Circularly Structured Oligonucleotides as Therapeutic Agents

Abstract

The present invention provides oligonucleotides, called circular structured oligonucleotides ("CSO"), that contain the functional domains, cyclization domains, and linker segments described herein, compositions containing same, and methods of using same. This design of the circular oligonucleotide maintains a circular form until it is in the presence of and hybridizes to the target RNA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of the filing date of U.S. Provisional Application No. 63 / 247,556, filed September 23, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Target RNA processing and translation can be regulated by antisense oligonucleotides through multiple mechanisms. These include cleavage of the target RNA by RNase H, regulation of aberrant splicing, increased processing and translation of the target RNA, and inhibition of translation by steric hindrance. The target RNA can be an mRNA or a non-coding RNA. In other mechanisms, the antisense strand of a duplex siRNA can be incorporated into an AGO to inhibit translation by the siRNA mechanism. In other mechanisms, such as models of the ADAR or CRISPR systems, the antisense can edit RNA or DNA, thereby regulating translation and processing.

[0003] Over the years, it has been found that antisense hybridization and affinity affect the selectivity of target RNA.In addition, for antisense nucleic acid to be used as a drug, the nuclease stability provided by the modification of internucleotide bond, for example phosphorothioate, is important.

[0004] Nuclease stability was hypothesized to be important for efficacy, and because antisense degradation was shown to be from the 3'-end, the focus was on modifying the 3'-end to slow degradation. These designs include capping the 3'-end and forming a hairpin loop at the 3'-end, creating oligos with secondary structures that include a 3'-3' linkage, or linking two antisense oligonucleotides at the 3'-end. These types of antisense showed increased nuclease stability, but did not improve antisense efficacy. Unfortunately, these modifications also increased the inflammatory response, thereby limiting the therapeutic index.

[0005] Both phosphorothioate-containing DNA and RNA and 2'-substituted RNA have been studied as antisense agents, offering different characteristics. DNA phosphorothioate antisense activates RNase H when hybridized to RNA, whereas RNA or 2'-substituted RNA antisense binds to RNA with higher affinity and does not activate RNase H. To further improve the antisense properties, mixtures of these two modifications have been used in antisense, commonly referred to as hybrid or gapmer antisense. In the most studied gapmer antisense, modified RNA segments are placed at both the 3'- and 5'-ends, whereas DNA is placed in the middle. Gapmer antisense is the most widely studied antisense, and drugs using this chemistry have been approved and are in clinical development.

[0006] One of the side effects of both DNA and RNA phosphorothioates is due to their interaction with proteins, more specifically, with the family of pattern recognition receptors (PRRs). These interactions result in the induction of immune cascades, thereby triggering off-target mechanisms of action and associated safety signals. Detailed structure-activity relationship studies show that accessibility of the 5'-end of DNA and RNA phosphorothioate antisense is necessary for immune activation. DNA and RNA phosphorothioates containing two 5'-ends have shown increased immunostimulatory activity. In contrast, DNA and RNA phosphorothioates containing two 3'-ends (lacking a 5'-end) have previously been shown to show minimal inflammatory responses.

[0007] In a continuing effort to improve the properties of DNA and modified RNA phosphorothioate antisense as therapeutic agents, structural changes in oligonucleotides have been explored. For example, previous studies have reported that oligodeoxynucleotide phosphorothioates containing a hairpin loop region at the 3'-end ("PS-oligonucleotides") are self-stabilizing oligonucleotides that resulted in increased in vivo nuclease stability and limited biological activity. To date, the focus has been on improving the stability of antisense by modifying the 3'-end with various modifications, including gapmer antisense.

[0008] Despite the progress made to date, there remains a need to develop antisense oligonucleotides with improved properties for use as therapeutic agents and in diagnostic applications. Summary of the Invention

[0009] The present invention provides a structural class of oligonucleotides referred to herein as "circularly structured oligonucleotides" (CSOs) or equivalently "cyclic oligos." In a CSO, two oligonucleotides are linked to each other (either directly or via a linker segment). One oligonucleotide, referred to as the "functional domain," provides function to the CSO (e.g., the functional segment can be an antisense oligonucleotide or an immunostimulatory oligonucleotide), and a second oligonucleotide, referred to as the "cyclization domain," contains nucleotide sequences complementary to the ends of the functional domain (e.g., Figures 1A-1C).

[0010] CSOs adopt an intramolecular cyclic structure as a result of the complementarity between the functional domain and the cyclization domain that form an intramolecular duplex. This intramolecular duplex formation changes both the shape of the functional domain and the accessibility to the termini of the oligonucleotide. This structure combines important attributes for creating optimal antisense and oligonucleotide- and nucleic acid-based therapeutics.

[0011] In gene and RNA expression regulation, this structure masks the 5' end, thereby reducing interactions with PRRs and allowing endosomal escape. Once inside the cytoplasm or nucleus, the circular structure opens in the presence of target RNA, since the affinity between the functional domain and the target RNA sequence is higher than that between the functional domain and the cyclization domain. When CSOs are in intramolecular cyclic form, there are fewer exposed phosphorothioate crosslinks, which may result in fewer polyanion-related side effects (e.g., complement activation and prolongation of partial thromboplastin time) that are known to occur with PS-oligonucleotides. CSOs also have reduced protein binding.

[0012] CSOs according to the invention can be made using standard techniques for the synthesis of constituent oligonucleotides, and are useful for all purposes for which functional oligonucleotides and nucleic acids are useful.

[0013] The above merely summarizes certain aspects of the invention and is not intended, nor should it be construed, as limiting the invention. All patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0014] [Figure 1A] 1A shows an embodiment of a circularly structured oligonucleotide according to the present invention. The dashed line represents the cyclization domain. The solid line represents the functional domain. L represents the link between the functional domain and the cyclization domain, either directly or via a linker. As shown in FIG. 1A, the circularly structured oligonucleotide maintains a circular form until it is in the presence of and hybridizes with the target RNA. In this embodiment, the functional domain is an antisense oligonucleotide complementary to the target RNA. The circularly structured oligonucleotide can act by various mechanisms of action depending on the nature of the oligonucleotide of the functional domain, as further described herein. [Figure 1B] FIG. 1B shows an embodiment of a circularly structured oligonucleotide according to the present invention. The dashed line represents the cyclization domain. The solid line represents the functional domain. L represents the linkage between the functional domain and the cyclization domain, either directly or via a linker. FIG. 1B shows an embodiment of a CSO according to the present invention, in which the oligonucleotide of the functional domain is a splitmer as described herein. [Figure 1C] 1A-1C show an embodiment of a circularly structured oligonucleotide according to the present invention. The dashed line represents the cyclization domain. The solid line represents the functional domain. L represents the link between the functional domain and the cyclization domain, either directly or via a linker. FIG. 1C shows an embodiment of a CSO according to the present invention, in which the oligonucleotide of the functional domain is a splicing oligonucleotide as described herein. [Figure 2A]2A shows an embodiment of the circularly structured oligonucleotide according to the present invention, in which the oligonucleotide of the functional domain is an siRNA. FIG. 2A shows an embodiment in which the cyclization domain is attached to the 5'-end of one strand of the siRNA. The cyclization domain can be attached to either the antisense strand or the sense strand of the siRNA. The short dashed line represents the cyclization domain. The solid line represents the sense strand of the siRNA. The dashed line represents the antisense strand of the siRNA. L represents a linker. [Figure 2B] FIG. 2B shows an embodiment of a circularly structured oligonucleotide according to the present invention, in which the oligonucleotide of the functional domain is an siRNA. FIG. 2B shows a further embodiment of a circularly structured oligonucleotide according to the present invention, in which the oligonucleotide of the functional domain is the antisense strand of an siRNA (dashed line) and a cyclization domain (solid line) is attached to the 5' end. In this embodiment, the sense strand is attached to the antisense strand and also acts as the cyclization domain. Alternatively, in FIG. 2B, the functional domain is the sense strand of an siRNA (dashed line) and a cyclization domain (solid line) is attached to the 5' end. In this embodiment, the antisense strand is attached to the sense strand and also acts as the cyclization domain. [Diagram 3] FIG. 1 shows the delivery of a CSO according to the present invention, in which the oligonucleotide of the functional domain is a gene regulatory oligonucleotide, and its release from the endosome to the cytoplasm. [Figure 4A] FIG. 1 shows an exemplary embodiment of a circularly structured oligonucleotide according to the invention, in which the oligonucleotide of the functional domain comprises an immunostimulatory oligonucleotide, and a circularization domain of various lengths is attached to the 3′ end. [Figure 4B] 4A-4C show exemplary embodiments of circularly structured oligonucleotides according to the present invention, in which the oligonucleotides of the functional domains comprise immunostimulatory oligonucleotides and cyclization domains of various lengths are attached to the 3'-end. FIG. 4B shows exemplary circular structures of compounds Nos. 53 and 54. [Diagram 5] FIG. 1 shows knockdown of PCSK9 in Hepa1-6 cells. [Figure 6A] FIG. 1 shows knockdown of PNPLA3 in HepG2 cells. [Figure 6B] FIG. 1 shows knockdown of PNPLA3 in HepG2 cells. [Figure 6C] FIG. 1 shows knockdown of PNPLA3 in HepG2 cells. [Figure 7] FIG. 1 shows the immunostimulatory effect of CSOs according to the invention, where the oligonucleotides of the functional domains are TLR9 agonists compared to acyclic TLR9 agonists. [Figure 8] FIG. 1 shows the effect of different linkers (i.e., direct conjugation versus triethylene glycol) on the immunostimulatory activity of CSOs according to the invention, where the oligonucleotides of the functional domains are TLR9 agonists. [Figure 9] FIG. 1 shows the effect of cyclization domains of different lengths on the immunostimulatory activity of CSOs according to the invention, where the oligonucleotides of the functional domains are TLR9 agonists. [Figure 10] FIG. 1 shows the effect of cyclization domains of different lengths and different linkers on the immunostimulatory activity of CSOs according to the invention, where the oligonucleotides of the functional domains are TLR9 agonists. [Figure 11] FIG. 1 shows the effect of conjugation of a cyclization domain 5′ or 3′ of a functional domain on the immunostimulatory activity of a CSO according to the invention, where the oligonucleotide of the functional domain is a TLR9 agonist. [Figure 12] FIG. 1 shows the effect of phosphodiester internucleotide linkages on the immunostimulatory activity of CSOs according to the invention, where the oligonucleotides of the functional domains are TLR9 agonists. [Figure 13] Figure 13 shows the activity of CSOs according to the present invention, where the oligonucleotides of the functional domains are TLR9 antagonists.As shown in Figure 13, these compounds did not elicit an immune response. [Figure 14]FIG. 1 shows the ability of CSOs according to the invention where oligonucleotides of functional domains are TLR9 antagonists that block the immunostimulatory effects of TLR9 agonists. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0016] As used herein, the use of the singular includes the plural unless otherwise stated. As used herein, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limiting. Additionally, terms such as "element" or "component" encompass both elements and components that include one unit and elements and components that include two or more subunits, unless otherwise stated.

[0017] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety and for the portions of the documents discussed herein.

[0018] Features may be described herein as part of the same or separate aspects or embodiments of the invention for clarity and concise description. Those skilled in the art will appreciate that the scope of the invention may include embodiments having all or partial combinations of the features described herein as part of the same or separate embodiments.

[0019] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as strictly limiting the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers having that range, such as 1, 2, 2.7, 3, 4, 5, 5,3, and 6. This applies regardless of the breadth of the range.

[0020] As used herein, the term "about" is understood by those skilled in the art and varies to some extent depending on the context in which it is used. When used herein when referring to a measurable value such as an amount, duration in time, etc., the term "about" is meant to encompass a variation of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the stated value, such variations being appropriate for carrying out the disclosed methods.

[0021] The present invention provides oligonucleotides called circular structured oligonucleotides ("CSO") that include a functional domain, a cyclization domain, and a linker segment. Unless otherwise specified, the functional domain and the cyclization domain are linked at their 5' ends via a 5'-5' bond. Alternatively, in some embodiments, the functional domain and the cyclization domain are linked at their 3' ends via a 3'-3' bond.

[0022] In an embodiment, the cyclization domain is linked to the functional domain at the 5' end with a 5'-5' linkage. In this configuration, the cyclization domain hybridizes to the 3'-end of the oligonucleotide of the functional domain, thereby forming a circular structure (e.g., Figures 1A-1C). This design of the cyclic oligonucleotide maintains the circular form until it is in the presence of the target RNA and the functional domain hybridizes to the target RNA. This structure allows for increased specificity.

[0023] In an embodiment, the cyclization domain is linked to the functional domain at the 3' end with a 3'-3' linkage. In this configuration, the cyclization domain hybridizes to the 5' end of the oligonucleotide of the functional domain, thereby forming a circular structure.

[0024] The functional domain provides the CSO with a desired function, for example, to regulate gene expression, an oligonucleotide of the functional domain is complementary to a target RNA.

[0025] In an embodiment, the oligonucleotides of the functional domain and the cyclization domain are DNA or RNA or a combination thereof. In an embodiment, the oligonucleotides of the functional domain are DNA or RNA or a combination thereof. In an embodiment, the oligonucleotides of the cyclization domain are DNA or RNA or a combination thereof.

[0026] In some embodiments, the oligonucleotides of the functional domains and / or the cyclization domain are unmodified. In some embodiments, the oligonucleotides of the functional domains are unmodified. In some embodiments, the oligonucleotides of the cyclization domain are unmodified. In some embodiments, the oligonucleotides of the functional domains and the cyclization domain are unmodified.

[0027] In an embodiment, at least one nucleotide of the oligonucleotide of the functional domain and / or the cyclization domain is modified. In an embodiment, two or more nucleotides of the oligonucleotide of the functional domain and / or the cyclization domain are modified.

[0028] In some embodiments, the oligonucleotides of the functional domain are modified. In embodiments, the oligonucleotides of the functional domain include modifications of internucleotide linkages, sugars, heterocyclic bases, or combinations thereof. These modifications can also be appropriately placed at specific positions within the oligonucleotides of the functional domain. Other chemistries and modifications are known in the field of oligonucleotides that can be readily used according to the present disclosure and are encompassed by the term "modified" as used herein in the context of oligonucleotides. As used herein, the terms "oligonucleotides of the functional domain" or "functional domain" are used interchangeably.

[0029] In an embodiment, the functional domain comprises an oligonucleotide having a length of 15 to 500 nucleotides. In an embodiment, the oligonucleotide of the functional domain is 17 and 300 nucleotides in length. In an embodiment, the oligonucleotide of the functional domain is 17 and 200 nucleotides in length. In an embodiment, the oligonucleotide of the functional domain is 17 and 100 nucleotides in length. In an embodiment, the oligonucleotide of the functional domain is 17 and 50 nucleotides in length.

[0030] In an embodiment, the oligonucleotides of the functional domains are between 50 and 250 nucleotides in length. In an embodiment, the oligonucleotides of the functional domains are between 50 and 150 nucleotides in length.

[0031] In an embodiment, the functional domain comprises an oligonucleotide between 15 and 50 nucleotides in length. In an embodiment, the functional domain comprises an oligonucleotide between 17 and 40 nucleotides in length. In an embodiment, the functional domain comprises an oligonucleotide between 17 and 25 nucleotides in length. In an embodiment, the oligonucleotides of the functional domain are between 17 and 22 nucleotides in length.

[0032] In embodiments, the oligonucleotides of the functional domain are 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In embodiments, the functional domain is 17 nucleotides in length. In embodiments, the functional domain is 18 nucleotides in length. In embodiments, the functional domain is 19 nucleotides in length. In embodiments, the functional domain is 20 nucleotides in length. In embodiments, the functional domain is 21 nucleotides in length. In embodiments, the functional domain is 22 nucleotides in length. In embodiments, the functional domain is 23 nucleotides in length. In embodiments, the functional domain is 24 nucleotides in length. In embodiments, the functional domain is 25 nucleotides in length. In embodiments, the functional domain is 26 nucleotides in length. In embodiments, the functional domain is 27 nucleotides in length. In embodiments, the functional domain is 28 nucleotides in length. In an embodiment, the functional domain is 29 nucleotides in length. In an embodiment, the functional domain is 30 nucleotides in length. In an embodiment, the functional domain is 31 nucleotides in length. In an embodiment, the functional domain is 32 nucleotides in length. In an embodiment, the functional domain is 33 nucleotides in length. In an embodiment, the functional domain is 34 nucleotides in length. In an embodiment, the functional domain is 35 nucleotides in length. In an embodiment, the functional domain is 36 nucleotides in length. In an embodiment, the functional domain is 37 nucleotides in length. In an embodiment, the functional domain is 38 nucleotides in length. In an embodiment, the functional domain is 39 nucleotides in length. In an embodiment, the functional domain is 40 nucleotides in length.

[0033] In embodiments, the functional domain includes, but is not limited to, an oligonucleotide selected from antisense oligonucleotides, microRNA (miRNA), siRNA, piRNA, hnRNA, ncRNA, snRNA, sgRNA, esiRNA, shRNA, lncRNA, CRISPR-based system, adenosine deaminase acting on RNA (ADAR) system, or splicing oligonucleotides. In embodiments, the functional domain includes, but is not limited to, an oligonucleotide selected from immunostimulatory oligonucleotides or immunoinhibitory oligonucleotides (also called immune antagonist oligonucleotides).

[0034] The only limitations on the nucleotides and internucleotide linkages of the oligonucleotides of the functional domain are that they do not (a) eliminate the ability of the cyclization domain to hybridize to form a duplex with the functional domain of the CSO under the desired conditions, and (b) the ability of the functional domain to perform its intended function (e.g., in the case of a functional domain that is an antisense oligonucleotide, to hybridize to a complementary RNA segment under physiological conditions to form a duplex, which duplex is a substrate for RNase H). Preferred nucleotides and internucleotide linkages are those that enhance the stability of the CSO against nucleases and other forms of chemical degradation and / or enhance the ability of the functional domain to perform its intended function.

[0035] In embodiments, the internucleotide linkages of the functional domains are phosphorothioate internucleotide linkages, phosphodiester internucleotide linkages, or a combination thereof.

[0036] The oligonucleotides of the cyclization domain are complementary to the sequence of nucleotides in the functional domain and are of the opposite polarity to the sequence of nucleotides in the functional domain to which they are complementary.

[0037] In some embodiments, the oligonucleotide of the cyclization domain is modified. In embodiments, the oligonucleotide of the cyclization domain includes a modification of the internucleotide bond, the sugar, the heterocyclic base, or a combination thereof. These modifications can also be located at specific positions within the oligonucleotide of the cyclization domain. As used herein, the terms "oligonucleotide of the cyclization domain" or "cyclization domain" are used interchangeably.

[0038] In embodiments, the internucleotide linkages of the cyclization domain are phosphorothioate internucleotide linkages, phosphodiester internucleotide linkages, or a combination thereof.

[0039] In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 400 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 100 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 75 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 50 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 40 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 30 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 25 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 12 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 10 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 4 and 6 nucleotides in length.

[0040] In an embodiment, the cyclization domain comprises an oligonucleotide 5-8 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 4 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 5 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 6 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 7 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 8 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 9 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 10 nucleotides in length.

[0041] In an embodiment, the cyclization domain comprises an oligonucleotide between 15 and 40 nucleotides in length. In an embodiment, the cyclization domain comprises an oligonucleotide between 17 and 30 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 15 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 16 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 17 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 18 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 19 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 20 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 21 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 22 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 23 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 24 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 25 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 26 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 27 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 28 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 29 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 30 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 31 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 32 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 33 nucleotides in length.In an embodiment, the cyclization domain oligonucleotide is 34 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 35 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 36 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 37 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 38 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 39 nucleotides in length. In an embodiment, the cyclization domain oligonucleotide is 40 nucleotides in length.

[0042] As used herein, the term "polarity" refers to the concept of directionality in primary structure (e.g., 3'→5' and 5'→3' for DNA and RNA, or N-terminus→C-terminus (or vice versa) for PNA). When the CSO of the present invention comprises an oligonucleotide that hybridizes in an antiparallel manner, for example by Watson-Crick base pairing, the cyclization domain is in a 5'→3' (or 2') configuration, and the sequence of nucleotides that are complementary in the functional domain is in a 3' (or 2')→5' configuration. The change in polarity of the CSO can occur anywhere in the CSO other than the cyclization domain and the nucleotide sequence of the functional domain to which the cyclization domain is complementary. In a preferred embodiment in which the CSO comprises an oligonucleotide, the functional domain is in a 3'→5' configuration, and the cyclization domain is in a 5'→3' configuration, such that the functional domain and the cyclization domain are linked via a 5'-5' bond. In some embodiments, the functional domain is in a 5'→3' configuration, and the cyclization domain is in a 3'→5' configuration, such that the functional domain and the cyclization domain are linked via a 3'-3' bond.

[0043] In embodiments, the functional domain and the cyclization domain are covalently linked to each other via a linker segment. In embodiments, the linker segment is a direct bond, a nucleotide or oligonucleotide of 2-5 nucleotides in length, or other chemical moiety, or a combination thereof. In some embodiments, the linker segment can be cleavable.

[0044] The only restriction on the linker segment is that it does not eliminate the essential functionality of the CSO, i.e., (a) the ability of the CSO to form an intramolecular ring structure under conditions of interest (e.g., physiological conditions) and (b) the ability of the functional domain to perform its intended function. Preferred "other chemical moiety" linkers include C 2 ~C 6 Alkyl, ethylene glycol, tri(ethylene glycol), tetra(ethylene glycol), penta(ethylene glycol), hexa(ethylene glycol) and -NH(CH 2 ) n Examples of linker segments include, but are not limited to, NH-, where n is 2, 3, 4, 5, or 6. Alternatively, the linker segment can be a combination of the above. In a preferred embodiment, the linker is a direct bond, where the functional domain and the cyclization domain are directly bonded. In one embodiment, the linker is ethylene glycol. In an embodiment, the linker is C 2 ~C 6 In an embodiment, the linker is C 2 In an embodiment, the linker is C 3 In an embodiment, the linker is C 4 In an embodiment, the linker is C 5 In an embodiment, the linker is C 6 It is an alkyl.

[0045] The oligonucleotides of the functional domain have terminal and linker ends. As the name implies, the linker end is the end of the oligonucleotide linked to the cyclic domain via a linker segment. Generally, the CSO is constructed such that the end of the functional domain forms a duplex with the cyclic domain, i.e., the cyclic domain is complementary to the end of the functional domain.

[0046] As used herein, the term "complementary" refers to a pair of nucleobases (or simply "bases") (or to the extent of complementarity as may be contextually required when assessing the "complementarity" of an oligonucleotide) in which hydrogen bonds preferentially bind to each other over other heterocyclic bases under selected (e.g., physiological) conditions. When the nucleobases are modified or unmodified, natural or synthetic purines and pyrimidines, the term "complementary" means complementary in the Watson-Crick sense.

[0047] If all bases in at least one strand of a nucleic acid pair are found opposite their complementary base pairs, such a strand is considered to be completely complementary to its sequence in the other strand. If one or more bases of such a strand are found in positions opposite any other base except its complementary base pair, such a base is considered a "mismatch" and the strand is considered to be partially complementary. Thus, strands can be partially complementary to varying degrees (e.g., 0% < x < 100% complementary) until the bases become misaligned, at which point they are non-complementary (e.g., 0% complementary). As will be readily understood and recognized by those skilled in the art, full (i.e., complete, 100%) complementarity is not required for hybridization of strands of nucleic acids (e.g., oligonucleotides, antisense or otherwise).

[0048] In embodiments, the target RNA can be mRNA, pre-mRNA, ncRNA, lncRNA, or microRNA. In embodiments, the target RNA is mRNA.

[0049] In an embodiment, a circularly structured oligonucleotide according to the invention is part of a pharmaceutical composition that includes a pharma- ceutically acceptable carrier.

[0050] The pharmaceutical composition comprising the circularly structured oligonucleotide of the present invention may further comprise any other drug or therapeutic agent useful for treating or preventing a disease or condition, which does not reduce the gene expression modulating effect of the circularly structured oligonucleotide according to the present invention.Agents useful for treating or preventing a disease or condition include, but are not limited to, small molecules, peptides, vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonists (e.g., TLR3 and / or TLR7 antagonists and / or TLR8 antagonists and / or TLR9 antagonists), chemotherapeutic agents (both traditional chemotherapy and modern targeted therapy), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and costimulatory molecules (e.g., cytokines, chemokines, protein ligands, transactivators, peptides or peptides containing modified amino acids), or combinations thereof. Alternatively, the circularly structured oligonucleotides according to the invention can be administered in combination with other compounds (e.g., formulated with lipids or liposomes, conjugated to peptides, antibodies or small molecules) to enhance the specificity or magnitude of gene expression modulation of the circularly structured oligonucleotides according to the invention.

[0051] In an embodiment, the oligonucleotides of the functional domains contain at least one phosphorothioate internucleotide linkage. In an embodiment, at least half of the internucleotide linkages are phosphorothioate. In an embodiment, all of the internucleotide linkages are phosphorothioate.

[0052] In some embodiments, the oligonucleotides of the functional domains are single-stranded.

[0053] In some embodiments, the oligonucleotides of the functional domains are at least 90% complementary to a portion of the target RNA over their entire length. In some embodiments, the oligonucleotides of the functional domains are at least 95% complementary to a portion of the target RNA over their entire length. In some embodiments, the oligonucleotides of the functional domains are at least 97% complementary to a portion of the target RNA over their entire length. In some embodiments, the oligonucleotides of the functional domains are at least 98% complementary to a portion of the target RNA over their entire length. In some embodiments, the oligonucleotides of the functional domains are at least 99% complementary to a portion of the target RNA over their entire length. In some embodiments, the oligonucleotides of the functional domains are at least 100% complementary to a portion of the target RNA over their entire length.

[0054] In embodiments where the oligonucleotide of the functional domain is an oligonucleotide of a CRISPR-based system or an Adenosine Deaminase Acting on RNA (ADAR) system, the portion of the oligonucleotide that is complementary to the target RNA (complementary domain) is at least 90% complementary, preferably at least 95% complementary, preferably at least 97% complementary, preferably at least 98% complementary, preferably at least 99% complementary, or preferably at least 100% complementary to the portion of the target RNA over its entire length.

[0055] In any of the compounds, compositions, or methods described herein, the CSO of the invention may have the sequence 3'-CGGTCACTCCTCCGTGCG-5'-5'-GCCAGT-3', 3'-CGGTCACTCCTCCGTGCG-5'-5'-GCGAAT-3', 5'-GCGTGCCTCCTCACTGGC-3'-3'-CGCACG-5', 5'-GCGTGCCTCCTCACGGC-3'-3'-GGAACC-5', 5'-GCGTG not an oligonucleotide having CCTCCTCACTGGC-3'-3'-CGCAC-5', 5'-GCGTGCCTCCTCACTGGC-3'-3'-GGAACCG-5', 5'-GCGTGCCTCCTCACTGGC-3'-3'-CGCACGGA-5', 5'-GCGTGCCTCCTCACTGGC-3'-3'-GGAAC-5', or 5'-GCGTGCCTCCTCACTGGC-3'-3'-GGAACAG-5'.

[0056] Functional Domains In various embodiments, the functional domain of the CSO is an oligonucleotide as further described below. The cyclization domain and linker of the CSO are as described above unless otherwise noted.

[0057] Inhibition of gene expression In embodiments, the invention provides a circular structured oligonucleotide (CSO) comprising a functional domain, a cyclization domain, and a linker, wherein the functional domain and the cyclization domain are linked at their 5' ends, the functional domain comprises an oligonucleotide 15-45 nucleotides in length and is complementary to a target RNA, the cyclization domain comprises an oligonucleotide 4-12 nucleotides in length and is complementary to a sequence of nucleotides in the functional domain and has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and the functional domain comprises a gene regulatory oligonucleotide. In some embodiments, the oligonucleotide of the functional domain is modified.

[0058] In embodiments, gene regulatory oligonucleotides of a functional domain (i.e., oligonucleotides capable of regulating expression of a target gene) include, but are not limited to, antisense oligonucleotides, microRNAs (miRNAs), piRNAs, hnRNAs, ncRNAs, snRNAs, sgRNAs, esiRNAs, shRNAs, or lncRNAs.

[0059] In some embodiments, modifications of the antisense oligonucleotides include at least one modified nucleobase, sugar and / or internucleotide linkage.

[0060] As shown herein, the CSO of the present invention, which comprises an antisense oligonucleotide linked to a cyclization domain via a 5'-5' linkage, surprisingly showed increased potency. This observation was in complete contrast to the previous hypothesis that increased stability (e.g., 3'-3' linked oligonucleotides) leads to increased potency. Furthermore, the antisense oligonucleotide of the CSO is less inflammatory when the 5'-end is absent. This design allows the antisense oligonucleotide to unfold into a linear structure and be active in cells where the target RNA is expressed.

[0061] When the CSO is an antisense oligonucleotide, it is in a circular form until a complementary target RNA is present, where it adopts a linear form and binds to the target RNA. The change from the circular form to the linear form could be confirmed by thermal melting and RNase H cleavage studies. In the linear form, the functional domain hybridizes (minimally, under physiological conditions) with the complementary target RNA to form a duplex. This duplex is a substrate for RNase H, and in the presence of RNase H and under appropriate conditions (e.g., physiological), the RNA strand of the duplex is cleaved by RNase H, thereby preventing expression.

[0062] CSOs containing antisense functional domains remain active in cell culture. The expected advantages of these CSOs are that their formation of intramolecular circular structures allows fewer interactions with non-target macromolecules (including nucleic acids and proteins), reduces polyanion-related side effects, and linearizes in the presence of target genes or RNA alone. Furthermore, due to the circular structure, these CSOs can escape endosomes due to the lack of 5'-end interactions.

[0063] The oligonucleotides of the present invention are isolated oligonucleotides. The term "isolated" means that they have been altered or removed from their natural state by human intervention. For example, an oligonucleotide naturally occurring in a living animal is not "isolated", whereas a synthetic oligonucleotide, or an oligonucleotide partially or completely separated from the coexisting materials of its natural state, is "isolated". An isolated oligonucleotide can be present in a substantially purified form or can be present in a non-natural environment, such as a cell to which the oligonucleotide is delivered. The oligonucleotides of the present invention can include partially purified DNA and / or RNA, substantially pure DNA and / or RNA, synthetic DNA and / or RNA, or recombinantly produced DNA and / or RNA, as well as modified DNA and / or RNA that differs from naturally occurring DNA and / or RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material, such as to the end of the oligonucleotide or to one or more internal nucleotides of the oligonucleotide, including modifications that render the oligonucleotide resistant to nuclease digestion.

[0064] The terms "microRNA," "miRNA," and "MiR" are interchangeable and refer to endogenous or artificial non-coding RNAs that can regulate gene expression. miRNAs are believed to function through RNA interference. The design of such microRNAs is within the skill of one of ordinary skill in the art.

[0065] The terms "piRNA" and "Piwi-interacting RNA" are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules are typically 26-31 nucleotides in length. The design of such PiRNAs is within the skill of one of ordinary skill in the art.

[0066] In an embodiment, the antisense oligonucleotide of the functional domain is as described in WO 2020 / 191177, which is incorporated herein by reference in its entirety. In an embodiment, the antisense oligonucleotide of the functional domain is a modified oligonucleotide comprising or consisting of an antisense oligonucleotide compound of 17-25 nucleotides in length, the antisense oligonucleotide compound comprising a 3' domain and a 5' domain, the 3' domain being 10-12 nucleotides in length, each nucleotide comprising a deoxyribonucleotide and a phosphodiester or phosphorothioate internucleotide linkage or a combination thereof, the 5' domain being 5-15 nucleotides in length, the 5' domain comprising unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or a combination thereof, with the proviso that the 5' domain comprises at least one modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. Such an antisense oligonucleotide is referred to as a "splitmer."

[0067] In an embodiment, a splitmer of a functional domain comprises 17-25 linked nucleotides having at least 12 adjacent nucleobases complementary to equal length portions of a target RNA.

[0068] In an embodiment, the modified ribonucleotides of the splitmer comprise 2'-substituted nucleotides, as described herein. In an embodiment, the 2'-substituted nucleotides are selected from 2'O-methyl ribonucleotides or 2'-MOE.

[0069] In some embodiments, the 3' domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3' end. In some embodiments, the 3' domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3' end. In some embodiments, the 3' domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

[0070] In some embodiments, when the 3' domain of the antisense oligonucleotide is 12 nucleotides in length, the antisense oligonucleotide of the invention has the formula (I): 5'-N m N 14 N 13 N 12 N 11 N 10 N 9 N 8 N 7 N 6 N 5 N 4 N 3 N 2 N 1 -3' is represented by During the ceremony, N is any nucleotide, N 13 ~N m comprises the 5' domain, N 1 ~N 12 comprises the 3' domain, m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0071] In some embodiments, when the 3' domain of the antisense oligonucleotide is 11 nucleotides in length, the antisense oligonucleotide of the invention has the formula (Ia): 5'-N m N 14 N 13 N 12 N 11 N 10 N 9 N 8 N 7 N 6 N 5 N 4 N 3 N 2 N 1 -3' is represented by N is any nucleotide, N 12 ~N m comprises the 5' domain, N 1 ~N 11 comprises the 3' domain, m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0072] In some embodiments, the antisense oligonucleotide of the invention has the formula (Ib): 5'-N m N 14 N 13 N 12 N 11 N 10 N 9 N 8 N 7 N 6 N 5 N 4 N 3 N 2 N 1 -3' is represented by N is any nucleotide, N 11 ~N m comprises the 5' domain, N 1 ~N 10 comprises the 3' domain, m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0073] In some embodiments, m is 0. In some embodiments, m is selected from 1, 2, 3, 4, 5, 6, or 7. In some embodiments, m is selected from 1, 2, 3, 4, 5, or 6. In some embodiments, m is selected from 1, 2, 3, 4, or 5. In some embodiments, m is selected from 1, 2, 3, or 4. In some embodiments, m is selected from 1, 2, or 3. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11.

[0074] In some embodiments, the antisense oligonucleotide compound of a functional domain is 17-25 nucleotides in length comprising at least 12 contiguous nucleobases complementary to equal length portions of a target RNA sequence, the antisense oligonucleotide compound comprises a 3' domain and a 5' domain adjacent to the 3' domain, the 3' domain being 10-12 nucleotides in length, each nucleotide comprising a deoxyribonucleotide and a phosphodiester or phosphorothioate intermolecular nucleotide linkage or a combination thereof, and The 5' domain is 5 to 15 nucleotides in length, and the 5' domain comprises unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or a combination thereof, with the proviso that the 5' domain comprises at least one modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone.

[0075] In an embodiment, the 3' domain is 12 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3' end (position 1 is the 3' end). In an embodiment, the 3' domain is 11 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3' end. In an embodiment, the 3' domain is 12 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

[0076] In embodiments, the nucleotides of the 3' domain contain natural nucleobases. In some embodiments, the nucleobases and sugars of the nucleotides of the 3' domain of the antisense oligonucleotide according to the invention are unmodified. In this respect, the nucleobases and sugars of the nucleotides of the 3' domain of the antisense oligonucleotide according to the invention are naturally occurring. Each of the nucleotides of the 3' domain contains a deoxyribonucleotide and a phosphodiester or phosphorothioate internucleotide linkage or a combination thereof. The nucleotides of the 3' domain contain a natural deoxyribose sugar and a phosphorothioate, phosphodiester or other phosphorus-based linkage or a combination thereof that is known to activate RNase H.

[0077] In an embodiment, at least one of the nucleotides of the 3' domain comprises a modified nucleobase.

[0078] In an embodiment, the nucleotide at position 9 or 10 from the 3' end is unmodified. In an embodiment, the nucleotide at position 11 from the 3' end is unmodified.

[0079] In an embodiment, the oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

[0080] In embodiments, at least half of the internucleotide linkages are phosphorothioate.

[0081] In embodiments, the antisense oligonucleotide is single stranded.

[0082] As used herein, the term "5' domain" refers to the nucleotides beginning with the first nucleotide following the 3' domain and proceeding toward the 5' end. The 5' domain hybridizes to the target RNA, but RNase H cannot cleave the target RNA in this domain. The term "5' domain" is generally 2-15 nucleotides in length and refers to the 11th to 25th nucleotides (the 1st nucleotide being the 3' end), the 12th to 25th nucleotides, or the 13th to 25th nucleotides of the antisense oligonucleotide measured from the 3' end, depending on the length of the 3' domain.

[0083] For example, an antisense oligonucleotide compound that is 17 nucleotides in length can include a 3' domain from positions 1 to 10 and a 5' domain from positions 11 to 17. The designation of modified nucleotides is position-specific as opposed to nucleotide-specific.

[0084] The 5' domain comprises nucleotides having non-RNase H activating modifications, such as modified sugars and / or modified backbones that do not activate RNase H. In some embodiments, the 5' domain comprises nucleotides comprising modified sugars. In some embodiments, the 5' domain comprises nucleotides comprising modified backbones. In some embodiments, the 5' domain comprises nucleotides comprising both modified sugars and modified backbones. In embodiments, the modified backbone is a non-phosphorus based backbone.

[0085] This design of the antisense allows for targeted RNA cleavage at specific sites toward the 5' end of the 3' domain.

[0086] In any of these embodiments, it is contemplated that the 5' domain comprises at least one nucleotide having a backbone modification or substitution and / or a sugar modification or substitution. In some embodiments, the nucleotide at one position in the 5' domain, at some positions in the 5' domain, or at all positions in the 5' domain comprises a backbone modification or substitution and / or a sugar modification or substitution. In one embodiment, the 5' domain comprises one nucleotide comprising a modified backbone and / or sugar. In one embodiment, the 5' domain comprises at least two nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5' domain comprises at least three nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5' domain comprises at least four nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5' domain comprises at least five nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5' domain comprises at least six nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5' domain comprises at least seven nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5' domain comprises at least 8 nucleotides comprising modified backbones and / or sugars, hi one embodiment, all of the nucleotides in the 5' domain are nucleotides comprising modified backbones and / or sugars.

[0087] It is specifically contemplated that the embodiments discussed herein may be implemented in the context of a particular nucleotide and position with respect to the position relative to the 3' end. For example, an antisense oligonucleotide having a modified nucleotide at position 13 refers to an antisense oligonucleotide having a modified nucleotide at position 13 from the 3' end of the antisense oligonucleotide.

[0088] In some embodiments, a functional domain antisense oligonucleotide is at least 90% complementary over its entire length to a portion of the target RNA.

[0089] In an embodiment, the oligonucleotide of the functional domain is a "gapmer". As used herein, a gapmer is a chimeric antisense oligonucleotide that comprises a central block of deoxynucleotide monomers of sufficient length to induce RNase H cleavage. Usually, the gapmer of the present invention is directed against one or more mRNAs that code for the target mRNA. The design of such gapmers is within the skill of the art.

[0090] In an embodiment, the antisense oligonucleotides of the functional domain are modified oligonucleotides that contain or consist of a region having a gapmer motif defined by two external regions or "wings" and a central or internal region or "gap". The three regions of the gapmer motif (5'-wing, gap, and 3'-wing) form a contiguous sequence of nucleosides in which at least a portion of the sugar moiety of each nucleoside of the wing is different from at least a portion of the sugar moiety of the nucleoside of the gap. Specifically, at least the sugar moiety of the nucleoside of each wing closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) is different from the sugar moiety of the adjacent gap nucleoside, thus defining the boundary between the wing and the gap (i.e., the wing / gap junction). In an embodiment, the sugar moieties within the gap are the same as each other. In an embodiment, the gap comprises one or more nucleosides having a sugar moiety that is different from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric gapmer).

[0091] In some embodiments, the gapmer wings independently comprise 1 to 6 nucleosides. In some embodiments, the gapmer wings independently comprise 1 to 5 nucleosides. In some embodiments, the gapmer wings comprise the same number of nucleosides. In some embodiments, the gapmer wings comprise 4 nucleosides. In some embodiments, each nucleoside in each wing of a gapmer is a modified nucleoside.

[0092] In certain embodiments, the gapmer gap comprises 7-24 nucleosides. In certain embodiments, the gapmer gap comprises 7-18 nucleosides. In certain embodiments, the gapmer gap comprises 9-14 nucleosides. In certain embodiments, the gapmer gap comprises 7-23 nucleosides. In certain embodiments, the gapmer gap comprises 9 nucleosides. In certain embodiments, the gapmer gap comprises 10 nucleosides. In certain embodiments, the gapmer gap comprises 11 nucleosides. In certain embodiments, the gapmer gap comprises 13 nucleosides. In certain embodiments, the gapmer gap comprises 14 nucleosides. In certain embodiments, the gapmer gap comprises 17 nucleosides. In certain embodiments, the gapmer gap comprises 18 nucleosides. In certain embodiments, each nucleoside in the gapmer gap is an unmodified 2'-deoxynucleoside.

[0093] In certain embodiments, the gapmer is a deoxy gapmer. In some embodiments, the nucleosides on the gap side of each wing / gap junction are unmodified 2'-deoxynucleosides and the nucleosides on the wing side of each wing / gap junction are modified nucleosides. In certain embodiments, each nucleoside of the gap is an unmodified 2'-deoxynucleoside. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0094] Here, the lengths (number of nucleosides) of the three regions of the gapmer may be provided using the notation [number of nucleosides in the 5'-wing]-[number of nucleosides in the gap]-[number of nucleosides in the 3'-wing]. Thus, a 5-10-5 gapmer consists of five linked nucleosides in each wing and 10 linked nucleosides in the gap. When such nomenclature is followed by a specific modification, the modification is a modification of the wing and the gap nucleoside contains an unmodified deoxynucleoside sugar. Thus, a 5-11-5 MOE or OMe gapmer consists of five linked MOE or OMe modified nucleosides in the 5'-wing, 11 linked deoxynucleosides in the gap, and five linked MOE or OMe nucleosides in the 3'-wing.

[0095] In some embodiments, the modified oligonucleotide is a 4-13-4 MOE or OMe gapmer. In some embodiments, the modified oligonucleotide is a 5-11-5 MOE or OME gapmer. In some embodiments, the modified oligonucleotide is a 3-15-3 BNA gapmer. In some embodiments, the modified oligonucleotide is a 3-15-3 LNA gapmer.

[0096] In any of the embodiments described herein, the modified oligonucleotide comprises or consists of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside throughout the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises or consists of a region having a fully modified sugar motif, where each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each of the uniformly modified nucleosides comprises the same 2'-modification. In certain embodiments, the uniformly modified sugar motif is 12-30 nucleosides in length. In certain embodiments, each nucleoside of the uniformly modified sugar motif is a 2'-substituted nucleoside, a sugar surrogate, or a bicyclic nucleoside. In certain embodiments, each nucleoside of a uniformly modified sugar motif is 2'-OCH 2 CH 2 OCH 3 group or 2'-OCH 3 In certain embodiments, modified oligonucleotides having at least one fully modified sugar motif can also have at least 1, at least 2, at least 3, or at least 4 2'-deoxynucleosides.

[0097] Examples of circularly structured oligonucleotides useful for gene silencing include, but are not limited to, the circularly structured oligonucleotides in Table 1. Oligonucleotide numbers 1-6 in Table 1 target proprotein convertase subtilisin / kexin type 9 (PCSK9). Oligonucleotide numbers 1512-1520, 1536-1545 and 1605 in Table 1 target patatin-like phospholipase domain-containing protein 3 (PNPLA3). Circularly structured oligonucleotides with functional domains directed to any other target of interest are well within the skill of the art. All internucleotide linkages are phosphorothioate linkages unless otherwise noted. [Table 1] TIFF2024541701000003.tif238159TIFF2024541701000004.tif70159

[0098] In some embodiments, the target nucleic acid (target RNA) is a targeted mouse sequence. In some embodiments, the target nucleic acid (target RNA) is a targeted human sequence.

[0099] In one embodiment, the PCSK9 nucleic acid is the mouse sequence set forth in GENBANK Accession No. NM_153565.2 (incorporated herein as SEQ ID NO: 57). In one embodiment, the PCSK9 nucleic acid is the human sequence set forth in GENBANK Accession No. NM_174936.3 (incorporated herein as SEQ ID NO: 58).

[0100] In some embodiments, the PNPLA3 nucleic acid is a mouse sequence set forth in GENBANK Accession No. NM_54088.3 (herein incorporated as SEQ ID NO: 80). In some embodiments, the PCSK9 nucleic acid is a human sequence set forth in GENBANK Accession No. NM_25225.2 (herein incorporated as SEQ ID NO: 81). In some embodiments, the PCSK9 nucleic acid is a rhesus sequence set forth in GENBANK Accession No. XM 1109144.3 (herein incorporated as SEQ ID NO: 82). In some embodiments, the PCSK9 nucleic acid is a cynomolgus sequence set forth in GENBANK Accession No. XM_5567051.2 (herein incorporated as SEQ ID NO: 83).

[0101] siRNA In an embodiment, the oligonucleotide of the functional domain is an siRNA. The siRNA comprises a short double-stranded RNA of about 15 to about 50 nucleotides, preferably about 18 to about 36 nucleotides, that targets an RNA.

[0102] In an embodiment, the present invention provides a circular structured oligonucleotide (CSO) comprising a functional domain, a cyclization domain, and a linker, wherein the functional domain and the cyclization domain are linked at their 5' or 3' ends, the functional domain comprises an oligonucleotide of 15-50 nucleotides in length and is complementary to a target RNA, the cyclization domain comprises an oligonucleotide of 4-100 nucleotides in length and is complementary to a sequence of nucleotides in the functional domain and has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and the functional domain comprises an siRNA.

[0103] In an embodiment, the cyclization domain is the same length as the sequence of the siRNA strand of the functional domain that it is complementary to.In an embodiment, the cyclization domain is longer than the sequence of the siRNA strand of the functional domain that it is complementary to.In an embodiment, the cyclization domain is shorter than the sequence of the siRNA strand of the functional domain that it is complementary to.

[0104] In embodiments, the cyclization domain comprises an oligonucleotide between 4 and 50 nucleotides in length. In embodiments, the cyclization domain comprises an oligonucleotide between 15 and 45 nucleotides in length. In embodiments, the cyclization domain comprises an oligonucleotide between 15 and 45 nucleotides in length. In embodiments, the cyclization domain oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0105] In embodiments, the cyclization domain comprises an oligonucleotide between 4 and 10 nucleotides in length. In embodiments, the cyclization domain comprises an oligonucleotide between 6 and 10 nucleotides in length. In embodiments, the cyclization domain comprises an oligonucleotide between 6 and 8 nucleotides in length. In embodiments, the oligonucleotide of the cyclization domain is 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.

[0106] In an embodiment, the cyclization domain is at least 95% complementary to the strand of the siRNA to which it is complementary. In an embodiment, the cyclization domain is at least 97% complementary to the strand of the siRNA to which it is complementary. In an embodiment, the cyclization domain is at least 98% complementary to the strand of the siRNA to which it is complementary. In an embodiment, the cyclization domain is at least 99% complementary to the strand of the siRNA to which it is complementary. In an embodiment, the cyclization domain is at least 100% complementary to the strand of the siRNA to which it is complementary.

[0107] The terms "siRNA" and "small interfering RNA" are interchangeable and refer to single-stranded or double-stranded RNA molecules capable of inducing RNA interference. siRNA molecules typically have a duplex region of 18-36 base pairs in length. Design of such siRNAs is within the skill of one of ordinary skill in the art.

[0108] In one embodiment, when the oligonucleotide of functional domain is siRNA, the cyclization domain is linked to the 5' end of the sense strand of siRNA via a linker segment (see Figure 2A).In one embodiment, when the oligonucleotide of functional domain is siRNA, the cyclization domain is linked to the 5' end of the antisense strand of siRNA via a linker segment.

[0109] In another embodiment, when the oligonucleotide of functional domain is the antisense strand of siRNA, cyclization domain is the sense strand of siRNA and is linked to either 3'-end or 5'-end of antisense strand (see Figure 2B and Figure 2C).In other words, in this embodiment, the sense strand of siRNA also acts as cyclization domain.

[0110] One or both strands of the siRNA of the present invention may also contain a 3'-overhang. "3' overhang" refers to at least one unpaired nucleotide extending from the 3'-end of the RNA strand. Thus, in one embodiment, the siRNA of the present invention contains at least one 3' overhang that is 1 to about 6 nucleotides in length (including ribonucleotides or deoxynucleotides), preferably 1 to about 5 nucleotides in length, more preferably 1 to about 4 nucleotides in length, and particularly preferably about 1 to about 2 nucleotides in length.

[0111] When both strands of siRNA molecule contain 3' overhang, the length of the overhang can be the same or different for each strand.In the most preferred embodiment, 3' overhang is present on both strands of siRNA and is 2 nucleotides long.To enhance the stability of the siRNA, 3' overhang can also be stabilized against degradation.In one embodiment, overhang is stabilized by including purine nucleotide, for example adenosine or guanosine nucleotide.

[0112] Alternatively, substitution of pyrimidine nucleotides with modified analogs, for example, substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, the absence of a 2' hydroxyl in 2'-deoxythymidine significantly enhances the nuclease resistance of the 3' overhangs in tissue culture medium.

[0113] The siRNA of the present invention can target any stretch of about 18-30, preferably 19-25 adjacent nucleotides of the target mRNA sequence. Techniques for selecting a target sequence for an siRNA are well known in the art. Thus, the sense strand of the present siRNA contains a nucleotide sequence identical to any adjacent stretch of about 18-30 nucleotides in the target mRNA.

[0114] Splicing Oligonucleotides In an embodiment, the invention provides a circular structured oligonucleotide (CSO) comprising a functional domain, a cyclization domain, and a linker, wherein the functional domain and the cyclization domain are linked at their 5' ends, the functional domain comprises an oligonucleotide 15-45 nucleotides in length and is complementary to a target RNA, the cyclization domain comprises an oligonucleotide 4-12 nucleotides in length and is complementary to a sequence of nucleotides in the functional domain and has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, the functional domain comprises a splicing oligonucleotide, and the oligonucleotide of the functional domain is modified.

[0115] As used herein, the term "splicing oligonucleotide" refers to an antisense oligonucleotide for regulating splicing. For splice regulation, the antisense binds to the target RNA and regulates splicing, thereby regulating protein expression. In a circularly structured oligonucleotide, the cyclization domain can be attached to either the 3'-end or the 5'-end to form a circular structure. In a preferred embodiment, the cyclization domain is attached to the 5'-end of the oligonucleotide of the functional domain. The circular structure reduces protein binding, reduces polyanionic properties, allows endosomal escape due to lack of terminal accessibility, and relieves interaction with pattern recognition receptors.

[0116] In an embodiment, the oligonucleotide for regulating splicing is snRNA. The terms "snRNA" and "small nuclear RNA" are interchangeable and refer to a class of small RNAs involved in various processes, including RNA splicing and regulating transcription factors. Also included is a subclass of small nucleolar RNA (snoRNA). This term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs. The design of such snRNAs is within the skill of a person skilled in the art.

[0117] In some embodiments, the oligonucleotides of the functional domains are as described in WO 2021 / 055011, which is incorporated herein by reference in its entirety. Specifically, in some embodiments, the oligonucleotides of the functional domains include an oligonucleotide comprising 14-30 linked nucleotides that are complementary to a target pre-mRNA that includes a retained intron, and the antisense oligonucleotides include 1-3 regions each independently comprising 2-5 contiguous deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. A CSO having a functional domain comprising an oligonucleotide that regulates splicing is useful for selecting a first mRNA transcript in a gene that comprises at least two mRNA transcripts, the oligonucleotide comprising at least 12 adjacent nucleobases complementary to equal length portions of the target pre-mRNA, the oligonucleotide targets a splice site of the pre-mRNA to the second mRNA transcript, thereby blocking the splice site to the second mRNA transcript and directing splicing of the pre-mRNA to the first mRNA transcript. In an embodiment, the 2'-substituted nucleotide is selected from 2'O-methyl ribonucleoside or 2'-methoxyethyl ribonucleoside (MOE).

[0118] In embodiments, the splicing oligonucleotide comprises one region comprising 2-5 contiguous deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. In embodiments, the splicing oligonucleotide comprises two regions independently comprising 2-5 contiguous deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. In embodiments, the splicing comprises three regions independently comprising 2-5 contiguous deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. In some embodiments, the contiguous deoxyribonucleotides are 2-4 nucleotides in length. In some embodiments, the contiguous deoxyribonucleotides are 4 nucleotides in length.

[0119] In an embodiment, the contiguous deoxyribonucleotides of the splicing oligonucleotide are at the 5' end of the antisense oligonucleotide, the 3' end of the splicing oligonucleotide, or adjacent to a 2'-substituted, non-ionic, or constrained sugar nucleotide, or a combination thereof. In an embodiment, the contiguous deoxyribonucleotides are at the 5' end of the splicing oligonucleotide. In an embodiment, the contiguous deoxyribonucleotides are at the 3' end of the splicing oligonucleotide. In an embodiment, the contiguous deoxyribonucleotides are adjacent to a 2'-substituted, non-ionic, or constrained sugar nucleotide, or a combination thereof.

[0120] In some embodiments, the splicing oligonucleotide of the functional domain comprises 14-30 linked nucleotides having at least 12 contiguous nucleobases complementary to an equal length portion of the target pre-mRNA including the retained intron, and the antisense oligonucleotide comprises 1-3 regions each independently comprising 2-5 contiguous deoxyribonucleotides, with the remaining nucleotides being 2'-substituted, non-ionic, or constrained sugar nucleotides, or combinations thereof.

[0121] In an embodiment, the 2'-substituted nucleotide is as described herein. In an embodiment, the 2'-substituted nucleotide is selected from 2'O-methyl ribonucleotides or 2'-MOE.

[0122] In some embodiments, the oligonucleotide of the functional domain comprises a region comprising 2-5 contiguous deoxyribonucleotides. In embodiments, the contiguous deoxyribonucleotides are at the 5' end of the splicing oligonucleotide, the 3' end of the antisense oligonucleotide, and are adjacent to 2'-substituted, non-ionic, or constrained sugar nucleotides, or combinations thereof. In embodiments, the contiguous deoxyribonucleotides are at the 5' end of the splicing oligonucleotide. In embodiments, the contiguous deoxyribonucleotides are at the 3' end of the splicing oligonucleotide.

[0123] In an embodiment, the consecutive deoxyribonucleotides are 2 to 4 nucleotides in length. In an embodiment, the consecutive deoxyribonucleotides are 4 nucleotides in length.

[0124] In embodiments, the exon is adjacent to the 5' splice site of the retained intron. In embodiments, the exon is adjacent to the 3' splice site of the retained intron. In embodiments, the exon is adjacent to the 5' splice site of the retained intron and the exon is adjacent to the 3' splice site of the retained intron.

[0125] Examples of cyclic oligonucleotides useful for splicing include, but are not limited to, the cyclic oligonucleotides in Table 2. The oligonucleotides in Table 2 target DMD. Circular oligonucleotides with functional domains directed to any other target of interest are well within the skill of the art. [Table 2] TIFF2024541701000006.tif126159

[0126] ADAR In an embodiment, the invention provides a circular structured oligonucleotide (CSO) comprising a functional domain, a cyclization domain, and a linker, wherein the functional domain and the cyclization domain are linked at their 5' ends, the functional domain comprises an oligonucleotide of 15-500 nucleotides in length and is complementary to a target RNA, the cyclization domain comprises an oligonucleotide of 4-12 nucleotides in length and is complementary to a sequence of nucleotides in the functional domain and has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and the functional domain comprises an antisense oligonucleotide of the adenosine deaminase acting on RNA (ADAR) system.

[0127] In some embodiments, the oligonucleotide of the functional domain is an antisense oligonucleotide that recruits endogenous ADAR (adenosine deaminase acting on RNA) enzymes to edit endogenous transcripts. ADAR is a group of enzymes that catalyze the conversion of adenosines (A's) to inosines (I's) in a process known as RNA editing. Although ADAR can act on various types of RNA, editing events in the coding region of mRNA are particularly interesting as I base pairs with guanosine (G). Thus, every A to I change catalyzed by ADAR is read as an A to G change during translation, potentially altering the sequence and function of the protein. This ability to recode makes ADAR an attractive therapeutic tool for correcting genetic mutations in mRNA.

[0128] The oligonucleotides of the ADAR system themselves contain one or more domains. One domain, known as the complementary domain, comprises a region of consecutive nucleotides that are complementary to the target RNA. In embodiments, the complementary domain is about 15 to about 120 nucleotides in length. In embodiments, the complementary domain is about 17 to about 60 nucleotides in length. Another domain, known as the recruitment domain, comprises a region of the oligonucleotide that recruits the ADAR enzyme.

[0129] CRISPR In an embodiment, the present invention provides a circular structured oligonucleotide (CSO) comprising a functional domain, a cyclization domain, and a linker, wherein the functional domain and the cyclization domain are linked at their 5' ends, the functional domain comprises an oligonucleotide between 17 and 500 nucleotides in length and is complementary to a target RNA, the cyclization domain comprises an oligonucleotide between 4 and 12 nucleotides in length and is complementary to a sequence of nucleotides in the functional domain and has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and the functional domain comprises an antisense oligonucleotide of a CRISPR-based system.

[0130] Functional domain oligonucleotides are antisense oligonucleotides that function as guide RNAs for CRISPR-based systems. The terms "sgRNA" and "guide RNA" are interchangeable and refer to specific RNA sequences that recognize a target DNA or RNA region of interest and direct an endonuclease there for editing. gRNAs are typically composed of two parts: crispr RNA (crRNA), a 17-30 nucleotide sequence complementary to the target DNA, and tracr RNA, which serves as a binding scaffold for Cas nucleases.

[0131] Any suitable engineered sgRNA, or crRNA and tracrRNA can be used as long as it is effective in recognizing the target DNA or RNA. The design of such sgRNA, or crRNA and tracrRNA is within the skill of a person skilled in the art.

[0132] Immunostimulatory Oligonucleotides In an embodiment, the invention provides a circular structured oligonucleotide (CSO) comprising a functional domain, a cyclization domain, and a linker, wherein the functional domain and the cyclization domain are linked at their 3' ends, the functional domain comprises an oligonucleotide between 11 and 400 nucleotides in length, the cyclization domain comprises an oligonucleotide between 4 and 400 nucleotides in length, which is complementary to a sequence of nucleotides in the functional domain and has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and the functional domain comprises an immunostimulatory oligonucleotide.

[0133] In embodiments, the immunostimulatory oligonucleotide is capable of inducing an interferon response in a vertebrate cell.

[0134] In an embodiment, the nucleotide sequence of the immunostimulatory oligonucleotide is not complementary to and does not bind to another nucleotide sequence, e.g., a target RNA. In this embodiment, the nucleotide sequence of the immunostimulatory oligonucleotide is not, e.g., an antisense oligonucleotide and does not have antisense activity.

[0135] In an embodiment, the immunostimulatory oligonucleotide is 11-40 nucleotides in length. In an embodiment, the immunostimulatory oligonucleotide is 15-28 nucleotides in length. In an embodiment, the immunostimulatory oligonucleotide is 17-25 nucleotides in length.

[0136] In embodiments, the cyclization domain is the same length as the sequence of the functional domain to which it is complementary. In embodiments, the cyclization domain is longer than the sequence of the functional domain to which it is complementary. In embodiments, the cyclization domain is shorter than the sequence of the functional domain to which it is complementary.

[0137] In an embodiment, the cyclization domain is 11 to 40 nucleotides in length. In an embodiment, the cyclization domain is 15 to 28 nucleotides in length. In an embodiment, the cyclization domain is 17 to 25 nucleotides in length. In an embodiment, the cyclization domain is 4 to 12 nucleotides in length. In an embodiment, the cyclization domain is 4 to 10 nucleotides in length. In an embodiment, the cyclization domain is 4 to 8 nucleotides in length.

[0138] In embodiments, the internucleotide linkages of the immunostimulatory oligonucleotides are phosphorothioate, phosphodiester, or combinations thereof.

[0139] In an embodiment the internucleotide linkages of the immunostimulatory oligonucleotides are phosphorothioate internucleotide linkages.

[0140] In an embodiment the internucleotide linkages of the immunostimulatory oligonucleotides are phosphodiester.

[0141] In embodiments, the internucleotide linkages of the immunostimulatory oligonucleotides are a combination of phosphorothioate and phosphodiester internucleotide linkages.

[0142] In embodiments, the internucleotide linkages of the cyclization domain are phosphorothioate, phosphodiester, or a combination thereof.

[0143] In embodiments, the internucleotide linkage of the cyclization domain is a phosphorothioate internucleotide linkage.

[0144] In embodiments, the internucleotide linkages of the cyclization domain are phosphodiester.

[0145] In embodiments, the internucleotide linkages of the cyclization domain are a combination of phosphorothioate and phosphodiester internucleotide linkages.

[0146] In some embodiments, the internucleotide linkages of the immunostimulatory oligonucleotide are phosphorothioate and the internucleotide linkages of the cyclization domain are phosphodiester, or vice versa.

[0147] Immunostimulatory oligonucleotides include, but are not limited to, oligonucleotides that induce immunostimulation via endosomal toll-like receptors, RIG-like receptors, STING, cGAS and inflammasomes. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that play a key role in initiating innate immune responses by detecting potential harmful pathogens. Each TLR has a broad range of specificity, for example, TLR1, 2, 4 and 6 recognize bacterial lipids, TLR3, 7 and 8 recognize viral RNA, TLR9 recognizes bacterial DNA containing CG motifs, and TLR5 and 10 recognize bacterial or parasitic proteins. The design of such immunostimulatory oligonucleotides is within the skill of the art.

[0148] In embodiments, when the oligonucleotide of the functional domain is an immunostimulatory oligonucleotide, the cyclization domain is attached to the 5'-end or 3'-end of the functional domain via a linker segment (see, e.g., FIG. 3). In some embodiments, the cyclization domain is attached to the 5'-end. In some embodiments, the cyclization domain is attached to the 3'-end.

[0149] In another embodiment, the oligonucleotide of the functional domain is an immunostimulatory oligonucleotide and the cyclization domain is another immunostimulatory oligonucleotide, which is complementary to a sequence of nucleotides in the functional domain, has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and is attached to the 3' end of the functional domain. In other words, in this embodiment, the cyclization domain also acts as an immunostimulatory oligonucleotide.

[0150] Examples of cyclic oligonucleotides useful for immunostimulation include, but are not limited to, the cyclic oligonucleotides in Table 3. The oligonucleotides in Table 3 contain CpG dinucleotides and activate Toll-like receptor 9 (TLR-9). Circular oligonucleotides with functional domains that contain immunostimulatory oligonucleotides directed to any other PRR or TLR of interest are well within the skill of the art. [Table 3] TIFF2024541701000008.tif230159

[0151] Immune antagonists In an embodiment, the invention provides a circular structured oligonucleotide (CSO) comprising a functional domain, a cyclization domain, and a linker, wherein the functional domain and the cyclization domain are linked at their 3' ends, the functional domain comprises an oligonucleotide between 11 and 400 nucleotides in length, the cyclization domain comprises an oligonucleotide between 4 and 400 nucleotides in length, which is complementary to a sequence of nucleotides in the functional domain and has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and the functional domain comprises an immune antagonist.

[0152] In embodiments, the immune antagonist oligonucleotides are capable of blocking the interferon response in vertebrate cells.

[0153] In embodiments, the nucleotide sequence of the immune antagonist oligonucleotide is not complementary to and does not bind to another nucleotide sequence, e.g., a target RNA. In embodiments, the nucleotide sequence of the immune antagonist oligonucleotide is not, e.g., an antisense oligonucleotide and does not have antisense activity.

[0154] In an embodiment, the immunostimulatory oligonucleotide is 11-30 nucleotides in length. In an embodiment, the immunostimulatory oligonucleotide is 15-28 nucleotides in length. In an embodiment, the immunostimulatory oligonucleotide is 17-25 nucleotides in length.

[0155] In embodiments, the cyclization domain is the same length as the sequence of the functional domain to which it is complementary. In embodiments, the cyclization domain is longer than the sequence of the functional domain to which it is complementary. In embodiments, the cyclization domain is shorter than the sequence of the functional domain to which it is complementary.

[0156] In an embodiment, the cyclization domain is 11 to 40 nucleotides in length. In an embodiment, the cyclization domain is 15 to 28 nucleotides in length. In an embodiment, the cyclization domain is 17 to 25 nucleotides in length. In an embodiment, the cyclization domain is 4 to 12 nucleotides in length. In an embodiment, the cyclization domain is 4 to 10 nucleotides in length. In an embodiment, the cyclization domain is 4 to 8 nucleotides in length.

[0157] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotides are phosphorothioate, phosphodiester, or combinations thereof.

[0158] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotides are phosphorothioate internucleotide linkages.

[0159] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotides are phosphodiester.

[0160] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotides are a combination of phosphorothioate and phosphodiester internucleotide linkages.

[0161] In embodiments, the internucleotide linkages of the cyclization domain are phosphorothioate, phosphodiester, or a combination thereof.

[0162] In embodiments, the internucleotide linkage of the cyclization domain is a phosphorothioate internucleotide linkage.

[0163] In embodiments, the internucleotide linkages of the cyclization domain are phosphodiester.

[0164] In embodiments, the internucleotide linkages of the cyclization domain are a combination of phosphorothioate and phosphodiester internucleotide linkages.

[0165] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotide are phosphorothioate and the internucleotide linkages of the cyclization domain are phosphodiester, or vice versa.

[0166] Immune antagonist oligonucleotides include, but are not limited to, oligonucleotides that block immune activation via endosomal toll-like receptors, RIG-like receptors, STING, cGAS and inflammasomes. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that play a key role in initiating innate immune responses by detecting potential harmful pathogens. Each TLR has a broad range of specificity, for example, TLR1, 2, 4 and 6 recognize bacterial lipids, TLR3, 7 and 8 recognize viral RNA, TLR9 recognizes bacterial DNA containing CG motifs, and TLR5 and 10 recognize bacterial or parasitic proteins. The design of such immune antagonist oligonucleotides is within the skill of one of ordinary skill in the art.

[0167] In another embodiment, the oligonucleotide of the functional domain is an immune antagonist oligonucleotide, and the cyclization domain is another immune antagonist oligonucleotide, which is complementary to the sequence of nucleotides in the functional domain, has the opposite polarity to the sequence of nucleotides in the functional domain to which it is complementary, and is linked to the 3'-end of the functional domain (see FIG. 3B). In other words, in this embodiment, the cyclization domain also acts as an immune antagonist oligonucleotide.

[0168] Examples of cyclic oligonucleotides useful for immunostimulation include, but are not limited to, the cyclic oligonucleotides in Table 4. The oligonucleotides in Table 4 contain a methylated C within the CpG dinucleotide and block activation of Toll-like receptor 9 (TLR-9). Circular oligonucleotides with functional domains that include immune antagonist oligonucleotides directed against any other PRR or TLR of interest are well within the skill of the art. [Table 4]

[0169] Examples of cyclization domains useful in the cyclic oligonucleotides described herein include, but are not limited to, the cyclization domains in Table 5. [Table 5] TIFF2024541701000011.tif186159

[0170] Pharmaceutical Compositions In some embodiments, the present specification describes a pharmaceutical composition comprising one or more CSO compounds of the present invention. In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable diluent or carrier. In some embodiments, the pharmaceutical composition comprises sterile saline and one or more CSO compounds. In some embodiments, the pharmaceutical composition consists of sterile saline and one or more CSO compounds. In some embodiments, the sterile saline is pharmaceutical grade saline. In some embodiments, the pharmaceutical composition comprises one or more CSO compounds and sterile water. In some embodiments, the pharmaceutical composition consists of a CSO compound and sterile water. In some embodiments, the sterile water is pharmaceutical grade water. In some embodiments, the pharmaceutical composition comprises one or more CSO compounds and phosphate buffered saline (PBS). In some embodiments, the pharmaceutical composition consists of one or more CSO compounds and sterile PBS. In some embodiments, the sterile PBS is pharmaceutical grade PBS.

[0171] In some embodiments, the pharmaceutical composition comprises one or more CSO compounds and one or more excipients. In some embodiments, the excipients are selected from water, salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0172] In some embodiments, the CSO compounds of the present invention can be mixed with pharma- ceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations.The composition and method for formulating pharmaceutical compositions depend on many criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0173] Conjugate Group In certain embodiments, the CSO according to the present invention further comprises one or more conjugate groups, if desired. The conjugate group is composed of one or more conjugate moieties and a conjugate linker that links the one or more conjugate moieties to the oligonucleotide. The conjugate group may be attached to either or both termini of the oligonucleotide and / or at any internal position. In certain embodiments, the conjugate group is attached to the 2' position of the nucleoside of the modified oligonucleotide. In certain embodiments, the conjugate group attached to either or both termini of the oligonucleotide is a terminal group. In certain such embodiments, the conjugate group or terminal group is attached to the 3' and / or 5' termini of the oligonucleotide. In certain such embodiments, the conjugate group is attached to the 3'-terminus of the oligonucleotide. In certain embodiments, the conjugate group is attached near the 3'-terminus of the oligonucleotide. In certain embodiments, the conjugate group is attached near the 5'-terminus of the oligonucleotide. In certain embodiments, the conjugate group is attached near the 5'-terminus of the oligonucleotide.

[0174] In any of the embodiments herein, the conjugate group comprises a GalNAc cluster comprising from 1 to 3 GalNAc ligands.

[0175] In any of the embodiments herein, the conjugate linker consists of a single bond.

[0176] In any of the embodiments herein, the conjugate linker is cleavable.

[0177] In any embodiment herein, the conjugate linker comprises 1 to 3 linker-nucleosides.

[0178] In any embodiment herein, the conjugate group is attached to the CSO at the 5' end of the functional domain. In any embodiment herein, the conjugate group is attached to the CSO at the 3' end of the functional domain. In any embodiment herein, the conjugate group is attached to the CSO at the 5' end of the cyclization domain. In any embodiment herein, the conjugate group is attached to the CSO at the 3' end of the cyclization domain.

[0179] In some embodiments, CSO is covalently bound to one or more conjugate groups.In some embodiments, conjugate group modifies one or more properties of CSO, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.In some embodiments, conjugate group gives new properties to CSO, such as fluorophore or reporter group that allows detection of oligonucleotide. Certain conjugate groups and moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids, 19 ...esters, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 4, 1053-1060), thioesters, such as hexyl-S-tritylthiol (Oberhauser et Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids, 1999, 20, 533-538). Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid, palmityl moieties (Mishra et al., Biochim. Biophys.Acta, 1995, 1264, 229-237), octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol groups (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc clusters (e.g., WO 2014 / 179620) have been previously described.

[0180] Conjugate moieties include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folates, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0181] In certain embodiments, the conjugate moiety comprises an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepines, indomethicine, barbiturates, cephalosporins, sulfa drugs, antidiabetics, antibacterial agents, or antibiotics.

[0182] The conjugate moiety is attached to the CSO via a conjugate linker. In some embodiments, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is directly attached to the CSO via a single bond). In some embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units, such as ethylene glycol, nucleoside, or amino acid units.

[0183] In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker comprises at least one neutral linking group.

[0184] In some embodiments, the conjugate linker, including the conjugate linker described above, is a bifunctional linking moiety, for example, one known in the art to be useful for attaching a conjugate group to a parent compound, such as an oligonucleotide provided herein. In general, the bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a specific site on the CSO, and the other is selected to bind to a conjugate group. Examples of functional groups used in the bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, the bifunctional linking moiety comprises one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0185] Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include substituted or unsubstituted C 1 ~C 10 Alkyl, substituted or unsubstituted C 2 ~C 10 Alkenyl or substituted or unsubstituted C 2 ~C 10 A non-limiting list of preferred substituents includes, but is not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0186] In some embodiments, the conjugate linker comprises 1-10 linker-nucleosides. In some embodiments, the conjugate linker comprises 2-5 linker-nucleosides. In some embodiments, the conjugate linker comprises exactly 3 linker-nucleosides. In some embodiments, the conjugate linker comprises a TCA motif. In some embodiments, such linker-nucleosides are modified nucleosides. In some embodiments, such linker-nucleosides comprise a modified sugar moiety. In some embodiments, the linker-nucleoside is unmodified. In some embodiments, the linker-nucleoside comprises an optionally protected heterocyclic base selected from a purine, a substituted purine, a pyrimidine, or a substituted pyrimidine. In some embodiments, the cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is usually desirable for the linker-nucleoside to be cleaved from the oligomeric compound after it reaches the target tissue. Thus, the linker-nucleoside is usually linked to each other and to the rest of the oligomeric compound via a cleavable bond. In some embodiments, such a cleavable bond is a phosphodiester bond.

[0187] As used herein, linker-nucleosides are not considered as part of CSO in general, or as part of cyclization domain or functional domain in particular. Thus, the nucleotides of linker-nucleosides are not counted against the length of CSO or its domain, and are not used to determine the percent complementarity of oligonucleotide to reference nucleic acid. Unless otherwise indicated, conjugate linkers contain 10 or less linker-nucleosides. In some embodiments, conjugate linkers contain 5 or less linker-nucleosides. In some embodiments, conjugate linkers contain 3 or less linker-nucleosides. In some embodiments, conjugate linkers contain 2 or less linker-nucleosides. In some embodiments, conjugate linkers contain 1 or less linker-nucleosides.

[0188] In some embodiments, it is desirable for the conjugate group to be cleaved from the CSO. For example, in some situations, CSOs containing certain conjugate moieties are better taken up by certain cell types, but it is desirable for the conjugate group to be cleaved to release the unconjugated or parent CSO once the CSO is taken up. Thus, some conjugate linkers may contain one or more cleavable moieties. In some embodiments, the cleavable moiety is a cleavable bond. In some embodiments, the cleavable moiety is an atomic group that includes at least one cleavable bond. In some embodiments, the cleavable moiety includes an atomic group that has one, two, three, four or more than four cleavable bonds. In some embodiments, the cleavable moiety is selectively cleaved inside a cell or intracellular compartment, such as a lysosome. In some embodiments, the cleavable moiety is selectively cleaved by an endogenous enzyme, such as a nuclease.

[0189] In some embodiments, the cleavable bond is selected from among amide, ester, ether, one or both esters of a phosphodiester, phosphate ester, carbamate, or disulfide. In some embodiments, the cleavable bond is one or both esters of a phosphodiester. In some embodiments, the cleavable moiety comprises a phosphate or a phosphodiester. In some embodiments, the cleavable moiety is a phosphate bridge between the oligonucleotide and the conjugate moiety or conjugate group.

[0190] use Circularly structured oligonucleotides can be useful for various mechanisms of action. These include antisense for mRNA, ncRNA, microRNA, lncRNA and splicing. Also, double-stranded circular structures can be used to deliver siRNA constructs. In addition, circular structures can provide a novel approach to deliver antisense for ADAR and CRISPR system mechanisms. In addition, circular structures can provide a novel approach to deliver antisense to disrupt the structure of RNA to increase translation. Circular structures also provide a defined structure to the nucleic acid, thereby allowing various degrees of interaction with PRRs and induced immune cascades.

[0191] For example, when the functional domain is an antisense oligonucleotide, the CSOs according to the invention are also useful in therapeutic approaches in which inhibition of gene expression is desired, which may include, for example, inhibition of endogenous genes (e.g., oncogenes) or exogenous genes (e.g., genes essential for the growth and / or metabolism of a pathogen).

[0192] In an embodiment, the invention provides a method of inhibiting gene expression comprising administering a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide as described herein.

[0193] In an embodiment, the present invention provides a method of inhibiting allele-specific gene expression comprising administering a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide as described herein.

[0194] The methods according to the invention are useful for treating a subject having a disease or disorder in which it would be beneficial to inhibit expression of the gene, in embodiments, the disease or disorder results from the aberrant expression or product of a cellular gene.

[0195] In some embodiments, a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide described herein is administered locally.

[0196] In some embodiments, a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide described herein is administered systemically.

[0197] In embodiments, a method of modulating RNA processing comprising administering a CSO compound as described herein, wherein the functional domain comprises an antisense oligonucleotide comprising 14-30 linked nucleotides having at least 12 adjacent nucleobases complementary to an equal length portion of a target RNA, and the antisense oligonucleotide comprises 1-3 regions each independently comprising 2-5 contiguous deoxyribonucleotides, the remaining nucleotides being 2'-substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. In embodiments, the processing of the RNA comprises splicing.

[0198] In an embodiment, the invention provides a method for selecting a first mRNA transcript in a gene that comprises at least two mRNA transcripts, comprising administering a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide as described herein.

[0199] In an embodiment, the present invention provides a method of treating a disease or disorder in a subject, where modulating RNA processing is beneficial to treat the subject, the method comprising administering a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide as described herein.

[0200] In embodiments, the invention provides a method of inducing nonsense-mediated mRNA decay of a target RNA comprising administering a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide as described herein.

[0201] In embodiments, the present invention provides a method of increasing the level of an mRNA encoding a protein or functional mRNA, increasing expression of the protein or functional mRNA, comprising administering a circularly structured oligonucleotide or a composition comprising a circularly structured oligonucleotide as described herein.

[0202] The circularly structured oligonucleotides of the present invention can be administered alone or in combination with any other drug or therapeutic agent. The drugs or therapeutic agents can be administered simultaneously or sequentially. Such drugs or therapeutic agents may be useful for treating or preventing a disease or condition and do not reduce the gene expression modulating effect of the cyclic oligonucleotides according to the present invention. Agents useful for treating or preventing a disease or condition include, but are not limited to, small molecules, peptides, vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonists (e.g., TLR3 and / or TLR7 antagonists and / or TLR8 antagonists and / or TLR9 antagonists), chemotherapeutic agents (both traditional chemotherapy and modern targeted therapy), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and costimulatory molecules (e.g., cytokines, chemokines, protein ligands, transactivators, peptides or peptides containing modified amino acids), or combinations thereof. Alternatively, cyclic oligonucleotides according to the invention can be administered in combination with other compounds (eg, lipids or liposomes) to enhance the specificity or magnitude of gene expression modulation of the cyclic oligonucleotides according to the invention.

[0203] The cyclic oligonucleotides of the present invention may be administered by any suitable route, including but not limited to parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intratumoral, intravenous, subcutaneous, intrathecal, intranasal, aerosol, intraocular, intratracheal, rectal, intravaginal, gene gun, skin patch, or in the form of eye drops or mouthwash. In any of the methods according to the present invention, the administration of the cyclic oligonucleotides according to the present invention, alone or in combination with any other agent, may be administered directly to a tissue or organ, including but not limited to the bladder, liver, lung, or kidney. In some embodiments, the administration of the cyclic oligonucleotides according to the present invention, alone or in combination with any other agent, is by intramuscular administration. In some embodiments, the administration of the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, is by mucosal administration. In some embodiments, the administration of the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, is by oral administration. In some embodiments, the administration of the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, is by rectal administration. In some embodiments, the administration of the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, is by intrathecal administration. In some embodiments, the administration of the antisense oligonucleotide according to the present invention, alone or in combination with any other drug, is by intratumoral administration.In some embodiments, the administration of the antisense oligonucleotide according to the present invention, alone or in combination with any other drug, is by parenteral administration.In some embodiments, the administration of the antisense oligonucleotide according to the present invention, alone or in combination with any other drug, is by subcutaneous administration.

[0204] In some embodiments, any of the circularly structured oligonucleotides described herein can be conjugated to a moiety that provides site-specific delivery of the CSO. In embodiments, such conjugates include, but are not limited to, antibodies, peptides, lipids, or small molecules.

[0205] In some embodiments, any of the circularly structured oligonucleotides described herein can be encapsulated with a moiety that provides site-specific delivery of the CSO. In embodiments, the CSO can be encapsulated, for example, in a lipid, lipid nanoparticle (LNP), or peptide macrocycle.

[0206] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol, methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting isotonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials. Administration of antisense oligonucleotides according to the present invention can be carried out using known procedures, using effective amounts and durations effective to reduce symptoms or surrogate markers of disease. For example, an effective amount of antisense oligonucleotides according to the present invention for treating disease and / or disorders can be the amount required to alleviate or reduce symptoms, or to delay or ameliorate tumors, cancer, or bacterial, viral, or fungal infections. In the context of administering a composition that regulates gene expression, an effective amount of the antisense oligonucleotide according to the present invention is sufficient to achieve desired regulation compared to gene expression in the absence of the antisense oligonucleotide according to the present invention.The effective amount for any particular application can vary depending on factors such as the disease or condition being treated, the specific oligonucleotide being administered, the size of the subject, or the severity of the disease or condition.Those skilled in the art can empirically determine the effective amount of a particular antisense oligonucleotide without the need for undue experimentation. synthesis

[0207] The CSOs described herein can be prepared by any suitable art-recognized method, including but not limited to H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate and phosphoramidite chemistry (i.e., H-phosphonate chemistry in some cycles and phosphoramidite chemistry in other cycles), which can be performed manually or by an automated synthesizer. The oligonucleotides of the present invention can also be modified in many ways without impairing their ability to hybridize to their targets (see, e.g., Agrawal and Gait, Advances in Nucleic Acid Therapeutics, (2019) https: / / doi.org / 10.1039 / 9781788015714).

[0208] definition Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout this disclosure are incorporated herein by reference in their entirety.

[0209] Unless otherwise indicated, the following terms have the following meanings: As used herein, "2'-deoxynucleoside" refers to a nucleoside that contains a 2'-H(H) furanosyl sugar moiety found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, a 2'-deoxynucleoside may contain a modified nucleobase or may contain an RNA nucleobase (uracil).

[0210] As used herein, "2'-substituted nucleoside" refers to a nucleoside that includes a 2'-substituted sugar moiety. As used herein, "2'-substituted" with respect to the sugar moiety means a sugar moiety that includes at least one 2'-substituent group other than H or OH.

[0211] As used herein, "5-methylcytosine" refers to a modified cytosine having a methyl group attached to position 5. 5-methylcytosine is a modified nucleobase.

[0212] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0213] As used herein, "administering" means providing a pharmaceutical agent to an animal.

[0214] As used herein, "animal" means a human or non-human animal.

[0215] As used herein, an "individual in need" refers to a human or non-human animal selected for treatment or therapy who is in need of such treatment or therapy.

[0216] As used herein, "antisense activity" refers to any detectable and / or measurable change that can be caused by the hybridization of antisense compound to its target nucleic acid.In some embodiments, antisense activity is the amount or expression of target nucleic acid or the protein encoded by such target nucleic acid is reduced compared to the target nucleic acid level or target protein level in the absence of antisense compound.In some embodiments, antisense activity is the amount or expression of target nucleic acid or the protein encoded by such target nucleic acid is increased compared to the target nucleic acid level or target protein level in the absence of antisense compound.

[0217] As used herein, "antisense compound" means an oligomeric compound capable of achieving at least one antisense activity.

[0218] As used herein, "improve" in relation to treatment means that at least one symptom is improved for the same symptom in the absence of treatment.In some embodiments, the improvement is the reduction in severity or frequency of symptoms, or the delay in onset or slowing down the progression of the severity or frequency of symptoms.In some embodiments, the symptoms or characteristics are ataxia, neuropathy, and aggregate formation.In some embodiments, the improvement of these symptoms results in the improvement of motor function, the reduction in neuropathy, or the reduction in the number of aggregates.

[0219] As used herein, "bicyclic nucleoside" or "BNA" refers to a nucleoside that includes a bicyclic sugar moiety. As used herein, "bicyclic sugar" or "bicyclic sugar moiety" refers to a modified sugar moiety that includes two rings, where the second ring is formed through a bridge that connects two of the atoms of the first ring, thereby forming a bicyclic structure. In some embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In some embodiments, the bicyclic sugar moiety does not include a furanosyl moiety.

[0220] As used herein, "chiral enriched population" refers to a plurality of molecules of the same molecular formula, and when a particular chiral center is stereo-random, the number or percentage of molecules in the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules that are expected to contain the same particular stereochemical configuration at the same particular chiral center in the population. A chiral enriched population of molecules with multiple chiral centers in each molecule may contain one or more stereo-random chiral centers. In some embodiments, the molecule is a modified oligonucleotide. In some embodiments, the molecule is a compound that contains a modified oligonucleotide.

[0221] As used herein, "cleavable moiety" means a bond or group that is cleaved under physiological conditions, eg, inside a cell, animal, or human.

[0222] As used herein, "complementary" in relation to oligonucleotide means that at least 70% of the nucleobase or one or more regions of the oligonucleotide and the nucleobase or one or more regions of another nucleic acid can hydrogen bond with each other when the nucleobase sequence of the oligonucleotide and the nucleobase sequence of the other nucleic acid are aligned in opposite directions.Complementary nucleobase refers to nucleobases that can form hydrogen bonds with each other.

[0223] Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids need not have nucleobase complementarity at every nucleoside. Rather, some mismatches are tolerated. As used herein, "fully complementary" or "100% complementary" with respect to an oligonucleotide means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at every nucleoside of the oligonucleotide.

[0224] As used herein, "conjugate group" refers to a group of atoms that is directly or indirectly attached to an oligonucleotide. A conjugate group includes a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

[0225] As used herein, "conjugate linker" means a group of atoms containing at least one bond that connects a conjugate moiety to an oligonucleotide.

[0226] As used herein, "conjugate moiety" means a group of atoms that is attached to an oligonucleotide via a conjugate linker.

[0227] As used herein, "adjacent" in the context of oligonucleotides refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to one another. For example, "adjacent nucleobases" means nucleobases that are immediately adjacent to one another in a sequence.

[0228] As used herein, "linker nucleoside" refers to a nucleoside that directly or indirectly links a CSO of the present invention to a conjugate moiety. A linker-nucleoside is located within the conjugate linker and is not considered part of the CSO compound even if it is adjacent to the CSO.

[0229] As used herein, "gapmer" refers to a modified oligonucleotide comprising an internal region having multiple nucleosides that support RNase H cleavage located between external regions having one or more nucleosides, the nucleosides comprising the internal region being chemically distinct from the nucleoside(s) comprising the external region. The internal region may be referred to as the "gap" and the external regions as the "wings". Unless otherwise indicated, "gapmer" refers to a sugar motif. Unless otherwise indicated, the sugar moieties of the nucleosides of the gapmer gap are unmodified 2'-deoxyfuranosyl. Thus, the term "MOE gapmer" refers to a gapmer having a 2'-MOE nucleoside sugar motif and a 2'-deoxynucleoside gap in both wings. Unless otherwise indicated, MOE gapmers may include one or more modified internucleoside linkages and / or modified nucleobases, and such modifications do not necessarily follow the gapmer pattern of sugar modifications.

[0230] As used herein, a "hot spot region" is a range of nucleobases on a target nucleic acid that is suitable for an oligomeric compound to reduce the amount or activity of the target nucleic acid, as demonstrated in the examples herein below.

[0231] As used herein, "hybridization" refers to the pairing or annealing of complementary oligonucleotides and / or nucleic acids.Although not limited to a specific mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleic acid bases.

[0232] As used herein, the term "internucleoside bond" refers to the covalent bond between adjacent nucleosides in an oligonucleotide.As used herein, "modified internucleoside bond" refers to any internucleoside bond other than phosphodiester internucleoside bond.A "phosphorothioate bridge" is a modified internucleoside bond in which one of the non-bridging oxygen atoms of phosphodiester internucleoside bond is replaced with a sulfur atom.

[0233] As used herein, the phrase "inhibition of expression or activity" refers to a reduction or blocking of expression or activity compared to the expression of the activity in an untreated or control sample, and does not necessarily indicate a complete elimination of expression or activity.

[0234] As used herein, "linker nucleoside" refers to a nucleoside that directly or indirectly links an oligonucleotide to a conjugate moiety. A linker-nucleoside is located within the conjugate linker of an oligomeric compound. A linker-nucleoside is not considered part of the oligonucleotide moiety of an oligomeric compound, even if it is adjacent to the oligonucleotide.

[0235] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety that includes a modification, e.g., a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

[0236] As used herein, "mismatch" or "non-complementary" means a nucleobase of a first oligonucleotide that is not complementary to the corresponding nucleobase of a second oligonucleotide or a target nucleic acid when the first and second oligomeric compounds are aligned.

[0237] As used herein, "MOE" means methoxyethyl. "2'-MOE" refers to a 2'-OCH substituted for the 2'OH group of the ribosyl sugar moiety. 2 CH 2 OCH 3 means a group.

[0238] As used herein, "motif" means a pattern of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages in an oligonucleotide.

[0239] As used herein, unless otherwise specified, "mRNA" means an RNA transcript that encodes a protein and includes pre-mRNA and mature mRNA.

[0240] As used herein, "nucleobase" refers to unmodified or modified nucleobase. As used herein, "unmodified nucleobase" refers to adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). As used herein, "modified nucleobase" refers to an atomic group other than unmodified A, T, C, U, or G that can pair with at least one unmodified nucleobase. "5-methylcytosine" is an example of a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. Modified bases, also called heterocyclic base moieties, include other nucleobases, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and cytosine. Examples of cytosines include cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines), 7-methylguanine and adenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.

[0241] In certain embodiments, the modified nucleobase is selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, other alkynyl derivatives of pyrimidine bases, 6-azapyrimidine ... Z-uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.In certain embodiments, the modified nucleobase is 5-methylcytosine.

[0242] Representative modified sugars include carbocyclic or acyclic sugars, sugars having a substituent at one or more of their 2', 3' or 4' positions, and sugars having a substituent in place of one or more hydrogen atoms of the sugar. In some embodiments, the sugar is modified by having a substituent at the 2' position. In further embodiments, the sugar is modified by having a substituent at the 3' position. In other embodiments, the sugar is modified by having a substituent at the 4' position. It is also contemplated that sugars may have modifications at more than one of their positions, or that antisense oligonucleotides may have one or more nucleotides with a sugar modification at one position and one or more nucleotides with sugar modifications at different positions.

[0243] Sugar modifications contemplated in oligonucleotides include, but are not limited to, sugar substituents selected from OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl are substituted or unsubstituted C 1 ~C 10 Alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. In some embodiments, these groups are O(CH 2 ) x OCH 3 , O((CH 2 ) x O) y CH 3 , O(CH 2 ) x NH 2 , O(CH 2 ) x CH 3 , O(CH 2 ) x ONH 2 , and O(CH 2 ) x ON((CH 2 )x CH 3 ) 2 wherein x and y are independently 1 to 10.

[0244] In some embodiments, the modified sugar is selected from the group consisting of: 1 ~C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , Cl, Br, CN, OCN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of antisense oligonucleotides, or groups for improving the pharmacodynamic properties of antisense oligonucleotides, and other substituents with similar properties. In one embodiment, the modification is 2'-methoxyethoxy (2'-O-CH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE). 2 CH 2 OCH 3 ) (Martin et al., 1995), i.e., an alkoxyalkoxy group. Another modification includes O(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE. 2 ) 2 ON(CH 3 ) 2 groups and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2- O-CH 2 -N(CH 3 ) 2 Includes:

[0245] Additional sugar substituents include allyl (-CH 2 -CH=CH 2 ), -O-allylCH 2 -CH=CH 2 ), methoxy (-O-CH 3 ), aminopropoxy (-OCH 2 CH 2 CH 2 NH 2 ), and fluoro (F). The sugar substituent at the 2' position (2'-) can be in the arabino (up) or ribo (down) position. One 2'-arabino modification is 2'-F. Other similar modifications can also be made at other positions on the oligomeric compound, particularly at the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. Oligomeric compounds can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Examples of U.S. patents disclosing the preparation of modified sugar structures include U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, which are incorporated by reference in their entireties.

[0246] Representative sugar substituents include those described in U.S. Patent Application Publication No. 2005 / 0261218, which is incorporated herein by reference. In certain embodiments, the sugar modification is a 2'-O-Me modification, a 2'F modification, a 2'H modification, a 2'amino modification, a 4'thioribose modification or a phosphorothioate modification on the carboxy group attached to the 6' carbon, or a combination thereof.

[0247] In one embodiment, the 2'-substituted non-bicyclic modified nucleoside is selected from the group consisting of F, OCH 3 , and O.C.H. 2 CH 2 OCH 3 The sugar moiety comprises a non-bridging 2'-substituent selected from:

[0248] Certain modified sugar moieties include a substituent that bridges two atoms of a furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'→2' bridging sugar substituents include 4'-CH 2 -2',4'-(CH 2 ) 2 -2',4'-(CH 2 ) 3 -2',4'-CH 2 -O-2' ("LNA"), 4'-CH 2 -S-2',4'-(CH 2 ) 2 -O-2' ("ENA"), 4'-CH(CH 3 )-O-2' (called "constrained ethyl" or "cEt"), 4'-CH 2 -O-CH 2 -2',4'-CH 2 -N(R)-2',4'-CH(CH 2 OCH 3)-O-2 ("constrained MOE" or "cMOE") and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845; Bhat et al., U.S. Pat. No. 7,569,686; Swayze et al., U.S. Pat. No. 7,741,457; and Swayze et al., U.S. Pat. No. 8,022,193), 4'-C(CH 3 )(CH 3 )-O-2' and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4'-CH 2 -N(OCH 3 )-2' and its analogs (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4'-CH 2 -ON(CH 3 )-2' (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4'-CH 2 -C(H)(CH 3 )-2' (e.g., Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH 2 -C(=CH 2 )-2' and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,426), 4'-C(R a R b )-N(R)-O-2', 4 , -C(R a R b )-ON(R)-2',4'-CH 2 -ON(R)-2' and 4'-CH 2 -N(R)-O-2' [where each R, R a and R b are independently H, a protecting group, or C 1 ~C 12 Examples of aryl groups include, but are not limited to, alkyl (see, for example, Imanishi et al., US Pat. No. 7,427,672).

[0249] In certain embodiments, such a 4'-2' bridge is independently -[C(Ra )(R b )] n -,-[C(R a )(R b )] n -O-, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a ) 2 -, -S(=O) x - and -N(R a )-, During the ceremony, x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R a and R b are independently H, a protecting group, a hydroxyl, C 1 ~C 12 Alkyl, substituted C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenyl, substituted C 2 ~C 12 Alkenyl, C 2 ~C 12 Alkynyl, Substituted C 2 ~C 12 Alkynyl, C 5 ~C 20 Aryl, Substituted C 5 ~C 20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C 5 ~C 7 Alicyclic radicals, substituted C 5 ~C 7 Alicyclic radicals, halogens, OJ 1 , N.J. 1 J 2 , S.J. 1 , N3, COOJ 1 , acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) 2 -J 1), or sulfoxyl (S(=O)-J 1 ), and each J 1 and J. 2 are independently H, C 1 ~C 12 Alkyl, substituted C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenyl, substituted C 2 ~C 12 Alkenyl, C 2 ~C 12 Alkynyl, Substituted C 2 ~C 12 Alkynyl, C 5 ~C 20 Aryl, Substituted C 5 ~C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C 1 ~C 12 Aminoalkyl, substituted C 1 ~C 12 aminoalkyl, or a protecting group.

[0250] Further bicyclic sugar moieties are known in the art, see, for example, Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; Albaek et al., J. Org. Chem., 2006, 71, 7731-7740; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J Am. Chem. Soc., 20017, 129, 8362-8379; Wengel et al., U.S. Pat. No. 7,053,207; Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al., U.S. Pat. No. 6,770,748; Imanishi et al., U.S. Pat.RE 44, 779; Wengel et al., U.S. Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengel et al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al., U.S. Pat. No. 6,525,191; Torsten et al., WO 2004 / 106356; Wengel et al., WO 1999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., U.S. Pat. No. 7,547, 684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat. Nos. 7,750, 131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; and U.S. Patent Application Publication Nos. Allerson et al., U.S. Pat. Application Publication No. 2008 / 0039618 and Migawa et al., U.S. Pat. Application Publication No. 2015 / 0191727.

[0251] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by their isomeric configuration. For example, LNA nucleosides (described herein) can be in the α-L or β-D configuration. [ka]

[0252] α-L-methyleneoxy (4'-CH2-0-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that have shown antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In this specification, the general description of bicyclic nucleosides includes both isomeric configurations. When the position of a particular bicyclic nucleoside (e.g., LNA or cEt) is specified in the illustrated embodiments of this specification, they are in the β-D configuration unless otherwise specified.

[0253] In certain embodiments, the modified sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (eg, 5'-substituted and 4'-2' bridging sugars).

[0254] In some embodiments, the modified sugar moiety is a sugar surrogate.In some such embodiments, the oxygen atom of the sugar moiety is replaced with, for example, sulfur, carbon or nitrogen atom.In some such embodiments, such modified sugar moieties also include bridging and / or non-bridging substituents as described herein.For example, some sugar surrogates include substitutions at the 4'-sulfur atom and at the 2' position (see, for example, Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No. 7,939,677) and / or 5' position.

[0255] In some embodiments, the sugar surrogate comprises a ring with more than five atoms. For example, in some embodiments, the sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include hexitol nucleic acid ("HNA", anitol nucleic acid ("ANA"), mannitol nucleic acid ("MNA") (e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA: [ka] ("F-HNA", see, e.g., Swayze et al., U.S. Pat. No. 8,088,904; Swayze et al., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No. 8,796,437; and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran), and a compound of the formula: [ka] Nucleosides containing additional modified THPs having wherein, independently for each of the modified THP nucleosides, Bx is a nucleobase moiety, T3 and T4 are each independently an internucleoside linking group that links a modified THP nucleoside to the remainder of the oligonucleotide, or one of T3 and T4 is an internucleoside linking group that links a modified THP nucleoside to the remainder of the oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5'- or 3'-terminal group; q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are each independently H, C 1 ~C 6 Alkyl, substituted C 1 ~C 6 Alkyl, C 2 ~C 6 Alkenyl, substituted C 2 ~C 6 Alkenyl, C 2 ~C 6 Alkynyl or substituted C 2 ~C 6 alkynyl, R 1 and R 2 Each of is independently hydrogen, halogen, substituted or unsubstituted alkoxy, NJ 1 J 2 , S.J. 1 , N3 , OC(=X)J 1 ,OC(=X)NJ 1 J 2 , N.J. 3 C(=X)NJ 1 J 2 and CN, where X is O, S or NJ 1 and each J 1 , J 2 and J. 3 are independently H or C 1 ~C 6 Examples of the aryl group include, but are not limited to, alkyl.

[0256] In one embodiment, q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 and each is H. In one embodiment, q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 At least one of q is other than H. 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 At least one of R 1 and R 2 In one embodiment, a modified THP nucleoside is provided, wherein one of R 1 is F and R 2 is H, and in some embodiments, R 1 is methoxy and R 2 is H, and in some embodiments, R 1 is methoxyethoxy, and R 2 is H.

[0257] In some embodiments, sugar surrogates contain rings with more than 5 atoms and more than 1 heteroatom.For example, their use in nucleosides and oligonucleotides containing morpholino sugar moieties has been reported (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat. No. 5,034,506).As used herein, the term "morpholino" refers to a sugar surrogate having the following structure: [ka]

[0258] In certain embodiments, morpholinos can be modified, for example, by adding or altering various substituents from the morpholino structures shown above. Such sugar surrogates are referred to herein as "modified morpholinos."

[0259] In some embodiments, the sugar substitute comprises an acyclic moiety. Examples of nucleosides and oligonucleotides that comprise such acyclic sugar substitutes include, but are not limited to, peptide nucleic acids ("PNAs", acyclic butyl nucleic acids (see, for example, Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and the nucleosides and oligonucleotides described in Manoharan et al., International Publication No. WO 2011 / 133876.

[0260] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

[0261] The nucleoside residues of the oligonucleotides of the functional domain or cyclization domain can be coupled to each other by any of a number of known internucleoside linkages. Two major classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphates (also called unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S"), and phosphorodithioates ("HS-P=S"), containing a phosphodiester bond ("P=O"). Representative non-phosphorus-containing internucleoside linkages include methylenemethylimino (-CH 2 -N(CH 3 )-O-CH 2 -), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH 2 -O-); and N,N'-dimethylhydrazine (CH 2 -N(CH 3 )-N(CH 3 Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those of skill in the art.

[0262] Such internucleoside linkages include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages.In some embodiments, the synthetic antisense oligonucleotides of the present invention can include a combination of internucleoside linkages.In some embodiments, the synthetic antisense oligonucleotides of the present invention are a combination of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages.In some embodiments, more than half, but less than all, of the internucleoside linkages are phosphorothioate internucleoside linkages.In some embodiments, all of the internucleoside linkages are phosphorothioate internucleoside linkages.

[0263] Modified oligonucleotides comprising internucleoside linkages with chiral centers can be prepared as a population of modified oligonucleotides comprising sterically random internucleoside linkages or as a population of modified oligonucleotides comprising phosphorothioate linkages, particularly stereochemical configurations. In some embodiments, the population of modified oligonucleotides comprises phosphorothioate internucleoside linkages, and all of the phosphorothioate internucleoside linkages are sterically random. Such modified oligonucleotides can be produced using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nevertheless, as will be appreciated by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereochemical configuration. In some embodiments, the population of modified oligonucleotides is enriched in modified oligonucleotides comprising one or more specific phosphorothioate internucleoside linkages of specific independently selected stereochemical configurations.

[0264] In certain embodiments, the phosphorothioate linkages may be a mixture of Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either the Rp or Sp form. In embodiments where the linkages are a mixture of Rp and Sp enantiomers, the Rp and Sp forms may be at predetermined locations within the oligonucleotide or may be randomly distributed throughout the oligonucleotide.

[0265] As used herein, "nucleobase sequence" means the order of adjacent nucleobases in a nucleic acid or oligonucleotide, independent of any sugar or internucleoside linkage modifications.

[0266] As used herein, "nucleoside" refers to a compound that includes a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each independently unmodified or modified. As used herein, "modified nucleoside" refers to a nucleoside that includes a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include basic nucleosides that lack a nucleobase. "Linked nucleosides" are nucleosides that are linked in a contiguous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0267] As used herein, "oligomeric compound" refers to an oligonucleotide and, optionally, one or more additional features, such as a conjugate group or a terminal group.An oligomeric compound may or may not be paired with a second oligomeric compound that is complementary to the first oligomeric compound.A "single-stranded oligomeric compound" is an unpaired oligomeric compound.

[0268] As used herein, "oligonucleotide" means a chain of linked nucleosides connected via internucleoside linkages, where each nucleoside linkage and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 8 to 50 linked nucleosides.

[0269] As used herein, "modified oligonucleotide" refers to an oligonucleotide in which at least one nucleoside or internucleoside bond is modified. As used herein, "unmodified oligonucleotide" refers to an oligonucleotide that does not contain any nucleoside or internucleoside modification.

[0270] As used herein, " pharmaceutically acceptable carrier or diluent " means any substance suitable for use in administering to animals. Such a carrier allows pharmaceutical compositions to be formulated, for example, as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral ingestion by subjects. In some embodiments, the pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.

[0271] As used herein, "pharmaceutical acceptable salts" refers to physiologically and pharma- ceutical acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.

[0272] As used herein, "pharmaceutical composition" refers to a mixture of substances suitable for administration to a subject.For example, pharmaceutical composition can include antisense compound and sterile aqueous solution.In some embodiments, pharmaceutical composition shows activity in free uptake assay in some cell line.

[0273] As used herein, "phosphorus moiety" refers to a group of atoms that includes a phosphorus atom. In certain embodiments, the phosphorus moiety includes a mono-, di-, or tri-phosphate, or a phosphorothioate.

[0274] As used herein, "prodrug" refers to an extracorporeal form of a therapeutic agent that is converted to a different form within an animal or its cells. Typically, the conversion of a prodrug within an animal is facilitated by the action of enzymes (e.g., endogenous or viral enzymes) or chemicals present in the cells or tissues and / or physiological conditions.

[0275] As used herein, "OMe" means methoxy. "2'-OMe" means a 2'-OCH substituted for the 2'OH group of the ribosyl sugar moiety. 3 means a group.

[0276] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0277] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, the range of 1 to 50 is understood to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0278] As used herein, "reducing or inhibiting the amount or activity" refers to a decrease or blocking of transcriptional expression or activity compared to transcriptional expression or activity in an untreated or control sample, and does not necessarily indicate a complete elimination of transcriptional expression or activity.

[0279] As used herein, "self-complementary" with respect to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself.

[0280] As used herein, "standard cell assay" refers to the assay described in Example 1 and reasonable variations thereof.

[0281] As used herein, "stereorandom chiral center" in the context of a population of molecules of the same molecular formula refers to a chiral center that has random stereochemical configuration. For example, in a population of molecules that contain stereorandom chiral centers, the number of molecules with stereorandom chiral center (S) configuration can be, but is not necessarily, the same as the number of molecules with stereorandom chiral center (R) configuration. The stereochemical configuration of the chiral center is considered to be random when it is the result of a synthetic method that is not designed to control the stereochemical configuration. In some embodiments, the stereorandom chiral center is a stereorandom phosphorothioate internucleoside linkage.

[0282] As used herein, "sugar moiety" refers to an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" refers to a 2'-OH(H) furanosyl moiety as found in RNA (an "unmodified RNA sugar moiety"), or a 2'-H(H) moiety as found in DNA (an "unmodified DNA sugar moiety"). An unmodified sugar moiety has one hydrogen at each of the 3' and 4' positions, an oxygen at the 3' position, and two hydrogens at the 5' position. As used herein, "modified sugar moiety" or "modified sugar" refers to a modified furanosyl sugar moiety or sugar surrogate. As used herein, a modified furanosyl sugar moiety refers to a furanosyl sugar that includes a non-hydrogen substituent in place of at least one hydrogen of the unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic and non-bicyclic sugars.

[0283] As used herein, "sugar surrogate" refers to a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, a conjugate group, or a terminal group in an oligonucleotide. Modified nucleosides that contain sugar surrogates can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides can hybridize to complementary oligomeric compounds or nucleic acids.

[0284] As used herein, "target nucleic acid" and "target RNA" refer to a nucleic acid that an antisense compound is designed to affect.

[0285] As used herein, "target region" means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

[0286] As used herein, "terminal group" means a chemical group or group of atoms that is covalently attached to the end of an oligonucleotide.

[0287] As used herein, a "therapeutically effective amount" refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal. For example, a therapeutically effective amount ameliorates a symptom of a disease.

[0288] As used herein, "treat," "treatment," or "treating" refers to the administration of a compound described herein to effect an alteration or amelioration of a disease, disorder, or symptom.

[0289] By "portion" is meant a defined number of adjacent (i.e. linked) nucleobases of a nucleic acid. In some embodiments, a portion is a defined number of adjacent nucleobases of a target nucleic acid. In some embodiments, a portion is a defined number of adjacent nucleobases of an antisense compound.

[0290] The term "co-administration" or "co-administered" generally refers to the administration of at least two different substances. Co-administration refers to the simultaneous administration of at least two different substances in any order, either in a single dose or in separate doses, as well as in orders separated in time by up to several days.

[0291] The term "in combination" generally refers to the administration of the oligonucleotide-based compound according to the present invention and another agent useful for treating a disease or condition that does not lose the activity of the compound during the course of treating a patient. Such administration can be in any order, including simultaneous administration and time-separated order, which can be from a few seconds to a few days apart. Such combined treatment can also include two or more administrations of the compound according to the present invention and / or the other agent independently. The administration of the compound according to the present invention and the other agent can be by the same or different routes.

[0292] The term "individual" or "subject" or "patient" generally refers to a mammal, such as a human. The term "mammal" is expressly intended to include warm-blooded vertebrates, including, but not limited to, humans, non-human primates, rats, mice, cats, dogs, horses, cows, bovines, pigs, sheep, and rabbits. As used herein, "individual in need" refers to a human or non-human animal selected for treatment or therapy who is in need of such treatment or therapy. As used herein, "inhibition of expression or activity" refers to a reduction or blocking of RNA or protein expression or activity, and does not necessarily indicate complete elimination of expression or activity.

[0293] Working Example Synthesis of CSOs containing antisense oligonucleotide functional domains Circularly structured oligonucleotides according to the invention can be synthesized by procedures well known in the art, such as phosphoramidate or H-phosphonate chemistry, which can be carried out manually or by an automated synthesizer. For example, the oligonucleotides of the invention can be synthesized by linear synthesis approaches.

[0294] The compounds used herein are synthesized using phosphoramidite chemistry. These protocols are described in detail, for example, in pubs.rsc.org / en / content / chapter / bk9781788012096-00453 / 978-1-78801-209-6, which is incorporated herein by reference.

[0295] Melting temperature of the CSO of the present invention To clarify that the cyclization domain hybridizes with the 3'-end of the oligonucleotide of the functional domain, thereby forming a circular structure, 2 μM samples of the CSO compounds were mixed in 10 mM sodium phosphate, 100 nM sodium chloride buffer (pH 7.2). The samples were heated to 95 °C for 5 min and then slowly cooled to room temperature. The melting temperature (Tm) was measured after the samples were stored in a refrigerator overnight. The cooling and heating curves from 20 to 90 °C are shown in Table 6. [Table 6]

[0296] Inhibition of target RNA by CSOs containing antisense oligonucleotide functional domains Circularly structured oligonucleotides targeted to PCSK9 nucleic acid were designed and tested for their effect on PCSK9 mRNA in vitro.

[0297] Hepa 1-6 cells were cultured in DMEM medium + 10% FBS and 100U / ml Pen / Strep (cells from ATCC). Cells were seeded and incubated overnight to be approximately 70% confluent at the time of transfection (approximately 100,000 cells / ml / 12-well plate). Cell medium was replaced and 900μl was added to each well. Oligonucleotides were mixed with lipofectamine in Opti-MEM medium and added to lipids (1:1 ratio) and incubated for 15-20 min. 100μl was added to each well to give an antisense concentration of 100nM. After a treatment period of approximately 16-48 hours, cells were harvested for RNA and / or protein analysis.

[0298] Culture supernatants were assayed for AK release cytotoxicity assay. Taqman probes for mPCSK9 and PPIB or HPRT1 (housekeeping control) were used (probes provided by ThermoFisher). Results of each experiment are shown in Figure 5.

[0299] Circularly structured oligonucleotides targeted to the PNPLA3 nucleic acid were designed and tested for their effects on PNPLA3 mRNA in vitro.

[0300] Human HepG2 cells were cultured according to ATCC recommended conditions and media (Eagle's Minimum Essential Medium with 10% FBS). Cells were plated at a density of 50K / well in PDL-coated 96-well plates and reverse transfected with 0.6 μl / well of RNAiMax and the indicated concentrations of oligonucleotide compounds. After 24 hours, cells are harvested using Cells to CT lysis reagent (ThermoFisher 4391851C). RNA was reverse transcribed into cDNA template using RT reagent kit (ThermoFisher A39110). Quantitative PCR was performed using qPCR Master Mix (ThermoFisher 4444964). PNPLA3 expression levels are quantified using a PNPLA3-FAM probe (ThermoFisher 4351368 Assay ID: Hs00228747) and normalized to the housekeeping gene POLR2A (ThermoFisher, 4448491, Assay ID: Hs01108291). Data are analyzed with GraphPad Prism.

[0301] To examine whether circularization of linear ASOs with modified RNA in splitmer format could provide additional efficacy, we compared the levels of PNPLA3 knockdown using oligonucleotides with the same base sequence in different formats and chemical configurations: linear ASO without modified RNA ID 1521, linear gapmer 1523, linear splitmer 1527, 1528, 1529, and circular splitmer ASOs 1542, 1543, 1544, and 1545 at concentrations of 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM (Figure 6A). As shown in Figure 6B and Figure 6C, the first generation format linear ASO 1521 was not active even at higher concentrations of 200 nM, the linear gapmer ASO 1523 was mildly active and could achieve approximately 50% knockout with an IC50 of 252 nM, the linear splitmer format ASOs 1527, 1528, and 1529 could achieve better knockdown activity than the gapmer format, and all the circularized splitmer ASOs (CSOs 1542, 1543, 1544, and 1545) were the most effective at reducing PNPLA3 mRNA levels 24 hours post-transfection.

[0302] Generation of immune responses by CSOs containing immunostimulatory oligonucleotide functional domains Mouse splenocyte restimulation assay Spleens from C57BL / 6J mice were mechanically dispersed into single cell suspensions and red blood cells were removed with ammonium chloride lysis buffer (catalog no. 420302, BioLegend, San Diego, CA). Cell viability was determined using vital dye staining and an automated cell counting system (Countess 3, Thermo Fisher, Waltham, MA). After counting, 1 × 10 5Viable mouse splenocytes were seeded into each well of a 96-well flat-bottom sterile tissue culture-treated plate (catalog no. 3596, Corning, Glendale, AZ) in RPMI 1640 (catalog no. A1049101, Thermo Fisher) containing 10% fetal bovine serum (catalog no. F2442, MilliporeSigma, Burlington, MA).

[0303] Immunostimulatory oligonucleotide (ISO) agonists for Toll-like receptor 9 (TLR9) and CSOs containing TLR9 immunostimulatory oligonucleotide (ISO) functional domains were synthesized using standard methodologies by Syngenis (Bentley, Australia) and provided as lyophilized preparations, which were then reconstituted in annealing buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 7.5) using a heat block incubation step (95°C, 5 min). After annealing, sample concentrations were determined using an absorbance method for oligonucleotide concentration determination using a NanoDrop spectrophotometer (Thermo Fisher). Control TLR9 agonists were obtained from Invivogen (San Diego, CA). The indicated doses of CSOs or control agonists were added to the indicated wells at the indicated concentrations, and cells were stimulated for 24 hours in a 37°C, 5% CO2 incubator. After the incubation period, cell culture supernatants were collected and analyzed using a custom multiplex cytokine / chemokine assay kit (U-PLEX Custom Biomarker Assay, Catalog No. K15069M-2, Meso Scale Diagnostics, Rockville, MD), data collection was performed on an MSD S600 Bioanalyzer (Meso Scale), and raw data analysis was performed on MSD Discovery Workbench (Meso Scale). The results are shown in Figures 7 to 12.

[0304] To assess the activity of CSOs containing immune antagonist oligonucleotide functional domains, such CSOs were incubated alone or with TLR9 agonists and cytokines were assessed 24 hours later.

[0305] The results are shown in Figures 13 and 14.

[0306] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A circularly structured oligonucleotide (CSO) comprising: a) a functional domain comprising an antisense oligonucleotide of 17-25 nucleotides in length, said antisense oligonucleotide comprising at least 12 consecutive nucleobases complementary to equal length portions of a target RNA sequence, and said antisense oligonucleotide comprising: i) a 3' domain comprising 10 to 12 deoxyribonucleotides, and ii) a 5' domain contiguous with the 3' domain, the 5' domain comprising at least four modified nucleotides, each of which is independently a modified deoxyribonucleotide or a modified ribonucleotide, wherein the modified nucleotides prevent cleavage by RNase H at the 5' domain. a functional domain comprising b) a cyclization domain that is complementary to the sequence of nucleotides in the functional domain and is of opposite polarity to the sequence of nucleotides in the functional domain; and c) a linker connecting the functional domain and the cyclization domain at the 5' end.

2. A CSO as described in claim 1, wherein the linker is a direct bond.

3. A CSO described in claim 1 or 2, wherein the cyclization domain hybridizes to a sequence of nucleotides in the 3' domain of the oligonucleotide of the functional domain, thereby forming a cyclic structure.

4. A CSO described in claim 1 or 2, wherein the 3' domain is 12 nucleotides in length and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3' end.

5. A CSO described in claim 1 or 2, wherein the 3' domain is 11 nucleotides in length and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3' end.

6. A CSO described in claim 1 or 2, wherein the 3' domain is 10 nucleotides in length and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

7. A CSO described in claim 1 or 2, wherein the 5' domain comprises modified nucleotides including a modified internucleotide bond, or a modification to the sugar, heterocyclic base, or a combination thereof.

8. A CSO described in claim 1 or 2, wherein at least four modified nucleotides in the 5' domain include modified ribonucleotides.

9. The CSO described in claim 8, wherein the modified ribonucleotide comprises a 2'-substituted ribonucleotide.

10. The CSO of claim 9, wherein the 2'-substituted ribonucleotide is a 2'-OMe ribonucleotide or a 2'MOE ribonucleotide.

11. A CSO described in claim 1 or 2, wherein each nucleotide in the 5' domain is independently a modified deoxyribonucleotide or a modified ribonucleotide.

12. A CSO as described in claim 11, wherein the nucleotides of the 5' domain are modified ribonucleotides.

13. The CSO described in claim 12, wherein the modified ribonucleotide comprises a 2'-substituted ribonucleotide.

14. The CSO of claim 13, wherein the 2'-substituted ribonucleotide comprises a 2'-OMe ribonucleotide or a 2'MOE ribonucleotide.

15. A CSO described in claim 1 or 2, wherein one or more conjugate groups are attached to the CSO.

16. The CSO described in claim 15, wherein the conjugate group is GalNAc or an antibody.

17. A pharmaceutical composition comprising the CSO of claim 1 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, further comprising one or more agents selected from the group consisting of small molecules, peptides, vaccines, antigens, antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonists, chemotherapeutic agents, targeted therapeutic agents, activated cells, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and costimulatory molecules, or combinations thereof.

19. Use of a CSO according to claim 1 or 2 or a pharmaceutical composition according to claim 17 for the manufacture of a medicament for inhibiting gene expression in a subject in need thereof.

20. Use of a CSO according to claim 1 or 2 or a pharmaceutical composition according to claim 17 for the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.

21. A CSO described in claim 1 or 2 or a pharmaceutical composition described in claim 17 for inhibiting gene expression in a subject in need thereof.

22. A CSO according to claim 1 or 2 or a pharmaceutical composition according to claim 17 for treating a disease or disorder in a subject in need thereof.

Images