Hybrid Oligonucleotide

Abstract

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

Description

Background Art

[0001] Cross - reference to related applications This application claims the benefit of 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, and the disclosure of each of these is hereby incorporated by reference in its entirety.

[0002] The recognition of specific RNA target nucleic acid sequences via complementary base - pairing oligonucleotides has been investigated as a method for treating various diseases and disorders. Both single - stranded and double - stranded oligonucleotides with various chemical modifications are designed to regulate gene expression, for example, via antisense mechanisms such as RNA interference (RNAi), gapmers, or steric blocks.

[0003] Two classes of internucleotide linkages are now predominant in clinically significant oligonucleotides: linkages with charged backbones such as phosphodiester and phosphorothioate (PS) (Stec et al., J. Am. Chem. Soc. 106(20), 1984, 6077 - 6079), and neutral linkages such as phosphorodiamidate linkages of morpholino oligonucleotide analogs where the ribose sugar of the nucleobase is replaced with a morpholino ring (PMO). PMO is widely used as a steric - block oligonucleotide that does not require RNase H activity (including, for example, as a splice - modulating oligonucleotide).

[0004] A gapmer comprises an oligonucleotide construct that includes a central gap region of approximately 8 to 12 nucleotides that supports RNase H activity and two flanking regions (also referred to as "wings") of approximately 1 to 5 modified nucleotides that provide stability and increase affinity for RNA. Typically, gapmers contain phosphorothioate (PS) linkages throughout the length of the oligonucleotide. The nucleotides in the central gap region typically contain phosphorothioate (PS)-linked DNA nucleotides, and the flanking wings typically contain sugar modifications linked by phosphorothioate (PS) linkages, such as 2'-O-methylation (2'-OMe), 2'-O-methoxyethyl (2'-MOE), or locked nucleic acid (LNA). In addition to supporting RNase H activity, the gap region is thought to facilitate transfection by dynamic covalent interactions with membrane proteins (Laurent et al., Angew. Chem. Int. Ed. 2021, 60, 1-6). Eight or nine continuous PS linkages in the gap 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 to 13 anionic PS linkages are thought to facilitate electrostatic and hydrophobic interactions with amino acid side chains, for example, for protein binding (Hyjek-Skladanowska, et al., (2020) J. Am. Chem. Soc., 142, 7456-7468). However, there is increasing evidence that PS is involved in unwanted interactions with cellular proteins that can lead to toxicity.

[0005] The selective chemical substitution of nucleotides in the gap region has been investigated to improve the therapeutic index and reduce the toxicity of gapmer oligonucleotides. Since many proteins prefer to bind to the 5’ wing (side) of the gapmer, it has been found that hydrophilic or neutral linkages at positions 2 to 4 from the 5’ end of the gap region tend to reduce toxicity. Other designs investigated have introduced unique chemistries into the side regions of the gapmer, such as charged N3’-P5’ or neutral phosphoryl guanidinium (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).

[0006] The use of neutral linkages, such as methylphosphonate, has also been reported but was found to have reduced solubility in aqueous solution and was associated with helix destabilization and synthetic challenges (Guo et al., Phosphorus, Sulfur and Silicon 1999, Vol 144-146, 363-366).

[0007] Recently, mixed backbone gapmers have been proposed to generate antisense oligonucleotides (ASOs) with lower toxicity without compromising activity in the central nervous system (CNS). A prodrug concept based on charge-neutralized phosphotriester modifications has also been proposed (Meade et al., Nat Biotechnol. 2014 32(12), 1256-1261).

[0008] Despite the individual success of morpholino and DNA-PS containing oligonucleotides, the chemical synthesis of oligonucleotide hybrids incorporating both morpholino and DNA-PS subunits is hampered by the synthetic strategies currently used to generate PMOs (Heera et al., J. Am. Chem. Soc., 142, 38:16240 (2020)). PMOs are typically prepared in the 6′ to 3′ direction on a polystyrene resin (Paul and Marvin, J. Am. Chem. Soc., 138:15663 (2016)), which is incompatible with the standard methods for the chemical synthesis of natural DNA / RNA or most analogs prepared in the stepwise 3′ to 5′ direction. (Figure 1). Also, PMO synthesis typically uses P(V) chemistry, whereas DNA or RNA synthesis uses P(III) phosphoramidite chemistry. For these and other reasons, PMO and DNA syntheses are considered to be incompatible.

[0009] Recent attempts in the synthesis of PMO-ASO chimeras include phosphorothioamidate (also referred to as phosphorothioate) morpholino (TMO)-oligonucleotides that combine a PS-charged linkage and a morpholino ring in the backbone. The synthesis of these oligonucleotides is achieved in the 3’ to 5’ direction by sequential addition of morpholino 3’-phosphorodiamidate P(III) nucleotides (Langner, et al., J. Am. Chem. Soc. 2020, 142(38), 16240-16253). Caruthers reported antisense oligonucleotides that can specifically hybridize to a target region in an exon of the human dystrophin gene to induce exon skipping, which are 8 to 50 nucleotides in length and contain at least 8 to 10 consecutive nucleotides complementary to the target region in the human dystrophin gene exon. The TMO contains a morpholino subunit, and 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, or to a TMO / DNA chimera by a nucleotide internucleotide linkage containing thiophosphate, and at least one nucleotide base contains a base other than uracil (Figures 2 and 3).

[0010] For example, there is still a need for a robust and flexible synthetic route to generate chimeras containing morpholino nucleotides and other therapeutically relevant modifications for use in new therapeutic agents with improved biological properties. SUMMARY OF THE INVENTION

[0011] The present disclosure provides a method of making a hybrid oligonucleotide, comprising assembling P(III) and P(V) nucleotide nucleotides on a support in the 6’ to 3’ or 5’ to 3’ direction, and using a PMO 6’-phosphoramidate P(III) or phosphoramidate P(V) as a first nucleotide from the support.

[0012] The present disclosure provides hybrid oligonucleotides synthesized by the methods of the present disclosure.

[0013] The present disclosure also provides (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 that is a morpholino nucleotide analog; (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 that is a deoxyribonucleotide or an analog thereof, or a ribonucleotide or an analog thereof; and (iii) at least one phosphorothioamidate linkage that links the terminal 3' nucleic acid residue of the first nucleotide sequence and the terminal 5' nucleic acid residue of the second nucleotide sequence and provides a hybrid oligonucleotide comprising the same.

[0014] The present disclosure also provides (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

Chemical formula

Chemical formula

[0015] In an embodiment, each 5'-nucleotide adjacent to each P(V) morpholino nucleotide analog is a P(III) or P(V) morpholino nucleotide analog.

[0016] In an embodiment, the hybrid oligonucleotide comprises two or more contiguous P(V) morpholino nucleotide analogs linked via phosphorodiamidate linkages.

[0017] In an embodiment, any P(V) morpholino nucleotide analog is linked via a phosphorothioamidate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0018] The present disclosure also relates to a compound comprising the hybrid oligonucleotide of the present disclosure and a cyclic peptide of formula (I):

Chemical formula

[0019] The present disclosure also relates to a compound comprising the hybrid oligonucleotide of the present disclosure and an EEV of formula (B):

Chemical formula

[0020] The present disclosure further relates to a composition comprising a compound of the present disclosure and a method of treating a disease with such a compound and composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

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Chemical formula

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Chemical formula

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Chemical formula

Chemical formula

Mode for Carrying Out the Invention

[0022] Although not wishing to be bound by theory, the disclosed hybrid oligonucleotides may have advantages including, but not limited to, reduced toxicity and increased uptake of free (non-formulated) oligonucleotides, for example, as a result of a decrease in the charge of the hybrid oligonucleotide and stabilization of the oligonucleotide termini from enzymatic degradation, compared to, for example, oligonucleotides consisting only of phosphorothioate (PS) or only of phosphorodiamidate (PMO). The properties of hybrid oligonucleotides having PMO compared to oligonucleotides having a phosphorothioate (PS)-only backbone or phosphoromorpholino oligonucleotides (PMO) are shown in FIG. 5. Hybrid oligonucleotides are useful for treating a variety of genetic diseases including, but not limited to, cancer, immune diseases or disorders, and neuromuscular diseases or disorders.

[0023] Definitions As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes a mixture of two or more such compositions, reference to “an agent” includes a mixture of two or more such agents, reference to “the component” includes a mixture of two or more such components, and the like.

[0024] As used herein, "oligonucleoside" refers to an oligonucleotide in which the internucleotide linkage does not contain a phosphorus atom.

[0025] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. The linkages in "oligonucleotides" may or may not contain a phosphorus atom. In embodiments, oligonucleotides are composed of natural and / or modified nucleobases, sugars, and covalent internucleotide linkages. In embodiments, oligonucleotides include, but are not limited to, one or more P(V) morpholino nucleotide analogs; one or more P(III) morpholino nucleotide analogs; and one or more deoxyribonucleotides (DNA) or ribonucleotide (RNA) analogs, including 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 embodiments, RNA nucleotides are 2'-modified RNA nucleotides. In embodiments, oligonucleotides contain one or more phosphorus-containing internucleotide linkages selected from phosphoramidates, phosphorodiamidates, phosphorothioamidates, phosphorothioates, phosphodiesters, phosphotriesters, methylphosphonates, and combinations thereof. In embodiments, oligonucleotides contain one or more non-phosphorus-containing internucleoside linkages selected from methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-), and combinations thereof. In embodiments, oligonucleotides contain one or more phosphorus-containing internucleotide linkages and one or more non-phosphorus-containing internucleotide linkages.

[0026] As used herein, the term "2'-modified" nucleotide refers to a nucleotide in which the ribose sugar contains a substituent at the 2'-position other than H or OH. 2'-modified monomers include 2'-substituents such as allyl, amino, azide, thio, O-allyl, O-C1-C 10 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-O-N(R m )(R n ), or O-CH2-C(=O)-N(R m )(R n )(wherein each R m and Rn is independently H or substituted or unsubstituted C1-C 10 alkyl), and include but are not limited to BNA and monomers (e.g., nucleosides and nucleotides). Examples of 2'-modified monomers include 2'-O-methyl (2'-OMe) P(III) RNA, inverted 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, inverted 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, 2'-fluoro (2'-F) P(III) RNA, inverted 2'-fluoro (2'-F) P(III) RNA, 2'-O-ethyl (cET) P(III) nucleotides, inverted 2'O-ethyl (cET) P(III) nucleotides, or combinations thereof, but are not limited thereto.

[0027] As used herein, the term "hybrid oligonucleotide" refers to an oligonucleotide containing at least one nucleotide, nucleoside, internucleotide linkage, or combination thereof that is different as compared to at least one other nucleotide(s), nucleoside(s), or internucleotide linkage(s) within the same oligonucleotide. In embodiments, hybrid oligonucleotides include nucleosides that differ at isolated positions. In embodiments, hybrid oligonucleotides include nucleosides that are grouped together in a region. In embodiments, the grouped nucleosides define a particular motif. As used herein, the term "mixed backbone oligonucleotide" refers to an oligonucleotide in which at least one internucleotide linkage is different from at least one other internucleotide linkage of the oligonucleotide.

[0028] As used herein, the term "gapmer" refers to a hybrid oligonucleotide that includes a central gap region that supports RNase H activity as well as upstream and downstream flank regions (also referred to as "wings"). In embodiments, the upstream and downstream flank regions increase oligonucleotide stability and the affinity of the gapmer for its target nucleic acid sequence. A "stereorandom gapmer" is a gapmer that possesses a mixture of (R) or (S) configurations at each of its stereocenters. In some embodiments, a stereorandom gapmer is a product from an extension reaction with morpholino or deoxyribonucleoside monomers. A "stereodefined gapmer" is a gapmer that possesses an (R) or (S) stereochemical configuration at each of its stereocenters, and the configuration is controlled. A stereodefined gapmer can be a product from a stereospecific extension reaction with a stereopure morpholino or deoxyribonucleoside monomer, and the phosphorus stereochemistry of the gapmer is controlled as an array of defined stereochemical (R) or (S) configurations.

[0029] The terms "mini-PEG", "PEG2", and "AEEA" are used interchangeably herein to refer to 2-[2-[2-aminoethoxy]ethoxy]acetic acid.

[0030] A "PMO-gapmer" is a gapmer that includes a flank region comprising morpholino monomers linked to each other by phosphorodiamidate linkages. The flank region may also be referred to as the wing region.

[0031] When referring to a reaction, "stereorandom" means that the reaction was carried out without preference for the resulting stereochemistry.

[0032] "R" and "S" are terms that represent isomers and are descriptors of the stereochemical configuration at asymmetrically substituted atoms including, but not limited to, carbon, sulfur, phosphorus, and quaternary nitrogen. The designation of an asymmetrically substituted atom as "R" or "S" is made by application of the Cahn-Ingold-Prelog priority rules, as is well known to those skilled in the art and as described in the International Union of Pure and Applied Chemistry (TUPAC) Rules for the Nomenclature of Organic Chemistry. Section E, Stereochemistry.

[0033] As used herein, the terms "upstream" and "downstream" refer to relative positions in an oligonucleotide. Naturally occurring oligonucleotides have a 5' end and a 3' end, which are named with respect to the carbon positions on the 5-membered deoxyribose (or ribose) ring. By convention, upstream refers to the relative position to the 5' end of the oligonucleotide, and downstream refers to the relative position to the 3' end of the oligonucleotide. For oligonucleotides containing nucleotides with 6-membered rings, such as morpholino nucleotide analogs, the "upstream" end of the oligonucleotide is referred to herein interchangeably as either the 6' end or the 5' end. The 3' end of an oligonucleotide containing a morpholino ring is the downstream end.

[0034] As used herein, the term "nucleobase" refers to a portion of a nucleoside or nucleotide capable of hydrogen bonding to a nucleobase of another nucleic acid. A nucleobase can include any atom or group of atoms capable of hydrogen bonding. In embodiments, the nucleobase is a nitrogenous base. A natural nucleobase is an unmodified nucleobase from its naturally occurring form found in RNA or DNA, which includes, for example, cytosine (C), guanine (G), adenine (A), thymine (T) (in DNA) or uracil (U) (in RNA). Modified DNA nucleobases include 5-methylcytidine (5mC). Modified RNA nucleobases include, but are not limited to, pseudouridine (Ψ), dihydrouridine (D), inosine (I), ribothymidine (rT), and 7-methylguanosine (m7G).

[0035] As used herein, the term "nucleoside" refers to a molecule that includes a nucleobase and a sugar but lacks a phosphate group. Nucleosides include, but are not limited to, natural nucleosides, non-base nucleosides, modified nucleosides, and nucleosides having mimetic bases and / or sugar groups. A "natural nucleoside" or "unmodified nucleoside" is a nucleoside that includes a natural nucleobase and a natural sugar. Natural nucleosides include RNA and DNA nucleosides.

[0036] As used herein, the term "nucleotide" refers to a molecule that includes a nucleobase, a sugar, and an internucleotide linkage. In embodiments, the internucleotide linkage includes a phosphate group. Nucleotides can be modified with any of a variety of substituents, which can include, for example, modified nucleobases, modified sugars, modified phosphate groups, or combinations thereof. A modified sugar is a sugar moiety modified from its naturally occurring form found in RNA (2'-OH) or DNA (2'-H), which includes ribose sugars having a 2'-modification and nucleotides in which the sugar moiety is replaced with an analog, such as a morpholine ring, but is not limited thereto. In embodiments, the phosphate group can be linked to the 2', 3' or 5'-hydroxyl moiety of the sugar. In embodiments, where the modified nucleoside includes a six-membered ring, such as a morpholino ring, the phosphate group can be linked to the 2', 3' or 6'-hydroxyl moiety. Modified nucleotides can be referred to herein as "nucleotide analogs", for example, modified deoxyribonucleotides can be referred to as deoxyribonucleotide analogs and modified ribonucleotides can be referred to as ribonucleotide analogs.

[0037] As used herein, the term "heterocyclic base moiety" refers to a nucleobase that includes a heterocycle.

[0038] As used herein, "inter-nucleotide linkage" refers to a covalent bond between adjacent nucleosides. Naturally occurring DNA and RNA contain 3'-to-5' phosphodiester (PO) inter-nucleotide linkages. Modified inter-nucleotide linkages can be used to alter, e.g., increase, the nuclease resistance of oligonucleotides as compared to natural phosphodiester bonds. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing inter-nucleotide linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphorothioamidate phosphoramidate, phosphorothioamidate, and phosphorothioate. Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, non-naturally occurring inter-nucleotide linkages include, but are not limited to, phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS) and phosphorylguanidine linkages.

[0039] A "morpholino" or "morpholino nucleotide analog" is a modified nucleotide analog that includes a base-pairing nucleobase (Pi), e.g., a puridine or pyrimidine base-pairing nucleobase, wherein the sugar moiety of the nucleotide is replaced with the morpholine ring shown below:

Chemical formula

[0040] A "morpholino oligonucleotide" is a polymeric molecule capable of hydrogen bonding to a target nucleic acid sequence, where the polymer contains at least one morpholino nucleotide analog coupled to an adjacent nucleotide or nucleotide analog via nitrogen in the morpholine ring. In embodiments, the morpholino oligomer includes morpholino nucleotide analogs linked by (thio)phosphorodiamidate linkages, and the morpholino nitrogen of one subunit is linked to the 5' or 6' carbon of an adjacent nucleotide or nucleotide analog. Morpholino oligomers are described, for example, in 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).

[0041] A "phosphorodiamidate" group includes phosphorus having two bonded oxygen atoms and two bonded nitrogen atoms, and may also refer herein to phosphorus having one bonded oxygen atom and three bonded nitrogen atoms. In the internucleotide linkages of the oligomers described herein (e.g., between the flank region and the gapmer), as shown in Formula II below, the first nitrogen is typically attached to the backbone chain and the second nitrogen is a ring nitrogen in the morpholine ring structure. Alternatively, or in addition, the nitrogen may be present at the 5'-exocyclic carbon as shown in Formulas III and IV below.

Chemical formula

[0042] In a thiophosphorodiamidate linkage, one oxygen atom, typically the oxygen attached to the backbone in the oligomers described herein, is replaced by sulfur.

[0043] 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, for example, FIGS. 29 and 30. P(V) nucleotides include nucleotides in which the phosphorus group is attached to the 5' or 6' carbon of a sugar (or sugar analog) and further nucleotides in which the phosphorus group is attached to the 3' carbon of a sugar (or sugar analog). A P(V) nucleotide in which the phosphorus group is attached to the 3' carbon of a sugar (or sugar analog) is referred to herein as an "inverse P(V) nucleotide".

[0044] As used herein, a "P(III)" nucleotide is a nucleotide in which the phosphorus group has three bonds. Examples of P(III) nucleotides include, but are not limited to, P(III)PMO (FIGS. 29 and 30), P(III)DNA, and P(III)RNA (FIGS. 29 and 31). P(III) nucleotides include nucleotides in which the phosphorus group is attached to the 5' or 6' carbon of a sugar (or sugar analog) and further nucleotides in which the phosphorus group is attached to the 3' carbon of a sugar (or sugar analog). A P(III) nucleotide in which the phosphorus group is attached to the 3' carbon of a sugar (or sugar analog) is referred to herein as an "inverse P(III) nucleotide".

[0045] As used herein, the term "mimetic" refers to a group that is substituted for a sugar, nucleobase, and / or internucleotide linkage in an oligonucleotide. Typically, mimetics are used in place of a sugar or a combination of sugar-internucleotide linkages, and the nucleobases are maintained for hybridization to a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics for combinations of sugar-internucleotide linkages include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral linkages. In some examples, mimetics are used in place of nucleobases. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those of skill in the art.

[0046] As used herein, the term "bicyclic nucleoside" or "BNA" refers to a nucleoside that includes a bridge connecting two atoms on the furanose ring of the nucleoside, thereby forming a bicyclic ring system. BNAs include, but are not limited to, α-L-LNA, β-D-LNA, ENA, oxyamino BNA (2'-O-N(CH3)-CH2-4'), and aminooxy BNA (2'-N(CH3)-O-CH2-4').

[0047] As used herein, the term "4'-to-2' bicyclic nucleoside" refers to a BNA in which a bridge connecting two atoms of the furanose ring bridges the 4' and 2' carbon atoms of the furanose ring, thereby forming a bicyclic ring system.

[0048] As used herein, "locked nucleic acid" or "LNA" refers to a nucleotide in which the 2'-hydroxyl group of the ribose sugar ring is linked via a methylene group to the 4'-carbon atom of the ribose sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage. LNAs include, but are not limited to, α-L-LNA and β-D-LNA.

[0049] "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 "protected by a base" which is bound to the solid support or to the growing oligomer chain on the solid support via its 5' (or 6') exocyclic carbon to prevent reaction or interference of the base pairing group during stepwise oligomer synthesis, and refers to the protection of the base pairing group on the morpholino subunit with a suitable protecting group, for example, the protection of a purine or pyrimidine base.

[0050] 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 embodiments, complementary nucleobases refer to the nucleobases of an oligonucleotide capable of base pairing with the nucleobases of its target nucleic acid. For example, when the nucleobase at a given position of an oligonucleotide is capable of hydrogen bonding with the nucleobase at a given position of the target nucleic acid, then the position of the hydrogen bond between the oligonucleotide and the target nucleic acid is considered complementary for that nucleobase pair.

[0051] As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds with each other or otherwise do not support hybridization.

[0052] As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid via nucleobase complementarity. In embodiments, an oligonucleotide and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bind to each other to enable a stable association between the oligonucleotide and the target. One of ordinary skill in the art will recognize that the inclusion of mismatches is possible without excluding the ability of the oligomeric compound to remain associated. Thus, described herein are oligonucleotides that can contain up to about 20% nucleotides that are mismatched (i.e., not nucleobases complementary to the corresponding nucleotides of the target). In embodiments, the oligonucleotide contains about 15% or less, about 10% or less, 5% or less mismatches, or no mismatches. The remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art will recognize that the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleobase complementary to the target nucleic acid.

[0053] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an oligonucleotide and its target nucleic acid). Without being limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonds between complementary nucleosides or nucleotide bases (nucleobases). For example, the natural base adenine (A) is a nucleobase complementary to the natural nucleobases thymidine (T) and uracil (U) that pair via the formation of hydrogen bonds. The natural base guanine (G) is a nucleobase complementary to the natural bases cytosine (C) and 5-methylcytosine (5mC). Hybridization can occur under a variety of circumstances.

[0054] As used herein, the term "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to a nucleic acid sequence with substantially higher affinity than it hybridizes to another nucleic acid sequence. In embodiments, the oligonucleotide specifically hybridizes to multiple target sites. In embodiments, the oligonucleotide specifically hybridizes to its target under stringent hybridization conditions.

[0055] As used herein, the term "sequence identity", when used in reference to two oligonucleotides, refers to the percentage of residues between two sequences that are the same at the same relative positions. Thus, a sequence has a given percentage of sequence identity when compared to another sequence. For sequence comparison, typically one sequence functions as a reference sequence, and this is compared to a test sequence. One of skill in the art will understand that two sequences are generally considered to be "substantially identical" when they contain the same residues at corresponding positions. In embodiments, sequence identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) in the version that exists as of the filing date. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of needle labeled "longest identity" (obtained using the -nobrief option) is used as the identity rate and is calculated as follows: (identical residues × 100) / (length of alignment - total number of gaps in the alignment).

[0056] In other embodiments, sequence identity can also be determined using the version of the Smith-Waterman algorithm that exists as of the filing date.

[0057] As is well known in the art, sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN, BLASTP, gapped BLAST, and PSI-BLAST as of the filing date. Exemplary such programs are described in 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 programs described above typically provide an indication of the degree of homology.

[0058] The terms “pre-mRNA” and “primary transcript” as used herein refer to newly synthesized eukaryotic mRNA molecules immediately after DNA transcription. Pre-mRNAs are typically capped with a 5′ cap, modified with a 3′ poly-A tail, and spliced to generate a mature mRNA sequence.

[0059] As used herein, the terms "target nucleic acid" and "target nucleic acid sequence" refer to a nucleic acid sequence to which a hybrid oligonucleotide binds or hybridizes. Target nucleic acids include, but are not limited to, RNA (including but not limited to pre-mRNA, mature mRNA, or mRNA including portions thereof), genomic DNA, cDNA derived from such RNA, and non-coding RNA, such as miRNA. In embodiments, the target nucleic acid can be a cellular gene (or mRNA transcribed from such a gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.

[0060] As used herein, the terms "splicing" and "processing" refer to the post-transcriptional modification of pre-mRNA in which introns are removed and exons are joined. Splicing occurs in a series of reactions catalyzed by a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs) called the spliceosome. Within an intron, a 3' splice site, a 5' splice site, and a branch site are required for splicing. The RNA components of the snRNPs can interact with the intron and participate in catalysis.

[0061] "Target pre-mRNA" is pre-mRNA that includes a target nucleic acid sequence to which an oligonucleotide hybridizes.

[0062] "Mature target mRNA" is an mRNA sequence resulting from the splicing of a target pre-mRNA sequence. In embodiments, the mature target mRNA encodes a functional protein. In embodiments, the mature target mRNA does not encode a functional protein. In embodiments, the mature target mRNA retains one or more intron sequences.

[0063] The term "target gene" refers to the gene encoding the target pre-mRNA.

[0064] "Target protein" refers to the amino acid sequence encoded by the target mRNA. In embodiments, the target protein may not be a functional protein.

[0065] As used herein, the term "expression" refers to all of the functions and processes by which the information encoded by a gene is converted into a structure that is present and functional in a cell. Such structures include, but are not limited to, the products of transcription and translation, such as proteins.

[0066] "Wild-type target protein" refers to the native, functional protein isoform produced by the "normal" or non-mutated version of the wild-type of the target gene.

[0067] "Respliced target protein", as used herein, refers to a protein encoded by an mRNA resulting from splicing of the target pre-mRNA to which an oligonucleotide hybridizes. The respliced target protein can be identical to the wild-type target protein, homologous to the wild-type target protein, a functional variant of the wild-type target protein, or an active fragment of the wild-type target protein.

[0068] As used herein, the term "modulate" refers to a change in function or activity as compared to the level of function or activity prior to modulation. For example, modulation includes either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.

[0069] The terms "inhibit", "inhibiting", or "inhibition" refer to a decrease in activity, expression, function, or other biological parameter, which may include, but need not require, complete ablation of the activity, expression, function, or other biological parameter. Inhibition may include, for example, at least about 10% reduction in activity, response, condition, or disease as compared to a control. In embodiments, the expression, activity, or function of a gene or protein is decreased by a statistically significant amount. In embodiments, the activity or function is decreased by at least about 10%, about 20%, about 30%, about 40%, about 50%, and up to about 60%, about 70%, about 80%, about 90%, or about 100%.

[0070] "Reduce" or other forms of the term, e.g., "reducing" or "reduction", mean a decrease in an event or feature. This is typically with respect to some standard or expected value, i.e., it is relative, although it is understood that it need not always be with respect to a stated standard or relative value.

[0071] As used herein, "treating", "treatment", "treat", and variations thereof refer to any administration of a disclosed compound that partially or completely alleviates, ameliorates, relieves, delays the onset of, reduces the severity of, and / or reduces the frequency of one or more symptoms or features of a disease described herein. With respect to a patient, the term "treatment" refers to the medical management of a patient intended to cure, ameliorate, stabilize, or prevent a disease, pathologic condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward the improvement of a disease, pathologic condition, or disorder, and also includes causal treatment, i.e., treatment directed toward the removal of the cause of the related disease, pathologic condition, or disorder. This term also includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, pathologic condition, or disorder; prophylactic treatment, i.e., treatment directed toward minimizing or partially or completely inhibiting the development of a related disease, pathologic condition, or disorder; and symptomatic treatment, i.e., treatment used to supplement another specific therapy directed toward the improvement of a related disease, pathologic condition, or disorder.

[0072] The term "therapeutically effective" refers to an amount of a disclosed compound and / or composition sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration need only involve a reduction or modification and need not necessarily involve elimination.

[0073] The term "pharmaceutically acceptable" refers to those compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with human and / or animal tissue without excessive toxicity, irritation, allergic response, or other problems or complications within the scope of reasonable medical judgment and commensurate with a reasonable benefit / risk ratio.

[0074] As used herein, the term "cap structure" or "terminal cap site" refers to a chemical modification incorporated at either end of an oligonucleotide.

[0075] As used herein, "subject" refers to an individual to be treated, and can include domesticated animals (such as cats, dogs, etc.), livestock (such as cows, horses, pigs, sheep, goats, etc.), laboratory animals (such as mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" can also include mammals, such as primates or humans. Thus, a subject can be a human or a veterinary patient. The term "patient" refers to a subject under the treatment of a clinician, such as a physician.

[0076] Mechanism of action of antisense oligonucleotides (ASO) In embodiments, the hybrid oligonucleotide is a single-stranded oligonucleotide. In embodiments, the hybrid oligonucleotide is a double-stranded oligonucleotide duplex comprising a sense and an antisense strand. In embodiments, the sense and antisense strands of the double-stranded oligonucleotide duplex are of the same length (i.e., there are no overhangs 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).

[0077] In embodiments, the hybrid oligonucleotide is complementary to the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide has a sequence that is complementary to the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide is sufficiently complementary to the target nucleic acid sequence. In embodiments, the hybrid oligonucleotide is not sufficiently complementary to the target nucleic acid sequence (i.e., the hybrid oligonucleotide sequence may contain one or more mismatches). The target nucleic acid sequence can be a coding or non-coding sequence. In embodiments, the target nucleic acid sequence includes DNA. In embodiments, the target nucleic acid sequence includes chromosomal DNA. In embodiments, the target nucleic acid sequence includes 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.

[0078] In embodiments, hybridization of the hybrid oligonucleotide to its target nucleic acid sequence modulates one or more aspects of protein transcription, translation, and expression. In embodiments, hybridization of the hybrid oligonucleotide to its target nucleic acid sequence blocks access to the target nucleic acid sequence by cellular machinery, such as splicing elements. In embodiments, hybridization of the hybrid oligonucleotide to its target nucleic acid sequence results in degradation of the target oligonucleotide, such as degradation of mRNA transcripts via RNase H. Other mechanisms are known and are reviewed by Agrawal, S. (1996) “Antisense Oligonucleotides: towards clinical trials.” Trends Biotechnol. 14(10):376-387.

[0079] In an embodiment, the hybridization of the hybrid oligonucleotide to the target nucleic acid sequence suppresses the expression of the target protein. In an embodiment, the hybridization of the hybrid oligonucleotide to the target nucleic acid sequence downregulates the expression of one or more wild-type target protein isoforms. In an embodiment, the hybridization of the hybrid oligonucleotide to the target nucleic acid sequence upregulates the expression of the target protein. In an embodiment, the hybridization of the hybrid oligonucleotide to the target nucleic acid sequence increases the expression of one or more wild-type target protein isoforms.

[0080] In an embodiment, the hybrid oligonucleotide stabilizes a target oligonucleotide, for example, a target mRNA. In an embodiment, the hybrid oligonucleotide increases the half-life of the target oligonucleotide. In an embodiment, the hybrid oligonucleotide increases the half-life of the target mRNA. In an embodiment, the hybrid oligonucleotide increases the expression of the protein product of the target mRNA.

[0081] In an embodiment, the hybrid oligonucleotide is an antisense oligonucleotide (ASO). In an embodiment, the hybrid oligonucleotide is a steric block oligonucleotide, a gapmer, a splice-switching 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 adapter. In an embodiment, the hybrid oligonucleotide is the sense strand of an siRNA. In an embodiment, the hybrid oligonucleotide is the antisense strand of an siRNA.

[0082] The effectiveness of a hybrid oligonucleotide can be evaluated by assessing any detectable and / or measurable activity resulting from hybridization of the hybrid oligonucleotide to its target nucleic acid. Detection, measurement, or both can be direct or indirect. In embodiments, the activity is evaluated by detecting, measuring, or both, the amount of target mRNA. In embodiments, the activity is evaluated by detecting, measuring, or both, the amount of re-spliced mRNA. In embodiments, the activity is evaluated by detecting, measuring, or both, the amount of target protein. In embodiments, the activity is evaluated by detecting, measuring, or both, the amount of target protein isoform.

[0083] Stereo block In embodiments, the hybrid oligonucleotide is a steric block antisense oligonucleotide (ASO). In embodiments, the transcription, translation, or expression of a target protein is regulated via the steric block. See, for example, Scharner and Aznarez (2020) “Clinical Applicatdions of Single-Stranded Oligonucleotides: Current Landscape of Approved and In-Development Therapeutics.” Mol. Therapy. 29(2):540-554. The steric block ASO is a short synthetic single-stranded oligonucleotide having a length in the range of about 8 to about 50, about 15 to about 30, about 20 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Unlike gapmers, steric block ASOs do not induce RNase-H-mediated degradation of the target nucleic acid sequence (e.g., target mRNA). As used herein, a “steric block” oligonucleotide binds to the target nucleic acid sequence via Watson-Crick base pairing and interferes with the binding of a trans-activating factor, such as a small nuclear RNA (snRNA), microRNA (miRNA), or RNA-binding protein, to the target oligonucleotide or prevents the formation of RNA secondary structure.

[0084] Splice switching In an embodiment, the hybrid oligonucleotide is a splice-switching oligonucleotide (also referred to as an exon-skipping oligonucleotide). The splice-switching oligonucleotide is a short synthetic single-stranded oligonucleotide having a length in the range of about 8 to about 50, about 15 to about 30, about 20 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The splice-switching oligonucleotide base pairs with pre-mRNA and disrupts the normal splicing repertoire of the transcript by blocking the RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. It is a type of steric-blocking oligonucleotide. In an embodiment, the splice-switching oligonucleotide does not induce RNase-H-mediated degradation of the target nucleic acid sequence (e.g., target mRNA).

[0085] Since pre-mRNA splicing is required for the proper expression of most protein-coding genes, targeting this process provides a means for manipulating protein production from genes. Splicing modulation can be used to treat diseases caused by mutations that result in disruption of normal splicing or when interfering with the normal splicing process of gene transcripts can be therapeutically beneficial. Such antisense oligonucleotides provide an effective and specific method for targeting and modifying splicing in a therapeutic modality. See, for example, Havens and Hastings (2016) “Splice-switching antisense oligonucleotides as therapeutic drugs.” Nucleic Acids Res. 44(14):6549-6563.

[0086] In embodiments, hybridization of the hybrid oligonucleotide to the target nucleic acid sequence within the target pre-mRNA modulates one or more aspects of pre-mRNA splicing. In embodiments, hybridization of the hybrid oligonucleotide causes one or more exons to be skipped (sometimes referred to as exon skipping). In embodiments, hybridization results in increased or decreased expression or activity of the target protein and / or downstream proteins regulated by the target gene. In embodiments, hybridization of the hybrid oligonucleotide to the target mRNA induces alternative splicing that results in an addition or deletion of nucleotides in the target transcript. In embodiments, hybridization induces alternative splicing that results in an addition or deletion of nucleotides within a single exon of the target transcript. In embodiments, hybridization induces alternative splicing that results in a deletion of nucleotides within a single exon of the target transcript. In embodiments, deletion of nucleotides within a single exon results in translation of a truncated protein. In embodiments, the truncated protein is less toxic to cells than the non-truncated protein.

[0087] As used herein, modulation of splicing refers to modifying the processing of a pre-mRNA transcript such that the spliced mRNA molecule contains either a deletion in one or more exons, or a deletion or addition of a sequence not normally found in the spliced mRNA (e.g., an intron sequence), as a result of different combinations of exons, exon skipping, or exon inclusion. In embodiments, hybridization of a hybrid oligonucleotide to a target pre-mRNA corrects a pre-mRNA sequence that has mutated native splicing. In embodiments, hybridization results in alternative splicing of the target pre-mRNA. In embodiments, hybridization results in exon inclusion or exon skipping of one or more exons. In embodiments, the skipped exon sequence contains a frameshift mutation, a nonsense mutation, or a missense mutation. In embodiments, the skipped exon sequence contains a nucleic acid deletion, substitution, or insertion. In embodiments, the skipped exon itself does not contain a sequence mutation, but the adjacent intron contains a mutation that results in a frameshift or nonsense mutation. In embodiments, hybridization of a hybrid oligonucleotide to a target nucleic acid sequence within a target pre-mRNA prevents inclusion of an intron sequence in the mature mRNA molecule. In embodiments, hybridization results in preferential expression of the wild-type target protein isoform. In embodiments, hybridization results in expression of a respliced target protein that includes an active fragment of the wild-type target protein.

[0088] In embodiments, the respliced target protein can rescue one or more phenotypes or symptoms of a disease associated with the transcription and translation of the target gene. In embodiments, the respliced target protein can rescue one or more phenotypes or symptoms of a disease associated with the expression of the target protein. In embodiments, the respliced target protein is an active fragment of the wild-type target protein. In embodiments, the respliced target protein functions in a manner substantially similar to the wild-type target protein. In embodiments, the respliced target protein causes the cell to function substantially similarly to a cell expressing the wild-type target protein. In embodiments, the respliced target protein does not cure a disease associated with the target gene or target protein, but improves one or more symptoms of the disease.

[0089] In embodiments, the respliced target protein can have one or more improved properties compared to the target protein. In embodiments, the respliced target protein can have one or more improved properties compared to the wild-type target protein. In embodiments, enzyme activity or stability can be enhanced by promoting different splicing of the target pre-mRNA. In embodiments, the respliced target protein can have the same or substantially the same sequence as a wild-type target protein variant having improved properties compared to another wild-type target protein variant.

[0090] In an embodiment, hybridization of the hybrid oligonucleotide to the mRNA target produces an mRNA encoding a truncated protein and / or a non-functional protein, for example, by introducing a frameshift mutation that results in a premature stop codon. In an embodiment, hybridization results in an mRNA encoding a truncated protein and / or a non-functional protein via alternative splicing. In an embodiment, the hybrid oligonucleotide causes degradation of the target transcript, for example, via nonsense-mediated decay. In an embodiment, hybridization of the hybrid oligonucleotide to the mRNA target produces an alternative mRNA isoform having beneficial properties.

[0091] In embodiments, the hybrid oligonucleotide induces the addition or deletion of one or more nucleotides in the resulting processed transcript, e.g., mRNA. If the number of nucleotides added or removed from the open reading frame is not divisible by 3 to obtain an integer, the resulting transcript may be translated into a functional or non-functional protein having more or fewer amino acids than the corresponding protein expressed from the transcript, but otherwise having the same amino acid sequence as the protein expressed from the transcript that would have had no added or removed nucleotides. If the number of nucleotides added or removed from the open reading frame is not divisible by 3 to obtain an integer, the open reading frame of the resulting processed transcript, e.g., mRNA, is shifted. For example, the number of nucleotides added or deleted to induce such a “frameshift” modification 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, the addition or deletion of a non-3-divisible number of nucleotides shifts the reading frame of the resulting processed transcript, e.g., mRNA, downstream of the frameshift. The shifted reading frame can result in nonsense-mediated decay, can result in premature stop codons within the nonsense downstream of the frameshift, and / or can result in the expression of a protein having a completely different sequence of amino acids downstream of the frameshift.

[0092] In embodiments, hybridization of the hybrid oligonucleotide induces the introduction of a premature termination codon (PTC) into the open reading frame. As used herein, a “premature termination codon” is a termination codon that is in frame with the transcription start codon and is located upstream of the physiological termination codon that is in frame with the translation start codon. Target transcripts having a PTC can be destabilized and degraded via various mechanisms including nonsense-mediated decay.

[0093] In an embodiment, the hybrid oligonucleotide induces exon skipping of an exon within the target transcript, where the exon has a number of nucleotides not divisible by 3. In an embodiment, the hybrid oligonucleotide induces exon skipping of an exon having a number of nucleotides not divisible by 3 that results in a premature termination codon (PTC) within the target transcript. In an embodiment, the hybrid oligonucleotide induces exon skipping of an exon having a number of nucleotides not divisible by 3 that results in a PTC within the target transcript that leads to nonsense-mediated decay of the target transcript. In an embodiment, inducing nonsense-mediated decay of the target transcript results in a decreased concentration of the target transcript. In an embodiment, inducing nonsense-mediated decay of the target transcript results in a decreased concentration of the target protein encoded by the target transcript. In an embodiment, inducing nonsense-mediated decay of the target transcript results in increased and / or decreased levels of the protein of a downstream gene controlled by the target gene.

[0094] Gapmer In an embodiment, the hybrid oligonucleotide is a gapmer. A gapmer is a short synthetic single-stranded oligonucleotide 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 in length, which includes a central "gap" region flanked by 3' wings (also referred to herein as 3' flanks) and 5' wings (also referred to herein as 5' flanks) that form adjacent sequences of monomer subunits. The central "gap" 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.

[0095] In embodiments, the gapmer forms a double-stranded RNA-DNA duplex that mimics an endogenous RNA-DNA hybrid that is naturally occurring and recognized and degraded via RNase H-mediated cleavage of an RNA strand that results in RNA degradation, such as during DNA replication, binding to a target RNA sequence in a sequence-specific manner by Watson-Crick base pairing.

[0096] In embodiments, the sugar groups of the 5'-side and 3'-side are different from the sugar groups of the central gap region. In embodiments, the sugar groups of each monomer subunit within the 5'-side are the same. In embodiments, the sugar groups of each monomer subunit within the 3'-side are the same. In embodiments, the sugar groups of each monomer subunit within the central gap are the same. In embodiments, not all of the sugar groups for the monomer subunits within the 5'-side are the same. In embodiments, not all of the sugar groups for the monomer subunits within the 3'-side are the same. In embodiments, not all of the sugar groups for the monomer subunits within the central gap are the same.

[0097] In embodiments, one or more nucleotides in the 3'-side, 5'-side, gap region, or combinations thereof include one or more modifications for increasing the nuclease resistance of the oligonucleotide, for reducing the immunogenicity of the oligonucleotide, for increasing the binding affinity, or combinations thereof. In embodiments, one or more nucleotides in the 5'-side, 3'-side, or both include modifications in the sugar ring of a DNA or RNA nucleotide. In embodiments, the 5'-side, 3'-side, central "gap", or combinations thereof include modifications to one or more internucleotide linkages. In embodiments, the gapmer may include modifications to one or more internucleotide linkages for increasing stability, for improving plasma protein binding, or combinations thereof.

[0098] RNA interference nucleic acid In an embodiment, the hybrid oligonucleotide is an RNA interference (RNAi) molecule or a small interfering RNA (siRNA) molecule.

[0099] Small interfering RNA (siRNA) is an oligonucleotide duplex having a sense strand (also referred to as the passenger strand) and an antisense strand (also referred to as the guide strand) that can associate with a cytoplasmic multi-protein complex known as the RNAi-induced silencing complex (RISC), thereby mediating the degradation of homologous mRNA transcripts. Thus, siRNA can be designed to knock down protein expression nodes.

[0100] In an embodiment, the siRNA compound is composed of a single molecule containing a duplex region formed by intrastrand pairing, such as a hairpin or panhandle structure.

[0101] MicroRNA (miRNA) In an embodiment, the hybrid oligonucleotide is a microRNA (「miRNA」) mimic. As used herein, the term 「miRNA mimic」 refers to a synthetic oligonucleotide that, for example, invades the RNAi pathway and mimics the gene silencing ability of miRNA by controlling gene expression. As used herein, microRNA (miRNA) is a small single-stranded molecule that targets the 5' or 3' untranslated (UTR) to control gene expression, for example, by suppressing or degrading mRNA translation. In an embodiment, the miRNA mimic interacts with the 3' UTR of the target mRNA to suppress expression. In an embodiment, the miRNA mimic interacts with the 5' UTR of the target mRNA to suppress expression.

[0102] In an embodiment, the miRNA mimic contains about 15 to about 30 nucleotides, or about 17 to about 25 nucleotides, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28 or 30 nucleotides.

[0103] miRNA inhibitor In embodiments, the hybrid oligonucleotide is a miRNA inhibitor (also referred to as an “antimir,” “microRNA inhibitor,” “miR inhibitor,” or “miRNA inhibitor”). As used herein, a “miRNA inhibitor” is an oligonucleotide that inhibits miRNA activity. In embodiments, miRNA inhibitors include one or more sequences that are reverse complements of mature miRNAs that are chemically modified to prevent RISC-induced cleavage, improve binding affinity, provide resistance to nuclease degradation, or combinations thereof. In embodiments, the miRNA inhibitor binds to the mature miRNA, sequesters the endogenous miRNA, and renders it unavailable for normal function.

[0104] Antagomir In embodiments, the hybrid oligonucleotide is an antagomir. An antagomir is an RNA-like oligonucleotide that possesses RNase protection and pharmacological properties, such as various modifications for improved tissue and cell uptake. In embodiments, the antagomir forms a duplex with the antagomir and the endogenous miRNA, thereby silencing the endogenous miRNA by preventing miRNA-induced gene silencing.

[0105] Supermir In an embodiment, the hybrid oligonucleotide is a supermir. A supermir is a ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or both, or a modified form thereof, single-stranded, double-stranded, or partially double-stranded oligomer or polymer having a nucleotide sequence that is substantially identical to a miRNA and is antisense to its target. In an embodiment, a supermir includes oligonucleotides composed of naturally occurring and non-naturally occurring nucleobases, sugar nucleotide internucleotide linkages, or combinations thereof. In an embodiment, a supermir is substantially single-stranded; for example, less than about 50% (e.g., less than about 40%, about 30%, about 20%, about 10%, or about 5%) of the supermir is duplexed to itself. In an embodiment, a supermir includes a hairpin segment that can self-hybridize to form double-stranded regions, e.g., at least about 1, about 2, about 3, or about 4 and less than about 8, about 7, about 6 nucleotides of double-stranded regions, e.g., at the 3' end.

[0106] Immunostimulatory oligonucleotide In an embodiment, the hybrid oligonucleotide is an immunostimulatory oligonucleotide. An immunostimulatory oligonucleotide is a single-stranded or double-stranded oligonucleotide capable of inducing an immune response when administered to a subject. In an embodiment, an immunostimulatory oligonucleotide includes a palindrome that forms a hairpin secondary structure or a CpG motif.

[0107] In embodiments, the immunostimulatory oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may or may not be methylated. In another embodiment, the immunostimulatory oligonucleotide comprises at least one CpG dinucleotide having methylated cytosine. In an embodiment, the immunostimulatory oligonucleotide comprises a single CpG dinucleotide, and the cytosine in the CpG dinucleotide is methylated. In an embodiment, the immunostimulatory oligonucleotide comprises at least two CpG dinucleotides, and at least one cytosine in the CpG dinucleotide is methylated. In an embodiment, each cytosine in the CpG dinucleotide is methylated. In an embodiment, the immunostimulatory oligonucleotide comprises a plurality of CpG dinucleotides, and at least one of the CpG dinucleotides comprises methylated cytosine.

[0108] Decoy oligonucleotide In embodiments, the hybrid oligonucleotide is a decoy oligonucleotide. Since transcription factors recognize their relatively short binding sequences even in the absence of surrounding genomic DNA, short oligonucleotides bearing the consensus binding sequences of specific transcription factors can be used as tools 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. Occupation of the DNA binding site of the transcription factor by the decoy renders it impossible for the transcription factor to later bind to the promoter region of the target gene. Decoys can be used as therapeutic agents to inhibit the expression of genes activated by transcription factors or to upregulate genes repressed by the binding of transcription factors.

[0109] U1 adapter In an embodiment, the hybrid oligonucleotide is a U1 adapter. The U1 adapter is a bifunctional oligonucleotide that inhibits the polyA site and has a "U1 domain" that binds to a target domain complementary to a site in the terminal exon of the target gene and to the U1 small nuclear RNA component of U1 snRNP (Goraczniak, et al., 2008, Nature Biotechnology, 27(3), 257-263 (which is hereby incorporated by reference in its entirety)). U1 snRNP is a ribonucleoprotein that functions primarily to direct an early step in spliceosome formation by binding to the exon-intron boundary of pre-mRNA (Brown and Simpson, 1998, Annu Rev Plant Physiol Plant Mol Biol 49:77-95). Nucleotides 2-11 at the 5' end of the U1 snRNA base pair bind to the 5'ss of pre mRNA. In one embodiment, the oligonucleotide of the present invention is a U1 adapter. In one embodiment, the Ul adapter can be administered in combination with at least one other iRNA agent.

[0110] Hybridization site The hybridization site of the hybrid oligonucleotide will vary depending on the target nucleic acid sequence and / or the disease being treated. In an embodiment, the target nucleic acid sequence is an RNA sequence. In an embodiment, the target nucleic acid sequence is in the target mRNA. In an embodiment, the target nucleic acid sequence is in the target pre-mRNA. In an embodiment, the target nucleic acid sequence is a DNA sequence. In an embodiment, the target nucleic acid sequence is a genomic DNA sequence.

[0111] Splice site In embodiments, the hybrid oligonucleotide hybridizes to at least a portion of a splice site (e.g., a splice acceptor or splice donor site), or a splicing element (SE) and / or a cis-acting splicing regulatory element (SRE), sterically blocks access thereto, thereby redirecting splicing to a cryptic or new splice site. In embodiments, the hybrid oligonucleotide prevents the binding of a trans-acting regulatory splicing factor at the target site and 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 effectively block or promote splicing. In embodiments, the hybrid oligonucleotide can be designed to base pair through the bases of a splicing regulatory stem-loop to strengthen the stem-loop structure.

[0112] Nucleotide repeat In embodiments, the hybrid oligonucleotide targets a nucleotide repeat (e.g., a trinucleotide repeat expansion, a pentanucleotide repeat expansion, or a hexanucleotide repeat expansion) in a target nucleic acid sequence, or targets a sequence having a nucleotide repeat. In embodiments, the hybrid oligonucleotide blocks the expansion of the nucleotide repeat. In embodiments, the hybrid oligonucleotide blocks the transcription of the nucleotide repeat.

[0113] In an embodiment, the hybrid oligonucleotide is complementary to a target nucleic acid sequence having a trinucleotide repeat expansion. In an embodiment, the hybrid oligonucleotide hybridizes to the trinucleotide repeat expansion of the target nucleic acid sequence. In an embodiment, the hybrid oligonucleotide is complementary to about 5 to 10 trinucleotide repeats in the target nucleic acid sequence. In an embodiment, 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.

[0114] In an embodiment, 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 an embodiment, 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 (such as CAG, CGG, GCC, GAA, or CUG repeats).

[0115] In an embodiment, the hybrid oligonucleotide degrades the trinucleotide repeat. In an embodiment, after binding of the hybrid oligonucleotide to the target nucleic acid sequence, the target nucleic acid sequence is degraded by RNase H.

[0116] Polyadenylation signal In an embodiment, the hybrid oligonucleotide targets a target polyadenylation signal (PAS). In an embodiment, the hybrid oligonucleotide inhibits polyadenylation of the gene transcript.

[0117] In embodiments, the hybrid oligonucleotide interacts with (e.g., binds to) a polyadenylation sequence element (PSE) that includes, but is not limited to, a polyadenylation signal (PAS), a cleavage site (CS), and a GU-rich downstream element (DSE). In embodiments, in some cases, the polyadenylation sequence element includes one or more of 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).

[0118] In embodiments, the PAS is an adenosine-rich hexamer sequence that includes canonical AATAAA hexamer or variants that differ by a single nucleotide (e.g., AAUAAA, AUUAAA, UAUAAA, AGUAAA, AAGAAA, AAUAUA, AAUACA, CAUAAA, GAUAAA, CAUAAA, GAUAAA, AAUGAA, UUUAAA, ACUAAA, AAUAGA, AAAAAG, AAAACA, 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). The hexamer sequences occur at various frequencies, with AAUAAA and AUUAAA being the most frequent (see the same reference). The PAS is typically found upstream of the CS. The hexamer sequence of the PAS functions as a binding site for cleavage and a cleavage and polyadenylation specificity factor (CPSF). The PAS can also be determined by the presence of other auxiliary elements, such as an upstream U-rich element (USE) (see Tian et al., Nuc.Acid.Res. (2005), 33(1):201-212 and Neve et al. (2017) RNA Biology, 2017, 14(7):865-890).

[0119] The DSE is a U-rich or U / G-rich element that functions as a binding site for the cleavage stimulatory factor (CstF). The DSE is typically found downstream of the CS. The DSE can be followed by an extension of three or more uracil bases downstream of the CS, often within 20-40 nucleotides of the CS. In mammals, CA and UA are the most frequent dinucleotides preceding the cleavage site (CS), although the actual cleavage site is known to be heterogeneous.

[0120] Two multi-subunit complexes, CPSF and CstF, cooperate with each other and two additional factors (cleavage factors I and II) to cleave the mRNA sequence. The poly(A) polymerase (PAP), a single-subunit enzyme, is also involved in the cleavage of most pre-mRNAs, like RNA polymerase II. CPSF and PAP, together with poly(A)-binding protein II and cleavage stimulatory factor (CstF), are involved in the addition of the poly(A) tail (Takagaki and Manley, Mol Cell Biol. (2000), 20(5):1515-1525).

[0121] Methods for identifying polyadenylation sequence elements are known and can include, but are not limited to, the methodologies described by, for example, 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. Biomolecules (2000), 10(915) doi:10.3390 / biom10060915; and Vickers et al. Nucleic Acids Research (2001) 29(6)1293-1299.

[0122] Method of production The present disclosure relates to a method of synthesizing hybrid oligonucleotides and cleaving them from a solid support. The solid support can be a universal solid support. P(V)PMO nucleotides can be used together with other resins typically used in PMO synthesis.

[0123] Synthesis is carried out from the 6’ or 5’ end to the 3’ end, involving 2’-deoxyribose, 2’-substituted ribose, LNA or other nucleotide-based PMO 6’-chlorophosphoroamidate P(V), PMO 6’-β-cyanoethyl amidite P(III), and reverse 5’-β-cyanoethyl amidite P(III).

[0124] The transition between the 6’ PMO flank and the gap is achieved by reacting a 5’ phosphoramidite with the N3 of the morpholino ring in the presence of an activator and by sulfurization. The result is a negatively charged N3’>P5’ thiophosphoramidate linker.

[0125] The transition between the gap and the 3’ PMO flank is achieved by coupling a PMO 6’-β-cyanoethyl amidite P(III) with the 3’ hydroxyl group in the presence of an activator and by sulfurization or oxidation. This results in the formation of a phosphorothioate PS or phosphodiester PO linker.

[0126] When PMO is introduced into the sequence later, 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.

[0127] The phosphate protecting groups can be O-cyanoethyl, O-methyl, O-ethyl, O-benzyl, O-allyl.

[0128] When PMO is introduced into the sequence later, cap A contains isobutyric anhydride, pivalic anhydride or benzoic anhydride instead of acetic anhydride.

[0129] A method for preparing a hybrid oligonucleotide, comprising assembling P(III) and P(V) nucleotide building blocks on a support in the 6'-to-3' or 5'-to-3' direction, and using PMO 6'-phosphoramidite P(III) or phosphoramidate P(V) as the first nucleotide from the support.

[0130] The present disclosure further relates to a method for preparing a hybrid oligonucleotide, comprising assembling P(III) and P(V) nucleotide building blocks on a support in the 6'-to-3' or 5'-to-3' direction, and using PMO 6'-phosphoramidite P(III) or phosphoramidate P(V) as the second nucleotide from the support, wherein the first nucleotide is reverse DNA (PIII).

[0131] The present disclosure also relates to 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 phosphorothioamidate 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 A hybrid oligonucleotide comprising relates to a method for preparing an oligonucleotide comprising

[0132] In an embodiment, the support can be a polystyrene resin.

[0133] In an embodiment, PMO6’-phosphoroamidite P(III) can be the first nucleotide from the support. PMO6’-phosphoramidate P(V) can be the first nucleotide from the support.

[0134] In an embodiment, the oligonucleotide prepared by the method can include phosphorothioester, phosphodiester linkage and / or phosphorodiamidate linkage and at least one phosphorothioamidate linkage. In an embodiment, the oligonucleotide prepared by the method can include phosphorothioester, phosphodiester linkage and / or phosphorodiamidate linkage and one phosphorothioamidate linkage.

[0135] In an embodiment, the oligonucleotide prepared by the method can include phosphorodiamidate linkage and at least one phosphorothioamidate linkage (FIG. 5). In an embodiment, the oligonucleotide prepared by the method has only phosphorodiamidate and phosphorothioamidate linkages. In an embodiment, the oligonucleotide prepared by the method can include phosphorodiamidate and phosphorothioester linkages and at least one phosphorothioamidate linkage.

[0136] In an embodiment, the phosphorothioester can link two reverse DNA amidites. In an embodiment, the phosphorothioester can link two reverse RNA amidites. In an embodiment, the phosphorothioester can link a reverse DNA amidite and a reverse DNA amidite. In an embodiment, the phosphorothioester links a reverse DNA amidite and PMO(III).

[0137] In an embodiment, the phosphorodiamidate can link PMO(III) and PMO(V). In an embodiment, the phosphorodiamidate can link two PMO(V)s.

[0138] In some embodiments, phosphorothioamidate can link PMO(V) to PMO(III). In some embodiments, phosphorothioamidate can link PMO(III) to reverse DNA P(III).

[0139] In some embodiments, phosphodiester can link reverse RNA and reverse DNA. In some embodiments, phosphodiester can link two reverse RNAs.

[0140] In embodiments, deoxyribonucleotide analogs, two or more ribonucleotide analogs can advance PMO.

[0141] The method can include coupling a first reverse amidite to the secondary amino group of the morpholino ring to form a phosphorothioamidate linkage.

[0142] Thus, the method provides for adding (PIII) and P(V) building blocks, which are PMO, reverse DNA, reverse RNA, and reverse LNA.

[0143] In embodiments, the method can further include adding at least one P(III) building block. In embodiments, the method can further include adding at least two P(III) building blocks. In embodiments, the building blocks can be added sequentially or as a chain of building blocks.

[0144] In embodiments, the P(III) building blocks can be continuous.

[0145] In embodiments, the method can further include adding at least one P(V) building block.

[0146] In an embodiment, the method may include adding at least two P(V) building blocks. The P(V) building blocks may be consecutive. In an embodiment, at least two P(V) building blocks may follow at least two P(III) building blocks. In an embodiment, at least two P(III) building blocks may follow at least two P(V) building blocks. In an embodiment, at least one P(III) building block and one 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 the same. In an embodiment, the P(III) building blocks may be different. In an embodiment, the P(V) building blocks may be the same. In an embodiment, the P(V) building blocks may be different. In an embodiment, the building blocks may be added continuously or as a group of building blocks.

[0147] In an embodiment, the method may include deprotecting a secondary amino group at the 3'-end of a first DNA monomer. The method may further include neutralizing the deprotected secondary amino group at the 3'-end of the first DNA monomer.

[0148] In an embodiment, the method may further include adding one or more additional DNA nucleotides to the first DNA nucleotide, and two or more DNA nucleotides are linked via a phosphorothioate (PS) linkage.

[0149] In an embodiment, the method may further include adding a second PMO 6'-phosphoramidite P(III) to the 3'-terminal DNA nucleotide of the hybrid oligonucleotide.

[0150] In an embodiment, the method is (i) a 6'-side comprising two or more morpholino nucleotide analogs, or a 5'-side comprising two or more ribonucleotides or ribonucleotide analogs; (ii) A gap region comprising five or more deoxyribonucleotides or deoxyribonucleotide analogs; and (iii) A 3'-side comprising two or more morpholino nucleotide analogs, or a 3'-side comprising two or more ribonucleotides or ribonucleotide analogs An oligonucleotide that may comprise a gapmer comprising the above may be provided.

[0151] In embodiments, the method may include extending an oligonucleotide by adding one or more additional DNA monomers to a first DNA monomer linked via phosphorothioate linkages (DNA-PS).

[0152] In embodiments, the method may further include adding PMO6'-phosphoramidite P(III) to the 3'-end of the oligonucleotide.

[0153] In embodiments, the 3'-PMO wing may be added when all cytosine-based nucleotides in the 5'-side and the gap can be protected by N-benzoyl, and cap A is based on isobutyric acid, pivalic acid or benzoic anhydride instead of acetic anhydride.

[0154] In embodiments, the methods of the present disclosure provide means for synthesizing the compounds disclosed herein.

[0155] In embodiments, the synthesis of the oligonucleotide can be automated. In embodiments, the synthesis of the oligonucleotide can be performed manually. In embodiments, the synthesis of the oligonucleotide can be performed via a combination of automated and manual synthesis. In embodiments, the synthesis of the oligonucleotide can be on a solid support. In embodiments, the synthesis of the oligonucleotide can be in solution phase. In embodiments, the synthesis of the oligonucleotide can be a combination of synthesis on a solid support and in solid phase.

[0156] In embodiments, the oligonucleotide can be assembled by stepwise coupling of individual monomers. In embodiments, the oligonucleotide is assembled by coupling linked monomers to individual monomers. In embodiments, the oligonucleotide is assembled by coupling a first set of linked monomers to a second set of linked monomers. Any method of coupling reaction for the synthesis of oligonucleotides can be used in the disclosed methods.

[0157] Hybrid Oligonucleotide Design The design of the hybrid oligonucleotide will depend on the target sequence. One of ordinary skill in the art can design, synthesize, and screen compounds of different nucleic acid base sequences to identify the sequence that provides the desired activity. In embodiments, the hybrid oligonucleotide includes one or more modified nucleotides, one or more modified internucleotide linkages, or combinations thereof. In embodiments, the hybrid oligonucleotide can be, but is not limited to, a gapmer, a splice-switching oligonucleotide, the sense strand of siRNA, the antisense strand of siRNA, a deoxyribozyme, a steric-blocking antisense oligonucleotide (ASO), a DNAzyme, an RNAzyme, an aptamer, an immunomodulatory oligonucleotide, or an antagomir.

[0158] In embodiments, the hybrid oligonucleotide includes one or more modified nucleotides. In embodiments, all of the nucleotides in the hybrid oligonucleotide are modified. In embodiments, one or more nucleotides include a modified nucleobase. In embodiments, one or more nucleotides include a modified sugar. In embodiments, the hybrid oligonucleotide includes one or more modified internucleotide linkages.

[0159] In an embodiment, 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 phosphorodiamidate linkage; and (iv) at least one phosphorothioamidate 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 hybrid oligonucleotide comprises two or more consecutive P(V) morpholino nucleotide analogs linked via a phosphorodiamidate linkage. In an embodiment, the hybrid oligonucleotide comprises 2 to 20, 3 to 10, or 4 to 6 consecutive P(V) morpholino nucleotide analogs linked via a phosphorodiamidate linkage. 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 via a phosphorodiamidate linkage. In an embodiment, any P(V) morpholino nucleotide analog is linked via a phosphorothioamidate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0160] In embodiments, the hybrid oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In embodiments, the hybrid oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs, or combinations thereof. In embodiments, the first nucleotide sequence comprises 2'-modified ribonucleotide analogs 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.

[0161] In embodiments, the hybrid oligonucleotide comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In embodiments, the hybrid oligonucleotide comprises at least one internucleotide linkage selected from methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments the hybrid oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS) and phosphorylguanidine linkages. In embodiments, the hybrid oligonucleotide comprises at least one phosphorothioate linkage.

[0162] 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, the first nucleotide sequence; (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, the second nucleotide sequence; and (iii) at least one phosphorothioamidate linkage that links 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 combinations thereof are each linked via internucleotide linkages.

[0163] 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.

[0164] In an embodiment, the first nucleotide sequence comprises two or more morpholino nucleotide analogs. In an embodiment, the first nucleotide sequence comprises 2 to 20, 3 to 10, or 4 to 6 morpholino nucleotide analogs. In an embodiment, 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 an embodiment, the first nucleotide sequence comprises two morpholino nucleotide analogs. In an embodiment, the first nucleotide sequence comprises three morpholino nucleotide analogs. In an embodiment, the first nucleotide sequence comprises four morpholino nucleotide analogs. In an embodiment, the first nucleotide sequence comprises five morpholino nucleotide analogs.

[0165] In an embodiment, the morpholino nucleotide analog is P(III)PMO or P(V)PMO. In an embodiment, the morpholino nucleotide analog is P(III)PMO. In an embodiment, P(III)PMO is selected from P(III)PMO-C, P(III)PMO-G, P(III)PMO-A, P(III)PMO-T, or P(III)PMO having a non-naturally occurring nucleobase. In an embodiment, the morpholino nucleotide analog is P(V)PMO. In an embodiment, (PV)PMO comprises P(V)PMO-C, P(V)PMO-G, P(V)PMO-A, P(V)PMO-T, or P(V)PMO having a non-naturally occurring nucleobase.

[0166] In an embodiment, two or more consecutive morpholino oligonucleotide analogs in the first nucleotide sequence are linked via phosphorodiamidate linkages. In an embodiment, 2 to 20, 3 to 10, or 4 to 6 consecutive morpholino oligonucleotide analogs in the first nucleotide sequence are linked via phosphorodiamidate linkages. In an embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive morpholino oligonucleotide analogs in the first nucleotide sequence are linked via phosphorodiamidate linkages. In an embodiment, the terminal 3' nucleic acid residue of the first nucleic acid sequence is a P(III) morpholino nucleotide analog. In an embodiment, the terminal 3' nucleic acid residue of the first nucleic acid sequence is a P(V) morpholino nucleotide analog. In an embodiment, the terminal 3' nucleic acid residue of the first nucleic acid sequence is bound to a solid substrate.

[0167] In an embodiment, the first nucleotide sequence of the hybrid oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In an embodiment, the deoxyribonucleotide analog or ribonucleotide analog is 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 comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In an embodiment, the deoxyribonucleotide analog or ribonucleotide analog is 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 2'-modified ribonucleotide analogs 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'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or combinations thereof.

[0168] In an embodiment, the first oligonucleotide sequence of the hybrid oligonucleotide comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In an embodiment, the first nucleotide sequence of the hybrid oligonucleotide comprises at least one internucleotide linkage selected from methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In an embodiment, the first oligonucleotide sequence of the hybrid oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS), and phosphorylguanidine linkage. In an embodiment, the first oligonucleotide sequence of the hybrid oligonucleotide comprises at least one phosphorothioate linkage.

[0169] In embodiments, the second nucleotide sequence of the hybrid oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, inverted P(IIII) DNA analogs, P(III) RNA analogs, inverted P(III) RNA analogs, or combinations thereof. In embodiments, the second nucleotide sequence of the hybrid oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In embodiments, the second oligonucleotide sequence of the hybrid oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, inverted P(IIII) DNA analogs, P(III) RNA analogs, inverted P(III) RNA analogs, or combinations thereof. In embodiments, the first nucleotide sequence comprises 2'-modified ribonucleotide analogs selected from 2'-O-methyl (2'-OMe) P(III) RNA, inverted 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, inverted 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, inverted 2'-fluoro P(III) RNA, or combinations thereof.

[0170] In embodiments, the second nucleotide sequence comprises one or more morpholino nucleotide analogs. In embodiments, the morpholino nucleotide analog is P(III)PMO. In embodiments, the P(III)PMO is selected from P(III)PMO-C, P(III)PMO-G, P(III)PMO-A, P(III)PMO-T, or P(III)PMO having a non-naturally occurring nucleobase. In embodiments, the morpholino nucleotide analog is P(V)PMO. In embodiments, the (PV)PMO comprises P(V)PMO-C, P(V)PMO-G, P(V)PMO-A, P(V)PMO-T, or P(V)PMO having a non-naturally occurring nucleobase.

[0171] In embodiments, the second oligonucleotide sequence of the hybrid oligonucleotide comprises at least one internucleotide linkage selected from phosphodiester, phosphorotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In embodiments, the second nucleotide sequence of the hybrid oligonucleotide comprises at least one internucleotide linkage selected from methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, the second oligonucleotide sequence of the hybrid oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS), and phosphorylguanidine linkage. In embodiments, the second oligonucleotide sequence of the hybrid oligonucleotide comprises at least one phosphorothiohydrooate linkage.

[0172] 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 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.

[0173] Hybrid gapmer design In embodiments, the hybrid oligonucleotide is a hybrid gapmer oligonucleotide. In embodiments, the hybrid gapmer oligonucleotide comprises (i) a first nucleotide sequence comprising a 5'-portion; (ii) a second oligonucleotide sequence comprising a gap oligonucleotide sequence; and (iii) a 3'-portion.

[0174] In an embodiment, the hybrid gapmer oligonucleotide comprises at least two consecutive morpholino nucleotide analogs linked via neutral phosphorodiamidate linkages. Each consecutive morpholino nucleotide analog in the hybrid gapmer oligonucleotide introduces n - 1 neutral linkages (n = the number of nucleotides in the hybrid gapmer oligonucleotide). In an embodiment, the 5' and 3' flanks each comprise at least two or three morpholino nucleotide analogs. Without wishing to be bound by theory, the morpholino nucleotide analogs in the flank regions are thought to improve oligonucleotide stability and binding affinity to a target nucleic acid sequence (e.g., target RNA).

[0175] 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 is noted that the 5' flank may also be referred to as the 6' flank to describe the six-membered morpholino ring.

[0176] In an embodiment, the 5' flank comprises 2 to 20, 2 to 10, or 2 to 5 morpholino nucleotide analogs. In an embodiment, the 5' flank comprises 2 to 20, 2 to 10, or 2 to 5 consecutive morpholino nucleotide analogs. In an embodiment, 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 an embodiment, 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.

[0177] In embodiments, two or more morpholino nucleotide analogs in the 5'-side portion are linked via phosphorodiamidate linkages. In embodiments, 2 to 20, 2 to 10, or 2 to 5 morpholino nucleotide analogs in the 5'-side portion are linked via phosphorodiamidate 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 in the 5'-side portion are linked via phosphorodiamidate linkages.

[0178] In embodiments, the 5'-side portion of the hybrid gapmer oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, inverted P(IIII) DNA analogs, P(III) RNA analogs, inverted P(III) RNA analogs, or combinations thereof. In embodiments, the 5'-side portion of the hybrid gapmer oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In embodiments, the 5'-side portion comprises a 2'-modified ribonucleotide analog selected from 2'-O-methyl (2'-OMe) P(III) RNA, inverted 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, inverted 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, inverted 2'-fluoro P(III) RNA, or combinations thereof.

[0179] In an embodiment, the 5'-side portion of the hybrid gapmer oligonucleotide comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In an embodiment, the 5'-side portion of the hybrid gapmer oligonucleotide comprises at least one internucleotide linkage selected from methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In an embodiment, the 5'-side portion of the hybrid gapmer oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS) and phosphorylguanidine linkage.

[0180] In embodiments, the hybrid gapmer oligonucleotide includes a 5'-side portion including morpholino nucleotide analogs linked by phosphorodiamidate linkages, a central gap region including DNA nucleotides or DNA nucleotide analogs linked by phosphorothioate linkages, and a 3'-side portion including morpholino nucleotide analogs linked by phosphorodiamidate linkages (6'-PMO-DNA-PMO-3'). In embodiments, the hybrid gapmer oligonucleotide includes a 5'-side portion including morpholino nucleotide analogs linked by phosphorodiamidate linkages, a central gap region including DNA nucleotides or DNA nucleotide analogs linked by phosphorothioate linkages, and a 3'-side portion having a structure including RNA nucleotides or RNA nucleotide analogs such as, for example, 2'-modified RNA nucleotide analogs (6'-PMO-DNA-RNA-3'). In embodiments, the hybrid gapmer oligonucleotide includes a 3'-side portion including RNA nucleotides or RNA nucleotide analogs such as, for example, 2'-modified RNA nucleotide analogs, a central gap region including DNA nucleotides or DNA nucleotide analogs linked by phosphorothioate linkages, and a 5'-side portion including morpholino nucleotide analogs linked by phosphorodiamidate linkages (5'-RNA-DNA-PMO-3').

[0181] In embodiments, the hybrid gapmer oligonucleotide includes a thiophosphoramidate linkage between the 5'-side portion and the central gap oligonucleotide. In embodiments, the 5' or 3'-side portion 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.

[0182] In embodiments, the gap oligonucleotide sequence includes at least 8 deoxyribonucleotides or deoxyribonucleotide analogs. In embodiments, the gap oligonucleotide sequence includes at least 8 consecutive deoxyribonucleotides or deoxyribonucleotide analogs. Without wishing to be bound by theory, a gap size of at least 8 deoxyribonucleotides or deoxyribonucleotide analogs is believed to be desirable for RNAse H activity.

[0183] 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 gapmer oligonucleotide have a negative charge. In embodiments, 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 at the 5'-side portion of the hybrid gapmer 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 gap oligonucleotide sequence of the hybrid gapmer oligonucleotide have a negative charge. In embodiments, 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 at the 3'-side portion of the hybrid gapmer oligonucleotide have a negative charge. In embodiments, the hybrid gapmer oligonucleotide includes 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.

[0184] In embodiments, the terminal residue at the first 5' (or 6') end of the 5' (or 6') side portion includes PMO6'-phosphoramidite P(III).

[0185] In an embodiment, the gap oligonucleotide sequence comprises 8 to 20 deoxyribonucleotides or ribonucleotide analogs. In an embodiment, the gap oligonucleotide sequence comprises at least 8 consecutive deoxyribonucleotides or deoxyribonucleotide analogs. In an embodiment, the gap oligonucleotide sequence comprises 8 to 20 consecutive deoxyribonucleotides or ribonucleotide analogs.

[0186] In an embodiment, the gap sequence of the hybrid gapmer oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In an embodiment, the deoxyribonucleotide analog or ribonucleotide analog is 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 gap sequence of the hybrid gapmer oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In an embodiment, the gap 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 combinations thereof.

[0187] In embodiments, the gap sequence of the hybrid gapmer oligonucleotide comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In embodiments, the gap sequence of the hybrid gapmer oligonucleotide comprises at least one internucleotide linkage selected from methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, the gap sequence of the hybrid gapmer oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, 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 gap oligonucleotide sequence are linked via phosphorothioate linkages. In embodiments, the gap sequence comprises at least two 2'-modified nucleotides linked by phosphorothioate linkages.

[0188] 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.

[0189] In embodiments, two or more morpholino nucleotide analogs at the 3'-side are linked via phosphorodiamidate linkages. In embodiments, 2 to 20, 2 to 10, or 2 to 5 morpholino nucleotide analogs at the 3'-side are linked via phosphorodiamidate 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 at the 3'-side are linked via phosphorodiamidate linkages.

[0190] In embodiments, the 3'-side of the hybrid gapmer oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs, or combinations thereof. In embodiments, the 3'-side of the hybrid gapmer oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In embodiments, the 3'-side 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'-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 combinations thereof.

[0191] In an embodiment, the 3'-side portion of the hybrid gapmer oligonucleotide comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In an embodiment, the 3'-side portion of the hybrid gapmer oligonucleotide comprises at least one internucleotide linkage selected from methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In an embodiment, the 3'-side portion of the hybrid gapmer oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS) and phosphorylguanidine linkage.

[0192] In an embodiment, the terminal residue at the second 5' or 6' end of the 3'-side portion comprises PMO 6'-phosphoramidite P(III).

[0193] In an embodiment, the gap oligonucleotide sequence is linked to the 3'-side portion via a phosphorothioate linkage.

[0194] In an embodiment, the hybrid gapmer oligonucleotide comprises 10 or fewer morpholino nucleotide analogs. In an embodiment, the hybrid gapmer oligonucleotide comprises 7 or fewer morpholino nucleotide analogs. In an embodiment, the hybrid gapmer oligonucleotide comprises 2 to 10, or 3 to 7 morpholino nucleotide analogs.

[0195] Steric block and splice-switching oligonucleotide In an embodiment, the hybrid oligonucleotide is a three-dimensional block oligonucleotide. In an embodiment, the three-dimensional block oligonucleotide is a splicing-switching oligonucleotide.

[0196] Without wishing to be bound by theory, the PMO sequence in the three-dimensional block oligonucleotide is thought to be able to modulate the binding energy of the oligonucleotide to the target nucleic acid sequence. Also, the PMO sequence in the three-dimensional block oligonucleotide is thought to be able to reduce the effect of the oligonucleotide on the innate immune system and / or alter the in vivo distribution, pharmacological properties, or cellular distribution of the oligonucleotide.

[0197] In an embodiment, the three-dimensional block oligonucleotide comprises 10 to 50, 15 to 40, or 20 to 30 nucleotide analogs in length. In an embodiment, the three-dimensional block oligonucleotide comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In an embodiment, the three-dimensional block 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 an embodiment, the three-dimensional block 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.

[0198] In embodiments, the three-dimensional block oligonucleotide comprises up to 25% morpholino nucleotide analogs and up to 75% 2'-modified RNA nucleotides. In embodiments, the three-dimensional block oligonucleotide comprises up to 75% morpholino nucleotide analogs and up to 25% 2'-modified RNA nucleotides. In embodiments, the three-dimensional block oligonucleotide comprises up to 30% morpholino nucleotide analogs and up to 70% 2'-modified RNA nucleotides. In embodiments, the three-dimensional block oligonucleotide comprises up to 70% morpholino nucleotide analogs and up to 30% 2'-modified RNA nucleotides. In embodiments, the three-dimensional block oligonucleotide comprises up to 40% morpholino nucleotide analogs and up to 60% 2'-modified RNA nucleotides. In embodiments, the three-dimensional block oligonucleotide comprises up to 60% morpholino nucleotide analogs and up to 40% 2'-modified RNA nucleotides. In embodiments, the three-dimensional block oligonucleotide comprises up to 50% morpholino nucleotide analogs and up to 50% 2'-modified RNA nucleotides.

[0199] In embodiments, the three-dimensional block oligonucleotide comprises at least two contiguous morpholino nucleotide analogs linked via a neutral phosphorodiamidate linkage. In embodiments, the three-dimensional block oligonucleotide comprises at least two or at least three morpholino nucleotide analogs. In embodiments, the three-dimensional block oligonucleotide comprises at least one thiophosphoramidate linkage between an upstream P(V) morpholino nucleotide analog and a downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0200] In embodiments, the three-dimensional block oligonucleotide comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs, or combinations thereof. In embodiments, the three-dimensional block oligonucleotide comprises at least one (1) 2'-modified P(III) ribonucleotide. In embodiments, the three-dimensional block oligonucleotide

[0201] comprises 2'-modified ribonucleotide analogs 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'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or combinations thereof.

[0202] In embodiments, the three-dimensional block oligonucleotide comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In embodiments, the three-dimensional block oligonucleotide comprises at least one internucleotide linkage selected from methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, the 5'-portion of the hybrid gapmer oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS) and phosphoryl guanidine linkage.

[0203] In an embodiment, the three-dimensional block 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 phosphorodiamidate linkage; and (iv) at least one phosphorothioamidate 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 three-dimensional block oligonucleotide comprises two or more consecutive P(V) morpholino nucleotide analogs linked via a phosphorodiamidate linkage. In an embodiment, the three-dimensional block oligonucleotide comprises 2 to 20, 3 to 10, or 4 to 6 consecutive P(V) morpholino nucleotide analogs linked via a phosphorodiamidate linkage. In an embodiment, the three-dimensional block 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 via a phosphorodiamidate linkage. In an embodiment, any P(V) morpholino nucleotide analog is linked via a phosphorothioamidate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0204] Short interfering RNA (siRNA) In an embodiment, the hybrid oligonucleotide forms the sense strand, the antisense strand, or both strands of short interfering RNA (siRNA). Without wishing to be bound by theory, the presence of a PMO oligonucleotide sequence in siRNA, particularly at the 5' or 3' end of the sense or antisense strand, is thought to reduce the enzymatic degradation of the siRNA oligonucleotide. Also, without wishing to be bound by theory, PMO in the sense strand rather than the antisense strand can introduce chemical asymmetry into the siRNA, enable discrimination between the antisense (guide) strand and the sense (passenger) strand, and is thought to be able to prevent the entry of the sense strand into the RNA-induced silencing complex (RISC). Further, including a PMO oligonucleotide sequence in the antisense strand is thought to be able to reduce the toxicity of the siRNA. The PMO oligonucleotide sequences in the sense and antisense strands can also improve their pharmacological profiles or alter the cellular distribution of the siRNA.

[0205] In an embodiment, siRNA is provided in which the antisense strand is a hybrid antisense oligonucleotide. In an embodiment, the antisense strand is about 10 to about 50, about 15 to about 30, or about 20 to about 30 nucleotides in length. In an embodiment, the hybrid antisense oligonucleotide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In an embodiment, the hybrid antisense oligonucleotide of the siRNA is about 21 nucleotides in length.

[0206] In an embodiment, siRNA is provided in which the sense strand is a hybrid sense oligonucleotide. In an embodiment, the sense strand is about 10 to about 50, about 15 to about 30, or about 20 to about 30 nucleotides in length. In an embodiment, the hybrid sense oligonucleotide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In an embodiment, the hybrid antisense oligonucleotide of the siRNA is about 21 nucleotides in length.

[0207] In embodiments, the double-stranded region of the siRNA duplex contains about 10 to about 50, about 15 to about 30, or about 20 to about 30 nucleotides. In embodiments, the double-stranded region of the siRNA duplex is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In embodiments, the double-stranded region of the siRNA duplex is about 21 nucleotides in length.

[0208] In embodiments, the double-stranded siRNA duplex contains a single-stranded or unpaired region at one or both ends of the molecule. In embodiments, the sense strand of the double-stranded siRNA duplex contains a 5' overhang, a 3' overhang, or both. In embodiments, the antisense strand of the double-stranded siRNA duplex contains a 5' overhang, a 3' overhang, or both. In embodiments, the overhang(s) are each independently about 1 to about 5 nucleotides in length, or about 1 to about 3 nucleotides in length, or 1, 2, 3, 4, or 5 nucleotides in length. The overhang can be the result of one strand being longer than the other, or of two strands of the same length being offset. In embodiments, the siRNA duplex contains at least one 3' overhang. In embodiments, the siRNA duplex contains at least one 5' overhang. In embodiments, both the sense and antisense strands of the siRNA duplex have a 3' overhang. In embodiments, both the sense and antisense strands of the siRNA duplex have a 5' overhang.

[0209] In an embodiment, the sense strand of the siRNA is of the same length as or shorter than the antisense strand, for example, 15 - 16 mer. In an embodiment, the morpholino nucleotide analog is included in the antisense strand, the sense strand, or both. In an embodiment, the morpholino nucleotide analog is included in only one of the strands of the siRNA duplex. In an embodiment, the morpholino nucleotide analog is included in the sense strand of the siRNA duplex. In an embodiment, the morpholino nucleotide analog is included in the antisense strand of the siRNA duplex. In an embodiment, the morpholino nucleotide analog is included in only the sense strand of the siRNA duplex.

[0210] In an embodiment, 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' termini of the sense strand. In an embodiment, the sense strand includes 2'-modified nucleotides at one or more of positions 1, 2, or 3 from the 5' or 3' terminus of the sense strand. In an embodiment, the siRNA sense strand has 1 - 5 2'-modified nucleotides.

[0211] In an embodiment, the antisense 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' termini of the antisense strand. In an embodiment, the antisense strand includes 2'-modified nucleotides at one or more of positions 1, 2, or 3 from the 5' or 3' terminus of the antisense strand. In an embodiment, the siRNA antisense strand has 1 - 5 2'-modified nucleotides.

[0212] In embodiments, the sense strand comprises at least two contiguous morpholino nucleotide analogs linked via neutral phosphorodiamidate linkages. In embodiments, the sense strand comprises at least two or at least three morpholino nucleotide analogs. In embodiments, the sense strand comprises at least one thiophosphoramidate linkage between an upstream P(V) morpholino nucleotide analog and a downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0213] In embodiments, the sense strand comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, inverted P(IIII) DNA analogs, P(III) RNA analogs, inverted P(III) RNA analogs, or combinations thereof. In embodiments, the sense strand comprises at least one (1) 2'-modified P(III) ribonucleotide. In embodiments, the sense strand comprises

[0214] a 2'-modified ribonucleotide analog selected from 2'-O-methyl (2'-OMe) P(III) RNA, inverted 2'-O-methyl (2'-OMe) P(III) RNA, 2'-O-methoxyethyl (2'-OMOE) P(III) RNA, inverted 2'-O-methoxyethyl (2'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, inverted 2'-fluoro P(III) RNA, or combinations thereof.

[0215] In an embodiment, the sense strand comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In an embodiment, the sense strand comprises at least one internucleotide linkage selected from methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N’-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In an embodiment, the 5'-portion of the hybrid gapmer oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS), and phosphorylguanidine linkages.

[0216] In embodiments, 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 phosphorothioamidate 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 sense strand comprises two or more contiguous P(V) morpholino nucleotide analogs linked via a phosphorodiamidate linkage. In embodiments, the sense strand comprises 2 to 20, 3 to 10, or 4 to 6 contiguous P(V) morpholino nucleotide analogs linked via a phosphorodiamidate linkage. In embodiments, the sense strand comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous P(V) morpholino nucleotide analogs linked via a phosphorodiamidate linkage. In embodiments, any P(V) morpholino nucleotide analog is linked via a phosphorothioamidate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0217] In embodiments, the antisense strand comprises at least two contiguous morpholino nucleotide analogs linked via a neutral phosphorodiamidate linkage. In embodiments, the antisense strand comprises at least two or at least three morpholino nucleotide analogs. In embodiments, the antisense strand comprises at least one thiophosphoramidate linkage between an upstream P(V) morpholino nucleotide analog and a downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0218] In embodiments, the antisense strand comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof. In embodiments, the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs, or combinations thereof. In embodiments, the antisense strand comprises at least one (1) 2'-modified P(III) ribonucleotide. In embodiments, 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.

[0219] In embodiments, the antisense strand comprises at least one internucleotide linkage selected from phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, phosphorodiamidate, phosphorothioamidate, or phosphorothioate. In embodiments, the antisense strand comprises at least one internucleotide linkage selected from methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In embodiments, the 5'-flank of the hybrid gapmer oligonucleotide comprises an internucleotide linkage selected from phosphorodiamidate, phosphorothioamidate, phosphorothioate (PS) and phosphorylguanidine linkages.

[0220] 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 phosphorodiamidate linkage; and (v) at least one phosphorothioamidate 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 two or more contiguous P(V) morpholino nucleotide analogs linked via phosphorodiamidate linkages. In embodiments, the antisense strand comprises 2 to 20, 3 to 10, or 4 to 6 contiguous P(V) morpholino nucleotide analogs linked via phosphorodiamidate linkages. In embodiments, the antisense strand comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous P(V) morpholino nucleotide analogs linked via phosphorodiamidate linkages. In embodiments, any P(V) morpholino nucleotide analog is linked via a phosphorothioamidate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

[0221] Endosomal Escape Vehicle (EEV) For example, provided herein are endosomal escape vehicles (EEVs) that can be used to transport a cargo through a cell membrane to deliver the cargo to the cytosol or nucleus of a cell. The cargo can include a therapeutic moiety (TM), such as a hybrid oligonucleotide described herein. The EEV can include a cell-penetrating peptide (CPP), such as a cyclic cell-penetrating peptide (cCPP) conjugated to an exocyclic peptide (EP). The EP can alternatively be referred to as a modulatory peptide (MP).

[0222] The EP may contain the sequence of a nuclear localization signal (NLS). The EP may be coupled to a cargo. The EP may be coupled to a cCPP. The EP may be coupled to both a cargo and a cCPP. The coupling between the EP, the cargo, the cCPP, or combinations thereof may be non-covalent or covalent. The EP may be coupled via a peptide bond to the N-terminus of the cCPP. The EP may be coupled via a peptide bond to the C-terminus of the cCPP. The EP may be coupled to the cCPP via the side chain of an amino acid in the cCPP. The EP may be coupled to the cCPP via the side chain of a lysine that can be conjugated to the side chain of glutamine in the cCPP. The EP may be conjugated to the 5' or 3' end of an oligonucleotide cargo. The EP may be coupled to a linker. The exocyclic peptide may be conjugated to the amino group of the linker. The EP may be coupled to the linker via the C-terminus of the EP and the cCPP via side chains on the cCPP and / or the EP. For example, the EP may contain a terminal lysine, which may then be coupled via an amide bond to a cCPP containing glutamine. If the EP contains a terminal lysine and the side chain of the lysine can be used to bind the cCPP, the C or N terminus may be bound to a linker on the cargo.

[0223] Exocyclic peptide In embodiments, compounds are provided that include a hybrid oligonucleotide, a cyclic cell-penetrating peptide (cCCP), and an exocyclic peptide (EP). The exocyclic peptide (EP) may include from 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 include from 6 to 9 amino acid residues. The EP may include from 4 to 8 amino acid residues.

[0224] 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 chemical moiety that is a homolog of a natural amino acid in that it has a structure similar to a natural amino acid so as to mimic the structure and reactivity of the natural amino acid. Non-natural amino acids can be one of the 20 common naturally occurring amino acids or a modified amino acid that is not the rare natural amino acid selenocysteine or pyrrolysine, and / or an amino acid analog. Non-natural amino acids can also be the D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloisoleucine, 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, their derivatives, or combinations thereof. These, and other amino acids, are listed in Table 1 along with their abbreviations as used herein. For example, the amino acid can be A, G, P, K, R, V, F, H, Nal, or citrulline.

[0225] The EP can include at least one positively charged amino acid residue, e.g., at least one lysine residue and / or a guanidine group, or at least one amino acid residue having a side chain that includes a protonated form thereof. The EP can include one or two amino acid residues having a side chain that includes a guanidine group, or a protonated form thereof. The amino acid residue having a side chain that includes a guanidine group can be an arginine residue. The protonated form can mean a salt thereof throughout the present disclosure.

[0226] EP may contain at least two, at least three, at least four or more lysine residues. EP may contain two lysine residues. EP may contain three lysine residues. EP may contain four lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group such as trifluoroacetyl (-COCF3), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), or (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) group. The amino group on the side chain of each lysine residue may be substituted with a trifluoroacetyl (-COCF3) group. The protecting group may be included to enable amide conjugation. The protecting group may be removed after EP is conjugated to cCPP.

[0227] EP may contain at least two amino acid residues having hydrophobic side chains. The amino acid residues having hydrophobic side chains may be selected from valine, proline, alanine, leucine, isoleucine, and methionine. The amino acid residue having a hydrophobic side chain may be valine or proline.

[0228] EP may contain at least one positively charged amino acid residue, for example, at least one lysine residue and / or at least one arginine residue. EP may contain at least two, at least three, at least four or more lysine residues and / or arginine residues.

[0229] EP may 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, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, KKKKR, KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG (wherein B is beta-alanine). The amino acids in EP may have D or L stereochemistry.

[0230] EP may contain 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 contain PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH (wherein B is beta-alanine). The amino acids in EP may have D or L stereochemistry.

[0231] EP can 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 can consist of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH (where B is beta-alanine). The amino acids in EP can have D or L stereochemistry.

[0232] EP can include an amino acid sequence identified in the art as a nuclear localization sequence (NLS). EP can consist of an amino acid sequence identified in the art as a nuclear localization sequence (NLS). EP can include an NLS containing the amino acid sequence PKKKRKV. EP can consist of an NLS containing the amino acid sequence PKKKRKV. EP can include an NLS selected from the amino acid sequences NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK. EP can consist of an NLS selected from the amino acid sequences NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK.

[0233] All extracyclic sequences may also contain an N-terminal acetyl group. Thus, for example, EP may have the structure: Ac-PKKKRKV.

[0234] Cell-penetrating peptide (CPP) Cell-penetrating peptides (CPPs) may contain from 6 to 20 amino acid residues. The cell-penetrating peptide may be a cyclic cell-penetrating peptide (cCPP). The cCPP is capable of crossing the cell membrane. The extracyclic peptide (EP) may be conjugated to the CPP, and the resulting construct may be referred to as an endosome escape vehicle (EEV). The cCPP may direct a cargo (e.g., a therapeutic moiety (TM), e.g., a hybrid oligonucleotide as described herein) to cross the cell membrane. The cCPP may deliver the cargo to the cytosol of the cell. The cCPP may deliver the cargo to the cellular location where the target (e.g., pre-mRNA) is located. To conjugate the cCPP to a cargo (e.g., a hybrid oligonucleotide as described herein), at least one bond or lone pair on the cCPP may be replaced.

[0235] The total number of amino acid residues in the cCPP ranges from 6 to 20 amino acid residues, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues (including all ranges and subranges therebetween). The cCPP may contain from 6 to 13 amino acid residues. The cCPPs disclosed herein may contain from 6 to 10 amino acids. By way of example, a cCPP containing 6 to 10 amino acid residues may have a structure according to any of Formulas I-A to I-E: [Chemical formula] (wherein AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and AA 10 are amino acid residues).

[0236] The cCPP may contain from 6 to 8 amino acids. The cCPP may contain 8 amino acids.

[0237] Each amino acid in cCPP can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic chemical moiety that is a homolog of a natural amino acid in that it has a structure similar to that of a natural amino acid so as to mimic the structure and reactivity of a natural amino acid. A non-natural amino acid can be a modified amino acid that is not one of the 20 common naturally occurring amino acids or the rare natural amino acid selenocysteine or pyrrolysine, and / or an amino acid analog. A non-natural amino acid can also be the D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, alloisoleucine, 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, their derivatives, or combinations thereof. These, and other amino acids, are listed in Table 1 along with their abbreviations as used herein.

[0238]

Table 1-1

Table 1-2

[0239] cCPP can contain from 6 to 20 amino acids, where (i) at least one amino acid has a side chain containing a guanidine group or its protonated form; (ii) at least one amino acid has no side chain or

Chemical formula

[0240] its protonated form; (iii) at least two amino acids independently have side chains containing an aromatic or heteroaromatic group. [Chemical] may have a side chain containing, or its protonated form. As used herein, when there is no side chain, the amino acid has two hydrogen atoms (e.g., -CH2-) on the carbon atom connecting the amine and the carboxylic acid.

[0241] The amino acid without a side chain can be glycine or b-alanine.

[0242] cCPP may contain 6 to 20 amino acid residues forming cCPP, (i) at least one amino acid can be glycine, b-alanine, or a 4-aminobutyric acid residue; (ii) at least one amino acid may have a side chain containing an aryl or heteroaryl group; (iii) at least one amino acid is a guanidine group, [Chemical] and may have a side chain containing, or its protonated form.

[0243] cCPP may contain 6 to 20 amino acid residues forming cCPP, (i) at least two amino acids can independently be beglycine, b-alanine, or a 4-aminobutyric acid residue; (ii) at least one amino acid may have a side chain containing an aryl or heteroaryl group; (iii) at least one amino acid is a guanidine group, [Chemical] and may have a side chain containing, or its protonated form.

[0244] cCPP may contain 6 to 20 amino acid residues forming cCPP, (i) at least three amino acids can independently be glycine, b-alanine, or a 4-aminobutyric acid residue; (ii) at least one amino acid may have a side chain containing an aromatic or heteroaromatic group; (iii) at least one amino acid is a guanidine group, [Chemical] and may have a side chain containing or its protonated form.

[0245] Glycine and related amino acid residues cCPP may contain (i) 1, 2, 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may contain (i) 2 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may contain (i) 3, 4, or 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof.

[0246] cCPP may contain (i) 1, 2, 3, 4, 5, or 6 glycine residues. cCPP may contain (i) 2 glycine residues. cCPP may contain (i) 3 glycine residues. cCPP may contain (i) 4 glycine residues. cCPP may contain (i) 5 glycine residues. cCPP may contain (i) 6 glycine residues. cCPP may contain (i) 3, 4, or 5 glycine residues. cCPP may contain (i) 3 or 4 glycine residues. cCPP may contain (i) 2 or 3 glycine residues. cCPP may contain (i) 1 or 2 glycine residues.

[0247] cCPP may include (i) 3, 4, 5, or 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may include (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may include (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may include (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may include (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may include (i) 3, 4, or 5 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof. cCPP may include (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or combinations thereof.

[0248] cCPP may include at least 3 glycine residues. cCPP may include (i) 3, 4, 5, or 6 glycine residues. cCPP may include (i) 3 glycine residues. cCPP may include (i) 4 glycine residues. cCPP may include (i) 5 glycine residues. cCPP may include (i) 6 glycine residues. cCPP may include (i) 3, 4, or 5 glycine residues. cCPP may include (i) 3 or 4 glycine residues.

[0249] In embodiments, none of the glycine, β-alanine, or 4-aminobutyric acid residues in cCPP are adjacent. Two or three glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent. Two glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent.

[0250] In embodiments, none of the glycine residues in cCPP are adjacent. Each glycine residue in cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be adjacent. Two glycine residues may be adjacent.

[0251] An amino acid side chain having an aromatic or heteroaromatic group cCPP may comprise 2, 3, 4, 5 or 6 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group. cCPP may comprise 2 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group. cCPP may comprise 3 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group. cCPP may comprise 4 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group. cCPP may comprise 5 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group. cCPP may comprise 6 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group. cCPP may comprise 2, 3, or 4 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group. cCPP may comprise 2 or 3 amino acid residues independently having a side chain containing an aromatic or heteroaromatic group.

[0252] cCPP may comprise 2, 3, 4, 5 or 6 amino acid residues independently having a side chain containing an aromatic group. cCPP may comprise 2 amino acid residues independently having a side chain containing an aromatic group. cCPP may comprise 3 amino acid residues independently having a side chain containing an aromatic group. cCPP may comprise 4 amino acid residues independently having a side chain containing an aromatic group. cCPP may comprise 5 amino acid residues independently having a side chain containing an aromatic group. cCPP may comprise 6 amino acid residues independently having a side chain containing an aromatic group. cCPP may comprise 2, 3, or 4 amino acid residues independently having a side chain containing an aromatic group. cCPP may comprise 2 or 3 amino acid residues independently having a side chain containing an aromatic group.

[0253] The aromatic group can be a 6- to 14-membered aryl. The aryl can be phenyl, naphthyl or anthracenyl, each of which is optionally substituted. The aryl can be phenyl or naphthyl, each of which is optionally substituted. The heteroaromatic group can be a 6- to 14-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S. The heteroaryl can be pyridyl, quinolyl, or isoquinolyl.

[0254] Amino acid residues having side chains containing an aromatic or heteroaromatic group can 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 acid having a side chain containing an aromatic or heteroaromatic group is

Chemical formula

[0255] Amino acid residues having side chains containing an aromatic or heteroaromatic group can each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. Amino acid residues having side chains containing an aromatic group can each independently be a residue 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-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine. Amino acid residues having side chains containing an aromatic group can each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, or homonaphthylalanine, each of which is optionally substituted with one or more substituents. Amino acid residues having side chains containing an aromatic group can each independently be a residue of phenylalanine, naphthylalanine, homophenylalanine, homonaphthylalanine, bis(homonaphthylalanine), or bis(homonaphthylalanine), each of which is optionally substituted with one or more substituents. Amino acid residues having side chains containing an aromatic group can each independently be a residue of phenylalanine or naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain containing an aromatic group can be a residue of phenylalanine. At least two amino acid residues having side chains containing an aromatic group can be residues of phenylalanine. Each amino acid residue having a side chain containing an aromatic group can be a residue of phenylalanine.

[0256] In an embodiment, none of the amino acids having a side chain containing an aromatic or heteroaromatic group are adjacent. Two amino acids having a side chain containing an aromatic or heteroaromatic group may be adjacent. The two adjacent amino acids may have opposite stereochemistry. The two adjacent amino acids may have the same stereochemistry. Three amino acids having a side chain containing an aromatic or heteroaromatic group may be adjacent. The three adjacent amino acids may have the same stereochemistry. The three adjacent amino acids may have alternating stereochemistry.

[0257] An amino acid residue containing an aromatic or heteroaromatic group may be an L-amino acid. An amino acid residue containing an aromatic or heteroaromatic group may be a D-amino acid. An amino acid residue containing an aromatic or heteroaromatic group may be a mixture of D- and L-amino acids.

[0258] Any substituent can be, for example, any atom or group that does not significantly reduce (e.g., by more than 50%) the cytoplasmic delivery efficiency of cCPP as compared to the same sequence without the substituent. Any substituent can be a hydrophobic substituent or a hydrophilic substituent. Any substituent can be a hydrophobic substituent. The substituent can increase the solvent-accessible surface area (as defined herein) of the hydrophobic amino acid. The substituent can be a halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboximidyl, alkoxycarbonyl, alkylthio, or arylthio. The substituent can be a halogen.

[0259] While not wishing to be bound by theory, amino acids having an aromatic or heteroaromatic group with a higher hydrophobicity value (i.e., amino acids having a side chain containing an aromatic or heteroaromatic group) are thought to be able to improve the cytoplasmic delivery efficiency of cCPP compared to amino acids having a lower hydrophobicity value. Each hydrophobic amino acid can independently have a hydrophobicity value higher than that of glycine. Each hydrophobic amino acid can independently be a hydrophobic amino acid having a hydrophobicity value higher than that of alanine. Each hydrophobic amino acid can independently have a hydrophobicity value of phenylalanine or higher. Hydrophobicity can be measured using hydrophobicity scales known in the art. Table B shows the hydrophobicity values for various amino acids reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S.A. 1984; 81(1): 140-144), Engleman, et al. (Ann. Rev. 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. U.S.A. 1981; 78(6): 3824-3828), and Janin (Nature. 1979; 277(5696): 491-492) (each of which is hereby incorporated by reference in its entirety). Hydrophobicity can be measured using the hydrophobicity scale reported by Engleman, et al.

[0260]

Table 2

[0261] An amino acid residue having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof As used herein, guanidine has the structure:

Chemical formula

[0262] As used herein, the protonated form of guanidine has the structure: [Chemical formula] and refers to.

[0263] A guanidine replacement group refers to a functional group on the side chain of an amino acid or the hydrogen bonding donating and accepting activity of a guanidinium group that will be positively charged at or above physiological pH.

[0264] The guanidine replacement group facilitates the cellular permeation and delivery of therapeutic agents while reducing the toxicity associated with the guanidine group or its protonated form. The cCPP may include at least one amino acid having a side chain containing a guanidine or guanidinium replacement group. The cCPP may include at least two amino acids having a side chain containing a guanidine or guanidinium replacement group. The cCPP may include at least three amino acids having a side chain containing a guanidine or guanidinium replacement group.

[0265] The guanidine or guanidinium group may be an isostere of guanidine or guanidinium. The guanidine or guanidinium replacement group may be less basic than guanidine.

[0266] As used herein, a guanidine replacement group [Chemical formula] or its protonated form and refers to.

[0267] 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 containing a guanidine group or its protonated form; (ii) at least one amino acid residue has no side chain or [Chemical formula] relates to a cCPP having a side chain containing or its protonated form; (iii) at least two amino acid residues independently have a side chain containing an aromatic or heteroaromatic group.

[0268] At least two amino acid residues have no side chain, or

Chemical Structure

[0269] The cCPP contains at least one amino acid having a side chain containing one of the following sites:

Chemical Structure

[0270] The cCPP contains at least two amino acids each independently having one of the following sites

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0271] cCPP may independently include 2, 3, 4, 5, or 6 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 2 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 3 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 4 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 5 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 6 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 2, 3, 4, or 5 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 2, 3, or 4 amino acid residues having a side chain containing a guanidine group, a guanidine replacement group, or a protonated form thereof. cCPP may independently include 2 or 3 amino acid residues having a side chain containing a guanidine group or a protonated form thereof. cCPP may include at least 1 amino acid residue having a side chain containing a guanidine group or a protonated form thereof. cCPP may include 2 amino acid residues having a side chain containing a guanidine group or a protonated form thereof. cCPP may include 3 amino acid residues having a side chain containing a guanidine group or a protonated form thereof.

[0272] An amino acid residue can independently have a side chain containing a non-adjacent guanidine group, guanidine replacement group, or its protonated form. Two amino acid residues can independently have a side chain containing an adjacent guanidine group, guanidine replacement group, or its protonated form. Three amino acid residues can independently have a side chain containing an adjacent guanidine group, guanidine replacement group, or its protonated form. Four amino acid residues can independently have a side chain containing an adjacent guanidine group, guanidine replacement group, or its protonated form. Adjacent amino acid residues can have the same stereochemistry. Adjacent amino acids can have alternating stereochemistry.

[0273] An amino acid residue independently having a side chain containing a guanidine group, guanidine replacement group, or its protonated form can be an L-amino acid. An amino acid residue independently having a side chain containing a guanidine group, guanidine replacement group, or its protonated form can be a D-amino acid. An amino acid residue independently having a side chain containing a guanidine group, guanidine replacement group, or its protonated form can be a mixture of L- or D-amino acids.

[0274] Each amino acid residue having a side chain containing a guanidine group or its protonated form can independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid or its protonated form. Each amino acid residue having a side chain containing a guanidine group or its protonated form can independently be a residue of arginine or its protonated form.

[0275] Each amino acid having a side chain containing a guanidine replacement group or its protonated form can independently be

Chemical formula

[0276] Without being bound by theory, the guanidine replacement group has a reduced basicity compared to arginine and in some cases is uncharged at physiological pH (e.g., -N(H)C(O)) and is hypothesized to be able to maintain a bidentate hydrogen bonding interaction with phospholipids on the cell membrane that facilitates effective membrane association and subsequent internalization. Removal of the positive charge is also thought to reduce the toxicity of cCPP.

[0277] One of ordinary skill in the art will understand that the N and / or C terminus of the above non-natural aromatic hydrophobic amino acids form amide bonds upon incorporation into the peptides disclosed herein.

[0278] cCPP can include a first amino acid having a side chain containing an aromatic or heteroaromatic group and a second amino acid having a side chain containing an aromatic or heteroaromatic group. The N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain containing an aromatic or heteroaromatic group, and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain containing an aromatic or heteroaromatic group. By convention, the term "first amino acid" often refers to the N-terminal amino acid of a peptide sequence, but as used herein, the "first amino acid" is used to distinguish the amino acid of interest from another amino acid in the CPP (e.g., the "second amino acid") such that the term "first amino acid" can refer to the amino acid located at the N-terminus of the peptide sequence.

[0279] cCPP can include the N-terminus of a second glycine that forms a peptide bond with an amino acid having a side chain containing an aromatic or heteroaromatic group, and the C-terminus of the second glycine forms a peptide bond with an amino acid having a side chain containing a guanidine group or its protonated form.

[0280] cCPP may include a first amino acid having a side chain containing a guanidine group or its protonated form, and a second amino acid having a side chain containing a guanidine group or its protonated form. The N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain containing a guanidine group or its protonated form, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain containing a guanidine group or its protonated form.

[0281] cCPP may include residues of asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine. cCPP may include a residue of asparagine. cCPP may include a residue of glutamine.

[0282] cCPP may include 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-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.

[0283] While not desiring to be bound by theory, the chirality of the amino acids in cCPP is thought to be able to affect cytoplasmic uptake efficiency. cCPP can include at least one D-amino acid. cCPP can include from 1 to 15 D-amino acids. cCPP can include from 1 to 10 D-amino acids. cCPP can include 1, 2, 3, or 4 D-amino acids. cCPP can include 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids having alternating D and L chirality. cCPP can include 3 adjacent amino acids having the same chirality. cCPP can include 2 adjacent amino acids having the same chirality. At least two of the amino acids can have opposite chirality. At least two amino acids having opposite chirality can be adjacent to each other. At least three amino acids can have alternating stereochemistry relative to each other. At least three amino acids having alternating chirality relative to each other can be adjacent to each other. At least four amino acids have alternating stereochemistry relative to each other. At least four amino acids having alternating chirality relative to each other can be adjacent to each other. At least two of the amino acids can have the same chirality. At least two amino acids having the same chirality can be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids having opposite chirality can be adjacent to at least two amino acids having the same chirality. Thus, the adjacent amino acids in cCPP can have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D. All of the amino acid residues forming cCPP can be L-amino acids. All of the amino acid residues forming cCPP can be D-amino acids.

[0284] At least two of the amino acids may have different chirality. At least two amino acids having different chirality may be adjacent to each other. At least three amino acids may have different chirality compared to adjacent amino acids. At least four amino acids may have different chirality compared to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chirality. One or more amino acid residues forming cCPP may be achiral. cCPP may contain a motif of 3, 4, or 5 amino acids, and two amino acids having the same chirality may be separated by an achiral amino acid. cCPP may contain the following sequences: D-X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L (where X is an achiral amino acid). The achiral amino acid may be glycine.

[0285]

Chemical formula

Chemical formula

Chemical formula

[0286] [Chemical formula] and at least two amino acids having side chains including or in its protonated form alternate with at least two amino acids having side chains including a guanidine group or in its protonated form.

[0287] cCPP has the structure of formula (A): [Chemical formula] or its protonated form (wherein, R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4, R5, R6, R7 are independently H or an amino acid side chain; At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, 3-(1-piperidinyl)alanine; AA SC is an amino acid side chain; q is 1, 2, 3, or 4) may include.

[0288] In embodiments, at least one of R4, R5, R6, and R7 is independently an uncharged non-aromatic side chain of an amino acid. In embodiments, at least one of R4, R5, R6, and R7 is independently H or the side chain of citrulline.

[0289] In embodiments, q is 1. In embodiments, q is 2. In embodiments, q is 3. In embodiments, q is 4.

[0290] In an embodiment, a compound comprising a cyclic peptide having 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, is provided. 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-naphthalen-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 having 6 to 12 amino acids, two amino acids of the cyclic peptide are arginine, at least two amino acids are aromatic hydrophobic amino acids selected from phenylalanine, naphthylalanine and combinations thereof, and at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine and combinations thereof.

[0291] cCPP has the structure of formula (I):

Chemical formula

[0292] R1, R2, and R3 can each independently be H, -alkylene-aryl, or -alkylene-heteroaryl. R1, R2, and R3 can each independently be H, -C 1-3 alkylene-aryl, or -C 1-3 alkylene-heteroaryl. 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 Alkylene can be methylene. Aryl can be 6- to 14-membered aryl. Heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl can be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl can be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R1, R2, and R3 can each independently be H, -C 1-3 alkylene-Ph or -C 1-3 alkylene-naphthyl. R1, R2, and R3 can each independently be H, -CH2Ph, or -CH2naphthyl. R1, R2, and R3 can each independently be H or -CH2Ph.

[0293] R1, R2, and R3 can each independently be the 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-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.

[0294] R1 can be the side chain of tyrosine. R1 can be the side chain of phenylalanine. R1 can be the side chain of 1-naphthylalanine. R1 can be the side chain of 2-naphthylalanine. R1 can be the side chain of tryptophan. R1 can be the side chain of 3-benzothienylalanine. R1 can be the side chain of 4-phenylphenylalanine. R1 can be the side chain of 3,4-difluorophenylalanine. R1 can be the side chain of 4-trifluoromethylphenylalanine. R1 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R1 can be the side chain of homophenylalanine. R1 can be the side chain of β-homophenylalanine. R1 can be the side chain of 4-tert-butyl-phenylalanine. R1 can be the side chain of 4-pyridinylalanine. R1 can be the side chain of 3-pyridinylalanine. R1 can be the side chain of 4-methylphenylalanine. R1 can be the side chain of 4-fluorophenylalanine. R1 can be the side chain of 4-chlorophenylalanine. R1 can be the side chain of 3-(9-anthryl)-alanine.

[0295] R2 can be the side chain of tyrosine. R2 can be the side chain of phenylalanine. R2 can be the side chain of 1-naphthylalanine. R1 can be the side chain of 2-naphthylalanine. R2 can be the side chain of tryptophan. R2 can be the side chain of 3-benzothienylalanine. R2 can be the side chain of 4-phenylphenylalanine. R2 can be the side chain of 3,4-difluorophenylalanine. R2 can be the side chain of 4-trifluoromethylphenylalanine. R2 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R2 can be the side chain of homophenylalanine. R2 can be the side chain of β-homophenylalanine. R2 can be the side chain of 4-tert-butyl-phenylalanine. R2 can be the side chain of 4-pyridinylalanine. R2 can be the side chain of 3-pyridinylalanine. R2 can be the side chain of 4-methylphenylalanine. R2 can be the side chain of 4-fluorophenylalanine. R2 can be the side chain of 4-chlorophenylalanine. R2 can be the side chain of 3-(9-anthryl)-alanine.

[0296] R3 can be the side chain of tyrosine. R3 can be the side chain of phenylalanine. R3 can be the side chain of 1-naphthylalanine. R3 can be the side chain of 2-naphthylalanine. R3 can be the side chain of tryptophan. R3 can be the side chain of 3-benzothienylalanine. R3 can be the side chain of 4-phenylphenylalanine. R3 can be the side chain of 3,4-difluorophenylalanine. R3 can be the side chain of 4-trifluoromethylphenylalanine. R3 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 can be the side chain of homophenylalanine. R3 can be the side chain of β-homophenylalanine. R3 can be the side chain of 4-tert-butyl-phenylalanine. R3 can be the side chain of 4-pyridinylalanine. R3 can be the side chain of 3-pyridinylalanine. R3 can be the side chain of 4-methylphenylalanine. R3 can be the side chain of 4-fluorophenylalanine. R3 can be the side chain of 4-chlorophenylalanine. R3 can be the side chain of 3-(9-anthryl)-alanine.

[0297] R4 can be H, -alkylene-aryl, or -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 Alkylene can be methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl can be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl can be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R4 can be H, -C 1-3 alkylene-Ph or -C 1-3It can be alkylene-naphthyl. R4 can be H or the 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 -CH2 naphthyl. R4 can be H or -CH2Ph.

[0298] 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 1-3 Alkylene can be methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl can be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl can be phenyl. Heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R5 can be H, -C 1-3 alkylene-Ph or -C 1-3 alkylene-naphthyl. R5 can be H or the 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 -CH2 naphthyl. R4 can be H or -CH2Ph.

[0299] 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 1-3The alkylene can be methylene. The aryl can be a 6- to 14-membered aryl. The heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R6 can be H, -C 1-3 Alkylene-Ph or -C 1-3 It can be alkylene-naphthyl. R6 can be H or the 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.

[0300] R7 can be H, -alkylene-aryl, -alkylene-heteroaryl. R7 can be H, -C 1-3 Alkylene-aryl, or -C 1-3 It can be alkylene-heteroaryl. R7 can be H or -alkylene-aryl. R7 can be H or -C 1-3 It can be alkylene-aryl. C 1-3 The alkylene can be methylene. The aryl can be a 6- to 14-membered aryl. The heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R7 can be H, -C 1-3 Alkylene-Ph or -C 1-3 It can be alkylene-naphthyl. R7 can be H or the side chain of an amino acid in Table A or Table C. R7 can be H or an amino acid residue having a side chain containing an aromatic group. R7 can be H, -CH2Ph, or -CH2naphthyl. R7 can be H or -CH2Ph.

[0301] One, two, or three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. One of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Two of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. At least one of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Four or fewer of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph.

[0302] 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.

[0303] One, two, or three of R1, R2, R3, R4, R5, R6, and R7 can be H. One of R1, R2, R3, R4, R5, R6, and R7 can be H. Two of R1, R2, R3, R4, R5, R6, and R7 are H. Three of R1, R2, R3, R5, R6, and R7 can be H. At least one of R1, R2, R3, R4, R5, R6, and R7 can be H. Three or fewer of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph.

[0304] 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.

[0305] At least one of R4, R5, R6, and R7 can be the side chain of 3-guanidino-2-aminopropionic acid. At least one of R4, R5, R6, and R7 can be the side chain of 4-guanidino-2-aminobutyric acid. At least one of R4, R5, R6, and R7 can be the side chain of arginine. At least one of R4, R5, R6, and R7 can be the side chain of homoarginine. At least one of R4, R5, R6, and R7 can be the side chain of N-methylarginine. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethylarginine. At least one of R4, R5, R6, and R7 can be the side chain of 2,3-diaminopropionic acid. At least one of R4, R5, R6, and R7 can be the side chain of 2,4-diaminobutyric acid, lysine. At least one of R4, R5, R6, and R7 can be the side chain of N-methyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N-ethyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least one of R4, R5, R6, and R7 can be the side chain of citrulline. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethyllysine, β-homoarginine. At least one of R4, R5, R6, and R7 can be the side chain of 3-(1-piperidinyl)alanine.

[0306] At least two of R4, R5, R6, and R7 can be the side chains of 3-guanidino-2-aminopropionic acid. At least two of R4, R5, R6, and R7 can be the side chains of 4-guanidino-2-aminobutyric acid. At least two of R4, R5, R6, and R7 can be the side chains of arginine. At least two of R4, R5, R6, and R7 can be the side chains of homoarginine. At least two of R4, R5, R6, and R7 can be the side chains of N-methylarginine. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethylarginine. At least two of R4, R5, R6, and R7 can be the side chains of 2,3-diaminopropionic acid. At least two of R4, R5, R6, and R7 can be the side chains of 2,4-diaminobutyric acid, lysine. At least two of R4, R5, R6, and R7 can be the side chains of N-methyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N-ethyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least two of R4, R5, R6, and R7 can be the side chains of citrulline. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine, β-homoarginine. At least two of R4, R5, R6, and R7 can be the side chains of 3-(1-piperidinyl)alanine.

[0307] At least three of R4, R5, R6, and R7 can be the side chains of 3-guanidino-2-aminopropionic acid. At least three of R4, R5, R6, and R7 can be the side chains of 4-guanidino-2-aminobutyric acid. At least three of R4, R5, R6, and R7 can be the side chains of arginine. At least three of R4, R5, R6, and R7 can be the side chains of homoarginine. At least three of R4, R5, R6, and R7 can be the side chains of N-methylarginine. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethylarginine. At least three of R4, R5, R6, and R7 can be the side chains of 2,3-diaminopropionic acid. At least three of R4, R5, R6, and R7 can be the side chains of 2,4-diaminobutyric acid, lysine. At least three of R4, R5, R6, and R7 can be the side chains of N-methyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N-ethyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least three of R4, R5, R6, and R7 can be the side chains of citrulline. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine, β-homoarginine. At least three of R4, R5, R6, and R7 can be the side chains of 3-(1-piperidinyl)alanine.

[0308] AA SC can be the side chain of a residue of asparagine, glutamine, or homoglutamine. AA SC can be the side chain of a residue of glutamine. cCPP is AA SC, for example, may further include a linker conjugated to a residue of asparagine, glutamine, or homoglutamine. Thus, cCPP may further include a linker conjugated to an asparagine, glutamine, or homoglutamine residue. cCPP may further include a linker conjugated to a glutamine residue.

[0309] 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.

[0310] m can be from 1 to 3. m can be 1 or 2. m can be 0. m can be 1. m can be 2. m can be 3.

[0311] The cCPP of formula (A) has the structure of formula (I)

Chemical formula

[0312] The cCPP of formula (A) has the structure of formula (I-a) or formula (I-b):

Chemical formula

[0313] The cCPP of formula (A) has the structure of formula (I-1), (I-2), (I-3) or (I-4):

Chemical formula

Chemical formula

[0314] The cCPP of formula (A) has the structure of formula (I-5) or (I-6):

Chemical formula

[0315] The cCPP may include one of the following sequences: FGFGRGR; GfFGrGr, FfΦGRGR; FfFGRGR; or FfΦGrGr. The cCPP may have one of the following sequences: FGFGRGRQ; GfFGrGrQ, FfΦGRGRQ; FfFGRGRQ; or FfΦGrGrQ.

[0316] The present disclosure also provides a structure of formula (II):

Chemical formula

Chemical formula

[0317] R 2a , R 2b , R 2c and R 2d at least two of which are

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0318] R 2a 、R 2b 、R 2c and R 2d All of which are

Chemical formula

Chemical formula

Chemical formula

[0319] R 2a 、R 2b 、R 2c and R 2d each can independently be the 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-aminobutanedioic acid, aspartic acid, glutamic acid, or homoglutamic acid.

[0320] AA SC is

Chem.

Chem.

[0321] R 1a 、R 1b 、and R 1c each can independently be a 6- to 14-membered aryl. R 1a 、R 1b 、and R 1c each can independently be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, or S. R 1a 、R 1b 、and R 1c are each independently selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl, or isoquinolyl. R 1a 、R 1b 、and R 1c can each independently be selected from phenyl, naphthyl, or anthracenyl. R 1a 、R 1b 、and R 1cmay each independently be phenyl or naphthyl. R 1a R 1b and R 1c may each independently be selected from pyridyl, quinolyl, or isoquinolyl.

[0322] 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.

[0323] 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.

[0324] 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'' may be 1, and each n' may be 3.

[0325] The cCPP of formula (II) may have the structure of formula (II-1):

Chemical formula

[0326] The CPP of formula (II)c has the structure of formula (IIa): [Chemical formula] (wherein R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , AA SC and n' are as defined herein) may have.

[0327] The cCPP of formula (II) has the structure of formula (IIb): [Chemical formula] (wherein R 2a , R 2b , AA SC , and n' are as defined herein) may have.

[0328] cCPP has the structure of formula (IIc): [Chemical formula] , or its protonated form (wherein AA SC and n' are as defined herein) may have.

[0329] cCPP has the structure of formula (III): [Chemical formula] (wherein AA SC is an amino acid side chain; R 1a , R 1b , and R 1c are each independently a 6- to 14-membered aryl or 6- to 14-membered heteroaryl; R 2a and R 2c are each independently H, [Chemical formula] or its protonated form; R 2b and R 2d are each independently guanidine or its protonated form; each n'' is independently an integer from 1 to 3; each n' is independently an integer from 1 to 5; each p' is independently an integer from 0 to 5) may have.

[0330] The cCPP of formula (III) has the structure of formula (III-1): [Chemical formula] (wherein, AA SC , R 1a , R 1b , R 1c , R 2a , R 2c , R 2b , R 2d , n', n'', and p' are as defined herein) may have.

[0331] The cCPP of formula (III) has the structure of formula (IIIa): [Chemical formula] (wherein, AA SC , R 2a , R 2c , R 2b , R 2d , n', n'', and p' are as defined herein) may have.

[0332] In formulas (III), (III-1), and (IIIa), R a and R c may be H. R a and R c may be H, R b and Rd can each independently be guanidine or its protonated form. R a can be H. R b can be H. p’ can be 0. R a and R c can be H and each p’ can be 0.

[0333] In Formulas (III), (III-1), and (IIIa), R a and R c can be H, R b and R d can each independently be guanidine or its protonated form, n’’ can be 2 or 3, and each p’ can be 0.

[0334] p’ can be 0. p’ can be 1. p’ can be 2. p’ can be 3. p’ can be 4. p’ can be 5.

[0335] cCPP has the structure:

Chemical formula

[0336] The cCPP of Formula (A)

[0337]

Table 3

[0338] The cCPP of Formula (A)

[0339]

Table 4

[0340] In an embodiment, cCPP

[0341]

Table 5

[0342] cCPP has the structure of formula (D) [Chemical formula] or its protonated form (wherein R1, R2, and R3 may each independently be an amino acid residue having a side chain containing H or an aromatic group; at least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; Y is [Chemical formula] and q is 1, 2, 3, or 4; each m is independently an integer of 0, 1, 2, or 3, each n is independently an integer of 0, 1, 2, or 3) and may include

[0343] The cCPP of formula (D) (wherein Y is [Chemical formula] ).

[0344] The cCPP of formula (D) (wherein Y is [Chemical formula] ).

[0345] The cCPP of formula (D) (wherein Y is [Chemical formula] ).

[0346] The cCPP of formula (D) wherein Y is

Chemical formula

[0347] The cCPP of formula (D) wherein Y is

Chemical formula

[0348] In an embodiment, AA SC can be conjugated to a linker.

[0349] Linker The cCPPs of the present disclosure can be conjugated to a linker. The linker can link a cargo to the cCPP. The linker can be attached to a side chain of an amino acid of the cCPP, and the cargo can be attached at a suitable position on the linker.

[0350] The linker can be any suitable moiety that conjugates the cCPP to one or more additional moieties, such as an exocyclic peptide (EP) and / or any suitable moiety that can conjugate to a cargo. Prior to conjugation to the cCPP and the one or more additional moieties, the linker has two or more functional groups, each of which is independently capable of forming a covalent bond to the cCPP and the one or more additional moieties. When the cargo is an oligonucleotide, the linker can be covalently attached to the 5'-end or the 3'-end of the cargo. The linker can be covalently attached to the 5'-end of the cargo. The linker can be covalently attached to the 3'-end of the cargo. When the cargo is a peptide, the linker can be covalently attached to the N-terminus or the C-terminus of the cargo. The linker can be covalently attached to the backbone of an oligonucleotide or peptide cargo. The linker can be any suitable moiety that conjugates the cCPPs described herein to a cargo, such as an oligonucleotide, peptide, or small molecule.

[0351] The linker can include a hydrocarbon linker.

[0352] The linker may include a cleavage site. The cleavage site can be a disulfide or a caspase-cleavage site (e.g., Val-Cit-PABC).

[0353] The linker may include (i) one or more D or L amino acids (each of which is optionally substituted); (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) one or more -(R 1- J-R 2 )z”-subunits (wherein each of R 1 and R 2 is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl in each occurrence, each J is independently C, NR 3 , -NR 3 C(O)-, S, and O, R 3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z” is an integer from 1 to 50); (viii) -(R 1- J)z”- or -(J-R 1 )z”-(wherein each of R 1 is independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl in each occurrence, each J is independently C, NR 3 , -NR 3 C(O)-, S, or O, 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 include one or more of (i) to (x).

[0354] The linker includes one or more D or L amino acids and / or -(R 1- J-R 2)z”-(wherein, R 1 and R 2 each is, in each occurrence, independently alkylene, and each J is independently C, NR 3 , -NR 3 C(O)-, S, and O, and R 4 is independently selected from H and alkyl, and z” is an integer from 1 to 50); or may include combinations thereof.

[0355] The linker may include, for example, as a spacer, -(OCH2CH2) z’ -(wherein z’ is an integer from 1 to 23, for example, 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’ may also be referred to as polyethylene glycol (PEG).

[0356] The linker may include one or more amino acids. The linker may include a peptide. The linker may include -(OCH2CH2) z’ -(wherein z’ is an integer from 1 to 23), and a peptide. The peptide may include 2 to 10 amino acids. The linker may further include a functional group (FG) capable of reacting via click chemistry. FG may be an azide or an alkyne, and a triazole is formed when the cargo is conjugated to the linker.

[0357] The linker may include (i) β-alanine residue and lysine residue; (ii) -(J-R 1 )z”; or (iii) combinations thereof. Each R 1 is independently optionally alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR 3 , -NR 3 C(O)-, S, or O, and 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 R1 can be alkylene, and each J can be O.

[0358] The linker can include (i) residues of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof; and (ii) -(R 1- J)z”- or -(J-R 1 )z”. Each R 1 can independently be alkylene, alkenylene, alkynylene, carbocyclic, or heterocyclic, each J can independently be C, NR 3 , -NR 3 C(O)-, S, or O, and R 3 is H, alkyl, alkenyl, alkynyl, carbocyclic, or heterocyclic, each of which is optionally substituted, and z” can be an integer from 1 to 50. Each R 1 can be alkylene, and each J can be O. The linker can include glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0359] The linker can be a trivalent linker. The linker can have the structure:

Chemical formula

[0360] The hydrocarbon can be a residue of glycine or beta-alanine.

[0361] The linker is bivalent and can link cCPP to the cargo. The linker is bivalent and can link cCPP to the exocyclic peptide (EP).

[0362] The linker is trivalent and can link cCPP to the cargo and EP.

[0363] The linker is bivalent or trivalent C1-C 50 alkylene (wherein 1 to 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 is bivalent or trivalent C1-C 50 alkylene (wherein 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or combinations thereof).

[0364] The linker has the structure:

Chemical formula

[0365] cCPP can be conjugated to cargo via a linker ("L"). The linker can be conjugated to cargo via a linking group ("M").

[0366] The linker has the structure:

Chemical formula

[0367] The linker has the structure:

Chemical formula

[0368] The linker has the structure:

Chemical formula

[0369] x can be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (including all ranges and sub - ranges therebetween).

[0370] x' can be an integer from 1 to 23, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 (including all ranges and sub - ranges therebetween). x' can be an integer from 5 to 15. x' can be an integer from 9 to 13. x' can be an integer from 1 to 5. x' can be 1.

[0371] y can be an integer from 1 to 5, for example, 1, 2, 3, 4, or 5 (including all ranges and sub - ranges therebetween). y can be an integer from 2 to 5. y can be an integer from 3 to 5. y can be 3 or 4. y can be 4 or 5. y can be 3. y can be 4. y can be 5.

[0372] z can be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (including all ranges and sub - ranges therebetween).

[0373] z' can be an integer from 1 to 23, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 (including all ranges and sub - ranges therebetween). z' can be an integer from 5 to 15. z' can be an integer from 9 to 13. z' can be 11.

[0374] As discussed above, a linker or M (where M is part of the linker) can be covalently attached to a cargo at any suitable position on the cargo. The linker or M (where M is part of the linker) can be covalently attached to the 3'-end of an oligonucleotide cargo or the 5'-end of an oligonucleotide cargo. The linker or M (where M is part of the linker) can be covalently attached to the N-terminus or C-terminus of a peptide cargo. The linker or M (where M is part of the linker) can be covalently attached to the backbone of an oligonucleotide or peptide cargo.

[0375] The linker can be attached to a side chain of 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) on cCPP. The linker can be attached to the side chain of lysine on cCPP.

[0376] The linker can be attached to a side chain of 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) on a peptide cargo. The linker can be attached to the side chain of lysine on a peptide cargo.

[0377] The linker has the structure:

Chemical formula

[0378] The linker has the structure: [Chemical formula] (wherein, M is a group that conjugates L to a cargo, such as an oligonucleotide; AA s is the side chain or terminus of an amino acid on cCPP; each AA x is independently an amino acid residue; o is an integer from 0 to 10; p is an integer from 0 to 5) may have.

[0379] M may contain alkylene, alkenylene, alkynylene, carbocyclic, or heterocyclic, each of which is optionally substituted. M is [Chemical formula] (wherein R is alkyl, alkenyl, alkynyl, carbocyclic, or heterocyclic) may be selected from.

[0380] M is [Chemical formula] (wherein R 10 is alkylene, cycloalkyl, or [Chemical formula] and a is from 0 to 10) may be selected from.

[0381] M is [Chemical formula] may be, and R 10 is [Chemical formula] may be, and a is from 0 to 10. M is [Chemical formula] may be.

[0382] M is a hetero-bifunctional crosslinker, for example, [Chem.] which may be, as 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).

[0383] M may be -C(O)-.

[0384] AA s may be the side chain or terminus of an amino acid on cCPP. AA s Non-limiting examples of AA include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or modified side chains of glutamine or asparagine (e.g., reduced side chains having an amino group). AA s is AA as defined herein SC may be.

[0385] Each AA x is independently a natural or non-natural amino acid. One or more AAs x may be natural amino acids. One or more AAs x may be non-natural amino acids. One or more AAs x may be a β-amino acid. The β-amino acid may be β-alanine.

[0386] o is an integer from 0 to 10, for example, 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.

[0387] p can be from 0 to 5, for example, 0, 1, 2, 3, 4, or 5. p can be 0. p can be 1. p can be 2. p can be 3. p can be 4. p can be 5.

[0388] The linker has the structure:

Chemical formula

[0389] r can be 0. r can be 1.

[0390] The linker has the structure:

Chemical formula

[0391] z” can 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 in between). z” can be an integer from 5 to 20. z” can be an integer from 10 to 15.

[0392] The linker has the structure:

Chemical formula

[0393] Other non-limiting examples of suitable linkers include [Chemical formula] [Chemical formula] (wherein M and AA s are as defined herein) are included.

[0394] As used herein, a compound comprising a cargosome comprising cCPP and a hybrid oligonucleotide, such as an antisense oligonucleotide complementary to a target in a pre-mRNA sequence, wherein the compound further comprises an L, and the linker is conjugated to the hybrid oligonucleotide via a linking group (M), and M is [Chemical formula] (wherein R 1 is alkylene, cycloalkyl, or [Chemical formula] and t’ is from 0 to 10, and each R is independently alkyl, alkenyl, alkynyl, carbocyclic, or heterocyclic, and R 1 is [Chemical formula] and t’ is 2) is provided.

[0395] The linker has the structure: [Chemical formula] (wherein AA s is as defined herein and m’ is from 0 to 10) may have.

[0396] The linker has the formula: [Chemical formula] It can be of the following.

[0397] The linker can be of the formula:

Chem.

[0398] The linker can be of the formula:

Chem.

[0399] The linker can be of the formula:

Chem.

[0400] The linker can be of the formula:

Chem.

[0401] The linker can be of the formula:

Chem.

[0402] The linker can be covalently attached to the cargo at any suitable position on the cargo. The linker is covalently attached to the 3'-end of the cargo or the 5'-end of an oligonucleotide cargo. The linker can be covalently attached to the backbone of the cargo.

[0403] The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine on cCPP, or the modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). The linker can be attached to the side chain of lysine on cCPP.

[0404] cCPP-linker conjugate cCPP can be conjugated to the linker as defined herein. The linker can be conjugated to the AA of cCPP as defined herein SC and can be conjugated.

[0405] The linker can include (e.g., as a spacer)-(OCH2CH2) z’ -subunit (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’ is also referred to as PEG. The cCPP-linker conjugate can have a structure selected from Table D:

[0406] [Table 6]

[0407] The linker can be -(OCH2CH2) z’ -subunit (wherein z' is an integer from 1 to 23) and can include a peptide subunit. The peptide subunit can include 2 to 10 amino acids. The cCPP-linker conjugate can have a structure selected from Table E:

[0408] [Table 7]

[0409] An enveloped extracellular vesicle (EEV) comprising an annular cell-penetrating peptide (cCPP), a linker, and an extracellular peptide (EP) is provided. The EEV has the structure of formula (B):

Chemical formula

[0410] R1, R2, R3, R4, R7, EP, m, q, y, x’, z’ are as described herein.

[0411] n can be 0. n can be 1. n can be 2.

[0412] The EEV has the structure of formula (B-a) or (B-b):

Chemical formula

[0413] EEV has the structure of formula (B-c): [Chemical formula] or its protonated form (where EP, R 1 , R 2 , R 3 , R 4 , and m are as defined above in formula (B); AA is an amino acid defined herein; M is as defined herein; n is an integer from 0 to 2; x is an integer from 1 to 10; y is an integer from 1 to 5; z is an integer from 1 to 10) may be included.

[0414] EEV has the structure of formula (B-1), (B-2), (B-3), or (B-4): [Chemical formula] [Chemical formula] or its protonated form (where EP is as defined above in formula (B)) may have.

[0415] EEV may include formula (B) and has the structure: Ac-PKKKRKV-AEEA-K(Cyclo[FGFGRGRQ])-PEG 12 -OH or Ac-PKKKRKV-AEEA-K(Cyclo[GfFGrGrQ])-PEG 12 -OH may have.

[0416] EEV has the formula:[[]] [Chemical formula] may include cCPP of.

[0417] EEV may include the formula: Ac-PKKKRKV-miniPEG2-Lys(Cyclo(FfFGRGRQ)-miniPEG2-K(N3).

[0418] EEV can be Ac-P-K(Tfa)-K(Tfa)-K(Tfa)-R-K(Tfa)-V-AEEA-K-(Cyclo[FGFGRGRQ])-PEG12-OH.

[0419] EEV is

Chem.

[0420] EEV can be Ac-PKKKRKV-AEEA-Lys-(Cyclo[FGFGRGRQ])-PEG12-OH. EEV is

Chem.

[0421] EEV is Ac-rr-miniPEG2-Dap[Cyclo(FfΦ-Cit-r-Cit-rQ)]-PEG12-OH Ac-frr-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rfr-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbfbr-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rrr-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbr-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbrbr-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hh-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hbh-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hbhbh-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rbhbh-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-hbrbh-PEG2-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH Ac-rr-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-frr-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rfr-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbfbr-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rrr-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbr-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbrbr-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hh-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hbh-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hbhbh-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-rbhbh-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-hbrbh-Dap(Cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH Ac-KKKK-miniPEG2-Lys(Cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KGKK-miniPEG2-Lys(Cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKGK-miniPEG2-Lys(Cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KGK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBKBK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PGKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKGKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKGRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKGKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRGV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKKRKG-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 and Ac-KRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 may be selected from.

[0422] EEV is Ac-PKKKRKV-Lys(cyclo[FfΦGrGrQ])-PEG 12 -K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦGrGrQ])-miniPEG2-K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRQ])-miniPEG2-K(N3)-NH2 Ac-KR-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2 Ac-PKKKGKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2 Ac-PKKKRKG-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2 Ac-KKKRK-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(cyclo[FFΦGRGRQ])-miniPEG2-K(N3)-NH2 Ac-PKKKRKV-miniPEG2-Lys(cyclo[βhFfΦGrGrQ])-miniPEG2-K(N3)-NH2 and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦSrSrQ])-miniPEG2-K(N3)-NH2 may be selected from.

[0423] EEV is Ac-PKKKRKV-miniPEG2-Lys(cyclo(GfFGrGrQ])-PEG 12 -O and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFKRKRQ])-PEG 12 -OH Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRGRGQ])-PEG 12 -OH Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRGRQ])-PEG 12 -OH Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRrRQ])-PEG 12 -OH Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRRRQ])-PEG 12 -OH and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRRRRQ])-PEG 12 -OH may be selected from

[0424] EEV is Ac-K-K-K-R-K-G-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-K-K-K-R-K-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-K-K-R-K-K-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-K-R-K-K-K-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-K-K-K-K-R-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-R-K-K-K-K-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH and Ac-K-K-K-R-K-PEG4-K(Cyclo[FGFGRGRQ])-PEG 12 -OH may be selected from.

[0425] EEV is Ac-PKKKRKV-PEG2-K(Cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2 Ac-PKKKRKV-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(Cyclo[GfFGrGrQ])-PEG2-K(N3)-NH2 and Ac-PKKKRKV-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH may be selected from.

[0426] The cargo may be AC and EEV is Ac-PKKKRKV-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(Cyclo[FfFGRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FfFGRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FfFGRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FfFGRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FfFGRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(Cyclo[FfFGRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(Cyclo[FfΦGrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(Cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(Cyclo[FfFGRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH and Ac-hbrbh-PEG2-K(Cyclo[FGFRRRRQ])-PEG12 -OH (wherein b is beta-alanine and the exocyclic sequence can be of D or L stereochemistry) can be selected from.

[0427] In embodiments, cCPP is

[0428] [Table 8] can be.

[0429] The cargo can be a protein and the EEV is Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rr-PEG2-K(cyclo[FGFGRGRQ])-PEG12 -OH Ac-rr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rhr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(Cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(Cyclo[FGFRRRRQ])-PEG12 -OH Ac-rbhbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH (wherein b is beta-alanine, and the exocyclic sequence may be of D or L stereochemistry) can be selected from.

[0430] Cargo 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 site. The cargo can be conjugated to the terminal carbonyl group 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 cCPP by a linker. The cargo can be conjugated to AA SC by a linker. At least one atom of cCPP can be replaced by the therapeutic site, or at least one lone pair of cCPP can form a bond to the therapeutic site. The hydroxyl group on the amino acid side chain of cCPP can be replaced by a bond to the cargo. The hydroxyl group on the glutamine side chain of cCPP can be replaced by a bond to the cargo. The cargo can be conjugated to cCPP by a linker. The cargo can be conjugated to AA SC by a linker.

[0431] The cargo can include one or more detectable sites, one or more therapeutic sites, one or more targeting sites, or any combination thereof. The cargo can be a hybrid oligonucleotide described herein.

[0432] Cyclic cell-penetrating peptide (cCPP) conjugated to a cargo site The cyclic cell-penetrating peptide (cCPP) can be conjugated to a cargo site.

[0433] The cargo site can be conjugated to cCPP via a linker. The cargo site can include a therapeutic site. The therapeutic site can include an oligonucleotide. The oligonucleotide can include a hybrid oligonucleotide described herein. The cargo site is conjugated to the linker with a terminal carbonyl group to form the following structure: [Chemical formula] (wherein, EP is an exocyclic peptide, M, AA SC , cargo, x’, y, and z’ are as defined above, and * is the point of attachment to AA SC ) can be provided. x’ can be 1. y can be 4. z’ can be 11. -(OCH2CH2) x’ - and / or -(OCH2CH2) z’ - can each independently be replaced with one or more amino acids including, for example, glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0434] An endosomal escape vehicle (EEV) can include a cyclic cell-penetrating peptide (cCPP), an exocyclic peptide (EP), and a linker, and be conjugated to a cargo to form a structure of formula (C):

Chemical formula

[0435] R1, R2, R3, R4, EP, cargo, m, n, x’, y, q, and z’ are as defined herein.

[0436] EEV can be conjugated to a cargo, and the EEV-conjugate has the structure of formula (C-a) or (C-b): [Chemical formula] or its protonated form (wherein EP, m, and z are as defined above in formula (C)).

[0437] EEV can be conjugated to a cargo, and the EEV-conjugate has the structure of formula (C-c): [Chemical formula] or its protonated form (wherein EP, R 1 , R 2 , R 3 , R 4 , and m are as defined above in formula (III); AA can be an amino acid as defined herein; n can be an integer from 0 to 2; x can be an integer from 1 to 10; y can be an integer from 1 to 5; z can be an integer from 1 to 10).

[0438] EEV can be conjugated to an oligonucleotide cargo, and the EEV-oligonucleotide conjugate has the structure of formula (C-1), (C-2), (C-3), or (C-4): [Chemical formula] [Chemical formula] may be included.

[0439] disease 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.

[0440] In embodiments, the disease is Duchenne muscular dystrophy.

[0441] 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 bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (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 (FRDA), fragile X-related tremor / ataxia syndrome (FXTAS), myoclonic epilepsy, oculopharyngeal muscular dystrophy (OPMD), or symptomatic or asymptomatic X-linked mental retardation.

[0442] In embodiments, the disease is Friedreich's ataxia (FRDA). In embodiments, the target gene is FXN, which encodes frataxin. In embodiments, the compounds provided herein include antisense oligonucleotides that target FXN.

[0443] Compositions and Methods of Administration 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 patients having or suspected of having a genetic disease or disorder.

[0444] The in vivo application of the disclosed compounds and compositions containing them can be achieved by any suitable methods and techniques known to those skilled in the art now or in the future. For example, the disclosed compounds can be formulated in a physiologically or pharmaceutically acceptable composition and administered by any suitable route known in the art, including oral and parenteral administration routes. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal, and intrathecal administration, for example, by injection. The administration of the disclosed compounds or compositions can be a single administration or at continuous or distinct intervals, as can be readily determined by those skilled in the art.

[0445] The compounds disclosed herein and compositions containing them can also be administered utilizing liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide a uniform dosage over a long period. The compounds can also be administered in their salt derivative forms or crystalline forms.

[0446] The compounds disclosed herein can be formulated into pharmaceutical compositions according to known methods for preparing pharmaceutically acceptable compositions. Formulation is well known and is described in detail in numerous references readily available to those skilled in the art. For example, Remington’s Pharmaceutical Science by E.W.Martin (1995) describes formulations that can be used in connection with the disclosed methods. Generally, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The form depends on the intended mode of administration and therapeutic use. The compositions also include conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein can advantageously include one or more totals of the subject compounds of about 0.1% to 100% by weight based on the total weight of the composition including the carrier or diluent.

[0447] Suitable formulations for administration may include aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending and thickening agents. The formulations can be provided in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored under lyophilized (freeze-dried) conditions that require only the addition of a sterile liquid carrier, for example, water for injection, immediately before use. Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules, tablets, etc. It should be understood that, in addition to the particularly mentioned ingredients, the compositions disclosed herein may include other agents conventional in the art, taking into account the type of formulation in question.

[0448] The compounds disclosed herein, and compositions containing them, can be delivered to cells either through direct contact with the cells or via carrier means. Carrier means for delivering the compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in liposomal moieties. Another means for delivering the compounds and compositions disclosed herein to cells involves binding the compound to a targeted protein or nucleic acid for delivery to the target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and enable the composition to move across a biological membrane. U.S. Application Publication No. 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. The compounds can also be incorporated into polymers, examples of which include poly(D-L-lactide-co-glycolide) polymers for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (used in Gliadel); chondroitin; chitin; and chitosan.

[0449] The compounds and compositions disclosed herein (including their pharmaceutically acceptable salts or prodrugs) can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salt can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0450] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing an active ingredient, adapted for the extemporaneous preparation of a sterile injectable or infusible solution or dispersion, optionally encapsulated in liposomes. The final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be, for example, a solvent or liquid dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the formation of liposomes, by maintenance of the required particle size in the case of a dispersion, or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, buffers, or sodium chloride, may be included. Prolonged absorption of injectable compositions can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0451] Sterile injectable solutions are prepared by incorporating the compounds and / or agents disclosed herein in the required amounts in a suitable solvent having the various other ingredients enumerated above, followed, if necessary, by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient in addition to any additional desired ingredients present in the previously sterile-filtered solution.

[0452] 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 extrapolation of effective dosages in humans from those in mice and other animals are known in the art.

[0453] The dosage range for administration of the composition is high enough to produce the desired effect on the condition or disorder. The dosage should not be so high as to cause harmful side effects, such as unwanted cross-reactions, anaphylactic reactions, etc. Usually, the dosage varies according to age, morbidity, gender, and the degree of the disease in the patient and can be determined by those skilled in the art. The dosage can be adjusted by the individual physician in case of any contraindications. The dosage can vary and can be administered daily in one or more doses for one day or several days.

[0454] Also disclosed are pharmaceutical compositions comprising the compounds disclosed herein in combination with a pharmaceutically acceptable carrier. Disclosed herein are pharmaceutical compositions adapted for oral, topical, or parenteral administration, including the amount of the compound. The dosage administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame without accompanying lethal toxicity and without causing side effects or pathological conditions that exceed acceptable levels. One skilled in the art will recognize that the dosage will depend on a variety of factors, including the condition (health) of the subject, the weight of the subject, the type of co-treatment, if any, the frequency of treatment, the therapeutic ratio, and the severity and stage of the pathological condition.

[0455] Also disclosed is a kit comprising the compounds disclosed herein and / or a pharmaceutical composition containing the same in one or more containers. The disclosed kit may optionally contain a pharmaceutically acceptable carrier and / or diluent. In one embodiment, the kit contains one or more other ingredients, accessories, or adjuvants described herein. In one embodiment, the kit contains instructions or packaging materials describing how to administer the compounds or compositions of the kit. The containers of the kit can be of any suitable material, such as glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, the compounds and / or agents disclosed herein are provided in the kit in solid form, such as tablets, pills, or powder form. In another embodiment, the compounds and / or agents disclosed herein are provided in the kit as a liquid or solution. In one embodiment, the kit contains an ampoule or syringe containing the compounds and / or agents disclosed herein in liquid or solution form.

[0456] Treatment method The present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering a compound disclosed herein and / or a composition containing the compound. In embodiments, the disease is a genetic disease. As used herein, "genetic disease" refers to any disease or disorder caused, in whole or in part, by a change in the genomic DNA sequence from the wild-type sequence. In embodiments, the compounds disclosed herein target genes or gene transcripts associated with genetic diseases. Treatment of the disease and / or symptoms of the disease can occur via a variety of molecular mechanisms such as those described herein.

[0457] In embodiments, the patient is identified as having or being at risk of having any of the diseases described herein.

[0458] In an embodiment, treatment refers to the partial or complete alleviation, improvement, mitigation, inhibition, delay in the onset thereof, reduction in the severity and / or frequency of occurrence of one or more symptoms in a subject.

[0459] In an embodiment, a method for modifying the expression, activity or a combination thereof of a target gene in a subject in need of modifying the expression, activity or a combination thereof of the target gene is provided, which comprises administering a compound disclosed herein. In an embodiment, treatment results in a reduced expression of the target protein from the target transcript. In an embodiment, treatment results in a reduced level of the target transcript. In an embodiment, treatment results in the regulation of splicing of the target transcript and / or downstream gene transcripts controlled by a protein binding to the target transcript. In an embodiment, the regulation of splicing of the downstream gene transcript results in an increase in the downstream transcript and / or downstream protein isoform associated with a healthy phenotype. In an embodiment, alternative splicing results in a decrease in the downstream transcript and / or downstream protein isoform associated with a disease phenotype.

[0460] In an embodiment, treatment results in a decrease in the level of the target transcript and / or the expression of the target transcript when compared to the average level of the target transcript or target protein in the subject prior to treatment, or when compared to one or more control individuals having a similar disease who have not been treated.

[0461] In an embodiment, treatment results in a decrease in the level of the downstream transcript and / or the expression of the downstream gene product associated with the disease phenotype when compared to the average level of the protein in the subject prior to treatment, or when compared to one or more control individuals having a similar disease who have not been treated.

[0462] In an embodiment, the treatment results in an increase in the level of downstream transcripts and / or the expression of downstream gene products associated with a healthy phenotype when compared to the average level of a protein in the subject prior to treatment or when compared to one or more control individuals having a similar disease who have not been treated.

[0463] In an embodiment, the treatment results in a decrease in the expression of a protein in the tissue of the subject when compared to the average level of the protein in the tissue of the subject prior to treatment or when compared to one or more control individuals having a similar disease who have not been treated.

[0464] In an embodiment, the treatment results in an increase in the expression of a selectively spliced downstream protein in the tissue of the subject when compared to the average level of the downstream protein in the tissue of the subject prior to treatment or when compared to one or more control individuals having a similar disease who have not been treated.

[0465] In an embodiment, the treatment results in an increase or decrease in the expression of a wild-type downstream protein isoform in the tissue of the subject when compared to the average level of the downstream protein in the tissue of the subject prior to treatment or when compared to one or more control individuals having a similar disease who have not been treated.

[0466] The terms "improve," "increase," "reduce," "decrease," etc., as used herein, refer to a value compared to a control. In an embodiment, a suitable control is a baseline measurement, e.g., a measurement in the same individual prior to the start of the treatment described herein or a measurement in a control individual (or control individuals) in the absence of the treatment described herein. A "control individual" is an individual having the same disease who is approximately the same age and / or gender as the individual being treated (to ensure that the disease stage in the treated and control individuals is comparable).

[0467] The individual being treated (also referred to as the "patient" or "subject") is an individual (fetus, infant, child, adolescent, or adult human) having or having the potential to develop a disease. The individual can have a disease mediated by abnormal gene expression or abnormal gene splicing. In embodiments, an individual having a disease can 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 having the disease. In embodiments, the individual can 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 protein expression or activity level in an individual not having the disease.

[0468] Nucleotide repeat expansion In embodiments, methods for treating diseases associated with nucleotide repeat expansions are provided. The disease can be associated with tri-, tetra-, penta-, hexa- and dodecanucleotide repeat expansions, including

[0469] [Table 9] See Paulson, H. (2018) “Repeat expansion disease,” Handb. Clin. Neurol. 147:105-123.

[0470] In embodiments, methods for treating diseases associated with trinucleotide repeat expansions are provided. In embodiments, the trinucleotide repeat expansion is in the 3' untranslated region of the gene / transcript. In embodiments, the trinucleotide repeat expansion is a CTG·CUG expansion. In embodiments, methods for treating myotonic dystrophy (DM1) are provided.

[0471] In embodiments, methods are provided for treating myotonic dystrophy type 1 (DM1) by reducing sequestration of at least one RNA-binding protein to pre-mRNA containing at least one expanded CUG repeat. In embodiments, methods are provided for treating DM1 by reducing the accumulation of pre-mRNA containing at least one expanded CUG repeat. In embodiments, methods are provided for treating DM1 by correcting splicing defects in downstream gene transcripts.

[0472] In embodiments, treatment results in a decrease in the number of CUG repeat RNA nuclear foci of the target gene when compared to the average level focus in the subject prior to treatment or one or more control individuals having a similar disease who have not been treated.

[0473] All publications, patents, and patent applications mentioned herein are indicative of the level of those of ordinary skill in the art to which the present 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 were specifically and individually indicated to be incorporated herein by reference.

Examples

[0474] Example 1: Synthetic method Method The synthesis of oligonucleotides was carried out on a Nitto UnyLinker support at 195 μmol / g on a 10 μmol scale. Three sets of ACGT monomers were used. The PMO 6'-amidite was purchased from Granlen Inc. The PMO 6'-phosphoramidate was purchased from Hongene Biotech Corporation. The reverse DNA or RNA amidites were purchased from ChemGenes Corporation or Hongene Biotech Corporation. For the sequences starting from PMO, the first monomer was the PMO amidite. All solid-phase syntheses were carried out using a Mermade6 automated synthesizer starting from the 5'-end. The PMO fragments were synthesized in an 11 mL glass vial or, on a larger scale, in a 25 mL glass peptide synthesizer with a frit.

[0475] Addition of the first morpholino monomer to the solid support: 1.0 g of the UnyLinker 195 μmol / g-loaded support was placed in a glass peptide synthesis vessel with a frit glass support. After detritylation and acetonitrile washing steps, a solution of the A-PMO amidite (600 mg, 0.77 mmol, 4 eq) monomer in acetonitrile (0.1 M, 7 mL) and solid 5-ethylthio-1H-tetrazole (ETT) (200 mg, 1.53 mmol) were added. The vessel was capped and stirred at room temperature for 30 minutes. After draining the solvent, the coupling step was repeated. Oxidation was carried out using a 0.05 M iodine-water (10%)-pyridine (90%) solution until discoloration no longer occurred. Acetonitrile washing removed traces of the oxidizing solution.

[0476] Detritylation solution (CYTFA): 100 mM 4-cyanopyridine, 100 mM TFA in dichloromethane:trifluoroethanol:ethanol (80:20:1). Neutralization solution (N): 5% DIPEA in dichloromethane:isopropanol (3:1). Coupling solution (C): 1.6 g of the PMO monomer, 9.0 mL of DMI, 1.0 mL of NEM. Washing solution (W): Dichloromethane. Storage solution (S): 30% trifluoroethanol in dichloromethane.

[0477]

Table 10

[0478] Addition of DNA monomer Addition of reverse DNA amidite to the terminal PMO monomer: The support (100 mg) was weighed into a 4 mL frit Mermade6 column and placed on a vacuum manifold for solid-phase extraction. The trityl group was removed using CYTFA (6×10 min) and neutralization solution (4×5 min). The column was transferred to Mermade6 for coupling with the first amidite (3×10 min) followed by sulfurization with 3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT) solution (2×3 min). The synthesis of the ASO fragment was continued in an automated manner and terminated with a dimethoxytrityl (DMT) protecting group. ASO synthesis on a 10 - 15 μmol scale ( Washing: Acetonitrile Deblocking: 3% trichloroacetic acid in DCM The amidite solution is 0.1 M in acetonitrile The activator solution is 0.2 M benzyl-S-tetrazole in acetonitrile. Oxidizing reagent: 0.05 M iodine, 10% water, 90% pyridine Sulfurizing reagent: 0.1 M DDTT in pyridine Cap A solution: 10% lutidine, 10% isobutyric anhydride, 80% ACN Cap B solution: 16% 1-methylimidazole, 85% THF Automated synthesis Start: ACN wash 2×1.5 mL Execution steps: Deblocking 4×1.2 mL ACN wash 4×1.5 mL Coupling 2 x 800 uL amidite solution + 800 uL activator (3 minutes for DNA, 10 minutes for RNA, reverse DNA, LNA) ACN wash 1.5 mL Oxidation 2 x 1000 uL oxidizing factor solution or 1000 uL sulfurizing solution ACN wash 1.5 mL Capping 2 x (A 400 uL + Cap B 400 uL) ACN wash 2 x 1.5 mL Finishing: Deblocking 4 x 1.2 mL ACN wash 4 x 1.5 mL

[0479] Deprotection and purification For simplicity of IEX purification, the trityl group was maintained. A suspension of the solid support in 15 mL of a 1 M 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) solution in dry acetonitrile (ACN) was shaken at room temperature for 1.5 hours. The clear solution was decanted and the process was repeated twice with fresh DBU solution. The support was rinsed with dry ACN, dried, and placed in a 30 mL flask with 15 ml of concentrated ammonia. The flask was heated at 50 °C overnight. The solution was filtered and analyzed by MALDI and IEX-HPLC. Purification yielded an oligonucleotide protected with 3'-trityl. The sample was desalted using a Sephadex column, collected in a 50 mL Falcon tube, and dried in vacuo.

[0480] The solid was dissolved in water (2 mL), followed by the addition of 20% H3PO4 (40 uL). The resulting suspension was left in a heating shaker at 50 °C for 30 minutes. Concentrated ammonia (1 mL) was added, and the suspension was filtered and then purified by IEX-HPLC. The fractions containing the deprotected product were pooled and desalted with Sephadex. Filtration and evaporation provided a fully deprotected PMO-DNA gapmer.

[0481] A solution of diethylamine (20%) in ACN was passed through a 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 left overnight at 50 °C. The flask was cooled to -20 °C and then aqueous methylamine (9 mL, 40%) was added. The flask was heated at 50 °C for over 6 hours. After analysis by HPLC and MALDI, IEX-HPLC purification was performed. Fractions containing the deprotected product were pooled and desalted with Sephadex. A sufficiently deprotected PMO-DNA gapmer was provided by filtration and evaporation.

[0482] Results A common misconception is that synthetic oligonucleotides can only be made in the 3’ to 5’ direction. Although not widely used, reverse ribose 5’-phosphoramidites are commercially available and have been used previously by others (Bhagat et al. J. Med. Chem. 2011, 54, 3027-3036, incorporated by reference as if fully set forth herein). Reverse DNA amidites as well as their 2’-F, 2’-OMe, 2’-OMOE congeners are available. The compatibility of PMO and phosphoramidite (PS) chemistries was tested using the 20 nucleotide (nt) long human DMPK sequence 5’-ACAGACAATAAATACCGAGG-3’ as a template. That length allows for comparative studies using 5-10-5 gapmers that are 20 nt long PMOs and testing of different chimeric designs. Synthesis of the PMO 6’-flanking domain was achieved by manual addition of PMO monomers to solid support B (Figures 18 and 19). The 3’-terminal secondary amino group was acid protected and neutralized to prepare a base for the addition of 5’-cyanophosphoramidite. Coupling of the first DNA monomer to the secondary amino group of the morpholino ring resulted in the formation of a stable thiophosphoramidate linkage. The DNA-PS was then extended in an automated fashion using standard DNA synthesis protocols to construct the gap sequence. Standard deprotection conditions (3% TCA in DCM) did not appear to affect the integrity of the phosphorodiamidate linkages even after multiple additions of DNA monomers. After completion of the DNA gap, a second PMO wing (flank) was added. PMO 6’-phosphoramidite P(III) was used as a “limpet” or first PMO nucleotide for a smooth transition between the DNA-PS domain and the PMO 3’ flank. Surprisingly, PMO synthesis was not successful and N-acetyl cleavage type oligonucleotides were detected instead of the complete construct. Acetyl protection of the more nucleophilic secondary amino group and an apparent shift of the cap group was likely. The cytidine N-acetyl protecting group was replaced with N-benzoyl. The steric bulk was increased by replacing acetic anhydride in the cap A solution with isobutyric anhydride to slow down the O-acyl group shift to the secondary amine.A similar strategy has been previously reported to prevent N - acetylation of "rapidly deprotected" nucleobases (Q. Zhu, M. O. Delaney, M. Geenberg Bioorganic & Medicinal Chemistry Letters 11, 2001, 1105 - 1107, incorporated herein by reference as if fully set forth herein). Thus, a simple cap A modification enabled the completion of PMO 3'-side synthesis. Commercially available P(III) PMO - G monomers showed low coupling efficiency. The addition of molecular sieves improved the yield, as did newly prepared cyanoethyl phosphoramidites. The addition of P(V) PMO - G monomers also resulted in lower coupling yields. In particular, first and second generation PMO - G (Figure 14) feature two nucleobase protecting groups that contribute to steric hindrance. Alternative solutions may include singly protected PMO - G (Figure 15) and allyl, methyl or benzyl groups as replacements for cyanoethyl phosphoramidites. For testing, a mixture - design gapmer 6'-PMO - DNA - RNA - 3' was used, which means that the 3'-side would be composed of 2'-OMOE or 2'-OMe RNA modifications typical of second - generation ASO designs.

[0483] In addition to introducing PMO(III) monomers at the first position on the 3'-side, similar substitutions at the first base on the 5'-side allowed the use of a universal solid support C (Figure 16) typical of conventional DNA, RNA synthesis. The solid supports used in trityl - based PMO synthesis employ secondary amine anchors decorated with PEG3 (Sarepta) A or sarcosine (Genetools) B (Figure 16). Fragments of those anchors remain at the 6'-end of the final product after ammonia deprotection. Switching to P(III) chemistry enables the synthesis of "clean" 6'-OH - terminated oligonucleotides.

[0484] Access to 6'-conjugation chemistry is available via conventional phosphoramidite approaches. Example 2: Condition Screening

[0485]

Table 11

[0486] Example 3: Oligonucleotide Library Method The synthesis of oligonucleotides was carried out on a Nitto UnyLinker support at 195 μmol / g on a 10 μmol scale. Three sets of ACGT monomers were used. PMO6'-phosphoramidite was purchased from Granlen Inc. PMO6'-phosphoramidate was procured from Hongene Biotech Corporation or Wuxi Corporation. Reverse β-cyanoethyl amidite was purchased from ChemGenes Corporation or Hongene Biotech Corporation. 0.1 M DDTT was used as a sulfurizing agent to oxidize the trialkyl phosphite intermediate. For the sequences starting from PMO, the first monomer was PMO6'-phosphoramidite. All solid-phase syntheses were performed using a Mermade6 automated synthesizer starting from the 5'-end. The PMO fragments were synthesized manually either in a glass peptide reaction vessel with a frit or in a 4 mL polyethylene Mermade6 column. First and second generation double-protected PMO-Gs were used for the synthesis of libraries 1 and 2. After synthesis, the oligonucleotides were deprotected, purified by IEX-HPLC on an Akta™ Avant 25 (Source15Q), and desalted on a Sephadex column (2.5 × 25 cm). The oligonucleotides were lyophilized, resuspended in sterile water, and the concentration was determined by measuring the UV absorbance at 260 nm. All oligonucleotides were characterized by HPLC (Agilent Technologies 1260 Infinity II), MALDI-ToF (Bruker Autoflex Max), and LCMS (in the case of molecular weight). LCMA characterization was performed by Novatia, LLC.

[0487] HPLC Method: Using an Agilent 1260 HPLC with a DNAPac 200 oligonucleotide HPLC column, a gradient of 20% acetonitrile solution in water and 1 M sodium perchlorate in TRIS buffer pH 8.0 was used and increased for analytical IEX injection.

[0488] Oligonucleotide purification: Using an Akta (trademark) Avant 25 HPLC with a Source 15Q column i.d. 10 mm × 200 mm, anion exchange chromatography (IEX-HPLC) was performed to elute at 8 mL / min using an increasing gradient of 2 M NaCl in 20% acetonitrile - water Tris buffer, pH 8.0 for preparative purification of trityl ON oligonucleotides. Fractions were analyzed by HPLC, pooled, desalted on a Sephadex column, and evaporated by a Genevac Elite 2.3 evaporator. 60 uL of 20% H3PO4 was added to a 2 mL aqueous solution of Tr ON oligonucleotides, and trityl-off oligonucleotides were generated by incubation at 50 °C for 30 minutes in a heating shaker. The suspension was treated with 1 mL of 28% ammonia in water, syringe filtered, and loaded onto a Source 15Q column. Gradient elution provided fully deprotected oligonucleotides.

[0489] Addition of the first morpholino monomer to the solid support: 1.0 g of UnyLinker 195 μmol / g loaded support was placed in a glass peptide synthesis vessel with a frit glass support. After detritylation and acetonitrile washing steps, a solution of A-PMO amidite (600 mg, 0.77 mmol, 4 eq) monomer in acetonitrile (0.1 M, 7 mL) and solid ETT (200 mg, 1.53 mmol) were added. The vessel was capped and stirred at room temperature for 30 minutes. After draining the solvent, the coupling step was repeated. Oxidation was performed using a 0.05 M iodine - water (10%) - pyridine (90%) solution until discoloration no longer occurred. Acetonitrile washing removed the remainder of the oxidizing solution.

[0490] Figure 18 shows an example of library processing and purification. Addition of PMO(V) monomer: Detritylation solution (CYTFA): 100 mM 4-cyanopyridine, 100 mM TFA in dichloromethane: trifluoroethanol: ethanol (80:20:1). Neutralization solution (N): 5% DIPEA in dichloromethane: isopropanol (3:1). Coupling solution (C): 1.6 g of PMO monomer, 9.0 mL of DMI, 1.0 mL of NEM. Washing solution (W): Dichloromethane. Storage solution (S): 30% trifluoroethanol in dichloromethane.

[0491] [Table 12]

[0492] Addition of conventional amidite to the terminal PMO monomer: The support (100 - 160 mg) was weighed into a 4 mL frit Mermade6 column and placed on a vacuum manifold for solid-phase extraction. The trityl was removed using CYTFA (6 x 10 min) and neutralization solution (4 x 5 min). The column was transferred to Mermade6 for coupling with the first deoxyribose phosphoramidite (2 x 15 min) followed by sulfurization with DDTT solution (2 x 3 min). The synthesis of the DNA-PS gap was continued in an automated manner and terminated with DMT-ON.

[0493] Deprotection and purification of the first library For the simplicity of IEX purification, the trityl group was maintained. A suspension of the solid support in 15 mL of 1 M DBU solution in dry ACN was shaken at room temperature for 1.5 h. The clear solution was decanted and the process was repeated twice with fresh DBU solution. The support was rinsed with dry ACN, dried, and placed into a 30 mL flask with 15 ml of aqueous concentrated ammonia. The flask was then heated at 50 °C overnight. The solution was filtered and analyzed by MALDI and IEX-HPLC. Purification yielded an oligonucleotide protected with 3'-N-trityl. The sample was desalted on Sephadex, collected in a 50 mL Falcon tube, and dried in vacuo. The resulting solid was dissolved in water (2 mL), followed by the addition of 20% H3PO4 (40 uL). The resulting suspension was placed in a heating shaker at 50 °C for 30 min. Aqueous concentrated ammonia (1 mL) was added and the suspension was syringe filtered and then purified by IEX-HPLC. Fractions containing the deprotected product were pooled and desalted on Sephadex. Filtration and evaporation provided a fully deprotected PMO-DNA gapmer.

[0494] Deprotection and Purification of the Second Library A solution of diethylamine (20%, 8 mL) in ACN was passed through the DMT-OFF support over 2 min. The support was air dried and placed into a 30 mL flask for ammonia deprotection. Concentrated aqueous ammonia (9 mL) was added and the suspension was left at 50 °C overnight. The flask was briefly cooled to -20 °C and then aqueous methylamine (9 mL, 40%) was added. The flask was heated at 50 °C for over 6 h. After analysis by HPLC and MALDI, IEX-HPLC purification was performed. Fractions containing the deprotected product were pooled and desalted on Sephadex. Filtration and evaporation provided a fully deprotected PMO-DNA gapmer.

[0495] [Table 13]

[0496] [Table 14]

[0497]

Table 15

[0498] Results Determining the optimal sizes of the PMO flanks and DNA gap by in vitro KD in HeLa cells. Two positive controls were synthesized: sequence oligo 33

Chemical formula

[0499] The requirements for the two-step purification of the first library promoted the use of 2'-O-MOE nucleotides at the 3'-side. Since the bulk of the neutral PMO was placed on the 6'-side, the replacement of two nucleotides at the 3'-side would have a negligible impact on the overall oligonucleotide charge. The role of the second library (Table 4) was to examine the effect of gradually increasing the 6'-side to 6-15 PMO nucleotides (ONs) and gradually decreasing the 3'-side from five to two 2'-O-MOE nucleotides. The size of the DNA-PS gap would shrink from 10 in oligo 1 to 3 nucleotides in oligo 25. RNAse H activity was improved with a gap of at least seven charged nucleotides. Indeed, oligos 22, 23, 24, and 25, characterized by short gaps 7-3 nt in length, showed knockdowns decreasing from 46% to 11%. Conversely, oligos 16, 17, 18, 19, 20, and 21, which had gap sizes of 8 or 10, provided the highest KD equivalent to or better than that of control oligo 14.

[0500] A library of 25 nt-long gapmers was prepared (Table 5). The gap size was maintained at the above levels, e.g., 8-10 nt, for RNAse H activity, while the PMO flank size was varied between 10-15 nt.

[0501] Lipofectamine delivery of PMO-DNA hybrids - DMPK knockdown in Hek293 cells.

[0502] Thermal denaturation studies The binding affinity of oligonucleotides can play a suggestive role in determining their therapeutic potential. Factors such as charge, the presence of hydrophobic groups, hydrogen bonding, sugar packers, etc. can affect the binding energy. Stereochemically restricted nucleotides have been developed to improve the binding affinity of ONs by entropy gain. Some synthetic ONs can increase the binding affinity, while steric random phosphorothioates decrease it. In addition to the backbone type, the number and positions of modifications within the sequence appear to play an important role in determining the binding affinity. Morpholino phosphoramidites as well as isosteric N3’-P5’ phosphoramidates and sulfur analogs have shown increased binding to complementary RNA. Thio phosphoramidate morpholino oligonucleotides (TMOs) have shown decreased binding to DNA (-12.6 °C) and increased binding to RNA targets (+10 °C). In contrast, alternating morpholino thio phosphoramidate and DNA-PS subunits increase the binding affinity for both DNA and RNA (6 - 10 °C). The alternation of subunits was hypothesized to result in increased flexibility relative to other rigid ONs that allow for efficient binding to the complementary strand. Motivated by these findings, the duplex formation properties of PMO-DNA chimeras with complementary RNA were investigated (Tables 3 - 5). The data presented in Table 3 show that the increase in thermal stability correlates with the size of the 6’ PMO wing. An increase in the number of PMO modifications from 2 to 10 (oligos 4 - 13) resulted in a modest Tm increase (+2.8 °C per base, or +0.35 °C). For oligos 20, 21, 22, 23, 24, and 25, an increase in the number of PMOs from 9 to 15 (Table 4) changed the Tm by +4.9 °C per base, or +0.82 °C. For 25 nt long gapmers of the same sequence (oligos 30, 31, 32) (Table 5), the change from 13 to 15 PMOs increased the Tm by +1.4 °C per base, or +0.7 °C. In contrast, decreasing the size of the 2’-MOE 3’ wing from 5 to 2 in oligos 16, 17, 18, 19, and 21 decreased the Tm by -4.0 °C per base, or -1.33 °C, despite increasing the size of the 6’ PMO wing by the same number of nucleotides.The same decrease in 2'-MOE size in a series of 25 nt long gapmers (oligos 27, 28, 29, and 30) resulted in a more modest decrease of -2.0 °C or -0.67 °C per base. The fully PMO-modified oligo 26 showed a Tm of 61.7 °C similar to the positive control (5-10-5) second generation gapmer sequence 126 (60.9 °C). These results indicate that the introduction of the block-like PMO does not modify the RNA-binding properties of the PMO-DNA chimera.

[0503] RNAse H activation by PMO-DNA(PS) hybrids The mechanism by which oligonucleotides can exert biological activity is complex. In contrast to steric blockers, RNase H-dependent oligonucleotides can inhibit protein expression by targeting substantially any region of mRNA. RNase H cleavage is catalytic and a single ASO can direct the cleavage of multiple copies of the target mRNA. Conversely, steric block oligonucleotides can bind and target only a single RNA molecule. To verify whether PMO-DNA hybrids can induce RNase H, heteroduplexes were prepared using synthetic 5'-fluorescein-labeled RNA and annealed with PMO-ASO oligonucleotides or DNA control sequences (oligo 33) at room temperature. An RNase H buffer (1 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.5) was prepared. Heteroduplexes at a concentration of 100 nM in RNase H buffer and U RNase H1 (Sigma Aldrich, MI) were mixed and incubated at 37 °C for various times. The enzyme reaction was terminated by heat inactivation at 65 °C for 20 minutes and then snap frozen in dry ice. RNase H activity was determined using gel electrophoresis with commercially available Invitrogen EX E-Gels (4% agarose, Sybrgold).

[0504] 3'-exonuclease (snake venom phosphodiesterase I, SVPDE) sensitivity assay To determine the exonuclease stability of hybrid oligonucleotides in situ, snake venom phosphodiesterase I (Crotalus Adamanteus) was used to demonstrate cleavage of phosphodiester bonds in a sequential manner starting from the 3'-position. Phosphodiester oligonucleotides such as DNA containing PO linkages are digested by SVDPE. Conversely, a control sequence (oligo 33) designed with PS linkages should show little or no cleavage by SVPDE. To perform enzymatic hydrolysis, a (13.3 mM) oligonucleotide hybrid was 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) at time points were heat inactivated (95 °C) and snap frozen in dry ice until analyzed by IEX-HPLC. Expansion and resolution of detectable peaks occurred for the second generation gapmer (oligo 33) over a 23-hour period. The major oligonucleotide peak at a retention time of 14 minutes was resolved into smaller peaks over time after being subjected to exonuclease conditions. SVPDE is Rp selective, which explains why the degradation of the sequence (oligo 33) slows down after hydrolysis of each nucleotide. No peak expansion was observed for (oligo 3), although some detectable degradation is likely due to the presence of impurities. The combination of morpholino units and phosphorothioates resulted in exceptional nuclease stability. Replacement of the terminal phosphorothioate with a non-ionic phosphoramidate in the PMO-ASO hybrid (oligo 6) represents one step in the direction of further increasing nuclease stability.

[0505] Conclusion PMO-DNA-RNA hybrids can be synthesized using solid-phase methods and commercially available nucleotides. Structurally diverse libraries were generated and evaluated in vitro and in vivo. These gapmers provide stability, result in increased knockdown of mRNA targets, and support RNAse H activity. PMO-DNA gapmers maintain or exceed the activity of second-generation ASOs. Reducing the overall charge not only improves pharmacological properties but also allows for reduced aggregation of conjugates with positively charged peptides. PMO chimeras can be used in other platforms, for example, DNAzyme, siRNA, or steric block oligonucleotides, to name a few.

[0506] Example 4: Testing of an Exemplary Library Method 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 μM. Cells were nucleofected with gapmers using the Lonza nucleofection kit and incubated for 24 hours. Oligo 1 (Table 6), which showed significant knockdown, was used as a reference.

[0507] [Table 16]

[0508] [Table 17]

[0509] [Table 18]

[0510] [Table 19]

[0511]

Table 20

[0512] Results Figure 20 shows the results of mRNA expression after electroporation of 15 μM of 20-mer oligonucleotides from Table 6.

[0513] Figure 21 shows the qPCR results of mRNA expression after electroporation of 15 μM of oligonucleotides from Table 7.

[0514] Tables 8 - 10 show the uptake rates of hybrid oligonucleotides with and without Lipofectamine.

[0515] The mRNA expression level decreases as the length of the PMO increases until it reaches, for example, the 10M-8-2 pattern.

[0516] Example 5: DMPK Knockdown Figures 22, 23, 24, and 25 show the DMPK knockdown results for the oligonucleotides shown in Tables 6 - 11 in HeLa cells (free or Lipofectamine delivery). Briefly, 24-well format HeLa cells were treated with PMO / ASO hybrids in OPTI-MEM medium for 48 hours. No significant toxicity was observed except for the peptide control at 100 μM (data not included). Using Lipofectamine transfection, all PMO / ASO hybrids and ASO gapmers show target K.D. For free uptake, ASO and PMO / ASO hybrid gapmers show K.D., but not the gapmer conjugated with EEV (Figure 25).

[0517]

Table 21

[0518]

Table 22

Chemical formula

[0519] Results Figure 28 shows that RNase H is active with the PMO-ASO gapmer duplex but not with the DNA / RNA control or PMO / RNA control.

[0520] Figure 29 is a demonstration of RNase H cleavage of PMO-ASO rather than the DNA / RNA control duplex with increasing units of RNase.

[0521] Figure 30 is a demonstration of RNase H cleavage of the experimental duplex control. RNA / PMO-ASO duplexes were generated using fluorescent RNA (25mer) and annealed to the PMO-ASO sequence. The duplexes were incubated in 8U RNase H buffer, and at various time points, aliquots were removed and heat shocked (65 °C) to inactivate the protein. The products were visualized using gel electrophoresis (E-gel, 4% agarose). Duplexes with short gaps indicate little or no degradation (oligos 25 and 23).

[0522] Example 8: Stability in Snake Venom Phosphodiesterase Digestion Figure 31 shows that snake venom phosphodiesterase digests oligonucleotides from the 3’ end. The hydrolytic enzyme snake venom phosphodiesterase (SVPDE Crotalus adamanteus) typically metabolizes extracellular nucleotides involved in intercellular signaling. Hydrolysis occurs at the 3’ end, releasing 5’ nucleotides.

[0523] Figure 32 shows that the ASO-PMO hybrid is stable in the presence of SVDPE.

[0524] Figure 33 shows IEX-HPLC data for an SVDPE assay using control gapmers with phosphorothioate (PS) backbone (oligo 37) and phosphodiester (oligo 38). Example 9: Stability in Phosphodiesterase II Method Phosphodiesterase II (5’-exonuclease) bovine spleen phosphodiesterase (Sigma, 10U). Digestion was performed with: 1.4 nmol oligonucleotide

Chemical formula

[0525] Results Figure 34 provides data showing that PMO-ASO (oligo 21) is stable during phosphodiesterase II digestion.

[0526] Figure 35 shows data for PO and PS controls. Example 10: Other hybrids

Chemical formula

[0527] Example 11: Examples of oligonucleotides, linking groups, and nucleosides Figures 5 and 6 show samples of hybrid gapmer oligonucleotides.

[0528] Figure 36 shows a hybrid gapmer oligonucleotide having a central gap region containing 5' and 3' gramps linked by phosphorothioamidate linkages and DNA P(III) nucleotides linked by phosphorothioate linkages.

[0529] Figure 10 shows P(III) morpholine analogs protected with Tr or DMT.

[0530] Figure 11 shows DMT-protected reverse DNA P(III) nucleotide analogs.

[0531] Figure 12 shows DMT-protected 2'-modified reverse RNA P(III) nucleoethyl nucleotide analogs.

[0532] Figure 13 shows DMT-protected 2'-modified reverse RNA P(III) nucleotide analogs having methylated C and U bases.

[0533] Figure 14 shows a Tr-protected P(V) morpholino nucleic acid base analog having a doubly protected G base.

[0534] Figure 15 shows a Tr-protected P(V) morpholino nucleic acid base analog.

[0535] Figure 7 shows the hybrid sense and antisense strands of siRNA and the nucleotides and linkages therein. Any combination of nucleotides and linkages can be used to prepare hybrid PMO oligonucleotides.

[0536] Example 12: Examples of Guide and Passenger RNAs The PMO can be at the end of the guide (antisense) or passenger (sense) strand or in the middle of the guide or passenger strand. [Chemical formula] [Chemical formula]

Claims

**Claim 1** A method for preparing a hybrid oligonucleotide, comprising assembling P(III) and P(V) nucleotides on a support in the 6'-to-3' or 5'-to-3' direction and using PMO 6'-phosphoroamidite P(III) or phosphoramidate P(V) as the first nucleotide from said support, said method. **Claim 2** The method according to claim 1, wherein PMO 6'-phosphoroamidite P(III) is the first nucleotide from said support. **Claim 3** The method according to claim 1, wherein PMO 6'-phosphoramidate P(V) is the first nucleotide from said support. **Claim 4** The hybrid oligonucleotide comprises (a) at least one phosphorothioamidate linkage; and (b) at least one phosphorothioester linkage, phosphodiester linkage, phosphorodiamidate linkage or a combination thereof The method according to any one of claims 1 to 3. **Claim 5** The method according to any one of claims 1 to 4, comprising coupling a first reverse amidite to a secondary amino group of a morpholino ring to form a phosphorothioamidate linkage. **Claim 6** The method according to any one of claims 1 to 5, further comprising adding at least one P(III) nucleotide. **Claim 7** The method according to any one of claims 1 to 6, comprising adding at least two P(III) nucleotides. **Claim 8** The method according to claim 7, wherein the P(III) nucleotides are consecutive. **Claim 9** The method according to any one of claims 1 to 8, further comprising adding at least one P(V) nucleotide. **Claim 10** The method according to any one of claims 1 to 9, comprising adding at least two P(V) nucleotides. **Claim 11** The method according to claim 10, wherein the P(V) nucleotides are consecutive. **Claim 12** The method according to any one of claims 1 to 11, wherein at least two P(III) nucleotides are followed by at least two P(V) nucleotides. **Claim 13** The method according to any one of claims 1 to 12, wherein at least two P(V) nucleotides are followed by at least two P(III) nucleotides. **Claim 14** The method according to 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 according to any one of claims 1 to 14, wherein the P(III) nucleotides are the same.

16. The method according to any one of claims 1 to 14, wherein the P(III) nucleotides are different.

17. The method according to any one of claims 1 to 16, wherein the P(V) nucleotides are the same.

18. The method according to any one of claims 1 to 16, wherein the P(V) nucleotides are different.

19. The method according to any one of claims 1 to 18, wherein the method comprises deprotecting and neutralizing a secondary amino group at the 3'-end of a first DNA monomer.

20. The method according to claim 19, wherein the method further comprises adding one or more additional DNA nucleotides to the first DNA nucleotide, and two or more DNA nucleotides are linked via a phosphorothioate (PS) linkage.

21. The method according to any one of claims 19 to 20, wherein the method further comprises adding a second PMO 6'-phosphoramidite P(III) to the 3'-end DNA nucleotide of the hybrid oligonucleotide.

22. The hybrid oligonucleotide is (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 gap region comprising five or more deoxyribonucleotides or deoxyribonucleotide analogs; and (iii) a 3'-flank comprising two or more morpholino nucleotide analogs, or a 3'-flank comprising two or more ribonucleotides or ribonucleotide analogs The method according to any one of claims 1 to 21, comprising a gapmer.

23. The method according to claim 22, wherein the method comprises extending the oligonucleotide by adding one or more additional DNA monomers to the first DNA monomer linked via a phosphorothioate bond (DNA-PS).

24. The method according to claim 23, further comprising adding PMO 6'-phosphoramidite P(III) to the 3' end of the oligonucleotide. **Claim 25** The method according to claim 24, comprising adding the PMO 6'-phosphoramidite P(III) after all cytidine-based nucleotides in the 5'-flank and the gap are protected with N-benzoyl, and cap A is based on isobutyric acid, pivalic acid or benzoic anhydride instead of acetic anhydride. **Claim 26** The method according to claim 23, wherein the oligonucleotide comprises 6'-PMO-DNA-PMO-3', 6'-PMO-DNA-RNA-3', 5'-RNA-DNA-PMO-3'. **Claim 27** The method according to any one of claims 23 to 26, wherein the nucleosides at the 3' and 5'-flanks are linked via an inter-subunit linkage. **Claim 28** A hybrid oligonucleotide synthesized by the method according to any one of claims 1 to 27. **Claim 29** The hybrid oligonucleotide according to claim 28, wherein the method further comprises purifying the oligonucleotide. **Claim 30** The hybrid oligonucleotide according to claim 28 or 29, wherein the method further comprises isolating the oligonucleotide. **Claim 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 its analog, or a ribonucleotide or its analog; and (iii) at least one phosphorothioamidate 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 **Claim 32** The hybrid oligonucleotide according to claim 31, comprising 10 to 50 nucleotides. **Claim 33** The hybrid oligonucleotide according to claim 31, comprising 15 to 30 nucleotides.

34. The hybrid oligonucleotide according to claim 31, comprising 20 to 30 nucleotides.

35. The hybrid oligonucleotide according to claim 31, wherein the first nucleotide sequence comprises two or more morpholino nucleotide analogs.

36. The hybrid oligonucleotide according to 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 according to claim 31, wherein the terminal 3' nucleic acid residue of the first nucleic acid sequence is a P(V) morpholino nucleotide analog.

38. The hybrid oligonucleotide according to claim 35, wherein the two or more morpholino oligonucleotide analogs are linked via phosphorodiamidate linkages.

39. The hybrid oligonucleotide according to claim 31, wherein the first nucleotide sequence comprises one or more nucleotide residues comprising deoxyribonucleotides or analogs thereof, or ribonucleotides or analogs thereof, or combinations thereof.

40. The hybrid oligonucleotide according to claim 31, wherein the second nucleotide sequence comprises two or more deoxyribonucleotide analogs, two or more ribonucleotide analogs, or combinations thereof.

41. The hybrid oligonucleotide according to claim 31, wherein the deoxyribonucleotide analog or ribonucleotide analog is selected from P(III) DNA analogs, reverse P(IIII) DNA analogs, P(III) RNA analogs, reverse P(III) RNA analogs, or combinations thereof.

42. The hybrid oligonucleotide according to claim 31, comprising 2'-modified ribonucleotide analogs 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'-MOE) P(III) RNA, 2'-fluoro P(III) RNA, reverse 2'-fluoro P(III) RNA, or combinations thereof.

43. The hybrid oligonucleotide according to claim 31, wherein the two or more deoxyribonucleotides or analogs, two or more ribonucleotides or analogs thereof, or combinations thereof are each linked via a nucleotide internucleoside linkage.

44. The hybrid oligonucleotide according to claim 43, wherein the nucleotide internucleoside linkage is a phosphorothioate, phosphodiester, or phosphorodiamidate linkage.

45. The hybrid oligonucleotide according to claim 31, wherein the second nucleotide sequence further comprises one or more PMOs.

46. The hybrid oligonucleotide according to claim 31, wherein the P(III)PMO is selected from P(III)PMO-C, P(III)PMO-G, P(III)PMO-A, P(III)PMO-T, or P(III)PMO having a non-naturally occurring nucleobase.

47. The hybrid oligonucleotide according to claim 31, wherein the (PV)PMO comprises P(V)PMO-C, P(V)PMO-G, P(V)PMO-A, P(V)PMO-T, or P(V)PMO having a non-naturally occurring nucleobase.

48. The hybrid oligonucleotide according to any one of claims 31 to 47, wherein less than 75% of the nucleotide internucleoside linkages have a negative charge.

49. The hybrid oligonucleotide according to any one of claims 31 to 47, wherein less than 50% of the nucleotide internucleoside linkages have a negative charge.

50. The hybrid oligonucleotide according to any one of claims 31 to 47, wherein less than 25% of the nucleotide internucleoside linkages have a negative charge.

51. A gapmer, wherein (i) the first nucleotide sequence comprises the 5'-portion of the gapmer, and the 5'-portion comprises 2 to 20 morpholino nucleotide analogs; (ii) the second oligonucleotide sequence comprises the gap oligonucleotide and the 3'-portion of the gapmer, the gap oligonucleotide sequence comprises 8 to 20 deoxyribonucleotides or ribonucleotide analogs, and the 3'-portion comprises 2 to 20 deoxyribonucleotides or ribonucleotide analogs. The hybrid oligonucleotide according to any one of claims 31 to 50, comprising the gapmer.

52. The hybrid oligonucleotide according to claim 51, wherein the 5'-side portion comprises 2 to 10 morpholino nucleotide analogs.

53. The hybrid oligonucleotide according to claim 51, wherein the 5'-side portion comprises 2 to 5 morpholino nucleotide analogs.

54. The hybrid oligonucleotide according to claim 51, wherein the morpholino nucleotide analogs in the 5'-side portion are linked via phosphorodiamidate linkages.

55. The hybrid oligonucleotide according to claim 51, wherein the first 5' or 6' end of the 5'-side portion comprises a linchpin residue containing PMO 6'-phosphoroamidite P(III).

56. The hybrid oligonucleotide according to claim 51, wherein the 3'-side portion comprises 2 to 10 morpholino nucleotide analogs.

57. The hybrid oligonucleotide according to claim 51, wherein the 3'-side portion comprises 2 to 5 morpholino nucleotide analogs.

58. The hybrid oligonucleotide according to claim 51, wherein the morpholino nucleotide analogs in the 3'-side portion are linked via phosphorodiamidate linkages.

59. The hybrid oligonucleotide according to claim 51, wherein the second 5' or 6' end of the 3'-side portion comprises a linchpin residue containing PMO 6'-phosphoroamidite P(III).

60. The hybrid oligonucleotide according to claim 51, wherein the gap comprises at least 8 consecutive deoxyribonucleotide analogs.

61. The hybrid oligonucleotide according to claim 51, comprising 10 or fewer morpholino nucleotide analogs.

62. The hybrid oligonucleotide according to claim 51, comprising 7 or fewer morpholino nucleotide analogs.

63. The hybrid oligonucleotide according to claim 51, wherein at least 50% of the deoxyribonucleotide analogs in the gap oligonucleotide sequence are linked via phosphorothioate linkages.

64. The hybrid oligonucleotide according to claim 51, wherein the gap oligonucleotide sequence is linked to the 3'-side via a phosphorothioate linkage.

65. The hybrid oligonucleotide according to claim 51, wherein the 5'-side comprises at least one deoxyribonucleotide analog or at least one ribonucleotide analog.

66. The hybrid oligonucleotide according to claim 51, wherein the 5'-side comprises at least one ribonucleic acid analog selected from 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 according to claim 51, wherein the 5'-side comprises at least one 2'-fluoro (2'-F) ribonucleotide.

68. The hybrid oligonucleotide according to claim 51, wherein the 5'-side comprises at least one deoxyribonucleotide analog.

69. The hybrid oligonucleotide according to claim 51, wherein the 3'-side comprises at least one deoxyribonucleotide analog or at least one ribonucleotide analog.

70. The hybrid oligonucleotide according to claim 69, wherein the 3'-side comprises at least one ribonucleic acid analog selected from locked nucleic acid (LNA) analog, at least one 2'-methoxy nucleic acid analog (2'-methoxy; 2'-OMe), at least one 2'-methoxyethyl nucleic acid analog (2'-O-(methoxyethyl; 2'-OMOE), at least one 2'-fluoro ribonucleic acid analog (2'-F), or a combination thereof.

71. The hybrid oligonucleotide according to claim 69, wherein the 3'-side comprises at least one 2'-fluoro (2'-F) RNA.

72. The hybrid oligonucleotide according to claim 69, wherein the 3'-side comprises at least one deoxyribonucleotide analog.

73. The hybrid oligonucleotide according to claim 69, wherein the gap 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 phosphoramidate linkage 【Chemical 1】 ; and (v) at least one phosphorothioamidate 【Chemical 2】 linkage the hybrid oligonucleotide.

75. The hybrid oligonucleotide according to claim 74, wherein each upstream nucleotide adjacent to each P(V) morpholino nucleotide analog is a P(III) or P(V) morpholino nucleotide analog.

76. The hybrid oligonucleotide according to claim 74 or 75, comprising two or more consecutive P(V) morpholino nucleotide analogs linked via a phosphoramidate linkage.

77. The hybrid oligonucleotide according to any one of claims 74 to 76, wherein any P(V) morpholino nucleotide analog is linked via a phosphorothioamidate linkage to any downstream nucleotide that is not a P(V) morpholino nucleotide analog.

78. The hybrid oligonucleotide according to any one of claims 74 to 77, comprising at least one 2'-modified P(III) ribonucleotide.

79. The hybrid oligonucleotide according to any one of claims 74 to 78, further comprising at least one phosphorothioate linkage 【Chemical Formula 3】 .

80. The hybrid oligonucleotide according to claim 74, wherein the oligonucleotide is selected from a gapmer, a splice-switching oligonucleotide, the sense strand of siRNA, the antisense strand of siRNA, a deoxyribozyme, a steric block antisense oligonucleotide (ASO), and an immunomodulatory oligonucleotide.

81. The hybrid oligonucleotide according to any one of claims 28 to 80, and a cyclic peptide of formula (I) 【Chemical 4】 , or a protonated form thereof (wherein R １ , R ２ , and R ３ is independently a residue of H or tyrosine, phenylalanine or tryptophan; R ４ and R ６ are, independently, H or an amino acid side chain; AA ＳＣ is an amino acid side chain; q is 1, 2, 3, or 4; each m is independently an integer from 0 to 3) a compound comprising.

82. The structure of formula (I) is a structure of formula (I-1) or (I-2): 【Chemical Formula 5】 or its protonated form (wherein AA ＳＣ is an amino acid side chain; each m is independently an integer from 0 to 3) The compound according to claim 81, having

83. The cyclic peptide is 【Chemical Formula 6】 The compound according to claim 81 or 82, which is

84. Said AA ＳＣ is a compound according to any one of claims 81 to 83, conjugated to a linker.

85. The linker is (i)-(OCH ２ CH ２ ) ｚ -subunit (wherein z is an integer of 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) The compound according to claim 84, comprising

86. The linker has the structure: 【Chemical Formula 7】 (wherein x is an integer from 2 to 20; y is an integer from 1 to 5; z is an integer from 2 to 20) The compound according to claim 84 or 85, having

87. The compound according to any one of claims 84 to 86, wherein an exocyclic peptide (EP) is conjugated to the linker.

88. The exocyclic peptide of the compound according to claim 87 contains 2 to 10 amino acid residues.

89. The hybrid oligonucleotide according to any one of claims 28 to 80, and EEV of formula (B): 【Chemical 8】 or its protonated form (wherein R １ , R ２ , and R ３ are each independently H or an aromatic or heteroaromatic side chain of an amino acid; R ４ and R ７ are, independently, H or an amino acid side chain; EP is an 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 from 1 to 4; z' is an integer from 1 to 23) A compound comprising

90. The EEV has a structure of formula (B-1), (B-2), (B-3), or (B-4): 【Chemical Formula 9-1】 【Chemical Formula 9-2】 or a compound according to claim 89, comprising its protonated form.

91. The EEV is Ac-PKKKRKVAEEA-K(Cyclo[FGFGGRGRQ])-PEG １２ -OH or Ac-PK-KKR-KV-AEEA-K(Cyclo[GfFGrGrQ])-PEG １２ The compound according to claim 89 or 90, comprising -OH.

92. The EEV is 【Chemical 10】 The compound according to claim 89 or 90, comprising

93. The EEV is 【Chemical 11】 The compound according to claim 89 or 90, comprising

94. A pharmaceutical composition comprising the compound according to any one of claims 81 to 93.

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