Cyclic antisense therapeutic drugs

Abstract

The present invention provides oligonucleotides called cyclic structured oligonucleotides ("CSOs") comprising a functional domain, a cyclization domain, and a linker segment as described herein, compositions containing the same, and methods of using the same. This design of the cyclic oligonucleotide maintains its cyclic form until it is in the presence of target RNA and hybridizes with the target RNA.

Description

Background Art

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 453,893, filed Mar. 22, 2023, and U.S. Provisional Patent Application No. 63 / 454,235, filed Mar. 23, 2023. The entire teachings of the above applications are incorporated herein by reference.

[0002] The processing and translation of target RNA can be regulated by antisense oligonucleotides through multiple mechanisms. These include cleavage of target RNA by RNase - H, regulation of aberrant splicing, enhancement of target RNA processing and translation, inhibition of translation by steric hindrance, etc. The target RNA may be mRNA or non - coding RNA. In other mechanisms, the antisense strand of double - stranded siRNA can be incorporated into AGO and inhibit translation by the siRNA mechanism. In other mechanisms such as ADAR - or CRISPR - based models, the antisense can edit RNA or DNA and thereby regulate translation and processing.

[0003] Over the years, it has been found that antisense hybridization and affinity affect the selectivity of target RNA. Furthermore, nuclease stability is important for using antisense nucleic acids as drugs, which has been achieved by modification of internucleotide linkages, such as phosphorothioate.

[0004] It was hypothesized that nuclease stability was important for efficacy, and since antisense degradation was shown to occur from the 3' end, efforts were focused on modifying the 3' end to slow down degradation. These designs included capping the 3' end, forming a hairpin loop at the 3' end, creating oligomers with secondary structures containing 3'-3' linkages, or attaching two antisense oligonucleotides to their 3' ends. 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 DNA and RNA containing phosphorothioate and 2'-substituted RNA have been studied as antisense agents, providing different characteristics. Phosphorothioate-type antisense DNA activates RNase H when hybridized to RNA, while antisense RNA or 2'-substituted RNA binds to RNA with higher affinity and does not activate RNase H. To further improve antisense properties, mixtures of these two modifications have been used in antisense, generally referred to as hybrid or gapmer-type antisense. In most of the studied gapmer-type antisense, the modified RNA segments are located at both the 3' and 5' ends, while the DNA is located in the middle. Gapmer-type antisense is the most widely studied antisense, and drugs utilizing this chemistry have been approved and are in clinical development.

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

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

[0008] Despite the progress made, there is still a need to develop antisense oligonucleotides with improved properties for use as therapeutic agents and in diagnostic applications. [Overview of the Initiative]

[0009] The present invention provides a structural class of oligonucleotides referred to herein as “cyclic structured oligonucleotides” (CSOs) or similarly, “cyclic oligos.” In a CSO, two oligonucleotides are linked to each other (directly or via a linker segment), the first oligonucleotide of the CSO being called the “functional domain” and providing a function to the CSO (for example, the functional segment may be an antisense oligonucleotide or an immunostimulant oligonucleotide), and the second oligonucleotide being called the “cyclization domain” and containing a nucleotide sequence complementary to a portion of the functional domain (for example, Figures 1A-1C). Preferably, the cyclization domain is complementary to the terminal nucleotide of the functional domain. Preferably, the cyclization domain is complementary to an equal-length portion of the terminal nucleotide of the functional domain.

[0010] CSOs adopt an intramolecular cyclic structure, forming an intramolecular double helix as a result of complementarity between the functional domain and the cyclization domain. This formation of the intramolecular double helix alters both the shape of the functional domain and the reachability of oligonucleotides to their ends. This structure combines properties crucial 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 interaction with PRRs and enabling endosomal escape. Upon entering the cytoplasm or nucleus, the cyclic structure opens in the presence of the target RNA because the affinity between the functional domain and the target RNA sequence is higher than the affinity between the functional domain and the cyclization domain. When the CSO is in its intramolecular cyclic form, there is less exposure of phosphorothioate bonds, which may result in fewer polyanion-related side effects known to occur with PS-oligonucleotides (e.g., complement activation and prolongation of partial thromboplastin time). Additionally, the CSO appears to have reduced protein binding.

[0012] The CSO according to the present invention can be prepared using standard techniques for synthesizing constituent oligonucleotides and is useful for all purposes where functional oligonucleotides and nucleic acids are useful.

[0013] The foregoing merely summarizes specific aspects of the present invention and is not intended to limit the invention in any way, nor should it be construed as limiting the invention. All patents, patent applications, and other publications listed herein are incorporated by reference in their entirety.

[0014] The aforementioned and other objects, features and advantages of the present invention will become apparent from the following more detailed description of preferred embodiments of the invention, as shown in the accompanying drawings, where similar reference numerals in different figures refer to the same parts. The drawings are not necessarily to scale and instead focus on illustrating the principles of the invention. [Brief explanation of the drawing]

[0015] [Figure 1A] This diagram illustrates various embodiments of the cyclic structured oligonucleotide according to the invention. Dashed lines represent the cyclization domain. Solid lines represent the functional domain. L represents direct or linker-mediated linkage between the functional domain and the cyclization domain. This diagram shows that the cyclic structured oligonucleotide maintains its cyclic form in the presence of target RNA and hybridizes with the target RNA. In this embodiment, the functional domain is an antisense oligonucleotide complementary to the target RNA. The cyclic structured oligonucleotide can act by various mechanisms of action depending on the properties of the oligonucleotide in the functional domain, as will be further described herein. [Figure 1B]This figure shows various embodiments of the cyclic structured oligonucleotide according to the present invention. Dashed lines represent the cyclization domain. Solid lines represent the functional domain. L represents a direct or linker-mediated linkage between the functional domain and the cyclization domain. This figure shows one embodiment of the CSO according to the present invention, in which the oligonucleotide of the functional domain is the splitmer described herein. [Figure 1C] This figure shows various embodiments of the cyclic structured oligonucleotide according to the present invention. Dashed lines represent the cyclization domain. Solid lines represent the functional domain. L represents a direct or linker-mediated linkage between the functional domain and the cyclization domain. This figure shows one embodiment of the CSO according to the present invention, in which the oligonucleotide of the functional domain is the splicing oligonucleotide described herein. [Figure 2] This figure shows the delivery of the CSO according to the present invention, in which the functional domain oligonucleotide is a gene regulatory oligonucleotide, and its release from the endosome into the cytoplasm. [Figure 3A] This graph shows the knockdown of angiopoietin-like protein 3 (ANGPTL3) in Hep3B cells. [Figure 3B] This table shows the knockdown of angiopoietin-like protein 3 (ANGPTL3) in Hep3B cells. The underlined segments of the control gapmer-type oligonucleotides represent the 2'-OME nucleotide. [Figure 4A] This graph shows the knockdown of apolipoprotein B (ApoB) in Hep3B cells. [Figure 4B] This table shows the knockdown of apolipoprotein B (ApoB) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 5A] This graph shows the knockdown of apolipoprotein C-III (APOC3) in Hep3B cells. [Figure 5B]This table shows the knockdown of apolipoprotein C-III (APOC3) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 6A] This graph shows the knockdown of diacylglycerol O-acyltransferase 2 (DGAT2) in Hep3B cells. [Figure 6B] This table shows the knockdown of diacylglycerol O-acyltransferase 2 (DGAT2) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 7A] This graph shows the knockdown of kallikrein B1 (KLKB1) in Hep3B cells. [Figure 7B] This table shows the knockdown of kallikrein B1 (KLKB1) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 8A] This graph shows the knockdown of the progenitor protein convertase subtilisin / kexin type 9 (PCSK9) in Hep3B cells. [Figure 8B] This table shows the knockdown of the progenitor protein convertase subtilisin / kexin type 9 (PCSK9) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 9A] This graph shows the knockdown of protein-tyrosine phosphatase 1B (PTP1B) in Hep3B cells. [Figure 9B] This table shows the knockdown of protein-tyrosine phosphatase 1B (PTP1B) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 10A]This graph shows signal transduction and the knockdown of activator 3 (STAT3) in Hep3B cells. [Figure 10B] This table shows the signal transduction and activator 3 (STAT3) knockdown in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 11A] This graph shows the knockdown of transthyretin (TTR) in Hep3B cells. [Figure 11B] This table shows the knockdown of transthyretin (TTR) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 12A] This graph shows the knockdown of superoxide dismutase 1 (SOD1) in Hep3B cells. [Figure 12B] This table shows the knockdown of superoxide dismutase 1 (SOD1) in Hep3B cells. [Figure 13A] This graph shows the knockdown of huntingtin (HTT) in U-251 MG cells. [Figure 13B] This table shows the knockdown of huntingtin (HTT) in U-251 MG cells. In the control gapmer-type oligonucleotides, "e" represents the 2'-MOE nucleotide, and "*" represents the phosphorothioate nucleotide bond. [Figure 14A] This graph shows the knockdown of huntingtin (HTT) in U-251 MG cells. [Figure 14B] This table shows the knockdown of huntingtin (HTT) in U-251 MG cells. In the control gapmer-type oligonucleotides, "e" represents the 2'-MOE nucleotide, and "*" represents the phosphorothioate nucleotide bond. [Figure 15A] This graph shows the knockdown of microtubule-associated tau protein (MAPT) in U-251 MG cells. [Figure 15B] This table shows the knockdown of microtubule-associated tau protein (MAPT) in U-251 MG cells. In the control gapmer-type oligonucleotides, "e" represents a 2'-MOE nucleotide, and "*" represents a phosphorothioate nucleotide bond. [Figure 16A] This graph shows the knockdown of superoxide dismutase 1 (SOD1) in U-251 MG cells. [Figure 16B] This table shows the knockdown of superoxide dismutase 1 (SOD1) in U-251 MG cells. In the control gapmer-type oligonucleotides, "e" represents the 2'-MOE nucleotide, and "*" represents the phosphorothioate nucleotide bond. [Figure 17A] This graph shows the knockdown of apolipoprotein C-III (APOC3) in Hep3B cells. [Figure 17B] This table shows the knockdown of apolipoprotein C-III (APOC3) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Figure 18A] This graph shows the knockdown of apolipoprotein C-III (APOC3) in Hep3B cells. [Figure 18B] This table shows the knockdown of apolipoprotein C-III (APOC3) in Hep3B cells. The underlined segments of the control gapmer oligonucleotides represent the 2'-OME nucleotide. [Modes for carrying out the invention]

[0016] Please understand that the general explanation above and the detailed explanation below are merely illustrative and descriptive, and not limiting.

[0017] In this specification, unless otherwise specified, the use of the singular form includes the plural form. Where used herein, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limited. Also, terms such as "element" or "component" include both elements and components containing one unit and elements and components containing multiple subunits, unless otherwise specified.

[0018] The headings of sections used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference with respect to parts and all of the documents described herein.

[0019] Features may be described herein as part of the same or different aspects or embodiments of the present invention for clarity and concise explanation. Those skilled in the art will understand that the scope of the present invention may include embodiments having all or some combinations of features described herein as part of the same or different embodiments.

[0020] Through this disclosure, various aspects of the present invention can be presented in the form of ranges. It should be understood that descriptions in the form of ranges are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of the invention. Therefore, a range description should be considered to specifically disclose all possible subranges, as well as the individual numerical values ​​within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, as well as the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0021] Where used herein, the term “about” is to be understood by those skilled in the art and varies to some extent depending on the context in which it is used. Where used herein when referring to measurable values ​​such as quantity, duration, etc., the term “about” means to include variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, such variations being suitable for performing the disclosed method.

[0022] The present invention provides oligonucleotides called cyclic structured oligonucleotides ("CSOs") comprising 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.

[0023] In several embodiments, the cyclization domain is bound to the functional domain at its 5' end by a 5'-5' bond. In this configuration, the cyclization domain hybridizes with the 3' end of the oligonucleotide of the functional domain, thereby forming a cyclic structure (e.g., Figures 1A-1C). This design of the cyclic oligonucleotide maintains its cyclic form in the presence of the target RNA until the functional domain hybridizes with the target RNA. This structure allows for increased specificity.

[0024] In several embodiments, the cyclization domain is attached to the functional domain at its 3' end by a 3'-3' bond. In this configuration, the cyclization domain hybridizes with the 5' end of the oligonucleotide of the functional domain, thereby forming a cyclic structure.

[0025] The functional domain provides the desired function to the CSO. For example, for gene expression regulation, the oligonucleotide of the functional domain is complementary to the target RNA.

[0026] In several embodiments, the oligonucleotides of the functional domain and the cyclization domain are DNA, RNA, or a combination thereof. In several embodiments, the oligonucleotides of the functional domain are DNA, RNA, or a combination thereof. In several embodiments, the oligonucleotides of the cyclization domain are DNA, RNA, or a combination thereof.

[0027] In multiple embodiments, the oligonucleotides of the functional domain and / or cyclization domain are unmodified. In multiple embodiments, the oligonucleotides of the functional domain are unmodified. In multiple embodiments, the oligonucleotides of the cyclization domain are unmodified. In multiple embodiments, the oligonucleotides of the functional domain and cyclization domain are unmodified.

[0028] In several embodiments, at least one nucleotide of the oligonucleotide in the functional domain and / or cyclization domain is modified. In several embodiments, two or more nucleotides of the oligonucleotide in the functional domain and / or cyclization domain are modified.

[0029] In several embodiments, the functional domain oligonucleotide is modified. In several embodiments, the functional domain oligonucleotide includes modifications of internucleotide bonds, sugars, heterocyclic bases, or combinations thereof. These modifications can also be appropriately positioned at specific locations within the functional domain oligonucleotide. Other chemistry and modifications are readily available in accordance with this disclosure and are known in the field of oligonucleotides, and are encompassed by the term “modified” as used herein in the context of oligonucleotides. Where used herein, the terms “functional domain oligonucleotide” and “functional domain” are interchangeable.

[0030] In several embodiments, the functional domain contains oligonucleotides with a nucleotide length of 15 to 50. In several embodiments, the functional domain contains oligonucleotides with a nucleotide length of 17 to 40. In several embodiments, the functional domain contains oligonucleotides with a nucleotide length of 17 to 25. In several embodiments, the oligonucleotides of the functional domain have a nucleotide length of 17 to 22.

[0031] In several embodiments, the oligonucleotides of the functional domain have a nucleotide length of 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. In several embodiments, the functional domain has a nucleotide length of 17. In several embodiments, the functional domain has a nucleotide length of 18. In several embodiments, the functional domain has a nucleotide length of 19. In several embodiments, the functional domain has a nucleotide length of 20. In several embodiments, the functional domain has a nucleotide length of 21. In several embodiments, the functional domain has a nucleotide length of 22. In several embodiments, the functional domain has a nucleotide length of 23. In several embodiments, the functional domain has a nucleotide length of 24. In several embodiments, the functional domain has a nucleotide length of 25. In several embodiments, the functional domain has a nucleotide length of 26. In multiple embodiments, the functional domain has a nucleotide length of 27. In multiple embodiments, the functional domain has a nucleotide length of 28. In multiple embodiments, the functional domain has a nucleotide length of 29. In multiple embodiments, the functional domain has a nucleotide length of 30. In multiple embodiments, the functional domain has a nucleotide length of 31. In multiple embodiments, the functional domain has a nucleotide length of 32. In multiple embodiments, the functional domain has a nucleotide length of 33. In multiple embodiments, the functional domain has a nucleotide length of 34. In multiple embodiments, the functional domain has a nucleotide length of 35. In multiple embodiments, the functional domain has a nucleotide length of 36. In multiple embodiments, the functional domain has a nucleotide length of 37. In multiple embodiments, the functional domain has a nucleotide length of 38. In multiple embodiments, the functional domain has a nucleotide length of 39. In multiple embodiments, the functional domain has a nucleotide length of 40.

[0032] In several embodiments, the functional domain includes, but is not limited to, oligonucleotides selected from antisense oligonucleotides, microRNAs (miRNAs), siRNAs, piRNAs, hnRNAs, ncRNAs, snRNAs, sgRNAs, esiRNAs, shRNAs, lncRNAs, CRISPR-based systems, RNA-specific adenosine deaminase (ADAR: Adenosine Deaminase acting on RNA) systems, or splicing oligonucleotides. In several embodiments, the functional domain includes, but is not limited to, oligonucleotides selected from immunostimulant oligonucleotides or immunoinhibitory oligonucleotides, also known as immune antagonist oligonucleotides.

[0033] The only limitations on the nucleotide and internucleotide bonding of oligonucleotides in the functional domain are that they do not preclude (a) the ability of the cyclizing domain to hybridize with the functional domain of the CSO under desired conditions to form a double helix, 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 with a complementary RNA segment under physiological conditions to form a double helix, which is a substrate of RNase H). Preferred nucleotide and internucleotide bonding enhances the stability of the CSO against nuclease and other forms of chemical degradation and / or enhances the ability of the functional domain to perform its intended function.

[0034] In several embodiments, the nucleotide-nucleotide linkages of the functional domain are phosphorothioate nucleotide linkages, phosphorodithioate nucleotide linkages, phosphodiester nucleotide linkages, or combinations thereof.

[0035] The oligonucleotides in the cyclization domain are complementary to the nucleotide sequence in the functional domain and have opposite polarity to the nucleotide sequence in the functional domain to which they are complementary.

[0036] In several embodiments, the oligonucleotide of the cyclization domain is modified. In several embodiments, the oligonucleotide of the cyclization domain includes modifications of internucleotide bonds, sugars, heterocyclic bases, or combinations 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” and “cyclization domain” are used interchangeably.

[0037] In several embodiments, the nucleotide-nucleotide bonds of the cyclization domain are phosphorothioate nucleotide bonds, phosphodiester nucleotide bonds, or a combination thereof.

[0038] In several embodiments, the cyclization domain contains oligonucleotides with a nucleotide length of 4 to 30. In several embodiments, the cyclization domain contains oligonucleotides with a nucleotide length of 4 to 25. In several embodiments, the cyclization domain contains oligonucleotides with a nucleotide length of 4 to 12. In several embodiments, the cyclization domain contains oligonucleotides with a nucleotide length of 4 to 10. In several embodiments, the cyclization domain contains oligonucleotides with a nucleotide length of 4 to 8. In several embodiments, the cyclization domain contains oligonucleotides with a nucleotide length of 4 to 6.

[0039] In several embodiments, the cyclization domain contains oligonucleotides with a nucleotide length of 5 to 8. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 4, 5, 6, 7, 8, 9, or 10. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 4. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 5. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 6. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 7. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 8. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 9. In several embodiments, the oligonucleotides of the cyclization domain have a nucleotide length of 10.

[0040] As used herein, the term “polarity” refers to the concept of orientation in the primary structure (e.g., 3'→5' and 5'→3' for DNA and RNA, or N-terminus→C-terminus (or vice versa) for PNA). If the CSO of the present invention comprises oligonucleotides that hybridize in an antiparallel manner by Watson-Crick base pairing, for example, the cyclization domain is in a 5'→3' (or 2') configuration, and the sequence of nucleotides to which it is complementary in the functional domain is in a 3' (or 2')→5' configuration. Changes in polarity in the CSO can occur anywhere in the CSO other than the cyclization domain and the sequence of nucleotides in the functional domain to which the cyclization domain is complementary. In preferred embodiments in which the CSO comprises oligonucleotides, the functional domain is in a 3'→5' configuration and the cyclization domain is in a 5'→3' configuration, so 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, so that the functional domain and the cyclization domain are linked via a 3'-3' bond.

[0041] In several embodiments, the functional domain and the cyclization domain are covalently bonded to each other via a linker. In several embodiments, the linker segment is a direct bond, a nucleotide or an oligonucleotide with a length of 2 to 5 nucleotides, or other chemical moieties, or a combination thereof. In some embodiments, the linker segment may be cleavable.

[0042] The only limitation on the linker segment is that it does not preclude the essential functions of the CSO, namely (a) the CSO's ability to form an intramolecular cyclic structure under desired conditions (e.g., physiological conditions), and (b) the ability of the functional domain to perform its intended function. Preferred “other chemical moieties” linkers include, but are not limited to, C2-C6 alkyl groups, ethylene glycol, tri(ethylene glycol), tetra(ethylene glycol), penta(ethylene glycol), hexa(ethylene glycol), and -NH(CH2). n Examples include NH- (wherein n is 2, 3, 4, 5, or 6). Alternatively, the linker segment may be a combination of the above. In preferred embodiments, the linker is a direct bond, in which case the functional domain and the cyclization domain are directly bonded. In one embodiment, the linker is ethylene glycol. In several embodiments, the linker is a C2-C6 alkyl. In several embodiments, the linker is a C2 alkyl. In several embodiments, the linker is a C3 alkyl. In several embodiments, the linker is a C4 alkyl. In several embodiments, the linker is a C5 alkyl. In several embodiments, the linker is a C6 alkyl.

[0043] In several embodiments, the linker is a branched linker, a fatty acid, or a lipid.

[0044] In the CSOs described herein, the oligonucleotide of the functional domain has a terminal and a linker terminal. As the name suggests, the linker terminal is the end of the oligonucleotide linked to the cyclization domain via a linker segment. Generally, CSOs are constructed such that the terminal of the functional domain forms a double helix with the cyclization domain, i.e., the cyclization domain is complementary to the terminal of the functional domain.

[0045] In multiple embodiments, the nucleotides of the cyclization domain are at least 90% complementary to a portion of the oligonucleotides of the functional domain over their entire length. In multiple embodiments, the nucleotides of the cyclization domain are at least 95% complementary to a portion of the oligonucleotides of the functional domain over their entire length. In multiple embodiments, the nucleotides of the cyclization domain are at least 97% complementary to a portion of the oligonucleotides of the functional domain over their entire length. In multiple embodiments, the nucleotides of the cyclization domain are at least 98% complementary to a portion of the oligonucleotides of the functional domain over their entire length. In multiple embodiments, the nucleotides of the cyclization domain are at least 99% complementary to a portion of the oligonucleotides of the functional domain over their entire length. In multiple embodiments, the nucleotides of the cyclization domain are at least 100% complementary to a portion of the oligonucleotides of the functional domain over their entire length.

[0046] If all the bases in at least one strand of a pair of nucleic acids are found on the opposite side of 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 at positions opposite any other base other than its complementary base pair, that base is considered a "mismatch", and the strand is considered to be partially complementary. Thus, a strand can be partially complementary to varying degrees (e.g., 0% < x < 100% complementary) until the bases become misaligned such that they are non-complementary (e.g., 0% complementary). As will be readily understood and recognized by those skilled in the art, overall complementarity (i.e., complete, 100% complementarity) is not required for hybridization of nucleic acid strands (e.g., oligonucleotides, antisense, or others).

[0047] In multiple embodiments, the target RNA can be mRNA, pre-mRNA, ncRNA, lncRNA, or microRNA. In multiple embodiments, the target RNA is mRNA.

[0048] In multiple embodiments, the cyclic structured oligonucleotide according to the present invention is part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0049] A pharmaceutical composition comprising the cyclic structured oligonucleotide of the present invention may further comprise any other agent or treatment useful for treating or preventing a disease or condition, provided that it does not diminish the gene expression regulatory effect of the cyclic 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 therapies), 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 containing peptides or modified amino acids), or combinations thereof. Alternatively, the cyclic structured oligonucleotides according to the present invention may be administered in combination with other compounds (for example, formulated with lipids or liposomes and conjugated to peptides, antibodies, or small molecules) to enhance the specificity or strength of gene expression regulation of the cyclic structured oligonucleotides according to the present invention.

[0050] In several embodiments, the oligonucleotide of the functional domain contains at least one phosphorothioate internucleotide bond. In several embodiments, at least half of the internucleotide bond is phosphorothioate. In several embodiments, all of the internucleotide bond is phosphorothioate.

[0051] In several embodiments, the oligonucleotide of the functional domain is single-stranded.

[0052] In multiple embodiments, the functional domain oligonucleotide is at least 90% complementary to a portion of the target RNA over its entire length. In multiple embodiments, the functional domain oligonucleotide is at least 95% complementary to a portion of the target RNA over its entire length. In multiple embodiments, the functional domain oligonucleotide is at least 97% complementary to a portion of the target RNA over its entire length. In multiple embodiments, the functional domain oligonucleotide is at least 98% complementary to a portion of the target RNA over its entire length. In multiple embodiments, the functional domain oligonucleotide is at least 99% complementary to a portion of the target RNA over its entire length. In multiple embodiments, the functional domain oligonucleotide is at least 100% complementary to a portion of the target RNA over its entire length.

[0053] Functional domain In various embodiments, the functional domain of CSO is an oligonucleotide as further described below. The cyclization domain and linker of CSO are as described above unless otherwise specified.

[0054] Inhibition of gene expression In several embodiments, the present invention provides a cyclic 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 with a nucleotide length of 15 to 45 and is complementary to a target RNA, the cyclization domain comprises an oligonucleotide with a nucleotide length of 4 to 12 and is at least 90% complementary to the nucleotide sequence in the functional domain and has the opposite polarity to the nucleotide sequence in the complementary functional domain, and the functional domain comprises a gene regulatory oligonucleotide. In several embodiments, the oligonucleotide of the functional domain is modified.

[0055] In several embodiments, the gene-regulating oligonucleotides of the functional domain (i.e., oligonucleotides capable of regulating the expression of a target gene) include, but are not limited to, antisense oligonucleotides, microRNAs (miRNAs), piRNAs, hnRNAs, ncRNAs, snRNAs, sgRNAs, esiRNAs, shRNAs, or lncRNAs.

[0056] In several embodiments, the gene-regulating oligonucleotide of the functional domain is an antisense oligonucleotide. In several embodiments, the modification of the antisense oligonucleotide includes at least one modified nucleic acid base, sugar, and / or internucleotide bond.

[0057] As will be recognized by those skilled in the art, the CSOs of the present invention can be used to improve prior art oligonucleotides. This disclosure provides improvements to the prior art by applying the CSOs described herein to previously reported oligonucleotide sequences, thereby providing improved compositions of previously reported oligonucleotides that may be useful for gene silencing.

[0058] Examples of previously reported antisense oligonucleotides useful as functional domains within cyclic structured oligonucleotides (CSOs) according to the present invention include, but are not limited to, the antisense oligonucleotides listed in Table 1. [Table 1]

[0059] In several embodiments, the antisense compound of a functional domain may have a defined identity percentage with respect to a specific nucleotide sequence of the Sequence ID provided herein (e.g., Tables 1, 2, and 4) or a portion thereof. When used herein, an antisense compound is identical to the sequence disclosed in Table 1 if it has the same nucleic acid base-pairing ability. For example, an RNA containing uracil instead of thymidine in the disclosed DNA sequence is considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shortened and extended forms of the antisense compounds described herein, as well as compounds having non-identical bases compared to the antisense compounds provided herein, are also contemplated. Non-identical bases may be adjacent to each other or may be dispersed throughout the antisense compound. The identity percentage of an antisense compound is calculated according to the number of bases that have identical base pairs with respect to the sequence it is being compared to.

[0060] In certain embodiments, the antisense compound or a portion thereof in a functional domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more antisense compounds or SEQ ID NOs disclosed herein, or a portion thereof.

[0061] As shown herein, the CSO of the present invention, comprising an antisense oligonucleotide linked to a cyclization domain via a 5'-5' linkage, surprisingly exhibited high potency. This observation contradicted the previous hypothesis that increased stability (e.g., 3'-3' linked oligonucleotides) would lead to increased potency. Furthermore, the absence of the 5'-terminus allows the antisense oligonucleotide of the CSO to suppress inflammation. This design allows the antisense oligonucleotide to unfold into a linear structure and be active in cells expressing the target RNA.

[0062] When a CSO is an antisense oligonucleotide, it remains in a circular form until it is present with a complementary target RNA and takes on a linear form to bind to the target RNA. The change from circular to linear form has been confirmed by studies of thermal fusion and RNase H cleavage. In the linear form, the functional domain hybridizes with the complementary target RNA (at least under physiological conditions) to form a double helix. This double helix 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 double helix is ​​cleaved by RNase H, thereby inhibiting expression.

[0063] CSOs containing antisense oligonucleotide functional domains maintain their activity in cell cultures. Expected advantages of these CSOs include reduced interaction with non-target macromolecules (including nucleic acids and proteins) due to the formation of an intramolecular cyclic structure, decreased polyanion-related side effects, and linearization in the presence of target genes or RNA alone. Furthermore, due to their cyclic structure, these CSOs can escape from endosomes due to the lack of 5'-terminal interaction.

[0064] The oligonucleotides of the present invention are isolated oligonucleotides. The term “isolated” means modified or removed from their natural state by human intervention. For example, oligonucleotides naturally present in living animals are not “isolated,” but synthetic oligonucleotides, or oligonucleotides partially or completely separated from their naturally occurring coexisting substances, are “isolated.” Isolated oligonucleotides may exist in a substantially purified form or in a non-natural environment, such as a cell to which the oligonucleotide is delivered. The oligonucleotides of the present invention may include partially purified DNA and / or RNA, substantially pure DNA and / or RNA, synthetic DNA and / or RNA, or DNA and / or RNA produced by recombinant 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 may include the addition of non-nucleotide substances to the terminal of the oligonucleotide or to one or more internal nucleotides of the oligonucleotide, including modifications that make the oligonucleotide resistant to nuclease digestion.

[0065] The terms "microRNA," "miRNA," and "MiR" are interchangeable and refer to endogenous or artificial non-coding RNAs that can regulate gene expression. miRNAs are thought to function through RNA interference. The design of such microRNAs is within the scope of the art of the art.

[0066] The terms "piRNA" and "Piwi-interacting RNA" are interchangeable and refer to a class of small RNA molecules involved in gene silencing. piRNA molecules are typically 26–31 nucleotides long. The design of such piRNAs is within the scope of the art of the art.

[0067] Splitmer In several embodiments, the chemical composition of the functional domain antisense oligonucleotide is described in International Publication No. 2020 / 191177, which is incorporated in its entirety herein by reference. In several embodiments, the functional domain antisense oligonucleotide is a modified oligonucleotide comprising or consisting of an antisense oligonucleotide compound having a nucleotide length of 17 to 25, the antisense oligonucleotide compound comprising a 3' domain and a 5' domain adjacent to the 3' domain, the 3' domain beginning with a 3' terminal nucleotide and having a nucleotide length of 10 to 12, each nucleotide comprising a deoxyribonucleotide and a phosphodiester nucleotide interbonding or a phosphorothioate nucleotide interbonding or a combination thereof, the 5' domain beginning with the first nucleotide following the 3' domain and continuing to a 5' terminal nucleotide, the 5' domain comprising an unmodified deoxyribonucleotide, an unmodified ribonucleotide, a modified deoxyribonucleotide, a modified ribonucleotide, or a combination thereof, provided that the 5' domain comprises at least one modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or a modified skeleton. Such antisense oligonucleotides are called "splitmers."

[0068] In several embodiments, the antisense oligonucleotide is an oligonucleotide selected from Table 1.

[0069] In several embodiments, the modified deoxyribonucleotides and / or modified ribonucleotides in the 5' domain do not need to be consecutive.

[0070] In several embodiments, the 5' domain-modified deoxyribonucleotide and / or modified ribonucleotide prevents RNase H cleavage at the 5' domain.

[0071] In several embodiments, the modified deoxyribonucleotide or modified ribonucleotide comprises a modified base, a modified sugar, and / or a modified skeleton.

[0072] In several embodiments, at least half of the nucleotides in the 5' domain include a modified deoxyribonucleotide or a modified ribonucleotide containing a modified sugar and / or a modified skeleton.

[0073] If fewer nucleotides are modified than the total number of nucleotides in the 5' domain, the modified and unmodified nucleotides are arranged such that two or fewer unmodified nucleotides are adjacent to each other in the 5' domain. For example, modified and unmodified nucleotides can alternate individually or in pairs within the 5' domain.

[0074] In several embodiments, all nucleotides in the 5' domain include modified deoxyribonucleotides or modified ribonucleotides containing modified sugars and / or modified skeletons.

[0075] In several embodiments, the functional domain splitmer comprises 17 to 25 ligated nucleotides having at least 12 consecutive nucleic acid bases complementary to equal-length portions of the target RNA.

[0076] In several embodiments, the modified ribonucleotide of the splitmer comprises a 2'-substituted nucleotide as described herein. In several embodiments, the 2'-substituted nucleotide is selected from 2'O-methyl ribonucleotide (2'-OME) or 2'-methoxyethyl ribonucleoside (2'-MOE).

[0077] In some embodiments, the 3' domain contains 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 contains 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 contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

[0078] In some embodiments, when the 3' domain of the antisense oligonucleotide is 12 nucleotides in length, the antisense oligonucleotide of the present invention has the formula (I): 5'-N m N 14 N 13 N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1-3' (wherein, N is any nucleotide, N 13 ~N m comprises the 5' domain, N1~N 12 comprises the 3' domain, and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11).

[0079] In some embodiments, when the 3' domain of the antisense oligonucleotide is 11 nucleotides in length, the antisense oligonucleotide of the present invention has the formula (Ia): 5'-N m N 14 N 13 N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1-3' (wherein, N is any nucleotide, N 12 ~N mThis includes the 5' domain, N1~N 11 It includes the 3' domain, and m is represented by (selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11).

[0080] In some embodiments, the antisense oligonucleotide of the present invention is of formula (Ib): 5'-N m N 14 N 13 N 12 N 11 N 10 N9N8N7N6N5N4N3N2N1-3' (In the formula, N is any nucleotide, N 11 ~N m This includes the 5' domain, N1~N 10 It includes the 3' domain, and m is represented by (selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11).

[0081] 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.

[0082] In several embodiments, the functional domain splitmer antisense oligonucleotide compound has a nucleotide length of 17 to 25 nucleotides and contains at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target RNA sequence, the antisense oligonucleotide compound comprises a 3' domain and a 5' domain adjacent to the 3' domain, the 3' domain starting with a 3' terminal nucleotide and having a nucleotide length of 10 to 12 nucleotides, and each nucleotide contains a deoxyribonucleotide and a phosphodiester or phosphothioate nucleotide interbond or a combination thereof. The 5' domain begins with the first nucleotide following the 3' domain and extends to the terminal nucleotide at the 5' end, and the 5' domain comprises an unmodified deoxyribonucleotide, an unmodified ribonucleotide, a modified deoxyribonucleotide, a modified ribonucleotide, or a combination thereof, provided that the 5' domain comprises at least one modified deoxyribonucleotide or modified ribonucleotide containing a modified sugar and / or a modified skeleton.

[0083] In several embodiments, the antisense oligonucleotide is an oligonucleotide selected from Table 1.

[0084] In several embodiments, the 5' domain-modified deoxyribonucleotide and / or modified ribonucleotide prevents RNase H cleavage in the 5' domain. In several embodiments, the modified deoxyribonucleotide or modified ribonucleotide includes a modified base, a modified sugar, and / or a modified skeleton. In several embodiments, the modified deoxyribonucleotide or modified ribonucleotide includes a modified sugar and / or a modified skeleton.

[0085] In several embodiments, at least half of the nucleotides in the 5' domain include a modified deoxyribonucleotide or a modified ribonucleotide containing a modified sugar and / or a modified skeleton.

[0086] If fewer nucleotides are modified than the total number of nucleotides in the 5' domain, the modified and unmodified nucleotides are arranged such that two or fewer unmodified nucleotides in the 5' domain are adjacent to each other.

[0087] In several embodiments, all nucleotides in the 5' domain include modified deoxyribonucleotides or modified ribonucleotides containing modified sugars and / or modified skeletons. In several embodiments, at least half of the nucleotides in the 5' domain include modified ribonucleotides containing modified sugars and / or modified skeletons.

[0088] In several embodiments, the 3' domain has a nucleotide length of 12 and contains 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 several embodiments, the 3' domain has a nucleotide length of 11 and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3' end. In several embodiments, the 3' domain has a nucleotide length of 12 and contains nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3' end.

[0089] In several embodiments, the nucleotides of the 3' domain contain native nucleic acid bases. In some embodiments, the nucleic acid bases and sugars of the nucleotides of the 3' domain of the antisense oligonucleotide according to the present invention are unmodified. In this respect, the nucleic acid bases and sugars of the nucleotides of the 3' domain of the antisense oligonucleotide according to the present invention are native. Each of the nucleotides of the 3' domain contains a deoxyribonucleotide and a phosphodiester nucleotide bond or a phosphorothioate nucleotide bond or a combination thereof. The nucleotides of the 3' domain contain a native deoxyribose sugar bonded to a phosphorothioate, phosphodiester, or other phosphate, or a combination thereof, and are known to activate RNase H.

[0090] In several embodiments, at least one of the nucleotides in the 3' domain contains a modified nucleic acid base.

[0091] In multiple embodiments, the nucleotides at positions 9 or 10 from the 3' end are unmodified. In multiple embodiments, the nucleotides at positions 9 and 10 from the 3' end are unmodified. In multiple embodiments, the nucleotides at positions 11 from the 3' end are unmodified. In multiple embodiments, the nucleotides at positions 9, 10, and 11 from the 3' end are unmodified. In multiple embodiments, the nucleotides at positions 12 from the 3' end are unmodified. In multiple embodiments, the nucleotides at positions 9, 10, 11, and 12 from the 3' end are unmodified.

[0092] In several embodiments, the oligonucleotide contains at least one phosphorothioate internucleotide bond. In several embodiments, at least half of the internucleotide bond is phosphorothioate. In several embodiments, all of the internucleotide bond is phosphorothioate.

[0093] In several embodiments, at least half of the internucleotide bonds are phosphodiesters. In several embodiments, all of the internucleotide bonds are phosphodiesters.

[0094] In several embodiments, the antisense oligonucleotide is single-stranded.

[0095] As used herein, the term “5' domain” refers to the nucleotides beginning with the first nucleotide following the 3' domain and extending toward the 5' end. The 5' domain hybridizes to the target RNA, but RNase H cannot cleave the target RNA within this domain. The term “5' domain” is generally 2–15 nucleotides long and, depending on the length of the 3' domain, refers to the 11th through 25th nucleotides (the 1st nucleotide being the 3' end), the 12th through 25th nucleotides, or the 13th through 25th nucleotides of the antisense oligonucleotide, measured from the 3' end.

[0096] For example, an antisense oligonucleotide compound with a nucleotide length of 21 may contain a 3' domain from position 1 to 12 and a 5' domain from position 13 to 21. The designation of modified nucleotides is position-specific, in contrast to nucleotide-specific.

[0097] The 5' domain contains a nucleotide having non-RNase H activating modifications, such as a modified sugar and / or a modified skeleton, which do not activate RNase H. In some embodiments, the 5' domain contains a nucleotide containing a modified sugar. In some embodiments, the 5' domain contains a nucleotide containing a modified skeleton. In some embodiments, the 5' domain contains a nucleotide containing both a modified sugar and a modified skeleton. In several embodiments, the modified skeleton is a non-phosphorus skeleton.

[0098] This antisense design allows for targeted RNA cleavage at a specific site toward the 5' end of the 3' domain. In multiple embodiments, the antisense design enables targeted RNA cleavage at the 9th, 10th, 11th, or 12th nucleotide position from the 3' end. In multiple embodiments, the antisense design enables targeted RNA cleavage at the 9th nucleotide position from the 3' end. In multiple embodiments, the antisense design enables targeted RNA cleavage at the 10th nucleotide position from the 3' end. In multiple embodiments, the antisense design enables targeted RNA cleavage at the 11th nucleotide position from the 3' end. In multiple embodiments, the antisense design enables targeted RNA cleavage at the 12th nucleotide position from the 3' end.

[0099] In any of these embodiments, at least half of the nucleotides in the 5' domain are intended to include modifications or substitutions of the skeleton and / or modifications or substitutions of sugars. In some embodiments, all nucleotides at positions within the 5' domain include modifications or substitutions of the skeleton and / or modifications or substitutions of sugars. In one embodiment, the 5' domain includes at least two nucleotides containing a modified skeleton and / or modified sugars. In one embodiment, the 5' domain includes at least three nucleotides containing a modified skeleton and / or modified sugars. In one embodiment, the 5' domain includes at least four nucleotides containing a modified skeleton and / or modified sugars. In one embodiment, the 5' domain includes at least five nucleotides containing a modified skeleton and / or modified sugars. In one embodiment, the 5' domain includes at least six nucleotides containing a modified skeleton and / or modified sugars. In one embodiment, the 5' domain includes at least seven nucleotides containing a modified skeleton and / or modified sugars. In one embodiment, the 5' domain includes at least eight nucleotides containing a modified skeleton and / or modified sugars. In one embodiment, all nucleotides in the 5' domain are nucleotides containing a modified skeleton and / or modified sugars.

[0100] The embodiments discussed herein are particularly intended to be implemented with respect to the position relative to the 3' end in the context of specific nucleotides and positions. 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.

[0101] In several embodiments, the antisense oligonucleotide of the functional domain is at least 90% complementary to a portion of the target RNA over its entire length.

[0102] Examples of split-mer antisense oligonucleotides useful as functional domains within cyclic structured oligonucleotides (CSOs) according to the present invention include, but are not limited to, the antisense oligonucleotides listed in Table 2. Unless otherwise specified, internucleotide bonds are phosphorothioate bonds. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0103] GCAT-DNA phosphorothioate linkage, G1 / C1 / A1 / T1 / U1-2'OME phosphorothioate linkage, A2 / T2 / C2 / G2-DNA phosphodiester linkage, G3 / C3 / A3 / T3 / U3-2'MOE phosphorothioate linkage, G4 / C4 / A4 / T4 / U4-2'MOE phosphodiester linkage. Lowercase g / c / a / t- indicates phosphodiester DNA.

[0104] Examples of useful cyclization domains in the cyclic oligonucleotides described herein include, but are not limited to, the cyclization domains listed in Table 3. Table 3 also provides functional domains (compound numbers(or more)) that are (some of) complementary to the cyclization domains. The nucleotide bonds in the oligonucleotides of Table 3 are phosphorothioate nucleotide bonds, phosphodiester nucleotide bonds, or combinations thereof. [Table 3-1] [Table 3-2]

[0105] Examples of cyclic structured oligonucleotides (CSOs) useful for gene silencing include, but are not limited to, the cyclic structured oligonucleotides listed in Table 4. All internucleotide bonds are phosphorothioate bonds unless otherwise specified. The sequence numbers for the cyclization domains and functional domains of the compounds shown below can be found in Tables 2 and 3 above. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0106] GCAT-DNA phosphorothioate linkage, G1 / C1 / A1 / T1 / U1-2'OME phosphorothioate linkage, A2 / T2 / C2 / G2-DNA phosphodiester linkage, G3 / C3 / A3 / T3 / U3-2'MOE phosphorothioate linkage, G4 / C4 / A4 / T4 / U4-2'MOE phosphodiester linkage, G5 / C5 / A5 / T5 / U5-2'OME phosphorothioate linkage, or 2'MOE phosphorothioate linkage; lowercase g / c / a / t- indicates phosphodiester DNA.

[0107] Pharmaceutical composition In certain embodiments, the pharmaceutical composition described herein comprises one or more CSO compounds of the present invention. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises sterile saline and one or more CSO compounds. In certain embodiments, the pharmaceutical composition consists of sterile saline and one or more CSO compounds. In certain embodiments, the sterile saline is pharmaceutical-grade saline. In certain embodiments, the pharmaceutical composition comprises one or more CSO compounds and sterile water. In certain embodiments, the pharmaceutical composition consists of one CSO compound and sterile water. In certain embodiments, the sterile water is pharmaceutical-grade water. In certain embodiments, the pharmaceutical composition comprises one or more CSO compounds and phosphate-buffered saline (PBS). In certain embodiments, the pharmaceutical composition consists of one or more CSO compounds and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical-grade PBS.

[0108] In certain embodiments, the pharmaceutical composition comprises one or more CSO compounds and one or more excipients. In certain embodiments, the excipients are selected from water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0109] In certain embodiments, the CSO compounds of the present invention may be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for formulating pharmaceutical compositions depend on many criteria, including the route of administration, the severity of the disease, or the dose administered, but are not limited to these.

[0110] conjugate group In certain embodiments, the CSO according to the present invention optionally further comprises one or more conjugate groups. The conjugate group consists of one or more conjugate moieties and a conjugate linker that links one or more conjugate moieties to an oligonucleotide. The conjugate group may be bonded to one or both ends and / or any internal position of the oligonucleotide. In certain embodiments, the conjugate group is bonded to the 2' position of the nucleoside of the modified oligonucleotide. In certain embodiments, the conjugate group bonded to one or both ends of the oligonucleotide is a terminal group. In certain such embodiments, the conjugate group or terminal group is bonded to the 3' and / or 5' ends of the oligonucleotide. In certain such embodiments, the conjugate group is bonded to the 3' end of the oligonucleotide. In certain embodiments, the conjugate group is bonded near the 3' end of the oligonucleotide. In certain embodiments, the conjugate group is bonded to the 5' end of the oligonucleotide. In certain embodiments, the conjugate group is bonded near the 5' end of the oligonucleotide.

[0111] In any embodiment described herein, the conjugate group comprises a GalNAc cluster containing one to three GalNAc ligands.

[0112] In any embodiment described herein, the conjugate linker consists of a single bond.

[0113] In any embodiment described herein, the conjugate linker is detachable.

[0114] In any embodiment described herein, the conjugate linker comprises one to three linker-nucleosides.

[0115] In any embodiment of this specification, the conjugate group is bound to the CSO at the 5' end of the functional domain. In any embodiment of this specification, the conjugate group is bound to the CSO at the 3' end of the functional domain. In any embodiment of this specification, the conjugate group is bound to the CSO at the 5' end of the cyclization domain. In any embodiment of this specification, the conjugate group is bound to the CSO at the 3' end of the cyclization domain.

[0116] In certain embodiments, the CSO is covalently bonded to one or more conjugate groups. In certain embodiments, the conjugate groups modify one or more properties of the CSO, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance. In certain embodiments, the conjugate groups confer new properties to the CSO, such as a fluorophore or reporter group that enables the detection of oligonucleotides. Specific conjugate groups and conjugate moieties, e.g., cholesterol moiety (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, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NYA Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992,20,533-538), aliphatic chains, e.g., 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, e.g., 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 Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chain (Manoharan et al.Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetate, palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc cluster (e.g., International Publication No. 2014 / 179620) have been previously described.

[0117] The conjugate portion includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin portions, polyethylene glycol, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid portions, folates, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, fluorophores, and pigments.

[0118] The conjugate portion also includes an antibody. In several embodiments, the antibody is a transferrin receptor (TFR) antibody. The use of the antibody conjugate can play a role in the delivery and / or cell targeting of the CPN of the present invention.

[0119] In certain embodiments, the conjugate portion includes an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethicine, barbiturate, cephalosporin, sulfonamide, antidiabetic agent, antibacterial agent, or antibiotic.

[0120] The conjugate moiety is bonded to the CSO via a conjugate linker. In certain embodiments, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is directly bonded to the CSO via a single bond). In certain embodiments, the conjugate linker comprises a chain structure such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, a nucleoside, or an amino acid unit.

[0121] In certain embodiments, the conjugate linker comprises one or more groups selected from alkyl groups, amino groups, oxo groups, amide groups, disulfide groups, polyethylene glycol groups, ether groups, thioether groups, and hydroxylamino groups. In certain such embodiments, the conjugate linker comprises groups selected from alkyl groups, amino groups, oxo groups, amide groups, and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl groups and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl groups 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.

[0122] In certain embodiments, conjugate linkers, including the above-described conjugate linker, are known in the art to be useful for conjugating a conjugate group to a difunctional linkage, such as a parent compound, like an oligonucleotide provided herein. Generally, a difunctional linkage includes at least two functional groups. One functional group is selected to bond to a specific site on the CSO, and the other is selected to bond to a conjugate group. Examples of functional groups used in a difunctional linkage include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, the difunctional linkage includes one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl groups.

[0123] 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, but are not limited to, substituted or unsubstituted C1-C11. 10 Alkyl, substituted, or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Examples of alkynyl substituents include hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl substituents. A non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl substituents.

[0124] In certain embodiments, the conjugate linker contains 1 to 10 linker-nucleosides. In certain embodiments, the conjugate linker contains 2 to 5 linker-nucleosides. In certain embodiments, the conjugate linker contains exactly 3 linker-nucleosides. In certain embodiments, the conjugate linker contains a TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments, such linker-nucleosides contain a modified sugar moiety. In certain embodiments, the linker-nucleosides are unmodified. In certain embodiments, the linker-nucleosides contain optionally protected heterocyclic bases selected from purines, substituted purines, pyrimidines, or substituted pyrimidines. In certain embodiments, the cleavable portion 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 typically desirable that the linker-nucleoside be cleaved from the oligomer compound after reaching the target tissue. Thus, the linker-nucleosides are typically linked to each other and to the rest of the oligomer compound via cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0125] As used herein, linker nucleosides are not generally considered part of the CSO, or in particular part of the cyclization domain or functional domain. Therefore, the nucleotides of linker nucleosides are not counted against the length of the CSO or its domains and are not used in determining the complementarity percentage of oligonucleotides to the reference nucleic acid. Unless otherwise indicated, a conjugate linker contains 10 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 5 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 3 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 2 or fewer linker nucleosides. In certain embodiments, a conjugate linker contains 1 or fewer linker nucleosides.

[0126] In certain embodiments, it is desirable that the conjugate group be cleaved from the CSO. For example, in certain circumstances, a CSO containing a particular conjugate moiety is better taken up by a particular cell type, but once the CSO is taken up, it is desirable that the conjugate group be cleaved to release the unconjugated CSO or the parent CSO. Therefore, a particular conjugate linker may contain one or more cleavable moieties. In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety is an atomic group containing at least one cleavable bond. In certain embodiments, the cleavable moiety includes an atomic group having one, two, three, four, or more than four cleavable bonds. In certain embodiments, the cleavable moiety is selectively cleaved within a cell or an intracellular compartment such as a lysosome. In certain embodiments, the cleavable moiety is selectively cleaved by an endogenous enzyme such as a nuclease.

[0127] In certain embodiments, the cleavable bond is selected from amides, esters, ethers, one or both phosphodiesters, phosphate esters, carbamates, or disulfides. In certain embodiments, the cleavable bond is one or both phosphodiesters. In certain embodiments, the cleavable moiety includes phosphate or a phosphodiester. In certain embodiments, the cleavable moiety is a phosphate bond between the oligonucleotide and the conjugate moiety or conjugate group.

[0128] use Cyclically structured oligonucleotides may be useful as antisense oligonucleotides for mRNA, ncRNA, microRNA, lncRNA, and splicing, or for delivering them (e.g., to a target or target environment (e.g., the cytoplasm)). Furthermore, the cyclic structure can provide a novel approach to delivering antisense to disrupt RNA structure and increase translation. For example, if the functional domain is an antisense oligonucleotide, the CSO according to the present invention is also useful in therapeutic approaches where inhibition of gene expression is desired. This may include, for example, inhibition of endogenous genes (e.g., oncogenes) or exogenous genes (e.g., genes essential for pathogen growth and / or metabolism).

[0129] In several embodiments, the present invention provides a method for inhibiting gene expression, comprising administering a cyclic structured oligonucleotide or a composition comprising a cyclic structured oligonucleotide as described herein.

[0130] In several embodiments, the present invention provides a method for inhibiting allele-specific gene expression, comprising administering a cyclic structured oligonucleotide or a composition comprising a cyclic structured oligonucleotide as described herein.

[0131] The methods according to the present invention are useful for treating subjects with a disease or disorder, and inhibiting gene expression is beneficial. In several embodiments, the disease or disorder is caused by abnormal expression or products of cellular genes.

[0132] In several embodiments, the cyclic structured oligonucleotide or composition comprising the cyclic structured oligonucleotide described herein is administered topically.

[0133] In several embodiments, the cyclic structured oligonucleotide or composition comprising the cyclic structured oligonucleotide described herein is administered systemically.

[0134] In several embodiments, the present invention provides a method for treating a disease or disorder of interest, wherein modulating RNA processing is beneficial for treating the subject, and the method comprises administering a cyclic structured oligonucleotide or a composition comprising a cyclic structured oligonucleotide as described herein.

[0135] In several embodiments, the present invention provides a method for inducing nonsense mutation-dependent degradation of a target RNA, comprising administering a cyclic structured oligonucleotide or a composition comprising a cyclic structured oligonucleotide as described herein.

[0136] The cyclic structured oligonucleotides of the present invention can be administered alone or in combination with any other agent or treatment. The agent or treatment can be administered concurrently or in combination. Such agents or treatments may be useful for treating or preventing a disease or condition and do not diminish the gene expression regulatory 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, peptide 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 therapies), 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 modified amino acids), or combinations thereof. Alternatively, the cyclic oligonucleotide according to the present invention may be administered in combination with other compounds (e.g., lipids or liposomes) to enhance the specificity or intensity of gene expression regulation of the cyclic oligonucleotide according to the present invention.

[0137] The cyclic oligonucleotides of the present invention may be administered by any suitable route, including but not limited to parenteral, transmucosal, oral, sublingual, transdermal, topical, inhalation, intratumoral, intravenous, subcutaneous, intrathecal, intranasal, aerosol, intraocular, intratracheal, rectal, vaginal, gene gun, skin patch, or in the form of eye drops or mouthwashes. In any of the methods according to the present invention, the cyclic oligonucleotides of the present invention may be administered directly to tissues or organs, such as the bladder, liver, lungs, or kidneys, either alone or in combination with any other agent, but not limited to these cases. In certain embodiments, the administration of the cyclic oligonucleotides of the present invention, either alone or in combination with any other agent, is by intramuscular administration. In certain embodiments, the administration of the antisense oligonucleotides of the present invention, either alone or in combination with any other agent, is by mucosal administration. In certain embodiments, the administration of the antisense oligonucleotides of the present invention, either alone or in combination with any other agent, is by oral administration. In certain embodiments, the administration of the antisense oligonucleotides of the present invention, either alone or in combination with any other agent, is by rectal administration. In certain embodiments, the administration of the antisense oligonucleotides of the present invention, either alone or in combination with any other agent, is by intrathecal administration. In certain embodiments, the antisense oligonucleotides according to the present invention are administered alone or in combination with any other agent by intratumoral administration. In certain embodiments, the antisense oligonucleotides according to the present invention are administered alone or in combination with any other agent by parenteral administration. In certain embodiments, the antisense oligonucleotides according to the present invention are administered alone or in combination with any other agent by subcutaneous administration.

[0138] In several embodiments, any of the cyclic structured oligonucleotides described herein can be conjugated with a moiety that provides site-specific delivery of a CSO. In several embodiments, such conjugates include, but are not limited to, antibodies, peptides, lipids, or small molecules.

[0139] In several embodiments, any of the cyclic structured oligonucleotides described herein can be encapsulated in a portion that provides site-specific delivery of the CSO. In several embodiments, the CSO can be encapsulated in, for example, lipids, lipid nanoparticles (LNPs), or macrocyclic peptide structures.

[0140] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, physiological saline, fixative oil, polyethylene glycol, or glycerin; propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate; and agents for adjusting isotonicity such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Administration of antisense oligonucleotides according to the present invention can be carried out by known procedures using effective amounts and durations that are effective in reducing the symptoms or surrogate markers of the disease. For example, an effective amount of antisense oligonucleotides according to the present invention for treating a disease and / or disorder may be the amount necessary to alleviate or reduce symptoms, or to delay or improve tumors, cancer, or bacterial, viral, or fungal infections. In relation to the administration of a composition that modulates gene expression, the effective amount of the antisense oligonucleotide according to the present invention is sufficient to achieve the desired modulation compared to gene expression in the absence of the antisense oligonucleotide according to the present invention. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the specific oligonucleotide 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 requiring excessive experimentation.

[0141] synthesis The CSOs described herein can be prepared by any suitable method recognized in the art, including but not limited to H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H-phosphonate chemistry in some cycles and phosphoramidite chemistry in other cycles), which can be done manually or by an automated synthesizer. The oligonucleotides of the present invention can also be modified in several ways without impairing their ability to hybridize to their targets (see, for example, Agrawal and Gait, Advances in Nucleic Acid Therapeutics, (2019) https: / / doi.org / 10.1039 / 9781788015714).

[0142] definition Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry described herein are well known and commonly used in the art. Where permitted, all patents, applications, published applications, and other publications and other data referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0143] Unless otherwise specified, the following terms have the following meanings: As used herein, “2'-deoxynucleoside” means a nucleoside containing a 2'-H(H) furanosyl sugar moiety found in naturally occurring deoxyribonucleic acid (DNA). In certain embodiments, the 2'-deoxynucleoside may contain a modified nucleic acid base or an RNA nucleic acid base (uracil).

[0144] As used herein, “2'-substituted nucleoside” means a nucleoside containing a 2'-substituted sugar moiety. As used herein, with respect to the sugar moiety, “2'-substituted” means a sugar moiety containing at least one 2'-substituent other than H or OH.

[0145] As used herein, "5-methylcytosine" refers to cytosine modified by the attachment of a methyl group at the 5-position. 5-methylcytosine is a modified nucleic acid base.

[0146] As used herein, the singular forms "a," "an," and "the" refer to both singular and plural objects unless otherwise explicitly indicated by the context.

[0147] As used herein, “administer” means to supply a drug to an animal.

[0148] As used herein, “animal” means human or non-human animal.

[0149] As used herein, “individual in need of it” refers to a human or non-human animal selected for treatment or therapy that requires such treatment or therapy.

[0150] As used herein, “antisense activity” means any detectable and / or measurable change resulting from the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of the target nucleic acid or the protein encoded by the target nucleic acid relative to the target nucleic acid level or target protein level in the absence of the antisense compound. In certain embodiments, antisense activity is an increase in the amount or expression of the target nucleic acid or the protein encoded by the target nucleic acid relative to the target nucleic acid level or target protein level in the absence of the antisense compound.

[0151] As used herein, “antisense compound” means an oligomeric compound capable of achieving at least one antisense activity.

[0152] As used herein, “improvement” in relation to treatment means improvement of at least one symptom compared to the same symptom in the absence of treatment. In certain embodiments, improvement is a reduction in the severity or frequency of a symptom, or a delay in the onset of a symptom or a slowing of its progression in terms of severity or frequency. In certain embodiments, the symptoms or features are ataxia, neuropathy, and aggregate formation. In certain embodiments, improvement of these symptoms results in improved motor function, a reduction in neuropathy, or a reduction in the number of aggregates.

[0153] As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside containing a bicyclic sugar moiety. As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety containing two rings, the second ring being formed via a bridge connecting two atoms in the first ring, thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not contain a furanosyl moiety.

[0154] As used herein, “chiral-enriched population” means multiple molecules of the same molecular formula where, if a particular chiral center is sterically random, the number or proportion of molecules in the population containing a particular stereochemistry at a particular chiral center is greater than the number or proportion of molecules in the population that would be expected to contain the same particular stereochemistry at the same particular chiral center. A chiral-enriched population of molecules having multiple chiral centers in each molecule may contain one or more sterically random chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound containing a modified oligonucleotide.

[0155] As used herein, “cleavable portion” means a bond or group of atoms that is cleaved under physiological conditions, for example, within a cell, animal, or human.

[0156] As used herein, the term “complementary” refers to a pair of nucleic acid bases (or simply “bases”) that, under selected (e.g., physiological) conditions, preferentially bond to each other with hydrogen than other heterocyclic bases (or the degree of complementarity that may be required in context when evaluating the “complementarity” of an oligonucleotide). If the nucleic acid bases are modified or unmodified, natural or synthetic purines and pyrimidines, the term “complementary” means complementary in the Watson-Crick sense. In some embodiments, if the nucleic acid base sequences of an oligonucleotide and the nucleic acid base sequences of another nucleic acid are aligned in opposite directions, at least 70% of the nucleic acid bases of the oligonucleotide or one or more regions and the nucleic acid bases of the other nucleic acid or one or more regions can bond to each other with hydrogen. Complementary nucleic acid bases refer to nucleic acid bases that can form hydrogen bonds with each other.

[0157] Complementary nucleic acid base pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to have nucleic acid base complementarity at each nucleoside; rather, mismatches are acceptable. As used herein, “perfectly complementary” or “100% complementary” with respect to an oligonucleotide means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

[0158] As used herein, “conjugate group” means an atomic group that is directly or indirectly bonded to an oligonucleotide. A conjugate group comprises a conjugate moiety and a conjugate linker that bonds the conjugate moiety to an oligonucleotide.

[0159] As used herein, “conjugate linker” means a group of atoms containing at least one bond that connects a conjugate portion to an oligonucleotide.

[0160] As used herein, “conjugate moiety” means the group of atoms bonded to an oligonucleotide via a conjugate linker.

[0161] As used herein, “consecutive” in the context of oligonucleotides refers to nucleosides, nucleic acid bases, sugar moieties, or internucleoside bonds that are directly adjacent to each other. For example, “consecutive nucleic acid bases” means nucleic acid bases that are directly adjacent to each other in a sequence.

[0162] As used herein, “linker-nucleoside” means a nucleoside that directly or indirectly links the CSO of the present invention to the conjugate portion. The linker-nucleoside is located within the conjugate linker and is not considered part of the CSO compound even if it is continuous with the CSO.

[0163] As used herein, “gapmer” means a modified oligonucleotide comprising an internal region having multiple nucleosides supporting RNase H cleavage, located between external regions having one or more nucleosides, wherein the nucleosides constituting the internal region are chemically distinct from the one or more nucleosides constituting the external region. The internal region may be referred to as the “gap,” and the external region as the “wing.” Unless otherwise indicated, “gapmer” refers to a sugar motif. Unless otherwise indicated, the sugar portion of the nucleoside in the gap of a gapmer is unmodified 2'-deoxyfuranosyl. Thus, the term “MOE gapmer” refers to a gapmer having a sugar motif of 2'-MOE nucleosides and a gap of 2'-deoxynucleosides in both wings. Unless otherwise indicated, a MOE gapmer may contain one or more modified internucleoside bonds and / or modified nucleic acid bases, the modifications not necessarily following a gapmer pattern of sugar modifications.

[0164] As used herein, “hotspot region” is a range of nucleic acid bases on a target nucleic acid suitable for an oligomeric compound to reduce the amount or activity of the target nucleic acid, as demonstrated in the following examples herein.

[0165] As used herein, “hybridization” means the pairing or annealing of complementary oligonucleotides and / or nucleic acids. While not limited to specific mechanisms, the most common mechanisms of hybridization involve hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between complementary nucleic acid bases.

[0166] As used herein, the term “nucleoside bond” refers to a covalent bond between adjacent nucleosides in an oligonucleotide. As used herein, “modified nucleoside bond” refers to any nucleoside bond other than a phosphodiester nucleoside bond. A “phosphothioate bond” is a modified nucleoside bond in which one of the non-bridged oxygen atoms of a phosphodiester nucleoside bond is replaced with a sulfur atom.

[0167] As used herein, the term "inhibition of expression or activity" refers to a reduction or blockage of expression or activity compared to the expression of activity in an untreated or control sample, and does not necessarily mean complete elimination of expression or activity.

[0168] As used herein, “linker-nucleoside” means a nucleoside that directly or indirectly links an oligonucleotide to a conjugate moiety. The linker-nucleoside is located within the conjugate linker of the oligomeric compound. The linker-nucleoside is not considered part of the oligonucleotide moiety of the oligomeric compound, even if it is contiguous with the oligonucleotide.

[0169] As used herein, “non-bicyclic modified sugar moiety” means a modified sugar moiety that includes a substituent, for example, a modification that does not form a bridge between two atoms of the sugar to form a second ring.

[0170] As used herein, “mismatch” or “non-complementary” means that, when the first oligomer compound and the second oligomer compound are aligned, the nucleic acid bases of the first oligonucleotide are not complementary to the corresponding nucleic acid bases of the second oligonucleotide or target nucleic acid.

[0171] As used herein, "MOE" means methoxyethyl. "2'-MOE" means the 2'-OCH2CH2OCH3 group in place of the 2'OH group in the ribosyl sugar moiety.

[0172] As used herein, “motif” means a pattern of unmodified and / or modified sugar moieties, nucleic acid bases, and / or nucleoside bonds in an oligonucleotide.

[0173] As used herein, "mRNA" means an RNA transcript that codes for a protein and includes pre-mRNA and mature mRNA, unless otherwise specified.

[0174] As used herein, “nucleic acid base” means either an unmodified nucleic acid base or a modified nucleic acid base. As used herein, “unmodified nucleic acid base” refers to adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). As used herein, “modified nucleic acid base” refers to an atomic group other than unmodified A, T, C, U, or G that can pair with at least one unmodified nucleic acid base. “5-methylcytosine” is an example of a modified nucleic acid base. A universal base is a modified nucleic acid base that can pair with any one of the five unmodified nucleic acid bases. Modified bases, also called heterocyclic base moieties, include other nucleic acid bases such as 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, as well as other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and Examples include thymine, 5-uracil (pseudracil), 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 cytosine), 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0175] In certain embodiments, modified nucleic acid bases are selected from universal bases, hydrophobic bases, indiscriminate bases, size-extended bases, and fluorinated bases as defined herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine and 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, and other alkynyl derivatives of pyrimidine bases. - Azouracil, cytosine and thymine, 5-uracil (pseudracil), 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 nucleic acid bases include tricyclic pyrimidines such as phenoxazinecytidine ([5,4-b][1,4]benzoxazine-2(3H)-one) and phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), and G-clamps such as substituted phenoxazinecytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one), and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one). Modified nucleic acid bases may also include those in which a purine base or pyrimidine base is substituted with another heterocycle, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. In certain embodiments, the modified nucleic acid base is 5-methylcytosine.

[0176] Typical modified sugars include carbocyclic or acyclic sugars, sugars having substituents on one or more of their 2', 3', or 4' positions, and sugars having substituents in place of one or more hydrogen atoms. In certain embodiments, sugars are modified by having a substituent at the 2' position. In additional embodiments, sugars are modified by having a substituent at the 3' position. In other embodiments, sugars are modified by having a substituent at the 4' position. It is also intended that sugars may have two or more modifications at those positions, or that antisense oligonucleotides may have one or more nucleotides with sugar modifications at one position, and one or more nucleotides with sugar modifications at different positions.

[0177] The sugar modifications intended for oligonucleotides include, but are not limited to, sugar substituents selected from the following: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl (where the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C12). 10 Alkyl or C2-C 10 (These may be alkenyls and alkynyls.) In some embodiments, these groups are O(CH2) x OCH3, O((CH2) x O) y CH3, O(CH2) x NH2, O(CH2) x CH3, O(CH2) x ONH2 and O(CH2) x ON((CH2) x CH3)2 (wherein x and y are independently between 1 and 10) may be selected.

[0178] In some embodiments, the modified sugars are as follows: C1~C 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, alkaryl groups, alkaryl groups, aralkyl groups, O-alkaryl groups or O-aralkyl groups, SH groups, SCH3 groups, Cl groups, Br groups, CN groups, OCN groups, CF3 groups, OCF3 groups, SOCH3 groups, SO2CH3 groups, ONO2 groups, NO2 groups, N3 groups, NH2 groups, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of antisense oligonucleotides, or groups for improving the pharmacodynamic properties of antisense oligonucleotides, and substituents selected from other substituents having similar properties. In one embodiment, the modification includes 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O-CH2CH2OCH3) (Martin et al., 1995), i.e., an alkoxyalkoxy group. Other modifications include the 2'-dimethylaminooxyethoxy group, also known as 2'-DMAOE, the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2O-CH2-N(CH3)2.

[0179] Further sugar substituents include allyl (-CH2-CH=CH2), -O-allyl (CH2-CH=CH2), methoxy (-O-CH3), aminopropoxy (-OCH2CH2CH2NH2), and fluoro (F). The 2'- (2'-) sugar substituent can be at the arabino (upper) or ribo (lower) 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 at the 5' position of the 2'-5' linked oligonucleotide and 5'-terminal nucleotide. The oligomeric compound may also have sugar mimetic molecules, such as cyclobutyl moieties, instead of pentofuranosyl sugars. Examples of U.S. patents disclosing the preparation of modified sugar structures include U.S. Patent 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; 5, This includes, but is not limited to, Nos. 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, all of which are incorporated herein by reference in their entirety.

[0180] Typical 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 may be 2'-O-Me modification, 2'F modification, 2'H modification, 2'amino modification, 4'thioribose modification, or phosphorothioate modification (on a carboxyl group bonded to the 6' carbon), or a combination thereof.

[0181] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety containing a non-crosslinked 2'-substituent selected from F, OCH3 and OCH2CH2OCH3.

[0182] Certain modified sugar moieties include substituents that bridge two atoms of the 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'-furanose ring atom and the 2'-furanose ring atom. Examples of such 4'-to-2' bridging sugar substituents include, but are not limited to, 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (called "restricted ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("restricted MOE" or "cMOE") and their analogues (e.g., Seth et al., U.S. Patent No. 7,399,845, Bhat et al., U.S. Patent No. 7,569,686, Swayze et al.) See, for example, al., U.S. Patent No. 7,741,457 and Swayze et al., U.S. Patent No. 8,022,193), 4'-C(CH3)(CH3)-O-2' and its analogues (see, for example, Seth et al., U.S. Patent No. 8,278,283), 4'-CH2-N(OCH3)-2' and its analogues (see, for example, Prakash et al., U.S. Patent No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, for example, Allerson et al., U.S. Patent No. 7,696,345 and Allerson et al., U.S. Patent No. 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, for example, Zhou, et al. al, J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and its analogues (see, for example, Seth et al., U.S. Patent No. 8,278,426), 4'-C(R a R b )-N(R)-O-2',4'-C(R a R b )-ON(R)-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2' (wherein each R, Ra and R b These are independently H, protecting groups, or C1-C 12 Examples include alkyl groups (see, for example, Imanishi et al., U.S. Patent No. 7,427,672).

[0183] In certain embodiments, such a bridging from 4' to 2' is independently performed by -[C(R a )(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 Includes 1 to 4 linked bases independently selected from )- [In the formula, x is 0, 1, or 2. n is 1, 2, 3, or 4. Each R a and R b These are independently H, protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl substitution C2~C 12 Alkenyl, C2~C 12 Alkinyl substitution C2~C 12 Alkinyl, C5~C 20 Aryl substitution C5~C 20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), where each J1 and J2 is independently H, C1-C 12Alkyl, substituted C1-C 12 Alkyl, C2~C 12 Alkenyl substitution C2~C 12 Alkenyl, C2~C 12 Alkinyl substitution C2~C 12 Alkinyl, C5~C 20 Aryl substitution C5~C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1~C 12 Aminoalkyl, substituted C1-C 12 [It is an aminoalkyl group or a protecting group.]

[0184] The bicyclic sugar moiety of さらなる, known in the technical field, であり, example, Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al. 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. 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, 2017, 129, 8362-8379; Wengel et al., U.S. Patent No. 7,053,207; Imanishi et al., U.S. Patent No. 6,268,490; Imanishi et al., U.S. Patent No. 6,770,748; Imanishi et al., U.S. Patent No. RE44,779; Wengel et al., U.S. Patent No. 6,794,499; Wengel et al., U.S. Patent No. 6,670,461; Wengel et al., U.S. Patent No. 7,034,133; Wengel et al., U.S. Patent No. 8,080,644; Wengel et al., U.S. Patent No. 8,034,909; Wengel et al., U.S. Patent No. 8,153,365; Wengel et al., U.S. Patent No. 7,572,582; およびRamasamy et al. al., U.S. Patent No. 6,525,191; Torsten et al., International Publication No. 2004 / 106356; Wengel et al., International Publication No. 1999 / 014226; Seth et al., International Publication No. 2007 / 134181; Seth et al. U.S. Patent No. 7,547,684; Seth et al. al., U.S. Patent No. 7,666,854; Seth Seth et al., U.S. Patent No. 8,088,746; Seth et al., U.S. Patent No. 7,750,131; Seth et al., U.S. Patent No. 8,030,467; Seth et al.See U.S. Patent No. 8,268,980; Seth et al., U.S. Patent No. 8,546,556; Seth et al., U.S. Patent No. 8,530,640; Migawa et al., U.S. Patent No. 9,012,421; Seth et al., U.S. Patent No. 8,501,805; and U.S. Patent Publication No. 2008 / 0039618 by Allerson et al. and U.S. Patent Publication No. 2015 / 0191727 by Migawa et al.

[0185] In certain embodiments, the bicyclic sugar moiety and the nucleoside incorporating such a bicyclic sugar moiety are further defined by the isomer configuration. For example, the LNA nucleoside (described herein) may be in an α-L configuration or a β-D configuration. [ka]

[0186] α-L-methyleneoxy(4'-CH2-0-2') or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that have exhibited antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In this specification, a general description of bicyclic nucleosides includes the configurations of both isomers. Where the position of a particular bicyclic nucleoside (e.g., LNA or cEt) is identified in the exemplary embodiments herein, they are in the β-D configuration unless otherwise specified.

[0187] In certain embodiments, the modified sugar moiety includes one or more non-crosslinked sugar substituents and one or more crosslinked sugar substituents (e.g., 5'-substituted sugars and 4'-2'-crosslinked sugars).

[0188] In certain embodiments, the modified sugar moiety is a sugar substitute. In certain such embodiments, the oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In certain such embodiments, such modified sugar moiety also includes crosslinked and / or non-crosslinked substituents as described herein. For example, certain sugar substitutes include substitutions of a 4'-sulfur atom and substitutions at the 2' position (see, for example, Bhat et al., U.S. Patent No. 7,875,733 and Bhat et al., U.S. Patent No. 7,939,677) and / or the 5' position.

[0189] In certain embodiments, the sugar substitute comprises a ring having more than five atoms. For example, in certain embodiments, the sugar substitute comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid ("HNA"), anitol nucleic acid ("ANA"), mannitol nucleic acid ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA: [ka]

[0190] (See "F-HNA," e.g., Swayze et al., U.S. Patent No. 8,088,904; Swayze et al., U.S. Patent No. 8,440,803; Swayze et al., U.S. Patent No. 8,796,437; and Swayze et al., U.S. Patent No. 9,005,906; F-HNA may also be called F-THP or 3'-fluorotetrahydropyran), and formula: [ka] [Here, independently of each of the modified THP nucleosides: Bx is the nucleic acid base portion, T3 and T4 are, independently, internucleoside linking groups that link a modified THP nucleoside to the rest of the oligonucleotide, or one of T3 and T4 is an internucleoside linking group that links a modified THP nucleoside to the rest 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. Nucleosides comprising additional modified THP compounds include q1, q2, q3, q4, q5, q6, and q7, each independently being H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl, and each of R1 and R2 being independently selected from hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN (wherein X is O, S, or NJ1, and each of J1, J2, and J3 is independently H or C1-C6 alkyl).

[0191] In certain embodiments, modified THP nucleosides are provided in which q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is not H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, modified THP nucleosides are provided in which one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.

[0192] In certain embodiments, the sugar substitutes include rings having more than five atoms and multiple heteroatoms. For example, their use in nucleosides and oligonucleotides containing morpholino sugar moieties has been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Patent No. 5,698,685; Summerton et al., U.S. Patent No. 5,166,315; Summerton et al., U.S. Patent No. 5,185,444; and Summerton et al., U.S. Patent No. 5,034,506). As used herein, the term “morpholino” means a sugar substitute having the following structure: [ka]

[0193] In certain embodiments, morpholino may be modified, for example, by adding or altering various substituents from the morpholino structure described above. Such sugar substitutes are referred to herein as “modified morpholino.” In some embodiments, morpholino or modified morpholino may further include a modified skeleton such as thiomorpholino or phosphorodiamidate morpholino (PMO), which is a morpholino nucleoside linked by internucleotide bonds of thiophosphoamidate or phosphorodiamidate.

[0194] In certain embodiments, the sugar substitute includes an acyclic moiety. Examples of nucleosides and oligonucleotides containing such acyclic sugar substitutes include, but are not limited to, peptide nucleic acids ("PNA"), acyclic butyl nucleic acids (e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described by Manoharan et al., International Publication No. 2011 / 133876.

[0195] Many other bicyclic and tricyclic sugars and sugar substitute ring systems that can be used in modified nucleosides are known in the art.

[0196] Nucleoside residues of oligonucleotides in functional or cyclization domains can be linked to one another by any of a number of known internucleoside bonds. Two main classes of internucleoside linkages are determined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside bonds include, but are not limited to, phosphodiester bonds ("P=O") containing phosphophosphates (also called unmodified or naturally occurring bonds), phosphotryesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S") and phosphorodithioates ("HS-P=S"). Typical non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-), siloxanes (-O-SiH2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside links are well known to those skilled in the art.

[0197] Such nucleoside interbondings include, but are not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, methylphosphonates, alkylphosphonates, alkylphosphonothioates, phosphotryesters, phosphoramidates, siloxanes, carbonates, carbolokics, acetamidates, carbamates, morpholinos, boranos, thioethers, cross-linked phosphoramidates, cross-linked methylenephosphonates, cross-linked phosphorothioates, and sulfone nucleoside interbondings. In some embodiments, the synthetic antisense oligonucleotides of the present invention may include combinations of nucleotide interbondings. In some embodiments, the synthetic antisense oligonucleotides of the present invention may include combinations of phosphorothioates and phosphodiester nucleotide interbondings. In some embodiments, phosphorothioate nucleotide interbondings are more than half of the nucleotide interbondings but less than all of the nucleotide interbondings. In some embodiments, all of the nucleotide interbondings are phosphorothioate nucleotide interbondings.

[0198] Modified oligonucleotides containing nucleoside-nucleoside bonds with chiral centers can be prepared as a population of modified oligonucleotides containing sterically random nucleoside-nucleoside bonds, or as a population of modified oligonucleotides containing phosphorothioate bonds, particularly stereochemical configurations. In certain embodiments, the population of modified oligonucleotides contains phosphorothioate nucleoside-nucleoside bonds, where all phosphorothioate nucleoside-nucleoside bonds are sterically random. Such modified oligonucleotides can be produced using synthetic methods that result in a random selection of the stereochemical configuration of each phosphorothioate bond. Nevertheless, as will be well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereochemical configuration. In certain embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides containing one or more specific phosphorothioate nucleoside-nucleoside bonds of specific independently selected stereochemical configurations.

[0199] In certain embodiments, the phosphorothioate bond may be a mixture of Rp and Sp enantiomers, or the phosphorothioate bond may be stereoregular or substantially stereoregular in either the Rp or Sp form. In embodiments where the bond is a mixture of Rp and Sp enantiomers, the Rp and Sp forms may be at predetermined positions within the oligonucleotide, or they may be randomly distributed throughout the oligonucleotide.

[0200] As used herein, “nucleic acid sequence” means the sequence of consecutive nucleic acid bases in a nucleic acid or oligonucleotide, independent of any modifications to sugar or nucleoside bonds.

[0201] As used herein, “nucleoside” means a compound comprising a nucleic acid base and a sugar moiety. The nucleic acid base and sugar moiety are, independently, either unmodified or modified. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleic acid base and / or a modified sugar moiety. Modified nucleosides include debasic nucleosides that lack a nucleic acid base. “Linked nucleosides” are nucleosides linked together in a continuous sequence (i.e., there are no additional nucleosides between linked nucleosides).

[0202] As used herein, “oligomer compound” means an oligonucleotide and, optionally, one or more additional features, such as a conjugate group or terminal group. The oligomer compound may or may not be paired with a second oligomer compound complementary to the first oligomer compound. “Single-stranded oligomer compound” is an unpaired oligomer compound.

[0203] As used herein, “oligonucleotide” means a chain of linked nucleosides connected via internucleoside bonds, where each nucleoside and internucleoside bond may be modified or unmodified. Unless otherwise indicated, an oligonucleotide consists of 8 to 50 linked nucleosides.

[0204] As used herein, “modified oligonucleotide” means an oligonucleotide in which at least one nucleoside or internucleoside bond has been modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not contain any nucleoside modifications or internucleoside modifications.

[0205] The terms “identity percentage,” “sequence identity,” “identity%,” “sequence identity%,” and “identity%” may be used interchangeably herein and refer to the quantitative measurement of similarity between two sequences (e.g., nucleic acids or amino acids). The identity percentage of genomic DNA sequences, intron and exon sequences, and amino acid sequences between humans and other species varies by species, with chimpanzees having the highest identity percentage with all other species in each category.

[0206] The percentage of identity between two nucleic acid sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Then, the nucleotides at the corresponding nucleotide positions are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences.

[0207] The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. For example, the percentage of identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 (each of which is incorporated herein by reference). For example, the percentage of identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0) using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Alternatively, the percentage of identity between two nucleotide sequences can be determined using the GAP program of the GCG software package using the NWSgapdna.CMP matrix. Commonly used methods for determining the percentage of identity between sequences include, but are not limited to, those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which are incorporated herein by reference.The techniques for determining identity are embodied in publicly available computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol., 215, 403(1990)).

[0208] Where a percent identity or range thereof (e.g., at least, more, etc.) is recited, unless otherwise specified, endpoints are included and a range (e.g., at least 70% identity) includes all ranges within the recited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity) and all increments thereof (e.g., one tenth of a percent (e.g., 0.1%), one hundredth of a percent (e.g., 0.01%), etc.).

[0209] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to an animal. Such specific carriers enable the pharmaceutical composition to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, or a sterile buffer solution.

[0210] As used herein, "pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a compound such as an oligomeric compound, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesirable toxicological effects.

[0211] As used herein, "pharmaceutical composition" means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition can include an antisense compound and a sterile aqueous solution. In certain embodiments, the pharmaceutical composition exhibits activity in a free uptake assay in a particular cell line.

[0212] As used herein, "phosphorus moiety" means an atomic group containing a phosphorus atom. In certain embodiments, the phosphorus moiety includes mono-, di- or triphosphate or phosphorothioate.

[0213] As used herein, "prodrug" means a therapeutic agent in an in vitro form that is converted into a different form in an animal or within its cells. Typically, the conversion of a prodrug in an animal is facilitated by the action of an enzyme (e.g., an endogenous or viral enzyme) or a chemical substance present in the cell or tissue and / or physiological conditions.

[0214] As used herein, "OMe" means methoxy. "2'-OMe" means a 2'-OCH3 group in place of the 2'OH group of the ribosyl sugar moiety.

[0215] The enumeration of numerical ranges by endpoints includes all numbers and fractions contained within each range, as well as the enumerated endpoints.

[0216] The ranges provided herein are understood to be abbreviated representations of all values ​​within the range. For example, the range from 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 any number, combination of numbers, or subrange from the group consisting of 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 any endpoint of the range are particularly intended. For example, nested subranges of the exemplary range 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.

[0217] As used herein, “reduction or inhibition of quantity or activity” means a reduction or blockage of transcriptional expression or activity compared to the transcriptional expression or activity in an untreated or control sample, and does not necessarily mean a complete elimination of transcriptional expression or activity.

[0218] As used herein, “self-complementary” with respect to oligonucleotides means oligonucleotides that hybridize at least partially with themselves.

[0219] As used herein, “standard cell assay” means the assay described in Example 1 and its reasonable variations.

[0220] As used herein, “sterically random chiral center” in the context of a group of molecules of the same molecular formula means a chiral center having a random stereochemical configuration. For example, in a group of molecules containing sterically random chiral centers, the number of molecules having the (S) configuration of the sterically random chiral center may be the same as, but not necessarily the same as, the number of molecules having the (R) configuration of the sterically random chiral center. The stereochemical configuration of a chiral center is considered random if it is the result of a synthetic method not designed to control the stereochemical configuration. In certain embodiments, a sterically random chiral center is a sterically random phosphorothioate nucleoside bond.

[0221] As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2'-OH(H) furanosyl moiety, such as that found in RNA (“unmodified RNA sugar moiety”), or a 2'-H(H) moiety, such as that found in DNA (“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” means a modified furanosyl sugar moiety or sugar substitute. As used herein, a modified furanosyl sugar moiety means a furanosyl sugar that contains a non-hydrogen substituent in place of at least one hydrogen of the unmodified sugar moiety. In certain embodiments, the modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and acyclic sugars.

[0222] As used herein, “sugar substitute” means a modified sugar moiety other than a furanosyl moiety that can link a nucleic acid base to another group, such as an internucleoside bond, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides containing sugar substitutes can be incorporated at one or more positions within an oligonucleotide, and such oligonucleotides can be hybridized to complementary oligomeric compounds or nucleic acids.

[0223] As used herein, “target nucleic acid” and “target RNA” mean nucleic acids that are designed to be affected by an antisense compound.

[0224] As used herein, “target region” means the portion of a target nucleic acid that is designed for the oligomeric compound to hybridize.

[0225] As used herein, “end group” means a chemical group or atomic group covalently bonded to the end of an oligonucleotide.

[0226] As used herein, “therapeutic dose” means the amount of a drug that provides a therapeutic benefit to an animal. For example, a therapeutic dose improves the symptoms of a disease.

[0227] As used herein, “treat,” “treatment,” or “treating” means administering any of the compounds described herein to bring about a change or improvement in a disease, disorder, or condition.

[0228] A “part” refers to a specified number of consecutive (i.e., linked) nucleic acid bases of a nucleic acid. In certain embodiments, a part is a specified number of consecutive nucleic acid bases of a target nucleic acid. In certain embodiments, a part is a specified number of consecutive nucleic acid bases of an antisense compound.

[0229] The terms "combined administration" or "administered in combination" generally refer to the administration of at least two different substances. Combined administration refers to the simultaneous administration of at least two different substances in any order, whether in single doses or separate doses, and in a sequence with a time interval of up to several days.

[0230] The term "in combination with" generally means administering an oligonucleotide-based compound according to the present invention and another agent useful for treating a disease or condition that does not inactivate the activity of the compound during the treatment of a patient. Such administration can be carried out in any order, including simultaneous administration and orders with open time intervals ranging from seconds to up to several days. Such combination therapies can also include multiple administrations of the compound according to the present invention and / or of the other agent independently. The administration of the compound according to the present invention and the other agent may be by the same route or by different routes.

[0231] The terms "individual" or "subject" or "patient" generally refer to mammals such as humans. The term "mammal" is clearly intended to include warm-blooded vertebrates including, but not limited to, humans, non-human primates, rats, mice, cats, dogs, horses, livestock cattle, cows, pigs, sheep and rabbits. As used herein, "an individual in need thereof" refers to a human or non-human animal selected for treatment or therapy that requires such treatment or therapy. As used herein, "inhibition of expression or activity" refers to a decrease or blockade of the expression or activity of RNA or protein and does not necessarily indicate complete elimination of expression or activity.

[0232] Example Synthesis of CSOs Containing Antisense Oligonucleotide Functional Domains The cyclic structured oligonucleotides according to the present 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 present invention can be synthesized by a linear synthetic approach.

[0233] The compounds used herein have been synthesized using phosphoramidite chemistry. These protocols are described in detail, for example, at pubs.rsc.org / en / content / chapter / bk9781788012096-00453 / 978-1-78801-209-6, which are incorporated herein by reference.

[0234] Inhibition of target RNA by CSOs containing antisense oligonucleotide functional domains. Cyclic structured oligonucleotides were designed targeting nucleotide sequences within APOB, TTR, APOC3, PTP1B, PCSK9, STAT3, ANGPTL3, KLKB1 DGAT2, HTT, MAPT, SOD1, or HPRT1, and their effects on mRNA expression in vitro were tested.

[0235] Hep3B cells (derived from human liver) or U-251 MG cells were cultured according to ATCC recommended conditions and medium (Eagle's Minimum Essential Medium containing 10% FBS). Cells were seeded in 96-well plates at a density of 20 K / well and reverse transfection was performed with 0.3 µl / well of RNAiMax and six different concentrations (25, 6.25, 1.56, 0.39, 0.10, and 0.024 nM) of cyclic structured antisense oligonucleotides (CSOs). Control wells were transfected with 0.3 µl / well of RNAiMax and medium alone or with 25 nM of non-template control antisense oligonucleotide, and incubated for 48 hours post-transfection. Gene expression was assayed using the Invitrogen® TaqMan® Fast Advanced Cells-to-CT® Kit according to the manufacturer's protocol (A35378, Invitrogen®). In short, cells were harvested using Cells to CT lysis reagent, and immediately afterward, total RNA was reverse transcribed to cDNA using RT reagent. Quantitative polymerase chain reaction (qPCR) was performed via multiplex reactions using qPCR Fast Advanced Master Mix and pre-designed primers and FAM-labeled probes (4351370, Invitrogen®) for the corresponding gene of interest (GOI), and normalized using pre-designed primers and VIC-labeled probes (number 4448486, Invitrogen®) for the reference gene hypoxanthine-guanine phosphoribosyltransferase (HPRT1). Data were plotted and analyzed using the "absolute IC50" nonlinear regression dose-response model in GraphPad Prism.

[0236] Figures 3–18 show a comparison of gapmer format controls with clinical candidate antisense oligonucleotide sequences, each possessing three different cyclization formats. Using the same sequences, linear gapmer format ASO and three different permutation cyclization formats CSO were designed. Hep3B cells or U-251 MG cells were transfected with the indicated oligonucleotides and concentrations (25, 6.25, 1.56, 0.39, 0.10, and 0.024 nM) and harvested after 48 hours for gene expression analysis. Data are plotted as a percentage of control cells (cells reverse-transfected with the vehicle alone). Tables of IC50 and IC80 values ​​calculated using the Graphpad Prism absolute IC50 nonlinear regression dose-response model are also provided. Gapmer non-target control: 5'- CCAAA TCTTATAATA ACTAC -3' (Sequence ID 307). Circular non-target control: 3'-ttgatg-5'-5'- CCAAATCT TATAATAACTAC-3' (SEQ ID NO: 308). The underlined part represents the 2'-MOE ribonucleotide.

[0237] While the present invention has been specifically illustrated and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made without departing from the scope of the invention as encompassed in the appended claims.

Claims

1. A cyclic structured oligonucleotide (CSO) comprising a functional domain and a cyclization domain, wherein the functional domain and the cyclization domain are linked at their 5' ends, the cyclization domain comprises an oligonucleotide having a nucleotide length of 4 to 12, complementary to the sequence of nucleotides in the functional domain, and having the opposite polarity to the sequence of nucleotides in the complementary functional domain, the cyclization domain hybridizes with the functional domain to form a cyclic structure, the functional domain comprises an antisense oligonucleotide, the antisense oligonucleotide is at least 80% identical to an antisense oligonucleotide selected from Table 1, and comprises at least 12 consecutive nucleic acid bases complementary to equal-length portions of a target RNA sequence.

2. The antisense oligonucleotide comprises a 3' domain and a 5' domain continuous with the 3' domain, The 3' domain begins with a 3' terminal nucleotide, has a nucleotide length of 10 to 12, each nucleotide is unmodified, and the internucleotide bonds between adjacent nucleosides are phosphodiester or phosphorothioate internucleotide bonds or a combination thereof. The CSO according to claim 1, wherein the 5' domain begins with the first nucleotide following the 3' domain and continues to the terminal nucleotide of the 5' end, at least three nucleotides of the 5' domain comprise a modified sugar and / or a modified skeleton, the modified deoxyribonucleotides and / or modified ribonucleotides of the 5' domain do not need to be consecutive, and the modified deoxyribonucleotides and / or modified ribonucleotides of the 5' domain prevent RNase H cleavage in the 5' domain.

3. The CSO according to claim 2, wherein at least half of the nucleotides of the 5' domain include a modified sugar and / or a modified skeleton.

4. The CSO according to claim 2, wherein all of the nucleotides in the 5' domain include a modified sugar and / or a modified skeleton.

5. The CSO according to any one of claims 1 to 4, wherein the cyclization domain is 4 to 8 nucleotides long.

6. The CSO according to any one of claims 1 to 5, wherein the nucleotide of the cyclization domain includes an unmodified deoxyribonucleotide, an unmodified ribonucleotide, a modified deoxyribonucleotide, a modified ribonucleotide, or a combination thereof.

7. The CSO according to claim 6, wherein the nucleotide of the cyclization domain includes an unmodified ribonucleotide.

8. The CSO according to claim 6, wherein the nucleotide of the cyclization domain includes an unmodified deoxyribonucleotide.

9. The CSO according to claim 6, wherein the nucleotide of the cyclization domain includes a modified ribonucleotide.

10. The CSO according to claim 9, wherein the modified ribonucleotide includes a 2'-substituted ribonucleotide.

11. The CSO according to claim 10, wherein the 2'-substituted ribonucleotide is a 2'-OMe ribonucleotide or a 2'-MOE ribonucleotide.

12. The CSO according to any one of claims 1 to 11, wherein the internucleotide bonds of the oligonucleotides of the functional domain and / or the cyclization domain include phosphorothioate internucleotide bonds, phosphodiester internucleotide bonds, or a combination thereof.

13. The CSO according to claim 12, wherein the oligonucleotide of the functional domain comprises at least one phosphorothioate nucleotide internucleotide bond.

14. The CSO according to claim 13, wherein at least half of the nucleotide internucleotide bonds are phosphorothioate bonds.

15. The CSO according to claim 13, wherein all of the nucleotide internucleotide bonds are phosphorothioate bonds.

16. The CSO according to claim 12, wherein all of the nucleotide interbonds are phosphodiester bonds.

17. The CSO according to claim 12, wherein the oligonucleotide of the cyclization domain includes at least one phosphorothioate nucleotide internucleotide bond.

18. The CSO according to claim 17, wherein at least half of the nucleotide internucleotide bonds are phosphorothioate bonds.

19. The CSO according to claim 17, wherein all of the nucleotide interbonds are phosphorothioate bonds.

20. The CSO according to claim 12, wherein all of the nucleotide interbonds are phosphodiester bonds.

21. The CSO according to any one of claims 1 to 19, wherein the functional domain comprises an antisense oligonucleotide that has at least 80% sequence identity with an antisense oligonucleotide selected from Table 2.

22. The CSO according to any one of claims 1 to 21, wherein the cyclization domain is selected from Table 3.

23. The CSO according to any one of claims 1 to 22, wherein the functional domain and the cyclization domain are linked via direct bonding at their 5' ends.

24. The CSO according to any one of claims 1 to 23, comprising an antisense oligonucleotide having at least 80% identity with an antisense oligonucleotide selected from Table 4.

25. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 24, and a pharmaceutically acceptable carrier.

26. A method for inhibiting gene expression, comprising administering a CSO according to any one of claims 1 to 24 or the composition according to claim 25.

27. The method according to claim 26, which is useful for treating a subject having a disease or disorder, and for which inhibiting gene expression is beneficial.

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