Hybrid oligonucleotides

Abstract

Provided herein are methods for preparing hybrid oligonucleotides, hybrid oligonucleotides, and compounds and compositions comprising hybrid oligonucleotides.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 389,775 filed on July 15, 2022, U.S. Provisional Patent Application No. 63 / 389,870 filed on July 16, 2022, and U.S. Provisional Patent Application No. 63 / 498,633 filed on April 27, 2023, the disclosures of each of which are incorporated herein by reference in their entirety. Background Art

[0003] Recognition of specific RNA target nucleic acid sequences via complementary base pairing of oligonucleotides has been studied as a method for treating a variety of diseases and disorders. Single-stranded and double-stranded oligonucleotides with different chemical modifications have been designed to regulate gene expression, for example, through antisense mechanisms such as RNA interference (RNAi), spacers, or steric blocking.

[0004] Two types of internucleotide linkages dominate clinically important oligonucleotides today: linkages with charged backbones, such as phosphodiesters and phosphorothioates (PS) (Stec et al., J. Am. Chem. Soc. 106 (20), 1984, 6077-6079), and neutral linkages, such as phosphorodiamidate linkages of, for example, morpholino oligonucleotide analogs, in which the ribose of the nucleobase is replaced with a morpholino ring (PMO). PMOs are widely used as steric blocking oligonucleotides that do not require RNase H activity, including, for example, as splicing regulatory oligonucleotides.

[0005] The spacer is an oligonucleotide construct including a central spacer region of about 8-12 nucleotides supporting RNase H activity and two flanking regions (also referred to as "wings") of about 1-5 modified nucleotides providing stability and increasing affinity for RNA. Typically, the spacer includes a phosphorothioate (PS) linkage along the entire length of the oligonucleotide. The nucleotides in the central spacer region typically include DNA nucleotides connected to phosphorothioate (PS), and the flanks typically include sugar modifications, such as 2'-O-methylation (2'-OMe), 2'-O-methoxyethyl (2'-MOE) or connected nucleic acid (LNA) (connected by phosphorothioate (PS) linkage). In addition to supporting RNase H activity, it is believed that the spacer promotes transfection by dynamic covalent interactions with membrane proteins (Laurent et al., Angew.Chem.Int.Ed.2021,60,1-6). It is believed that 8 or 9 consecutive PS linkages in the spacer region are required for RNase H activity (Shen et al., (2014) Nucleic Acids Res., 42, 8648-8662; Brown et al., (1994) J. Biol. Chem., 269, 26801-26805), and 10-13 anionic PS linkages promote electrostatic and hydrophobic interactions with amino acid side chains, such as for protein binding (Hyjek-Skladanowska et al., (2020). J. Am. Chem. Soc., 142, 7456-7468). However, there is increasing evidence that PS interacts unfavorably with cellular proteins, which may lead to toxicity.

[0006] The selective chemical substitution of nucleotides in the spacer region has been studied to improve the therapeutic index and reduce the toxicity of the spacer polymer oligonucleotide. Because many proteins are conducive to being attached to the 5' wing (flank) of the spacer polymer, it has been found that the hydrophilic or neutral linkages at positions 2-4 from the 5' end of the spacer region tend to reduce toxicity. Other designs that have been studied introduce unique chemical components into the side-joined regions of the spacer polymer, such as charged N3'-P5' or neutral guanidine (PN) linkages, which improve pharmacology and gene silencing (Kupryushkin et al., Phosphoryl Guanidines:A New Type of Nucleic Acid Analogues.Acta Nat.2014,6,116-118; Gryaznov et al., Nucleic Acids Res.,24:1508-1514,1996).

[0007] The use of neutral linkages, such as methylphosphonates, has also been reported, but was found to have reduced solubility in aqueous solution and was associated with destabilization of the helix and synthetic challenges (Guo et al., Phosphorus, Sulfur and Silicon 1999, Vol. 144-146, 363-366).

[0008] Hybrid backbone spacers have recently been proposed to generate less toxic antisense oligonucleotides (ASOs) without compromising activity in the central nervous system (CNS). Prodrug concepts based on charge neutralizing phosphotriester modifications have also been proposed (Meade et al., Nat Biotechnol. 2014 32(12), 1256-1261).

[0009] Despite individual successes with oligonucleotides containing morpholinos and DNA-PS, chemical synthesis of oligonucleotide hybrids that incorporate both morpholinos and DNA-PS subunits is hampered by current synthetic strategies for generating PMOs (Heera et al., J. Am. Chem. Soc., 142, 38: 16240 (2020)). PMOs are typically prepared in a 6' to 3' direction on polystyrene resins (Paul and Marvin, J. Am. Chem. Soc., 138: 15663 (2016)), which is incompatible with standard methods for chemical synthesis of natural DNA / RNA or most analogs, which are prepared in a stepwise 3' to 5' direction. (Figure 1). In addition, PMO synthesis typically uses P(V) chemistry, while DNA or RNA synthesis uses P(III) phosphorimidate chemistry. For these and other reasons, PMO and DNA synthesis are considered incompatible.

[0010] Recent attempts to synthesize PMO-ASO chimeras include thiophosphoroamidate (also known as phosphorothioamidate) morpholino (TMO)-oligonucleotides that combine PS charged linkages and morpholino rings in the backbone. The synthesis of these oligonucleotides is achieved by sequentially adding morpholino 3'-diimidophosphoroate P (III) nucleotides in a 3' to 5' direction (Langner et al., J. Am. Chem. Soc. 2020, 142 (38), 16240-16253). Caruthers reported an antisense oligonucleotide that can specifically hybridize to a target region in an exon of a human dystrophin gene to induce exon skipping of 8 to 50 nucleotides in length, the antisense oligonucleotide comprising a thiomorpholino nucleotide (TMO) comprising at least 8 to 10 consecutive nucleotides complementary to a target region in an exon of a human dystrophin gene, wherein the TMO comprises a morpholino subunit, wherein the morpholino nitrogen of the morpholino subunit is linked to the 5′-exocyclic carbon of an adjacent nucleotide or the 6′-exocyclic carbon of an adjacent morpholino subunit by an internucleotide linkage containing a phosphorothioate, or a TMO / DNA chimera, wherein at least one nucleotide base comprises a base other than uracil ( Figure 2 and Figure 3 ).

[0011] There remains a need for a robust and flexible synthetic route to generate chimeras containing morpholino nucleotides and other therapeutically relevant modifications, for example for use in new therapeutics with improved biological properties. Summary of the invention

[0012] The present disclosure provides a method for preparing a hybrid oligonucleotide, the method comprising assembling P(III) and P(V) nucleotides in a 6' to 3' or 5' to 3' direction on a support, and using PMO 6'-phosphoramidate P(III) or phosphoramidate P(V) as the first nucleotide from the support.

[0013] The present disclosure provides a hybrid oligonucleotide synthesized by the method of the present disclosure.

[0014] The present disclosure also provides a hybrid oligonucleotide comprising:

[0015] (i) a first nucleotide sequence comprising a first 5' or 6' end and a first 3' end, wherein the first 3' end comprises a terminal 3' nucleic acid residue, and the terminal 3 nucleic acid residue is a morpholino nucleotide analog;

[0016] (ii) a second nucleotide sequence comprising a second 5' end and a second 3' end, wherein the second 5' end comprises a terminal 5' nucleic acid residue, and the terminal 5 nucleic acid residue is a deoxyribonucleotide or an analog thereof, or a ribonucleotide or an analog thereof; and

[0017] (iii) at least one phosphorothioate linkage connecting the terminal 3' nucleic acid residue of the first nucleotide sequence and the terminal 5' nucleic acid residue of the second nucleotide sequence.

[0018] The present disclosure also provides a hybrid oligonucleotide comprising:

[0019] (i) at least one P(III) morpholino nucleotide analog;

[0020] (ii) at least one P(V) morpholino nucleotide analog;

[0021] (iii) at least one P(III) ribonucleotide, at least one P(III) deoxyribonucleotide analog, or a combination thereof;

[0022] (iv) at least one phosphorodiamidate linkage as well as

[0023] (v) at least one phosphorothioate Key link.

[0024] In an embodiment, each 5' nucleotide adjacent to each P(V) morpholino nucleotide analog is a P(III) or P(V) morpholino nucleotide analog.

[0025] In an embodiment, the hybrid oligonucleotide comprises two or more consecutive P(V) morpholino nucleotide analogs linked by phosphorodiamidate linkages.

[0026] In an embodiment, any P(V) morpholino nucleotide analog is linked to any downstream nucleotide that is not a P(V) morpholino nucleotide analog via a phosphorothioate linkage.

[0027] The present disclosure also relates to a compound comprising: a hybrid oligonucleotide of the present disclosure, and a cyclic peptide of formula (I):

[0028] or its protonated form,

[0029] in:

[0030] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0031] R4 and R6 are independently H or an amino acid side chain;

[0032] AA SC is the amino acid side chain;

[0033] q is 1, 2, 3, or 4; and

[0034] Each m is independently an integer from 0 to 3.

[0035] The present disclosure also relates to a compound comprising: a hybrid oligonucleotide of the present disclosure, and an EEV of formula (B):

[0036] or a protonated form thereof, wherein:

[0037] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0038] R4 and R7 are independently H or an amino acid side chain;

[0039] EP is an exocyclic peptide as defined herein;

[0040] Each m is independently an integer from 0 to 3;

[0041] n is an integer from 0 to 2;

[0042] x' is an integer from 1 to 20;

[0043] y is an integer from 1 to 5;

[0044] q is 1-4; and

[0045] z' is an integer from 1 to 23.

[0046] The present disclosure also relates to compositions comprising the disclosed compounds and methods of treating disease using such compounds and compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A to Figure 1B . Compare schematic diagrams of: (A) a typical spacer structure comprising 5' and 3' ribonucleic acid (RNA) flanking regions in which RNA monomer units are linked by phosphorothioate (PS) linkages (RNA-PS), and a central spacer region containing deoxyribonucleotides (DNA) in which DNA monomer units are linked by phosphorothioate (PS) linkages (DNA-PS); and (B) a hybrid spacer oligonucleotide as described herein comprising 5' and 3' diaminophosphorothioate morpholino oligomer (PMO) flanking regions and a central spacer region containing DNA in which DNA monomer units are linked by phosphorothioate (PS) linkages (DNA-PS).

[0048] Figure 2 . Table comparing the properties of hybrid oligonucleotides with a PMO-ASO backbone with oligonucleotides with a PMO-only backbone or a PS-only backbone.

[0049] Figure 3 Schematic diagram comparing the common direction of synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs) (6' to 3') with the typical direction of synthesis of deoxyribonucleotide-containing antisense oligonucleotides (ASOs) (3' to 5').

[0050] Figure 4 . Schematic diagram showing the reverse direction of synthesis (5' to 3') of ASOs according to the methods described herein.

[0051] Figure 5 A to Figure 5I. Schematic representation of sample oligonucleotides. (A) Types of monomers: P(V) morpholino; P(III) morpholino; reverse DNA P(III) and reverse 2' modified RNA P(III) ribonucleotides with 6' or 5' phosphoro linking groups (e.g., reverse 2'-O-methyl (2'-OMe) P(III) or 2'-O-methoxyethyl (2'-MOE) P(III) ribonucleotides); (B) Types of internucleotide linkages: phosphorodiamidate; phosphorothioamidate; and phosphorothioate; (C) hybrid spacer oligonucleotides having 5' and 3' flanks comprising reverse RNA P(III) nucleotide monomers and reverse DNA P(III) ribonucleotides joined by phosphorothioate linkages. (D) a traditional PMO oligonucleotide comprising a P(V) morpholino nucleotide analog linked via a phosphorodiamidate linkage; (E) a hybrid spacer oligonucleotide having 6' and 3' flanks comprising a P(V) morpholino nucleotide analog linked via a phosphorodiamidate linkage; a central spacer region comprising reverse DNA P(III) nucleotides linked via phosphorothioate linkages, wherein the 5'(6') terminal nucleotide in each of the 5' and 3' flanks is a P(III) morpholino nucleotide analog and the 5' flank comprises a series of reverse morpholino P(III) nucleotides linked via phosphorothioate linkages; (F) a hybrid spacer oligonucleotide having 6' and 3' flanks comprising a P(V) morpholino nucleotide analog linked via a phosphorodiamidate linkage; a reverse DNA P(III) nucleotide linked via a phosphorothioate linkage; (G) a hybrid spacer oligonucleotide having a 5' flank comprising a P(V) morpholino nucleotide analog linked by a phosphorodiamidate linkage, wherein the 5' terminal nucleotide is a P(III) morpholino nucleotide analog; a central spacer region comprising reverse DNA P(III) nucleotides linked by phosphorothioate linkages, wherein the 5' (6') terminal nucleotide in each of the 5' and 3' flanks is a P(III) morpholino nucleotide analog and the 5' flank is linked to the spacer region by a phosphorothioate linkage; (G) a hybrid spacer oligonucleotide having a 5' flank comprising a P(V) morpholino nucleotide analog linked by a phosphorodiamidate linkage, wherein the 5' terminal nucleotide is a P(III) morpholino nucleotide analog; a central spacer region comprising reverse DNA P(III) nucleotides linked by phosphorothioate linkages, wherein the 5' flank is linked to the spacer region by a phosphorothioate linkage, and a 3' flank comprising reverse RNA P(III) nucleotides linked by a phosphorothioate linkage;(H) a hybrid oligonucleotide comprising a P(III) morpholino linked by a phosphorodiamidate to a series of P(V) morpholinos linked by a phosphorodiamidate linkage, and a series of reverse RNA P(III) nucleotides linked by a phosphorothioate linkage, wherein the upstream RNA(III) nucleotide is linked by a phosphorothioate linkage to the downstream P(V) morpholino; (I) a hybrid oligonucleotide comprising a P(III) morpholino linked by a phosphorodiamidate to a series of P(V) morpholinos linked by a phosphorodiamidate linkage, and a series of reverse morpholino P(III) nucleotides linked by a phosphorothioate linkage, wherein the upstream morpholino(III) nucleotide is linked by a phosphorothioate linkage to the downstream P(V) morpholino.;

[0052] Figure 6 A to Figure 6F. Schematic diagram of sample oligonucleotides. (A) Types of monomers: P(V) morpholino; P(III) morpholino; reverse DNA with 6' or 5' phosphate linkages P(III), reverse 2' modified RNA P(III) (e.g., reverse 2'-O-methyl (2'-OMe) P(III), reverse 2'-O-methoxyethyl (2'-MOE) P(III), and reverse 2'-fluoro (2'-F) P(III) ribonucleotides); (B) types of internucleotide linkages: diaminophosphorothioate; thiophosphorothioate; phosphorothioate and phosphodiester; (C) a hybrid oligonucleotide comprising two or more regions comprising P(V) morpholino nucleotide analogs linked by diaminophosphorothioate linkages, wherein each region independently comprises a 5' end and a 3' end, wherein the terminal 5'(6') nucleotide of each region is a P(III) morpholino nucleotide analog (referred to herein as a "pin" nucleotide), and the 3' end of each upstream region is linked to the 5' end of an adjacent downstream region via a thiophosphorothioate linkage; (D) a hybrid spacer oligonucleotide as in (C), comprising one or more reverse RNA oligonucleotides comprising a thiophosphorothioate linkage. wherein the 5' end of the RNA P(III) region is linked to an upstream P(V) morpholino region via a P(III) morpholino nucleotide analog ("pin"), and the P(III) morpholino nucleotide analog is linked to the upstream P(V) morpholino region and the downstream RNA P(III) region via a phosphorothioate aminothioate linkage; (E) and (F) are examples of hybrid oligonucleotides comprising: one or more P(V) morpholino nucleotide analogs, one or more P(III) morpholino nucleotide analogs, or a combination thereof; and one or more DNA or RNA nucleotide analogs selected from the group consisting of: reverse DNA P(III), reverse 2' modified RNA; P(III) or a combination thereof, wherein the adjacent 5' nucleotide of each P(V) morpholino nucleotide analog is a P(III) or a P(V) morpholino nucleotide analog, wherein two or more consecutive P(V) morpholino nucleotide analogs are linked by a phosphorodiamidate linkage, and wherein any P(V) morpholino nucleotide analog is linked to a downstream nucleotide that is not a P(V) morpholino nucleotide analog by a phosphorothioate linkage.

[0053] Figure 7 A to Figure 7C. Schematic representation of siRNA duplexes. (A) Types of monomers: P(V) morpholino; P(III) morpholino; reverse DNA P(III), reverse 2' modified RNA P(III) with 6' or 5' phosphate linkage groups (e.g., reverse 2'-O-methyl (2'-OMe) P(III), reverse 2'-O-methoxyethyl (2'-MOE) P(III), and reverse 2'-fluoro (2'-F) P(III) ribonucleotides); (B) Types of internucleotide linkages: phosphorodiamidate; phosphorothioate; phosphorothioate; (C) Schematic representation of siRNA duplexes, wherein the sense and antisense strands are hybrid oligonucleotides as described herein.

[0054] Figure 8 .One of the conventional siRNA designs.

[0055] Fig. 9 .Introduce the PMO to the siRNA strand at a terminal or internal position.

[0056] Fig.10 . Structures of triphenylmethyl or "trityl" (Tr) and dimethoxytriphenylmethyl or "dimethoxytrityl" (DMT) protected P(III) morpholino nucleotide analogs (A, C, G and T) that may be included in the hybrid oligonucleotides as described herein.

[0057] Fig.11 . Structure of dimethoxytrityl (DMT) protected inverse DNA P(III) nucleotide analogs (A, C, G and T) that may be included in hybrid oligonucleotides as described herein.

[0058] Fig.12 . Structures of dimethoxytrityl (DMT) protected reverse RNA P(III) nucleotide analogs (A, C, G and T) that may include 2' modifications (2'-F, 2-'O-Me, 2'-OMOE) in hybrid oligonucleotides as described herein.

[0059] Fig.13 . Structures of dimethoxytrityl (DMT) protected 2' modified reverse RNA P(III) nucleotide analogs with methylated C and U bases that may be included in hybrid oligonucleotides as described herein.

[0060] Fig.14 . Trityl (Tr) protected P(V) morpholino nucleobase analogs with modified G bases that may be included in hybrid oligonucleotides as described herein.

[0061] Fig.15. Trityl (Tr) protected P(V) morpholino nucleobase analogs (G, T, A, C) that may be included in hybrid oligonucleotides as described herein.

[0062] Fig.16 .Examples of solid supports commonly used for the synthesis of PMOs (A and B) and charged oligonucleotide C.

[0063] Fig.17 A to Fig.17 F. Structures of monomers that can be used in hybrid oligonucleotides: (A) P(V) morpholino nucleotide analogs; (B) P(III) morpholino nucleotide analogs; (C) reverse DNA P(III) deoxyribonucleotides; (D) reverse 2' modified RNA P(III) ribonucleotides (e.g., reverse 2'-O-methyl (2'-OMe) P(III) or 2'-O-methoxyethyl (2'-MOE) P(III) ribonucleotides); (E) reverse P(III) locked nucleic acids (LNA); and (F) reverse 2'-fluoro P(III) ribonucleotides.

[0064] Fig.18 . Schematic representation of the method used to process and purify hybrid oligonucleotides.

[0065] Fig.19 Schematic diagram showing an example of a hybrid gapmer oligonucleotide on a solid support with a PMO containing 6' and 3' flanking regions and a central DNA-PS spacer region. Regions synthesized by manual and automated means are marked on the sequence.

[0066] Fig. 20 . Graph showing the mRNA expression levels of DMPK following a 15 μM dose of the PMO / ASO hybrid oligonucleotides generated in the first library compared to positive, negative controls and no treatment (NT). Oligonucleotides were delivered by electroporation.

[0067] Fig.21 . Graph showing the mRNA expression levels of DMPK following administration of 15 μM of a 20-mer ASO / PMO hybrid oligonucleotide. Oligonucleotides were delivered by electroporation.

[0068] Fig. 22 . Graph showing DMPK expression in HeLa cells after liposome-facilitated uptake of 100 nM PMO / ASO hybrid oligonucleotides.

[0069] Fig.23 . Graph showing DMPK expression in HeLa cells after liposome-facilitated uptake of 100 nM PMO / ASO hybrid oligonucleotides.

[0070] Fig.24. A graph showing DMPK expression in HeLa cells after free uptake of 100 μM of PMO / ASO hybrid oligonucleotide.

[0071] Fig.25 . Graph showing DMPK expression in HeLa cells after treatment with PMO / ASO hybrid oligonucleotides, ASO spacers and endosomal escape vehicle (EEV)-conjugated spacers (liposomal and free uptake).

[0072] Fig.26 . Graph showing exon skipping efficacy based on RT-PCR of mixed backbone EGFP PMO oligonucleotides: Oligonucleotide 49: 5'- GCT ATTACCTTAACCCAG-3', oligonucleotide 50: 5'-GCTATT ACCTTAACCCAG -3', oligonucleotide 51: 5'-G CTATTACCTTAACCCAG-3', oligonucleotide 52: 5'-GCTATTACCTTAAC CCAG-3', oligonucleotide 53 5'-GCTATTACCTTAACCCAG-3', and control sequence: oligonucleotide 48: 5'- GCTATTACCTTAACCCAG -3' and oligonucleotide 54: 5'-GCTATTACCTTAACCCAG-3'; wherein ATCG indicates PMO P(V), AT CG indicates 2'-OMe ribonucleotide. Oligonucleotide 50 showed 66% exon skipping and oligonucleotide 53 showed 8% exon skipping, controls showed 37-40% exon skipping. Oligonucleotides were delivered to HeLa eGFP cells using nucleofection.

[0073] Fig. 27 . Graph showing the viability of Hela and HepG2 cells exposed to: Conventional spacer (oligonucleotide 14): PMO spacer (oligonucleotide 17): and PMO spacer (oligonucleotide 55): ATCG indicates PMO, ATCG indicates DNA, and the bold underlined is 2'-OMOE RNA. The PMO spacer (oligonucleotide 17) showed no toxicity up to 1 mM concentration. The conventional spacer (oligonucleotide 14) showed toxicity in both cell lines above 100 mM.

[0074] Fig.28 . Gel showing RNase H digestion activity when fluorescent RNA forms a duplex in the presence of ASO-PMO hybrid oligonucleotides compared to conventional spacer controls and PMO controls.

[0075] Fig.29. shows that compared with the conventional spacer control, when the fluorescent RNA was in the presence of the ASO-PMO hybrid oligonucleotides oligonucleotide 11 (5'-ACAGACAATAAATACCGAGG-3') and oligonucleotide 55 Gel showing the concentration dependence of RNase H digestion activity upon duplex formation.

[0076] Fig.30 . Gel showing RNase H digestion activity when fluorescent RNA is contacted with different ASO-PMO hybrid oligonucleotides.

[0077] Fig.31 . HPLC trace results (IEX-HPLC) of snake venom phosphodiesterase (SVDPE) assay using a control spacer (0053).

[0078] Fig.32 HPLC trace showing stability of ASO-PMO hybrid oligonucleotides in the presence of snake venom phosphodiesterase (SVDPE). Oligonucleotide 3: Oligonucleotide 6: 5'-ACAGACAATAAATACCGAGG-3' (ATCG indicates PMO, ATCG indicates DNA, underlined is 2'-OMe RNA, bold underlined is 2'-OMOE RNA).

[0079] Fig.33 HPLC trace showing stability of PMO-ASO (oligonucleotide 21) in phosphodiesterase II digestion.

[0080] Fig.34 HPLC traces showing phosphodiesterase II digestion with control oligonucleotides having phosphodiester (PO) and phosphorothioate (PS) backbones.

[0081] Fig.35 HPLC trace results of snake venom phosphodiesterase (SVDPE) assay using a control spacer (oligonucleotide 37) with a phosphorothioate (PS) backbone (IEX-HPLC).

[0082] Fig.36 . Schematic diagram comparing the backbone of a thiomorpholino oligonucleotide (TMO) and its oligonucleotide DNA hybrid in the present invention.

[0083] Fig.37 . The structure of a P(III)PMO suitable for use in the transition between an ASO and a PMO monomeric subunit.

[0084] Fig.38. Graph showing exon skipping efficacy of RT-PCR based on mixed backbone EGFP PMO with PS linkage; Oligonucleotide 55: Oligonucleotide 56: Morpholino control (oligonucleotide 57) 5'-GCTATTACCTTAACCCAG-3' and its conjugate with EEV-3 (Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH): oligonucleotide 55-EEV3 and oligonucleotide 56-EEV3 and oligonucleotide 57-EEV3; wherein A TCG indicates PMO P(V), ATCG indicates PMO P(III), and oligonucleotide 55 shows higher efficacy than oligonucleotide 56. As observed for oligonucleotide 55-EEV3 and oligonucleotide 56-EEV3, the addition of EEV3 to PMO appears to improve efficacy. HeLa eGFP cells were treated with 50 μM formulations (free intake) for a duration of 24 hours in 24-well plates, n=3. The cell density was 0.1E+06 cells / well. DETAILED DESCRIPTION

[0085] While not wishing to be bound by theory, it is believed that the disclosed hybrid oligonucleotides have advantages, e.g., relative to phosphorothioate-only (PS) oligonucleotides or phosphorodiamidate-only (PMO) oligonucleotides, including, but not limited to, reduced toxicity, increased uptake of free (unformulated) oligonucleotides, e.g., due to reduced charge of the hybrid oligonucleotides, and stabilization of the oligonucleotide termini from enzymatic degradation. Figure 5 The properties of hybrid oligonucleotides with phosphorothioate (PS) backbones compared to oligonucleotides or phosphomorpholino oligonucleotides (PMOs) with PMOs alone are shown in Hybrid oligonucleotides can be used to treat a variety of genetic diseases, including but not limited to cancer, immune diseases or disorders, and neuromuscular diseases or disorders.

[0086] definition

[0087] 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 mixtures of two or more such compositions, reference to "an agent" includes mixtures of two or more such agents, reference to "the component" includes mixtures of two or more such components, etc.

[0088] As used herein, "oligonucleoside" refers to an oligonucleotide in which the internucleotide linkages do not contain a phosphorus atom.

[0089] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of connected nucleotides or nucleosides. The linkage in an "oligonucleotide" may or may not contain a phosphorus atom. In an embodiment, an oligonucleotide is composed of natural and / or modified nucleobases, sugars, and covalent internucleotide linkages. In an embodiment, an oligonucleotide comprises one or more P (V) morpholino nucleotide analogs; one or more P (III) morpholino nucleotide analogs; and one or more deoxyribonucleotides (DNA) or ribonucleotides (RNA) analogs, including but not limited to: DNA P (III) nucleotides, reverse DNA P (III) nucleotides, RNA P (III) nucleotides, reverse RNA P (III) nucleotides, P (V) morpholino nucleotide analogs, P (III) morpholino nucleotide analogs, or combinations thereof. In an embodiment, an RNA nucleotide is an RNA nucleotide modified by 2'. In an embodiment, an oligonucleotide comprises one or more of the following phosphorus-containing internucleotide linkages: phosphoramidate, diaminophosphoroester, thiophosphoramidate, thiophosphate, phosphodiester, phosphotriester, methylphosphonate, and combinations thereof. In an embodiment, the oligonucleotide comprises one or more non-phosphorus-containing internucleoside linkages selected from the group consisting of methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-) and combinations thereof. In an embodiment, the oligonucleotide comprises one or more phosphorus-containing internucleoside linkages and one or more non-phosphorus-containing internucleoside linkages.

[0090] As used herein, the term "2'-modified" nucleotide refers to a nucleotide in which the ribose contains a substituent other than H or OH at the 2' position. 2'-modified monomers include, but are not limited to, BNAs and monomers (e.g., nucleosides and nucleotides) having 2'-substituents such as allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ) or O-CH2-C(=O)-N(R m )(R n ), where each R m and Rn are independently H or substituted or unsubstituted C1-C 10Examples of alkyl 2'-modified monomers include, but are not limited to, 2'-O-methyl (2'-OMe) P (III) RNA, reverse 2'-O-methyl (2'-OMe) P (III) RNA, 2'-O-methoxyethyl (2'-OMOE) P (III) RNA, reverse 2'-O-methoxyethyl (2'-OMOE) P (III) RNA, 2'-fluoro (2'-F) P (III) RNA, reverse 2'-fluoro (2'-F) P (III) RNA, 2'-O-ethyl (cET) P (III) nucleotides, reverse 2'O-ethyl (cET) P (III) nucleotides, or combinations thereof.

[0091] As used herein, the term "hybrid oligonucleotide" refers to an oligonucleotide containing at least one nucleotide, nucleoside, internucleotide linkage or a combination thereof that is different from at least one other nucleotide, nucleoside or internucleotide linkage within the same oligonucleotide. In an embodiment, a hybrid oligonucleotide includes different nucleosides in a separated position. In an embodiment, a hybrid oligonucleotide includes different nucleosides grouped together in a region. In an embodiment, the nucleosides of the grouping determine a specific motif. As used herein, the term "mixed backbone oligonucleotide" refers to an oligonucleotide wherein at least one internucleotide linkage is different from at least one other internucleotide linkage of an oligonucleotide.

[0092] As used herein, the term "spacer" refers to a heterozygous oligonucleotide including a central spacer region and upstream and downstream flanking regions (also referred to as "wings") that support RNase H activity. In an embodiment, the upstream and downstream flanking regions increase the affinity of oligonucleotide stability and spacer polymers to their target nucleic acid sequences. "Stereo-random spacer" is a spacer polymer with a mixture of (R) or (S) configuration at each of its stereocenters. In some embodiments, a stereo-random spacer polymer is the product of an extension reaction in the presence of a morpholino or deoxyribonucleoside monomer. "Stereo-defined spacer" is a spacer polymer with (R) or (S) stereochemical configuration at each of its stereocenters, wherein the configuration is controlled. Stereo-defined spacer polymers can be the product of a stereo-specific extension reaction in the case of a stereo-pure morpholino or deoxyribonucleoside monomer, wherein the phosphorus stereochemistry of the spacer polymer is controlled to a series of defined stereochemical (R) or (S) configurations.

[0093] The terms "miniPEG," "PEG2," and "AEEA" are used interchangeably herein to refer to 2-[2-[2-aminoethoxy]ethoxy]acetic acid.

[0094] A "PMO-gapmer" is a gapmer that includes flanking regions comprising morpholino monomers linked to each other by phosphorodiamidate bonds. The flanking regions may also be referred to as wing regions.

[0095] "Stereorandom" when referring to a reaction means that the reaction is carried out without preference for the resulting stereochemistry.

[0096] "R" and "S" are terms describing isomers that describe the stereochemical configuration at an asymmetrically substituted atom, including, but not limited to, carbon, sulfur, phosphorus, and quaternary nitrogen. Asymmetrically substituted atoms are designated "R" or "S" by application of the Cahn-Ingold-Prelog priority rules, which are well known to those skilled in the art and described in the International Union of Pure and Applied Chemistry (TUPAC) Rules for the Nomenclature of Organic Chemistry. Section E, Stereochemistry.

[0097] As used herein, the terms "upstream" and "downstream" refer to the relative position in an oligonucleotide. Naturally occurring oligonucleotides have 5' and 3' ends designated for the carbon positions on a 5-membered deoxyribose (or ribose) ring. As a rule, upstream refers to the relative position toward the 5' end of an oligonucleotide, and downstream refers to the relative position toward the 3' end of an oligonucleotide. For oligonucleotides including nucleotides comprising a 6-membered ring, such as morpholino nucleotide analogs, the "upstream" end of an oligonucleotide is interchangeably referred to as the 6' end or the 5' end in this article. The 3' end of an oligonucleotide including a morpholino ring is the downstream end.

[0098] As used herein, the term "nucleobase" refers to the part of the nucleobase that can hydrogen bond to another nucleic acid in a nucleoside or nucleotide. The nucleobase may include any atom or atom group that can hydrogen bond. In an embodiment, the nucleobase is a nitrogenous base. A natural nucleobase is an unmodified nucleobase compared to the naturally occurring form found in RNA or DNA, and includes, for example, cytosine (C), guanine (G), adenine (A), thymine (T) (in DNA) or uracil (U) (in RNA). The modified DNA nucleobase includes 5-methylcytidine (5mC). The modified RNA nucleobase includes, but is not limited to, pseudouridine (Ψ), dihydrouridine (D), inosine (I), ribothymidine (rT) and 7-methylguanosine (m7G).

[0099] As used herein, the term "nucleoside" refers to a molecule comprising a nucleobase and a sugar but lacking a phosphate group. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides with simulated bases and / or sugar groups. "Natural nucleosides" or "unmodified nucleosides" are nucleosides comprising natural nucleobases and natural sugars. Natural nucleosides include RNA and DNA nucleosides.

[0100] As used herein, the term "nucleotide" refers to a molecule comprising a nucleobase, a sugar and an internucleotide linkage. In an embodiment, the internucleotide linkage comprises a phosphate group. Nucleotide can be modified with any one of a variety of substituents, and can include, for example, a modified nucleobase, a modified sugar, a modified phosphate group or a combination thereof. The modified sugar is a sugar moiety modified in comparison to the naturally occurring form found in RNA (2'-OH) or DNA (2'-H), and includes but is not limited to a nucleotide with a ribose modified by 2' and wherein the sugar moiety is replaced with an analog (such as a morpholine ring). In an embodiment, a phosphate group can be connected to the 2', 3' or 5' hydroxyl moiety of the sugar. In an embodiment, when the modified nucleoside includes a six-membered ring (such as a morpholino ring), a phosphate group can be connected to the 2', 3' or 6' hydroxyl moiety. The modified nucleotide may be referred to as a "nucleotide analog" herein, and for example, a modified deoxyribonucleotide may be referred to as a deoxyribonucleotide analog and a modified ribonucleotide may be referred to as a ribonucleotide analog.

[0101] As used herein, the term "heterocyclic base moiety" refers to a nucleobase comprising a heterocyclic ring.

[0102] As used herein, "internucleotide linkage" refers to the covalent linkage between adjacent nucleosides. Naturally occurring DNA and RNA include 3' to 5' phosphodiester (PO) internucleotide linkages. Modified internucleotide linkages compared to natural phosphodiester linkages can be used to change (e.g., increase) the nuclease resistance of oligonucleotides. Two major types of internucleoside linkage groups are defined according to the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleotide linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, thiophosphoramidate phosphoramidate, thiophosphoramidate and thiophosphate. Representative non-phosphorus-containing internucleoside linkage groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, non-naturally occurring internucleotide linkages include, but are not limited to, phosphorodiamidate, phosphorothioate, phosphorothioate (PS), and phosphorylguanidine linkages.

[0103] "Morpholino" or "morpholino nucleotide analog" refers to a modified nucleotide analog comprising a base-pairing nucleobase (Pi), such as a purine or pyrimidine base-pairing nucleobase, wherein the sugar molecule of the nucleotide is replaced with a morpholine ring as shown below:

[0104]

[0105] "Morpholino oligonucleotide" refers to a polymeric molecule that can be hydrogen-bonded to a target nucleic acid sequence, wherein the polymer contains at least one morpholino nucleotide analog, and the at least one morpholino nucleotide analog is coupled to an adjacent nucleotide or nucleotide analog by the nitrogen in the morpholine ring. In an embodiment, a morpholino oligomer comprises a morpholino nucleotide analog connected by a (thio) diaminophosphorothioate linkage, wherein the morpholino nitrogen of one of the subunits is connected to the 5' or 6' carbon of an adjacent nucleotide or nucleotide analog. Morpholino oligomers are described in, for example, U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063 and 5,506,337, each of which is incorporated herein by reference in its entirety.

[0106] A "phosphorodiamidate" group comprises a phosphorus with two attached oxygen atoms and two attached nitrogen atoms, and may also refer herein to a phosphorus with one attached oxygen atom and three attached nitrogen atoms. In the inter-subunit linkages of the oligomers described herein (e.g., between the wing regions and the spacer), one nitrogen is typically flanked to the backbone, and the second nitrogen is a ring nitrogen in a morpholino ring structure, as shown in Formula II below. Alternatively or in addition, nitrogen may be present at a 5'-exocyclic carbon, as shown in Formulas III and IV below.

[0107]

[0108] In a phosphorothiodiamidate linkage, one oxygen atom (usually one pendant to the backbone in the oligomers described herein) is replaced with sulfur.

[0109] As used herein, a "P(V)" nucleotide is a nucleotide in which the phosphorus group has five bonds. Examples of P(V) nucleotides include, but are not limited to, P(V)PMO. See, e.g. Fig.29 , Fig.30 P(V) nucleotides include nucleotides in which the phosphorus group is attached to the 5' or 6' carbon of the sugar (or sugar analog) and nucleotides in which the phosphorus group is attached to the 3' carbon of the sugar (or sugar analog). P(V) nucleotides in which the phosphorus group is attached to the 3' carbon of the sugar (or sugar analog) are referred to herein as "inverted P(V) nucleotides".

[0110] As used herein, a "P(III)" nucleotide is a nucleotide in which the phosphorus group has 3 bonds. Examples of P(III) nucleotides include, but are not limited to: P(III)PMO( Fig.29 , Fig.30 ), P(III)DNA and P(III)RNA( Fig.29 , Fig.31). P(III) nucleotides include nucleotides in which the phosphorus group is attached to the 5' or 6' carbon of the sugar (or sugar analog) and nucleotides in which the phosphorus group is attached to the 3' carbon of the sugar (or sugar analog). P(III) nucleotides in which the phosphorus group is attached to the 3' carbon of the sugar (or sugar analog) are referred to herein as "inverted P(III) nucleotides".

[0111] As used herein, the term "simulations" refers to groups that are substituted for sugars, nucleobases, and / or internucleotide linkages in oligonucleotides. Typically, a simulation is used to replace sugar or a sugar-nucleotide linkage combination, and the nucleobase is maintained to hybridize with a selected target. Representative examples of sugar simulations include, but are not limited to, cyclohexenyl or morpholino. Representative examples of simulations of sugar-nucleotide linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups connected by uncharged non-chiral linkages. In some cases, a simulation is used to replace a nucleobase. Representative nucleobase simulations 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). The synthetic methods of sugar, nucleoside, and nucleobase simulations are well known to those skilled in the art.

[0112] 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 to form a bicyclic system. BNAs include, but are not limited to, α-L-LNA, β-D-LNA, ENA, oxyamino BNA (2'-ON(CH3)-CH2-4') and aminooxy BNA (2'-N(CH3)-O-CH2-4').

[0113] As used herein, the term "4' to 2' bicyclic nucleoside" refers to a BNA in which the bridge connecting the two atoms of the furanose ring bridges the 4' carbon atom and the 2' carbon atom of the furanose ring to form a bicyclic ring system.

[0114] As used herein, "locked nucleic acid" or "LNA" refers to a nucleotide modified so that the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the ribosyl sugar ring via a methylene group to form a 2'-C,4'-C-oxymethylene linkage. LNA includes, but is not limited to, α-L-LNA and β-D-LNA.

[0115] "Solid phase supported morpholino subunit" refers to the first or any subsequent morpholino subunit monomer incorporated into a morpholino oligomer by solid phase stepwise synthesis as described herein. The subunit is attached to a solid support or an oligomer chain growing on a solid support via its 5' (or 6') exocyclic carbon. "Base protection" refers to the protection of base pairing groups (e.g., purine or pyrimidine bases) on a morpholino subunit with a protecting group suitable for preventing the reaction or interference of the base pairing group during stepwise oligomer synthesis.

[0116] As used herein, the term "nucleobase complementarity" refers to the ability of a nucleobase to base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T), and in RNA, adenine (A) is complementary to uracil (U). In an embodiment, a complementary nucleobase refers to a nucleobase in an oligonucleotide that can base pair with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an oligonucleotide can hydrogen bond with a nucleobase at a certain position of a target nucleic acid, the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at the nucleobase pair.

[0117] As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not hydrogen bond with each other or otherwise support hybridization.

[0118] As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize with another oligomeric compound or nucleic acid through nucleobase complementarity. In an embodiment, when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond to each other, thereby allowing stable association between an oligonucleotide and a target, an oligonucleotide and its target are complementary to each other. It is recognized by those skilled in the art that, without eliminating the ability of an oligomeric compound to maintain association, it is possible to include mismatches. Therefore, oligonucleotides that can contain up to about 20% mismatched nucleotides (i.e., nucleobases that are not complementary to the corresponding nucleotides of the target) are described herein. In an embodiment, an oligonucleotide contains no more than about 15%, no more than about 10%, no more than 5% mismatches or does not contain mismatches. The remaining nucleotides are nucleobase complementary or do not otherwise destroy hybridization (e.g., universal bases). It will be recognized by those of ordinary skill in the art that the compounds provided herein are complementary to target nucleic acids 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.

[0119] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., oligonucleotides and their target nucleic acids). Although not limited by a particular mechanism, the most common pairing mechanism involves hydrogen bonding between complementary nucleosides or nucleotide bases (nucleobases), which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding. For example, the natural base adenine (A) is a nucleobase complementary to the natural nucleobases thymidine (T) and uracil (U), which pair by forming hydrogen bonds. The natural base guanine (G) is a nucleobase complementary to the natural bases cytosine (C) and 5-methylcytosine (5mC). Hybridization can be performed under different circumstances.

[0120] As used herein, the term "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to a nucleic acid sequence with substantially greater affinity than it hybridizes to another nucleic acid sequence. In embodiments, an oligonucleotide specifically hybridizes to more than one target site. In embodiments, an oligonucleotide specifically hybridizes to its target under stringent hybridization conditions.

[0121] As used herein, when used in the context of two oligonucleotides, the term "sequence identity" refers to the percentage of residues that are identical and in the same relative position between two sequences. Therefore, a sequence has a certain sequence identity percentage compared to another sequence. In sequence comparison, a sequence usually serves as a reference sequence, and the reference sequence is compared with a test sequence. It will be appreciated by those skilled in the art that if two sequences contain identical residues in corresponding positions, they are generally considered to be "substantially identical". In an embodiment, sequence identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is implemented in the Needle program of the EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), using the version existing as of the submission date. The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Use the Needle output (obtained using the -nobrief option) labeled as "longest identity" as the identity percentage, and calculate as follows: (identical residues × 100) / (total number of gaps in the alignment length-alignment).

[0122] In other embodiments, sequence identity may also be determined using the Smith-Waterman algorithm with the version existing as of the submission date.

[0123] As is well known in the art, sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN, BLASTP, Gapped BLAST, and PSI-BLAST as of the submission date. Exemplary such programs are described in the following references: 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., Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above-mentioned programs generally provide an indication of the degree of homology.

[0124] As used herein, the terms "pre-mRNA" and "primary transcript" refer to newly synthesized eukaryotic mRNA molecules directly following DNA transcription. Pre-mRNA is typically capped with a 5' cap, modified with a 3' poly A tail, and spliced ​​to produce a mature mRNA sequence.

[0125] As used herein, the terms "target nucleic acid" and "target nucleic acid sequence" refer to a nucleic acid sequence that binds or hybridizes to a hybrid oligonucleotide. Target nucleic acids include, but are not limited to, RNA (including, but not limited to, mRNA, including pre-mRNA, mature mRNA, or portions thereof), genomic DNA, cDNA derived from such RNA, and non-translated RNA, such as miRNA. In embodiments, the target nucleic acid may be a cellular gene (or mRNA transcribed from such a gene) that is expressed and is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.

[0126] As used herein, the terms "splicing" and "processing" refer to the modification of pre-mRNA after transcription, in which introns are removed and exons are joined. Splicing is carried out in a series of reactions catalyzed by a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs) called spliceosomes. Within introns, 3' splice sites, 5' splice sites, and branching sites are required for splicing. The RNA component of snRNP interacts with introns and can participate in catalysis.

[0127] A "target pre-mRNA" is a pre-mRNA comprising a target nucleic acid sequence to which an oligonucleotide hybridizes.

[0128] "Mature target mRNA" is an mRNA sequence produced by splicing of a target pre-mRNA sequence. In an embodiment, the mature target mRNA encodes a functional protein. In an embodiment, the mature target mRNA does not encode a functional protein. In an embodiment, the mature target mRNA retains one or more intron sequences.

[0129] The term "target gene" refers to a gene encoding a target pre-mRNA.

[0130] "Target protein" refers to an amino acid sequence encoded by a target mRNA. In an embodiment, the target protein may not be a functional protein.

[0131] As used herein, the term "expression" refers to all functions and steps that convert the coding information of a gene into structures that exist and operate in a cell. Such structures include, but are not limited to, products of transcription and translation, such as proteins.

[0132] "Wild-type target protein" refers to the native functional protein isoform produced by the wild-type, "normal," or non-mutated version of the target gene.

[0133] As used herein, "re-splicing target protein" refers to a protein encoded by an mRNA generated by splicing of a target pre-mRNA hybridized to an oligonucleotide. The re-splicing 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.

[0134] As used herein, the term "modulate" refers to a disturbance in a function or activity when compared to the level of the function or activity before the modulation. For example, modulation includes altering (increasing (stimulating or inducing) or decreasing (inhibiting or decreasing)) gene expression.

[0135] The term "inhibit / inhibiting / inhibition" refers to reducing activity, expression, function or other biological parameters, and may include but does not require complete elimination of the activity, expression, function or other biological parameters. Inhibition may include, for example, a reduction in activity, response, illness or disease by at least about 10% compared to a control. In embodiments, the expression, activity or function of a gene or protein is reduced by a statistically significant amount. In embodiments, the activity or function is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50% and at most about 60%, about 70%, about 80%, about 90% or about 100%.

[0136] "Reduce" or other forms of the word, such as "reducing" or "reduction" means that an event or characteristic is reduced. It should be understood that this is usually relative to a certain standard or expected value, in other words it is relative, but it is not always necessary to mention a standard or relative value.

[0137] As used herein, "treat", "treating", "treatment" and variations thereof refer to any administration of a disclosed compound that partially or completely relieves, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a disease as described herein. With respect to a patient, the term "treatment" refers to the medical management of a patient with the intent to cure, improve, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed to improving a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed to removing the cause of the disease, pathological condition, or disorder in question. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, or disorder; preventive treatment, i.e., treatment directed to minimizing or partially or completely inhibiting the development of a disease, pathological condition, or disorder in question; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to improving a disease, pathological condition, or disorder in question.

[0138] The term "therapeutically effective" refers to an amount of the disclosed compound and / or composition sufficient to improve one or more causes or symptoms of a disease or disorder. Such improvement only requires reduction or modification, not necessarily elimination.

[0139] 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 / or animals without excessive toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable benefit / risk ratio.

[0140] As used herein, the term "cap structure" or "terminal capping moiety" refers to a chemical modification that has been incorporated at either end of an oligonucleotide.

[0141] As used herein, "subject" refers to an individual to be treated and may include domestic animals (e.g., cats, dogs, etc.), livestock (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, a subject may be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician (e.g., a physician).

[0142] Mechanism of action of antisense oligonucleotides (ASOs)

[0143] In embodiments, the hybrid oligonucleotide is a single-stranded oligonucleotide. In embodiments, the hybrid oligonucleotide is a double-stranded oligonucleotide duplex comprising sense and antisense strands. In embodiments, the sense and antisense strands of the double-stranded oligonucleotide duplex are of the same length (i.e., there is no overhang at either end of the duplex). In embodiments, the sense and antisense strands of the double-stranded oligonucleotide duplex are not of the same length (i.e., there is an overhang at one or more of the 3' end of the sense strand, the 5' end of the sense strand, the 3' end of the antisense strand, and the 5' end of the antisense strand).

[0144] In embodiments, the hybrid oligonucleotide is complementary to the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide has a sequence complementary to the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide is completely complementary to the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide is not completely complementary to the target nucleic acid sequence (i.e., the hybrid oligonucleotide sequence may contain one or more mismatches). The target nucleic acid sequence may be a coding sequence or a non-coding sequence. In embodiments, the target nucleic acid sequence comprises DNA. In embodiments, the target nucleic acid sequence comprises chromosomal DNA. In embodiments, the target nucleic acid sequence comprises RNA. In embodiments, the target nucleic acid sequence is in mRNA. In embodiments, the target nucleic acid sequence is in pre-mRNA. In embodiments, the target nucleic acid sequence is in mature mRNA. In embodiments, the hybrid oligonucleotide hybridizes to the target nucleic acid sequence by Watson-Crick base pairing.

[0145] In an embodiment, a hybrid oligonucleotide hybridizes with its target nucleic acid sequence to regulate one or more aspects of protein transcription, translation and expression. In an embodiment, a hybrid oligonucleotide hybridizes with its target nucleic acid sequence to block access to the target nucleic acid sequence by a cellular mechanism (e.g., a splicing factor). In an embodiment, a hybrid oligonucleotide hybridizes with its target nucleic acid sequence to degrade the target oligonucleotide, e.g., mRNA transcripts are degraded via RNase H. Other mechanisms are known and reviewed in Agrawal, S. (1996) "Antisense Oligonucleotides: toward clinical trials." Trends Biotechnol. 14 (10): 376-387.

[0146] In an embodiment, hybridization of a hybrid oligonucleotide to a target nucleic acid sequence inhibits the expression of a target protein. In an embodiment, hybridization of a hybrid oligonucleotide to a target nucleic acid sequence downregulates the expression of one or more wild-type target protein isoforms. In an embodiment, hybridization of a hybrid oligonucleotide to a target nucleic acid sequence upregulates the expression of a target protein. In an embodiment, hybridization of a hybrid oligonucleotide to a target nucleic acid sequence increases the expression of one or more wild-type target protein isoforms.

[0147] In embodiments, the hybrid oligonucleotide stabilizes a target oligonucleotide (e.g., a target mRNA). In embodiments, the hybrid oligonucleotide increases the half-life of a target oligonucleotide. In embodiments, the hybrid oligonucleotide increases the half-life of a target mRNA. In embodiments, the hybrid oligonucleotide increases the expression of a protein product of a target mRNA.

[0148] In embodiments, the hybrid oligonucleotide is an antisense oligonucleotide (ASO). In embodiments, the hybrid oligonucleotide is a steric blocking oligonucleotide, a spacer, a splice switch oligonucleotide, an exon skipping oligonucleotide, a microRNA (miRNA), an antagomir, an aptamer, a ribozyme, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a miRNA mimic, a miRNA inhibitor, or a U1 adaptor. In embodiments, the hybrid oligonucleotide is the sense strand of a siRNA. In embodiments, the hybrid oligonucleotide is the antisense strand of a siRNA.

[0149] The efficacy of a hybrid oligonucleotide can be assessed by assessing any detectable and / or measurable activity attributable to hybridization of the hybrid oligonucleotide with its target nucleic acid. Detection, measurement, or both can be direct or indirect. In embodiments, activity is assessed by detecting, measuring the amount of target mRNA, or both. In embodiments, activity is assessed by detecting, measuring the amount of re-splicing mRNA, or both. In embodiments, activity is assessed by detecting, measuring the amount of target protein, or both. In embodiments, activity is assessed by detecting, measuring the amount of target protein isomers, or both.

[0150] Space blocking

[0151] In an embodiment, the hybrid oligonucleotide is a steric blocking antisense oligonucleotide (ASO). In an embodiment, the transcription, translation or expression of the target protein is regulated by steric blocking. See, for example, Scharner and Aznarez (2020) "Clinical Applications of Single-Stranded Oligonucleotides: Current Landscape of Approved and In-Development Therapeutics." Mol. Therapy. 29 (2): 540-554. Spatial blocking ASO is a short synthetic single-stranded oligonucleotide, the length of which is about 8 to about 50, about 15 to about 30, about 20 to about 30 nucleotides or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Different from spaced polymers, steric blocking ASO does not trigger RNAse H-mediated degradation of target nucleic acid sequences (e.g., target mRNA). As used herein, a "steric blocking" oligonucleotide refers to an oligonucleotide that binds to a target nucleic acid sequence via Watson and Crick base pairing and hinders the binding of a transactivator (e.g., small nuclear RNA (snRNA), micro RNA (miRNA), or RNA binding protein) to the target oligonucleotide or prevents the formation of RNA secondary structure.

[0152] Splice switch

[0153] In an embodiment, the hybrid oligonucleotide is a splice switch oligonucleotide (also referred to as an exon skipping oligonucleotide). The splice switch oligonucleotide is a short synthetic single-stranded oligonucleotide having a length of about 8 to about 50, about 15 to about 30, about 20 to about 30 nucleotides or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. The splice switch oligonucleotide is a class of steric blocking oligonucleotides that base-pair with pre-mRNA and that disrupt the normal splicing spectrum of transcripts by blocking the RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing mechanism and pre-mRNA. In an embodiment, the splice switch oligonucleotide does not trigger the degradation of a target nucleic acid sequence (e.g., target mRNA) mediated by RNase H.

[0154] Because splicing of pre-mRNA is required for proper expression of most protein-coding genes, targeting the process provides a means of manipulating the production of proteins from genes. Splicing regulation can be used to treat diseases caused by mutations that disrupt normal splicing or when interference with the normal splicing process of gene transcripts can be therapeutic. Such antisense oligonucleotides provide an effective and specific way to target and alter splicing in a therapeutic manner. See, for example, Havens and Hastings (2016) "Splice-switching antisense oligonucleotides as therapeutic drugs." Nucleic Acids Res. 44(14): 6549-6563.

[0155] In an embodiment, a hybrid oligonucleotide hybridizes with a target nucleic acid sequence in a target pre-mRNA to regulate one or more aspects of pre-mRNA splicing. In an embodiment, the hybridization of a hybrid oligonucleotide causes one or more exons to be skipped (sometimes referred to as exon skipping). In an embodiment, hybridization causes the expression or activity of a target protein and / or a downstream protein regulated by a target gene to increase or decrease. In an embodiment, a hybrid oligonucleotide hybridizes with a target mRNA to induce alternative splicing, and the alternative splicing causes nucleotides to be added or deleted in a target transcript. In an embodiment, hybridization induces alternative splicing, and the alternative splicing causes nucleotides to be added or deleted in a single exon of a target transcript. In an embodiment, hybridization induces alternative splicing, and the alternative splicing causes nucleotides to be deleted in a single exon of a target transcript. In an embodiment, deletion of nucleotides in a single exon causes translation of truncated proteins. In an embodiment, compared with untruncated proteins, truncated proteins are less toxic to cells.

[0156] As used herein, the regulation of splicing refers to changing the processing of pre-mRNA transcripts so that the spliced ​​mRNA molecules contain different exon combinations due to exon skipping or exon inclusion, deletion in one or more exons, or deletion or addition of sequences (e.g., intron sequences) not usually found in the spliced ​​mRNA. In an embodiment, the hybrid oligonucleotide hybridizes with the target pre-mRNA to restore the natural splicing of the mutated pre-mRNA sequence. In an embodiment, hybridization causes the alternative splicing of the target pre-mRNA. In an embodiment, hybridization causes the exon inclusion or exon skipping of one or more exons. In an embodiment, the exon sequence of the jump includes a frameshift mutation, a nonsense mutation, or a missense mutation. In an embodiment, the exon sequence of the jump includes a nucleic acid deletion, substitution, or insertion. In an embodiment, the exon of the jump itself does not include a sequence mutation, but the adjacent intron includes a mutation that causes a frameshift mutation or a nonsense mutation. In an embodiment, the hybrid oligonucleotide hybridizes with the target nucleic acid sequence in the target pre-mRNA to prevent the inclusion of an intron sequence in the mature mRNA molecule. In an embodiment, hybridization allows the wild-type target protein isomer to be preferentially expressed. In embodiments, the hybridization results in expression of a re-spliced ​​target protein comprising an active fragment of a wild-type target protein.

[0157] In an embodiment, the re-splicing target protein can rescue one or more phenotypes or symptoms of a disease associated with the transcription and translation of the target gene. In an embodiment, the re-splicing target protein can rescue one or more phenotypes or symptoms of a disease associated with the expression of the target protein. In an embodiment, the re-splicing target protein is an active fragment of a wild-type target protein. In an embodiment, the re-splicing target protein acts in a manner substantially similar to the wild-type target protein. In an embodiment, the re-splicing target protein allows a cell to act substantially similarly to a similar cell expressing a wild-type target protein. In an embodiment, the re-splicing target protein does not cure a disease associated with a target gene or with a target protein, but improves one or more symptoms of the disease.

[0158] 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 the wild-type target protein. In embodiments, the enzyme activity or stability may be enhanced by promoting different splicing of the target pre-mRNA. In embodiments, the re-spliced ​​target protein may have the same or substantially similar sequence as a wild-type target protein isoform having improved properties compared to another wild-type target protein isoform.

[0159] In an embodiment, hybrid oligonucleotides hybridize to mRNA targets, for example, by introducing frameshift mutations that produce premature stop codons to generate mRNAs encoding truncated proteins and / or non-functional proteins. In an embodiment, hybridization generates mRNAs encoding truncated proteins and / or non-functional proteins by alternative splicing. In an embodiment, hybrid oligonucleotides trigger degradation of target transcripts, for example, by nonsense-mediated decay. In an embodiment, hybrid oligonucleotides hybridize to mRNA targets to generate alternate mRNA isoforms with beneficial properties.

[0160] In an embodiment, the hybrid oligonucleotide induces the addition or deletion of one or more nucleotides in the resulting processed transcript (such as mRNA). If the number of nucleotides added or removed from the open read segment is divisible by three to obtain an integer, the resulting transcript can be translated into a functional or non-functional protein, which has more or less amino acids than the corresponding protein expressed from the transcript, but has the same amino acid sequence as the protein expressed from the transcript without the added or removed nucleotides in other aspects except the added or deleted amino acids. If the number of nucleotides added or removed from the open reading frame is not divisible by three to obtain an integer, the open reading frame of the resulting processed transcript (such as mRNA) is shifted. For example, the number of nucleotides added or deleted to induce such a "frameshift" change can be 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, etc. Due to the triplet nature of the genetic code, adding or deleting a number of nucleotides that is not divisible by three shifts the reading frame of the resulting processed transcript (such as mRNA) downstream of the frameshift. The shifted reading frame can cause nonsense-mediated decay, can cause premature termination codons within the nonsense downstream of the frameshift, and / or can cause the expression of a protein with a completely different amino acid sequence downstream of the frameshift.

[0161] In an embodiment, hybridization induction of the hybrid oligonucleotide introduces a premature stop codon (PTC) into the open reading frame. As used herein, a "premature stop codon" is a stop codon that is in phase with the translation start codon and is located upstream of a physiological stop codon that is in phase with the translation start codon. Target transcripts with PTCs can be destabilized and degraded by various mechanisms including nonsense-mediated decay.

[0162] In embodiments, the hybrid oligonucleotide induces exon skipping of an exon in a target transcript, wherein the exon has a number of nucleotides that is not divisible by three. In embodiments, the hybrid oligonucleotide induces exon skipping of an exon with a number of nucleotides that is not divisible by three, producing a preliminary termination codon (PTC) in the target transcript. In embodiments, the hybrid oligonucleotide induces exon skipping of an exon with a number of nucleotides that is not divisible by three, producing a PTCT in the target transcript, which causes nonsense-mediated decay of the target transcript. In embodiments, the nonsense-mediated decay of the target transcript is induced so that the concentration of the target transcript is reduced. In embodiments, the nonsense-mediated decay of the target transcript is induced so that the concentration of the target protein encoded by the target transcript is reduced. In embodiments, the nonsense-mediated decay of the target transcript is induced so that the level of the protein of the downstream gene regulated by the target gene is increased and / or reduced.

[0163] Gap polymer

[0164] In embodiments, the hybrid oligonucleotide is a spacer. Spacers are short, synthetic single-stranded oligonucleotides having a length in the range of about 10 to about 30, about 20 to about 30, or about 15 to about 20, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and include a central "spacer" region flanked by 3' wings (also referred to herein as 3' wings) and 5' wings (also referred to herein as 5' wings), thereby forming a continuous sequence of monomeric subunits. The central "spacer" region typically includes about 5 to about 20, or about 8 to about 10, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 deoxyribonucleotides or deoxyribonucleotide analogs. The 3' and 5' flanks typically include about 2 to about 20, about 2 to about 10, or about 4 to about 6 nucleotides.

[0165] In an embodiment, the gapmer binds to the target RNA sequence in a sequence-specific manner through Watson and Crick base pairing to form a double-stranded RNA-DNA duplex that mimics endogenous RNA-DNA hybrids that naturally occur, for example, during DNA replication, and is recognized and degraded via RNase H-mediated RNA strand cleavage that causes RNA degradation.

[0166] In an embodiment, the sugar groups of the 5' flank and the 3' flank are different from the sugar groups of the central spacer region. In an embodiment, the sugar groups of each monomer subunit in the 5' flank are the same. In an embodiment, the sugar groups of each monomer subunit in the 3' flank are the same. In an embodiment, the sugar groups of each monomer subunit in the central spacer are the same. In an embodiment, not all sugar groups of the monomer subunits in the 5' flank are the same. In an embodiment, not all sugar groups of the monomer subunits in the 3' flank are the same. In an embodiment, not all sugar groups of the monomer subunits in the central spacer are the same.

[0167] In embodiments, one or more nucleotides in the 3' flank, 5' flank, spacer region, or a combination thereof include one or more modifications to increase nuclease resistance of the oligonucleotide, reduce immunogenicity of the oligonucleotide, increase binding affinity, or a combination thereof. In embodiments, one or more nucleotides in the 5' flank, 3' flank, or both include modifications in the sugar ring of the DNA or RNA nucleotide. In embodiments, the 5' flank, 3' flank, central "spacer," or a combination thereof include modifications to one or more internucleotide linkages. In embodiments, the spacer polymer may include modifications to one or more internucleotide linkages to increase stability, improve plasma protein binding, or a combination thereof.

[0168] RNA interference nucleic acid

[0169] In an embodiment, the hybrid oligonucleotide is an RNA interference (RNAi) molecule or a small interfering RNA (siRNA) molecule.

[0170] Small interfering RNA (siRNA) is an oligonucleotide duplex having a sense strand (also called a follower strand) and an antisense strand (also called a guide strand) that can associate with a cytoplasmic multiprotein complex called the RNAi-induced silencing complex (RISC) and thereby mediate the degradation of homologous mRNA transcripts. Thus, siRNAs can be designed to knock down protein expression.

[0171] In an embodiment, the siRNA compound consists of a single molecule that includes a duplex region formed by intrastrand pairing, such as a hairpin or coiled-handle structure.

[0172] MicroRNA (miRNA)

[0173] In embodiments, the hybrid oligonucleotide is a microRNA ("miRNA") mimic. As used herein, the term "miRNA mimic" refers to a synthetic oligonucleotide that simulates the gene silencing ability of miRNA, for example, by participating in RNAi pathways and regulating gene expression. As used herein, microRNA (miRNA) is a small single-stranded molecule that targets 5' or 3' untranslated regions (UTRs) to control gene expression, for example, by translational inhibition or degradation of mRNA. In embodiments, miRNA mimics interact with the 3'UTR of the target mRNA to inhibit expression. In embodiments, miRNA mimics interact with the 5'UTR of the target mRNA to inhibit expression.

[0174] In embodiments, the miRNA mimic comprises about 15 to about 30 nucleotides, or about 17 to about 25 nucleotides, or about 15, about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, or 30 nucleotides.

[0175] miRNA inhibitors

[0176] In embodiments, the hybrid oligonucleotide is a miRNA inhibitor (also referred to as "antimir", "microRNA inhibitor", "miR inhibitor" or "miRNA inhibitor"). As used herein, a "miRNA inhibitor" is an oligonucleotide that hinders the activity of a miRNA. In embodiments, the miRNA inhibitor includes one or more sequences of the reverse complement of a mature miRNA that are chemically modified to prevent RISC-induced cleavage, enhance binding affinity, provide resistance to nucleolytic degradation, or a combination thereof. In embodiments, the miRNA inhibitor binds to a mature miRNA and chelates endogenous miRNA, making it unavailable for normal function.

[0177] Antagomir

[0178] In embodiments, the hybrid oligonucleotide is an antagomir. Antagomirs are RNA-like oligonucleotides with various modifications for RNA enzyme protection and pharmacological properties such as enhanced tissue and cell uptake. In embodiments, antagomirs silence endogenous miRNAs by forming a duplex comprising the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing.

[0179] Supermir

[0180] In an embodiment, the hybrid oligonucleotide is a supermir. Supermir refers to a single-stranded, double-stranded or partially double-stranded oligomer or a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or a modified form thereof, and its nucleotide sequence is substantially identical to miRNA and is antisense with respect to its target. In an embodiment, supermir includes an oligonucleotide consisting of naturally occurring and non-naturally occurring nucleobases, sugar nucleotide inter-linkages or a combination thereof. In an embodiment, supermir is substantially single-stranded, such as less than about 50% (e.g., less than about 40%, about 30%, about 20%, about 10% or about 5%) of supermir itself forming a duplex. In an embodiment, supermir includes, for example, a hairpin segment at the 3' end, which can self-hybridize and form a duplex region, such as at least about 1, about 2, about 3 or about 4 and less than about 8, about 7, about 6 or a duplex region of nucleotides.

[0181] Immunostimulatory oligonucleotides

[0182] In an embodiment, the hybrid oligonucleotide is an immunostimulatory oligonucleotide. An immunostimulatory oligonucleotide is a single-stranded or double-stranded oligonucleotide that can induce an immune response when administered to a subject. In an embodiment, the immunostimulatory oligonucleotide includes a palindromic sequence that forms a hairpin secondary structure or a CpG motif.

[0183] In embodiments, the immunostimulatory oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide can be unmethylated or methylated. In another embodiment, the immunostimulatory oligonucleotide comprises at least one CpG dinucleotide with methylated cytosine. In embodiments, the immunostimulatory oligonucleotide comprises a single CpG dinucleotide, wherein the cytosine in the CpG dinucleotide is methylated. In embodiments, the immunostimulatory oligonucleotide comprises at least two CpG dinucleotides, wherein at least one cytosine in the CpG dinucleotide is methylated. In embodiments, each cytosine in the CpG dinucleotide is methylated. In embodiments, the immunostimulatory oligonucleotide comprises a plurality of CpG dinucleotides, wherein at least one of the CpG dinucleotides comprises a methylated cytosine.

[0184] Decoy oligonucleotide

[0185] In embodiments, the hybrid oligonucleotide is a decoy oligonucleotide. Because transcription factors recognize their relatively short binding sequences even in the absence of surrounding genomic DNA, short oligonucleotides with a common binding sequence of a specific transcription factor can be used as a tool for manipulating gene expression in living cells. This strategy involves the intracellular delivery of such "decoy oligonucleotides", which are then recognized and bound by the target factor. The decoy occupies the DNA binding site of the transcription factor so that the transcription factor cannot subsequently bind to the promoter region of the target gene. The decoy can be used as a therapeutic agent to suppress the expression of genes activated by transcription factors, or to raise genes that are suppressed by binding to transcription factors.

[0186] U1 Adaptor

[0187] In an embodiment, the hybrid oligonucleotide is a U1 adapter. The U1 adapter inhibits the poly A site and is a bifunctional oligonucleotide having a target domain complementary to a site in the terminal exon of the target gene and a 'U1 domain' of a U1 smaller nuclear RNA component bound to the U1 snRNP (Goraczniak et al., 2008, Nature Biotechnology, 27 (3), 257-263, which reference is expressly incorporated herein by reference in its entirety). The U1 snRNP is a ribonucleoprotein complex whose main function is to guide the early steps in the formation of the spliceosome by binding to the exon-intron boundary of the pre-mRNA (Brown and Simpson, 1998, Annu Rev Plant Physiol Plant Mol Biol 49: 77-95). The nucleotides 2-11 at the 5' end of the U1 snRNA base pair bind to the 5'ss of the pre-mRNA. In one embodiment, the oligonucleotide of the present invention is a U1 adapter. In one embodiment, the U1 adapter can be administered in combination with at least one other iRNA agent.

[0188] Hybridization site

[0189] The hybridization site of the hybrid oligonucleotide will vary depending on the target nucleic acid sequence and / or the disease being treated. In embodiments, the target nucleic acid sequence is an RNA sequence. In embodiments, the target nucleic acid sequence is in a target mRNA. In embodiments, the target nucleic acid sequence is in a target pre-mRNA. In embodiments, the target nucleic acid sequence is a DNA sequence. In embodiments, the target nucleic acid sequence is a genomic DNA sequence.

[0190] Splice site

[0191] In an embodiment, the hybrid oligonucleotide hybridizes to and spatially blocks at least a portion of a splicing site (e.g., splicing acceptor or splicing donor site) or a splicing element (SE) and / or a cis-acting splicing regulatory element (SRE), thereby redirecting splicing to a hidden or de novo splicing site. In an embodiment, the hybrid oligonucleotide is targeted to a splicing enhancer sequence (e.g., ESE and / or ISE) or a splicing silencer sequence (e.g., ESS and / or ISS) to prevent binding of a trans-acting regulatory splicing factor at the target site and effectively block or promote splicing. In an embodiment, the hybrid oligonucleotide can be designed to perform base pairing on the bases of the splicing regulatory stem loop to strengthen the stem-loop structure.

[0192] Nucleotide repeats

[0193] In an embodiment, the hybrid oligonucleotide targets nucleotide repeats (e.g., trinucleotide repeat expansion, pentanucleotide repeat expansion, or hexanucleotide repeat expansion) in a target nucleic acid sequence, or targets a sequence with nucleotide repeats. In an embodiment, the hybrid oligonucleotide blocks the amplification of nucleotide repeats. In an embodiment, the hybrid oligonucleotide blocks the transcription of nucleotide repeats.

[0194] In embodiments, the hybrid oligonucleotide is complementary to a target nucleic acid sequence having trinucleotide repeat expansion. In embodiments, the hybrid oligonucleotide is hybridized to a trinucleotide repeat expansion of the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide is complementary to about 5 to 10 trinucleotide repeats in the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide is complementary to 5 to 15 trinucleotide repeats in the target nucleic acid sequence or about 5, about 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 trinucleotide repeats in the target nucleic acid sequence.

[0195] In embodiments, the hybrid oligonucleotide is complementary to a trinucleotide repeat, such as a CAG repeat, a CGG repeat, a GCC repeat, a GAA repeat, or a CUG repeat. In embodiments, the target nucleic acid sequence comprises at least 5, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000 trinucleotide repeats (e.g., CAG, CGG, GCC, GAA, or CUG repeats).

[0196] In an embodiment, the hybrid oligonucleotide degrades a trinucleotide repeat. In an embodiment, after the hybrid oligonucleotide binds to the target nucleic acid sequence, the target nucleic acid sequence is degraded by RNase H.

[0197] polyadenylation signal

[0198] In embodiments, the hybrid oligonucleotide targets the polyadenylation signal (PAS) of a target. In embodiments, the hybrid oligonucleotide inhibits polyadenylation of a gene transcript.

[0199] In an embodiment, the hybrid oligonucleotide interacts with (e.g., binds to) a polyadenylation sequence element (PSE), which includes, but is not limited to, a polyadenylation signal (PAS), a cleavage site (CS), and a GU-rich downstream element (DSE). In various embodiments and in various cases, the polyadenylation sequence element includes one or more of the following: an auxiliary upstream element (USE), a G-rich sequence (GRS), an auxiliary downstream element (AUX DSE), and / or a sequence downstream of a core U-rich element (URE) (see, e.g., Chen and Wilusz (1998) Nuc. Acid. Rec. 1998 26(12): 2891-2898).

[0200] In an embodiment, PAS is an adenosine-rich hexamer sequence comprising a canonical AATAAA hexamer or variants that differ by a single nucleotide (e.g., AAUAAA, AUUAAA, UAUAAA, AGUAAA, AAGAAA, A AUAUA, AAUACA, CAUAAA, GAUAAA, CAUAAA, GAUAAA A, AAUGAA, UUUAAA, ACUAAA, AAUAGA, AAAAAG, AA AACA, GGGGCU; Marsollier et al. Int. J. Mol. Sci., (2018), 19, 1347, doi: 10.3390 / ijms19051347; Beaudoing et al. Genome Res. (2000), 10, 1001-1010; and Tian, ​​B. et al., Nucleic Acids Res. (2005), 33, 201-212). Hexamer sequences exist at different frequencies, with AAUAAA and AUUAAA being the most frequent (see, supra). PAS is usually found upstream of CS. The hexamer sequence of PAS serves as a binding site for the cleavage and polyadenylation specificity factor (CPSF). PAS can also be determined by the presence of other auxiliary elements, such as upstream U-rich elements (USE) (see Tian et al., Nuc. Acid. Res. (2005), 33 (1): 201-212 and Neve et al. (2017) RNA Biolog y, 2017, 14 (7): 865-890.

[0201] DSE is a U-rich or U / G-rich element that acts as a binding site for the cleavage stimulating factor (CstF). DSE is usually found downstream of the CS. The DSE may be followed by a stretch of three or more uracil bases present downstream of the CS, often within 20 to 40 nucleotides of the CS. In mammals, CA and UA are the most frequent dinucleotides before the cleavage site (CS), although the actual cleavage site is known to be heterogeneous.

[0202] CPSF and CstF are two multi-subunit complexes that cooperate with each other and with two other factors (cleavage factors I and II) to cleave mRNA sequences. The single-subunit enzyme poly(A) polymerase (PAP) is also involved in the cleavage of most pre-mRNAs, as is RNA polymerase II. CPSF and PAP, together with poly(A) binding protein II and cleavage stimulating factor (CstF), participate in the addition of poly(A) tails (Takagaki and Manley, Mol Cell Biol. (2000), 20(5): 1515-1525).

[0203] Methods for identifying polyadenylation sequence elements are known and may include, but are not limited to, methods described, for example, by the following references: Tian et al., Nuc. Acid. Res. (2005) 33(1): 201-212; Beaudoing, et al., Genome Res. (2000), 10, 1001-1010; Marsollier et al., Int. J. Mol. Sci. (2018), 19, 1347, doi: 10.3390 / ijms19051347; Chen, Molec. Therapy (2016), 24(8) 1405-1411; Venkataraman et al., Genes and Dev. (2005) 19: 1315-1327; Nourse et al., Biomol. ecules (2000), 10(915) doi:10.3390 / biom10060915; and Vickers et al. Nucleic Acids Research (2001) 29(6) 1293-1299.

[0204] Preparation method

[0205] The present disclosure relates to methods for synthesizing hybrid oligonucleotides and cleaving hybrid oligonucleotides from solid supports. The solid support can be a general solid support. The P(V)PMO nucleotides can be used with other resins commonly used for PMO synthesis.

[0206] The synthesis proceeds from the 6' or 5' end towards the 3' end and involves PMO 6'-chlorophosphoramidate P(V), PMO 6'-β-cyanoethylimidate P(III) and reverse 5'-β-cyanoethylimidate P(III) based on 2'-deoxyribose, 2'-substituted ribose LNA or other nucleotides.

[0207] The transition between the 6' PMO flank and the spacer is achieved by reacting the 5' phosphoroimidate with the N3 of the morpholino ring in the presence of an activating agent and thiolation. The result is a negatively charged N3'>P5' phosphorothioamidate linker.

[0208] The transition between the spacer and the 3' PMO flank is achieved by coupling the PMO 6'-β-cyanoethylimidate P(III) to the 3' hydroxyl group in the presence of an activating agent and sulfonation or oxidation. This results in the formation of a phosphorothioate PS or a phosphodiester PO linker.

[0209] If a PMO is subsequently introduced into the sequence, labile protecting groups on the nucleobases should be avoided. The adenosine and cytidine protecting groups can be N-benzoyl, and the guanosine protecting group can be N-isobutyryl.

[0210] The phosphate protecting group can be O-cyanoethyl, O-methyl, O-ethyl, O-benzyl, O-allyl.

[0211] If the PMO is introduced later in the sequence, then cap A contains isobutyric anhydride, pivalic anhydride, or benzoic anhydride, but not acetic anhydride.

[0212] The method of making oligonucleotides comprises assembling P(III) and P(V) nucleotide building blocks in the 6' to 3' or 5' to 3' direction on a support and using PMO 6'-phosphorimidate P(III) or phosphoramidate P(V) as the first nucleotide from the support.

[0213] The present disclosure also relates to a method of preparing an oligonucleotide comprising assembling P(III) and P(V) nucleotide building blocks in the 6' to 3' or 5' to 3' direction on a support, and using PMO 6'-phosphoramidate P(III) or phosphoramidate P(V) as the second nucleotide from the support, wherein the first nucleotide is reverse DNA (PIII).

[0214] The present disclosure also relates to a method for preparing an oligonucleotide, the method comprising:

[0215] a first nucleotide sequence comprising a first 5' or 6' end and a first 3' end, wherein the terminal 3' nucleic acid residue of the first nucleotide sequence is a morpholino nucleotide analog;

[0216] (ii) a second nucleotide sequence comprising a second 5' or 6' end and a second 3' end, wherein the terminal 5' nucleic acid residue of the second nucleotide sequence is a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof; and

[0217] (iii) at least one phosphorothioate linkage connecting the terminal 3' nucleic acid residue of the first nucleotide sequence and the terminal 5' nucleic acid residue of the second nucleotide sequence.

[0218] In an embodiment, the support may be a polystyrene resin.

[0219] In an embodiment, the PMO 6'-phosphoramidate P(III) can be the first nucleotide from the vector. The PMO 6'-phosphoramidate P(V) can be the first nucleotide from the vector.

[0220] In embodiments, the oligonucleotide prepared by the method may comprise a thiophosphate, a phosphodiester linkage and / or a phosphorodiamidate linkage and at least one thiophosphoramidate linkage. In embodiments, the oligonucleotide prepared by the method may comprise a thiophosphate, a phosphodiester linkage and / or a phosphorodiamidate linkage and a thiophosphoramidate linkage.

[0221] In an embodiment, the oligonucleotide prepared by the method may comprise a phosphorodiamidate linkage and at least one phosphorothioate linkage ( Figure 5 ). In an embodiment, the oligonucleotide prepared by the method has only phosphorodiamidate and phosphorothioate linkages. In an embodiment, the oligonucleotide prepared by the method may comprise phosphorodiamidate and phosphorothioate linkages and at least one phosphorothioate linkage.

[0222] In embodiments, phosphorothioate can connect two reverse DNA amidoesters. In embodiments, phosphorothioate can connect two reverse RNA amidoesters. In embodiments, phosphorothioate can connect reverse DNA amidoester and reverse DNA amidoester. In embodiments, phosphorothioate connects reverse DNA amidoester and PMO (III).

[0223] In embodiments, a phosphorodiamidate may link PMO (III) and PMO (V). In embodiments, a phosphorodiamidate may link two PMO (V).

[0224] In some embodiments, the phosphorothioate can link PMO (V) to PMO (III). In some embodiments, the phosphorothioate can link PMO (III) to inverse DNA P (III).

[0225] In some embodiments, a phosphodiester can link the reverse RNA and the reverse DNA. In some embodiments, a phosphodiester can link the two reverse RNAs.

[0226] In embodiments, a deoxyribonucleotide analog, two or more ribonucleotide analogs may precede the PMO.

[0227] The method can include coupling a first reverse amidite to a secondary amino group of the morpholino ring to form a phosphorothioate linkage.

[0228] Thus, the method adds (PIII) and P(V) building blocks, wherein the building blocks are PMO, reverse DNA, reverse RNA and reverse LNA.

[0229] In an embodiment, the method may further comprise adding at least one P(III) building block. In an embodiment, the method may further comprise adding at least two P(III) building blocks. In an embodiment, the building blocks may be added consecutively or may be added as a series of building blocks.

[0230] In embodiments, the P(III) building blocks may be continuous.

[0231] In embodiments, the method may further comprise adding at least one P(V) building block.

[0232] In an embodiment, the method may include adding at least two P(V) building blocks. The P(V) building blocks may be continuous. In an embodiment, at least two P(III) building blocks may be followed by at least two P(V) building blocks. In an embodiment, at least two P(V) building blocks may be followed by at least two P(III) building blocks. In an embodiment, at least one P(III) building block and one P(V) building block may alternate to form a P(III)-P(V)-P(III) or P(V)-P(III)-P(V) motif. In an embodiment, the P(III) building blocks may be identical. In an embodiment, the P(III) building blocks may be different. In an embodiment, the P(V) building blocks may be identical. In an embodiment, the P(V) building blocks may be different. In an embodiment, the building blocks may be added continuously or may be added as a group of building blocks.

[0233] In an embodiment, the method may include deprotecting the 3'-terminal secondary amino group of the first DNA monomer. The method may also include neutralizing the deprotected 3'-terminal secondary amino group of the first DNA monomer.

[0234] In embodiments, the method may include further adding one or more additional DNA nucleotides to the first DNA nucleotide, wherein the two or more DNA nucleotides are linked via a phosphorothioate (PS) linkage.

[0235] In embodiments, the method may further comprise adding a second PMO 6'-phosphorimidate P(III) to the 3' terminal DNA nucleotide of the hybrid oligonucleotide.

[0236] In embodiments, the method may provide an oligonucleotide that may comprise a gapmer comprising:

[0237] (i) a 6' flank comprising two or more morpholino nucleotide analogs, or a 5' flank comprising two or more ribonucleotides or ribonucleotide analogs;

[0238] (ii) a spacer region comprising 5 or more deoxyribonucleotides or deoxyribonucleotide analogs; and

[0239] (iii) a 3' flank comprising two or more morpholino nucleotide analogues, or a 3' flank comprising two or more ribonucleotides or ribonucleotide analogues.

[0240] In embodiments, the method may further comprise extending the oligonucleotide by adding one or more additional DNA monomers to the first DNA monomer, the one or more additional DNA monomers being linked via a phosphorothioate bond (DNA-PS).

[0241] In embodiments, the method may further comprise adding PMO 6'-phosphorimidate P(III) to the 3' end of the oligonucleotide.

[0242] In an embodiment, if all cytidine-based nucleotides in the 5' flank and gap can be N-benzoyl protected, and cap A is based on isobutyric anhydride, pivalic anhydride, or benzoic anhydride instead of acetic anhydride, then a 3'-PMO wing can be added.

[0243] In embodiments, the methods of the present disclosure provide means for synthesizing the compounds disclosed herein.

[0244] In embodiments, the synthesis of oligonucleotides can be automated. In embodiments, the synthesis of oligonucleotides can be performed manually. In embodiments, the synthesis of oligonucleotides can be performed via a combination of automation and manual synthesis. In embodiments, the synthesis of oligonucleotides can be performed on a solid support. In embodiments, the synthesis of oligonucleotides can be performed in liquid phase. In embodiments, the synthesis of oligonucleotides can be a combination of synthesizing on a solid support and in a solid phase.

[0245] In an embodiment, oligonucleotides are assembled by stepwise coupling of individual monomers. In an embodiment, oligonucleotides are assembled by coupling connected monomers to individual monomers. In an embodiment, oligonucleotides are assembled by coupling a first group of connected monomers to a second group of connected monomers. Any method for the coupled reaction of synthetic oligonucleotides can be used in the disclosed method.

[0246] Hybrid oligonucleotide design

[0247] The design of heterozygous oligonucleotide will depend on the sequence targeted. Those skilled in the art can design, synthesize and screen compounds of different core base sequences to identify sequences producing desired activity. In embodiments, heterozygous oligonucleotide includes one or more modified nucleotides, one or more modified nucleotides inter-linked or a combination thereof. In embodiments, heterozygous oligonucleotide can be but is not limited to a spacer, a splicing switch oligonucleotide, the sense strand of siRNA, the antisense strand of siRNA, a deoxyribozyme, a steric blocking antisense oligonucleotide (ASO), a DNA enzyme, an RNA enzyme, an aptamer, an immunomodulatory oligonucleotide or an antagomir.

[0248] In an embodiment, a hybrid oligonucleotide comprises one or more modified nucleotides. In an embodiment, all nucleotides in a hybrid oligonucleotide are modified. In an embodiment, one or more nucleotides comprise a modified nucleobase. In an embodiment, one or more nucleotides comprise a modified sugar. In an embodiment, a hybrid oligonucleotide comprises one or more modified internucleotide linkages.

[0249] In embodiments, the hybrid oligonucleotide comprises: (i) at least one P (III) morpholino nucleotide analog; (ii) at least one P (V) morpholino nucleotide analog; (iii) at least one P (III) ribonucleotide, at least one P (III) deoxyribonucleotide analog or a combination thereof; (iv) at least one diaminophosphoester linkage; and (iv) at least one thioimidophosphoester linkage. In embodiments, each upstream nucleotide adjacent to each P (V) morpholino nucleotide analog is a P (III) or P (V) morpholino nucleotide analog. In embodiments, the hybrid oligonucleotide comprises 2 or more continuous P (V) morpholino nucleotide analogs connected by diaminophosphoester linkages. In embodiments, the hybrid oligonucleotide comprises 2 to 20, 3 to 10 or 4 to 6 continuous P (V) morpholino nucleotide analogs connected by diaminophosphoester linkages. In an embodiment, the hybrid oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive P(V) morpholino nucleotide analogs linked by phosphorodiamidate linkages. In an embodiment, any P(V) morpholino nucleotide analog is linked by a phosphorothioate aminothioate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0250] In an embodiment, the hybrid oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the hybrid oligonucleotide comprises one or more nucleotide residues, and the one or more nucleotide residues comprise a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof or a combination thereof. In an embodiment, the deoxyribonucleotide analog or the ribonucleotide analog is selected from: a P(III) DNA analog, a reverse P(IIII) DNA analog, a P(III) RNA analog, a reverse P(III) RNA analog, or a combination thereof. In an embodiment, the first nucleotide sequence comprises a 2'-modified ribonucleotide analog selected from: 2'O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0251] In embodiments, the hybrid oligonucleotide comprises at least one internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, thiophosphoramidate or thiophosphate. In embodiments, the hybrid oligonucleotide comprises at least one internucleotide linkage selected from the group consisting of methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, the hybrid oligonucleotide comprises an internucleotide linkage selected from the group consisting of phosphorodiamidate, phosphorothioate, phosphorothioate (PS) and phosphorylguanidine linkage. In embodiments, the hybrid oligonucleotide comprises at least one thiophosphate linkage.

[0252] In an embodiment, the hybrid oligonucleotide comprises: (i) a first nucleotide sequence comprising a first 5' or 6' end and a first 3' end, wherein the terminal 3' nucleic acid residue of the first nucleotide sequence is a morpholino nucleotide analog; (ii) a second nucleotide sequence comprising a second 5' or 6' end and a second 3' end, wherein the terminal 5' nucleic acid residue of the second nucleotide sequence is a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof; and (iii) at least one thiophosphoramidate linkage, wherein the at least one thiophosphoramidate linkage connects the terminal 3' nucleic acid residue of the first nucleotide sequence and the terminal 5' nucleic acid residue of the second nucleotide sequence. In an embodiment, two or more deoxyribonucleotides or analogs, two or more ribonucleotides or analogs thereof, or a combination thereof are each connected by an internucleotide linkage.

[0253] In an embodiment, the hybrid oligonucleotide comprises 10 to 50 nucleotides, 15 to 30 nucleotides, or 20 to 30 nucleotides. In an embodiment, the hybrid oligonucleotide comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0254] In embodiments, the first nucleotide sequence comprises two or more morpholino nucleotide analogs. In embodiments, the first nucleotide sequence comprises 2 to 20, 3 to 10 or 4 to 6 morpholino nucleotide analogs. In embodiments, the first nucleotide sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 morpholino nucleotide analogs. In embodiments, the first nucleotide sequence comprises 2 morpholino nucleotide analogs. In embodiments, the first nucleotide sequence comprises 3 morpholino nucleotide analogs. In embodiments, the first nucleotide sequence comprises 4 morpholino nucleotide analogs. In embodiments, the first nucleotide sequence comprises 5 morpholino nucleotide analogs.

[0255] In an embodiment, the morpholino nucleotide analog is a P(III)PMO or a P(V)PMO. In an embodiment, the morpholino nucleotide analog is a P(III)PMO. In an embodiment, the P(III)PMO is selected from: P(III)PMO-C, P(III)PMO-G, P(III)PMO-A, P(III)PMO-T, or a P(III)PMO with a non-naturally occurring nucleobase. In an embodiment, the morpholino nucleotide analog is a P(V)PMO. In an embodiment, the (PV)PMO comprises: P(V)PMO-C, P(V)PMO-G, P(V)PMO-A, P(V)PMO-T, or a P(V)PMO with a non-naturally occurring nucleobase.

[0256] In embodiments, 2 or more continuous morpholino oligonucleotide analogs in the first nucleotide sequence are connected by diaminophosphoester linkages. In embodiments, 2 to 20, 3 to 10 or 4 to 6 continuous morpholino oligonucleotide analogs in the first nucleotide sequence are connected by diaminophosphoester linkages. In embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 continuous morpholino oligonucleotide analogs in the first nucleotide sequence are connected by diaminophosphoester linkages. In embodiments, the terminal 3' nucleic acid residues of the first nucleic acid sequence are P (III) morpholino nucleotide analogs. In embodiments, the terminal 3' nucleic acid residues of the first nucleic acid sequence are P (V) morpholino nucleotide analogs. In embodiments, the terminal 3' nucleic acid residues of the first nucleic acid sequence are attached to a solid substrate.

[0257] In an embodiment, the first nucleotide sequence of the hybrid oligonucleotide comprises one or more nucleotide residues, and the one or more nucleotide residues comprise deoxyribonucleotides or analogs thereof or ribonucleotides or analogs thereof or combinations thereof. In an embodiment, the deoxyribonucleotide analogs or ribonucleotide analogs are selected from: P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs or combinations thereof. In an embodiment, the first nucleotide sequence of the hybrid oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the first oligonucleotide sequence of the hybrid oligonucleotide comprises one or more nucleotide residues, and the one or more nucleotide residues comprise deoxyribonucleotides or analogs thereof or ribonucleotides or analogs thereof or combinations thereof. In an embodiment, the deoxyribonucleotide analogs or ribonucleotide analogs are selected from: P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs or combinations thereof. In an embodiment, the first nucleotide sequence comprises a 2'-modified ribonucleotide analog selected from the group consisting of 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0258] In an embodiment, the first oligonucleotide sequence of a hybrid oligonucleotide comprises at least one internucleotide linkage selected from the following: phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, diaminophosphorothioate, phosphoramidite or thiophosphate. In an embodiment, the first nucleotide sequence of a hybrid oligonucleotide comprises at least one internucleotide linkage selected from the following: methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethyl trap (-CH2-N(CH3)-N(CH3)-). In an embodiment, the first oligonucleotide sequence of a hybrid oligonucleotide comprises an internucleotide linkage selected from the following: diaminophosphorothioate, phosphoramidite, phosphorothioate (PS) and phosphorylguanidine linkage. In an embodiment, the first oligonucleotide sequence of a hybrid oligonucleotide comprises at least one phosphorothioate linkage.

[0259] In an embodiment, the second nucleotide sequence of the hybrid oligonucleotide comprises one or more nucleotide residues, and the one or more nucleotide residues comprise deoxyribonucleotides or analogs thereof or ribonucleotides or analogs thereof or combinations thereof. In an embodiment, the deoxyribonucleotide analogs or ribonucleotide analogs are selected from: P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs or combinations thereof. In an embodiment, the second nucleotide sequence of the hybrid oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the second oligonucleotide sequence of the hybrid oligonucleotide comprises one or more nucleotide residues, and the one or more nucleotide residues comprise deoxyribonucleotides or analogs thereof or ribonucleotides or analogs thereof or combinations thereof. In an embodiment, the deoxyribonucleotide analogs or ribonucleotide analogs are selected from: P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs or combinations thereof. In an embodiment, the first nucleotide sequence comprises a 2'-modified ribonucleotide analog selected from the group consisting of 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0260] In an embodiment, the second nucleotide sequence comprises one or more morpholino nucleotide analogs. In an embodiment, the morpholino nucleotide analog is a P(III)PMO. In an embodiment, the P(III)PMO is selected from: P(III)PMO-C, P(III)PMO-G, P(III)PMO-A, P(III)PMO-T or a P(III)PMO with a non-naturally occurring nucleobase. In an embodiment, the morpholino nucleotide analog is a P(V)PMO. In an embodiment, the (PV)PMO comprises: P(V)PMO-C, P(V)PMO-G, P(V)PMO-A, P(V)PMO-T or a P(V)PMO with a non-naturally occurring nucleobase.

[0261] In embodiments, the second oligonucleotide sequence of the hybrid oligonucleotide comprises at least one internucleotide linkage selected from the following: phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, diaminophosphoroester, thiophosphoramidate or thiophosphate. In embodiments, the second nucleotide sequence of the hybrid oligonucleotide comprises at least one internucleotide linkage selected from the following: methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethyl trap (-CH2-N(CH3)-N(CH3)-). In embodiments, the second oligonucleotide sequence of the hybrid oligonucleotide comprises an internucleotide linkage selected from the following: diaminophosphoroester, thiophosphoramidate, thiophosphate (PS) and phosphorylguanidine linkage. In embodiments, the second oligonucleotide sequence of the hybrid oligonucleotide comprises at least one thiophosphate linkage.

[0262] In embodiments, less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the internucleotide linkages in the hybrid oligonucleotide have a negative charge. In embodiments, less than less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the internucleotide linkages in the first oligonucleotide sequence of the hybrid oligonucleotide have a negative charge. In embodiments, less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the internucleotide linkages in the second oligonucleotide sequence of the hybrid oligonucleotide have a negative charge. In embodiments, the hybrid oligonucleotide comprises less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 charged internucleotide linkages.

[0263] Hybrid spacer design

[0264] In an embodiment, the hybrid oligonucleotide is a hybrid gapmer oligonucleotide.In an embodiment, the hybrid gapmer oligonucleotide comprises: (i) a first nucleotide sequence comprising a 5' flank; (ii) a second oligonucleotide sequence comprising a gapper oligonucleotide sequence; and (iii) a 3' flank.

[0265] In an embodiment, the hybrid spacer polymer oligonucleotide comprises at least two continuous morpholino nucleotide analogs connected via neutral diaminophosphorothioate linkages. Each continuous morpholino nucleotide analog in the hybrid spacer polymer oligonucleotide introduces n-1 neutral linkages (wherein the number of nucleotides in the n=hybrid spacer polymer oligonucleotide). In an embodiment, the 5' and 3' flanks each comprise at least 2 or 3 morpholino nucleotide analogs. Although not wishing to be limited by theory, it is believed that the morpholino nucleotide analogs in the flank regions improve oligonucleotide stability and binding affinity to target nucleic acid sequences (e.g., target RNA).

[0266] In an embodiment, the 5' flank comprises 2 to 20 nucleotides. In an embodiment, the 5' flank comprises at least one P(V) morpholino nucleotide analog. In an embodiment, the 5' flank comprises at least one P(III) ribonucleotide. It should be noted that the 5' flank may also be referred to as the 6' flank in order to explain the 6-membered morpholino ring.

[0267] In embodiments, the 5' flank comprises 2 to 20, 2 to 10, or 2 to 5 morpholino nucleotide analogs. In embodiments, the 5' flank comprises 2 to 20, 2 to 10, or 2 to 5 consecutive morpholino nucleotide analogs. In embodiments, the 5' flank comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 morpholino nucleotide analogs. In embodiments, the 5' flank comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive morpholino nucleotide analogs.

[0268] In embodiments, 2 or more morpholino nucleotide analogs of the 5' flank are connected by diaminophosphoester linkages. In embodiments, 2 to 20, 2 to 10 or 2 to 5 morpholino nucleotide analogs of the 5' flank are connected by diaminophosphoester linkages. In embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 morpholino nucleotide analogs of the 5' flank are connected by diaminophosphoester linkages.

[0269] In an embodiment, the 5' flank of the hybrid spacer oligonucleotide comprises one or more nucleotide residues comprising a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof or a combination thereof. In an embodiment, the deoxyribonucleotide analog or the ribonucleotide analog is selected from: a P(III) DNA analog, a reverse P(IIII) DNA analog, a P(III) RNA analog, a reverse P(III) RNA analog, or a combination thereof. In an embodiment, the 5' flank of the hybrid spacer oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the 5' flank comprises a 2' modified ribonucleotide analog selected from the group consisting of 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0270] In embodiments, the 5' flank of the hybrid spacer polymer oligonucleotide comprises at least one internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, diaminophosphorothioate, phosphoramidite, or thiophosphate. In embodiments, the 5' flank of the hybrid spacer polymer oligonucleotide comprises at least one internucleotide linkage selected from the group consisting of methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, the 5' flank of the hybrid spacer polymer oligonucleotide comprises an internucleotide linkage selected from the group consisting of diaminophosphorothioate, phosphoramidite, phosphorothioate (PS), and phosphorylguanidine linkage.

[0271] In embodiments, the hybrid spacer oligonucleotide comprises: a 5' flank comprising a morpholino nucleotide analog connected by a diaminophosphoester linkage; a central spacer region comprising a DNA nucleotide or a DNA nucleotide analog connected by a thiophosphate linkage; and a 3' flank comprising a morpholino nucleotide analog connected by a diaminophosphoester linkage (6'-PMO-DNA-PMO-3'). In embodiments, the hybrid spacer oligonucleotide comprises a structure having the following: a 5' flank comprising a morpholino nucleotide analog connected by a diaminophosphoester linkage; a central spacer region comprising a DNA nucleotide or a DNA nucleotide analog connected by a thiophosphate linkage; and a 3' flank comprising an RNA nucleotide or an RNA nucleotide analog, including, for example, a 2' modified RNA nucleotide analog (6'-PMO-DNA-RNA-3'). In an embodiment, the hybrid spacer oligonucleotide comprises: a 3' flank comprising RNA nucleotides or RNA nucleotide analogs, including, for example, 2' modified RNA nucleotide analogs; a central spacer region comprising DNA nucleotides or DNA nucleotide analogs linked by phosphorothioate linkages; and a 5' flank comprising a morpholino nucleotide analog linked by a phosphorodiamidate linkage (5'-RNA-DNA-PMO-3').

[0272] In an embodiment, the hybrid spacer oligonucleotide comprises a phosphorothioate linkage between the 5' flank and the central spacer oligonucleotide. In an embodiment, the 5' or 3' flank may include a variety of nucleotide analogs, including but not limited to morpholino nucleotide analogs, 2'-O-methyl (2'-OMe) nucleotide analogs, 2'-O-methoxyethyl (2'-OMOE) nucleotide analogs, locked nucleic acids (LNA), or combinations thereof.

[0273] In an embodiment, the spacer oligonucleotide sequence comprises at least 8 deoxyribonucleotides or deoxyribonucleotide analogs. In an embodiment, the spacer oligonucleotide sequence comprises at least 8 consecutive deoxyribonucleotides or deoxyribonucleotide analogs. Although not wishing to be limited by theory, it is believed that the spacing size of at least 8 deoxyribonucleotides or deoxyribonucleotide analogs is ideal for RNase H activity.

[0274] In embodiments, less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the internucleotide linkages in a hybrid spacer polymer oligonucleotide have a negative charge. In embodiments, less than less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the internucleotide linkages in the 5′ flank of a hybrid spacer polymer oligonucleotide have a negative charge. In embodiments, less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the internucleotide linkages in the spacer oligonucleotide sequence of a hybrid spacer polymer oligonucleotide have a negative charge. In embodiments, less than less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of the internucleotide linkages in the 3' flank of the hybrid gapper oligonucleotide have a negative charge. In embodiments, the hybrid gapper oligonucleotide comprises less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 charged internucleotide linkages.

[0275] In an embodiment, the terminal residue at the first 5' (or 6') end of the 5' (or 6') flank comprises PMO 6'-phosphorimidate P(III).

[0276] In an embodiment, the spacer oligonucleotide sequence comprises 8 to 20 deoxyribonucleotides or ribonucleotide analogs. In an embodiment, the spacer oligonucleotide sequence comprises at least 8 consecutive deoxyribonucleotides or deoxyribonucleotide analogs. In an embodiment, the spacer oligonucleotide sequence comprises 8 to 20 consecutive deoxyribonucleotides or ribonucleotide analogs.

[0277] In an embodiment, the spacer sequence of the hybrid spacer oligonucleotide comprises one or more nucleotide residues, and the one or more nucleotide residues comprise a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof or a combination thereof. In an embodiment, the deoxyribonucleotide analog or the ribonucleotide analog is selected from: a P(III) DNA analog, a reverse P(IIII) DNA analog, a P(III) RNA analog, a reverse P(III) RNA analog or a combination thereof. In an embodiment, the spacer sequence of the hybrid spacer oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the spacer sequence comprises a 2'-modified ribonucleotide analog selected from the group consisting of 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0278] In embodiments, the spacer sequence of the hybrid spacer oligonucleotide comprises at least one internucleotide linkage selected from the following: phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, diaminophosphorothioate, phosphoramidite or thiophosphate. In embodiments, the spacer sequence of the hybrid spacer oligonucleotide comprises at least one internucleotide linkage selected from the following: methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethyl trap (-CH2-N(CH3)-N(CH3)-). In embodiments, the spacer sequence of the hybrid spacer oligonucleotide comprises an internucleotide linkage selected from the following: diaminophosphorothioate, phosphoramidite, phosphorothioate (PS) and phosphorylguanidine linkage. In embodiments, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the deoxyribonucleotide analogs of the spacer oligonucleotide sequence are linked by phosphorothioate linkages. In embodiments, the spacer sequence comprises at least two 2' modified nucleotides linked by a phosphorothioate linkage.

[0279] In embodiments, the 3' flank comprises 2 to 20, 2 to 10, or 2 to 5 morpholino nucleotide analogs. In embodiments, the 3' flank comprises 2 to 20, 2 to 10, or 2 to 5 consecutive morpholino nucleotide analogs. In embodiments, the 3' flank comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 morpholino nucleotide analogs. In embodiments, the 3' flank comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive morpholino nucleotide analogs.

[0280] In embodiments, 2 or more morpholino nucleotide analogs of the 3' flank are connected by diaminophosphoester linkages. In embodiments, 2 to 20, 2 to 10 or 2 to 5 morpholino nucleotide analogs of the 3' flank are connected by diaminophosphoester linkages. In embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 morpholino nucleotide analogs of the 3' flank are connected by diaminophosphoester linkages.

[0281] In an embodiment, the 3' flank of the hybrid spacer oligonucleotide comprises one or more nucleotide residues, the one or more nucleotide residues comprising a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof or a combination thereof. In an embodiment, the deoxyribonucleotide analog or the ribonucleotide analog is selected from: a P(III) DNA analog, a reverse P(IIII) DNA analog, a P(III) RNA analog, a reverse P(III) RNA analog or a combination thereof. In an embodiment, the 3' flank of the hybrid spacer oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the 3' flank comprises a 2' modified ribonucleotide analog selected from the group consisting of 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-MOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0282] In an embodiment, the 3' flank of the hybrid spacer polymer oligonucleotide comprises at least one internucleotide linkage selected from the following: phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, diaminophosphorothioate, phosphoramidite or thiophosphate. In an embodiment, the 3' flank of the hybrid spacer polymer oligonucleotide comprises at least one internucleotide linkage selected from the following: methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In an embodiment, the 3' flank of the hybrid spacer polymer oligonucleotide comprises an internucleotide linkage selected from the following: diaminophosphorothioate, phosphoramidite, phosphorothioate (PS) and phosphorylguanidine linkage.

[0283] In an embodiment, the terminal residue at the second 5' or 6' end of the 3' flank comprises PMO 6'-phosphorimidate P(III).

[0284] In an embodiment, the spacer oligonucleotide sequence is linked to the 3' flank via a phosphorothioate linkage.

[0285] In an embodiment, the hybrid gapped polymer oligonucleotide comprises 10 or less morpholino nucleotide analogs. In an embodiment, the hybrid gapped polymer oligonucleotide comprises 7 or less morpholino nucleotide analogs. In an embodiment, the hybrid gapped polymer oligonucleotide comprises 2 to 10 or 3 to 7 morpholino nucleotide analogs.

[0286] Steric blocking and splice switch oligonucleotides

[0287] In an embodiment, the hybrid oligonucleotide is a steric blocking oligonucleotide.In an embodiment, the steric blocking oligonucleotide is a splice switch oligonucleotide.

[0288] Although not wishing to be bound by theory, it is believed that the PMO sequence in a steric blocking oligonucleotide can modulate the binding energy of the oligonucleotide to the target nucleic acid sequence. It is also believed that the PMO sequence in a steric blocking oligonucleotide can reduce the effect of the oligonucleotide on the innate immune system, or change the biodistribution, pharmacological properties or cellular distribution of the oligonucleotide.

[0289] In embodiments, the length of the steric blocking oligonucleotide comprises 10 to 50, 15 to 40, or 20 to 30 nucleotides. In embodiments, the length of the steric blocking oligonucleotide comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In embodiments, the steric blocking oligonucleotide comprises at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% morpholino nucleotide analogs. In embodiments, the steric blocking oligonucleotide comprises at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% 2'-modified RNA nucleotides.

[0290] In an embodiment, the spatial blocking oligonucleotide comprises at most 25% morpholino nucleotide analogs and at most 75% 2'-modified RNA nucleotides. In an embodiment, the spatial blocking oligonucleotide comprises at most 75% morpholino nucleotide analogs and at most 25% 2'-modified RNA nucleotides. In an embodiment, the spatial blocking oligonucleotide comprises at most 30% morpholino nucleotide analogs and at most 70% 2'-modified RNA nucleotides. In an embodiment, the spatial blocking oligonucleotide comprises at most 70% morpholino nucleotide analogs and at most 30% 2'-modified RNA nucleotides. In an embodiment, the spatial blocking oligonucleotide comprises at most 40% morpholino nucleotide analogs and at most 60% 2'-modified RNA nucleotides. In an embodiment, the spatial blocking oligonucleotide comprises at most 60% morpholino nucleotide analogs and at most 40% 2'-modified RNA nucleotides. In an embodiment, the spatial blocking oligonucleotide comprises at most 50% morpholino nucleotide analogs and at most 50% 2'-modified RNA nucleotides.

[0291] In embodiments, the steric blocking oligonucleotide comprises at least two continuous morpholino nucleotide analogs connected via neutral diaminophosphorothioate linkages. In embodiments, the steric blocking oligonucleotide comprises at least 2 or at least 3 morpholino nucleotide analogs. In embodiments, the steric blocking oligonucleotide comprises at least one thiophosphorothioate linkage between an upstream P (V) morpholino nucleotide analog and a downstream nucleotide that is not a P (V) morpholino nucleotide analog.

[0292] In an embodiment, the steric blocking oligonucleotide comprises one or more nucleotide residues, and the one or more nucleotide residues comprise a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof or a combination thereof. In an embodiment, the deoxyribonucleotide analog or the ribonucleotide analog is selected from: a P(III) DNA analog, a reverse P(IIII) DNA analog, a P(III) RNA analog, a reverse P(III) RNA analog, or a combination thereof. In an embodiment, the steric blocking oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the steric blocking oligonucleotide comprises a 2'-modified ribonucleotide analog selected from:

[0293] 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0294] In an embodiment, the steric blocking oligonucleotide comprises at least one internucleotide linkage selected from the following: phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, diaminophosphorothioate, phosphoramidite or thiophosphate. In an embodiment, the steric blocking oligonucleotide comprises at least one internucleotide linkage selected from the following: methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethyl trap (-CH2-N(CH3)-N(CH3)-). In an embodiment, the 5' flank of the hybrid spacer oligonucleotide comprises an internucleotide linkage selected from the following: diaminophosphorothioate, phosphoramidite, phosphorothioate (PS) and phosphorylguanidine linkage.

[0295] In embodiments, the steric blocking oligonucleotide comprises: (i) at least one P(III) morpholino nucleotide analog; (ii) at least one P(V) morpholino nucleotide analog; (iii) at least one P(III) ribonucleotide, at least one P(III) deoxyribonucleotide analog or a combination thereof; (iv) at least one diaminophosphoester linkage; and (iv) at least one thioimidophosphoester linkage. In embodiments, each upstream nucleotide adjacent to each P(V) morpholino nucleotide analog is a P(III) or P(V) morpholino nucleotide analog. In embodiments, the steric blocking oligonucleotide comprises 2 or more continuous P(V) morpholino nucleotide analogs connected by diaminophosphoester linkages. In embodiments, the steric blocking oligonucleotide comprises 2 to 20, 3 to 10 or 4 to 6 continuous P(V) morpholino nucleotide analogs connected by diaminophosphoester linkages. In an embodiment, the steric blocking oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive P(V) morpholino nucleotide analogs linked by phosphorodiamidate linkages. In an embodiment, any P(V) morpholino nucleotide analog is linked by a phosphorothioate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0296] Short interfering RNA (siRNA)

[0297] In an embodiment, the hybrid oligonucleotide forms the sense strand, the antisense strand, or both strands of a short interfering RNA (siRNA). Although not wishing to be bound by theory, it is believed that the presence of a PMO oligonucleotide sequence in the siRNA, particularly at the 5' or 3' end of the sense or antisense strand, reduces enzymatic degradation of the siRNA oligonucleotide. Again, although not wishing to be bound by theory, it is believed that a PMO in the sense strand but not in the antisense strand can introduce chemical asymmetry into the siRNA, thereby allowing the antisense (guide) to be distinguished from the sense (follower) strand and can prevent the sense strand from entering the RNA-induced silencing complex (RISC). It is also believed that including a PMO oligonucleotide sequence in the antisense strand can reduce the toxicity of the siRNA. PMO oligonucleotide sequences in the sense and antisense strands can also improve the pharmacological profile of their siRNAs, or change cellular distribution.

[0298] In embodiments, siRNAs are provided wherein the antisense strand is a hybrid antisense oligonucleotide. In embodiments, the length of the antisense strand is about 10 to about 50, about 15 to about 30, or about 20 to about 30 nucleotides. In embodiments, the length of the hybrid antisense oligonucleotide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In embodiments, the length of the hybrid antisense oligonucleotide of the siRNA is about 21 nucleotides.

[0299] In an embodiment, a siRNA is provided wherein the sense strand is a hybrid sense oligonucleotide. In an embodiment, the length of the sense strand is about 10 to about 50, about 15 to about 30, or about 20 to about 30 nucleotides. In an embodiment, the length of the hybrid sense oligonucleotide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In an embodiment, the length of the hybrid antisense oligonucleotide of the siRNA is about 21 nucleotides.

[0300] In embodiments, the double-stranded region of the siRNA duplex comprises about 10 to about 50, about 15 to about 30, or about 20 to about 30 nucleotides. In embodiments, the length of the double-stranded region of the siRNA duplex is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In embodiments, the length of the double-stranded region of the siRNA duplex is about 21 nucleotides.

[0301] In an embodiment, a double-stranded siRNA duplex includes a single strand or unpaired region at one or both ends of the molecule. In an embodiment, the sense strand of a double-stranded siRNA duplex includes a 5' overhang, a 3' overhang, or both. In an embodiment, the antisense strand of a double-stranded siRNA duplex includes a 5' overhang, a 3' overhang, or both. In an embodiment, one or more overhangs are each independently about 1 to about 5 nucleotides in length, or about 1 to about 3 nucleotides in length, or about 1, 2, 3, 4, or 5 nucleotides in length. The overhang can be the result of one chain being longer than the other, or the result of two chains of the same length being staggered. In an embodiment, the siRNA duplex includes at least one 3' overhang. In an embodiment, the siRNA duplex includes at least one 5' overhang. In an embodiment, the sense and antisense strands of the siRNA duplex both have 3' overhangs. In an embodiment, the sense and antisense strands of the siRNA duplex both have 5' overhangs.

[0302] In embodiments, the sense strand of siRNA is the same length as the antisense strand or is shorter than the antisense strand, for example, 15-16 polymers. In embodiments, morpholino nucleotide analogs are included in the antisense strand, the sense strand or both. In embodiments, morpholino nucleotide analogs are included only in one strand of the siRNA duplex. In embodiments, morpholino nucleotide analogs are included in the sense strand of the siRNA duplex. In embodiments, morpholino nucleotide analogs are included in the antisense strand of the siRNA duplex. In embodiments, morpholino nucleotide analogs are included only in the sense strand of the siRNA duplex.

[0303] In embodiments, the sense strand includes 2' modified nucleotides, such as 2'F, 2'O-MOE, or 2'OMe modifications, at one or both of the 5' and 3' ends of the sense strand. In embodiments, the sense strand includes 2' modified nucleotides at one or more of positions 1, 2, or 3 at the 5' or 3' ends of the sense strand. In embodiments, the siRNA sense strand has 1 to 5 2' modified nucleotides.

[0304] In embodiments, the antisense strand includes 2' modified nucleotides at one or both of the 5' and 3' ends of the antisense strand, such as 2'F, 2'O-MOE or 2'OMe modifications. In embodiments, the antisense strand includes 2' modified nucleotides at one or more of positions 1, 2 or 3 at the 5' end or 3' end of the antisense strand. In embodiments, the siRNA antisense strand has 1 to 5 2' modified nucleotides.

[0305] In embodiments, the sense strand comprises at least two continuous morpholino nucleotide analogs connected via neutral diaminophosphorothioate linkages. In embodiments, the sense strand comprises at least 2 or at least 3 morpholino nucleotide analogs. In embodiments, the sense strand comprises at least one thiophosphorothioate linkage between an upstream P(V) morpholino nucleotide analog and a downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0306] In an embodiment, the sense strand comprises one or more nucleotide residues comprising a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof or a combination thereof. In an embodiment, the deoxyribonucleotide analog or the ribonucleotide analog is selected from: a P(III) DNA analog, a reverse P(IIII) DNA analog, a P(III) RNA analog, a reverse P(III) RNA analog, or a combination thereof. In an embodiment, the sense strand comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the sense strand comprises a 2'-modified ribonucleotide analog selected from:

[0307] 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or a combination thereof.

[0308] In an embodiment, the sense strand comprises at least one internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, thiophosphoramidate or thiophosphate. In an embodiment, the sense strand comprises at least one internucleotide linkage selected from the group consisting of methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In an embodiment, the 5' flank of the hybrid spacer oligonucleotide comprises an internucleotide linkage selected from the group consisting of phosphorodiamidate, phosphorothioate, phosphorothioate (PS) and phosphorylguanidine linkage.

[0309] In an embodiment, the sense strand comprises: (i) at least one P(III) morpholino nucleotide analog; (ii) at least one P(V) morpholino nucleotide analog; (iii) at least one P(III) ribonucleotide, at least one P(III) deoxyribonucleotide analog or a combination thereof; (iv) at least one phosphorodiamidate linkage; and (iv) at least one phosphorothioimidate linkage. In an embodiment, each upstream nucleotide adjacent to each P(V) morpholino nucleotide analog is a P(III) or P(V) morpholino nucleotide analog. In an embodiment, the sense strand comprises 2 or more continuous P(V) morpholino nucleotide analogs connected by phosphorodiamidate linkages. In an embodiment, the sense strand comprises 2 to 20, 3 to 10 or 4 to 6 continuous P(V) morpholino nucleotide analogs connected by phosphorodiamidate linkages. In an embodiment, the sense strand comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive P(V) morpholino nucleotide analogs connected by phosphorodiamidate linkages. In an embodiment, any P(V) morpholino nucleotide analog is connected to any downstream nucleotide that is not a P(V) morpholino nucleotide analog by a phosphorothioate linkage.

[0310] In embodiments, the antisense strand comprises at least two continuous morpholino nucleotide analogs connected via neutral diaminophosphorothioate linkages. In embodiments, the antisense strand comprises at least 2 or at least 3 morpholino nucleotide analogs. In embodiments, the antisense strand comprises at least one thiophosphorothioate linkage between an upstream P (V) morpholino nucleotide analog and a downstream nucleotide that is not a P (V) morpholino nucleotide analog.

[0311] In an embodiment, the antisense strand comprises one or more nucleotide residues, and the one or more nucleotide residues comprise deoxyribonucleotides or analogs thereof or ribonucleotides or analogs thereof or combinations thereof. In an embodiment, the deoxyribonucleotide analogs or ribonucleotide analogs are selected from: P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs or combinations thereof. In an embodiment, the antisense strand comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the antisense strand comprises a 2'-modified ribonucleotide analog selected from: 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA or combinations thereof.

[0312] In an embodiment, the antisense strand comprises at least one internucleotide linkage selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, thiophosphoramidate or thiophosphate. In an embodiment, the antisense strand comprises at least one internucleotide linkage selected from the group consisting of methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbonylcarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In an embodiment, the 5' flank of the hybrid spacer oligonucleotide comprises an internucleotide linkage selected from the group consisting of phosphorodiamidate, phosphorothioate, phosphorothioate (PS) and phosphorylguanidine linkage.

[0313] In embodiments, the antisense strand comprises: (i) at least one P(III) morpholino nucleotide analog; (ii) at least one P(V) morpholino nucleotide analog; (iii) at least one P(III) ribonucleotide, at least one P(III) deoxyribonucleotide analog or a combination thereof; (iv) at least one diaminophosphoester linkage; and (iv) at least one thioimidophosphoester linkage. In embodiments, each upstream nucleotide adjacent to each P(V) morpholino nucleotide analog is a P(III) or P(V) morpholino nucleotide analog. In embodiments, the antisense strand comprises 2 or more continuous P(V) morpholino nucleotide analogs connected by diaminophosphoester linkages. In embodiments, the antisense strand comprises 2 to 20, 3 to 10 or 4 to 6 continuous P(V) morpholino nucleotide analogs connected by diaminophosphoester linkages. In an embodiment, the antisense strand comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive P(V) morpholino nucleotide analogs connected by phosphorodiamidate linkages. In an embodiment, any P(V) morpholino nucleotide analog is connected to any downstream nucleotide that is not a P(V) morpholino nucleotide analog by a phosphorothioate linkage.

[0314] Endosomal escape vector (EEV)

[0315] Provided herein are endosomal escape vehicles (EEVs) that can be used to transport cargo across a cell membrane, e.g., to deliver cargo to the cytosol or nucleus of a cell. The cargo can include a therapeutic moiety (TM), e.g., a hybrid oligonucleotide as described herein. The EEV can include a cell penetrating peptide (CPP), e.g., a cyclic cell penetrating peptide (cCPP), conjugated to an extracyclic peptide (EP). EPs can be interchangeably referred to as regulatory peptides (MPs).

[0316] EP may comprise a sequence of a nuclear localization signal (NLS). EP may be coupled to cargo. EP may be coupled to cCPP. EP may be coupled to cargo and cCPP. The coupling between EP, cargo, cCPP or a combination thereof may be non-covalent or covalent. EP may be attached to the N-terminus of cCPP via a peptide bond. EP may be attached to the C-terminus of cCPP via a peptide bond. EP may be attached to cCPP via the side chain of an amino acid in cCPP. EP may be attached to cCPP via the side chain of lysine, which may be conjugated to the side chain of glutamine in cCPP. EP may be conjugated to the 5' or 3' end of an oligonucleotide cargo. EP may be coupled to a linker. An exocyclic peptide may be conjugated to the amino group of a linker. EP may be coupled to a linker via the C-terminus of EP and may be coupled to cCPP via the side chain on cCPP and / or EP. For example, the EP may comprise a terminal lysine, which can then be coupled to a cCPP containing glutamine via an amide bond. When the EP contains a terminal lysine and the side chain of the lysine is available for attachment to the cCPP, the C-terminus or N-terminus can be attached to a linker on the cargo.

[0317] Exocyclic peptide

[0318] In an embodiment, a compound comprising a hybrid oligonucleotide, a cyclic cell penetrating peptide (cCCP) and an extracyclic peptide (EP) is provided. The extracyclic peptide (EP) may comprise 2 to 10 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues, including all ranges and values ​​therebetween. The EP may comprise 6 to 9 amino acid residues. The EP may comprise 4 to 8 amino acid residues.

[0319] Each amino acid in the exocyclic peptide can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid, because it has a structure similar to that of a natural amino acid, so that it simulates the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and / or amino acid analogs, and the modified amino acids and / or amino acid analogs are not one of 20 common naturally occurring amino acids or rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, allo-isoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, its derivatives or their combinations. These and other amino acids are listed in Table 1 together with the abbreviations used in this article. For example, the amino acid can be A, G, P, K, R, V, F, H, NaI, or citrulline.

[0320] The EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue, and / or at least one amino acid residue comprising a side chain containing a guanidine group or a protonated form thereof. The EP may comprise 1 or 2 amino acid residues, wherein the 1 or 2 amino acid residues comprise a side chain containing a guanidine group or a protonated form thereof. The amino acid residue comprising a side chain containing a guanidine group may be an arginine residue. Throughout this disclosure, a protonated form may refer to a salt thereof.

[0321] EP may comprise at least two, at least three or at least four or more lysine residues. EP may comprise 2 lysine residues. EP may comprise 3 lysine residues. EP may comprise 4 lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group, the protecting group including, for example, trifluoroacetyl (-COCF3), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexyl)ethyl (Dde) or (4,4-dimethyl-2,6-dioxocyclohexyl-1-ylidene-3)-methylbutyl (ivDde). The amino group on the side chain of each lysine residue may be substituted with a trifluoroacetyl (-COCF3). A protecting group may be included to achieve amide conjugation. The protecting group may be removed after EP is conjugated to cCPP.

[0322] EP may contain at least 2 amino acid residues with hydrophobic side chains. The amino acid residue with a hydrophobic side chain may be selected from valine, proline, alanine, leucine, isoleucine and methionine. The amino acid residue with a hydrophobic side chain may be valine or proline.

[0323] EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and / or at least one arginine residue. EP may comprise at least two, at least three or at least four or more lysine residues and / or arginine residues.

[0324] EP can contain 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, K KHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, KKKKR, KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, wherein B is beta-alanine. The amino acids in the EP can have D or L stereochemistry.

[0325] 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. The EP may comprise PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, wherein B is β-alanine. The amino acids in the EP may have D or L stereochemistry.

[0326] 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, wherein B is β-alanine. The amino acids in EP may have D or L stereochemistry.

[0327] The EP may comprise an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. The EP may consist of an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. The EP may comprise an NLS comprising an amino acid sequence PKKKRKV. The EP may consist of an NLS comprising an amino acid sequence PKKK RKV. The EP may comprise an NLS comprising an amino acid sequence selected from the group consisting of NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RM RKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VS RKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDE VAKKKSKK, and RKCLQAGMNLEARKTKK. The EP may be composed of an NLS comprising an amino acid sequence selected from the group consisting of NLSKRPAAIKKAGQAKKKK, PAAKR VKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVEL R, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKK KFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEAR KTKK.

[0328] All exocyclic sequences may also contain an N-terminal acetyl group. Thus, for example, EP may have the following structure: Ac-PKKKRKV.

[0329] Cell Penetrating Peptides (CPP)

[0330] The cell penetrating peptide (CPP) may comprise 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). The cCPP is capable of penetrating the cell membrane. An extracyclic peptide (EP) may be conjugated to the cCPP, and the resulting construct may be referred to as an endosome escape vector (EEV). The cCPP may guide cargo (e.g., a therapeutic moiety (TM), such as a hybrid oligonucleotide as described herein) to penetrate the cell membrane. The cCPP may deliver cargo to the cytosol of the cell. The cCPP may deliver cargo to a cell location where a target (e.g., pre-mRNA) is located. In order to conjugate the cCPP to cargo (e.g., a hybrid oligonucleotide as described herein), at least one bond or lone electron pair on the cCPP may be replaced.

[0331] The total number of amino acid residues in the cCPP is in the range of 6 to 20 amino acid residues, such as 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. The cCPP may comprise 6 to 13 amino acid residues. The cCPP disclosed herein may comprise 6 to 10 amino acids. For example, a cCPP comprising 6-10 amino acid residues may have a structure according to any one of Formulas IA to IE:

[0332]

[0333] Among them, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9 and AA 10 is an amino acid residue.

[0334] cCPP may comprise 6 to 8 amino acids. cCPP may comprise 8 amino acids.

[0335] Each amino acid in cCPP can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid, because it has a structure similar to that of a natural amino acid, so that it simulates the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of 20 common naturally occurring amino acids or rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, allo-isoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, derivatives thereof, or combinations thereof. These and other amino acids are listed in Table 1 together with the abbreviations used herein.

[0336] Table 1. Amino acid abbreviations

[0337]

[0338]

[0339] *Single letter abbreviations: When shown herein in upper case letters, it indicates the L-amino acid form, and when shown herein in lower case letters, it indicates the D-amino acid form.

[0340] The cCPP may comprise 6 to 20 amino acids, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid has no side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acids independently have a side chain comprising an aromatic or heteroaromatic group.

[0341] At least two amino acids may have no side chains or have Or its protonated form of the side chain. As used herein, when no side chain is present, the amino acid has two hydrogen atoms (eg, -CH2-) on the carbon atom or carbon atoms connecting the amine and the formic acid.

[0342] The amino acid without a side chain may be glycine or β-alanine.

[0343] The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least one amino acid may be a glycine, β-alanine or 4-aminobutyric acid residue; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.

[0344] The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least two amino acids may independently be glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.

[0345] The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least three amino acids may independently be glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aromatic or heteroaromatic group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.

[0346] Glycine and related amino acid residues

[0347] cCPP may contain (i) 1, 2, 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 2 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.

[0348] 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.

[0349] cCPP may contain (i) 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.

[0350] 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 may comprise (i) 3 or 4 glycine residues.

[0351] In an embodiment, none of the glycine, β-alanine or 4-aminobutyric acid residues in the cCPP are consecutive. Two or three glycine, β-alanine, 4- or 4-aminobutyric acid residues may be consecutive. Two glycine, β-alanine or 4-aminobutyric acid residues may be consecutive.

[0352] In an embodiment, none of the glycine residues in the cCPP are consecutive. Each glycine residue in the cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be consecutive. Two glycine residues may be consecutive.

[0353] Amino acid side chains with aromatic or heteroaromatic groups

[0354] 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.

[0355] 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.

[0356] The aromatic group may be a 6- to 14-membered aryl group. The aryl group may be a phenyl, naphthyl or anthracenyl group, each of which is optionally substituted. The aryl group may be a phenyl or naphthyl group, 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. The heteroaryl group may be a pyridyl, quinolyl or isoquinolyl group.

[0357] The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be bis(homonaphthylalanine), homonaphthylalanine, naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine, 3-(1,1′-biphenyl-4-yl)-alanine, 3-(3-benzothienyl)-alanine or tyrosine, each of which is optionally substituted with one or more substituents. The amino acids having side chains containing aromatic or heteroaromatic groups may each independently be selected from:

[0358]

[0359] and

[0360] wherein the H at the N-terminus and / or the H at the C-terminus are replaced by a peptide bond.

[0361] The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis(homonaphthylalanine), tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be 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-anthryl)-alanine. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, homophenylalanine, homonaphthylalanine, bis(homonaphthylalanine) or bis(homonaphthylalanine), each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups 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 containing an aromatic group may be a residue of phenylalanine. At least two amino acid residues having a side chain containing an aromatic group may be residues of phenylalanine. Each amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine.

[0362] In an embodiment, none of the amino acids having a side chain comprising an aromatic or heteroaromatic group are consecutive. Two amino acids having a side chain comprising an aromatic or heteroaromatic group may be consecutive. Two consecutive amino acids may have opposite stereochemistry. Two consecutive amino acids may have the same stereochemistry. Three amino acids having a side chain comprising an aromatic or heteroaromatic group may be consecutive. Three consecutive amino acids may have the same stereochemistry. Three consecutive amino acids may have alternating stereochemistry.

[0363] The amino acid residue containing an aromatic or heteroaromatic group can be an L-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a D-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a mixture of a D-amino acid and an L-amino acid.

[0364] The optional substituents may be any atom or group that does not significantly reduce (e.g., reduce by more than 50%) the cytoplasmic delivery efficiency of the cCPP, for example, compared to an otherwise identical sequence without the substituent. The optional substituent may be a hydrophobic substituent or a hydrophilic substituent. The optional substituent may be a hydrophobic substituent. The substituent may increase the solvent accessible surface area (as defined herein) of the hydrophobic amino acid. The substituent may be a halogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, a cycloalkynyl, a heterocyclyl, an aryl, a heteroaryl, an alkoxy, an aryloxy, an acyl, an alkylcarbamoyl, an alkylcarboxamido, an alkoxycarbonyl, an alkylthio, or an arylthio. The substituent may be a halogen.

[0365] Although not wishing to be bound by theory, it is believed that amino acids with aromatic or heteroaromatic groups (i.e., amino acids with side chains comprising aromatic or heteroaromatic groups) having higher hydrophobicity values ​​can improve the cytoplasmic delivery efficiency of cCPPs relative to amino acids with lower hydrophobicity values. Each hydrophobic amino acid can independently have a hydrophobicity value greater than that of glycine. Each hydrophobic amino acid can independently be a hydrophobic amino acid having a hydrophobicity value greater than that of alanine. Each hydrophobic amino acid can independently have a hydrophobicity value greater than or equal to that of phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table B lists the hydrophobicity values ​​of various amino acids as reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. USA 1984; 81(1): 140-144), Engleman et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986; 1986(15): 321-53), Kyte and Doolittle (J. Mol. Biol. 1982; 157(1): 105-132), Hoop and Woods (Proc. Natl. Acad. Sci. USA 1981; 78(6): 3824-3828), and Janin (Nature. 1979; 277(5696): 491-492), each of which is incorporated herein by reference in its entirety. Hydrophobicity can be measured using the hydrophobicity scale reported in Engleman et al.

[0366] Table B. Amino Acid Hydrophobicity

[0367]

[0368]

[0369] Amino acid residues having a side chain containing a guanidine group, a guanidine replacement group or a protonated form thereof

[0370] As used herein, guanidine refers to the following structure:

[0371]

[0372] As used herein, the protonated form of guanidine refers to the following structure:

[0373]

[0374] Guanidine replacement groups refer to those functional groups on the side chain of amino acids that are active in donating and accepting hydrogen bonds to positively charged functional groups or reproducible guanidinium groups at physiological pH or above.

[0375] Although reduced toxicity is associated with a guanidine group or its protonated form, the guanidine replacement group facilitates cellular penetration and delivery of therapeutic agents. The cCPP may comprise at least one amino acid having a side chain comprising a guanidine or guanidinium replacement group. The cCPP may comprise at least two amino acids having side chains comprising a guanidine or guanidinium replacement group. The cCPP may comprise at least three amino acids having side chains comprising a guanidine or guanidinium replacement group.

[0376] The guanidine or guanidinium salt group may be an isostere of guanidine or guanidinium salt. The guanidine or guanidinium salt replacement group may be less basic than guanidine.

[0377] As used herein, a guanidine replacement group refers to or its protonated form.

[0378] The present disclosure relates to a cCPP comprising 4 to 20 amino acid residues, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid residue has no side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acid residues independently have a side chain comprising an aromatic or heteroaromatic group.

[0379] At least two amino acid residues may have no side chains or have Or its protonated form of the side chain. As used herein, when no side chain is present, the amino acid residue has two hydrogen atoms (eg, -CH2-) on the carbon atom or carbon atoms connecting the amine and the formic acid.

[0380] The cCPP may comprise at least one amino acid having a side chain comprising one of the following moieties: or its protonated form.

[0381] The cCPP may comprise at least two amino acids, each of which independently has one of the following moieties: or a protonated form thereof. At least two amino acids may have a side chain comprising the same moiety selected from: or its protonated form. At least one amino acid may have or their protonated side chains. At least two amino acids may have or their protonated forms. One, two, three or four amino acids may have a or its protonated form. An amino acid may have a side chain comprising or their protonated side chains. The two amino acids may have or its protonated form. Or its protonated form may be attached to the terminus of the amino acid side chain. Attachment can be made to the terminus of an amino acid side chain.

[0382] The cCPP may comprise (iii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3, 4 or 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3 or 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) two amino acid residues having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) three amino acid residues having a side chain comprising a guanidine group or a protonated form thereof.

[0383] The amino acid residues may independently have side chains comprising discontinuous guanidine groups, guanidine replacement groups, or protonated forms thereof. Two amino acid residues may independently have side chains comprising continuous guanidine groups, guanidine replacement groups, or protonated forms thereof. Three amino acid residues may independently have side chains comprising continuous guanidine groups, guanidine replacement groups, or protonated forms thereof. Four amino acid residues may independently have side chains comprising continuous guanidine groups, guanidine replacement groups, or protonated forms thereof. Continuous amino acid residues may have the same stereochemistry. Continuous amino acids may have alternating stereochemistry.

[0384] The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group, or a protonated form thereof can be an L-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a D-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a mixture of L-amino acids or D-amino acids.

[0385] Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof. Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be a residue of arginine or a protonated form thereof.

[0386] Each amino acid having a side chain containing a guanidine replacement group or a protonated form thereof may independently be or its protonated form.

[0387] Without being bound by theory, it is hypothesized that the guanidine replacement group has reduced basicity relative to arginine and in some cases is uncharged at physiological pH (e.g., -N(H)C(O)), and is able to maintain bidentate hydrogen bonding interactions with phospholipids on the plasma membrane, which is believed to facilitate efficient membrane association and subsequent internalization. Removal of the positive charge is also believed to reduce the toxicity of cCPP.

[0388] It will be understood by those skilled in the art that the N-terminus and / or C-terminus of the above non-natural aromatic hydrophobic amino acids form an amide bond upon incorporation into the peptides disclosed herein.

[0389] The cCPP may comprise a first amino acid having a side chain comprising an aromatic or heteroaromatic group and a second amino acid having a side chain comprising an aromatic or heteroaromatic group, wherein the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain comprising an aromatic or heteroaromatic group. Although by convention, the term "first amino acid" often refers to the N-terminal amino acid of a peptide sequence, as used herein, "first amino acid" is used to distinguish the indicated amino acid from another amino acid in the cCPP (e.g., "second amino acid"), such that the term "first amino acid" may be or may refer to an amino acid located at the N-terminus of the peptide sequence.

[0390] The cCPP may comprise an N-terminus of a second glycine forming a peptide bond with an amino acid having a side chain comprising an aromatic or heteroaromatic group, and a C-terminus of a second glycine forming a peptide bond with an amino acid having a side chain comprising a guanidino group or a protonated form thereof.

[0391] The cCPP may comprise a first amino acid having a side chain comprising a guanidine group or a protonated form thereof and a second amino acid having a side chain comprising a guanidine group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidine 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 guanidine group or a protonated form thereof.

[0392] The cCPP may comprise residues of asparagine, aspartic acid, glutamine, glutamic acid or homoglutamine. The cCPP may comprise residues of asparagine. The cCPP may comprise residues of glutamine.

[0393] 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-anthryl)-alanine.

[0394] Although not wishing to be bound by theory, it is believed that the chirality of the amino acids in the cCPP can affect the efficiency of cytoplasmic uptake. The cCPP may include at least one D amino acid. The cCPP may include one to fifteen D amino acids. The cCPP may include one to ten D amino acids. The cCPP may include 1, 2, 3 or 4 D amino acids. The cCPP may include 2, 3, 4, 5, 6, 7 or 8 consecutive amino acids with alternating D and L chirality. The cCPP may include three consecutive amino acids with the same chirality. The cCPP may include two consecutive amino acids with the same chirality. At least two of the amino acids may have opposite chirality. At least two amino acids with opposite chirality may be adjacent to each other. At least three amino acids may have alternating stereochemistry relative to each other. At least three amino acids with alternating chirality relative to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry relative to each other. At least four amino acids with alternating chirality relative to each other may be adjacent to each other. At least two of the amino acids may have the same chirality. At least two amino acids with the same chirality may be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Thus, the adjacent amino acids in a cCPP may have any of the following sequences: DL; LD; DLLD; LDDL; LDLLD; DLDDL; DLLDL; or LDDLD. The amino acid residues forming the cCPP may all be L-amino acids. The amino acid residues forming the cCPP may all be D-amino acids.

[0395] At least two of the amino acids may have different chirality. At least two amino acids with different chirality may be adjacent to each other. At least three amino acids may have different chirality relative to adjacent amino acids. At least four amino acids may have different chirality relative to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chirality. One or more amino acid residues forming the cCPP may be achiral. The cCPP may comprise a motif of 3, 4 or 5 amino acids, wherein two amino acids with the same chirality may be separated by an achiral amino acid. The cCPP may comprise the following sequence: DXD; DXDX; DXDXD; LXL; LXLX; or LXLXL, wherein X is an achiral amino acid. The achiral amino acid may be glycine.

[0396] Contains

[0397]

[0398] 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 guanidine or a protonated form thereof may be adjacent to at least one amino acid having a side chain comprising guanidine or a protonated form thereof. An amino acid having a side chain comprising guanidine or a protonated form thereof may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Two amino acids having side chains comprising guanidine or its protonated form may be adjacent to each other. Two amino acids having side chains comprising guanidine or its protonated form are adjacent to each other. A cCPP may comprise at least two consecutive amino acids having side chains that may comprise an aromatic or heteroaromatic group and at least two amino acids having side chains comprising or non-adjacent amino acids with side chains comprising aromatic or heteroaromatic groups. The cCPP may comprise at least two consecutive amino acids with side chains comprising aromatic or heteroaromatic groups and at least two consecutive amino acids with side chains comprising or non-adjacent amino acids with side chains in their protonated form. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other amino acid combinations may have any arrangement of D and L amino acids, such as any of the sequences described in the previous paragraph.

[0399] Contains

[0400]

[0401] or a protonated form thereof alternates with at least two amino acids having a side chain comprising a guanidine group or a protonated form thereof.

[0402] The cCPP may comprise a structure of formula (A):

[0403]

[0404] or its protonated form,

[0405] in:

[0406] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0407] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0408] R4, R5, R6, and R7 are independently H or an amino acid side chain;

[0409] At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-alanine, 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, or 3-(1-piperidinyl)alanine;

[0410] AA SC is an amino acid side chain; and

[0411] q is 1, 2, 3, or 4.

[0412] In an embodiment, at least one of R4, R5, R6, R7 is independently an uncharged non-aromatic side chain of an amino acid. In an embodiment, at least one of R4, R5, R6, R7 is independently H or a side chain of citrulline.

[0413] In an embodiment, q is 1. In an embodiment, q is 2. In an embodiment, q is 3. In an embodiment, q is 4.

[0414] In an embodiment, a compound is provided including a cyclic peptide with 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 an embodiment, at least two charged amino acids of the cyclic peptide are arginine. In an embodiment, at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine (3-naphthyl-2-yl-alanine) or a combination thereof. In an embodiment, at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, glycine or a combination thereof. In an embodiment, the compound is a cyclic peptide with 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 a combination thereof, and at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine and a combination thereof.

[0415] The cCPP may comprise the structure of formula (I):

[0416]

[0417] or its protonated form,

[0418] in:

[0419] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0420] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0421] R4 and R7 are independently H or an amino acid side chain;

[0422] AA SC is the amino acid side chain;

[0423] q is 1, 2, 3, or 4; and

[0424] Each m is independently an integer of 0, 1, 2 or 3.

[0425] R1, R2 and R3 may each independently be H, -alkylene-aryl or -alkylene-heteroaryl. R1, R2 and R3 may each independently be H, -C 1-3 Alkylene-aryl or -C 1-3 R1, R2 and R3 can each independently be H or -alkylene-aryl. R1, R2 and R3 can each independently be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. 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. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R1, R2 and R3 may each independently be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R1, R2 and R3 may each independently be H, -CH2Ph ​​or -CH2Naphthyl. R1, R2 and R3 may each independently be H or -CH2Ph.

[0426] R1, R2 and R3 can 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, or 3-(9-anthryl)-alanine.

[0427] R1 may be a side chain of tyrosine. R1 may be a side chain of phenylalanine. R1 may be a side chain of 1-naphthylalanine. R1 may be a side chain of 2-naphthylalanine. R1 may be a side chain of tryptophan. R1 may be a side chain of 3-benzothienylalanine. R1 may be a side chain of 4-phenylphenylalanine. R1 may be a side chain of 3,4-difluorophenylalanine. R1 may be a side chain of 4-trifluoromethylphenylalanine. R1 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R1 may be a side chain of homophenylalanine. R1 may be a side chain of β-homophenylalanine. R1 may be a side chain of 4-tert-butyl-phenylalanine. R1 may be a side chain of 4-pyridylalanine. R1 may be a side chain of 3-pyridylalanine. R1 may be a side chain of 4-methylphenylalanine. R1 may be a side chain of 4-fluorophenylalanine. R1 may be a side chain of 4-chlorophenylalanine. R1 may be the side chain of 3-(9-anthryl)-alanine.

[0428] R2 may be a side chain of tyrosine. R2 may be a side chain of phenylalanine. R2 may be a side chain of 1-naphthylalanine. R1 may be a side chain of 2-naphthylalanine. R2 may be a side chain of tryptophan. R2 may be a side chain of 3-benzothienylalanine. R2 may be a side chain of 4-phenylphenylalanine. R2 may be a side chain of 3,4-difluorophenylalanine. R2 may be a side chain of 4-trifluoromethylphenylalanine. R2 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R2 may be a side chain of homophenylalanine. R2 may be a side chain of β-homophenylalanine. R2 may be a side chain of 4-tert-butyl-phenylalanine. R2 may be a side chain of 4-pyridylalanine. R2 may be a side chain of 3-pyridylalanine. R2 may be a side chain of 4-methylphenylalanine. R2 may be a side chain of 4-fluorophenylalanine. R2 may be a side chain of 4-chlorophenylalanine. R2 may be the side chain of 3-(9-anthryl)-alanine.

[0429] R3 may be a side chain of tyrosine. R3 may be a side chain of phenylalanine. R3 may be a side chain of 1-naphthylalanine. R3 may be a side chain of 2-naphthylalanine. R3 may be a side chain of tryptophan. R3 may be a side chain of 3-benzothienylalanine. R3 may be a side chain of 4-phenylphenylalanine. R3 may be a side chain of 3,4-difluorophenylalanine. R3 may be a side chain of 4-trifluoromethylphenylalanine. R3 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 may be a side chain of homophenylalanine. R3 may be a side chain of β-homophenylalanine. R3 may be a side chain of 4-tert-butyl-phenylalanine. R3 may be a side chain of 4-pyridylalanine. R3 may be a side chain of 3-pyridylalanine. R3 may be a side chain of 4-methylphenylalanine. R3 may be a side chain of 4-fluorophenylalanine. R3 may be a side chain of 4-chlorophenylalanine. R3 may be the side chain of 3-(9-anthryl)-alanine.

[0430] R4 can be H, -alkylene-aryl, -alkylene-heteroaryl. R4 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R4 can be H or -alkylene-aryl. R4 can be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. 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. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R4 may be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R4 can be H or a side chain of an amino acid in Table A or Table C. R4 can be H or an amino acid residue having a side chain containing an aromatic group. R4 can be H, -CH2Ph ​​or -CH2naphthyl. R4 can be H or -CH2Ph.

[0431] R5 can be H, -alkylene-aryl, -alkylene-heteroaryl. R5 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R5 can be H or -alkylene-aryl. R5 can be H or -C 1-3 Alkylene-aryl. C 1-3The alkylene group may be a methylene group. 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. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R5 may be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R5 can be H or a side chain of an amino acid in Table 1 or Table 3. R4 can be H or an amino acid residue having a side chain containing an aromatic group. R5 can be H, -CH2Ph ​​or -CH2naphthyl. R4 can be H or -CH2Ph.

[0432] R6 can be H, -alkylene-aryl, -alkylene-heteroaryl. R6 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R6 can be H or -alkylene-aryl. R6 can be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. 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. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R6 may be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R6 can be H or a side chain of an amino acid in Table A or Table C. R6 can be H or an amino acid residue having a side chain containing an aromatic group. R6 can be H, -CH2Ph ​​or -CH2naphthyl. R6 can be H or -CH2Ph.

[0433] R7 can be H, -alkylene-aryl, -alkylene-heteroaryl. R7 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R7 can be H or -alkylene-aryl. R7 can be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. 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. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R7 may be H, -C 1-3 Alkylene -Ph or -C 1-3Alkylene-naphthyl. R7 may be H or a side chain of an amino acid in Table A or Table C. R7 may be H or an amino acid residue having a side chain containing an aromatic group. R7 may be H, -CH2Ph ​​or -CH2naphthyl. R7 may be H or -CH2Ph.

[0434] One, two or three of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. One of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. Two of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. Three of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. At least one of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. No more than four of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph.

[0435] One, two or three of R1, R2, R3 and R4 are -CH2Ph. One of R1, R2, R3 and R4 is -CH2Ph. Two of R1, R2, R3 and R4 are -CH2Ph. Three of R1, R2, R3 and R4 are -CH2Ph. At least one of R1, R2, R3 and R4 is -CH2Ph.

[0436] One, two or three of R1, R2, R3, R4, R5, R6 and R7 may be H. One of R1, R2, R3, R4, R5, R6 and R7 may be H. Two of R1, R2, R3, R4, R5, R6 and R7 are H. Three of R1, R2, R3, R5, R6 and R7 may be H. At least one of R1, R2, R3, R4, R5, R6 and R7 may be H. No more than three of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph.

[0437] One, two or three of R1, R2, R3 and R4 are H. One of R1, R2, R3 and R4 is H. Two of R1, R2, R3 and R4 are H. Three of R1, R2, R3 and R4 are H. At least one of R1, R2, R3 and R4 is H.

[0438] At least one of R4, R5, R6 and R7 may be a side chain of 3-guanidino 2-alanine. At least one of R4, R5, R6 and R7 may be a side chain of 4-guanidino 2-aminobutyric acid. At least one of R4, R5, R6 and R7 may be a side chain of arginine. At least one of R4, R5, R6 and R7 may be a side chain of homoarginine. At least one of R4, R5, R6 and R7 may be a side chain of N-methylarginine. At least one of R4, R5, R6 and R7 may be a side chain of N,N-dimethylarginine. At least one of R4, R5, R6 and R7 may be a side chain of 2,3-diaminopropionic acid. At least one of R4, R5, R6 and R7 may be a side chain of 2,4-diaminobutyric acid, lysine. At least one of R4, R5, R6 and R7 may be a side chain of N-methyllysine. At least one of R4, R5, R6 and R7 may be a side chain of N, N-dimethyllysine. At least one of R4, R5, R6 and R7 may be a side chain of N-ethyllysine. At least one of R4, R5, R6 and R7 may be a side chain of N, N, N-trimethyllysine or 4-guanidinophenylalanine. At least one of R4, R5, R6 and R7 may be a side chain of citrulline. At least one of R4, R5, R6 and R7 may be a side chain of N, N-dimethyllysine or β-homoarginine. At least one of R4, R5, R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.

[0439] At least two of R4, R5, R6 and R7 may be the side chain of 3-guanidino 2-alanine. At least two of R4, R5, R6 and R7 may be the side chain of 4-guanidino 2-aminobutyric acid. At least two of R4, R5, R6 and R7 may be the side chain of arginine. At least two of R4, R5, R6 and R7 may be the side chain of homoarginine. At least two of R4, R5, R6 and R7 may be the side chain of N-methylarginine. At least two of R4, R5, R6 and R7 may be the side chain of N,N-dimethylarginine. At least two of R4, R5, R6 and R7 may be the side chain of 2,3-diaminopropionic acid. At least two of R4, R5, R6 and R7 may be the side chain of 2,4-diaminobutyric acid, lysine. At least two of R4, R5, R6 and R7 may be the side chain of N-methyllysine. At least two of R4, R5, R6 and R7 may be the side chain of N, N-dimethyllysine. At least two of R4, R5, R6 and R7 may be the side chain of N-ethyllysine. At least two of R4, R5, R6 and R7 may be the side chain of N, N, N-trimethyllysine, 4-guanidinophenylalanine. At least two of R4, R5, R6 and R7 may be the side chain of citrulline. At least two of R4, R5, R6 and R7 may be the side chain of N, N-dimethyllysine, β-homoarginine. At least two of R4, R5, R6 and R7 may be the side chain of 3-(1-piperidinyl)alanine.

[0440] At least three of R4, R5, R6 and R7 may be the side chain of 3-guanidino 2-alanine. At least three of R4, R5, R6 and R7 may be the side chain of 4-guanidino 2-aminobutyric acid. At least three of R4, R5, R6 and R7 may be the side chain of arginine. At least three of R4, R5, R6 and R7 may be the side chain of homoarginine. At least three of R4, R5, R6 and R7 may be the side chain of N-methylarginine. At least three of R4, R5, R6 and R7 may be the side chain of N,N-dimethylarginine. At least three of R4, R5, R6 and R7 may be the side chain of 2,3-diaminopropionic acid. At least three of R4, R5, R6 and R7 may be the side chain of 2,4-diaminobutyric acid, lysine. At least three of R4, R5, R6 and R7 may be the side chain of N-methyllysine. At least three of R4, R5, R6 and R7 may be the side chain of N,N-dimethyllysine. At least three of R4, R5, R6 and R7 may be the side chain of N-ethyllysine. At least three of R4, R5, R6 and R7 may be the side chain of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least three of R4, R5, R6 and R7 may be the side chain of citrulline. At least three of R4, R5, R6 and R7 may be the side chain of N,N-dimethyllysine, β-homoarginine. At least three of R4, R5, R6 and R7 may be the side chain of 3-(1-piperidinyl)alanine.

[0441] AA SC Can be the side chain of an asparagine, glutamine or homoglutamine residue. SC The cCPP may also contain conjugated AA SC Thus, the cCPP may also comprise a linker conjugated to an asparagine, glutamine or homoglutamine residue. The cCPP may also comprise a linker conjugated to a glutamine residue.

[0442] q can be 1, 2, or 3. q can be 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.

[0443] m may be 1-3. m may be 1 or 2. m may be 0. m may be 1. m may be 2. m may be 3.

[0444] The cCPP of formula (A) may comprise the structure of formula (I):

[0445] or its protonated form, wherein AA SC , R1, R2, R3, R4, R 7、 m and q are as defined herein.

[0446] The cCPP of formula (A) may comprise the structure of formula (Ia) or formula (Ib):

[0447]

[0448] or its protonated form, wherein AA SC , R1, R2, R3, R4 and m are as defined herein.

[0449] The cCPP of formula (A) may comprise the structure of formula (I-1), (I-2), (I-3) or (I-4):

[0450]

[0451] or its protonated form, wherein AA SC and m are as defined herein. The cCPP of formula (A) may comprise a structure of formula (I-5) or (I-6):

[0452]

[0453] (I-6) or its protonated form, wherein AA SC As defined herein.

[0454] cCPP may comprise one of the following sequences: FGFGRGR; GfFGrGr, FfΦGRGR; FfFGRGR; or FfΦGrGr. cCPP may have one of the following sequences: FGFGRGRQ; GfFGrGrQ, FfΦGRGRQ; FfFGRGRQ; or FfΦGrGrQ.

[0455] The present disclosure also relates to a cCPP having a structure of formula (II):

[0456]

[0457] in:

[0458] AA SC is the amino acid side chain;

[0459] R 1a , R 1b and R 1c are each independently a 6- to 14-membered aryl group or a 6- to 14-membered heteroaryl group;

[0460] R 2a , R 2b , R 2c and R 2d independently, an amino acid side chain;

[0461] R 2a , R 2b, R 2c and R 2d At least one of or its protonated form;

[0462] R 2a , R 2b , R 2c and R 2d At least one of is guanidine or a protonated form thereof;

[0463] Each n" is independently an integer 0, 1, 2, 3, 4 or 5;

[0464] Each n' is independently an integer 0, 1, 2 or 3; and

[0465] If n' is 0, then R 2a , R 2b , R 2b or R 2d Does not exist.

[0466] R 2a , R 2b , R 2c and R 2d At least two of them can be or its protonated form. 2a , R 2b , R 2c and R 2d Two or three of the or its protonated form. 2a , R 2b , R 2c and R 2d One of them can be or its protonated form. 2a , R 2b , R 2c and R 2d At least one of can be or its protonated form, and R 2a , R 2b , R 2c and R 2d The remainder of R may be guanidine or its protonated form. 2a , R 2b , R 2c and R 2d At least two of them can be or its protonated form, and R 2a , R 2b , R 2c and R2d The remainder of may be guanidine or a protonated form thereof.

[0467] R 2a , R 2b , R 2c and R 2d But all of them are or its protonated form. 2a , R 2b , R 2c and R 2d At least one of them can be or its protonated form, and R 2a , R 2b , R 2c and R 2d The remainder of may be guanidine or a protonated form thereof. 2a , R 2b , R 2c and R 2d The group can be or its protonated form, and R 2a , R 2b , R 2c and R 2d The remainder of the is guanidine or its protonated form.

[0468] R 2a , R 2b , R 2c and R 2d Each of can independently be a side chain of 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homolysine, serine, homoserine, threonine, allothreonine, histidine, 1-methylhistidine, 2-aminosuccinic acid, aspartic acid, glutamic acid or homoglutamic acid.

[0469] AA SC Can be Wherein t can be an integer from 0 to 5. SC Can be Wherein t may be an integer from 0 to 5. t may be from 1 to 5. t may be 2 or 3. t may be 2. t may be 3.

[0470] R 1a , R 1b and R 1c R may each independently be a 6- to 14-membered aryl group. 1a , R 1b and R 1c R and R are each independently a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O or S.1a , R 1b and R 1c R may be independently selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl or isoquinolyl. 1a , R 1b and R 1c R may be independently selected from phenyl, naphthyl or anthracenyl. 1a , R 1b and R 1c R may be independently phenyl or naphthyl. 1a , R 1b and R 1c They may each be independently selected from pyridyl, quinolyl or isoquinolyl.

[0471] 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.

[0472] Each n" may independently be an integer from 1 to 3. Each n" may independently be 2 or 3. Each n" may be 2. Each n" may be 3. At least one n" may be 0. At least one n" may be 1. At least one n" may be 2. At least one n" may be 3.

[0473] 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.

[0474] The cCPP of formula (II) may have the structure of formula (II-1):

[0475]

[0476] Where R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d AA SC , n′ and n″ are as defined herein.

[0477] The cCPP of formula (II) may have the structure of formula (IIa):

[0478]

[0479] Where R 1a , R 1b , R1c , R 2a , R 2b , R 2c , R 2d AA SC and n' are as defined herein.

[0480] The cCPP of formula (II) may have the structure of formula (IIb):

[0481]

[0482] Where R 2a , R 2b AA SC and n' are as defined herein.

[0483] The cCPP may have the structure of formula (IIc):

[0484] or its protonated form,

[0485] in:

[0486] AA SC and n' are as defined herein.

[0487] The cCPP may have the structure of formula (III):

[0488]

[0489] in:

[0490] AA SC is the amino acid side chain;

[0491] R 1a , R 1b and R 1c are each independently a 6- to 14-membered aryl group or a 6- to 14-membered heteroaryl group;

[0492] R 2a and R 2c Each independently is H, or its protonated form;

[0493] R 2b and R 2d each independently is guanidine or a protonated form thereof;

[0494] Each n" is independently an integer from 1 to 3;

[0495] Each n' is independently an integer from 1 to 5; and

[0496] Each p' is independently an integer from 0 to 5.

[0497] The cCPP of formula (III) may have the structure of formula (III-1):

[0498]

[0499] in:

[0500] AA SC , R 1a , R 1b , R 1c , R 2a , R 2c , R 2b , R 2d n', n", and p' are as defined herein.

[0501] The cCPP of formula (III) may have the structure of formula (IIIa):

[0502]

[0503] in:

[0504] AA SC , R 2a , R 2c , R 2b , R 2d n', n", and p' are as defined herein.

[0505] In formula (III), (III-1) and (IIIa), R a and R c It can be H. R a and R c Can be H and R b and R d R and R are independently guanidine or a protonated form thereof. a It can be H. R b It can be H. p′ can be 0. R a and R c can be H and each p′ can be 0.

[0506] In formula (III), (III-1) and (IIIa), R a and R c Can be H, R b and R d each independently may be guanidine or a protonated form thereof, n" may be 2 or 3, and each p' may be 0.

[0507] p' can be 0. p' can be 1. p' can be 2. p' can be 3. p' can be 4. p' can be 5.

[0508] cCPP can have the following structure:

[0509]

[0510] The cCPP of formula (A) may be selected from:

[0511] CPP sequence (FfΦRrRrQ) (FfΦCit-r-Cit-rQ) (FfΦGrGrQ) (FfFGRGRQ) (FGFGRGRQ) (GfFGrGrQ) (FGFGRRRQ) (FGFRRRRQ)

[0512] The cCPP of formula (A) may be selected from:

[0513]

[0514]

[0515] In an embodiment, the cCPP is selected from:

[0516]

[0517] Φ = L-naphthylalanine; φ = D-naphthylalanine; Ω = L-norleucine cCPP may include a structure of formula (D) or its protonated form,

[0518] in:

[0519] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0520] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0521] R4 and R6 are independently H or an amino acid side chain;

[0522] AA SC is the amino acid side chain;

[0523] Y is

[0524] q is 1, 2, 3, or 4;

[0525] Each m is independently an integer 0, 1, 2 or 3, and

[0526] Each n is independently an integer 0, 1, 2 or 3.

[0527] cCPP of formula (D), wherein Y is

[0528]

[0529] cCPP of formula (D), wherein Y is:

[0530] cCPP of formula (D), wherein Y is:

[0531] cCPP of formula (D), wherein Y is:

[0532] cCPP of formula (D), wherein Y is:

[0533] In the embodiment, AA SC Can be conjugated to a linker.

[0534] Connectors

[0535] The cCPP of the present disclosure can be conjugated to a linker. The linker can connect the cargo to the cCPP. The linker can be attached to the side chain of the amino acid of the cCPP, and the cargo can be attached at a suitable position on the linker.

[0536] The linker can be any suitable part that can conjugate the cCPP to one or more other parts, such as an exocyclic peptide (EP) and / or a cargo. Before being conjugated to the cCPP and one or more other parts, the linker has two or more functional groups, each of which is independently capable of forming a covalent bond with the cCPP and one or more other parts. If the cargo is an oligonucleotide, the linker can be covalently bound to the 5' end of the cargo or the 3' end of the cargo. The linker can be covalently bound to the 5' end of the cargo. The linker can be covalently bound to the 3' end of the cargo. If the cargo is a peptide, the linker can be covalently bound to the N-terminus or C-terminus of the cargo. The linker can be covalently bound to the backbone of the oligonucleotide or peptide cargo. The linker can be any suitable part that conjugates the cCPP described herein to cargo, such as an oligonucleotide, a peptide or a small molecule.

[0537] The linker may comprise a hydrocarbon linker.

[0538] The linker may comprise a cleavage site. The cleavage site may be a disulfide or a caspase cleavage site (eg, Val-Cit-PABC).

[0539] The linker 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- JR 2 )z"-subunit, where R 1 and R 2 Each of the groups in each instance is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, and each J is independently C, NR 3 、-NR 3C(O)-, S and O, among which 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-(JR 1 )z"-, where each R 1 In each instance, is independently alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, and each J is independently C, NR 3 、-NR 3 C(O)-, S or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; or (ix) the linker may comprise one or more of (i) to (x).

[0540] The linker may comprise one or more D or L amino acids and / or -(R 1- JR 2 )z”-, where R 1 and R 2 Each of the above is independently alkylene in each instance, and each J is independently C, NR 3 、-NR 3 C(O)-, S and O, among which R 4 is independently selected from H and alkyl, and z" is an integer from 1 to 50; or a combination thereof.

[0541] The linker may contain -(OCH2CH2) z’ -(OCH2CH2)z' may also be referred to as polyethylene glycol (PEG).

[0542] The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise -(OCH2CH2) z’ - (wherein z' is an integer from 1 to 23) and a peptide. The peptide may contain 2 to 10 amino acids. The linker may also contain a functional group (FG) that can react by click chemistry. FG may be an azide or an alkyne, and a triazole is formed when the cargo is conjugated to the linker.

[0543] The linker may comprise (i) a β-alanine residue and a lysine residue; (ii) -(JR 1 )z”; or (iii) any combination thereof. Each R 1can be independently alkylene, alkenylene, alkynylene, carbocyclic or heterocyclic, each J is independently C, NR 3 、-NR 3 C(O)-, S or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R 1 may be alkylene and each J may be O.

[0544] The linker may comprise (i) a residue of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof; and (ii) -(R 1- J)z"-or-(JR 1 )z". Each R 1 can be independently alkylene, alkenylene, alkynylene, carbocyclic or heterocyclic, each J is independently C, NR 3 、-NR 3 C(O)-, S or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R 1 may be an alkylene group and each J may be O. The linker may comprise glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof.

[0545] The linker can be a trivalent linker. The linker can have the following structure: Wherein A1, B1 and C1 can be independently a hydrocarbon linker (e.g. NRH-(CH2) n -COOH), PEG linkers (e.g. NRH-(CH2O) n -COOH, wherein R is H, methyl or ethyl) or one or more amino acid residues, and Z is independently a protecting group. The linker may also incorporate a cleavage site, including a disulfide [NH2-(CH2O) n -SS-(CH2O) n -COOH] or caspase cleavage site (Val-Cit-PABC).

[0546] The hydrocarbon may be the residue of glycine or beta-alanine.

[0547] The linker may be bivalent and connects the cCPP to the cargo.The linker may be bivalent and connects the cCPP to the exocyclic peptide (EP).

[0548] The linker can be trivalent and connects the cCPP to the cargo and the EP.

[0549] The linker can be a divalent or trivalent C1-C 50 Alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl or cycloalkenyl. The linker can be a divalent or trivalent C1-C 50 Alkylene wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.

[0550] The joint can have the following structures:

[0551] Where: each AA is independently an amino acid residue; * is attached to AA SC point, and AA SC is a side chain of an amino acid residue of a 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, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, and 6-aminohexanoic acid.

[0552] The cCPP can be attached to the cargo via a linker ("L"). The linker can be conjugated to the cargo via a bonding group ("M").

[0553] The joint can have the following structures:

[0554] 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 a residue attached to AA SC point, and AA SC is the side chain of an amino acid residue of cCPP; and M is a bonding group as defined herein.

[0555] The joint can have the following structures:

[0556]

[0557] Where: 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 attached to AA SC point, and AA SC is the side chain of an amino acid residue of cCPP; and M is a bonding group as defined herein.

[0558] The joint can have the following structures:

[0559]

[0560] Where: 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 attached to AA SC point, and AA SC It is the side chain of the amino acid residue of cCPP.

[0561] x can 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.

[0562] x' 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. 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.

[0563] 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.

[0564] z can 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.

[0565] 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.

[0566] As discussed above, the linker or M (wherein M is part of a linker) can be covalently bound to the cargo at any suitable position on the cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the 3' end of the oligonucleotide cargo or the 5' end of the oligonucleotide cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the N-terminus or C-terminus of the peptide cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the backbone of the oligonucleotide or peptide cargo.

[0567] The linker may be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (eg, a reduced side chain having an amino group). The linker may be bound to the side chain of lysine on the cCPP.

[0568] The linker can be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the peptide cargo, or a modified side chain of glutamine or asparagine (eg, a reduced side chain with an amino group). The linker can be bound to the side chain of lysine on the peptide cargo.

[0569] The joint can have the following structures:

[0570]

[0571] in

[0572] M is a group that conjugates L to a cargo (eg, an oligonucleotide); AA s is the side chain or end of the amino acid on the cCPP; each AA x are independently amino acid residues;

[0573] o is an integer from 0 to 10; and

[0574] p is an integer from 0 to 5.

[0575] The joint can have the following structures:

[0576]

[0577] in

[0578] M is a group that conjugates L to a cargo (eg, an oligonucleotide); AA s is the side chain or end of the amino acid on the cCPP; each AA x are independently amino acid residues;

[0579] o is an integer from 0 to 10; and

[0580] p is an integer from 0 to 5.

[0581] M may comprise an alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, each of which is optionally substituted. M may be selected from:

[0582]

[0583] wherein R is an alkyl, alkenyl, alkynyl, carbocyclic or heterocyclic group.

[0584] M can be selected from:

[0585]

[0586]

[0587] Where: R 10 is an alkylene group, a cycloalkyl group or Where a is 0 to 10.

[0588] M can be R 10 Can be and a is 0 to 10. M can be

[0589] M may be a heterobifunctional cross-linker, such as Such heterobifunctional cross-linkers are 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.

[0590] M may be -C(O)-.

[0591] AA s It can be the side chain or the end of an amino acid on cCPP. s Non-limiting examples include aspartic acid, glutamic acid, glutamine, asparagine or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). s May be AA as defined herein SC .

[0592] Each AA x are independently natural or unnatural amino acids. One or more AA x Can be natural amino acids. One or more AA x Can be unnatural amino acids. One or more AA x The β-amino acid may be β-alanine.

[0593] 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.

[0594] 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.

[0595] The joint can have the following structures:

[0596]

[0597] Among them, M, AA s , each-(R 1- JR 2 )z"-, o and z" are as defined herein; r can be 0 or 1.

[0598] r can be 0. r can be 1.

[0599] The joint can have the following structures:

[0600]

[0601] Among them, M, AA s Each of "o, p, q, r and z" may be as defined herein.

[0602] z" may be an integer from 1 to 50, for example, 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 5-20. z" may be an integer from 10-15.

[0603] The joint can have the following structures:

[0604]

[0605] in:

[0606] M, AA s and o are as defined herein.

[0607] Other non-limiting examples of suitable linkers include:

[0608]

[0609]

[0610] Among them, M and AA s As defined in this article

[0611] Provided herein is a compound comprising a cCPP and a cargo, wherein the cargo comprises a hybrid oligonucleotide, such as an antisense oligonucleotide, which is complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein a linker is conjugated to the hybrid oligonucleotide via a bonding group (M), wherein M is selected from:

[0612]

[0613] Where: R 1 is an alkylene group, a cycloalkyl group or wherein t' is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, wherein R 1 yes And t' is 2.

[0614] The joint can have the following structures:

[0615]

[0616] Among them, AA s As defined herein, and m' is 0-10.

[0617] The linker may have the following structural formula:

[0618]

[0619] The linker may have the following structural formula: where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0620] The linker may have the following structural formula:

[0621] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0622] The linker may have the following structural formula:

[0623] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0624] The linker may have the following structural formula: where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0625] The linker may have the following structural formula:

[0626]

[0627] The linker can be covalently bound to the cargo at any suitable position on the cargo. The linker is covalently bound to the 3' end of the cargo or the 5' end of the oligonucleotide cargo. The linker can be covalently bound to the backbone of the cargo.

[0628] The linker may be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (eg, a reduced side chain having an amino group). The linker may be bound to the side chain of lysine on the cCPP.

[0629] cCPP-linker conjugate

[0630] The cCPP may be conjugated to a linker as defined herein. The linker may be conjugated to the AA of the cCPP as defined herein. SC .

[0631] The linker may contain -(OCH2CH2) 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. "-(OCH2CH2) z’ Also known as PEG. The cCPP-linker conjugate may have a structure selected from Table D:

[0632] Table D: cCPP-linker conjugates

[0633] <![CDATA[Cyclo(FfФ-4gp-r-4gp-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Cit-r-Cit-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Pia-r-Pia-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Dml-r-Dml-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Cit-r-Cit-rQ)-PEG 12 -OH]]> <![CDATA[Cyclo(fФR-Cit-R-Cit-Q)-PEG 12 -OH]]>

[0634] The linker may contain -(OCH2CH2) z’ -subunit (wherein z' is an integer from 1 to 23) and a peptide subunit. The peptide subunit may contain 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table E:

[0635] Table E: cCPP-linker conjugates

[0636]

[0637]

[0638] Provided are EEVs comprising a cyclic cell penetrating peptide (cCPP), a linker and an exocyclic peptide (EP). The EEV may comprise a structure of formula (B):

[0639] or its protonated form,

[0640] in:

[0641] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0642] R4 and R7 are independently H or an amino acid side chain;

[0643] EP is an exocyclic peptide as defined herein;

[0644] Each m is independently an integer from 0 to 3;

[0645] n is an integer from 0 to 2;

[0646] x' is an integer from 1 to 20;

[0647] y is an integer from 1 to 5;

[0648] q is 1-4; and

[0649] z' is an integer from 1 to 23.

[0650] R1, R2, R3, R4, R7, EP, m, q, y, x', z' are as described herein.

[0651] n can be 0. n can be 1. n can be 2.

[0652] The EEV may comprise a structure of formula (Ba) or (Bb):

[0653]

[0654] or its protonated form, wherein EP, R 1 , R 2 , R 3 , R 4 , m and z' are as defined above in formula (B).

[0655] The EEV may comprise a structure of formula (Bc):

[0656]

[0657] or its protonated form, wherein EP, R 1 , R 2 , R 3 , R 4 and m are as defined above in formula (B); AA is an amino acid as defined herein; M is as defined herein; n is an integer from 0 to 2; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10.

[0658] The EEV may have a structure of formula (B-1), (B-2), (B-3) or (B-4):

[0659]

[0660]

[0661] or a protonated form thereof, wherein EP is as defined above in formula (B).

[0662] The EEV may comprise formula (B) and may have the following structure: Ac-PKKKRKV-AEEA-K(cyclo[FGFGRGRQ])-PEG 12 -OH or Ac-PKKKRKV-AEEA-K(cyclo[GfFGr GrQ])-PEG 12 -OH.

[0663] The EEV may comprise a cCPP of the formula:

[0664]

[0665] The EEV may comprise the formula Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3).

[0666] The EEV can be Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-AEEA-K-(cyclo[FGFGRGRQ])-PEG12-OH.

[0667] An EEV can be:

[0668]

[0669] The EEV may be Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH. The EEV may be:

[0670]

[0671] EEV can be selected from

[0672] Ac-rr-miniPEG2-Dap[Cycle(FfΦ-Cit-r-Cit-rQ)]-PEG 12 -OH

[0673] Ac-frr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0674] Ac-rfr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0675] Ac-rbfbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0676] Ac-rrr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0677] Ac-rbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0678] Ac-rbrbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0679] Ac-hh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0680] Ac-hbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0681] Ac-hbhbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0682] Ac-rbhbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0683] Ac-hbrbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0684] Ac-rr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0685] Ac-frr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0686] Ac-rfr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0687] Ac-rbfbr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0688] Ac-rrr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0689] Ac-rbr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0690] Ac-rbrbr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0691] Ac-hh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0692] Ac-hbh-Dap(cyclo(FfΦ-Cit-r-Git-rQ))-b-OH

[0693] Ac-hbhbh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0694] Ac-rbhbh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0695] Ac-hbrbh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0696] Ac-KKKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0697] Ac-KGKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0698] Ac-KKGK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0699] Ac-KKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0700] Ac-KK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0701] Ac-KGK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0702] Ac-KBK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0703] Ac-KBKBK-miniPEG2-Lys(cyclo(Fr-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0704] Ac-KR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0705] Ac-KBR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0706] Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0707] Ac-PKKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0708] Ac-PGKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0709] Ac-PKGKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ])-miniPEG2-K(N3)-NH2

[0710] Ac-PKKGRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0711] Ac-PKKKGKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0712] Ac-PKKKRGV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0713] Ac-PKKKRKG-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ])-miniPEG2-K(N3)-NH2

[0714] Ac-KKKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0715] Ac-KKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 and

[0716] Ac-KRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2.

[0717] EEV can be selected from:

[0718] Ac-PKKKRKV-Lys(cyclo[FfΦGrGrQ])-PEG 12 -K(N3)-NH2

[0719] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦGrGrQ])-miniPEG2-K(N3)-NH2

[0720] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRQ])-miniPEG2-K(N3)-NH2

[0721] Ac-KR-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0722] Ac-PKKKGKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0723] Ac-PKKKRKG-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0724] Ac-KKKRK-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0725] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FFΦGRGRQ])-miniPEG2-K(N3)-NH2

[0726] Ac-PKKKRKV-miniPEG2-Lys(cyclo[βhFfΦGrGrQ])-miniPEG2-K(N3)-NH2 and

[0727] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦSrSrQ])-miniPEG2-K(N3)-NH2.

[0728] EEV can be selected from:

[0729] Ac-PKKKRKV-miniPEG2-Lys(cyclo(GfFGrGrQ])-PEG 12 -OH

[0730] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFKRKRQ])-PEG 12 -OH

[0731] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRGRGQ])-PEG 12 -OH

[0732] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRGRQ])-PEG 12 -OH

[0733] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRrRQ])-PEG 12 -OH

[0734] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRRRQ])-PEG 12 -OH and

[0735] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRRRRQ])-PEG 12 -OH.

[0736] EEV can be selected from:

[0737] Ac-KKKRKG-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0738] Ac-KKKRK-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0739] Ac-KKRKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0740] Ac-KRKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0741] Ac-KKKKR-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0742] Ac-RKKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH and

[0743] Ac-KKKRK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH.

[0744] EEV can be selected from:

[0745] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0746] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0747] Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH2 and

[0748] Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH.

[0749] Cargo can be AC ​​and EEV can be selected from:

[0750] Ac-PKKKRKV-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0751] Ac-PKKKRKV-PEG2-K(cyclo[FfΦCit-r-Cit-rQ]])-PEG 12 -OH

[0752] Ac-PKKKRKV-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0753] Ac-PKKKRKV-PEG2-K(cyclic[FGFGRGRQ])-PEG 12 -OH

[0754] Ac-PKKKRKV-PEG2-K(cyclic[GfFGrGrQ])-PEG 12 -OH

[0755] Ac-PKKKRKV-PEG2-K(cyclic[FGFGRRRQ])-PEG 12 -OH

[0756] Ac-PKKKRKV-PEG2-K(cyclic[FGFRRRRQ])-PEG 12 -OH

[0757] Ac-rr-PEG2-K(cyclic[FfΦGrGrQ])-PEG 12 -OH

[0758] Ac-rr-PEG2-K(cyclic[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0759] Ac-rr-PEG2-K(cyclic[FfF-GRGRQ])-PEG 12 -OH

[0760] Ac-rr-PEG2-K(cyclic[FGFGRGRQ])-PEG 12 -OH

[0761] Ac-rr-PEG2-K(cyclic[GfFGrGrQ])-PEG 12 -OH

[0762] Ac-rr-PEG2-K(cyclic[FGFGRRRQ])-PEG 12 -OH

[0763] Ac-rr-PEG2-K(cyclic[FGFRRRRQ])-PEG 12 -OH

[0764] Ac-rrr-PEG2-K(cyclic[FfΦGrGrQ])-PEG 12 -OH

[0765] Ac-rrr-PEG2-K(cyclic[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0766] Ac-rrr-PEG2-K(cyclic[FfFGRGRQ])-PEG 12-OH

[0767] Ac-rrr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0768] Ac-rrr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0769] Ac-rrr-PFG2-K(cyclo[FGFGRRRQ])-PFG 12 -OH

[0770] Ac-rrr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0771] Ac-rhr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0772] Ac-rhr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0773] Ac-rhr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0774] Ac-rhr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0775] Ac-rhr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0776] Ac-rhr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0777] Ac-rhr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0778] Ac-rbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0779] Ac-rbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0780] Ac-rbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0781] Ac-rbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0782] Ac-rbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0783] Ac-rbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0784] Ac-rbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0785] Ac-rbrbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0786] Ac-rbrbr-PEG2-K(cyclo{FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0787] Ac-rbrbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0788] Ac-rbrbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0789] Ac-rbrbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0790] Ac-rbrbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0791] Ac-rbrbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0792] Ac-rbhbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0793] Ac-rbhbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH

[0794] Ac-rbhbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0795] Ac-rbhbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0796] Ac-rbhbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0797] Ac-rbhbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0798] Ac-rbhb-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0799] Ac-hbrbh-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0800] Ac-hbrbh-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0801] Ac-hbrbh-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0802] Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0803] Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0804] Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH and

[0805] Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH,

[0806] wherein b is β-alanine and the exocyclic sequence can be of D or L stereochemistry.

[0807] In an embodiment, the cCPP may be

[0808]

[0809]

[0810] The cargo may be a protein and the EEV may be selected from:

[0811] Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0812] Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0813] Ac-PKKKRKV-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0814] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0815] Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0816] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0817] Ac-PKKKRKV-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0818] Ac-rr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0819] Ac-rr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0820] Ac-rr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0821] Ac-rr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0822] Ac-rr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0823] Ac-rr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0824] Ac-rr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0825] Ac-rrr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0826] Ac-rrr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0827] Ac-rrr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0828] Ac-rrr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0829] Ac-rrr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0830] Ac-rrr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0831] Ac-rrr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0832] Ac-rhr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0833] Ac-rhr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0834] Ac-rhr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0835] Ac-rh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0836] Ac-rhr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0837] Ac-rhr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0838] Ac-rhr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0839] Ac-rbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0840] Ac-rbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0841] Ac-rbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0842] Ac-rbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0843] Ac-rbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0844] Ac-rbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0845] Ac-rbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0846] Ac-rbrbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0847] Ac-rbrbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0848] Ac-rbrbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0849] Ac-rbrbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0850] Ac-rbrbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0851] Ac-rbrbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0852] Ac-rbrbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0853] Ac-rbhbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0854] Ac-rbhbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0855] Ac-rbhbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0856] Ac-rbhbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0857] Ac-rbhbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0858] Ac-rbhbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0859] Ac-rbhbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0860] Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0861] Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0862] Ac-hbrbh-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0863] Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0864] Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0865] Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0866] Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0867] wherein b is β-alanine and the exocyclic sequence can be of D or L stereochemistry.

[0868] goods

[0869] A cell penetrating peptide (CPP), such as a cyclic cell penetrating peptide (e.g., cCPP), can be conjugated to a cargo. The cargo can be a therapeutic moiety. The cargo can be conjugated to the terminal carbonyl of a linker. At least one atom of the cyclic peptide can be replaced by the cargo or at least one lone pair can form a bond to the cargo. The cargo can be conjugated to the cCPP via a linker. The cargo can be conjugated to the AA via a linker. SC At least one atom of the cCPP can be replaced by a therapeutic moiety or at least one lone pair of the cCPP forms a bond to the therapeutic moiety. A hydroxyl group on an amino acid side chain of the cCPP can be replaced by a bond to a cargo. A hydroxyl group on a glutamine side chain of the cCPP can be replaced by a bond to a cargo. A cargo can be conjugated to the cCPP via a linker. A cargo can be conjugated to an AA via a linker. SC .

[0870] The cargo may comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof.The cargo may be a hybrid oligonucleotide as described herein.

[0871] Cyclic cell penetrating peptide (cCPP) conjugated to a cargo moiety

[0872] A cyclic cell penetrating peptide (cCPP) can be conjugated to a cargo moiety.

[0873] The cargo moiety can be conjugated to the cCPP via a linker. The cargo moiety can comprise a therapeutic moiety. The therapeutic moiety can comprise an oligonucleotide. The oligonucleotide can comprise a hybrid oligonucleotide as described herein. The cargo moiety can be conjugated to the linker at the terminal carbonyl to provide the following structure:

[0874] in:

[0875] EP is an exocyclic peptide and M, AA SC , Goods, x', y and z' are as defined above, * is attached to AA SC point. x' can be 1. y can be 4. z' can be 11. -(OCH2CH2) x’ -and / or-(OCH2CH2) z’ - may be independently substituted with one or more amino acids, including, for example, glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminocaproic acid, or a combination thereof.

[0876] An endosomal escape vehicle (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP), and a linker, and may be conjugated to a cargo to form an EEV-conjugate comprising a structure of formula (C):

[0877]

[0878] or its protonated form,

[0879] in:

[0880] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0881] R4 is H or an amino acid side chain;

[0882] EP is an exocyclic peptide as defined herein;

[0883] Goods are Parts as defined herein;

[0884] Each m is independently an integer from 0 to 3;

[0885] n is an integer from 0 to 2;

[0886] x' is an integer from 2 to 20;

[0887] y is an integer from 1 to 5;

[0888] q is an integer from 1 to 4; and

[0889] z' is an integer from 2 to 20.

[0890] R1, R2, R3, R4, EP, cargo, m, n, x', y, q and z' are as defined herein.

[0891] The EEV may be conjugated to a cargo and the EEV-conjugate may comprise a structure of formula (Ca) or (Cb):

[0892]

[0893] or a protonated form thereof, wherein EP, m and z are as defined above in formula (C).

[0894] The EEV may be conjugated to a cargo and the EEV-conjugate may comprise a structure of formula (Cc):

[0895] or its protonated form,

[0896] Among them, EP, R 1 , R 2 , R 3 , R 4 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.

[0897] The EEV may be conjugated to an oligonucleotide cargo and the EEV-oligonucleotide conjugate may comprise a structure of formula (C-1), (C-2), (C-3), or (C-4):

[0898]

[0899]

[0900]

[0901] disease

[0902] In embodiments, the disease is a genetic disease or disorder. In embodiments, the disease is an inflammatory disease. In embodiments, the disease is an autoimmune disease. In embodiments, the disease is cancer. In embodiments, the disease is a neurological disease. In embodiments, the disease is a cardiovascular disease. In embodiments, the disease is a metabolic disease. In embodiments, the disease is an infectious disease. In embodiments, the disease is a hematological disease. In embodiments, the disease is a musculoskeletal disease. In embodiments, the disease is a degenerative neurological disease. In embodiments, the disease is a respiratory disease. In embodiments, the disease is a gastrointestinal disease. In embodiments, the disease is an endocrine disease. In embodiments, the disease is an ophthalmic disease. In embodiments, the disease is a kidney disease.

[0903] In an embodiment, the disease is Duchenne muscular dystrophy.

[0904] In embodiments, the disease is Huntington's disease (HD), Huntington's disease-like 2 (HDL2), myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), spinocerebellar ataxia (e.g., spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), or spinocerebellar ataxia type 3 (SCA3)), spinal and bulbar muscular atrophy (SBMA), dentate-rubral-pallidular atrophy with Lewy bodies (DRPLA), amyotrophic lateral sclerosis, frontotemporal dementia, fragile X syndrome, fragile X mental retardation 1 (FMR1), fragile X mental retardation 2 (FMR2), fragile XE mental retardation (FRAXE), Friedreich's ataxia (Friedreich's ataxia (FRDA), fragile X-associated tremor / ataxia syndrome (FXTAS), myoclonic epilepsy, oculopharyngeal muscular dystrophy (OPMD), or syndromic or nonsyndromic X-linked mental retardation.

[0905] In an embodiment, the disease is Friedreich's ataxia (FRDA). In an embodiment, the target gene is FXN, which encodes a frataxin protein. In an embodiment, the compound provided herein comprises an antisense oligonucleotide targeted to FXN.

[0906] Compositions and methods of administration

[0907] The compounds of the present disclosure can be formulated into compositions suitable for in vivo use. The compounds and / or compositions can be administered to a patient suffering from or suspected of suffering from a genetic disease or disorder.

[0908] The in vivo application of the disclosed compound and the composition containing the disclosed compound can be realized by any suitable method and technology known to those skilled in the art at present or in the future. For example, the disclosed compound can be formulated into a physiologically or pharmaceutically acceptable composition and applied 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 compound or composition can be a single administration, or can be easily determined by those skilled in the art to be performed at continuous or different intervals.

[0909] The compounds disclosed herein and compositions comprising the compounds disclosed herein can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide uniform dosages over extended periods of time. The compounds can also be administered in the form of their salt derivatives or in crystalline form.

[0910] The compounds disclosed herein can be formulated into pharmaceutical compositions according to methods known for preparing pharmaceutically acceptable compositions. Formulations are described in detail in many sources well known to those skilled in the art and readily available. For example, Remington's Pharmaceutical Science (1995) by EW Martin describes formulations that can be used in conjunction with the disclosed methods. In general, the compounds disclosed herein can be formulated so that an effective amount of the compound is combined with a suitable carrier to facilitate the effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semisolid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions and sprays. The form depends on the intended mode of administration and therapeutic application. The composition also includes conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents used with the compounds include ethanol, dimethyl sulfoxide, glycerol, aluminum oxide, starch, saline and equivalent carriers and diluents. To provide for administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein may advantageously contain a total of about 0.1% to 100% by weight of one or more of the compounds of the present invention, based on the weight of the total composition including carrier or diluent.

[0911] Preparations suitable for administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The preparation may be present in unit dose or multi-dose containers (e.g., sealed ampoules and vials), and may be stored under freeze drying (lyophilization) conditions that require only sterile liquid carriers (e.g., water for injection) before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, etc. It should be understood that, in addition to the ingredients specifically mentioned above, the compositions disclosed herein may also include other conventional agents in the art regarding the type of preparation discussed.

[0912] Compounds disclosed herein and compositions comprising compounds disclosed herein can be delivered to cells by direct contact with cells or via carrier means. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome portion. Another means for delivering compounds disclosed herein and compositions to cells includes attaching the compound to a protein or nucleic acid targeted to be delivered to a target cell. U.S. Patent 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 the composition to be translocated through a biological membrane. U.S. Application Publication No. 20020035243 also describes compositions for transporting biological parts through a cell membrane for intracellular delivery. The compounds may also be incorporated into polymers, examples of which include poly(DL-lactide-co-glycolide) polymers for intracranial tumors; 20:80 molar ratio of poly[bis(p-carboxyphenoxy)propane:sebacic acid] (as used in GLIADEL); chondroitin; chitin; and chitosan.

[0913] The compounds and compositions disclosed herein (including pharmaceutically acceptable salts or prodrugs thereof) can be administered intravenously, intramuscularly or intraperitoneally by infusion or injection. Solutions of active agents or their salts can be prepared in water, optionally mixed with nontoxic surfactants. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations may contain preservatives to prevent microbial growth.

[0914] The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or sterile powder comprising an active ingredient, and the dosage form is suitable for temporary preparation of sterile injectable or infusible solutions or dispersions optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under manufacturing and storage conditions. The liquid carrier or vehicle can be a solvent or liquid dispersion medium, comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and their suitable mixtures. Suitable fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using a surfactant. Optionally, prevention of microbial action can be achieved by various other antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents may include, for example, sugar, buffer or sodium chloride. The extended absorption of injectable compositions can be achieved by including an agent (such as aluminum monostearate and gelatin) that delays absorption.

[0915] Sterile injectable solutions are prepared by combining the desired amount of the compounds and / or agents disclosed herein with the various other ingredients listed above in an appropriate solvent, followed by filtered sterilization as required. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze drying techniques which produce a powder of the active ingredient plus any other desired ingredients present in a previously sterile-filtered solution.

[0916] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.

[0917] The dosage range of the composition is large enough to produce the desired effect of the symptom or condition. The dosage should not be large enough to cause harmful side effects, such as undesirable cross-reactions, allergic reactions, etc. Usually, the dosage will vary with the age, condition, sex and degree of disease of the patient, and can be determined by those skilled in the art. In the case of any contraindications, the dosage can be adjusted by individual physicians. The dosage can vary and can be administered once or multiple times a day for one or more days.

[0918] Also disclosed are pharmaceutical compositions comprising a combination of a compound disclosed herein and a pharmaceutically acceptable carrier. Disclosed herein are pharmaceutical compositions suitable for oral, topical or parenteral administration, comprising a certain amount of a compound. The dosage 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 without causing side effects or morbidity exceeding acceptable levels. Those skilled in the art will recognize that dosage will depend on a variety of factors, including the patient's condition (health), the patient's weight, the type of concurrent treatment (if any), the frequency of treatment, the treatment ratio, and the severity and stage of the pathological condition.

[0919] Also disclosed is a medicine box including one or more containers for containing compounds disclosed herein and / or pharmaceutical compositions containing compounds disclosed herein. The disclosed medicine box may optionally include a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the medicine box includes one or more other components, auxiliary materials or adjuvants as described herein. In one embodiment, the medicine box includes instructions or packaging materials describing how to apply the compound or composition of the medicine box. The container of the medicine box can be any suitable material, such as glass, plastic, metal, etc., and has any suitable size, shape or configuration. In one embodiment, the compounds disclosed herein and / or agents are provided in a medicine box in a solid form (such as tablets, pills or powder forms). In another embodiment, the compounds disclosed herein and / or agents are provided in a medicine box in a liquid or solution form. In one embodiment, the medicine box comprises an ampoule or syringe containing compounds disclosed herein and / or agents in a liquid or solution form.

[0920] Treatment

[0921] The present disclosure provides a method for treating a disease in a subject in need thereof, the method comprising administering a compound disclosed herein and / or a composition containing the compound. In an embodiment, the disease is a genetic disease. As used herein, "genetic disease" refers to any disease or condition caused in whole or in part by a change in a genomic DNA sequence compared to a wild-type sequence. In an embodiment, the compound disclosed herein targets a gene or gene transcript associated with a genetic disease. The treatment of disease and / or disease symptoms can be carried out by a variety of molecular mechanisms (such as those described herein).

[0922] In embodiments, the patient is identified as having or being at risk of having any disease as described herein.

[0923] In embodiments, treating refers to partially or completely alleviating, ameliorating, alleviating, suppressing, delaying the onset of, reducing the severity and / or incidence of one or more symptoms of a subject.

[0924] In an embodiment, a method for changing the expression, activity, or a combination thereof of a target gene in a subject in need is provided, the method comprising administering a compound disclosed herein. In an embodiment, treatment reduces the expression of a target protein from a target transcript. In an embodiment, treatment reduces the level of a target transcript. In an embodiment, treatment causes regulation of the splicing of downstream gene transcripts regulated by target transcripts and / or proteins bound to target transcripts. In an embodiment, regulation of the splicing of downstream gene transcripts increases downstream transcripts and / or downstream protein isoforms associated with a healthy phenotype. In an embodiment, alternative splicing reduces downstream transcripts and / or downstream protein isoforms associated with a disease phenotype.

[0925] In embodiments, treatment results in a decrease in the level of a target transcript and / or expression of a target transcript compared to the average level of the target transcript or target protein in the subject prior to treatment or compared to one or more untreated control individuals with a similar disease.

[0926] In embodiments, treatment results in a decrease in the level of a downstream transcript and / or expression of a downstream gene product associated with a disease phenotype, compared to the average level of the protein in the subject prior to treatment or compared to one or more untreated control individuals with a similar disease.

[0927] In embodiments, treatment results in an increase in the level of a downstream transcript and / or expression of a downstream gene product associated with a healthy phenotype compared to the average level of the protein in the subject prior to treatment or compared to one or more untreated control individuals with a similar disease.

[0928] In embodiments, treatment results in reduced expression of the protein in the tissue of the subject of interest compared to the average level of the protein in the tissue of the subject of interest prior to treatment or compared to one or more untreated control individuals with a similar disease.

[0929] In embodiments, treatment results in increased expression of the alternatively spliced ​​downstream protein in the tissue of the subject of interest compared to the average level of the downstream protein in the tissue of the subject of interest prior to treatment or compared to one or more untreated control individuals with a similar disease.

[0930] In embodiments, treatment results in an increase or decrease in expression of a wild-type downstream protein isoform in a tissue of the subject of interest compared to the average level of the downstream protein in the tissue of the subject of interest prior to treatment or compared to one or more untreated control individuals with a similar disease.

[0931] As used herein, the terms "improve", "increase", "decrease", "lower", etc. 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 a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein. A "control individual" is an individual with the same disease who is approximately the same age and / or sex as the individual being treated (to ensure that the stage of disease in the individual being treated and the one or more control individuals is similar).

[0932] The individual treated (also referred to as a "patient" or "subject") is an individual (fetus, infant, child, adolescent, or adult) who suffers from a disease or has the potential to develop a disease. An individual may suffer from a disease mediated by abnormal gene expression or abnormal gene splicing. In embodiments, an individual suffering from a disease may have a downstream protein expression or activity level that is less than about 1% to less than about 99% of the normal wild-type protein expression or activity level in an individual not suffering from the disease. In embodiments, an individual may have a downstream protein expression or activity level that is more than about 10% or more than about 500% higher than the normal wild-type target protein expression or activity level in an individual not suffering from the disease.

[0933] Nucleotide repeat expansion

[0934] In an embodiment, a method for treating a disease associated with nucleotide repeat expansion is provided. The disease may be associated with trinucleotide repeat expansion, tetranucleotide repeat expansion, pentanucleotide repeat expansion, hexanucleotide repeat expansion, and dodecanucleotide repeat expansion and includes, but is not limited to:

[0935]

[0936] See Paulson, H. (2018) “Repeat expansion disease,” Handb. Clin. Neurol. 147: 105-123.

[0937] In embodiments, a method for treating a disease associated with a trinucleotide repeat expansion is provided. In embodiments, the trinucleotide repeat expansion is carried out in the 3' untranslated region of a gene / transcript. In embodiments, the trinucleotide repeat expansion is a CTG.CUG expansion. In embodiments, a method for treating myotonic dystrophy (DM1) is provided.

[0938] In embodiments, a method for treating myotonic dystrophy type 1 (DM1) by reducing the sequestration of at least one RNA binding protein to a pre-mRNA comprising at least one expanded CUG repeat is provided. In embodiments, a method for treating DM1 by reducing the accumulation of a pre-mRNA comprising at least one expanded CUG repeat is provided. In embodiments, a method for treating DM1 by correcting splicing defects of downstream gene transcripts is provided.

[0939] In embodiments, treatment results in a decrease in the number of CUG repeat RNA nuclear foci of the target gene compared to the average foci level in the subject prior to treatment or compared to one or more untreated control individuals with a similar disease.

[0940] All publications, patents and patent applications mentioned in this specification are indicative of the skill levels of those skilled in the art to which the invention pertains. All publications, patents and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference.

[0941] Example

[0942] Example 1: Synthesis method

[0943] method

[0944] The synthesis of oligonucleotides was carried out on a Nitto UnyLinker carrier 195 μmol / g at a 10 μmol scale. Three sets of ACGT monomers were used. PMO 6'-imidamide was purchased from Granlen Co., Ltd. PMO 6'-phosphoramidate was purchased from Hongene Biotech. Reverse DNA or RNA imidamide was purchased from ChemGenes or Hongene Biotech. For sequences starting with PMO, the first monomer was a PMO imidamide. All solid phase syntheses were carried out starting from the 5' end using a Mermade 6 automated synthesizer. PMO fragments were synthesized on a larger scale in 11 mL glass vials or 25 mL glass peptide synthesizers with sinter.

[0945] The first morpholino monomer is added to the solid support: 1.0g of UnyLinker 195μmol / g loading support is placed in a glass peptide synthesis container with a sintered glass support. After the trityl removal and acetonitrile washing steps, a solution of A-PMO imide ester (600mg, 0.77mmol, 4 equivalents) monomer in acetonitrile (0.1M, 7mL) and solid 5-ethylthio 1H-tetrazole (ETT) (200mg, 1.53mmol) are added. The container is capped and stirred at room temperature for 30 minutes. After the solvent is discharged, the coupling step is repeated. Oxidation is carried out using 0.05M iodine-water (10%)-pyridine (90%) solution until no more discoloration occurs. Acetonitrile washing removes traces of oxidizing solution.

[0946] Detritylation solution (CYTFA) : 100 mM 4-cyanopyridine, 100 mM TFA in dichloromethane:trifluoroethanol:ethanol (80:20:1).

[0947] Neutralizing solution (N) : 5% DIPEA in dichloromethane:isopropanol (3:1).

[0948] Coupling solution (C) : 1.6 g of PMO monomer, 9.0 mL of DMI, 1.0 mL of NEM.

[0949] Washing solution (W) : Dichloromethane.

[0950] Storage solution(S) : 30% trifluoroethanol in dichloromethane.

[0951] Table 1. Conditions for manual PMO synthesis.

[0952]

[0953]

[0954] Add DNA monomer

[0955] Reverse DNA imidase is added to the terminal PMO monomer: The carrier (100 mg) is weighed in a 4 mL sintered Mermade 6 column and placed in a vacuum manifold for solid phase extraction. The trityl group is removed using CYTFA (6x10 min) and neutralization solution (4x5 min). The column is transferred to a Mermade 6 for coupling with the first imidase (3x10 min), followed by sulfurization with 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) solution (2x3 minutes). The synthesis of the ASO fragment is continued in an automated manner and terminated with a dimethoxytrityl (DMT) protecting group.

[0956] 10-15 μmol scale ASO synthesis (

[0957] Washing: Acetonitrile

[0958] Deblocking: 3% trichloroacetic acid in DCM

[0959] The imidate solution is a 0.1M acetonitrile solution.

[0960] The activator solution is a 0.2 M solution of benzyl-S-tetrazole in acetonitrile.

[0961] Oxidizing agent: 0.05M iodine, 10% water, 90% pyridine

[0962] Sulfidation reagent: 0.1 M DDTT in pyridine

[0963] Capping solution A: 10% lutidine, 10% isobutyric anhydride, 80% ACN

[0964] Capping solution B: 16% 1-methylimidazole, 85% THF

[0965] Automated synthesis

[0966] initialization:

[0967] ACN wash, 2x1.5mL

[0968] Run steps:

[0969] Deblocking, 4x1.2mL

[0970] ACN wash, 4x1.5mL

[0971] Coupling, 2x800uL of imidoester solution + 800uL of activator (3 minutes for DNA, 10 minutes for RNA, reverse DNA, LNA)

[0972] ACN wash, 1.5 mL

[0973] Oxidation, 2x1000uL of oxidant solution or 1000uL of sulfide solution

[0974] ACN wash, 1.5 mL

[0975] Cap, 2x (A 400uL + Cap B 400uL)

[0976] ACN wash, 2x1.5mL

[0977] termination:

[0978] Deblocking, 4x1.2mL

[0979] ACN wash, 4x1.5mL

[0980] Deprotection and purification

[0981] In order to simplify IEX purification, retain trityl. The suspension of the solid support in 15mL of 1M1,8-diazabicyclo [5.4.0] 11-7-ene (DBU) anhydrous acetonitrile (ACN) solution was vibrated at room temperature for 1.5 hours. The clear solution was poured out, and the process was repeated twice with fresh DBU solution. The support was rinsed with anhydrous ACN, dried and placed in a 30mL flask containing 15ml concentrated ammonia. The flask was heated at 50°C overnight. The solution was filtered and analyzed by MALDI and IEX-HPLC. Purification produces 3'-trityl protected oligonucleotides. The sample was desalted using a Sephadex column, collected in a 50mL Falcon tube and vacuum dried.

[0982] The solid was dissolved in water (2 mL) and 20% H3PO4 (40 uL) was subsequently added. The resulting suspension was placed in a heated oscillator at 50 ° C for 30 minutes. Concentrated aqueous ammonia (1 mL) was added and the suspension was filtered and then purified on IEX-HPLC. The fractions containing the deprotected product were pooled and desalted on a dextran gel. Filtration and evaporation provided a fully deprotected PMO-DNA spacer polymer.

[0983] A solution of diethylamine (20%) in ACN was passed through the support (8 mL) over 2 minutes. The support was air dried and placed in a 30 mL flask for ammonia deprotection. Concentrated ammonia (9 mL) was added, and the suspension was placed at 50 ° C overnight. The flask was cooled to -20 ° C, and then an aqueous solution of methylamine (9 mL, 40%) was added. The flask was heated at 50 ° C for another 6 hours. Analyzed by HPLC and MALDI, followed by IEX-HPLC purification. The fractions containing the deprotected product were pooled and desalted on a dextran gel. Filtration and evaporation provided a fully deprotected PMO-DNA spacer polymer.

[0984] result

[0985] A common misunderstanding is that synthetic oligonucleotides can only be prepared in 3' to 5' directions. Although not widely used, reverse ribose 5'-phosphoimidate is commercially available and has been used by others before (Bhagat et al. J.Med.Chem.2011,54,3027-3036, the reference is incorporated herein by reference, as fully described herein). Reverse DNA imidates and its 2'-F, 2'-OMe, 2'-OMOE congeners are available. The compatibility of PMO and phosphoroimidate (PS) chemistry is tested as template using 20 nucleotide (nt) long human DMPK sequence 5'-ACAGACAATAAATACCGAGG-3'. The length allows 5-10-5 interval polymers, 20nt long PMO comparative studies and test different chimeric designs. The synthesis of PMO 6'-flank domains is realized by manually adding PMO monomers to solid carrier B ( Fig.18 and Fig.19). The 3'-terminal secondary amino group is deprotected with acid and neutralized to prepare for the addition of 5'-cyanophosphorimidate. The first DNA monomer is coupled to the secondary amino group of the morpholino ring so as to form a stable thiophosphoramidate linkage. The DNA-PS is then extended in an automated manner using a standard DNA synthesis protocol to construct a spacer sequence. Standard deprotection conditions (3% TCA in DCM solution) do not seem to affect the integrity of the diaminophosphoroester linkage even after repeated additions of DNA monomers. After the DNA interval is completed, the second PMO wing (flank) is added. In order to make a smooth transition between the DNA-PS domain and the PMO 3'-flank, PMO 6'-phosphorimidate P (III) is used as a "pin" or the first PMO nucleotide. Surprisingly, PMO synthesis is unsuccessful, and oligonucleotides with N-acetyl truncations are detected, rather than complete constructs. Acetyl protection and capping groups may have migrated to more nucleophilic secondary amino groups. Cytidine N-acetyl protecting groups are replaced with N-benzoyl. In order to slow down the migration of O-acyl groups to secondary amines, the steric volume of the cap A solution was increased by replacing the acetic anhydride with isobutyric anhydride. A similar strategy was previously reported to prevent N-acetylation of "fast deprotection" nucleobases (Q. Zhu, MO Delaney, M. Geenberg Bioorganic & Medicinal Chemistry Letters 11, 2001, 1105-1107, which is incorporated herein by reference as if fully described herein). Therefore, simple cap A modification allows the completion of PMO 3'-flank synthesis. Commercially available P(III)PMO-G monomers exhibit low coupling efficiency. The addition of molecular sieves improves yields as well as newly prepared cyanoethyl phosphoramidites. The addition of P(V)PMO-G monomers also results in lower coupling yields. It is worth noting that the 1st and 2nd generation PMO-G ( Fig.14 ) has two nucleobase protecting groups that contribute to steric hindrance. Alternatives may include a single protected PMO-G ( Fig.15 ) and allyl, methyl or benzyl as replacements for cyanoethyl phosphorimidate. For testing, a hybrid design spacer 6'-PMO-DNA-RNA-3' was used, meaning that the 3'-flank would consist of either 2'-OMOE or 2'-OMe RNA modifications that are characteristic of the 2nd generation ASO design.

[0986] In addition to the introduction of the PMO (III) monomer at the first position of the 3'-flank, similar substitutions in the first base of the 5'-flank allow the use of the universal solid support C ( Fig.16 The solid supports used in the synthesis of trityl-based PMOs were decorated with secondary amine anchors (PEG3 (Sarepta) A or sarcosine (Genetools) B). Fig.16 ). A fragment of the anchor is still present at the 6' end of the final product after ammonia deprotection. Switching to P(III) chemistry allows the synthesis of "clean" 6'-OH terminated oligonucleotides.

[0987] The 6'-conjugation chemistry can be obtained via conventional phosphorimidate methodology.

[0988] Example 2: Condition screening

[0989]

[0990]

[0991] Example 3: Oligonucleotide library

[0992] method

[0993] Oligonucleotide synthesis was performed on a Nitto UnyLinker vector 195 μmol / g at a 10 μmol scale. Three sets of ACGT monomers were used. PMO 6'-phosphoramidate was purchased from Granlen Co., Ltd. PMO 6'-phosphoramidate was from Hongene Biotech or Wuxi. Reverse β-cyanoethyl imide was purchased from ChemGenes or HongeneBiotech. 0.1M DDTT was used as a sulfurizing agent to oxidize the trialkylphosphite intermediate. For sequences starting with PMO, the first monomer was PMO 6'-phosphoramidate. All solid phase syntheses were performed starting from the 5' end using a Mermade 6 automated synthesizer. PMO fragments were manually synthesized in a glass peptide reaction vessel with a sinter or in a 4 mL polyethylene Mermade 6 column. Libraries 1 and 2 were synthesized using 1st and 2nd generation double protected PMO-G. After synthesis, the oligonucleotides were deprotected and IEX-HPLC purification on Avant 25 (Source15Q) and desalting on a dextran gel column (2.5x 25cm). Oligonucleotides are freeze-dried and resuspended in sterile water and concentration is determined by measuring UV absorbance at 260nm. All oligonucleotides are characterized by HPLC (Agilent Technologies 1260Infinity II), MALDI-ToF (BrukerAutoflex Max) and LCMS for molecular weight. LCMA characterization is carried out by Novatia Limited Liability Company.

[0994] HPLC Method:

[0995] Analytical IEX injections were performed on a DNAPac 200 oligonucleotide HPLC column on an Agilent 1260 HPLC using 1 M sodium perchloride in 20% acetonitrile in water, TRIS buffer pH 8.0 and increasing gradient.

[0996] Oligonucleotide Purification:

[0997] exist Trityl ON oligonucleotides were purified preparatively by anion exchange chromatography (IEX-HPLC) on an Avant 25HPLC using a Source 15Q column (10 mm x 200 mm internal diameter), eluting at 8 mL / min with an increasing gradient of 2M NaCl / 20% acetonitrile-water Tris buffer (pH 8.0). Fractions were analyzed by HPLC, pooled, desalted on a Sephadex column and evaporated in a Genevac Elite 2.3 evaporator. We produced oligonucleotides without trityl by adding 60 uL of 20% H3PO4 to a 2 mL aqueous solution of Tr ON oligonucleotides and incubating in a heated oscillator at 50°C for 30 min. The suspension was treated with 1 mL of water containing 28% ammonia, syringe filtered and loaded into a Source 15Q column. Gradient elution provided fully deprotected oligonucleotides.

[0998] The first morpholino monomer is added to the solid support: 1.0 g of UnyLinker 195 μmol / g loading support is placed in a glass peptide synthesis container with a sintered glass support. After the detritylation and acetonitrile washing steps, a solution of A-PMO imide ester (600 mg, 0.77 mmol, 4 equivalents) monomer in acetonitrile (0.1 M, 7 mL) and solid ETT (200 mg, 1.53 mmol) are added. The container is capped and stirred at room temperature for 30 min. After the solvent is discharged, the coupling step is repeated. Oxidation is performed using 0.05 M iodine-water (10%)-pyridine (90%) solution until no more discoloration occurs. The acetonitrile washing removes the remaining oxidation solution.

[0999] Fig.18 Examples of processing and purification of libraries are shown.

[1000] Addition of PMO(V) monomer:

[1001] Detritylation solution (CYTFA ): 100 mM 4-cyanopyridine, 100 mM TFA in dichloromethane:trifluoroethanol:ethanol (80:20:1).

[1002] Neutralizing solution (N): 5% DIPEA in dichloromethane:isopropanol (3:1).

[1003] Coupling solution (C): 1.6 g of PMO monomer, 9.0 mL of DMI, 1.0 mL of NEM.

[1004] Washing solution (W): dichloromethane.

[1005] Storage solution(S): 30% trifluoroethanol in dichloromethane.

[1006] Table 2. Conditions for manual PMO synthesis.

[1007] step step Reagents Time [min] Temperature [℃] 1 washing W 3x1 Room temperature 2 Detritylation CYTFA 6x10 Room temperature 3 washing W 3x1 Room temperature 4 Neutralization N 4x5 Room temperature 5 washing W 3x1 Room temperature 6 Coupling C 1x120 50 7 washing W 3x1 Room temperature

[1008] Conventional imide esters are added to terminal PMO monomers: carrier (100-160mg) is weighed in 4mL sintered Mermade 6 posts and is placed in a vacuum manifold for solid phase extraction. CYTFA (6x10min) and neutralization solution (4x5min) are used to remove trityl. Post is transferred to Mermade 6 to couple with the first deoxyribose phosphorimidate (2x15min), then sulfided with DDTT solution (2x3min). Synthesize DNA-PS at intervals and terminate with DMT-ON in an automated manner.

[1009] Deprotection and purification of the first library

[1010] In order to simplify IEX purification, the trityl group is retained. The suspension of the solid support in 15mL of 1M DBU anhydrous ACN solution is shaken at room temperature for 1.5 hours. The clear solution is poured out and the process is repeated twice with fresh DBU solution. The support is rinsed with anhydrous ACN, dried and placed in a 30mL flask containing 15ml concentrated ammonia. The flask is then heated overnight at 50°C. The solution is filtered and analyzed by MALDI and IEX-HPLC. Purification produces 3'-N-trityl protected oligonucleotides. The sample is desalted on a dextran gel, collected in a 50mL falcon tube and vacuum dried. The resulting solid is dissolved in water (2mL) and 20% H3PO4 (40uL) is subsequently added. The resulting suspension is placed in a heated oscillator at 50°C for 30min. Concentrated ammonia (1mL) is added and the suspension is syringe filtered and then purified on IEX-HPLC. The fractions containing the deprotected product are collected and desalted on a dextran gel. Filtration and evaporation afforded fully deprotected PMO-DNA spacer polymers.

[1011] Deprotection and purification of the second library

[1012] A solution of diethylamine (20%, 8 mL) in ACN was passed through the carrier without DMT over 2 minutes. The carrier was air dried and placed in a 30 mL flask for ammonia deprotection. Concentrated ammonia (9 mL) was added and the suspension was left overnight at 50 ° C. The flask was briefly cooled to -20 ° C and then an aqueous solution of methylamine (9 mL, 40%) was added. The flask was heated at 50 ° C for another 6 hours. Analyzed by HPLC and MALDI, followed by IEX-HPLC purification. The fractions containing the deprotected product were pooled and desalted on a dextran gel. Filtration and evaporation provided fully deprotected PMO-DNA spacer polymers.

[1013] Table 3. Library of 20 nt chimeric oligonucleotides tested in HeLa cells 1.

[1014]

[1015] a ATCG indicates PMO, ATCG indicates DNA, the underlined is 2'-OMe RNA, and the bold underlined is 2'-OMOE RNA.

[1016] Table 4. Library 2 of 20 nt long chimeric oligonucleotides tested in HeLa cells.

[1017]

[1018] a ATCG indicates PMO, ATCG indicates DNA, the underlined is 2'-OMe RNA, and the bold underlined is 2'-OMOE RNA.

[1019] Table 5. Library of 25 nt long chimeric oligonucleotides tested in HeLa cells.

[1020]

[1021] a ATCG indicates PMO, ATCG indicates DNA, the underlined is 2'-OMe RNA, and the bold underlined is 2'-OMOE RNA.

[1022] result

[1023] Determination of the optimal size of the PMO flanks and DNA spacer by in vitro KD in HeLa cells

[1024] Two positive controls were synthesized: sequence oligonucleotide 33 (5'- ACAGA CAATAAATAC CGAGG-3'), (2'-OMe)5-10-5 spacer and (2'-OMOE)5-10-5 spacer control sequence (oligonucleotide 14). They were all nuclear transfected into HeLa cells for 24h. In contrast to sequence 33 (43% KD), oligonucleotide 14 showed 56% DMPK knockdown and was therefore selected as a positive reference in future experiments. The missense sequence (oligonucleotide 15) was selected as a negative control. The first library was a library of 20nt long PMO-DNA-PMO spacers prepared by gradually increasing the number of 6'-PMO wings (flanks) from 2 nucleotides to 10 nucleotides. In contrast, the size of the 3'-PMO flank was initially limited to two nucleotides because it was considered sufficient to provide nuclease protection. The PMO-DNA spacer was initially delivered to HeLa cells at 15μM by electroporation to eliminate differences in uptake rates due to different PS contents. Oligonucleotide 1 and oligonucleotides 9, 10, 11, 12 and 13 with high PMO content in one or both flanks showed increased knockdown compared to 56% of the positive control spacer sequence (oligonucleotide 14) (Table 3). Oligonucleotide 2, which differs from control oligonucleotide 33 by the additional two PMOs in the left flank, showed similar knockdown. PMO replacement of the last two 3' flank nucleotides (oligonucleotide 3) resulted in reduced activity (29%) compared to oligonucleotide 14. Similarly, the low PMO content and wing size in oligonucleotides 4, 5, 6 and 7 proved insufficient to knockdown DMPK.

[1025] The requirement for two-step purification of the first library prompted the use of 2'-O-MOE nucleotides in the 3'-flank. Most of the neutral PMO is located in the 6'-flank, so the effect of replacing the two nucleotides in the 3'-flank on the total oligonucleotide charge is negligible. The role of the second library (Table 4) is to detect the effect of gradually increasing the 6'-flank from 6 PMO nucleotides (ON) to 15 and gradually reducing the 3'-flank from five 2'-O-MOE nucleotides to two. The size of the DNA-PS interval will be shortened from 10 nucleotides in oligonucleotide 1 to 3 nucleotides in oligonucleotide 25. RNase H activity improves with an interval of at least 7 charged nucleotides. In fact, oligonucleotides 22, 23, 24 and 25 with short intervals of 7-3nt in length showed a knockdown from 46% to 11%. In contrast, oligonucleotides 16, 17, 18, 19, 20 and 21 with interval sizes of 8 or 10 provide the highest KD equal to or better than the control oligonucleotide 14.

[1026] Libraries of 25 nt long gapmers were prepared (Table 5). The gap size was maintained at the level of RNase H activity mentioned above, e.g., 8-10 nt, while the PMO flank size was varied between 10 nt and 15 nt.

[1027] Liposomal delivery of PMO-DNA hybrids-DMPK knockdown in HEK293 cells.

[1028] Thermal denaturation studies

[1029] The binding affinity of an oligonucleotide can be indicative in determining its therapeutic potential. Factors such as charge, the presence of hydrophobic groups, hydrogen bonding, sugar folds, etc. can affect the binding energy. Conformationally restricted nucleotides have been developed to improve the binding affinity of ONs by increasing entropy. Some synthetic ONs can increase binding affinity, while stereo-random thiophosphates reduce it. In addition to the backbone type, the number of modifications and their position within the sequence also appear to play a key role in determining binding affinity. Morpholino imidophosphorothioates and isoelectronic N3'-P5'phosphorothioates and sulfur analogs show increased binding to complementary RNA. Thiophosphorothioate morpholino oligonucleotides (TMOs) show reduced binding to DNA (-12.6°C) and increased binding to RNA targets (+10°C). In contrast, alternating morpholino thiophosphorothioates and DNA-PS subunits increase the binding affinity to DNA and RNA (6-10°C). It is assumed that the alternation of subunits can increase the flexibility of otherwise rigid ON, thereby allowing effective binding to complementary strands. Encouraged by these findings, the duplex formation properties of PMO-DNA chimeras for complementary RNA were studied (Tables 3 to 5). The data presented in Table 3 show that the increase in thermal stability is related to the size of the 6'PMO wing. The number of PMO modifications increased from 2 to 10 (oligonucleotides 4-13) caused a moderate Tm increase (+2.8°C or +0.35°C per base). For oligonucleotides 20, 21, 22, 23, 24 and 25, the number of PMOs increased from 9 to 15 (Table 4) to change Tm by +4.9°C or +0.82°C per base. For iso-sequence 25nt long spacers (oligonucleotides 30, 31, 32) (Table 5), the change of PMO from 13 to 15 increased Tm by +1.4°C or +0.7°C per base. In contrast, reducing the size of the 2'-MOE 3' wing from 5 to 2 in oligonucleotides 16, 17, 18, 19 and 21 reduced the Tm by -4.0°C or -1.33°C per base, despite increasing the size of the 6' PMO wing by the same number of nucleotides. The same reduction in 2'-MOE size in a series of 25nt long spacers (oligonucleotides 27, 28, 29 and 30) caused a more modest decrease of -2.0°C or -0.67°C per base. The fully PMO-modified oligonucleotide 26 showed a Tm of 61.7°C, similar to the positive control (5-10-5) 2nd generation spacer sequence 126 (60.9°C). These results indicate that the introduction of PMOs in a block manner does not alter the RNA binding properties of the PMO-DNA chimeras.

[1030] Activation of RNase H by PMO-DNA(PS) hybrids

[1031] The mechanism that enables oligonucleotides to exert biological activity is complex. Compared with steric blockers, RNase H dependent oligonucleotides can inhibit protein expression by targeting virtually any region of mRNA. RNase H degradation is catalytic and a single ASO can directly degrade multiple copies of target mRNA. On the contrary, steric blocking oligonucleotides can only bind and target a single RNA molecule. In order to verify whether PMO-DNA hybrids can direct RNase H, heteroduplexes are prepared with synthetic 5'-fluorescein-labeled RNA, and annealed with PMO-ASO oligonucleotides or DNA control sequences (oligonucleotide 33) at room temperature. Prepare RNase H buffer (1mM MgCl2, 50mM KCl, 10mM Tris-HCl pH 8.5). The heteroduplex and URNase H1 (Sigma Aldrich, MI) at a concentration of 100nM in RNase H buffer are mixed and incubated at 37°C at different time points. The enzymatic reaction is terminated by heat inactivation at 65°C for 20 minutes, and then quickly frozen in dry ice. RNase H activity was determined using gel electrophoresis using commercially available Invitrogen EX E-gels (4% agarose, Sybrgold).

[1032] 3′-Exonuclease (Snake Venom Phosphodiesterase I, SVPDE) Sensitivity Assay

[1033] To determine the exonuclease stability of the hybrid oligonucleotides in situ, snake venom phosphodiesterase I (Crotalus Adamanteus) was used to demonstrate the sequential cleavage of phosphodiester bonds starting from the 3' position. Phosphodiester oligonucleotides (such as PO linkages containing DNA) are digested by SVDPE. In contrast, by designing a control sequence (oligonucleotide 33) with a PS linkage, only little or no SVPDE cleavage should occur. For enzymatic hydrolysis, the (13.3 mM) oligonucleotide hybrids were incubated at 37°C in a reaction mixture containing 100 mM Tris-HCl buffer (pH 8.5), 14 mM MgCl2, 72 mM NaCl and SVPDE enzyme (0.1 U / mL). Aliquots (45 mL) of the time points were heat inactivated (95°C) and snap frozen in dry ice until analysis by IEX-HPLC. The second generation spacer (oligonucleotide 33) experienced detectable peak broadening and degradation within 23h. The large oligonucleotide peak at 14min retention time degraded to a small peak over time after being subjected to exonuclease conditions. SVPDE is Rp selective, which explains why the sequence (oligonucleotide 33) degradation slowed down after each nucleotide hydrolysis. No peak broadening was observed for (oligonucleotide 3), but some detectable degradation may occur due to the presence of impurities. The combination of morpholino units and thiophosphates produces exceptional nuclease stability. The replacement of the terminal thiophosphate in the PMO-ASO hybrid (oligonucleotide 6) with a nonionic diaminophosphorothioate represents a further increase in nuclease stability.

[1034] in conclusion

[1035] PMO-DNA-RNA hybrids can be synthesized using solid phase methods and commercially available nucleotides. Structurally diverse libraries have been prepared and evaluated in vitro and in vivo. These spacer polymers provide stability, resulting in increased knockdown of mRNA targets and support for RNase H activity. PMO-DNA spacer polymers maintain or outperform the activity of 2nd generation ASOs. Reducing the overall charge not only improves pharmacological properties, but also simultaneously allows for reduced aggregation of conjugates with positively charged peptides. PMO chimeras can be used in other platforms such as DNA enzymes, siRNAs, or steric blocking oligonucleotides (to name a few).

[1036] Example 4: Testing of exemplary libraries

[1037] method

[1038] The synthesis of the library disclosed in this example was prepared as described above. In vitro DMPK knockdown using ASO / PMO hybrids was tested in vitro using HeLa cells at a dose of 15 uM. Cells were nucleofected with spacer polymers using the Lonza nucleofection kit and incubated for 24 hours. Oligonucleotide 1 (Table 6), which showed significant knockdown, was used as a reference.

[1039] Table 6

[1040]

[1041] ATCG indicates PMO, ATCG indicates DNA, the underlined is 2′-OMe RNA, and the bold underlined is 2′-OMOE RNA.

[1042] Table 7

[1043]

[1044] ATCG indicates PMO, ATCG indicates DNA, underlined is 2'-OMe RNA, and bold underlined is 2'-OMOE RNA.

[1045] Table 8

[1046]

[1047] ATCG indicates PMO, ATCG indicates DNA, underlined is 2'-OMe RNA, and bold underlined is 2'-OMOE RNA.

[1048] Table 9

[1049]

[1050] ATCG indicates PMO, ATCG indicates DNA, underlined is 2'-OMe RNA, and bold underlined is 2'-OMOE RNA.

[1051] ACTG =2'-MOE RNA ACTG=PMO NA=at NT level

[1052] Table 10

[1053]

[1054] result

[1055] Fig. 20 Results for mRNA expression following electroporation of 15 μM of the 20-mer oligonucleotides from Table 6 are shown.

[1056] Fig.21Shown are qPCR results of mRNA expression after electroporation of 15 μM of oligonucleotides from Table 7.

[1057] Tables 8 to 10 show the percentage of uptake of the hybrid oligonucleotide in the presence and absence of liposomes.

[1058] The mRNA expression level decreased with increasing PMO length until it reached the 10M-8-2 pattern.

[1059] Example 5: DMPK knockdown

[1060] Fig. 22 , Fig.23 , Fig.24 and Fig.25 Results of DMPK knockdown in HeLa cells for the oligonucleotides shown in Tables 6 to 11 are shown (free or liposome delivered). Briefly, HeLa cells in a 24-well format were treated with OPTI-MEM medium containing PMO / ASO hybrids for 48 hours. No significant toxicity was observed except for the peptide control at 100 μM (data not included). In the case of liposome transfection, all PMO / ASO hybrids and ASO spacers showed the target KD. For free uptake, ASO and PMO / ASO hybrid spacers showed KD, but EEV-conjugate spacers did not show KD ( Fig.25 ).

[1061] Table 11

[1062]

[1063] ATCG indicates PMO, ATCG indicates DNA, underlined indicates 2′-OMe modified RNA, and bold underlined indicates 2′-OMOE modified RNA.

[1064] EEV1: CPP12-Q-PEG12-K(Mal)

[1065] EEV2: Ac-CPEG12-K(cyclo(FfΦRrRrQ))-NH2

[1066] Peptide 1: Cyclic (FfΦRrRrQ)-PEG8-OH

[1067] Example 6: Examples of siRNA

[1068] Table 12

[1069]

[1070] ACUG=2'-OMe RNA; ACTG = PMO; actg = 2'-F RNA; SS = sense strand; AS = antisense strand

[1071] The pairing of siRNA duplexes is:

[1072] Oligonucleotide 42 and Oligonucleotide 47

[1073] Oligonucleotide 44 and Oligonucleotide 46

[1074] Oligonucleotide 43 and Oligonucleotide 46

[1075] Oligonucleotide 42 and Oligonucleotide 46

[1076] Oligonucleotide 45 and Oligonucleotide 46

[1077] Example 7: ASO-PMO / RNA duplex is a substrate for RNase H

[1078] method

[1079] RNase H 1, 5, 8, 10, 20 or 40 U activity (buffer 10 mM Tris-HCl, 1 mM MgCl2, 50 mM KCl)

[1080] 5.6ug, diluted to 65uL or 85uL

[1081] Incubate at 37°C for 1 hour, 8 minutes, or 16 minutes

[1082] The proteins were heat-shocked at 65°C and then reacted on e-gel (4% agarose) for 15 minutes with 10 uL per reaction.

[1083] ASO / PMO is oligonucleotide 11 (5'-ACAGACAATAAATACCGAGG-3'):

[1084] result

[1085] Fig.28 This indicates that RNase H is active in the PMO-ASO spacer duplex, but not in the DNA / RNA control or the PMO / RNA control.

[1086] Fig.29 RNase H cleavage of the PMO-ASO but not the DNA / RNA control duplex is demonstrated with increasing RNase units.

[1087] Fig.30RNase H cleavage of the control of the experimental duplex is shown. RNA / PMO-ASO duplexes were prepared using fluorescent RNA (25-mer) and annealed to the PMO-ASO sequence. The duplexes were incubated in 8U RNase H buffer, at various time points, aliquots were removed, and heat shocked (65°C) to inactivate the RNase H protein. The products were visually observed using gel electrophoresis (E-gel, 4% agarose). The duplexes with short intervals showed little degradation (oligonucleotides 25 and 23).

[1088] Example 8: Stability under snake venom phosphodiesterase digestion

[1089] Fig.31 Snake venom phosphodiesterases were shown to digest oligonucleotides from the 3' end. The hydrolase snake venom phosphodiesterase (SVPDE Eastern Diamondback Crotalus) metabolizes extracellular nucleotides that are often involved in intercellular signaling. Hydrolysis occurs at the 3' end and releases the 5' nucleotide.

[1090] Fig.32 This indicates that the ASO-PMO hybrid is stable in the presence of SVPDE.

[1091] Fig.33 Shown are IEX-HPLC data of SVDPE assays using a control gapmer (oligonucleotide 37) and a phosphodiester (oligonucleotide 38) with a phosphorothioate (PS) backbone.

[1092] Example 9: Stability in the context of phosphodiesterase II

[1093] method

[1094] Phosphodiesterase II (5'-exonuclease) bovine spleen phosphodiesterase (Sigma, 10 U).

[1095] Use the following for digestion:

[1096] 1.4 nmol oligonucleotide (oligonucleotide 21: 5'-ACAGACAATAAATACCGA GG -3')

[1097] 100 mM ammonium acetate

[1098] 1mM EDTA

[1099] 1 mM Tween 80, pH 6-7

[1100] Final enzyme concentration 10mU / μL

[1101] At 37°C, the cells were collected at various time points, heat inactivated at 65°C and snap frozen.

[1102] result

[1103] Fig.34 Data are presented showing that PMO-ASO (oligonucleotide 21) is stable during phosphodiesterase II digestion.

[1104] Fig.35 Data for PO and PS controls are shown.

[1105] Example 10: Other hybrids

[1106] Oligonucleotide 48 5'- GCU AUU ACC UUA ACC CAG -3'

[1107] Oligonucleotide 49 5'-GCT AUU ACC UUA ACC CAG -3'

[1108] Oligonucleotide 50 5'-GCT ATT ACC UUA ACC CAG -3'

[1109] Oligonucleotide 51 5'-GCT ATT ACC UUA ACC CAG -3'

[1110] Oligonucleotide 52 5'-GCT ATT ACC TTA ACC CAG -3'

[1111] Oligonucleotide 53 5'-GCT ATT ACC TTA ACC CAG -3'

[1112] Oligonucleotide 54 5'-GCT ATT ACC TTA ACC CAG-3

[1113] ACUG =2'-OMe RNA; ACTG = PMO

[1114]

[1115] Control: 5'- ACAGA CAATAAATAC CGAGG -3'

[1116] ACUG =2'-OMe RNA; ACTG=PMO; ACTG=2'-OMOE RNA; ATCG=DNA

[1117] Example 11: Examples of Oligonucleotides, Linkers and Nucleosides

[1118] Figure 5 and Figure 6A sample of a hybrid gapmer oligonucleotide is shown.

[1119] Fig.36 Shown are hybrid gapmer oligonucleotides having 5' and 3' flanks linked by phosphorothioate linkages and a central spacer region comprising DNA P(III) nucleotides linked by phosphorothioate linkages.

[1120] Fig.10 Shown are TR or DMT protected P(III) morpholine analogs.

[1121] Fig.11 DMT-protected reverse DNA P(III) nucleotide analogs are shown.

[1122] Fig.12 DMT protected 2' modified reverse RNA P(III) nucleotide analogs are shown.

[1123] Fig.13 Shown are DMT protected 2' modified reverse RNA P(III) nucleotide analogs with methylated C and U bases.

[1124] Fig.14 A Tr protected P(V) morpholino nucleobase analog with a doubly protected G base is shown.

[1125] Fig.15 Tr protected P(V) morpholino nucleobase analogs are shown.

[1126] Figure 7 Hybrid sense and antisense strands of siRNA and the nucleotides and linkages therein are shown.Any combination of nucleotides and linkages can be used to prepare a hybrid PMO oligonucleotide.

[1127] Example 12: Examples of Guide and Follower RNA

[1128] The PMO can be at the end of the guide (antisense) or follower (sense) strand or in the middle of the guide or follower strand ( ACUG =2'-OMe RNA; ACTG=PMO; actg=2'-F RNA).

[1129] Follower 3'-TT U u A g A g U u U u A g U a G g G -5'

[1130] Guide 5'-a A aA u C u C a A a A u CA u C c C u-3'

[1131] Zui 3'-TT U u A g A g U u U u A g U a G g G -5'

[1132] Direction 5'-a A a A u C u C a A a A u CA u C cCU-3'

[1133] Zui 3'- T t U u A g A g U UUu A g U a G g G -5'

[1134] Direction 5'-a A a A u C u C a A a A u CA u C c C u-3'

[1135] 3'-TtUuAgAgUuUuAgUaGgG-5'

[1136] Direction 5'-a A a U c U c A a A a U CAu C c C u-3'

[1137] Zui 3'- T tU u A g A g U u U u A g U a G GG-5’

[1138] Guide 5’-a A a U c U c A a A a U CAu C c C u-3’。

Claims

1. A method for preparing a hybrid oligonucleotide, the method comprising assembling P(III) and P(V) nucleotides in a 6' to 3' or 5' to 3' direction on a support and using PMO 6'-phosphorimidate P(III) or phosphoramidate P(V) as the first nucleotide from the support.

2. The method of claim 1, wherein PMO 6'-phosphorimidate P(III) is the first nucleotide from the vector.

3. The method of claim 1, wherein PMO 6'-phosphoramidate P(V) is the first nucleotide from the vector.

4. The method of any one of claims 1 to 3, wherein the hybrid oligonucleotide comprises: (a) at least one phosphorothioate linkage; and (b) at least one phosphorothioate linkage, phosphodiester linkage, phosphorodiamidate linkage, or a combination thereof.

5. The method of any one of claims 1-4, wherein the method comprises coupling a first reverse amidoester to a secondary amino group of a morpholino ring to form a thiophosphoramidate linkage.

6. The method of any one of claims 1-5, further comprising adding at least one P(III) nucleotide.

7. The method of any one of claims 1 to 6, comprising adding at least two P(III) nucleotides.

8. The method of claim 7, wherein the P(III) nucleotides are consecutive.

9. The method of any one of claims 1-8, further comprising adding at least one P(V) nucleotide.

10. The method of any one of claims 1 to 9, comprising adding at least two P(V) nucleotides.

11. The method of claim 10, wherein the P(V) nucleotides are consecutive.

12. The method of any one of claims 1 to 11, wherein at least two P(III) nucleotides are followed by at least two P(V) nucleotides.

13. The method of any one of claims 1 to 12, wherein at least two P(V) nucleotides are followed by at least two P(III) nucleotides.

14. The method of any one of claims 1 to 13, wherein at least one P(III) nucleotide and one P(V) nucleotide alternate to form a P(III)-P(V)-P(III) or P(V)-P(III)-P(V) motif.

15. The method of any one of claims 1-14, wherein the P(III) nucleotides are identical.

16. The method of any one of claims 1-14, wherein the P(III) nucleotides are different.

17. The method of any one of claims 1-16, wherein the P(V) nucleotides are identical.

18. The method of any one of claims 1-16, wherein the P(V) nucleotides are different.

19. The method of any one of claims 1 to 18, wherein the method comprises deprotecting and neutralizing the 3'-terminal secondary amino group of the first DNA monomer.

20. The method of claim 19, wherein the method comprises further adding one or more other DNA nucleotides to the first DNA nucleotide, wherein two or more DNA nucleotides are linked via phosphorothioate (PS) linkages.

21. The method of any one of claims 19-20, wherein the method further comprises adding a second PMO 6'-phosphorimidate P(III) to the 3' terminal DNA nucleotide of the hybrid oligonucleotide.

22. The method of any one of claims 1 to 21, wherein the hybrid oligonucleotide comprises a gapmer comprising: (i) a 6' flank comprising two or more morpholino nucleotide analogs, or a 5' flank comprising two or more ribonucleotides or ribonucleotide analogs; (ii) a spacer region comprising 5 or more deoxyribonucleotides or deoxyribonucleotide analogs; and (iii) a 3' flank comprising two or more morpholino nucleotide analogues, or a 3' flank comprising two or more ribonucleotides or ribonucleotide analogues.

23. The method of claim 22, wherein the method comprises extending the oligonucleotide by adding one or more further DNA monomers to the first DNA monomer, the one or more further DNA monomers being linked via phosphorothioate bonds (DNA-PS).

24. The method of claim 23, wherein the method further comprises adding PMO 6'-phosphorimidate P(III) to the 3' end of the oligonucleotide.

25. The method of claim 24, comprising adding the PMO 6'-phosphorimidate P(III) after all cytidine-based nucleotides in the 5' flank and the gap are N-benzoyl protected and cap A is based on isobutyric anhydride, pivalic anhydride or benzoic anhydride instead of acetic anhydride.

26. The method of claim 23, wherein the oligonucleotide comprises: 6'-PMO-DNA-PMO-3', 6'-PMO-DNA-RNA-3', 5'-RNA-DNA-PMO-3'.

27. The method of any one of claims 23-26, wherein the nucleosides of the 3' and 5' flanks are linked by an intersubunit linkage.

28. A hybrid oligonucleotide synthesized by the method according to any one of claims 1 to 27.

29. The hybrid oligonucleotide of claim 28, wherein the method further comprises purifying the oligonucleotide.

30. The hybrid oligonucleotide of claim 28 or 29, wherein the method further comprises isolating the oligonucleotide.

31. A hybrid oligonucleotide comprising: (i) a first nucleotide sequence comprising a first 5' or 6' end and a first 3' end, wherein the terminal 3' nucleic acid residue of the first nucleotide sequence is a morpholino nucleotide analog; (ii) a second nucleotide sequence comprising a second 5' or 6' end and a second 3' end, wherein the terminal 5' nucleic acid residue of the second nucleotide sequence is a deoxyribonucleotide or an analog thereof or a ribonucleotide or an analog thereof; and (iii) at least one phosphorothioate linkage connecting the terminal 3' nucleic acid residue of the first nucleotide sequence and the terminal 5' nucleic acid residue of the second nucleotide sequence.

32. The hybrid oligonucleotide of claim 31 , comprising 10 to 50 nucleotides.

33. The hybrid oligonucleotide of claim 31 , comprising 15 to 30 nucleotides.

34. The hybrid oligonucleotide of claim 31 , comprising 20 to 30 nucleotides.

35. The hybrid oligonucleotide of claim 31, wherein the first nucleotide sequence comprises two or more morpholino nucleotide analogs.

36. The hybrid oligonucleotide of claim 31, wherein the terminal 3' nucleic acid residue of the first nucleic acid sequence is a P(III) morpholino nucleotide analog.

37. The hybrid oligonucleotide of claim 31, wherein the terminal 3' nucleic acid residue of the first nucleic acid sequence is a P(V) morpholino nucleotide analog.

38. A hybrid oligonucleotide as described in claim 35, wherein the two or more morpholino oligonucleotide analogs are linked by a phosphorodiamidate linkage.

39. The hybrid oligonucleotide of claim 31, wherein the first nucleotide sequence further comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof or ribonucleotides or analogs thereof or combinations thereof.

40. The hybrid oligonucleotide of claim 31, wherein the second nucleotide sequence comprises two or more deoxyribonucleotide analogs, two or more ribonucleotide analogs, or a combination thereof.

41. The hybrid oligonucleotide of claim 31, wherein the deoxyribonucleotide analog or ribonucleotide analog is selected from: a P(III) DNA analog, a reverse p(IIII) DNA analog, a P(III) RNA analog, a reverse P(III) RNA analog, or a combination thereof.

42. A hybrid oligonucleotide as described in claim 31, wherein the hybrid oligonucleotide comprises a 2'-modified ribonucleotide analog selected from the following: 2'-O-methyl (2'-OMe) P(III) RNA, reverse 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, reverse 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA or a combination thereof.

43. The hybrid oligonucleotide of claim 31, wherein the two or more deoxyribonucleotides or analogs, two or more ribonucleotides or analogs thereof, or a combination thereof are each linked by an internucleotide linkage.

44. A hybrid oligonucleotide as described in claim 43, wherein the internucleotide linkage is a phosphorothioate, phosphodiester or phosphorodiamidate linkage.

45. The hybrid oligonucleotide of claim 31, wherein the second nucleotide sequence further comprises one or more PMOs.

46. ​​The hybrid oligonucleotide of claim 31, wherein the P(III)PMO is selected from the group consisting of: P(III)PMO-C, P(III)PMO-G, P(III)PMO-A, P(III)PMO-T, or a P(III)PMO with a non-naturally occurring nucleobase.

47. The hybrid oligonucleotide of claim 31, wherein the (PV)PMO comprises: P(V)PMO-C, P(V)PMO-G, P(V)PMO-A, P(V)PMO-T, or a P(V)PMO with a non-naturally occurring nucleobase.

48. The hybrid oligonucleotide of any one of claims 31-47, wherein less than 75% of the internucleotide linkages have a negative charge.

49. The hybrid oligonucleotide of any one of claims 31-47, wherein less than 50% of the internucleotide linkages have a negative charge.

50. The hybrid oligonucleotide of any one of claims 31-47, wherein less than 25% of the internucleotide linkages have a negative charge.

51. A hybrid oligonucleotide as described in any one of claims 31-50, comprising a gapmer, wherein: (i) the first nucleotide sequence comprises a 5' flank of the gapmer, wherein the 5' flank comprises 2 to 20 morpholino nucleotide analogs; (ii) the second oligonucleotide sequence comprises a spacer oligonucleotide and a 3' flank of the spacer polymer, wherein the spacer oligonucleotide sequence comprises 8 to 20 deoxyribonucleotides or ribonucleotide analogues and the 3' flank comprises 2 to 20 deoxyribonucleotides or ribonucleotide analogues.

52. The hybrid oligonucleotide of claim 51, wherein the 5' flank comprises 2 to 10 morpholino nucleotide analogs.

53. The hybrid oligonucleotide of claim 51, wherein the 5' flank comprises 2 to 5 morpholino nucleotide analogs.

54. The hybrid oligonucleotide of claim 51, wherein the 5'-flanking morpholino nucleotide analog is linked via a phosphorodiamidate linkage.

55. The hybrid oligonucleotide of claim 51, wherein the first 5' or 6' end of the 5' flank comprises a pin residue comprising PMO 6'-phosphorimidate P(III).

56. The hybrid oligonucleotide of claim 51, wherein the 3' flank comprises 2 to 10 morpholino nucleotide analogs.

57. The hybrid oligonucleotide of claim 51, wherein the 3' flank comprises 2 to 5 morpholino nucleotide analogs.

58. The hybrid oligonucleotide of claim 51, wherein the 3'-flanking morpholino nucleotide analog is linked via a phosphorodiamidate linkage.

59. The hybrid oligonucleotide of claim 51, wherein the second 5' or 6' end of the 3' flank comprises a pin residue comprising PMO 6'-phosphorimidate P(III).

60. The hybrid oligonucleotide of claim 51, wherein the gap comprises at least 8 consecutive deoxyribonucleotide analogs.

61. The hybrid oligonucleotide of claim 51, comprising 10 or fewer morpholino nucleotide analogs.

62. The hybrid oligonucleotide of claim 51, comprising 7 or fewer morpholino nucleotide analogs.

63. The hybrid oligonucleotide of claim 51, wherein at least 50% of the deoxyribonucleotide analogs of the spacer oligonucleotide sequence are linked by phosphorothioate linkages.

64. The hybrid oligonucleotide of claim 51, wherein the spacer oligonucleotide sequence is linked to the 3' flank via a phosphorothioate linkage.

65. The hybrid oligonucleotide of claim 51, wherein the 5' flank comprises at least one deoxyribonucleotide analog or at least one ribonucleotide analog.

66. The hybrid oligonucleotide of claim 51, wherein the 5' flank comprises at least one ribonucleic acid analog selected from the group consisting of locked nucleic acid (LNA), 2' methoxy nucleic acid (2'-methoxy; 2'-OMe), 2' methoxyethyl nucleic acid (2'-O-(methoxyethyl; 2'-OMOE), 2' fluoro ribonucleic acid analog (2'-F), or a combination thereof.

67. The hybrid oligonucleotide of claim 51, wherein the 5' flank comprises at least one 2' fluoro (2'-F) ribonucleotide.

68. The hybrid oligonucleotide of claim 51, wherein the 5' flank comprises at least one deoxyribonucleotide analog.

69. The hybrid oligonucleotide of claim 51, wherein the 3' flank comprises at least one deoxyribonucleotide analog or at least one ribonucleotide analog.

70. The hybrid oligonucleotide of claim 69, wherein the 3' flank comprises at least one ribonucleic acid analogue selected from the group consisting of a locked nucleic acid (LNA) analogue, at least one 2' methoxy nucleic acid analogue (2'-methoxy; 2'-OMe), at least one 2' methoxyethyl nucleic acid analogue (2'-O-(methoxyethyl; 2'-OMOE), at least one 2' fluoro ribonucleic acid analogue (2'-F), or a combination thereof.

71. The hybrid oligonucleotide of claim 69, wherein the 3' flank comprises at least one 2' fluoro (2'-F) RNA.

72. The hybrid oligonucleotide of claim 69, wherein the 3' flank comprises at least one deoxyribonucleotide analogue.

73. The hybrid oligonucleotide of claim 69, wherein the spacer sequence comprises at least two 2' modified nucleotides linked by a phosphorothioate linkage.

74. A hybrid oligonucleotide comprising: (i) at least one P(III) morpholino nucleotide analog; (ii) at least one P(V) morpholino nucleotide analog; (iii) at least one P(III) ribonucleotide, at least one P(III) deoxyribonucleotide analog, or a combination thereof; (iv) at least one phosphorodiamidate linkage as well as (v) at least one phosphorothioate Key link.

75. A hybrid oligonucleotide as described in claim 74, wherein each upstream nucleotide adjacent to each P(V) morpholino nucleotide analogue is a P(III) or P(V) morpholino nucleotide analogue.

76. A hybrid oligonucleotide as described in claim 74 or 75, comprising two or more consecutive P(V) morpholino nucleotide analogues connected by diamidophosphorothioate linkages.

77. A hybrid oligonucleotide as described in any one of claims 74 to 76, wherein any P(V) morpholino nucleotide analogue is linked to any downstream nucleotide that is not a P(V) morpholino nucleotide analogue via a phosphorothioate aminothioate linkage.

78. The hybrid oligonucleotide of any one of claims 74-77, comprising at least one 2' modified P(III) ribonucleotide.

79. The hybrid oligonucleotide of any one of claims 74-78, further comprising at least one phosphorothioate linkage.

80. The hybrid oligonucleotide of claim 74, wherein the oligonucleotide is selected from the group consisting of a gapmer, a splice switch oligonucleotide, a sense strand of an siRNA, an antisense strand of an siRNA, a deoxyribozyme, a steric blocking antisense oligonucleotide (ASO), and an immunomodulatory oligonucleotide.

81. A compound comprising: a hybrid oligonucleotide as described in any one of claims 28-80, and Cyclic peptide of formula (I) or its protonated form, in: R1, R2 and R3 are each independently H or a residue of tyrosine, phenylalanine or tryptophan; R4 and R6 are independently H or an amino acid side chain; AA SC is the amino acid side chain; q is 1, 2, 3, or 4; and Each m is independently an integer from 0 to 3.

82. The compound of claim 81, wherein the structure of formula (I) has the structure of formula (I-1) or (I-2): or its protonated form, in: AA SC is an amino acid side chain; and Each m is independently an integer from 0 to 3.

83. The compound of claim 81 or 82, wherein the cyclic peptide is:

84. The compound of any one of claims 81-83, wherein the AA SC Conjugation to a linker.

85. The compound of claim 84, wherein the linker comprises: (i)-(OCH2CH2) z -subunit, wherein z is an integer from 2 to 20; (ii) one or more amino acid side chains, such as the side chains of glycine, b-alanine, 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminopentanoic acid, or combinations thereof; or (iii) A combination of (i) and (ii).

86. The compound of claim 84 or 85, wherein the linker has the structure: in: x is an integer from 2 to 20; y is an integer from 1 to 5; and z is an integer from 2 to 20.

87. The compound of any one of claims 84-86, wherein an exocyclic peptide (EP) is conjugated to the linker.

88. The compound of claim 87, wherein the exocyclic peptide comprises 2 to 10 amino acid residues.

89. A compound comprising: a hybrid oligonucleotide as described in any one of claims 28-80, and EEV of formula (B): or its protonated form, in: R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; R4 and R7 are independently H or an amino acid side chain; EP is an exocyclic peptide as defined herein; Each m is independently an integer from 0 to 3; n is an integer from 0 to 2; x' is an integer from 1 to 20; y is an integer from 1 to 5; q is 1-4; and z' is an integer from 1 to 23.

90. The compound of claim 89, wherein the EEV comprises a structure of formula (B-1), (B-2), (B-3), or (B-4): or its protonated form.

91. The compound of claim 89 or 90, wherein the EEV comprises: Ac-PKKKRKVAEEA-K(cyclo[FGFGRGRQ])-PEG 12 -OH or Ac-PK-KKR-KV-AEEA-K(cyclo[GfFGrGrQ])-PEG 12 -OH.

92. The compound of claim 89 or 90, wherein the EEV comprises 93. The compound of claim 89 or 90, wherein the EEV comprises 94. A pharmaceutical composition comprising a compound as described in any one of claims 81-93.

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