% identical to the amino acid sequence of the wild-type target protein. In embodiments, the amino acid sequence of the re-spliced target protein is at least 75% identical to the amino acid sequence of the wild-type target protein. In embodiments, the amino acid sequence of the re-spliced target protein is at least 90% identical to the amino acid sequence of the wild-type target protein. In embodiments, the re-spliced target protein is a shortened form of the wild-type target protein.
In embodiments, the re-splicing of the target protein can rescue one or more phenotypes or symptoms of the disease associated with transcription and translation of the target gene. In embodiments, the re-splicing of the target protein may rescue one or more phenotypes or symptoms of the disease associated with the expression of the target protein. In embodiments, the re-spliced target protein is an active fragment of a wild-type target protein. In embodiments, the re-spliced target protein functions in a substantially similar manner as the wild-type target protein. In embodiments, the re-splicing of the target protein allows the cell to function substantially similarly to a similar cell expressing the wild-type target protein. In embodiments, the re-splicing of the target protein does not cure the disease associated with the target gene or target protein, but ameliorates one or more symptoms of the disease. In embodiments, re-splicing the target protein results in an increase in target protein function of at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 205, 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% or about 95%, and up to about 100%.
In embodiments, the alternative splicing target protein can have an amino acid sequence that is reduced by about 1 or more amino acids from the size of the wild-type target protein, for example by 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 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, or about 180 or more amino acids.
In embodiments, the re-spliced target protein may have one or more properties that are improved relative to the target protein. In embodiments, the re-spliced target protein may have one or more properties that are improved relative to a wild-type target protein. In embodiments, enzymatic activity or stability may be enhanced by promoting different splicing of target pre-mRNA. In embodiments, the re-spliced target protein may have a sequence identical or substantially similar to a wild-type target protein isomer, with improved properties compared to another wild-type target protein isomer.
In embodiments, one or more properties of the target protein are absent (eliminated) or reduced in the re-spliced target protein. In embodiments, one or more properties of the wild-type target protein are absent (eliminated) or reduced in the re-spliced target protein. Non-limiting examples of properties that may be reduced or eliminated include immunogenicity, angiogenesis, thrombosis, aggregation, and ligand binding activity.
In embodiments, the re-spliced target protein contains one or more amino acid substitutions as compared to the wild-type target protein. In embodiments, the substitution may be a conservative substitution or a non-conservative substitution. Examples of conservative amino acid substitutions include substitution of one amino acid for another amino acid in one of the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). In embodiments, structurally similar amino acids are substituted to reverse the charge of the residue (e.g., glutamine for glutamic acid, or vice versa, aspartic acid for asparagine, or vice versa). In embodiments, tyrosine replaces phenylalanine, or vice versa. Other non-limiting examples of amino acid substitutions are described, for example, by H.Neurath and R.L.Hill,1979 in The Proteins, ACADEMIC PRESS, new York. Common substitutions are Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu and Asp/Gly.
In embodiments, the re-spliced target protein may comprise substitutions, deletions, and/or insertions at one or more (e.g., several) positions as compared to the wild-type target protein. In embodiments, the number of amino acid substitutions, deletions and/or insertions in the amino acid sequence of the re-spliced target protein is no more than 200, no more than 150, no more than 100, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10.
Therapeutic method
In embodiments, the amount of the active ingredient is about 0.1mg/kg to about 1000mg/kg, e.g., about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, About 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg, about 33mg/kg, about, About 34mg/kg, about 35mg/kg, about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about, About 57mg/kg, about 58mg/kg, about 59mg/kg, about 60mg/kg, about 61mg/kg, about 62mg/kg, about 63mg/kg, about 64mg/kg, about 65mg/kg, about 66mg/kg, about 67mg/kg, about 68mg/kg, about 69mg/kg, about 70mg/kg, about 71mg/kg, about 72mg/kg, about 73mg/kg, about 74mg/kg, about 75mg/kg, about 76mg/kg, about 77mg/kg, about 78mg/kg, about 79mg/kg, about, About 80mg/kg, about 81mg/kg, about 82mg/kg, about 83mg/kg, about 84mg/kg, about 85mg/kg, about 86mg/kg, about 87mg/kg, about 88mg/kg, about 89mg/kg, about 90mg/kg, about 91mg/kg, about 92mg/kg, about 93mg/kg, about 94mg/kg, about 95mg/kg, about 96mg/kg, about 97mg/kg, about 98mg/kg, about 99mg/kg, about 100mg/kg, about 110mg/kg, about 120mg/kg, about, About 130mg/kg, about 140mg/kg, about 150mg/kg, about 160mg/kg, about 170mg/kg, about 180mg/kg, about 190mg/kg, about 200mg/kg, about 210mg/kg, about 220mg/kg, about 230mg/kg, about 240mg/kg, about 250mg/kg, about 260mg/kg, about 270mg/kg, about 280mg/kg, about 290mg/kg, about 300mg/kg, about 310mg/kg, about 320mg/kg, about 330mg/kg, about 340mg/kg, about 350mg/kg, about 360mg/kg, about 370mg/kg, about 380mg/kg, about 390mg/kg, about 400mg/kg, about 410mg/kg, about 420mg/kg, about 430mg/kg, about 440mg/kg, about 450mg/kg, about 460mg/kg, about 470mg/kg, about 480mg/kg, about 490mg/kg, about 500mg/kg, about 510mg/kg, about 520mg/kg, about 530mg/kg, about 540mg/kg, about, About 550mg/kg, about 560mg/kg, about 570mg/kg, about 580mg/kg, about 590mg/kg, about 600mg/kg, about 610mg/kg, about 620mg/kg, about 630mg/kg, about 640mg/kg, about 650mg/kg, about 660mg/kg, about 670mg/kg, about 680mg/kg, about 690mg/kg, about 700mg/kg, about 710mg/kg, about 720mg/kg, about 730mg/kg, about 740mg/kg, about 750mg/kg, about 760mg/kg, about 770mg/kg, about 780mg/kg, about 790mg/kg, about 800mg/kg, about 810mg/kg, about 820mg/kg, about 830mg/kg, about 840mg/kg, about 850mg/kg, about 860mg/kg, about 870mg/kg, about 880mg/kg, about 890mg/kg, about 900mg/kg, about 910mg/kg, about 920mg/kg, about 930mg/kg, about 940mg/kg, about 950mg/kg, about 960mg/kg, a dose of about 970mg/kg, about 980mg/kg, about 990mg/kg, or about 1000mg/kg (including all values and ranges therebetween) is administered to a patient diagnosed with Duchenne Muscular Dystrophy (DMD) AC of the present disclosure.
The present disclosure provides a method of treating Duchenne Muscular Dystrophy (DMD) in a subject in need thereof comprising administering a compound disclosed herein. In some embodiments, the target gene is DMD. In embodiments, the target sequence comprises at least a portion of exon 44 of the DMD, at least a portion of exon 44 flanking the 3 'intron of the DMD, at least a portion of exon 44 flanking the 5' intron of the DMD, or a combination thereof.
In various embodiments, treatment refers to a partial or complete alleviation, improvement, alleviation, inhibition, delay of onset, severity, and/or reduction of incidence of one or more symptoms in a subject.
In embodiments, a method for altering expression of a target gene in a subject in need thereof is provided, the method comprising administering a compound disclosed herein. In embodiments, the treatment results in reduced expression of the target protein. In embodiments, the treatment results in expression of the alternatively spliced target protein. In embodiments, the treatment results in preferential expression of the wild-type target protein isomer.
In embodiments, treatment according to the present disclosure results in a reduction in expression of a target protein in a subject by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the target protein in a pre-treatment subject or in one or more control subjects with a similar disease that are untreated. In embodiments, treatment according to the present disclosure results in an increase in expression of the re-spliced target protein in the subject of more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, as compared to the average level of the target protein in the subject prior to treatment or in one or more control subjects without treatment having a similar disease. In embodiments, treatment according to the present disclosure results in an increase or decrease in expression of a wild-type target protein isomer in a subject by more than about 5%, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%, compared to the average level of the target protein in a pre-treatment subject or in one or more control subjects with no treatment for a similar disease.
As used herein, the terms "improve," "increase," "decrease," and the like indicate values relative to a control. In embodiments, a suitable control is a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A "control individual" is an individual suffering from the same disease that is about the same age and/or sex as the individual being treated (to ensure that the disease stages of the treated individual and the control individual are comparable).
The treated individual (also referred to as a "patient" or "subject") is an individual (fetus, infant, child, adolescent or adult) who has a disease or has the potential to develop a disease. An individual may have a disease mediated by aberrant gene expression or aberrant gene splicing. In various embodiments, an individual with a disease may have a wild-type target protein expression or activity level that is about 1% to 99% of the normal protein expression or activity level in an individual not having the disease. In embodiments, the ranges include, but are not limited to, about 80-99%, about 65-80%, about 50-65%, about 30-50%, about 25-30%, about 20-25%, about 15-20%, about 10-15%, about 5-10%, or about 1-5% of the normal thymidine phosphorylase expression or activity level. In embodiments, the individual may have a target protein expression or activity level that is about 1% to about 500% higher than the normal wild-type target protein expression or activity level. In embodiments, the ranges include, but are not limited to, about 1-10%, about 10-50%, about 50-100%, about 100-200%, about 200-300%, about 300-400%, about 400-500%, or about 500-1000% higher target protein expression or activity levels.
In embodiments, the individual is the most recently diagnosed with the disease. Typically, early treatment (starting treatment as soon as possible after diagnosis) is important to minimize the impact of the disease and maximize therapeutic benefit.
In embodiments, the efficacy of the compounds of the present disclosure and AC on DMD is evaluated in an animal model of DMD. Animal models are a valuable resource for studying disease pathogenesis and provide a means for testing dystrophin-related activity. In embodiments, mdx mice and dystrophy gold beagle dogs (GRMD) that are both dystrophin negative (see, e.g., collins & Morgan, int J Exp pathl 84:165-172, 2003) are used to evaluate compounds of the present disclosure. In embodiments, compounds of the present disclosure are evaluated using a C57BL/10ScSn-Dmdmdx/J (Bl 10/mdx) or D2.B10-Dmdmdx/J (D2/mdx) mouse model. In embodiments, transgenic mice carrying the human DMD gene and lacking the mouse Dmd gene (hmdmd/Dmd-free mice) are used to evaluate compounds of the present disclosure. Such mice can be generated by crossing male hDMD mice (available from Jackson Laboratory, bar Harbor, ME) with female DMD-free mice. Each of the following references describes these models and is incorporated by reference herein in its entirety as international publication No. WO2019014772 to ：J Neuromuscul Dis.2018;5(4)：407-417.;Proc Natl Acad Sci U S A.1984;81(4)：1189-92.;Am J Pathol.2010;176(5)：2414-24.;J Clin Invest.2009;119(12)：3703-12;. These and other animal models can be used to measure the functional activity of various dystrophins.
In embodiments, in vitro models are used to evaluate the efficacy of the compositions of the present disclosure. In embodiments, the in vitro model is an immortalized muscle cell model. The model is described in the following articles, which are incorporated herein by reference in their entirety: nguyen et al J Pers Med.2017, month 12; 7 (4): 13.
Preparation method
The compounds described herein may be prepared in a number of ways known to those skilled in the art of organic synthesis or in variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. The optimal reaction conditions may vary with the particular reactants or solvents used, but such conditions may be determined by one skilled in the art.
Variations of the compounds described herein include addition, subtraction, or movement of the various components as described for each compound. Similarly, the chirality of a molecule may change when one or more chiral centers are present in the molecule. In addition, 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 one skilled in the art. The chemical nature of the protecting groups can be found, for example, in Wuts and Greene, protective Groups in Organic Synthesis, 4 th edition, wiley & Sons,2006, which is incorporated herein by reference in its entirety.
Starting materials and reagents for preparing the disclosed compounds and compositions are available from commercial suppliers such as Aldrich Chemical Co.,(Milwaukee,WI)、Acros Organics(Morris Plains,NJ)、Fisher Scientific(Pittsburgh,PA)、Sigma(St.Louis,MO)、Pfizer(New York,NY)、GlaxoSmithKline(Raleigh,NC)、Merck(Whitehouse Station,NJ)、Johnson&Johnson(New Brunswick,NJ)、Aventis(Bridgewater,NJ)、AstraZeneca(Wilmington,DE)、Novartis(Basel,Switzerland)、Wyeth(Madison,NJ)、Bristol-Myers-Squibb(New York,NY)、Roche(Basel,Switzerland)、Lilly(Indianapolis,IN)、Abbott(Abbott Park,IL)、Schering Plough(Kenilworth,NJ) or Boehringer Ingelheim (Ingelheim, germany) or are prepared by methods known to those skilled in the art following procedures set forth in the references, such as 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 supplements (ELSEVIER SCIENCE Publishers, 1989); organic Reactions, volumes 1-40 (John Wiley and Sons, 1991); march' S ADVANCED Organic Chemistry, (John Wiley and Sons, 4 th edition); larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, are available from commercial sources.
The reactions to produce the compounds described herein may be carried out in solvents that may be selected by one skilled in the art of organic synthesis. The solvent may be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) under which the reaction is carried out. The reaction may be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation may be monitored according to any suitable method known in the art. For example, product formation may be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible) or mass spectrometry, or by chromatography such as High Performance Liquid Chromatography (HPLC) or thin layer chromatography.
The disclosed compounds can be prepared by solid phase peptide synthesis in which the amino acid α -N-terminus is protected by an acid or base protecting group. Such protecting groups should have properties that are stable to the conditions of peptide bond formation while being easily removable without disrupting the growing peptide chain or racemizing any chiral centers contained therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenyl isopropoxycarbonyl, t-pentyloxycarbonyl, isobornyloxycarbonyl, α -dimethyl-3, 5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-t-butoxycarbonyl and the like. A9-fluorenylmethoxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. For side chain amino groups such as lysine and arginine, other preferred side chain protecting groups are 2,5,7, 8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, cbz, boc and adamantyloxy carbonyl; for tyrosine are benzyl, o-bromobenzyloxy-carbonyl, 2, 6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl and acetyl (Ac); for serine are tert-butyl, benzyl and tetrahydropyranyl; for histidine are trityl, benzyl, cbz, p-toluenesulfonyl and 2, 4-dinitrophenyl; for tryptophan is formyl; benzyl and tert-butyl for aspartic acid and glutamic acid, and triphenylmethyl (trityl) for cysteine. In the solid phase peptide synthesis method, the α -C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful in the above synthesis are those materials which are inert to the reagents and reaction conditions of the progressive condensation-deprotection reaction and insoluble in the medium used. The solid support used for the synthesis of the α -C-terminal carboxy peptide is a 4-hydroxymethylphenoxymethyl-co (styrene-1% divinylbenzene) or 4- (2 ',4' -dimethoxyphenyl-Fmoc-aminomethyl) phenoxyacetamido ethyl resin available from Applied Biosystems (Foster City, calif.). The α -C-terminal amino acid is coupled to the resin via coupling with or without 4-Dimethylaminopyridine (DMAP), 1-Hydroxybenzotriazole (HOBT), benzotriazole-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) or bis (2-oxo-3-oxazolidinyl) phosphine chloride (BOPCl) in a solvent such as dichloromethane or DMF at a temperature between 10 ℃ and 50 ℃ for about 1 to about 24 hours via N, N ' -Dicyclohexylcarbodiimide (DCC), N ' -Diisopropylcarbodiimide (DIC) or O-benzotriazol-1-yl-N, N ' -tetramethyluronium Hexafluorophosphate (HBTU). When the solid support is a 4- (2 ',4' -dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine (preferably piperidine) prior to coupling with the α -C-terminal amino acid as described above. One method for coupling with the deprotected 4- (2 ',4' -dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (HBTU, 1 eq.) and 1-hydroxybenzotriazole (HOBT, 1 eq.) in DMF. The coupling of the consecutive protected amino acids can be performed in an automated polypeptide synthesizer. In one example, fmoc is used to protect the alpha-N-terminus in the amino acid of the growing peptide chain. Removal of the Fmoc protecting group from the α -N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about a 3-fold molar excess and preferably coupled in DMF. The coupling agent may be O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate (HBTU, 1 eq.) and 1-hydroxybenzotriazole (HOBT, 1 eq.). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either continuously or in a single operation. Removal and deprotection of the polypeptide can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising anisole, water, ethylene dithiol and trifluoroacetic acid. In the case where the α -C-terminus of the polypeptide is an alkylamide, the resin is cleaved by ammonolysis with the alkylamine. Alternatively, the peptide may be removed by transesterification (e.g., with methanol), followed by ammonolysis, or by direct transamidation. The protected peptide may be purified at this point or used directly in the next step. The removal of the side chain protecting groups can be accomplished using the cleavage mixtures described above. The fully deprotected peptide may be purified by a series of chromatographic steps using any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography (e.g., on Sephadex G-25, LH-20 or counter-current distribution); high Performance Liquid Chromatography (HPLC), particularly reverse phase HPLC on octyl-silica or octadecylsilyl-silica bonded phase column packing.
The above polymers, such as PEG groups, may be attached to AC under any suitable conditions for reacting the protein with the activated polymer molecules. Any method known in the art may be used, including other chemoselective conjugation/attachment methods via acylation, reductive alkylation, michael addition, thiol alkylation, or by reactive groups on the PEG moiety (e.g., aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazino) to reactive groups on AC (e.g., aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazino). Activating groups useful for attaching the water-soluble polymer to one or more proteins include, but are not limited to, sulfones, maleimides, thiols, triflates (tresylates), aziridines (azidirine), oxiranes, 5-pyridinyl, and alpha-halo acyl groups (e.g., alpha-iodoacetic acid, alpha-bromoacetic acid, alpha-chloroacetic acid). If attached to AC by reductive alkylation, the polymer selected should have a single reactive aldehyde in order to control the degree of polymerization. See, for example, kinstler et al, adv. Drug. Delivery Rev.54:477-485 (2002); roberts et al, adv. Drug Delivery Rev.54:459-476 (2002); and Zalipsky et al, adv. Drug Delivery Rev.16:157-182 (1995).
To covalently attach AC directly to a CPP, the appropriate amino acid residue of the CPP can be reacted with an organic derivatizing agent capable of reacting with selected side chains or N-or C-termini of the amino acid. Reactive groups on the peptide or conjugate moiety include, for example, aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazine groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimidyl ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, or other agents known in the art.
Methods of synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited to methods of synthesizing AC. In embodiments, provided herein are compounds having reactive phosphorus groups that can be used to form internucleoside linkages (including, for example, phosphodiester and phosphorothioate internucleoside linkages). The methods of preparation and/or purification of the precursor or antisense compound are not limited to the compositions or methods provided herein. Methods for the synthesis and purification of DNA, RNA and antisense compounds are well known to those skilled in the art.
The oligomerization of modified and unmodified nucleosides can be carried out routinely according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, agrawal et al (1993), humana Press) and/or RNA (Scaringe, methods (2001), 23, 206-217.Gait et al Applications of Chemically synthesized RNA in RNA: protein Interactions, smith et al (1998), 1-36.Gallo et al, tetrahedron (2001), 57, 5707-5713).
Antisense compounds provided herein can be conveniently and routinely prepared by well known solid phase synthesis techniques. The equipment used for such synthesis is sold by several suppliers including, for example, applied Biosystems (Foster City, calif.). Any other method known in the art for such synthesis may additionally or alternatively be used. The use of similar techniques to prepare oligonucleotides such as phosphorothioates and alkylated derivatives is well known. The invention is not limited by the synthetic method of antisense compounds.
Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analytical methods include Capillary Electrophoresis (CE) and electrospray mass spectrometry. Such synthetic and analytical methods can be performed in multiwell plates. The method of the present invention is not limited to oligomer purification methods.
Application method
In vivo application of the disclosed compounds and compositions containing them may be accomplished by any suitable method and technique currently or contemplated to be known to those skilled in the art. For example, the disclosed compounds may be formulated in a physiologically or pharmaceutically acceptable form and administered by any suitable route known in the art, including, for example, oral and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal and intrathecal administration, such as by injection. The administration of the disclosed compounds or compositions may be a single administration, or at successive or different intervals, as readily determinable by one of skill in the art.
The compounds disclosed herein and compositions comprising them may also be administered using liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide uniform doses over an extended period of time. The compounds may also be administered in the form of their salt derivatives or in crystalline form.
The compounds disclosed herein may be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in many sources well known and readily available to those skilled in the art. For example, remington's Pharmaceutical Science, e.w. martin (1995) describes formulations that can be used in conjunction with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound. The compositions used may also take various forms. These forms include, for example, solid, semi-solid, and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The composition also preferably comprises 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, dimethylsulfoxide, glycerol, alumina, starch, saline and equivalent carriers and diluents. To provide for administration of such doses for the desired therapeutic treatment, the compositions disclosed herein may advantageously comprise between about 0.1% and 100% by weight of one or more of the subject compounds, in total, based on the weight of the total composition comprising the carrier or diluent.
Formulations suitable for administration include, for example, sterile injectable aqueous solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only a sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets and the like. It should be understood that the compositions disclosed herein may contain other conventional agents in the art regarding the type of formulation in question, in addition to the ingredients specifically mentioned above.
The compounds disclosed herein and compositions comprising them may be delivered to cells by direct contact with the cells or via carrier means. Carrier means for delivering the compounds and compositions to cells are known in the art and include, for example, encapsulation of the compositions in a liposomal fraction. Another means for delivering the compounds and compositions disclosed herein to a cell includes attaching the compounds to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Pat. No.6,960,648 and U.S. application publication nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow translocation of the composition across a biological membrane. U.S. application publication number 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. The compounds may also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymers for intracranial tumors; poly [ bis (p-carboxyphenoxy) propane: sebacic acid ] in a molar ratio of 20:80 (as used in GLIADEL); chondroitin; chitin; and chitosan.
The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, may be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or salt thereof may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these formulations may contain preservatives to prevent microbial growth.
Pharmaceutical dosage forms suitable for injection or infusion may comprise sterile aqueous solutions or dispersions or sterile powders containing the active ingredient which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Optionally, the action of microorganisms may be prevented by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the compounds and/or agents disclosed herein in the required amounts with various other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solution thereof.
Useful dosages of the compounds and agents disclosed herein and pharmaceutical compositions can be determined by comparing their in vitro and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.
The dosage range in which the composition is administered is a dosage range large enough to produce the desired effect affecting the symptom or condition. The dosage should not be so large as to cause adverse side effects such as undesired cross-reactions, allergic reactions, etc. Generally, the dosage will vary with the age, condition, sex and degree of disease of the patient and can be determined by one skilled in the art. In the case of any contraindications, the dosage can be adjusted by the individual physician. The dosage may vary, and may be administered in one or more doses per day for one or more days.
Also disclosed are pharmaceutical compositions comprising a combination of a compound disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions suitable for oral, topical or parenteral administration comprising an amount of a compound constitute a preferred aspect. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient within a reasonable time frame without lethal toxicity, and preferably cause no more than an acceptable level of side effects or morbidity. Those skilled in the art will recognize that the dosage will depend on a variety of factors including the condition (health) of the subject, the weight of the subject, the type of concurrent therapy (if any), the frequency of treatment, the rate of treatment, and the severity and stage of the pathological condition.
Also disclosed are kits comprising compounds disclosed herein in one or more containers. The disclosed kits may optionally include a pharmaceutically acceptable carrier and/or diluent. In one embodiment, the kit comprises one or more other components, adjuvants or adjuvants as described herein. In another embodiment, the kit includes one or more anti-cancer agents, such as those described herein. In one embodiment, the kit includes instructions or packaging materials describing how to administer the compounds or compositions of the kit. The container of the kit may be of any suitable material, such as glass, plastic, metal, etc., and may be of any suitable size, shape or configuration. In one embodiment, the compounds and/or agents disclosed herein are provided in the kit as a solid (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 comprises an ampoule or syringe containing a compound and/or agent disclosed herein in liquid or solution form.
Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Certain definitions
As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a mixture of two or more such compositions, reference to "an agent" includes a mixture of two or more such agents, reference to "the component" includes a mixture of two or more such components, and the like.
The term "about" when immediately preceding a numerical value means a range (e.g., plus or minus 10% of the stated value). For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc., unless the context of the present disclosure indicates otherwise, or is inconsistent with such interpretation. For example, in a list of values such as "about 49, about 50, about 55,", about 50 "means a range extending to less than half the interval between the front and back values, e.g., greater than 49.5 to less than 52.5. Furthermore, the phrase "less than about" value or "greater than about" value should be understood in accordance with the definition of the term "about" provided herein. Similarly, the term "about" when preceding a series of values or ranges of values (e.g., "about 10, 20, 30" or "about 10-30") refers to all values in the series or endpoints of the range, respectively.
As used herein, the term "cyclic cell penetrating peptide" or "cCPP" refers to a peptide that facilitates delivery of an antisense compound.
The terms "miniPEG", "PEG2" and "AEEA" are used interchangeably herein to refer to 2- [2- [ 2-aminoethoxy ] ethoxy ] acetic acid.
As used herein, the term "endosomal escape vector" (EEV) refers to cCPP conjugated to a linker and/or an Exocyclic Peptide (EP) by chemical linkage (i.e., covalent or non-covalent interactions). The EEV may be an EEV of formula (B).
As used herein, the term "EEV-conjugate" refers to an endosomal escape carrier as defined herein conjugated to AC by chemical linkage (i.e., covalent or non-covalent interactions). AC may be delivered into cells via EEV. The EEV-conjugate may be an EEV-conjugate of formula (C).
As used herein, the terms "exocyclic peptide" (EP) and "modulator peptide" (MP) are used interchangeably to refer to two or more amino acid residues joined by peptide bonds that can be conjugated to a cyclic cell penetrating peptide (cCPP) as disclosed herein. When conjugated to the cyclic peptides disclosed herein, EP may alter the tissue distribution and/or retention of the compound. Typically, an EP comprises at least one positively charged amino acid residue, e.g. at least one lysine residue and/or at least one arginine residue. Non-limiting examples of EPs are described herein. An EP may be a peptide 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 viral large T antigen, whose smallest functional units are the seven amino acid sequence PKKKRKV, the double-typed nucleoplasmin NLS with sequence NLSKRPAAIKKAGQAKKKK, the c-myc nuclear localization sequence with amino acid sequence PAAKRVKLD or RQRRNELKRSF, the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of the input protein-alpha, the sequences VSRKRPRP and PPKKARED of the myoma T protein, the sequence PQPKKKPL of human p53, the sequence SALIKKKKKMAP of mouse c-ablIV, the sequences DRLRR and PKQKKRK of influenza virus NS1, the sequence RKLKKKIKKL of hepatitis virus delta antigen and the sequence REKKKFLKRR of mouse Mxl protein, the sequence KRKGDEVDGVDEVAKKKSKK of human poly (ADP-ribose) polymerase and the sequence RKCLQAGMNLEARKTKK of the steroid hormone receptor (human) glucocorticoid. Additional examples of NLS are described in International publication No. 2001/038547 and incorporated herein by reference in its entirety.
As used herein, "linker" or "L" refers to a moiety that covalently binds one or more moieties (e.g., exocyclic Peptides (EP) and AC) to a cyclic cell penetrating peptide (cCPP). The linker may comprise a natural or unnatural amino acid or polypeptide. The linker may be a synthetic compound containing two or more suitable functional groups suitable for binding cCPP to AC to form the compounds disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. cCPP can be covalently bound to AC via a linker.
As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides of the oligonucleotide may be modified. The oligonucleotides may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides may be composed of natural and/or modified nucleobases, sugars, and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.
The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked to an alpha amino group of one amino acid through a carboxyl group of another amino acid. Two or more amino acid residues may be linked to an alpha amino group through the carboxyl group of one amino acid. Two or more amino acids of a polypeptide may be joined by peptide bonds. A polypeptide may include peptide backbone modifications in which two or more amino acids are covalently attached by bonds other than peptide bonds. The polypeptide may include one or more unnatural amino acids, amino acid analogs, or other synthetic molecules that are capable of being integrated into the polypeptide. The term polypeptide includes naturally occurring and artificially occurring amino acids. The term polypeptide includes, for example, peptides comprising about 2 to about 100 amino acid residues as well as proteins comprising more than about 100 amino acid residues or more than about 1000 amino acid residues, including but not limited to therapeutic proteins such as antibodies, enzymes, receptors, soluble proteins, and the like.
The term "therapeutic polypeptide" refers to a polypeptide that has therapeutic, prophylactic or other biological activity. The therapeutic polypeptide may be produced in any suitable manner. For example, the therapeutic polypeptide may be isolated or purified from a naturally occurring environment, may be chemically synthesized, may be recombinantly produced, or a combination thereof.
The term "small molecule" refers to an organic compound that is pharmacologically active 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 typically manufactured by chemical synthesis.
As used herein, the term "contiguous" refers to two amino acids joined by a covalent bond. For example, in a representative cyclic cell penetrating peptide (cCPP) such asAA 1/AA2、AA2/AA3、AA3/AA4, and AA 5/AA1, illustrate contiguous pairs of amino acids.
As used herein, a residue of a chemical refers to a derivative of a chemical that is present in a particular product. To form a product, at least one atom of the substance is replaced with a bond to another moiety, such that the product contains a derivative or residue of the chemical substance. For example, the cyclic cell penetrating peptides (cCPP) described herein have amino acids (e.g., arginine) incorporated therein by formation of one or more peptide bonds. The amino acid incorporated into cCPP may be referred to as a residue, or simply as an amino acid. Thus, arginine or arginine residues refer to
The term "protonated form thereof" refers to a protonated form of an amino acid. For example, the guanidinium group on the arginine side chain may be protonated to form a guanidinium group. The structure of the protonated form of arginine is
As used herein, the term "chiral" refers to a molecule having more than one stereoisomer that differs in the three-dimensional arrangement of atoms, wherein one stereoisomer is a non-superimposable mirror image of the other stereoisomer. In addition to glycine, amino acids have a chiral carbon atom adjacent to a carboxyl group. The term "enantiomer" refers to a chiral stereoisomer. Chiral molecules may be amino acid residues having the "D" and "L" enantiomers. Molecules without chiral centers, such as glycine, may be referred to as "achiral".
As used herein, the term "hydrophobic" refers to a moiety that is insoluble or has minimal solubility in water. Typically, the neutral and/or non-polar moiety, or predominantly neutral and/or non-polar moiety, is hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein below.
As used herein, "aromatic" refers to an unsaturated ring molecule having 4n+2 pi electrons, wherein n is any integer. The term "non-aromatic" refers to any unsaturated ring molecule that does not fall within the definition of aromatic.
"Alkyl", "alkyl chain" or "alkyl group" refers to a fully saturated, straight or branched hydrocarbon chain group having one to forty carbon atoms and attached to the remainder of the molecule by a single bond. Including alkyl groups containing any number of carbon atoms from 1 to 40. The alkyl group containing up to 40 carbon atoms is a C 1-C40 alkyl group, the alkyl group containing up to 10 carbon atoms is a C 1-C10 alkyl group, the alkyl group containing up to 6 carbon atoms is a C 1-C6 alkyl group, and the alkyl group containing up to 5 carbon atoms is a C 1-C5 alkyl group. C 1-C5 alkyl includes C 5 alkyl, C 4 alkyl, C 3 alkyl, C 2 alkyl, and C 1 alkyl (i.e., methyl). C 1-C6 alkyl includes all of the moieties described above for C 1-C5 alkyl, but also includes C 6 alkyl. C 1-C10 alkyl includes all of the moieties described above for C 1-C5 alkyl and C 1-C6 alkyl, but also includes C 7、C8、C9 and C 10 alkyl. Similarly, C 1-C12 alkyl includes all of the foregoing moieties, but also includes C 11 and C 12 alkyl. Non-limiting examples of C 1-C12 alkyl groups include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless 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 group having one to forty carbon atoms. Non-limiting examples of C 2-C40 alkylene include ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. Unless specifically stated otherwise in the specification, the alkylene chain may be optionally substituted.
"Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain group having two to forty carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the remainder of the molecule by a single bond. Including alkenyl groups containing any number of carbon atoms from 2 to 40. Alkenyl containing up to 40 carbon atoms is C 2-C40 alkenyl, alkenyl containing up to 10 carbon atoms is C 2-C10 alkenyl, alkenyl containing up to 6 carbon atoms is C 2-C6 alkenyl, and alkenyl containing up to 5 carbon atoms is C 2-C5 alkenyl. C 2-C5 alkenyl includes C 5 alkenyl, C 4 alkenyl, C 3 alkenyl and C 2 alkenyl. C 2-C6 alkenyl includes all moieties described above with respect to C 2-C5 alkenyl, but also includes C 6 alkenyl. C 2-C10 alkenyl includes all of the moieties described above for C 2-C5 alkenyl and C 2-C6 alkenyl, but also includes C 7、C8、C9 and C 10 alkenyl. Similarly, C 2-C12 alkenyl includes all of the foregoing moieties, but also includes C 11 and C 12 alkenyl. Non-limiting examples of C 2-C12 alkenyl include vinyl (ethenyl/vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless specifically stated otherwise in the specification, an alkyl group may be optionally substituted.
"Alkenylene", "alkenylene" or "alkenylene group" refers to a straight or branched divalent hydrocarbon chain radical having two to forty carbon atoms and having one or more carbon-carbon double bonds. Non-limiting examples of C 2-C40 alkenylene groups include ethylene, propylene, butene, and the like. Unless specifically stated otherwise in the specification, alkenylene chains may be optional.
"Alkoxy" OR "alkoxy group" refers to the group-OR, wherein 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, wherein R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, as defined herein. Unless specifically stated otherwise in the specification, an acyl group may be optionally substituted.
"Alkylcarbamoyl" or "alkylcarbamoyl group" refers to the group-O-C (O) -NR aRb, wherein R a and R b are the same or different and are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl as defined herein, or R aRb may together form a cycloalkyl group or a heterocyclyl group as defined herein. Unless specifically stated otherwise in the specification, alkylcarbamoyl groups may be optionally substituted.
"Alkylcarboxamide" or "alkylcarboxamide group" refers to the group-C (O) -NR aRb, wherein R a and R b are the same or different and are independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl or heterocyclyl group as defined herein, or R aRb may together form a cycloalkyl group as defined herein. Unless specifically stated otherwise in the specification, the alkylcarboxamido groups may be optionally substituted.
"Aryl" refers to a hydrocarbon ring system group comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from acetate (ACEANTHRYLENE), acenaphthylene (ACENAPHTHYLENE), acetenyl (acetenyl), anthracene, azulene (azulene), benzene, chrysene (chrysene), fluoranthene (fluoranthene), fluorene, asymmetric indacene (as-indacene), symmetric indacene (s-indacene), indane, indene, naphthalene, phenalene (phenalene), phenanthrene, heptaidiene (pleiadiene), pyrene, and benzophenanthrene. Unless specifically stated otherwise in the specification, the term "aryl" is intended to include optionally substituted aryl groups.
"Heteroaryl" refers to a group of a5 to 20 membered ring system comprising a hydrogen atom, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, heteroaryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxacycloheptenyl, 1, 4-benzodioxanyl, benzonaphtofuranyl, benzoxazolyl, benzodioxolyl, benzodioxanyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothienyl, isothiazolyl, imidazolyl, furanyl, furanonyl, isothiazolyl, imidazolyl indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxopyridyl, 1-oxopyrimidinyl, 1-oxopyrazinyl, 1-oxopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless specifically stated otherwise in the specification, heteroaryl groups may be optionally substituted.
The term "substituted" as used herein means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamide, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom is replaced by a non-hydrogen atom such as, but not limited to: halogen atoms such as F, cl, br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in a group such as a thiol group, a thioalkyl group, a sulfone group, a sulfonyl group, and a sulfoxide group; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylaryl amines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups and triarylsilyl groups; and other heteroatoms in various other groups. "substituted" also means any of the above groups in which one or more atoms are replaced with Gao Jiejian (e.g., double or triple bonds) to heteroatoms (such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles). For example, "substituted" includes any of the above groups in which one or more atoms are replaced by -NRgRh、-NRgC(＝O)Rh、-NRgC(＝O)NRgRh、-NRgC(＝O)ORh、-NRgSO2Rh、-OC(＝O)NRgRh、-ORg、-SRg、-SORg、-SO2Rg、-OSO2Rg、-SO2ORg、＝NSO2Rg and-SO 2NRgRh. "substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by -C(＝O)Rg、-C(＝O)ORg、-C(＝O)NRgRh、-CH2SO2Rg、-CH2SO2NRgRh. In the above, R g and R h are the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. "substituted" also means any of the foregoing groups in which one or more atoms is replaced with an amino, cyano, hydroxy, imino, nitro, oxo, thio, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and/or heteroarylalkyl group. "substituted" may also mean an amino acid in which one or more atoms in the side chain are replaced by alkyl, alkenyl, alkynyl, acyl, alkylcarboxamide, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl or heteroaryl groups. In addition, each of the foregoing substituents may also be optionally substituted with one or more of the foregoing substituents.
As used herein, "subject" means an individual. Thus, a "subject" may include domestic animals (e.g., cats, dogs, etc.), farm animals (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "subject" may also include mammals, such as primates or humans. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under treatment by a clinician (e.g., physician).
The term "inhibition" refers to a decrease in activity, response, illness, disease or other biological parameter. This may include, but is not limited to, complete elimination of an activity, response, disorder or disease. This may also include, for example, a 10% reduction in activity, response, illness or disease compared to a natural or control level. Thus, the reduction may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any reduction therebetween, as compared to a native or control level.
"Reduction" or other forms of the word, such as "reduction", means a decrease in an event or feature (e.g., tumor growth). It will be appreciated that this is typically associated with some standard or expected value, in other words it is relative, but reference to a standard or relative value is not always required. For example, "reducing tumor growth" means reducing the growth rate of a tumor relative to a standard or control (e.g., untreated tumor).
The term "treatment" refers to the medical management of a patient with the aim of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term includes active therapies, i.e. therapies directed specifically to ameliorating a disease, pathological condition or disorder, and also includes causal therapies, i.e. therapies directed to abrogating the etiology of the associated disease, pathological condition or disorder. In addition, the term includes palliative treatment, i.e., treatment intended to alleviate symptoms rather than cure a disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a related disease, pathological condition, or disorder; and supportive treatment, i.e., treatment for supplementing another specific therapy aimed at ameliorating the associated disease, pathological condition or disorder.
The term "therapeutically effective" means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of the disease or disorder. Such improvements need only be reduced or altered and need not be eliminated.
The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
The term "carrier" means a compound, composition, substance or structure that, when combined with a compound or composition, facilitates or facilitates the preparation, storage, administration, delivery, availability, selectivity or any other characteristic of the compound or composition for its intended use or purpose. For example, the carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers may include sugars such as lactose.
As used herein, the term "sequence identity" refers to the percentage of amino acids that are identical between two polypeptide sequences and in the same relative position. Thus, a polypeptide sequence has a certain percentage of sequence identity compared to another polypeptide sequence. For sequence comparison, one sequence is typically used as a reference sequence, which is compared to a test sequence. One of ordinary skill in the art will appreciate that two sequences are generally considered "substantially identical" if they contain the same residues at the corresponding positions. In embodiments, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.mol. Biol. 48:443-453) (version present by the date of filing) implemented in the Needle program of the EMBOSS package (EMBOSS: the European Molecular Biology Open Software Suite, rice et al, 2000,Trends Genet.16:276-277). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. The Needle output labeled "longest identity" (obtained using the-nobrief option) was used as the percent identity and calculated as follows: (identical residue. Times.100)/(alignment Length-total number of gaps in the alignment)
In embodiments, sequence identity may be determined using the Smith-Waterman algorithm with versions that exist by the date of submission.
As used herein, "sequence homology" refers to the percentage of amino acid homology between two polypeptide sequences and in the same relative position. Thus, a polypeptide sequence has a certain percentage of sequence homology as compared to another polypeptide sequence. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Or homologous residues may be different residues having suitably similar structural and/or functional characteristics. For example, as is well known to those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non-polar" side chains, and amino acid substitutions of one amino acid for another amino acid of the same type can generally be considered "homologous" substitutions.
As is well known in the art, any of a variety of algorithms may be used to compare amino acid sequences, including those available in commercial computer programs, such as BLASTP, gapped BLAST, and PSI-BLAST, which exist by the date of filing. Exemplary such procedures are described below: altschul et al, basic local ALIGNMENT SEARCH tool, J.mol.biol.,215 (3): 403-410, 1990; altschul et al Methods in Enzymology; altschul et al ,"Gapped BLAST and PSI-BLAST：a new generation of protein database search programs",Nucleic Acids Res.25：3389-3402,1997;Baxevanis et al, bioinformation A PRACTICAL Guide to THE ANALYSIS of Genes and Proteins, wiley,1998; and Misener et al, (ed), bioinformatics Methods and Protocols (Methods in Molecular Biology, vol.132), humana Press,1999. In addition to identifying homologous sequences, the above procedure typically provides an indication of the degree of homology.
As used herein, the terms "antisense compound" and "AC" are used interchangeably to refer to a polymeric nucleic acid structure (also referred to as an oligonucleotide or polynucleotide) that is at least partially complementary to a target nucleic acid molecule to which it (AC) hybridizes. AC may be a short (in embodiments, less than 50 base pairs) polynucleotide or a polynucleotide homolog comprising a sequence complementary to a target sequence in a target pre-mRNA strand. AC may be formed from natural nucleic acids, synthetic nucleic acids, nucleic acid homologs, or any combination thereof. In embodiments, the AC comprises an oligonucleotide. In embodiments, the AC comprises an antisense oligonucleotide. In embodiments, AC comprises a conjugate group. Non-limiting examples of AC include, but are not limited to, primers, probes, antisense oligonucleotides, external Guide Sequence (EGS) oligonucleotides, alternative splicers, siRNA, oligonucleotides, oligonucleotide analogs, oligonucleotide mimics, and chimeric combinations of these. Thus, these compounds may be introduced in single-stranded, double-stranded, cyclic, branched or hairpin form, and may contain structural elements such as internal or terminal bulges or loops. The oligomeric double-stranded compound may be two strands that hybridize to form a double-stranded compound, or a single strand that has sufficient self-complementarity to allow hybridization and formation of a complete or partial double-stranded compound. In embodiments, AC modulates (increases, decreases, or alters) expression of a target nucleic acid. Various modifications may be made to the polymeric nucleic acid structure, such as Phosphorodiamidate Morpholino (PMO). Thus, AC as used herein encompasses any modification described herein, such as PMO.
As used herein, the terms "pre-mRNA" and "primary transcript" refer to eukaryotic mRNA molecules that are newly synthesized directly after transcription of DNA. The pre-mRNA must be capped with a 5 'cap, modified with a 3' poly A tail, and spliced to produce the mature mRNA sequence.
As used herein, the term "targeting" or "targeting" refers to the association of an Antisense Compound (AC) with a target nucleic acid molecule or target nucleic acid molecule region. In embodiments, the AC is capable of hybridizing to the target nucleic acid under physiological conditions. In embodiments, the AC targets a particular portion or site within the target nucleic acid, e.g., a portion of the target nucleic acid having at least one identifiable structure, function, or feature, such as a particular exon or intron, or a selected nucleobase or motif within an exon or intron.
As used herein, the terms "target nucleic acid" and "target sequence" refer to a nucleic acid molecule having a nucleic acid sequence that binds or hybridizes to an antisense compound. Target nucleic acids include, but are not limited to, RNAs (including but not limited to pre-mrnas and mrnas or portions thereof), cdnas derived from such RNAs, and untranslated RNAs such as mirnas. For example, in embodiments, a target nucleic acid may be a nucleic acid molecule that expresses a cellular gene (or mRNA transcribed from such gene) associated with a particular disorder or disease condition, or an infectious agent. In embodiments, the target nucleic acid is a target RNA. In embodiments, the target nucleic acid is a target mRNA. In embodiments, the target nucleic acid is a target pre-mRNA.
As used herein, the terms "splicing" and "processing" refer to the modification of post-transcriptional pre-mRNA in which introns are removed and exons are joined. Splicing occurs in a series of reactions catalyzed by a large RNA-protein complex (called a spliceosome) composed of five small nuclear ribonucleoproteins (snrnps). Within the intron, splicing requires a3 'splice site, a 5' splice site, and a branching site. The RNA component of snRNP interacts with introns and may be involved in catalysis
As used herein, the term "exon" refers to a portion of pre-mRNA that is typically contained in mature mRNA after splicing.
As used herein, the term "intron" refers to a portion of a pre-mRNA that is typically not included in the mature mRNA after splicing.
As used herein, the term "flanking" refers to an intron immediately upstream (5 ') or downstream (3') of the associated exon. For example, a 5 'flanking intron of exon 44 refers to an intron immediately upstream of exon 44 (i.e., directly coupled to the 5' end of exon 44). For example, a 3 'flanking intron of exon 44 refers to an intron immediately downstream of exon 44 (i.e., directly coupled to the 5' end of exon 44).
A "target pre-mRNA" is a pre-mRNA comprising a target sequence that hybridizes to AC.
A "target mRNA" is an mRNA sequence resulting from splicing of a target pre-mRNA sequence. In embodiments, the target mRNA does not encode a functional protein. In embodiments, the target mRNA retains one or more intron sequences.
As used herein, the term "gene" refers to a nucleic acid molecule having a nucleic acid sequence that encompasses the 5 'promoter region, as well as any introns and exons, associated with expression of a gene product, and the 3' untranslated region ("UTR") associated with expression of a gene product.
The "target gene" of the present disclosure refers to a gene encoding a target pre-mRNA.
"Target protein" refers to the amino acid sequence encoded by the target mRNA. In embodiments, the target protein may not be a functional protein.
"Wild-type target protein" refers to a natural functional protein isoform produced by a wild-type, normal or unmutated form of a target gene. Wild-type target protein also refers to a protein produced from a target pre-mRNA that has been spliced correctly.
As used herein, the term "transcript" refers to an RNA molecule transcribed from DNA and includes, but is not limited to, mRNA, mature mRNA, pre-mRNA, and partially processed RNA.
As used herein, "re-spliced target protein" refers to a protein encoded by mRNA produced by splicing of target pre-mRNA hybridized to AC. The re-spliced target protein may be identical to the wild-type target protein, may be homologous to the wild-type target protein, may be a functional variant of the wild-type target protein, or may be an active fragment of the wild-type target protein.
As used herein, "functional fragment" or "active fragment" refers to a portion of a eukaryotic wild-type target protein that exhibits activity, such as one or more activities of a full-length wild-type target protein, or has another activity. In embodiments, a re-spliced target protein sharing at least one biological activity of a wild-type target protein is considered an active fragment of the wild-type target protein. The activity may be any percentage (i.e., more or less) of the activity of the full-length wild-type target protein, including but not limited to about 1% of the activity compared to the wild-type target protein, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 200%, about 300%, about 400%, about 500% or more (including all values and ranges between these values). Thus, in embodiments, the active fragment may retain at least a portion of one or more biological activities of the wild-type target protein. In embodiments, the active fragment may enhance one or more biological activities of the wild-type target protein.
As used herein, the term "nucleoside" means a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides and nucleosides having mimicking bases and/or sugar groups. A "natural nucleoside" or "unmodified nucleoside" is a nucleoside comprising a natural nucleobase and a natural sugar. Natural nucleosides include RNA nucleosides and DNA nucleosides.
As used herein, the term "natural sugar" refers to a sugar of a nucleoside that has not been modified for its naturally occurring form in RNA (2 '-OH) or DNA (2' -H).
As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group covalently linked to a sugar. The nucleotide may be modified with any of a variety of substituents.
As used herein, the term "nucleobase" refers to a nucleoside or a base portion of a nucleotide. A nucleobase may comprise any atom or group of atoms capable of hydrogen bonding with a base of another nucleic acid. A natural nucleobase is a nucleobase that has not been modified for its natural form of presence in RNA or DNA.
As used herein, the term "heterocyclic base moiety" refers to a nucleobase comprising a heterocycle.
As used herein, "oligonucleotide" refers to an oligonucleotide in which internucleoside linkages do not contain a phosphorus atom.
As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. In certain embodiments, one or more nucleotides of the oligonucleotide are modified. In embodiments, the oligonucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In embodiments, the oligonucleotides are comprised of natural and/or modified nucleobases, sugars, and covalent internucleoside linkages, and may also include non-nucleic acid conjugates.
As used herein, "internucleoside linkage" refers to covalent linkage between adjacent nucleosides.
As used herein, "natural internucleoside linkage" refers to 3 'to 5' phosphodiester linkage.
As used herein, the term "modified internucleoside linkage" refers to any linkage between nucleosides or nucleotides other than naturally occurring internucleoside linkages.
As used herein, the term "chimeric antisense compound" or "chimeric AC" refers to an antisense compound having at least one sugar, nucleobase, and/or internucleoside linkage that is differentially modified compared to other sugar, nucleobase, and internucleoside linkages within the same oligomeric compound. The remaining sugar, nucleobase and internucleoside linkages can be independently modified or unmodified. In general, chimeric oligomeric compounds will have modified nucleosides that can be located at separate positions or combined together in regions that will define a particular motif. Any combination of modifying and/or mimicking groups may comprise the chimeric oligomeric compounds as described herein.
As used herein, the term "mixed backbone antisense oligonucleotide" refers to an antisense oligonucleotide in which at least one internucleoside linkage of the antisense oligonucleotide is different from at least one other internucleoside linkage of the antisense oligonucleotide.
As used herein, the term "nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (a) is complementary to thymine (T). For example, in RNA adenine (A) is complementary to uracil (U). In embodiments, complementary nucleobases refer to nucleobases in an antisense compound that are capable of base pairing with nucleobases of their target nucleic acids. For example, if a nucleobase at a position of an antisense compound is capable of hydrogen bonding with a nucleobase at a position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds with each other or 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 by nucleobase complementarity. In embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond to each other to allow stable association between the antisense compound and the target. One skilled in the art recognizes that it is possible to include mismatches without eliminating the ability of the oligomeric compounds to remain associated. Thus, described herein are antisense compounds that can comprise up to about 20% mismatched nucleotides (i.e., not nucleobase complementary to the corresponding nucleotide of the target). Preferably, the antisense compound contains no more than about 15%, more preferably no more than about 10%, most preferably no more than 5% mismatch or no mismatch. The remaining nucleotides are nucleobase complementary or do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art will recognize that the compounds provided herein 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% nucleobases complementary to a target nucleic acid.
As used herein, "hybridization" means pairing of complementary oligomeric compounds (e.g., antisense compounds and their target nucleic acids). Although not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, huo Siting, or reverse Huo Siting hydrogen bonding between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is a nucleobase complementary to the natural nucleobases thymidine and uracil, which pair by forming hydrogen bonds. The natural base guanine is a nucleobase complementary to the natural bases cytosine and 5-methylcytosine. Hybridization may occur under different conditions.
As used herein, the term "specific hybridization" 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 more than one target site. In embodiments, the oligomeric compounds specifically hybridize to their targets under stringent hybridization conditions.
The term "modulation" refers to a perturbation of expression, function or activity when compared to the level of expression, function or activity prior to modulation. Modulation may include an increase (stimulation or induction) or decrease (inhibition or decrease) in expression, function or activity. In one embodiment, modulation may include perturbing splice site selection during pre-mRNA processing.
The term "inhibit/inhibiting/inhibition" refers to a decrease in activity, expression, function, or other biological parameter, and may include, but does not require, complete elimination of the activity, expression, function, or other biological parameter. Inhibition may include, for example, at least about a 10% reduction in activity, response, disorder or disease as compared to a control. In embodiments, expression, activity, or function of the gene or protein is reduced by a statistically significant amount.
As used herein, the term "expression" refers to all functions and steps that convert the coding information of a gene into the structure that is present and manipulated in a cell. Such structures include, but are not limited to, products of transcription and translation.
As used herein, the term "2' -modified" or "2' -substituted" means a sugar comprising a substituent other than H or OH at the 2' position. 2 '-modified monomers include, but are not limited to, BNA and monomers (e.g., nucleosides and nucleotides) having a 2' -substituent such as allyl, amino, azido, thio, O-allyl, O-C 1-C10 alkyl 、-OCF3、O-(CH2)2-O-CH3、2'-O(CH2)2SCH3、O-(CH2)2-O-N(Rm)(Rn), or O-CH 2-C(＝O)-N(Rm)(Rn, wherein each Rm and Rn is independently H or a substituted or unsubstituted C 1-C10 alkyl.
As used herein, the term "MOE" refers to a 2' -O-methoxyethyl substituent.
As used herein, the term "high affinity modified nucleotide" refers to a nucleotide having at least one modified nucleobase, internucleoside linkage, or sugar moiety such that the modification increases the affinity of an antisense compound comprising the modified nucleotide to a target nucleic acid. High affinity modifications include, but are not limited to, BNA, LNA, and 2' -MOE.
As used herein, the term "mimetic" refers to a group that replaces sugar, nucleobase, and/or internucleoside linkages in AC. In general, mimetics are used in place of sugar or sugar-internucleoside linkage combinations and maintain nucleobases for hybridization to a selected target. Representative examples of glycomimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide Nucleic Acids (PNAs) and morpholino groups linked by uncharged achiral linkages. In some cases, a mimetic is used instead of a nucleobase. Representative nucleobase mimics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al, nuc Acid res.2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.
As used herein, the term "bicyclic nucleoside" or "BNA" refers to a nucleoside in which the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring, thereby forming a bicyclic ring system. BNA includes, but is not limited to, alpha-L-LNA, beta-D-LNA, ENA, oxyBNA (2 '-O-N (CH 3)-CH2 -4') and aminooxyBNA (2 '-N (CH 3)-O-CH2 -4').
As used herein, the term "4 'to 2' bicyclic nucleoside" refers to BNA in which the bridge connecting two atoms of the furanose ring bridges the 4 'carbon atom and the 2' carbon atom of the furanose ring, thereby forming a bicyclic ring system.
As used herein, "locked nucleic acid" or "LNA" refers to a nucleotide that is modified such that the 2 '-hydroxyl group of the ribosyl sugar ring is attached to the 4' carbon atom of the sugar ring via a methylene group, thereby forming a 2'-C,4' -C-oxymethylene linkage. LNAs include, but are not limited to, alpha-L-LNA and beta-D-LNA.
As used herein, the term "cap structure" or "terminal cap portion" refers to a chemical modification that has been incorporated into either end of an AC.
As used herein, the term "dosage unit" refers to a form that provides a pharmaceutical agent. In embodiments, the dosage unit is a vial comprising a lyophilized antisense oligonucleotide. In embodiments, the dosage unit is a vial comprising a reconstituted antisense oligonucleotide.
Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Other embodiments are within the scope of the following claims.
All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications, patents and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Examples
Example 1 conjugation of oligonucleotides to cell penetrating peptides.
As shown in FIG. 1A, an oligonucleotide having a (NH 2- (CH 2) 5 -CH 2-) linker at the 5' phosphorothioate terminus is conjugated to a CPP via a carboxylate or N-hydroxysuccinimide ester (NHS ester) functionality on the peptide. As shown in fig. 1B, the oligonucleotide was conjugated to a Cell Penetrating Peptide (CPP) via amide bond formation (left) or click chemistry. The linker/CPP is mounted on either the 5 'or 3' end of the oligonucleotide.
As shown in fig. 2A and 2B, oligonucleotide-peptide conjugates were synthesized that did not have (fig. 2A) and that had (fig. 2B) a PEG (polyethylene glycol) linker inserted between the oligonucleotide moiety and the peptide. In the figure, "R" represents palmitoyl.
Exemplary Antisense Compounds (ACs) targeting exon 44 of DMD or AC shown in table 7, e.g., those in tables 6A-6M or the reverse complement thereof, are conjugated to CPPs or EEVs disclosed herein. In embodiments, AC is conjugated to a cyclic peptide having the amino acid sequence FfΦ RrRrQ (EEV-1). In embodiments, AC is conjugated to a cyclic peptide having the amino acid sequence fΦ RrRrQ, which is conjugated to a lipid group (R1) (EEV-1 (R1)). In embodiments, AC is conjugated to an Endosomal Escape Vector (EEV) having a sequence loop (Ff Φ RrRrQ) -PEG 12-OH. In embodiments, AC is conjugated to a cyclic peptide having amino acid sequence FGFGRGRQ. In embodiments, the AC is conjugated to EEV having the sequence Ac-PKKKRKV-AEEA-Lys- (cyclo [ FGFGRGRQ ]) -PEG 12-OH.
Table 7: DMD-targeted exon 44 AC
Example 2. Cell penetrating peptides conjugated to oligonucleotides were used to correct for exon 23 splicing of dystrophin in DMD mouse models.
The present study uses MDX mice (Sicinski et al (1989)"The molecular basis of muscular dystrophy in the mdx mouse：a point mutation.Science.244(4912)：1578-80),, which contain a C-to-T mutation, resulting in a stop codon at position 2983 within exon 23 of the dystrophin muscular dystrophy gene (Dmd) on the X chromosome mice expressing this mutant allele produce truncated dystrophin and are therefore a model of Duchenne muscular dystrophy ("DMD").
MDX mice were used to evaluate the ability of the composition to jump exon 23 to treat DMD. AC used in this study was Phosphorodiamidate Morpholino Oligomer (PMO) with the following sequence: 5'-GCTATTACCTTAACCCA-3' (PMO-MDX-23), which targets exon 23.PMO-MDX-23 was conjugated to an Endosomal Escape Vector (EEV) having the sequence: c-ring (FfΦ RrRrQ) -PEG12-OH (EEV-1) to form EEV-PMO conjugate (EEV-PMO-MDX-23). PMO without EEV is called PMO-MDX-23. The preparation of EEV-PMO-MDX-23 is schematically shown in FIG. 4A.
EEV-PMO-MDX-23 and PMO-MDX-23 are administered Intramuscularly (IM) or Intravenously (IV) to MDX mice at the following doses: 1mpk, 3mpk, 10mpk, 30mpk; (mpk: milligrams of compound/kilogram of body weight). Total RNA was extracted from tissue samples and analyzed by RT-PCR to visualize the efficiency of splice correction.
As shown in FIG. 4B, MDX mice treated with EEV-PMO-MDX-23 (1 mpk, IM or 3mpk, IM) produced dystrophin lacking exon 23, whereas MDX mice treated with PMO-MDX-23 alone produced only dystrophin containing exon 23 (similar to untreated controls).
MDX mice were administered Intravenously (IV) 10mpk PMO-MDX-23 and EEV-PMO-MDX-23. The dystrophin products in each muscle group (e.g., quadriceps, tibialis anterior, diaphragmatia, and heart) are shown in fig. 4C, demonstrating that delivery of EEV-PMO-MDX-23 to MDX mice results in correction of dystrophin splicing (e.g., dystrophin excision of exon 23) in quadriceps, tibialis anterior, diaphragmatia, and heart, as compared to delivery of PMO-MDX-23 alone.
Fig. 5A-5D show the effect of the following IV dose regimen on exon skipping efficacy: 10mpk or 30mpk, once every 1 week. Notably, a single 30mpk EEV-PMO-MDX-231 week dose resulted in the highest percentage of exon skipping in all four tissues: quadriceps (fig. 5A), tibialis anterior (fig. 5B), diaphragm (fig. 5C), and heart (fig. 5D).
Figures 6A-6D show the percentage of exon 23 splice correction (as determined by RT-PCR) in tibialis anterior (figure 6A), quadriceps (figure 6B), diaphragm (figure 6C) and heart (figure 6D) following administration of EEV-PMO-MDX-23 at 10mpk twice a week, 10mpk once a week and 30mpk once a week.
Figures 7A-7D show the corrected amounts of dystrophin by exon-23 in quadriceps (figure 7A), tibialis Anterior (TA) (figure 7B), diaphragm (figure 7C) and heart (figure 7D) detected by western blotting after delivery of PMO-MDX-23 or EEV-PMO-MDX-23.
Figures 8A-8D show western blots of corrected dystrophin and α -actin by exon 23 in the diaphragm (figure 8A), heart (figure 8B), quadriceps (figure 8C) and tibialis anterior (figure 8D) following intravenous delivery of 10mpk or 30mpk EEV-PMO-MDX-23-1.
FIGS. 9A-9B show dystrophin levels in two weeks (FIG. 9A) and four weeks (FIG. 9B) of MDX mice after treatment with 30mpk EEV-PMO-MDX-23 or 30mpk PMO-MDX-23.
FIGS. 10A-10D show the percentage of exon 23 correction in the tibialis anterior (FIG. 10A), quadriceps (FIG. 10B), diaphragm (FIG. 10C) and heart (FIG. 10D) of MDX mice administered 30mpk PMO-MDX-23 or 30mpk EEV-PMO-MDX-23. Mice administered EEV-PMO-MDX-23 exhibited enhanced splice correction compared to mice administered PMO-MDX-23 alone.
Fig. 11A-11C show the high and sustained levels of exon 23 skipping and dystrophin correction observed in MDX mice at most 8 weeks after a single IV dose (40 mg/kg) of EEV-PMO-MDX-23 in heart (fig. 11A), tibialis anterior (fig. 11B) and diaphragm (fig. 11C).
Example 3 cell penetrating peptides conjugated to oligonucleotides were used to correct for exon 23 splicing of dystrophin in a D2-mdx mouse model of DMD.
The study used a D2-mdx mouse model. (Fukada et al (2010)"Genetic background affects properties of satellite cells and mdx phenotypes.Am.J.Path.176(5)：2414-24).
Robust exon 23 skipping was observed in different muscle groups (n=6) after repeated monthly 20mg/kg doses of EEV-PMO-MDX-23-2 (eev=ac-PKKKRKV-miniPEG-K (loop (GfFGrGrQ)) -PEG 12 -OH; pmo= 5'-GGCCAAACCTCGGCTTACCTGAAAT-3') or PMO-MDX-23-2 (5'-GGCCAAACCTCGGCTTACCTGAAAT-3'). Fig. 12A-12D show that D2-mdx mice exhibited extensive recovery of dystrophin expression and muscle integrity in the heart (fig. 12A), diaphragm (fig. 12B), tibialis anterior (fig. 12C), and triceps (fig. 12D).
The myoglycans interact with dystrophin and other dystrophin-related proteins to form dystrophin-related glycoprotein complexes (DAGCs). DAGC protects the myomembrane from damage caused by shrinkage. In the D2-mdx mouse model, the deletion of dystrophin resulted in the deletion of alpha-muscle glycans. D2-MDX mice received treatment with 4xQ4W (20 mg/kg) IV injection of PMO-MDX-23-2 or EEV-PMO-MDX-23-2. EEV-PMO-MDX-23-2 treated muscles showed almost complete recovery of dystrophin and alpha-inosine, while PMO-MDX-23-2 treated mice showed limited recovery of dystrophin or alpha-inosine (data not shown).
The D2-MDX mice were administered 20mg/kg of PMO-MDX-23-2 or EEV-PMO-MDX-23-2 (monthly). Creatine Kinase (CK) levels (fig. 13A) and functional readings (line hang time (fig. 13B) and normalized grip strength (fig. 13C)) were compared to wild-type (DBA/2J) and vehicle (saline) treated mice. Treatment with EEV-PMO-MDX-23 normalized serum CK levels and significantly improved muscle function when compared to PMO-MDX-23-2 alone (fig. 13A-13C).
Example 4: duration and repeat dose effects on D2MDX mice after EEV-PMO-MDX-23-2 administration
The method comprises the following steps: administering 20, 40 or 80mpk EEV-PMO-mx-23-2 (eev=ac-PKKKRKV-miniPEG-K (loop (GfFGrGrQ)) -PEG 12 -OH; pmo= 5'-GGCCAAACCTCGGCTTACCTGAAAT-3') or PMO-MDX-23-2 to D2/MDX mice
(5'-GGCCAAACCTCGGCTTACCTGAAAT-3') exon 23 skipping was induced in the D2/MDX mouse model. Tissues were harvested at 1,2,4 and 8 weeks to test single dose duration effects and single dose range findings. D2/MDX mice were dosed weekly with 40mpk for 4 weeks and mice were sacrificed 1 week after the final dose to test for repeat dose effects.
Results: exon skipping was observed in all 4 tissues after a single dose (fig. 14A to 14D), as well as dystrophin production. Exon skipping peaks at 2 weeks post injection and is maintained in skeletal muscle for at least 8 weeks: fig. 15A (triceps) and fig. 15B (tibialis anterior). After 4 and 8 weeks, exon skipping was observed in the diaphragm (fig. 15C) and heart (fig. 15D). After 4 weekly administrations, cumulative exon skipping was observed in all 4 tissues, especially in the myocardium (fig. 16) (tissues were collected 1 week after the last dose).
Example 5: functional assays in D2MDX
The method comprises the following steps: 6 groups of male D2/MDX and DBA/2J (wild type) mice (n=8 per group) were dosed intravenously every 2 weeks for a total of 6 doses, including vehicle (saline), PMO-MDX-2 (5'-GGCCAAACCTCGGCTTACCTGAAAT-3') or one of two EEV-PMO constructs targeting exon 23 (EEV-PMO-MDX 23-2:eev=ac-PKKK RKV-miniPEG-K (loop (GfFGrGrQ)) -PEG 12 -OH; pmo= 5'-GGCCAAA CCTCGGCTTACCTGAAAT-3'; and EEV-PMO-MDX-23-3:eev= A c-PKKKRKV-Lys (ffΦ GrGrQ) -PEG 12-K(N3)-NH2; pmo=j =3)
5'-GGCCAAACCTCGGCTTACCTGAAAT-3' -C4 COT). C4cot = cycloocta-2-yne-1-O- (CH 2)4 -O-C (O)) doses are listed in the figure, creatine kinase levels, grip strength and line hang time were measured once every 4 weeks for a total of 4 determinations.
Results: the line-on-time of EEV-PMO-MDX-23-2 80mpk q2w treatment was slightly higher than the other groups at 2 weeks after the first injection and continued to show statistically significant improvement, and increased compared to the vehicle d2.MDX group at both 4 and 8 weeks after the first injection (fig. 17). After 12 weeks of treatment, EEV-PMO-MDX-23-2 80mpk Q2W was statistically indistinguishable from WT animals (FIG. 17). EEV-PMO-MDX-23-2 40pmk Q2W and EEV-PMO-MDX-23-3 15mpk Q2W treatments showed significantly higher line-on-time from 8 weeks after the first treatment compared to vehicle D2.MDX group, stabilized until 12 weeks of treatment, at which point signs of phenotype improvement became apparent for the first time (FIG. 17). The PMO-MDX-23-2 alone treatment appears to follow the same trend as vehicle d2.MDX group and vehicle control group.
Serum Creatine Kinase (CK) levels were measured at 4 time points: pre-dose, and at 4,8 and 12 weeks. Mice treated with EEV-PMO-MDX-23-2 or EEV-PMO-MDX-23-3 showed a significant decrease in serum CK, approaching the wild type. Mice treated with PMO-MDX-23-2 showed no significant drop at all time points after treatment (fig. 18A-18C).
Grip strength was measured before dosing (fig. 19A) and at 12 weeks (fig. 19B). For the treated mice, a dose-dependent increase in grip strength was observed. Vehicle and PMO treated mice did not show significant improvement.
Example 6 correction of hDMD exon 44 splicing Using cell penetrating peptide conjugated to oligonucleotide
The purpose is as follows: the present study uses the hmdmd and CD1 mouse models and the NHP model to study the effect of compounds comprising antisense compounds and cell penetrating peptides. Each compound contains the exocyclic sequence PKKKRKV.
The compound evaluated: the study evaluated EEV-PMO-DMD44-1, EEV-PMO-DMD44-2 and EEV-PMO-DMD44-3. The sequence information for these compounds is shown in table 8.
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Compound synthesis and purification: compounds were synthesized according to the following procedure. The TFA-lysine protected cCPP was reacted with AC of Table 8, followed by deprotection to provide cCPP-AC conjugates. Briefly, cCPP was pre-activated by reacting it with HATU (2.0 eq) and DIPEA (2.0 eq) in DMSO (10 mm,1.8 ml). After 10min at room temperature, the pre-activated solution was combined with a solution of AC in DMSO (10 mm,1.8 ml) and thoroughly mixed. The reaction was incubated at room temperature for 2 hours. The reaction was monitored by LCMS (Q-TOF) using BEH C18 column @1.7 Μm,2.1 mm. Times.50 mm), buffer A: water (0.1% fa), buffer B: acetonitrile (0.1% fa), flow rate: 0.4mL/min, starting from 2% buffer B and gradually increasing to 98% over 3.4 min. Upon completion, in situ deprotection of TFA protected lysine was initiated by dilution of the reaction mixture with 0.2M KCl (aqueous) pH 12 (36 mL). Using the analytical method described above, the reaction was monitored by LCMS (Q-TOF). The crude mixture was loaded directly onto a C18 reverse phase column (Oligo purity column, 150mm x 21.2 mm). The crude product was then purified using a gradient of 5% -20% over 60min using water containing 0.1% fa and acetonitrile as solvent and a flow rate of 20 mL/min. The fractions containing the desired product were combined and the pH of the solution was adjusted to 7 using 0.5M NaOH. The solution was frozen and lyophilized to give a white powder. Formate was exchanged with chloride by reconstitution of cCPP-AC conjugate in 1M aqueous NaCl solution and repeated washing (centrifugation at 3500rpm for 20-40 min) through 3-kD MW-cut-off amicon tube. The procedure was performed three times with 1M NaCl and three times with saline (0.9% NaCl, sterile, endotoxin free). The conductivity of the final filtrate was evaluated to confirm the appropriate salt concentration. The solution was further diluted with saline to the desired formulation concentration and sterile filtered in a biosafety cabinet. The concentration of each formulation was re-measured after filtration.
EEV-PMO-DMD44-1 was obtained in 74% yield. The purity and identity of each formulation was assessed by liquid chromatography-mass spectrometry quadrupole time of flight mass spectrometry (QTOF-LCMS). EEV-PMO-DMD44-1 was 99% pure by RP-FA and 78% pure by CEX. MW calculated for C 411H661N173O130P24 is 10849.26. MW identified by QTOF-LCMS is 10850.95. The endotoxin amount, residual free peptide, FA content and pH of the formulation were further determined.
EEV-PMO-DMD44-2 was obtained in a yield of 70%. Purity and identity of each formulation was assessed by QTOF-LCMS. EEV-PMO-DMD44-2 was 99% pure by RP-FA and 78% pure by CEX. MW calculated for C 411H661N173O130P24 is 10849.26. MW identified by QTOF-LCMS is 10850.88.
EEV-PMO-DMD44-3 was obtained in a yield of 68%. Purity and identity of each formulation was assessed by QTOF-LCMS. EEV-PMO-DMD44-3 was 86.3% pure (impurity was unreacted AC) as determined by RP-FA. MW calculated for C 422H669N173O130P24 is 10989.45. MW identified by QTOF-LCMS is 10990.07.
HDMD mouse model: hDMD mice were ordered from Jackson Lab (STOCK Tg (DMD) 72Thoen/J; accession number: 018900) and bred in the house. The hemizygous mice were further genotyped at Transnetyx. All groups were dosed by intravenous (iv) injection at 5mL/kg per animal and sacrificed 5 days after injection. All animals were euthanized by CO 2 asphyxiation, followed by terminal blood sampling via cardiac puncture. The maximum available volume of whole blood was collected into heparin lithium tubes and processed into plasma. A portion of the plasma was analyzed for clinical chemistry by TESTING FACILITY (idex) and the remaining portion was stored frozen at nominally-70 ℃. Tissues (triceps, TA, diaphragm, heart, kidney, liver, brain) were harvested and flash frozen in liquid nitrogen and stored at-80 ℃ for further evaluation of exon skipping and drug concentration measurements. Animals were age matched and assigned to eight (8) treatment groups according to table 9. Groups 1-1 (3 homozygous hDMD mice, 6 weeks old), groups 1-2 (3 homozygous hDMD mice, 6 weeks old), groups 1-3 (1 male, 1 female, hemizygous hDMD,11 weeks old), groups 1-4 (1 male, 1 female, hemizygous DMD,11 weeks old) received 10, 20, 40 and 80 milligrams per kilogram body weight (mpk) of EEV-PMO-DMD44-1, respectively. Groups 2-1 (3 homozygous hDMD mice, 6 weeks old), groups 2-2 (3 homozygous hDMD mice, 6 weeks old), groups 2-3 (1 male, 1 female, hemizygous hDMD,11 weeks old), groups 2-4 (1 male, 1 female, hemizygous DMD,11 weeks old) received 10, 20, 40 and 80mpk of EEV-PMO-DMD44-2, respectively. All animals survived until their predetermined euthanasia time. Tissue was collected according to the protocol. The amounts of AC and cCPP-AC in the various tissue samples were quantified by LC-MS. Exon skipping in different tissues was analyzed by RT-PCR and exon 44 correction was quantified.
Table 9: experimental design of hDMD experiment
CD1 mouse model: tolerance of EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 was evaluated using 7 week old CD1 male mice. They were ordered from CHARLES RIVER Lab and were adapted for 5 days after receiving before injection. Animals were age matched and assigned to nine (9) treatment groups according to table 10. Group 1 (3 mice, saline); group 2-1 (3 mice), group 2-2 (2 mice), group 2-3 (2 mice), group 2-4 (2 mice), group 2-5 (3 mice), group 2-6 (3 mice) received EEV-PMO-DMD44-1 at 80, 100, 120, 160, 200 and 300mpk, respectively. Group 3-1 (3 mice), group 3-2 (2 mice), group 3-3 (2 mice), group 3-4 (2 mice), group 3-5 (3 mice), group 3-6 (3 mice) received EEV-PMO-DMD44-2 at 80, 100, 120, 160, 200 and 300mpk, respectively.
Table 10: experimental design of tolerance study in CD1 mice
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NHP model: one female animal was administered a 60 minute IV infusion according to each compound (EEV-PMO-DMD 44-1 and EEV-PMO-DMD 44-2) according to Table 11 at a dose volume of 10 mL/kg. Each test article was formulated at 4mg/mL in saline. Blood and urine were collected at the times shown in table 12 for further PK analysis. Biceps biopsies were taken 2 days after injection. Animals were sacrificed on day 7 post injection and skeletal muscles (quadriceps, diaphragm, biceps, deltoid, tibialis anterior (TiA), smooth muscles (esophagus, aorta, colon) and cardiac muscles (ventricle, atrium), crushed and stored at-80 ℃ for evaluation of exon skipping and biodistribution in tissues.
Table 11: experimental design of NHP study
Table 12: NHP study time point
Bioassay sample analysis: tissues were thawed, weighed and homogenized (w/v, 1/5) with RIPA buffer spiked with 1 Xprotease inhibitor cocktail (ThermoFisher Scientific, reference number 1860932). The homogenate was centrifuged at 5000rpm for 5 minutes at 4 ℃. The supernatant was precipitated with a mixture of H 2 O, acetonitrile and MeOH and centrifuged at 15000rpm for 15 min at 4 ℃. The supernatant was transferred to an injection plate and LC-MS/MS analysis was performed using Shimadzu UPLC integrated with Triple Quad Sciex 4500 instrument. The dynamic range of LC-MS/MS assay is 25ng/g tissue to 50,000ng/g tissue. Details of the LC-MS/MS method are summarized here and in table 13. Briefly, UPLC was operated using the following conditions: waters Acquity UPLC BEH C4, 300A,1.7um, 2.1X105 mm; buffer a: h 2 O,0.2% FA; buffer B:95% acetonitrile in H 2 O,0.2% fa; flow rate (0.3 mL/min) and column temperature of 50 ℃. The 10min run starts with 2% buffer B and gradually increases to 35% for 3.5min, followed by 90% for 1min, stay at 90% gradient for 2.5min and finally run at 2% gradient for 2min. The MRM method for 7.5min duration was established as follows: positive polarity; a turbine spray ion source; curtain gas: 25, a step of selecting a specific type of material; collision gas: 6, preparing a base material; ion spray voltage: 5500; temperature: 500; ion source gas 1:60; ion source gas 2:60. the underlined lines of Table 13 were used to quantify the intact and corresponding metabolites (235-PEG 12).
Table 13: LC-MS/MS assay
Exon skipping analysis by RT-PCR: hDMD mice and NHPs express full length human dystrophin mRNA. Delivery of AC may alter splicing and produce shortened dystrophin mRNA following exon 44 skipping. Tissue was homogenized using 1mL RLT lysis buffer (Qiagen, cat No. 79216). The detection of the splicing correction process was measured by RT-PCR, in which RNA extracted from the tissue was first reverse transcribed into cDNA and further analyzed by one-step RT-PCR using the following primer set: forward primer 5'-GCTCAGGTCGGATTGACATT-3' and reverse primer 5'-GGGCAACTCTTCCACCAGTA-3'. RT-PCR reads of tissue without splice correction yielded 641bp gene fragments, and a new 493bp gene fragment appeared after splice correction. Quantification of the relative intensities of the bands corresponding to the hopped and non-hopped transcripts was performed to assess AC-induced exon-44 hopping efficacy. The degree of splice correction (percent) detected by RT-PCR was calculated using the following equation: % correction = (intensity of 493bp fragment band)/(intensity of 493bp fragment band + intensity of 641bp fragment band).
Results: efficacy of EEV-PMO-DMD44-1, EEV-PMO-DMD44-2, and EEV-PMO-DMD44-3 in patient myotubes (FIG. 21): the DMD exon 44 skipping in DMD patient-derived myocytes was evaluated for EEV-PMO-DMD44-1, EEV-PMO-DMD44-2, and EEV-PMO-DMD44-3, each targeting human dystrophin (DMD) exon 44. Briefly, patient-derived myoblasts with exon 45 deletion (DMDΔ45) were treated with 1 μM, 3 μM and 10 μM of EEV-PMO-DMD44-1, EEV-PMO-DMD44-2 and EEV-PMO-DMD44-3 in PromoCell skeletal muscle cell growth medium supplemented with 2% horse serum and 1% chick embryo extract for 24 hours. After 24 hours, the growth medium containing the compounds was replaced with DMEM/2% horse serum and incubated for 5 days to promote myoblast fusion and differentiation into myotubes. Cells were washed and harvested for RNA extraction to assess exon 44 skipping, or SIMPLE WESTERN analysis for protein extraction and dystrophin recovery in RIPA buffer containing protease inhibitors. Dystrophin levels were normalized to HSP90 and expressed relative to untreated healthy samples. Data are expressed as mean ± SD, n=3-4. Untreated DMD delta 45 patient-derived cells express about 10% spontaneous DMD exon 44 skipping and about 4% dystrophin at baseline. All three compounds resulted in robust exon skipping and dystrophin recovery in a dose-dependent manner.
Fig. 22A and 22B show exon skipping in hmmd mice administered EEV-PMO-DMD44-1 (fig. 22A) and EEV-PMO-DMD44-2 (fig. 22B) via IV injection. No serious side effects were observed. Mice were normal after injection, 24 hours post injection and prior to the day of sacrifice. The clinical chemistry for measuring liver and kidney toxicity (alkaline phosphatase (ALP), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), albumin, blood Urea Nitrogen (BUN), creatinine, calcium, phosphorus, chlorine, potassium, sodium, BUN/creatinine, magnesium) and hemolysis and lipidemia index was evaluated 5 days after IV injection. No significant toxicity was detected in EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 treated mice by clinical chemistry evaluation. Tissue concentrations and exon skipping in various muscle groups were assessed 5 days after 10, 20, 40 and 80mpk IV doses. For each dose of EEV-PMO-DMD44-1, the following exon skipping was achieved in heart/triceps/TiA/diaphragmatic tissue, respectively: 10mpk (0%, 6%, 12%, 6%); 20mpk (0%, 22%, 36%, 33%); 40mpk (20%, 94%, 99%, 82%); 80mpk (79%, 97%, 99%, 98%). For each dose of EEV-PMO-DMD44-2, the following exon skipping was achieved in heart/triceps/TiA/diaphragmatic tissue, respectively: 10mpk (0%, 17%, 22%, 14%); 20mpk (2%, 44%, 58%, 35%); 40mpk (17%, 92%, 95%, 83%); 80mpk (79%, 98%, 99%). In the transgenic murine model carrying the full-size human DMD gene, strong dose-dependent accumulation and efficient exon skipping of both EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 were observed in the myocardium and skeletal muscle. At lower doses of 10mpk and 20mpk, EEV-PMO-DMD44-2 drug exposure and efficacy were slightly higher than EEV-PMO-DMD44-1. However, the effect starts to diminish at a dose of 40mpk, with both compounds resulting in the same high level of exon skipping (greater than 80%) in all skeletal muscles. The corresponding tissue concentration of EEV-PMO-DMD44-1 is in the range of 100-300ng/g tissue, while for EEV-PMO-DMD44-2 the range is shifted to slightly higher values, 300-500ng/g tissue concentration. Interestingly, the minimum effective dose of both EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 in the heart was achieved with 40mpk, corresponding to tissue concentrations of 170ng and 350ng, respectively.
EEV-PMO-DMD44-1 was very well tolerated in all doses administered to CD1 mice at 80, 100, 160, 200 and 300mpk doses. Only transient symptoms were observed, which completely resolved 1 hour after injection. No biomarker abnormalities were observed at 1 day and 7 days post injection. EEV-PMO-DMD44-2 is less tolerant. At the highest dose of EEV-PMO-DMD44-2 of 300mpk, one of the three mice died within 1-3 hours after injection. At the lower dose of EEV-PMO-DMD44-2, 200mpk, one of the three mice had severe symptoms (no response to stimulus, pulled back of the ear, slow breathing, difficulty returning to normal). These symptoms worsen gradually and they combine with muscle twitches. No symptoms were observed for the lower doses of 160mpk and 80 mpk. Surprisingly, at 100mpk, one of the three mice showed delayed symptoms at 2 hours post injection; they were completely normal at 1 day and 7 days after injection.
To further demonstrate the efficacy of exon skipping of cCPP-AC conjugates, NHP was utilized. Specifically, 60 min IV infusions of EEV-PMO-DMD44-1 or EEV-PMO-DMD44-2 administered at 40mg/kg to cynomolgus monkeys with intact muscle tissue are well tolerated. More specifically, the animals experienced nausea at 45 minutes of treatment initiation, which resolved significantly about 3 hours after treatment, and the animals were more alert, no longer bent down to humpback and consumed the provided product. At about 20 hours after dosing, the animals became (motor, alert and responsive such that the animals were phenotypically "normal") (BAR), no biscuits remained in the cages, and edible products were observed.
No abnormalities in the clinical chemistry group were observed at 2 and 7 days post injection. The percent exon 44 skipping in different tissues was analyzed according to standard protocols. Fig. 23A-23B depict exon skipping (fig. 23A) and drug exposure (fig. 23B) of EEV-PMO-DMD 44-1. Fig. 24A-24B depict exon skipping (fig. 24A) and drug exposure (fig. 24B) of EEV-PMO-DMD 44-2. Both compounds showed excellent exon skipping levels in the different muscle groups given 40mpk by IV 7 days post injection. From an efficacy standpoint, EEV-PMO-DMD44-1 performs better in TiA, the diaphragm, and less significantly in the ventricles and atria. In all skeletal muscles, exon skipping of over 78% was achieved, with a maximum of 98.4% being achieved in the diaphragm. EEV-PMO-DMD44-1 at 40mpk produced 31.9% and 23.4% in the heart tissue in the ventricle and atrium, respectively. Of the smooth muscles, the esophagus showed 57.1% of the highest efficacy. Both EEV-PMO-DMD44-1 and EEV-PMO-DMD44-2 are distributed in various tissues at pharmacologically relevant concentrations. In some cases, such as the ventricles and atria in cardiac tissue and more notably the esophagus and colon, the same tissue concentration does not translate into the same functional delivery. This may indicate that endosomal escape levels in different tissues may be different. However, in skeletal muscle, approximately 200 ng/g tissue concentration is associated with more than 80% of robust exon skipping, whereas in cardiac tissue, 800-1000 ng/g tissue concentration is associated with 50% of exon skipping in the rough combination of atria and ventricles. Only a single dose of 40mpk of the cCPP-AC conjugate was very encouraging in 50% exon skipping, as cardiac tissue was more challenging tissue for delivery and tissue critical for treatment of neuromuscular disorders (such as DMD).
Patient-derived myocytes: compounds for exon 44 skipping (EEV-PMO-DMD 44-1) were added to DMD patient-derived myocytes and administered to hmd transgenic mice expressing the full-length human dystrophin gene to test DMD transcription correction of human sequence-specific PMOs. FIG. 25A shows robust dose-dependent exon skipping and recovery of dystrophin in muscle cells from DMD patients treated with EEV-PMO-DMD 44-1. Dose-dependent exon 44 skipping and dystrophin recovery (up to 100% and 43.7%, respectively) were observed in DMD patient-derived myocytes treated with EEV-PMO-DMD44-1 compared to untreated patient-derived cells and healthy cells. FIG. 25B shows dose-dependent tissue exposure and exon skipping in the myocardium and skeletal muscle of transgenic mice carrying an integrated copy of the full-length human DMD gene following administration of increasing IV doses of EEV-PMO-DMD44-1 at various levels ranging from 10mg/kg to 80 mg/kg. Exon skipping and tissue exposure were assessed five days after dosing, respectively. A dose-dependent level of tissue exposure of up to 80% and exon skipping of up to 100% at the translation-related dose was observed.
After Intravenous (IV) administration of EEV-PMO-DMD44-1 at 10, 20, 40 and 80mg/kg, dose-dependent tissue exposure and exon skipping were observed in the heart (fig. 26A), tibialis anterior (fig. 26B) and diaphragm (fig. 26C) of hDMD transgenic mice.
A single 30mg/kg IV dose (1 hour infusion) of EEV-PMO-DMD44-1 was administered to the NHP, and an extended circulation half-life of EEV-PMO-DMD44-1 of up to 50 hours was observed in the plasma of the NHP (data not shown). This pharmacokinetic profile suggests the opportunity for anticipated tissue exposure, target binding, and pharmacodynamic effects. 7 days after IV infusion, robust exon 44 skipping was observed in the different muscle groups isolated from EEV-PMO-DMD44-1 treated NHP (FIG. 11B). Thus, an extended half-life and high levels (almost 90% in biceps) of exon skipping were observed in NHPs administered with EEV-PMO-DMD 44-1.
FIG. 27 shows that the circulation half-life of EEV-PMO-DMD44-1 is observed to be prolonged in NHP.
FIG. 28 shows that a single 30mg/kg IV dose of EEV-PMO-DMD44-1 resulted in a significant level of exon skipping in both skeletal muscle and heart of NHP, which provided confidence in translation potential. Robust exon 44 skipping was observed in different muscle groups isolated from EEV-PMO-DMD44-1 treated NHP 7 days after 1 hour of infusion at 30mg/kg IV.
These results represent a robust set of translation data. Exon skipping translates into promising dystrophin production in heart and skeletal muscle. Dystrophin production is sufficient to result in improved function. Dystrophin production persists for 4+ weeks after a single injection.
Hmdmd mice: human dystrophy mice IV are dosed with 15mg/kg EEV-PMO-DMD44-1 or R6 (polyarginine) linear peptide conjugated to the same exon 44 jump PMO. In mice treated with EEV-PMO-DMD-44, exon skipping in the heart (fig. 29A), diaphragm (fig. 29B) and triceps (fig. 29C) was between 60% and about 95%, whereas in mice administered R6-PMO, exon skipping was less than 20%.
EXAMPLE 7 pharmacokinetic Studies of EEV-PMO-DMD44-1 in CD1 mice
Plasma, renal and tibialis anterior drug exposure (AUC) of EEV-PMO-DMD44-1 and PMO-DMD44-1 (the major metabolite of EEV-PMO-DMD 44-1) were studied using a CD1 mouse model.
Five to seven week old mice were treated via intravenous injection of 80mpk EEV-PMO-DMD44-1 or PMO-DMD 44-1. Mice were bled and/or sacrificed at different time points.
Tables 14, 15 and 16 show the pharmacokinetic properties observed in plasma, kidney and tibialis anterior, respectively. For the table: AUC last = area under the curve from zero to the last quantifiable concentration; d = dose; c max = maximum serum or plasma concentration; t max = time to reach C max; CL = total plasma, serum or blood clearance; t 1/2 = elimination half-life; v ss = apparent distribution volume at equilibrium; q h = liver blood flow (ml/min/kg).
The AUC values for metabolites in tibialis anterior were approximately 1000-fold lower compared to the kidneys. The Mean Residence Time (MRT) value of the metabolite in the plasma may be directly related to the tissue MRT value, as it is transferred from the tissue to the plasma prior to urine excretion.
TABLE 14 plasma pharmacokinetic Properties
EEV-PMO-DMD44-1 PMO-DMD44-1
AUClast(nM*hr) 7768 522
AUClast/D 1053 71
Cmax(nM) 9322 6
Cmax/D 1264 0.8
Tmax(hr) 0.1 48
CL(mL/min/kg) 16 -
Qh(％) 18 -
t1/2(hr) 21 56
MRTlast(hr) 1.0 60.4
Vss(mL/kg) 983 -
TABLE 15 renal pharmacokinetic Properties
EEV-PMO-DMD44-1 PMO-DMD44-1
AUClast(pmol/g*hr) 36171 8508417
AUClast/D 4905 1153875
Cmax(pmol/g) 4805 84778
Cmax/D 652 11497
Tmax(hr) 4 72
t1/2(hr) 6 102
MRTlast(hr) 9 75
TABLE 16 tibialis anterior pharmacokinetic profile
EEV-PMO-DMD44-1 PMO-DMD44-1
AUClast(pmol/g*hr) - 8126
AUClast/D - 1102
Cmax(pmol/g) 10 118
Cmax/D 1 16
Tmax(hr) 4 24
t1/2(hr) - 113
MRTlast(hr) - 61
Example 8: efficacy study of hDMD
The method comprises the following steps: 6 groups (n=5) of male hDMD mice were treated with 80mpk of EEV-PMO (EEV-PMO-DMD 44-1). Tissues were collected 1 week, 2 weeks, and 4 weeks after dosing. Exon skipping in 5 tissues was determined by RT-PCR: triceps, TA, diaphragm, heart and gastrocnemius.
Results: exon skipping was maintained for at least 12 weeks in diaphragm, TA, gastrocnemius and triceps (fig. 30B to 30E). Exon skipping was observed to decrease in the heart at 4 weeks (fig. 30A).
Example 9: duration effect on NHP
The method comprises the following steps: 6 NHPs were dosed via a1 hour IV infusion with a single dose of 45mpk EEV-PMO-DMD 44-1. Bicep biopsies were performed at the time points shown in fig. 31. Determination of exon skipping by RT-PCR
Results: as shown in fig. 31, exon skipping was observed after 2 days, and peaked around 1 week. Exon skipping lasted up to 12 weeks (apparent t 1/2 = 14 days) after a single IV dose. Near peak efficacy was observed 2 days post dosing.
Example 10: positioning
Subcellular localization of three constructs was determined: PMO alone (PMO), PMO conjugated to EEV (EEV-PMO), and PMO conjugated to EEV and nuclear localization signal (EEV-NLS-PMO). The sequence information for these constructs is shown in table 17.
TABLE 17 constructs
Briefly, THP-1 monocytes were contacted with 3. Mu.M PMO, EEV-PMO or EEV-NLS-PMO and incubated for 24 hours and examined by LC-MS.
FIG. 32A shows whole cell uptake of PMO with EEV-PMO and EEV-NLS-PMO. Both EEV-PMO and EEV-NLS-PMO showed a significant increase in cellular uptake compared to PMO alone.
EEV-PMO and PMO: about 3 times
EEV-NLS-PMO and PMO: about 58 times
EEV-NLS-PMO and EEV-PMO: about 19 times
FIG. 32B shows subcellular localization of PMO and EEV-NLS-PMO in THP cells assayed using LC-MS/MS. As shown in FIG. 32B, EEV-PMO exhibits improved cell permeability compared to PMO alone. The addition of NLS further increases cell permeability. Fig. 32C shows nuclear uptake of the three constructs.
Claim (modification according to treaty 19)
1. A compound comprising formula (C):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
R 4 and R 6 are independently H or an amino acid side chain;
Wherein both of R 1、R2、R3 and R 4 contain phenylalanine side chains
EP is a cyclic exopeptide comprising 4 to 8 amino acid residues comprising 1 or 2 amino acid residues comprising a side chain comprising a guanidine group or protonated form thereof and 2, 3 or 4 lysine residues;
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;
AC is an antisense compound complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence.
2. The compound of claim 1, wherein q is 1.
3. The compound of claim 1 or 2, wherein the EP has the structure: ac-PKKKRKV.
4. The compound of any one of claims 1-3, wherein the cyclic peptide comprises FGFGRGRQ.
5. The compound of any one of claims 1-4, comprising: ac-PKKKRKV-PEG 2 -K (cyclo [ FGFGRGRQ ]) -PEG 12 -OH.
6. The compound of any one of claims 1-5, wherein the AC comprises the sequence: 5'-AAACGCCGCCATTTCTCAACAGATC-3'.
7. The compound of any one of claims 1-5, wherein the AC comprises the sequence: -ACTGTTCAGCTTCTGTTAGCCACTG-3'.
8. The compound of any one of claims 1-5, wherein the AC comprises the sequence: 5'-AACGCCGCCATTTCTCAACAGATCT-3'.
9. The compound of any one of claims 1-5, wherein the AC comprises a sequence selected from the group consisting of SEQ ID nos: 1, consisting of the following sequence: 5'-TGTTCAGCTTCTGTTAGCCACTGAT-3'.
10. The compound of any one of claims 1-5, wherein the AC comprises a sequence selected from the group consisting of SEQ ID nos: 1, consisting of 25 consecutive nucleotides starting at position 6 of the sequence: 5' -CCGCCATTTCTCAACAGATCTGTCA-3'5'.
11. The compound of claim 1, wherein the AC comprises at least one modified nucleotide or nucleic acid comprising Phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino nucleotides (PMO), locked Nucleic Acids (LNA), peptide Nucleic Acids (PNA), nucleotides comprising a 2' -O-methyl (2 ' -OMe) modified backbone, 2' O-methoxy-ethyl (2 ' -MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides, or 2' -deoxy-2 ' -fluoro- β -D-arabinonucleic acid (2 ' f-ANA).
12. The compound of claim 1, wherein the AC comprises 15-30 nucleic acids.
13. The compound of claim 1, wherein the cyclic peptide is conjugated to the 3' end of the AC.
14. The compound of claim 1, wherein the cyclic peptide is conjugated to the 5' end of the AC.
15. The compound of claim 1, wherein the cyclic peptide is conjugated to the backbone of the AC.
16. The compound of any one of claims 1-15, further comprising a linker that conjugates the cyclic peptide to the AC.
17. The compound of claim 16, wherein the linker is covalently bound to the 5' end of the AC.
18. The compound of claim 16, wherein the linker is covalently bound to the 3' end of the AC.
19. The compound of claim 16, wherein the linker is covalently bound to the backbone of the AC.
20. The compound of any one of claims 16-19, wherein the linker is covalently bound to a side chain of an amino acid residue on the cyclic peptide.
21. The compound of any one of claims 16-20, wherein the linker is a divalent or trivalent C 1-C50 alkylene group, wherein 1-25 methylene groups are optionally and independently replaced by-N (H) -, -N (C 1-C4 alkyl) -, -N (cycloalkyl) -, -O-, -C (O) O-, -S (O) 2-、-S(O)2N(C1-C4 alkyl) -, -S (O) 2 N (cycloalkyl) -, -N (H) C (O) -, -N (C 1-C4 alkyl) C (O) -, -N (cycloalkyl) C (O) -, -C (O) N (H) -, -C (O) N (C 1-C4 alkyl), -C (O) N (cycloalkyl), aryl, heteroaryl, cycloalkyl, or cycloalkenyl.
22. The compound of any one of claims 16-21, wherein the linker comprises:
(i) One or more D or L amino acid residues, each of which is optionally substituted;
(ii) Optionally substituted alkylene;
(iii) Optionally substituted alkenylene;
(iv) Optionally substituted alkynylene;
(v) Optionally substituted carbocyclyl;
(vi) Optionally substituted heterocyclyl;
(vii) One or more- (R 1-J-R2) z "-subunits, wherein each of R 1 and R 2 is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S, and O, wherein R 3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50;
(viii) - (R 1- J) z "-or- (J-R 1) z" -, wherein each of R 1 is independently in each occurrence alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S, or O, wherein R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z "is an integer from 1 to 50; or (ix) combinations thereof.
23. The compound of claim 22, wherein the linker comprises:
(i) Beta alanine and lysine residues;
(ii)-(J-R1)z；
(iii) - (J-R 2) x, or
(Iv) A combination thereof.
24. The compound of claim 22 or 23, wherein each R 1 and R 2 is independently alkylene, each J is O, each x is independently an integer from 1 to 20, and each z is independently an integer from 1 to 20.
25. The compound of any one of claims 16-20, wherein the linker comprises:
(i) - (OCH 2CH2)x -subunit wherein x is an integer from 1 to 20;
(ii) One or more residues of glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminopentanoic acid or combinations thereof; or (b)
(Iii) A combination of (i) and (ii).
26. The compound of any one of claims 16-25, wherein the linker has the structure:
Wherein:
x is an integer from 1 to 20;
y is an integer from 1 to 5;
z is an integer from 1 to 20;
m is a binding moiety; and
AA SC is an amino acid residue of the cyclic peptide.
27. The compound of claim 26, wherein M is:
-C(O)-、 wherein R is alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl; or wherein M is:
wherein: r 1 is alkylene, cycloalkyl or Wherein m is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, and wherein each B is independently selected from nucleobases.
28. The compound of claim 27, wherein M is-C (O) -.
29. The compound of any one of claims 26-28, wherein z is 11.
30. The compound of any one of claims 26-29, wherein x is 1.
31. The compound of any one of claims 26-30, comprising an Exocyclic Peptide (EP) conjugated to the linker at the amino group of the linker.
32. The compound of claim 31, wherein the EP comprises 2 to 10 amino acid residues.
33. The compound of claim 1, wherein the amino group on the side chain of each lysine residue is substituted with a trifluoroacetyl (-COCF 3) group, an allyloxycarbonyl (Alloc), a 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl (Dde) or a (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene-3) -methylbutyl (ivDde) group.
34. The compound of any one of claims 1-33, wherein the Exocyclic Peptide (EP) comprises at least 2 amino acid residues with hydrophobic side chains.
35. The compound of claim 34, wherein the amino acid residue having a hydrophobic side chain is selected from valine, proline, alanine, leucine, isoleucine and methionine.
36. The compound of any one of claims 1-35, wherein the exocyclic peptide comprises one of the following sequences:
PKKKRKV;KR;RR;KKK;KGK;KBK;KBR;KRK;KRR;RKK;RRR;KKKK;KKRK;KRKK;KRRK;RKKR;RRRR;KGKK;KKGK;KKKKK;KKKRK;KBKBK;KKKRKV;PGKKRKV;PKGKRKV;PKKGRKV;PKKKGKV;PKKKRGV; Or PKKKRKG.
37. The compound of any one of claims 1-36, comprising the structure:
Wherein:
x is an integer from 1 to 20;
y is an integer from 1 to 5;
z is an integer from 1 to 20;
EP is a cyclic exopeptide;
m is a binding moiety;
AC is an antisense compound; and
AA SC is an amino acid residue of the cyclic peptide.
38. The compound of any one of claims 1-37, wherein the cyclic peptide comprises 4 to 20 amino acid residues, wherein at least two amino acid residues comprise a side chain comprising a guanidine group or a protonated form thereof, and at least two amino acid residues independently comprise a hydrophobic side chain.
39. The compound of any one of claims 1-38, wherein the cyclic peptide comprises 1, 2,3, or 4 acid residues containing a guanidino group or protonated form thereof.
40. The compound of claim 38 or 39, wherein the cyclic peptide comprises 2,3 or 4 amino acid residues comprising a hydrophobic side chain.
41. The compound of any one of claims 38-40, wherein the cyclic peptide comprises at least one amino acid comprising a side chain comprising Or a protonated form thereof.
42. The compound of any one of claims 38-41, wherein the cyclic peptide comprises 1, 2, 3, or 4 amino acids comprising a side chain selected from the group consisting of Or a protonated form thereof.
43. The compound of any one of claims 38-42, wherein the cyclic peptide comprises at least one glycine residue.
44. The compound of any one of claims 38-43, wherein the cyclic peptide comprises 1, 2, 3, or 4 glycine residues.
45. The compound of any one of claims 1-44, wherein the cyclic peptide comprises formula (I):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1,2, 3 or 4; and
Each m is independently an integer of 0,1, 2 or 3.
46. The compound of claim 45, wherein the side chain comprising aryl is that of phenylalanine.
47. The compound of any one of claims 45 or 46, wherein both of R 1、R2、R3 and R 4 are H.
48. The compound of any one of claims 45-47, wherein the cyclic peptide comprises a structure of formula (I-1), (I-2), (I-3), or (I-4):
or a protonated form thereof,
Wherein:
AA SC is an amino acid side chain; and
Each m is independently an integer from 0 to 3.
49. The compound of any one of claims 45-48, wherein the cyclic peptide comprises the structure:
or a protonated form thereof,
Wherein:
AA SC is an amino acid side chain; and
Each m is independently an integer from 0 to 3.
50. The compound of any one of claims 45-49, wherein AA SC comprises a side chain of an asparagine residue, an aspartic acid residue, a glutamine residue, a glutamic acid residue, a homoglutamic acid residue, or a homoglutamine residue.
51. The compound of any one of claims 45-50, wherein AA SC comprises a side chain of glutamine.
52. The compound of any one of claims 1-51, wherein the AC comprises a sequence from tables 6A-6M, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto.
53. The compound of any one of claims 1-51, wherein the cyclic peptide comprises GfFGrGrQ.
54. The compound of any one of claims 1-51, wherein the cyclic peptide comprises Ff Φ GRGRQ.
55. The compound of any one of claims 1-53 comprising a structure of formula (C-1), formula (C-2), formula (C-3), or formula (C-4):
Or a protonated form thereof;
Wherein a sequence comprising 15-30 nucleic acids is complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence.
56. A pharmaceutical composition comprising a compound of any one of claims 1 to 55.
57. A cell comprising a compound of any one of claims 1 to 55.
58. A method of treating DMD, the method comprising administering to a patient in need thereof a compound of any one of claims 1 to 55.

Claims (58)

1. A compound, the compound comprising:
(a) A cyclic peptide; and
(B) An Antisense Compound (AC) complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence.
2. The compound of claim 1, wherein the AC comprises at least one modified nucleotide or nucleic acid comprising Phosphorothioate (PS) nucleotides, phosphorodiamidate morpholino nucleotides (PMO), locked Nucleic Acids (LNA), peptide Nucleic Acids (PNA), nucleotides comprising a 2' -O-methyl (2 ' -OMe) modified backbone, 2' O-methoxy-ethyl (2 ' -MOE) nucleotides, 2',4' constrained ethyl (cEt) nucleotides, or 2' -deoxy-2 ' -fluoro- β -D-arabinonucleic acid (2 ' f-ANA).
3. The compound of claim 1, wherein the AC comprises 15-30 nucleic acids.
4. The compound of claim 1, wherein the cyclic peptide is conjugated to the 3' end of the AC.
5. The compound of claim 4, wherein the cyclic peptide is conjugated to the 5' end of the AC.
6. The compound of claim 4, wherein the cyclic peptide is conjugated to the backbone of the AC.
7. The compound of any one of claims 4-6, further comprising a linker that conjugates the cyclic peptide to the AC.
8. The compound of claim 7, wherein the linker is covalently bound to the 5' end of the AC.
9. The compound of claim 7, wherein the linker is covalently bound to the 3' end of the AC.
10. The compound of claim 7, wherein the linker is covalently bound to the backbone of the AC.
11. The compound of any one of claims 7-10, wherein the linker is covalently bound to a side chain of an amino acid residue on the cyclic peptide.
12. The compound of any one of claims 7-11, wherein the linker is a divalent or trivalent C 1-C50 alkylene group, wherein 1-25 methylene groups are optionally and independently replaced by-N (H) -, -N (C 1-C4 alkyl) -, -N (cycloalkyl) -, -O-, -C (O) O-, -S (O) 2-、-S(O)2N(C1-C4 alkyl) -, -S (O) 2 N (cycloalkyl) -, -N (H) C (O) -, -N (C 1-C4 alkyl) C (O) -, -N (cycloalkyl) C (O) -, -C (O) N (H) -, -C (O) N (C 1-C4 alkyl), -C (O) N (cycloalkyl), aryl, heteroaryl, cycloalkyl, or cycloalkenyl.
13. The compound of any one of claims 7-12, wherein the linker comprises:
(i) One or more D or L amino acid residues, each of which is optionally substituted;
(ii) Optionally substituted alkylene;
(iii) Optionally substituted alkenylene;
(iv) Optionally substituted alkynylene;
(v) Optionally substituted carbocyclyl;
(vi) Optionally substituted heterocyclyl;
(vii) One or more- (R 1-J-R2) z "-subunits, wherein each of R 1 and R 2 is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S, and O, wherein R 3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50;
(viii) - (R 1- J) z "-or- (J-R 1) z" -, wherein each of R 1 is independently in each occurrence alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR 3、-NR3 C (O) -, S, or O, wherein R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z "is an integer from 1 to 50; or (ix) combinations thereof.
14. The compound of claim 13, wherein the linker comprises:
(i) Beta alanine and lysine residues;
(ii)-(J-R1)z；
(iii) - (J-R 2) x, or
(Iv) A combination thereof.
15. The compound of claim 13 or 14, wherein each R 1 and R 2 is independently alkylene, each J is O, each x is independently an integer from 1 to 20, and each z is independently an integer from 1 to 20.
16. The compound of any one of claims 7-11, wherein the linker comprises:
(i) - (OCH 2CH2)x -subunit wherein x is an integer from 1 to 20;
(ii) One or more residues of glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminopentanoic acid or combinations thereof; or (b)
(Iii) A combination of (i) and (ii).
17. The compound of any one of claims 7-16, wherein the linker has the structure:
Wherein:
x is an integer from 1 to 20;
y is an integer from 1 to 5;
z is an integer from 1 to 20;
M is a bonding moiety; and
AA SC is an amino acid residue of the cyclic peptide.
18. The compound of claim 17, wherein M is:
-C(O)-、 wherein R is alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl; or wherein M is:
wherein: r 1 is alkylene, cycloalkyl or Wherein m is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, and wherein each B is independently selected from nucleobases.
19. The compound of claim 18, wherein M is-C (O) -.
20. The compound of any one of claims 17-19, wherein z is 11.
21. The compound of any one of claims 17-20, wherein x is 1.
22. The compound of any one of claims 17-21, comprising an Exocyclic Peptide (EP) conjugated to the linker at the amino group of the linker.
23. The compound of claim 22, wherein the EP comprises 2 to 10 amino acid residues.
24. The compound of claim 22 or 23, wherein the EP comprises 4 to 8 amino acid residues.
25. The compound of any one of claims 22-24, wherein the EP comprises 1 or 2 amino acids comprising a side chain comprising a guanidino group or protonated form thereof.
26. The compound of any one of claims 22-25, wherein the EP comprises 1,2, 3 or 4 lysine residues.
27. The compound of claim 26, wherein the amino group on the side chain of each lysine residue is substituted with a trifluoroacetyl (-COCF 3) group, an allyloxycarbonyl (Alloc), a 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) ethyl (Dde) or a (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene-3) -methylbutyl (ivDde) group.
28. The compound of any one of claims 22-27, wherein the Exocyclic Peptide (EP) comprises at least 2 amino acid residues with hydrophobic side chains.
29. The compound of claim 28, wherein the amino acid residue having a hydrophobic side chain is selected from valine, proline, alanine, leucine, isoleucine and methionine.
30. The compound of any one of claims 23-29, wherein the exocyclic peptide comprises one of the following sequences:
PKKKRKV;KR;RR;KKK;KGK;KBK;KBR;KRK;KRR;RKK;RRR;KKKK;KKRK;KRKK;KRRK;RKKR;RRRR;KGKK;KKGK;KKKKK;KKKRK;KBKBK;KKKRKV;PGKKRKV;PKGKRKV;PKKGRKV;PKKKGKV;PKKKRGV; Or PKKKRKG.
31. The compound of any one of claims 22-30, wherein the exocyclic peptide has the structure: ac-P-K-K-K-R-K-V-.
32. The compound of any one of the preceding claims, comprising the structure:
Wherein:
x is an integer from 1 to 20;
y is an integer from 1 to 5;
z is an integer from 1 to 20;
EP is a cyclic exopeptide;
M is a bonding moiety;
AC is an antisense compound; and
AA SC is an amino acid residue of the cyclic peptide.
33. The compound of any one of claims 1-32, wherein the cyclic peptide comprises 4 to 20 amino acid residues, wherein at least two amino acid residues comprise a side chain comprising a guanidine group or a protonated form thereof, and at least two amino acid residues independently comprise a hydrophobic side chain.
34. The compound of any one of claims 1-33, wherein the cyclic peptide comprises 1, 2, 3, or 4 acid residues containing a guanidino group or protonated form thereof.
35. The compound of claim 33 or 34, wherein the cyclic peptide comprises 2, 3, or 4 amino acid residues comprising a hydrophobic side chain.
36. The compound of any one of claims 33-35, wherein the cyclic peptide comprises at least one amino acid comprising a side chain comprising Or a protonated form thereof.
37. The compound of any one of claims 33-36, wherein the cyclic peptide comprises 1, 2, 3, or 4 amino acids comprising a side chain selected from the group consisting of Or a protonated form thereof.
38. The compound of any one of claims 33-37, wherein the cyclic peptide comprises at least one glycine residue.
39. The compound of any one of claims 33-38, wherein the cyclic peptide comprises 1, 2, 3, or 4 glycine residues.
40. The compound of any one of the preceding claims, wherein the cyclic peptide comprises formula (I):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
At least one of R 1、R2 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 an amino acid side chain;
q is 1,2, 3 or 4; and
Each m is independently an integer of 0,1, 2 or 3.
41. The compound of claim 40, wherein R 4 is H or a side chain comprising an aryl or heteroaryl group.
42. The compound of claim 40 or 41, wherein the side chain comprising aryl is a side chain of phenylalanine.
43. The compound of any one of claims 40-42, wherein both of R 1、R2、R3 and R 4 comprise a side chain of phenylalanine.
44. The compound of any one of claims 40-43, wherein both of R 1、R2、R3 and R 4 are H.
45. The compound of any one of claims 40-44, wherein the cyclic peptide comprises a structure of formula (I-1), (I-2), (I-3), or (I-4):
or a protonated form thereof,
Wherein:
AA SC is an amino acid side chain; and
Each m is independently an integer from 0 to 3.
46. The compound of any one of claims 40-44, wherein the cyclic peptide comprises the structure:
or a protonated form thereof,
Wherein:
AA SC is an amino acid side chain; and
Each m is independently an integer from 0 to 3.
47. The compound of any one of claims 40-46, wherein AA SC comprises a side chain of an asparagine residue, an aspartic acid residue, a glutamine residue, a glutamic acid residue, a homoglutamic acid residue, or a homoglutamine residue.
48. The compound of any one of claims 40-47, wherein AA SC comprises a side chain of glutamine.
49. The compound of any one of the preceding claims, wherein the AC comprises a sequence from tables 6A-6M, its reverse complement, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity thereto.
50. The compound of any one of claims 1-49, wherein the AC comprises the sequence: 5'-AAACGCCGCCATTTCTCAACAGATC-3'.
51. The compound of any one of claims 1-50, wherein the cyclic peptide comprises FGFGRGRQ.
52. The compound of any one of claims 1-50, wherein the cyclic peptide comprises GfFGrGrQ.
53. The compound of any one of claims 1-50, wherein the cyclic peptide comprises Ff Φ GRGRQ.
54. The compound of any one of claims 1-53, having the structure of formula (C):
or a protonated form thereof,
Wherein:
R 1、R2 and R 3 may each independently be H or an amino acid residue having a side chain comprising an aromatic group;
R 4 and R 6 are independently H or an amino acid side chain;
EP is a cyclic exopeptide as defined herein;
Each m is independently an integer from 0 to 3;
n is an integer from 0 to 2;
x' is an integer from 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;
Wherein the AC comprises a sequence of 15-30 nucleic acids that is complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in a pre-mRNA sequence.
55. The compound of any one of claims 1-53 comprising a structure of formula (C-1), formula (C-2), formula (C-3), or formula (C-4):
Or a protonated form thereof;
Wherein a sequence comprising 15-30 nucleic acids is complementary to a target sequence comprising at least a portion of exon 44 of the DMD gene in the pre-mRNA sequence.
56. A pharmaceutical composition comprising a compound of any one of claims 1 to 55.
57. A cell comprising a compound of any one of claims 1 to 55.
58. A method of treating DMD, the method comprising administering to a patient in need thereof a compound of any one of claims 1 to 55.
CN202280066121.9A 2021-09-01 2022-08-30 Compounds and methods for skipping exon 44 in duchenne muscular dystrophy Pending CN118215504A (en)

Applications Claiming Priority (13)

Application Number Priority Date Filing Date Title
US63/239,645 2021-09-01
US63/239,671 2021-09-01
US63/290,960 2021-12-17
US63/292,685 2021-12-22
US63/298,565 2022-01-11
US63/268,580 2022-02-25
US63/268,577 2022-02-25
US63/362,294 2022-03-31
US63/362,423 2022-04-04
US63/337,560 2022-05-02
US202263354456P 2022-06-22 2022-06-22
US63/354,456 2022-06-22
PCT/US2022/075691 WO2023034817A1 (en) 2021-09-01 2022-08-30 Compounds and methods for skipping exon 44 in duchenne muscular dystrophy

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