Delivery of RNA therapeutics using ring-shaped nucleic acids

Abstract

The present invention provides oligonucleotides referred to as ring-shaped oligonucleotides ("RSO") as described herein, compositions comprising same, and methods of using same.

Description

[0001] Delivery of RNA Therapeutics Using Ring-Shaped Nucleic Acids

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 522,878, filed on June 23, 2023 and U.S. Provisional Application No. 63 / 524,009, filed on June 29, 2023. The entire teachings of the above applications are incorporated herein by reference.

[0004] BACKGROUND

[0005] Processing and Translation of targeted RNA can be modulated by antisense oligonucleotides by multiple mechanisms. These include cleaving the targeted RNA by RNase-H, modulating aberrant splicing, processing of targeted RNA and increased translation, inhibiting the translation by steric hindrance, etc. Targeted RNA could be mRNA or noncoding RNA. In other mechanisms, the antisense strand of a duplex siRNA could be incorporated in AGO and inhibit translation by the siRNA mechanism. In other mechanisms, like adenosine deaminase acting on RNA (ADAR) based or CRISPR-based models, antisense could edit RNA or DNA and thereby modulate translation and processing. In other mechanisms, modified mRNA or AAV could be delivered to produce a novel protein.

[0006] Over the years, it has been learned that antisense hybridization and affinity influence selectivity for the targeted RNA. In addition, to use antisense nucleic acids as drugs, nuclease stability is important which has been provided by the modification of internucleotide linkages, for example, phosphorothioate.

[0007] It was postulated that nuclease stability is key for potency and since the degradation of antisense was shown to be from the 3 ’-end, the focus was to modify the 3 ’-end to slow down degradation. These designs include capping on the 3 ’-end, and a hairpin loop on the 3’- end, creating oligos having secondary structures comprising 3 ’-3’ linkages or attaching two (2) antisense oligonucleotides at their 3’ ends. These types of antisense oligonucleotides showed increased nuclease stability but antisense potency was not improved. Unfortunately, these modifications also increased inflammatory responses thereby limiting the therapeutic index.

[0008] Both DNA and RNA and 2 ’-substituted RNA containing phosphorothioate have been studied as antisense agents and provide different characteristics. DNA phosphorothioate antisense, when hybridized to RNA, activates RNase H, whereas RNA or 2’ -substituted RNA antisense binds to RNA with higher affinity and does not activate RNase H. To further improve antisense characteristics, a mixture of these two modifications has been employed in antisense, generally referred to as a hybrid or “gapmer” antisense oligonucleotide. In most studied gapmer antisense, modified RNA segment is placed on both the 3’- and 5’- ends whereas DNA is placed in the middle. Gapmer antisense is the most widely studied antisense design and drugs employing this chemistry are approved and are in clinical development.

[0009] One of the side effects of both DNA and RNA phosphorothioate is due to the interaction with proteins and, more specifically, with the family of Pattern Recognition Receptors (PRRs). These interactions result in the induction of an immune cascade, thereby causing an off-target mechanism of action and related safety signals. Detailed structureactivity relationship studies have shown that the accessibility of the 5 ’-end of DNA and RNA, phosphodiester, and phosphorothioate antisense is required for immune activation. DNA and RNA phosphorothioate containing two 5 ’-ends have shown increased immunostimulatory activity. In contrast, it has been previously shown that DNA and RNA phosphorothioate, which contain two 3 ’-ends (and lack 5’-), show minimal inflammatory responses.

[0010] In continuing efforts to improve the properties of DNA and RNA, unmodified and phosphorothioate antisense as therapeutic agents, structural changes in oligonucleotides were considered. For example, in earlier studies, self-stabilized oligonucleotides were reported wherein oligodeoxynucleotide phosphorothioates (“PS-oligonucleotide”) containing a hairpin loop region at the 3 '-end provided increased in vivo nuclease stability and limited biological activity. To date, the focus has been to improve the stability of antisense by modifying 3’- end by various modifications, including in gapmer antisense.

[0011] WO 2023 / 049275 provides an example of cyclic structured oligonucleotides ("CSOs") comprising a functional domain and a cyclizing domain. The CSO is a single oligonucleotide comprising a functional domain and a cyclizing domain linked at their 5’ or 3’ ends; wherein the cyclizing domain is complementary to and of opposite polarity to a sequence of nucleotides within the functional domain; and wherein the cyclizing domain hybridizes with the functional domain, thereby forming a cyclic structure.

[0012] International application PCT / US24 / 20897 provides an example of a structural class of oligonucleotides referred to herein as “circular prodrug nucleic acid” (CPN). A CPN is also a single oligonucleotide comprising a functional domain and a circularizing domain, wherein the circularizing domain comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the 5’ end of the oligonucleotide of the functional domain is linked to the first nucleic acid molecule and the 3’ end of the oligonucleotide of the functional domain is linked to the second nucleic acid molecule; wherein the first nucleic acid molecule and the second nucleic acid molecule of the circularizing domain are complementary to each other and of opposite polarity to each other and hybridize to form a double-stranded section.

[0013] Despite the advances that have been made, there is still a desire to develop antisense oligonucleotides and RNA therapeutics that have improved properties for use as therapeutic agents and in diagnostic applications.

[0014] SUMMARY OF THE INVENTION

[0015] The present invention provides a structural class of oligonucleotides referred to herein as “ring-shaped oligonucleotide” (RSO) (also referred to as “ring-shaped nucleic acids” or “ring-shaped structures” (RSS)). A RSO comprises two oligonucleotides, wherein the first oligonucleotide comprises a functional oligonucleotide (also referred to herein as “the functional oligonucleotide”) and the second oligonucleotide comprises a complementary oligonucleotide (also referred to herein as “the complementary oligonucleotide” or the “anchor oligonucleotide”), wherein the anchor oligonucleotide comprises a 5’ region, a linker segment, and a 3’ region, wherein the 5’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 5’ end of the first oligonucleotide and the 3’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 3’ end of the first oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ring-shape (see e.g., Fig. 1). In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are independently 4 to 300 nucleotides in length, wherein the nucleotides of the 5’ region and the 3’ region of the anchor oligonucleotide are independently selected from RNA, DNA, or a combination thereof and the linker segment is as defined herein. The first oligonucleotide and the second oligonucleotide are further described herein.

[0016] The functional oligonucleotide of the “first oligonucleotide” provides a function to the RSO.

[0017] RSOs of the invention adopt an intermolecular ring-shaped structure due to the complementarity between the first oligonucleotide and the second oligonucleotide. This intermolecular duplex formation changes the functional oligonucleotide’s shape and accessibility to the oligonucleotide's 5’ and 3’ ends. This structure combines key attributes to create optimal nucleic acid-based therapeutics. Improving RNA therapeutics to improve the delivery of nucleic acids to cytoplasm and nucleus in a stable form can improve the usefulness of RNA therapeutics. The 3’ - and 5’- ends of nucleic acids bind to pattern recognition receptors and exonucleases, thereby impacting RNA therapeutics’ availability. Therefore, delivering RNA therapeutic agents to the appropriate compartment by making the ends inaccessible to these factors would help improve RNA therapeutics. The conditional release of RNA therapeutics intracellularly by factors present in nature would permit the agent to be available in the desired compartment.

[0018] In gene and RNA expression modulation, this structure of the RSO masks the 5 ’-end of the functional oligonucleotide thereby reducing the interaction with PRRs and permitting endosomal escape. The structure of RSO also masks the 3 ’-end of the functional oligonucleotide to provide nuclease stability. According to some embodiments, once in the cytoplasm or nucleus, one or both of the double-stranded sections formed between the first oligonucleotide and the anchor oligonucleotide will be cleaved by RNase H, Dicer, restriction enzymes, or other intracellular factors and the ring-shaped structure will open, thereby presenting the functional oligonucleotide and allowing it to perform its function. According to some embodiments, the linker segment is a nucleotide linker as further described herein and the linker segment is cleaved by intracellular endonucleases or other intracellular factors and the ring-shaped structure will open, thereby presenting the functional oligonucleotide and allowing it to perform its function. This process of cleaving the RSO to present the functional oligonucleotide is referred to herein as “zw situ activation”.

[0019] In embodiments, once cleaved, the first oligonucleotide and the second oligonucleotide cannot re-hybridize, thereby preventing the re-circularization of the oligonucleotides of the RSO.

[0020] According to some embodiments, for example, when the functional oligonucleotide is an antisense oligonucleotide, it is in the ring-shaped form until it is in the presence of complementary target RNA where the functional oligonucleotide has a higher affinity for the target RNA resulting in the release of the second oligonucleotide and allowing the functional oligonucleotide to adopt a linear form and bind to the target RNA. The changes from ringshaped form to linear form could be confirmed by thermal melting and RNase H cleavage studies. In the linear form, the functional oligonucleotide hybridizes (under physiological conditions, at a minimum) with the complementary target RNA to form a duplex. This duplex is a substrate for RNase H, and, in the presence of RNase H and under the proper conditions (e.g., physiological), the RNA strand of the duplex will be cleaved by RNase H, thereby preventing expression. In other embodiments, when the functional oligonucleotide is an antisense oligonucleotide, the antisense oligonucleotide could also be a steric blocker or splice modulator and the target RNA could be coding RNA or non-coding RNA.

[0021] In other embodiments, the functional oligonucleotide can be a modified mRNA or AAV, wherein the modified mRNA or AAV could be delivered to produce a novel protein.

[0022] When the RSO is in the ring-shaped form, it may exhibit fewer of the polyanionic- related side effects (e.g., complement activation and prolongation of partial thromboplastin time) known to occur with PS-oligonucleotides, because there are fewer exposed phosphorothioate linkages. Also, RSOs in ring-shaped form would have reduced protein binding, reduced off-target interactions with non-targeted RNAs, and increased nuclease stability and endosomal escape.

[0023] RSOs according to the invention can be made using standard techniques for synthesis of the constituent oligonucleotides and are useful for all purposes for which the functional oligonucleotide is useful.

[0024] The foregoing merely summarizes certain aspects of the invention and is not intended, nor should it be construed, as limiting the invention in any manner. All patents, patent applications, and other publications recited in this specification are hereby incorporated by reference in their entirety.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0027] Fig. 1 A and Fig. IB depict embodiments of the ring-shaped oligonucleotide (RSO) (100) according to the invention. Line (101) represents the first oligonucleotide (i.e., the functional oligonucleotide) and line (102) represents the second oligonucleotide (i.e., the anchor oligonucleotide). The functional oligonucleotide can be single-stranded (Fig. 1 A) or double-stranded (Fig. IB). A sequence of nucleotides at the 5’ end of the anchor oligonucleotide (e.g., the 5’ region) is complementary to a sequence of nucleotides at the 5’ end of the functional oligonucleotide and a sequence of nucleotides at the 3’ end of the anchor oligonucleotide (e.g., the 3’ region) is complementary to a sequence of nucleotides at the 3’ end of the functional oligonucleotide. Hybridization between the first oligonucleotide and the second oligonucleotide brings the 5’ end of the first oligonucleotide into proximity with the 3’ end of the first oligonucleotide, thereby resulting in a “ring shape”. An optional linker can be present between the 5’ region and the 3’ region of the anchor oligonucleotide as long as the linker does not interfere with the ability of the first and second oligonucleotides to hybridize with each.

[0028] Fig. 2A through Fig. 2D depict embodiments of the ring-shaped oligonucleotide (RSO) (200) according to the invention. Line (201) represents the first oligonucleotide (i.e., the functional oligonucleotide), dashed line 203 represents a first extension segment, dashed line 204 represents a second extension segment, and line (202) represents the second oligonucleotide (i.e., the anchor oligonucleotide). Dashed line 205 represents an extension segment. The functional oligonucleotide can be single-stranded (Fig. 2A and Fig. 2B) or double-stranded (Fig. 2C and Fig. 2D). A sequence of nucleotides within the anchor oligonucleotide is complementary to a sequence of nucleotides in the extension segments. For example, a sequence of nucleotides at the 5’ end of the anchor oligonucleotide (e.g., the 5’ region) is complementary to a sequence of nucleotides in the first extension segment and a sequence of nucleotides at the 3’ end of the anchor oligonucleotide (e.g., the 3’ region) is complementary to a sequence of nucleotides in the second extension segment. Hybridization between the first and second extension segments and the second oligonucleotide brings the 5’ end of the first oligonucleotide into proximity with the 3’ end of the first oligonucleotide, thereby resulting in a “ring shape”. An optional linker can be present between the 5’ region and the 3’ region of the anchor oligonucleotide as long as the linker does not interfere with the ability of the first and second extension segments and the second oligonucleotide to hybridize with each other.

[0029] Fig. 3 A through Fig. 3E depict non-limiting examples of RSOs according to the invention. 301 represents a linker segment (L) as defined herein. The sequences on either side of L, and including L, represent a second oligonucleotide (i.e., the anchor oligonucleotide). In Fig. 3C and Fig. 3D, 302 represents a first oligonucleotide (i.e., the functional oligonucleotide) of any length as described herein. The sequences added to the 5’ and 3’ ends of the functional oligonucleotide (302) represent a first extension segment and a second extension segment.

[0030] Fig. 4A through Fig. 4H depict a comparison of RSO compounds with an antisense oligonucleotide targeting apolipoprotein C-III (APOC3) as the first (functional) oligonucleotide. Data is plotted as the percent of knockdown of APOCIII RNA in treated cells compared to control untreated cells (cells reverse-transfected with vehicle alone). The compound numbers for the data provided herein are based on the sequence identifier for the compound. For example, compound 43 represents the oligonucleotide identified by SEQ ID NO: 43. Fig. 4A shows that an anchor oligonucleotide according to the invention alone did not have activity. Fig. 4B shows the activity of SEQ ID NO: 48 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 48 + SEQ ID NO: 43 or SEQ ID NO: 44). Fig. 4C shows the activity of SEQ ID NO: 49 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 49 + SEQ ID NO: 43 or SEQ ID NO: 44). Fig. 4D shows the activity of SEQ ID NO: 50 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 50 + SEQ ID NO: 43 or SEQ ID NO: 44). Fig. 4E shows the activity of SEQ ID NO: 51 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 51 + SEQ ID NO: 45 or SEQ ID NO: 46). Fig. 4F shows the activity of SEQ ID NO: 52 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 52 + SEQ ID NO: 45 or SEQ ID NO: 46). Fig. 4G shows the activity of SEQ ID NO: 53 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 53 + SEQ ID NO: 45 or SEQ ID NO: 46). Fig. 4H shows the activity of SEQ ID NO: 54 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 54 + SEQ ID NO: 43 or SEQ ID NO: 44).

[0031] Fig. 5 A through Fig. 5D depict a comparison of RSO compounds with an antisense oligonucleotide targeting microtubule-associated protein tau (MAPT) as the first (functional) oligonucleotide. Data is plotted as percent knockdown of MAPT in treated cells compared to control untreated cells (cells reverse-transfected with vehicle alone). The compound numbers for the data provided herein are based on the sequence identifier for the compound. For example, compound 43 represents the oligonucleotide identified by SEQ ID NO: 43. Fig. 5 A shows the activity of SEQ ID NO: 57 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 57 + SEQ ID NO: 43 or SEQ ID NO: 44). Fig. 5B shows the activity of SEQ ID NO: 58 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 58 + SEQ ID NO: 43 or SEQ ID NO: 44). Fig. 5C shows the activity of SEQ ID NO: 59 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 59 + SEQ ID NO: 43 or SEQ ID NO: 44). Fig. 5D shows the activity of SEQ ID NO: 60 alone or in combination with an anchor oligonucleotide to form an RSO of the invention (i.e., SEQ ID NO: 60 + SEQ ID NO: 43 or SEQ ID NO: 44). DETAILED DESCRIPTION

[0032] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0033] Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0034] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

[0035] Features may be described herein as part of the same or separate aspects or embodiments of the present invention for clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

[0036] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and increments and / or portions therein, as well as individual numerical values within that range. For example, description of a range such as from 6 to 30 should be considered to include subranges such as from 6 to 27, from 6 to 25, from 6 to 15, from 8 to 20, from 8 to 15, from 10 to 15, etc., as well as individual numbers with that range, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 and further, as applicable, any portions or increments between or of a number, for example, tenths, hundredths, etc. This applies regardless of the breadth of the range.

[0037] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0038] The present invention provides oligonucleotides referred to as ring-shaped oligonucleotides (“RSOs”). In some embodiments, the RSO comprises two oligonucleotides, wherein the first oligonucleotide comprises a functional oligonucleotide (also referred to herein as “the functional oligonucleotide”) and the second oligonucleotide comprises a anchor oligonucleotide (also referred to herein as “the complementary oligonucleotide”), wherein the anchor oligonucleotide comprises a 5’ region and a 3’ region, wherein the 5’ region and the 3’ region are linked, directly or through a linker segment, and wherein the 5’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 5’ end of the first oligonucleotide and the 3’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 3’ end of the first oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ring-shape (see e.g., Fig. 1).

[0039] In some embodiments, the RSO comprises two oligonucleotides, wherein the first oligonucleotide comprises a functional oligonucleotide (also referred to herein as “the functional oligonucleotide”) and the second oligonucleotide comprises a anchor oligonucleotide (also referred to herein as “the complementary oligonucleotide”), wherein the first oligonucleotide further comprises a first extension segment at the 5’ end and a second extension segment at the 3’ end (also referred to herein as the “first extension segment” and the “second extension segment”, respectively), wherein the anchor oligonucleotide comprises a 5’ region and a 3’ region, wherein the 5’ region and the 3’ region are linked, directly or through a linker segment, and wherein the 5’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the first extension segment of the first oligonucleotide and the 3’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the second extension segment of the first oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ring-shape (see e.g., Fig. 2). In some embodiments, the RSO comprises two oligonucleotides, wherein the first oligonucleotide comprises a functional oligonucleotide and the second oligonucleotide comprises an anchor oligonucleotide, wherein the first oligonucleotide further comprises an extension segment at either the 5’ end or the 3’ end, wherein the anchor oligonucleotide comprises a 5’ region and a 3’ region, wherein the 5’ region and the 3’ region are linked, directly or through a linker segment, and wherein one region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the extension segment of the first oligonucleotide and the other region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the first oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ringshape structure (see e.g., Fig. 2B).

[0040] Preferably, at least one of the duplexes formed between the first oligonucleotide and the second oligonucleotide (e.g., directly or through an extension segment) is cleaved via in situ activation and degradation, as described herein, to release the functional oligonucleotide from the RSO shape.

[0041] As used herein, the terms “complementary segment”, “first complementary segment”, “second complementary segment” “extension segment”, “first extension segment”, and “second extension segment” refer to a number of additional nucleotides that are added to the 5’ end and / or 3’ end functional oligonucleotide of the first oligonucleotide and do not contribute to the function of first oligonucleotide. Rather, these extension segments provide a nucleotide sequence that hybridizes with nucleotides of the second oligonucleotide to form a double-stranded section (duplex) to provide the ring shape of the RSOs of the invention.

[0042] In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are independently 4 to 300 nucleotides in length, wherein the nucleotides of the 5’ region and the 3’ region are independently selected from RNA, DNA, or a combination thereof.

[0043] In embodiments, the first extension segment and the second extension segment are independently 4 to 300 nucleotides in length, wherein the nucleotides of the first extension segment and the second extension segment are independently selected from RNA, DNA, or a combination thereof.

[0044] In embodiments, the extension segments (i.e., the extension segment, the first extension and the second extension segment) are each, independently, are between 4 and 300 nucleotides in length. In embodiments, the extension segments each, independently, are between 4 and 200 nucleotides in length. In embodiments, the extension segments each, independently, are between 4 and 150 nucleotides in length. In embodiments, the extension segments each, independently, are between 4 and 100 nucleotides in length. In embodiments, the extension segments each, independently, are between 4 and 50 nucleotides in length.

[0045] In embodiments, the extension segments each, independently, are between 4 and 25 nucleotides in length. In embodiments, the extension segments independently comprise between 4 and 12 nucleotides in length. In embodiments, the extension segments independently comprise between 4 and 10 nucleotides in length. In embodiments, the extension segments independently comprise between 4 and 8 nucleotides in length.

[0046] In embodiments, the extension segments are, independently, 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, or 30 nucleotides in length. In embodiments, the extension segments are, independently, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. In embodiments, the extension segments are, independently, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In embodiments, the extension segments are, independently, 4, 5, 6, 7, or 8 nucleotides in length. In embodiments, the extension segments are 13 nucleotides in length. In embodiments, the extension segments are 12 nucleotides in length. In embodiments, the extension segments are 11 nucleotides in length. In embodiments, the extension segments are 10 nucleotides in length. In embodiments, the extension segments are 9 nucleotides in length. In embodiments, the extension segments are 8 nucleotides in length. In embodiments, the extension segments are 7 nucleotides in length. In embodiments, the extension segments are 6 nucleotides in length. In embodiments, the extension segments are 5 nucleotides in length.

[0047] In embodiments, the first extension segment and the second extension segment are the same length. In embodiments, the first extension segment and the second extension segment are different lengths.

[0048] In embodiments, the first extension segment and / or the second extension segment are the same length as the region of the second oligonucleotide to which they are complementary. In embodiments, the first extension segment and / or the second extension segment are different lengths than the region of the second oligonucleotide to which they are complementary.

[0049] In embodiments, the extension segment is the same length as the region of the second oligonucleotide to which it is complementary. In embodiments, the extension segment is a different length as the region of the second oligonucleotide to which it is complementary.

[0050] The first oligonucleotide and the second oligonucleotide are further described herein. In embodiments, the 3’ end of the first extension segment is linked to the 5’-end of the functional oligonucleotide with a 5 ’-3’ linkage and the 5’ end of the second extension segment is linked to the 3’ of the oligonucleotide with a 3’-5’ linkage.

[0051] The design of RSO maintains a ring-shaped form until one or both of the double stranded regions or the linker segment are cleaved by intracellular factors, such as RNase H and Dicer, endonucleases, and others or the ring is melted opened in the presence of the target RNA. This structure allows for increased stability, specificity and release of functional oligonucleotide in the appropriate compartment.

[0052] The functional oligonucleotide provides a desired function to the RSO. For example, for gene expression modulation, the functional oligonucleotide is complementary to a targeted RNA. As used herein, the terms “first oligonucleotide” or the “functional oligonucleotide” are used interchangeably.

[0053] RSOs of the invention adopts an intermolecular ring-shaped structure due to the complementarity between the oligonucleotide and the second oligonucleotide, which form an intermolecular duplex.

[0054] In embodiments wherein the first oligonucleotide comprises the first and second extension segments, the nucleotides of the functional oligonucleotide of the first oligonucleotide do not participate in the formation of the intermolecular duplex.

[0055] The nucleotides of the first extension segment and the second extension segment are not part of the functional oligonucleotide and, therefore, do not provide the desired function to the RSO.

[0056] In embodiments, the nucleotides of the functional oligonucleotide are DNA or RNA or combinations thereof.

[0057] In embodiments, the nucleotides of the functional oligonucleotide are unmodified.

[0058] In embodiments, the nucleotides of the anchor oligonucleotide are unmodified. In embodiments, the nucleotides of the 5’ region and the 3’ region of the anchor oligonucleotide are unmodified.

[0059] By “unmodified” it is intended that the nucleotides of the of nucleotides of the first oligonucleotide and / or the second oligonucleotide comprise naturally occurring nucleobases, sugars, and internucleotide backbones.

[0060] In embodiments, at least one nucleotide of the functional oligonucleotide is modified. In embodiments, two or more nucleotides of the functional oligonucleotide are modified. In embodiments, at least half of the nucleotides of the functional oligonucleotide are modified. In embodiments, all of the nucleotides of the functional oligonucleotide are modified. By “unmodified” it is intended that one or more nucleotides of the functional oligonucleotide comprises a modification of the inter-nucleotide linkage, sugar, heterocyclic base, or a combination thereof. These modifications could also be appropriately placed at specific positions within the functional oligonucleotide. Other chemistries and modifications are known in the field of oligonucleotides that can be readily used in accordance with the disclosure and are encompassed within the term ‘modified’ as used in the context of an oligonucleotide herein.

[0061] Except as otherwise described herein, the functional oligonucleotide comprises between 15 and 5000 nucleotides in length. In embodiments, the functional oligonucleotide comprises between 15 and 500 nucleotides in length. In embodiments, the functional oligonucleotide is 17 and 300 nucleotides in length. In embodiments, the functional oligonucleotide is 17 and 200 nucleotides in length. In embodiments, the functional oligonucleotide is 17 and 100 nucleotides in length. In embodiments, the functional oligonucleotide is 17 and 50 nucleotides in length. In embodiments, the functional oligonucleotide is 17 and 25 nucleotides in length.

[0062] In embodiments, the functional oligonucleotide is between 50 and 250 nucleotides in length. In embodiments, the functional oligonucleotide is between 50 and 150 nucleotides in length.

[0063] In embodiments, the functional oligonucleotide comprises between 15 and 50 nucleotides in length. In embodiments, the functional oligonucleotide comprises between 17 and 40 nucleotides in length. In embodiments, the functional oligonucleotide comprises between 17 and 25 nucleotides in length. In embodiments, the functional oligonucleotide is between 17 and 22 nucleotides in length.

[0064] In embodiments, the functional oligonucleotide is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In embodiments, the functional oligonucleotide is 17 nucleotides in length. In embodiments, the functional oligonucleotide is 18 nucleotides in length. In embodiments, the functional oligonucleotide is 19 nucleotides in length. In embodiments, the functional oligonucleotide is 20 nucleotides in length. In embodiments, the functional oligonucleotide is 21 nucleotides in length. In embodiments, the functional oligonucleotide is 22 nucleotides in length. In embodiments, the functional oligonucleotide is 23 nucleotides in length. In embodiments, the functional oligonucleotide is 24 nucleotides in length. In embodiments, the functional oligonucleotide is 25 nucleotides in length. In embodiments, the functional oligonucleotide is 26 nucleotides in length. In embodiments, the functional oligonucleotide is 27 nucleotides in length. In embodiments, the functional oligonucleotide is 28 nucleotides in length. In embodiments, the functional oligonucleotide is 29 nucleotides in length. In embodiments, the functional oligonucleotide is 30 nucleotides in length. In embodiments, the functional oligonucleotide is 31 nucleotides in length. In embodiments, the functional oligonucleotide is 32 nucleotides in length. In embodiments, the functional oligonucleotide is 33 nucleotides in length. In embodiments, the functional oligonucleotide is 34 nucleotides in length. In embodiments, the functional oligonucleotide is 35 nucleotides in length. In embodiments, the functional oligonucleotide is 36 nucleotides in length. In embodiments, the functional oligonucleotide is 37 nucleotides in length. In embodiments, the functional oligonucleotide is 38 nucleotides in length. In embodiments, the functional oligonucleotide is 39 nucleotides in length. In embodiments, the functional oligonucleotide is 40 nucleotides in length.

[0065] In embodiments, the functional oligonucleotide includes, but is not limited to, an oligonucleotide selected from an antisense oligonucleotide, a microRNA (miRNA), an siRNA, a piRNA, an hnRNA, an ncRNA, an snRNA, a miRNA mimic, an sgRNA, an esiRNA, an shRNA, a IncRNA, an mRNA, a guide RNA for a CRISPR-based system, a guide RNA for prime editing, a guide RNA for an adenosine deaminase acting on RNA (ADAR) system, or a splicing oligonucleotide.

[0066] In embodiments, the functional oligonucleotide includes, but is not limited to, an oligonucleotide selected from an immunostimulatory oligonucleotide or an immune- inhibitory oligonucleotide (also referred to as an immune antagonist oligonucleotide).

[0067] The only limitation on the nucleotides and intemucleotide linkages of the functional oligonucleotide is that they do not eliminate the ability of the functional oligonucleotide to carry out its intended function (e.g., in the case of an antisense oligonucleotide, to hybridize to and form a duplex with a complementary RNA segment under physiological conditions, which duplex is a substrate for RNase H). Preferred nucleotides and internucleotide linkages are those that will enhance the stability of the RSO to nucleases and other forms of chemical degradation and / or enhance the ability of the functional oligonucleotide to carry out its intended function.

[0068] In embodiments, the internucleotide linkages of the functional oligonucleotide are phosphorothioate intemucleotide linkages, phosphodiester intemucleotide linkages or a combination thereof.

[0069] In embodiments, one or more nucleotides of the second oligonucleotide comprise a modification of the inter-nucleotide linkage, sugar, heterocyclic base, or a combination thereof. These modifications could also be placed at specific positions within the anchor oligonucleotide.

[0070] As used herein, the terms “the second oligonucleotide” or the “complementary oligonucleotide” or the “anchor oligonucleotide” are used interchangeably.

[0071] In embodiments, the internucleotide linkages of the anchor oligonucleotide are phosphorothioate intemucleotide linkages, phosphodiester intemucleotide linkages or a combination thereof. In embodiments, the internucleotide linkages of the anchor oligonucleotide can be modified as known by one skilled in the art provided that it remains a substrate for RNase H or Dicer or other intracellular proteins. In embodiments, the intemucleotide linkages of the anchor oligonucleotide are phosphodiester intemucleotide linkages.

[0072] In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide each, independently, are between 4 and 300 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide each, independently, are between 4 and 200 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide each, independently, are between 4 and 150 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide each, independently, are between 4 and 100 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide each, independently, are between 4 and 50 nucleotides in length.

[0073] In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide each, independently, are between 4 and 25 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide independently comprises between 4 and 12 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide independently comprises between 4 and 10 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide independently comprises between 4 and 8 nucleotides in length.

[0074] In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, 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, or 30 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, 4, 5, 6, 7, or 8 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 5 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 6 nucleotides in length.

[0075] In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 15 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 14 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 13 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 12 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 11 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 10 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are

[0076] 9 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 8 nucleotides in length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are 7 nucleotides in length.

[0077] Preferably, the 5’ region and the 3’ region of the anchor oligonucleotide are linked through a linker segment wherein the linker segment is a nucleotide sequence between, and including, 1 and 50 nucleotides in length. Preferably, linker segment is between, and including, 1 and 40 nucleotides in length. Preferably, linker segment is between, and including, 1 and 30 nucleotides in length. Preferably, linker segment is between, and including, 1 and 20 nucleotides in length. Preferably, linker segment is between, and including, 1 and 10 nucleotides in length. Preferably, linker segment is between, and including, 1 and 5 nucleotides in length.

[0078] Preferably, when the linker segment is longer, for example between, and including, 30 and 50 nucleotides in length then the linker segment can form a secondary structure such as a hairpin structure, wherein the duplex section of the hairpin structure is optionally cleavable. In embodiments, the duplex section of the hairpin structure is cleavable.

[0079] Preferably, the linker segment is shorter, for example between, and including, 1 and

[0080] 10 nucleotides in length; preferably between, and including, 1 and 5 nucleotides in length. In embodiments, the linker segment is 10 nucleotides in length. In embodiments, the linker segment is 9 nucleotides in length. In embodiments, the linker segment is 8 nucleotides in length. In embodiments, the linker segment is 7 nucleotides in length. In embodiments, the linker segment is 6 nucleotides in length. In embodiments, the linker segment is 5 nucleotides in length. In embodiments, the linker segment is 4 nucleotides in length. In embodiments, the linker segment is 3 nucleotides in length. In embodiments, the linker segment is 2 nucleotides in length. In embodiments, the linker segment is 1 nucleotide in length.

[0081] In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are the same length. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are different lengths.

[0082] As used herein, the term “polarity” refers to the concept of directionality in primary structure (e.g., 3 ' — 5' and 5'— >3' in the case of DNA and RNA, or N-terminal^C-terminal (or vice versa) in the case of PNAs). In the case of the RSOs of the invention where the 5’ region and nucleotides at the 5’ end of the first oligonucleotide, or the first extension segment, which, for example, hybridize by Watson-Crick base pairing in anti-parallel fashion, the 5’ region can be in the 5'— >3 ' (or 2') configuration and the sequence of nucleotides to which it is complementary in the first oligonucleotide, or first extension segment, can be in the 3' (or 2')— >5’ configuration.

[0083] In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, at least 95% complementary to the corresponding nucleotides within the first oligonucleotide, or the first extension segment when present. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, at least 97% complementary to the corresponding nucleotides within the first oligonucleotide, or the first extension segment when present. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, at least 98% complementary to the corresponding nucleotides within the first oligonucleotide, or the first extension segment when present. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, at least 99% complementary to the corresponding nucleotides within the first oligonucleotide, or the first extension segment when present. In embodiments, the 5’ region and the 3’ region of the anchor oligonucleotide are, independently, at least 100% complementary to the corresponding nucleotides within the first oligonucleotide, or the first extension segment when present.

[0084] In embodiments, the 5’ region of the anchor oligonucleotide is linked to the 3’ region of the anchor oligonucleotide through a direct bond.

[0085] In embodiments, the 5’ region of the anchor oligonucleotide is linked to the 3’ region of the anchor oligonucleotide through a linker segment. In embodiments, the linker segment is a nucleotide, an oligonucleotide between 2 and 50 nucleotides in length, or another chemical moiety, or combinations thereof. In some embodiments, the linker segment is cleavable. In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are independently selected from RNA, DNA, or a combination thereof and the nucleotides of the 3’ region of the second oligonucleotide are independently selected from RNA, DNA, or a combination thereof.

[0086] In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are DNA and the nucleotides of the 3’ region of the second oligonucleotide are DNA. In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are DNA and the nucleotides of the 3’ region of the second oligonucleotide are RNA. In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are RNA and the nucleotides of the 3’ region of the second oligonucleotide are DNA. In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are RNA and the nucleotides of the 3’ region of the second oligonucleotide are RNA.

[0087] In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are a combination of RNA and DNA and the nucleotides of the 3’ region of the second oligonucleotide are RNA. In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are a combination of RNA and DNA and the nucleotides of the 3’ region of the second oligonucleotide are DNA.

[0088] In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are RNA and the nucleotides of the 3’ region of the second oligonucleotide are a combination of RNA and DNA. In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are DNA and the nucleotides of the 3’ region of the second oligonucleotide are a combination of RNA and DNA.

[0089] In embodiments, the nucleotides of the 5’ region of the second oligonucleotide are a combination of RNA and DNA and the nucleotides of the 3’ region of the second oligonucleotide are a combination of RNA and DNA.

[0090] Preferably, it is required that at least one of the double-stranded sections formed by the hybridization between the first oligonucleotide and the second oligonucleotide, e.g., between the 5’ region of the second oligonucleotide and the first oligonucleotide or the first extension segment and / or between the 3’ region of the second oligonucleotide and the first oligonucleotide or the second extension segment, is cleavable. For example, if the nucleotides of the first oligonucleotide are RNA then, preferably, at least one of the 5’ region or the 3’ region of the second oligonucleotide is DNA thereby forming at least one RNA / DNA duplex, which is cleavable by RNase H. Likewise, if the nucleotides of the first oligonucleotide are DNA then, preferably, at least one of the 5’ region or the 3’ region of the second oligonucleotide is RNA thereby forming at least one RNA / DNA duplex, which is cleavable by RNase H. Cleavage of the duplex allows for the release of functional oligonucleotide from the RSO shape and allows the functional oligonucleotide to perform its function.

[0091] If the nucleotides of the first oligonucleotide are RNA, the 5’ region and / or the 3’ region of the second oligonucleotide can also be RNA thereby forming an RNA / RNA duplex. Such an RNA / RNA duplex is cleavable, for example, by dicer.

[0092] The nucleotides of the first oligonucleotide and / or the 5’ region and / or the 3’ region of the second oligonucleotide can be a combination of RNA / DNA so long as at least one of the duplexes maintains the ability to be cleaved.

[0093] In embodiments where the first oligonucleotide comprises a first extension segment and a second extension segment, it is preferable that at least one of the double-stranded sections formed by the hybridization between the second oligonucleotide and the first extension segment and the second extension segment is cleavable. For example, if the nucleotides of the first extension segment and the nucleotides of the second extension segment are DNA then at least one of the regions of the second anchor oligonucleotide is preferably RNA thereby forming at least one RNA / DNA duplex, which is cleavable by RNase H. If the nucleotides of the first extension segment and the nucleotides of the second extension segment are RNA then at least one of the regions of the second anchor oligonucleotide is preferably DNA thereby forming at least one RNA / DNA duplex, which is cleavable by RNase H.

[0094] If the nucleotides of the second anchor oligonucleotide is RNA then at least one of the nucleotides of the first extension segment and / or the nucleotides of the second extension segment are DNA then at least one of the regions of thereby forming at least one RNA / DNA duplex, which is cleavable by RNase H. If the nucleotides of the second anchor oligonucleotide is DNA then at least one of the nucleotides of the first extension segment and / or the nucleotides of the second extension segment are RNA then at least one of the regions of thereby forming at least one RNA / DNA duplex, which is cleavable by RNase H.

[0095] If the nucleotides of the second anchor oligonucleotide is RNA, the nucleotides of the first extension segment and / or the nucleotides of the second extension segment can also be RNA thereby forming an RNA / RNA duplex. Such an RNA / RNA duplex is cleavable by dicer.

[0096] The nucleotides of the first extension segment, the second extension segment and / or the 5’ region and / or the 3’ region of the second oligonucleotide can be a combination of RNA / DNA so long as at least one of the duplexes formed between the second anchor oligonucleotide and the first and / or second extension segments maintains the ability to be cleaved.

[0097] Cleavage of at least one of the duplexes (e.g., by RNase H or dicer) allows for the release of functional oligonucleotide from the RSO shape and allows the functional oligonucleotide to perform its function.

[0098] If the duplex formed between the second oligonucleotide and the first oligonucleotide, directly or through a first and / or second extension segment cannot be cleaved, for example if the duplex is DNA / DNA, preferably the second oligonucleotide comprises a cleavable linker segment, which, when cleave, will release the functional oligonucleotide from the RSO shape.

[0099] The only limitation on the linker segment is that it does not eliminate the essential functions of the RSO, namely (a) the ability of the first and second oligonucleotides of the RSO to form an interm olecular ring-shaped structure under the conditions of interest (e.g., physiological conditions) and (b) the ability of the functional oligonucleotide to carry out its intended function.

[0100] Preferred “other chemical moiety” linkers include, but are not limited to, C2-C6 alkyl, ethylene glycol, tri (ethylene glycol), tetra (ethylene glycol), penta (ethylene glycol), hexa (ethylene glycol) and -NH(CH2)nNH-, wherein n is 2, 3, 4, 5, or 6. Alternatively, the linker segment can be a combination of the foregoing.

[0101] In embodiments, the linker segment is an oligonucleotide between 2 and 50 nucleotides in length, or another chemical moiety, or combinations thereof.

[0102] In an embodiment, the linker is ethylene glycol. In embodiments, the linker is a C2-C6 alkyl. In embodiments, the linker is a C2 alkyl. In embodiments, the linker is a C3 alkyl. In embodiments, the linker is a C4 alkyl. In embodiments, the linker is a Cs alkyl. In embodiments, the linker is a Ce alkyl.

[0103] When every base in at least one strand of a pair of nucleic acids is found opposite its complementary base pair, such strand is considered fully complementary to its sequence in the other strand. When one, or more, bases of such a strand is found in a position where it is opposite any other base excepting its complementary base pair, that base is considered “mismatched” and the strand is considered partially complementary. Accordingly, strands can be varying degrees of partially complementary (e.g., 0%<x<100% complementary), until no bases align, at which point they are non-complementary (e.g., 0% complementary). As is readily understood and recognized by one of skill in the art, full (i.e., complete, 100%) complementarity is not required for hybridization of strands of nucleic acids e.g., oligonucleotides, antisense or otherwise).

[0104] In embodiments, the RSO according to the invention is part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0105] The pharmaceutical composition comprising the RSO of the invention may further comprise any other agent or therapy useful for treating or preventing a disease or condition and does not diminish the function of the RSO according to the invention. Agent(s) useful for treating or preventing the disease or condition includes, but is not limited to, small molecules, peptides, vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonist (e.g. antagonists of TLR3 and / or TLR7 and / or antagonists of TLR8 and / or antagonists of TLR9), chemotherapeutic agents (both traditional chemotherapy and modem 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, trans-activating factors, peptides or peptides comprising modified amino acids), or combinations thereof. Alternatively, the RSO according to the invention can 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 magnitude of the gene expression modulation of the RSO according to the invention.

[0106] In embodiments, the functional oligonucleotide comprises at least one phosphorothioate internucleotide linkage. In embodiments, at least half of the internucleotide linkages are phosphorothioate. In embodiments, all of the internucleotide linkages are phosphorothi oate .

[0107] In embodiments, the functional oligonucleotide is single-stranded.

[0108] In embodiments, the functional oligonucleotide is at least 90% complementary over its entire length to a portion of a target RNA. In embodiments, the functional oligonucleotide is at least 95% complementary over its entire length to a portion of the target RNA. In embodiments, the functional oligonucleotide is at least 97% complementary over its entire length to a portion of the target RNA. In embodiments, the functional oligonucleotide is at least 98% complementary over its entire length to a portion of the target RNA. In embodiments, the functional oligonucleotide is at least 99% complementary over its entire length to a portion of the target RNA. In embodiments, the functional oligonucleotide is at least 100% complementary over its entire length to a portion of the target RNA. In embodiments, the target RNA may be an mRNA, pre-mRNA, ncRNA, IncRNA, or microRNA. In embodiments, the target RNA is mRNA.

[0109] In embodiments, wherein the functional oligonucleotide is a guide oligonucleotide of a CRISPR-based system or an adenosine deaminase acting on RNA (ADAR) system, then the portion of the oligonucleotide that is complementary to the target RNA (complementary domain) is at least 90% complementary over its entire length to a portion of the target RNA, preferably at least 95% complementary, preferably at least 97% complementary, preferably at least 98% complementary, preferably at least 99% complementary, or preferably at least 100% complementary.

[0110] Functional Oligonucleotides

[0111] In any of the embodiments described herein the functional oligonucleotide of the RSO can be an oligonucleotide as further described below. The anchor oligonucleotide of the RSO is as described above unless otherwise noted.

[0112] Inhibition of Gene Expression

[0113] In embodiments, the invention provides a ring-shape oligonucleotide (RSO) comprising a first oligonucleotide comprising a gene modulating oligonucleotide (i.e., an oligonucleotide that can modulate the expression of a target gene). Such oligonucleotides can include, but are not limited to, an antisense oligonucleotide, a microRNA (miRNA), a miRNA mimic, a piRNA, a hnRNA, a ncRNA, a siRNA, a snRNA, a sgRNA, an esiRNA, an shRNA, or a IncRNA.

[0114] In embodiments, the invention provides a ring-shaped oligonucleotide (RSO) wherein the first oligonucleotide is a gene modulating oligonucleotide between 15 and 45 nucleotides in length and complementary to target RNA.

[0115] In embodiments, the 5’ region and the 3’ region of the second oligonucleotide are independently 4 to 30 nucleotides in length, and the 5’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 5’ end of the first oligonucleotide and the 3’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 3’ end of the first oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ring-shape.

[0116] In embodiments, the nucleotides of the functional oligonucleotide are modified. In embodiments, the invention provides a ring-shaped oligonucleotide (RSO) wherein the first oligonucleotide is a gene modulating oligonucleotide between 15 and 45 nucleotides in length and complementary to target RNA, wherein the functional oligonucleotide further comprises a first extension segment attached to its 5’ end and a second extension segment attached to its 3’ end. In embodiments, the 5’ region and the 3’ region of the second oligonucleotide are independently 4 to 30 nucleotides in length, wherein the 5’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the first extension segment and the 3’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the second extension segment, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ring-shape.

[0117] In embodiments, the nucleotides of the functional oligonucleotide are modified.

[0118] In embodiments, the modification of the functional oligonucleotide comprises at least one modified nucleobase, sugar and / or internucleotide linkage.

[0119] As shown herein, the RSOs of the invention comprising a gene modulating oligonucleotide as the functional oligonucleotide demonstrated increased potency. Furthermore, because the gene modulating oligonucleotide of the RSO lacks a free 5’ end, the RSO may be less inflammatory. This design permits gene modulating oligonucleotides (e.g., antisense oligonucleotides) to unfold, or to be cleaved, to linear structure and to be active upon in situ activation in cells where the target RNA is expressed.

[0120] In embodiments, where the functional oligonucleotide of the RSO is an antisense oligonucleotide, it is in the ring-shape until it is in the cytoplasm or the nucleus. Upon entering the cytoplasm or nucleus, the second oligonucleotide is cleaved by RNase H or Dicer, thereby linearizing the RSO and allowing for the antisense oligonucleotide of the functional oligonucleotide to bind to the target RNA. Additionally, or alternatively, upon entering the cytoplasm or nucleus, the first and second oligonucleotides “melt” apart, due to the higher affinity of the first oligonucleotide for its target RNA, thereby linearizing the RSO and allowing for the antisense oligonucleotide of the functional oligonucleotide to bind to the target RNA.

[0121] The changes from ring-shaped form to linear form could be confirmed by RNase H and / or Dicer cleavage studies or temperature melting studies. In the linear form, the functional oligonucleotide hybridizes (under physiological conditions, at a minimum) with the complementary target RNA to form a duplex. Depending on the intended mechanism of action of the functional oligonucleotide, the duplex formed would lead to modulation of translation. For example, if the duplex is a substrate for RNase H, and, in the presence of RNase H and under the proper conditions (e.g., physiological), the target RNA strand of the duplex will be cleaved by the RNase H, thereby preventing expression.

[0122] RSOs comprising an antisense oligonucleotide as the functional oligonucleotide maintain activity in cell cultures. The advantage foreseen with these RSOs is that their formation of intermolecular ring-shaped structures allows for less interaction with nontargeted macromolecules (including nucleic acids and proteins), have reduced polyanionic- related side effect. Additionally, due to ring-shaped structure, these RSOs can escape from endosomes due to a lack of a free 5’ end of the oligonucleotide to interaction with pattern recognition receptors.

[0123] The oligonucleotides of the invention are isolated oligonucleotides. The term “isolated” means altered or removed from the natural state through human intervention. For example, an oligonucleotide naturally present in a living animal is not “isolated,” but a synthetic oligonucleotide, or an oligonucleotide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated oligonucleotide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the oligonucleotide has been delivered. The oligonucleotides of the invention can comprise partially purified DNA and / or RNA, substantially pure DNA and / or RNA, synthetic DNA and / or RNA, or recombinantly produced DNA and / or RNA, as well as altered DNA and / or RNA that differs from naturally occurring DNA and / or RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the oligonucleotide or to one or more internal nucleotides of the oligonucleotide, including modifications that make the oligonucleotide resistant to nuclease digestion.

[0124] The terms “microRNA,” “miRNA,” and “MiR” are interchangeable and refer to endogenous or artificial non-coding RNAs that can regulate gene expression. It is believed that miRNAs function via RNA interference. The design of such microRNAs is within the skill of ordinary artisans.

[0125] The terms “piRNA” and “Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length. The design of such piRNAs is within the skill of ordinary artisans. Splitmer

[0126] In embodiments, the antisense oligonucleotide of the functional oligonucleotide is as described in W02020 / 191177, which is incorporated herein by reference in its entirety. In embodiments, the antisense oligonucleotide of the functional oligonucleotide is a modified oligonucleotide comprising or consisting of an antisense oligonucleotide compound 17 to 25 nucleotides in length, wherein the antisense oligonucleotide compound comprises a 3’ domain and a 5’ domain, which is contiguous with the 3’ domain, wherein the 3’ domain begins at the terminal nucleotide at the 3’ end and is 10 to 12 nucleotides in length and each nucleotide comprises a deoxyribonucleotide and a phospodiester or phosphothioate internucleotide linkage or combinations thereof; and wherein the 5’ domain begins at the first nucleotide following the 3’ domain and continues to the terminal nucleotide at the 5’ end, wherein the 5’ domain comprises unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or combinations thereof, provided that the 5’ domain comprises at least 3 modified deoxyribonucleotide or modified ribonucleotide, wherein the modified deoxyribonucleotides and / or modified ribonucleotides of the 5’ domain need not be consecutive; and wherein the modified deoxyribonucleotides and / or modified ribonucleotides of the 5’ domain prevent RNase H cleavage in the 5’ domain.

[0127] In embodiments, the 5’ domain comprises unmodified ribonucleotides, modified ribonucleotides, or combinations thereof, provided that the 5’ domain comprises at least 3 modified ribonucleotide, wherein the modified ribonucleotides of the 5’ domain need not be consecutive; and wherein the modified ribonucleotides of the 5’ domain prevent RNase H cleavage in the 5’ domain.

[0128] In embodiments, the modified deoxyribonucleotide or modified ribonucleotide comprise a modified base, a modified sugar and / or modified backbone. In embodiments, the modified deoxyribonucleotide or modified ribonucleotide comprise a modified sugar and / or modified backbone.

[0129] In embodiments, at least four of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at 5 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 6 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 7 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 8 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 9 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 10 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone.

[0130] In embodiments, at least half of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least half of the nucleotides of the 5’ domain comprise a modified ribonucleotide comprising a modified sugar and / or backbone. Such antisense oligonucleotides are referred to as “splitmer”.

[0131] In embodiments, all of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, all of the nucleotides of the 5’ domain comprise a modified ribonucleotide comprising a modified sugar and / or backbone.

[0132] When less than all of the nucleotides of the 5’ domain are modified, the modified and unmodified nucleotides are arranged so that no more than 2 unmodified nucleotides in the 5’ domain are next to each other.

[0133] In embodiments, the splitmer of the functional oligonucleotide comprises 17 to 25 linked nucleotides having at least 12 contiguous nucleobases complementary to an equal length portion of a target RNA.

[0134] In embodiments, the modified ribonucleotides of the splitmer comprise 2 ’-substituted nucleotides are as described herein. In embodiments, the 2’ -substituted nucleotides are selected from 2’ O-methyl ribonucleosides (2’-0ME) or 2 ’-methoxy ethyl ribonucleosides (2’ -MOE).

[0135] In some embodiments, the 3’ domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3’ end. In some embodiments, the 3’ domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3’ end. In some embodiments, the 3’ domain comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3’ end. In some embodiments, when the 3’ domain of the antisense oligonucleotide is 12 nucleotides in length, the antisense oligonucleotides of the invention are represented by Formula (I):

[0136] 5 ’ -NmN14N13N12Nl 1N10N9N8N7N6N5N4N3N2N1-3 ’ wherein

[0137] N is any nucleotide;

[0138] N13 through Nm comprises the 5’ domain;

[0139] Ni through N12 comprises the 3’ domain; and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0140] In some embodiments, when the 3’ domain of the antisense oligonucleotide is 11 nucleotides in length, the antisense oligonucleotides of the invention are represented by Formula (la):

[0141] 5 ’ -NmN14N13N12Nl 1N10N9N8N7N6N5N4N3N2N1-3 ’ wherein

[0142] N is any nucleotide;

[0143] N12 through Nm comprises the 5’ domain;

[0144] Ni through N11 comprises the 3’ domain; and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0145] In some embodiments, the antisense oligonucleotides of the invention are represented by Formula (lb):

[0146] 5 ’ -NmN14N13N12Nl 1N10N9N8N7N6N5N4N3N2N1-3 ’ wherein

[0147] N is any nucleotide;

[0148] N11 through Nm comprises the 5’ domain;

[0149] Ni through N10 comprises the 3’ domain; and m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0150] 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. In embodiments, the splitmer antisense oligonucleotide compound of the functional oligonucleotide further comprises a first extension segment attached to the 5’ end of the splitmer and a second extension segment attached to the 3' end of the splitmer.

[0151] In embodiments, the splitmer antisense oligonucleotide compound of the functional oligonucleotide is 17 to 25 nucleotides in length comprising at least 12 contiguous nucleobases complementary to an equal length portion of a target RNA sequence, wherein the antisense oligonucleotide compound comprises a 3’ domain and a 5’ domain, which is contiguous with the 3’ domain, wherein the 3’ domain begins at the terminal nucleotide at the 3’ end and is 10 to 12 nucleotides in length and each nucleotide comprises a deoxyribonucleotide and a phosphodiester or phosphothioate internucleotide linkage or combinations thereof; and wherein the 5’ domain begins at the first nucleotide following the 3’ domain and continues to the terminal nucleotide at the 5’ end, wherein the 5’ domain comprises unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or combinations thereof, provided that the 5’ domain comprises at least 3 modified deoxyribonucleotide or modified ribonucleotide, wherein the modified deoxyribonucleotides and / or modified ribonucleotides of the 5’ domain need not be consecutive; and wherein the modified deoxyribonucleotides and / or modified ribonucleotides of the 5’ domain prevent RNase H cleavage in the 5’ domain. In embodiments, the modified deoxyribonucleotide or modified ribonucleotide comprise a modified base, a modified sugar and / or modified backbone. In embodiments, the modified deoxyribonucleotide or modified ribonucleotide comprise a modified sugar and / or modified backbone.

[0152] In embodiments, at least four of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at 5 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 6 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 7 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 8 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 9 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least 10 of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone.

[0153] In embodiments, at least half of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone. In embodiments, at least half of the nucleotides of the 5’ domain comprise a modified ribonucleotide comprising a modified sugar and / or backbone.

[0154] In embodiments, all of the nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone.

[0155] When less than all of the nucleotides of the 5’ domain are modified, the modified and unmodified nucleotides are arranged so that no more than 2 unmodified nucleotides in the 5’ domain are next to each other.

[0156] In embodiments, the splitmer antisense oligonucleotide compound of the functional oligonucleotide further comprises a first extension segment attached to the 5’ end of the splitmer and a second extension segment attached to the 3' end of the splitmer.

[0157] In embodiments, the 3’ domain is 12 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3’ end (position 1 is the 3’ end). In embodiments, the 3’ domain is 11 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3’ end. In embodiments, the 3’ domain is 12 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3’ end.

[0158] In embodiments, the nucleotides of the 3’ domain comprise a natural nucleobase. In some embodiments, the nucleobases and sugars of the nucleotides of the 3’ domain of the antisense oligonucleotide according to the invention are unmodified. In this respect, the nucleobases and sugars of the nucleotides of the 3’ domain of the antisense oligonucleotide according to the invention are naturally occurring. Each of the nucleotides of the 3’ domain comprise a deoxyribonucleotide and a phosphodiester or phosphorothioate internucleotide linkage or combinations thereof. The nucleotides of the 3 ’-domain comprise natural deoxyribose sugar and phosphorothioate, phosphodiester or other phosphorus-based linkages or combinations thereof, which are known to activate RNase H.

[0159] In embodiments, at least one of the nucleotides of the 3’ domain comprises a modified nucleobase. In embodiments, the nucleotides at the 9thor 10thpositions from the 3’ end are not modified. In embodiments, the nucleotides at the 9thand 10thpositions from the 3’ end are not modified. In embodiments, the nucleotide at the 11thposition from the 3’ end is not modified. In embodiments, the nucleotides at the 9th, 10th, and 11thpositions from the 3’ end are not modified. In embodiments, the nucleotide at the 12thposition from the 3’ end is not modified. In embodiments, the nucleotides at the 9th, 10th, 11th, and 12thpositions from the 3’ end are not modified.

[0160] In embodiments, the splitmer oligonucleotide comprises at least one phosphorothioate internucleotide linkage. In embodiments, at least half of the internucleotide linkages are phosphorothioate. In embodiments, all of the internucleotide linkages are phosphorothioate. In embodiments, at least half of the internucleotide linkages are phosphodiester. In embodiments, all of the internucleotide linkages are phosphodiester.

[0161] In embodiments, the splitmer is single stranded.

[0162] As used here, the term “5’ domain” refers to the nucleotides beginning at the first nucleotide following the 3’ domain and goes to the 5’ end. The 5’ domain hybridizes to the target RNA but does not allow RNase H to excise the target RNA in this domain. The term “5’ domain” is generally 2 to 15 nucleotides in length and refers to the 11ththrough the 25thnucleotides (the 1stnucleotide is the 3’ end), 12ththrough the 25thnucleotides, or 13ththrough the 25thnucleotides of the antisense oligonucleotide as measured from the 3’ end depending on the length of the 3’ domain. In embodiments, the 5’ domain refers to the 11thnucleotide through the 5’ terminal nucleotide (the 1stnucleotide is the terminal nucleotide at the 3’ end) of the splitmer oligonucleotide of the functional oligonucleotide. In embodiments, the 5’ domain refers to the 12thnucleotide through the 5’ terminal nucleotide of the splitmer oligonucleotide of the functional oligonucleotide. In embodiments, the 5’ domain refers to the 13thnucleotide through the 5’ terminal nucleotide of the splitmer oligonucleotide of the functional oligonucleotide.

[0163] For example, a splitmer compound that is 20 nucleotides in length may comprise a 3’ domain from position 1 to position 12 and a 5’ domain from position 13 to position 20. Alternatively, for example, a splitmer compound that is 20 nucleotides in length may comprise a 3’ domain from position 1 to position 11 and a 5’ domain from position 12 to position 20. The designation of the modified nucleotide is position-specific, as opposed to nucleotide-specific.

[0164] The 5’ domain comprises nucleotides having non-RNase H activating modifications such as modified sugars and / or modified backbones that do not activate RNase H. In some embodiments, the 5’ domain comprises nucleotides comprising a modified sugar. In some embodiments, the 5’ domain comprises nucleotides comprising a modified backbone. In some embodiments, the 5’ domain comprises nucleotides comprising both a modified sugar and modified backbone. In embodiments, the modified backbone is a non-phosphorus-based backbone.

[0165] This design of antisense allows for targeted RNA cleavage at the specific sites towards the 5’ end of 3’ domain.

[0166] In any of these embodiments it is contemplated that at least half of the nucleotides of the 5’ domain comprise a backbone modification or substitution and / or a sugar modification or substitution. In some embodiments, the nucleotides at all positions within the 5’ domain comprises a backbone modification or substitution and / or a sugar modification or substitution. In one embodiment, the 5’ domain comprises at least four nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5’ domain comprises at least five nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5’ domain comprises at least six nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5’ domain comprises at least seven nucleotides comprising a modified backbone and / or sugar. In one embodiment, the 5’ domain comprises at least eight nucleotides comprising a modified backbone and / or sugar. In one embodiment, all of the nucleotides of the 5’ domain are nucleotides comprising a modified backbone and / or sugar.

[0167] It is specifically contemplated that embodiments discussed herein, in the context of a specific nucleotide and position, may be implemented with respect to a position relative to the 3' end. For example, an antisense oligonucleotide with 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.

[0168] In embodiments, the antisense oligonucleotide of the functional oligonucleotide is at least 90% complementary over its entire length to a portion of the target RNA.

[0169] Gapmer

[0170] In embodiments, the functional oligonucleotide is a “gapmer”. As used herein, a gapmer is a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage. Usually, the gapmers of the invention are directed against one or more mRNA encoding a target mRNA. The design of such gapmers is within the skill of ordinary artisans. In embodiments, the functional oligonucleotide is a modified oligonucleotide comprising or consisting of a region having a gapmer motif, which is defined by two external regions or "wings" and a central or internal region or "gap." The three regions of a gapmer motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3 '-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing / gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5'- wing differs from the sugar motif of the 3 '-wing (asymmetric gapmer).

[0171] In certain embodiments, the wings of a gapmer independently comprise 1-6 nucleosides. In certain embodiments, the wings of a gapmer independently comprise 1-5 nucleosides. In certain embodiments, the wings of a gapmer comprise the same number of nucleosides. In certain embodiments, the wings of a gapmer comprise 4 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0172] In certain embodiments, the gap of a gapmer comprises 7-24 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-18 nucleosides. In certain embodiments, the gap of a gapmer comprises 9-14 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-23 nucleosides. In certain embodiments, the gap of a gapmer comprises 9 nucleosides. In certain embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiments, the gap of a gapmer comprises 11 nucleosides. In certain embodiments, the gap of a gapmer comprises 13 nucleosides. In certain embodiments, the gap of a gapmer comprises 14 nucleosides. In certain embodiments, the gap of a gapmer comprises 17 nucleosides. In certain embodiments, the gap of a gapmer comprises 18 nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is an unmodified 2'-deoxy nucleoside.

[0173] In certain embodiments, the gapmer is a deoxy gapmer. In embodiments, the nucleosides on the gap side of each wing / gap junction are unmodified 2'-deoxy nucleosides and the nucleosides on the wing sides of each wing / gap junction are modified nucleosides. In certain embodiments, each nucleoside of the gap is an unmodified 2'-deoxy nucleoside. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0174] Herein, the lengths (number of nucleosides) of the three regions of a gapmer may be provided using the notation [5 of nucleosides in the 5'-wing] - [10 of nucleosides in the gap] - [5 of nucleosides in the 3'-wing], Thus, a 5-10-5 gapmer consists of 5 linked nucleosides in each wing and 10 linked nucleosides in the gap. Where such nomenclature is followed by a specific modification, that modification is the modification in the wings and the gap nucleosides comprise unmodified deoxynucleosides sugars. Thus, a 5-11-5 MOE or OMe gapmer consists of 5 linked MOE or OMe modified nucleosides in the 5'-wing, 11 linked deoxynucleosides in the gap, and 5 linked MOE or OMe nucleosides in the 3 '-wing.

[0175] In certain embodiments, modified oligonucleotides are 4-13-4 MOE or OMe gapmers. In certain embodiments, modified oligonucleotides are 5-11-5 MOE or OME gapmers. In certain embodiments, modified oligonucleotides are 3-15-3 BNA gapmers. In certain embodiments, modified oligonucleotides are 3-15-3 LNA gapmers. In certain embodiments, modified oligonucleotides in the gapmer comprise mesyl phosphonate nucleotides and / or phosphoramidate nucleotides.

[0176] In any of the embodiments described herein, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2'-modification. In certain embodiments, the uniformly modified sugar motif is 12 to 30 nucleosides in length. In certain embodiments, each nucleoside of the uniformly modified sugar motif is a 2 ’-substituted nucleoside, a sugar surrogate, or a bicyclic nucleoside. In certain embodiments, each nucleoside of the uniformly modified sugar motif comprises either a 2’-OCH2CH2OCH3 group or a 2’-OCH3 group. In certain embodiments, modified oligonucleotides having at least one fully modified sugar motif may also have at least 1, at least 2, at least 3, or at least 4 2’- deoxynucleosides. Examples of gene modulating oligonucleotides suitable for use as the functional oligonucleotide (i.e., the first oligonucleotide) of a ring-shaped oligonucleotide (RSO) for gene silencing of the invention include, but are not limited to, the gene modulating oligonucleotides of Table 1. Functional oligonucleotides directed to any other target of interest are well within the skill of one in the art. The first and second extension segments (lowercase letters) depicted in the sequences of the first oligonucleotides below can be RNA, DNA, or combinations thereof. Additionally, the first and second extension segments (lowercase letters) depicted in the sequences of the first oligonucleotides below, can be added to any other functional oligonucleotide of interest. Table 1

[0177] Uppercase G / C / A / T- DNA with phosphorothioate linkage; Underlined Uppercase G / C / A / T or G1 / C1 / A1 / U1 - 2’0ME or 2’ MOE ribonucleotide; A2 / T2 / C2 / G2 - DNA phosphodiester linkage; lowercase G / C / A / T - first and second extension segment; Underlined lowercase G / C / A / T - first and / or second extension segment RNA phosphodiester linkage. Examples of second oligonucleotides useful in the RSOs described herein include, but are not limited to, the nucleic acids of Table 2. For example, the second oligonucleotides of Table 2 bind to complementary sequences within the first oligonucleotides of Table 1 to provide the RSO of the invention. Further second oligonucleotides of the RSO, complementary to functional oligonucleotides directed to any other target of interest are well within the skill of one in the art.

[0178] Table 2 L is a nucleotidic or non-nucleotidic linker segment. Sequences can be DNA, RNA or combinations thereof. Underlined in SEQ ID NO: 44 and 46 = RNA

[0179] The hybridization between a first oligonucleotide and a second oligonucleotide provides for the RSO structure of the invention. For example, selecting SEQ ID NO: 49 from Table 1 and SEQ ID NO: 43 from Table 2: (5’-anchor oligonucleotide-3)

[0180] 5’ -gccagactctcaatcaggactctc-3’ (SEQ ID NO: 43)

[0181] 5’-gagagtctggcGiCiTiTiCiTTGTCCAGCTTiTiAiTiTigagagtcctga-3’ (SEQ ID NO: 49)

[0182] (5 ’-first extension segment-FUNCTIONAL OLIGO-second extension segment-3 ’)_ shows that the first 11 nucleotides at the 5’ end (5’ region) of the anchor oligonucleotide (i.e., 5’-gccagactctc-3’) are complementary to the first extension segment (3’-cggtctgagag-5’) and that the last 11 nucleotides at the 3’ end (3’ region) of the anchor oligonucleotide (i.e., 5’- tcaggactctc-3’) are complementary to the second extension segment (3’-agtcctgagag-5’). In this example, there is a 2 nucleotide linker segment (i.e., aa) between the 5’ region and the 3’ region of the anchor oligonucleotide. The hybridization between the first and second oligonucleotides brings the 5’ end and the 3’ end of the first oligonucleotide into proximity with each other giving a circular, ring shape. A depiction of an RSO comprising SEQ ID NO: 43 as the anchor oligonucleotide and SEQ ID NO: 49 as the first oligonucleotide is shown in Fig. 3E, wherein 302 is the functional oligonucleotide (i.e., 5’- G1C1T1T1C1TTGTCCAGCTT1T1A1T1T1-3’), 301 is the linker segment (i.e., aa), and hybridization between the 5’ and 3’ regions of the anchor oligonucleotide and the first and second extension segments are shown.

[0183] Splicing Oligonucleotides

[0184] In embodiments, the invention provides a ring shaped oligonucleotide (RSO) comprising a splicing oligonucleotide as the functional oligonucleotide. In embodiments, the splicing oligonucleotide is modified.

[0185] As used herein, the term “splicing oligonucleotide” refers to an antisense oligonucleotide for modulating splicing. For splice modulation, the antisense oligonucleotide binds to the target RNA and modulates splicing, thereby the expression of a protein. The ring-shaped structure allows reduced protein binding, reduced polyanionic characteristic, and lack of accessibility to the ends which permits endosomal escape and mitigates interaction with pattern recognition receptors.

[0186] In embodiments, the oligonucleotide for modulating splicing is a snRNA. The terms “snRNA” and “small nuclear RNA” are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors. The subclass of small nucleolar RNAs (snoRNAs) is also included. The term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs. The design of such snRNA is within the skill of ordinary artisans.

[0187] In embodiments, the splicing oligonucleotide is as described in WO 2021 / 055011, which is incorporated herein by reference in its entirety. Specifically, in embodiments, the splicing oligonucleotide comprises an oligonucleotide comprising 14 to 30 linked nucleotides complementary to a target pre-mRNA comprising a retained intron, wherein the antisense oligonucleotide comprises 1 to 3 DNA regions each region independently comprising from 2 to 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2 ’-substituted, nonionic or constrained sugar nucleotides, or combinations thereof. In embodiments, the 2’- substituted nucleotides are selected from 2’ O-methyl ribonucleosides or 2 ’-methoxy ethyl ribonucleosides (MOE). A RSO having a functional oligonucleotide comprising an oligonucleotide that modulates splicing is useful for selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, wherein the oligonucleotide comprises at least 12 contiguous nucleobases complementary to an equal length portion of a target pre-mRNA; wherein the oligonucleotide targets a splice site of the pre-mRNA for a second mRNA transcript thereby blocking the splice site for the second mRNA transcript and directing splicing of the pre- mRNA to the first mRNA transcript.

[0188] In embodiments, the splicing oligonucleotide comprises 1 region comprising from 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2 ’-substituted, nonionic or constrained sugar nucleotides, or combinations thereof. In embodiments, the splicing oligonucleotide comprises 2 regions independently comprising from 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2 ’-substituted, non-ionic, or constrained sugar nucleotides, or combinations thereof. In embodiments, the splicing comprises 3 regions independently comprising from 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2 ’-substituted, non-ionic, or constrained sugar nucleotides, or combinations thereof. In some embodiments, the consecutive deoxyribonucleotides are 2- 4 nucleotides in length. In some embodiments, the consecutive deoxyribonucleotides are 4 nucleotides in length.

[0189] In embodiments, the region of consecutive deoxyribonucleotides of the splicing oligonucleotide are at the 5’ end of the antisense oligonucleotide, at the 3’ end of the splicing oligonucleotide, or flanked by the 2 ’-substituted, non-ionic, or constrained sugar nucleotides, or combinations thereof. In embodiments, the consecutive deoxyribonucleotides are at the 5’ end of the splicing oligonucleotide. In embodiments, the consecutive deoxyribonucleotides are at the 3’ end of the splicing oligonucleotide. In embodiments, the consecutive deoxyribonucleotides are flanked by the 2 ’-substituted, non-ionic, or constrained sugar nucleotides, or combinations thereof.

[0190] In embodiments, the splicing oligonucleotide comprises 14 to 30 linked nucleotides having at least 12 contiguous nucleobases complementary to an equal length portion of a target pre- mRNA comprising a retained intron, wherein the antisense oligonucleotide comprises 1 to 3 DNA regions each region independently comprising from 2 to 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2’ -substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. In embodiments, the 2 ’-substituted nucleotides are as described herein. In embodiments, the 2 ’-substituted nucleotides are selected from 2’ O-methyl ribonucleotides or 2’ -MOE.

[0191] In embodiments, the splicing oligonucleotide comprises 1 region comprising from 2 to 5 consecutive deoxyribonucleotides. In embodiments, the consecutive deoxyribonucleotides are at the 5’ end of the splicing oligonucleotide, at the 3’ end of the antisense oligonucleotide, flanked by at the 2 ’-substituted, non-ionic, or constrained sugar nucleotides, or combinations thereof. In embodiments, the consecutive deoxyribonucleotides are at the 5’ end of the splicing oligonucleotide. In embodiments, the consecutive deoxyribonucleotides are at the 3’ end of the splicing oligonucleotide.

[0192] In embodiments, the consecutive deoxyribonucleotides are 2-4 nucleotides in length. In embodiments, the consecutive deoxyribonucleotides are 4 nucleotides in length.

[0193] In embodiments, an exon flanks the 5’ splice site of the retained intron. In embodiments, an exon flanks the 3’ splice site of the retained intron. In embodiments, an exon flanks the 5’ splice site of the retained intron and an exon flanks the 3’ splice site of the retained intron.

[0194] ADAR

[0195] In embodiments, the invention provides a ring-shaped oligonucleotide (RSO) comprising a functional oligonucleotide comprising an antisense oligonucleotide (e.g., guide RNA) of an adenosine deaminase acting on RNA (ADAR) system. ADARs are a group of enzymes that catalyze the conversion of adenosines (A’s) to inosines (I’s) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I’s base pair like guanosines (G’s). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. This ability to re-code makes ADAR an attractive therapeutic tool to correct genetic mutations within mRNA.

[0196] The recognition domain recognizes and binds to specific double-stranded RNA (dsRNA) and the catalytic domain converts adenosine to inosine in the target dsRNA through deamination. In RNA, inosine functions similarly to guanosine for translation and replication, for example because of its similarity to guanosine inosine will hybridize and / or bind to cytosine whereas prior to the deamination of the adenosine to inosine, the corresponding nucleotide pair would be thymine. Additionally, inosine is most often found to mimic guanosine for translational purposes. Thus, conversion of adenosine to inosine in an mRNA can result in a codon change that may lead to changes to the encoded protein and its functions. There are three known ADAR proteins expressed in humans, AD ARI, ADAR2, and ADAR3. AD ARI and ADAR2 are expressed throughout the body whereas ADAR3 is expressed only in the brain.

[0197] ADAR proteins are naturally expressed proteins in various cells, tissues, organs and / or organism. It has been reported that some ADAR proteins, e.g., AD ARI and ADAR2, can edit adenosine through deamination, converting adenosine to inosine which can provide a number of functions including being read as or similar to G during translation. Mechanism of ADAR-mediated mRNA editing (e.g., deamination) has been reported. For example, ADAR proteins are reported to catalyze conversion of adenosine to inosine on double-stranded RNA substrates with mismatches. As appreciated by those skilled in the art, inosine can be recognized as guanosine by cellular translation and / or splicing machinery. ADAR can thus be used for functional adenosine to guanosine editing of nucleic acids, e.g., pre-mRNA and mRNA substrates.

[0198] Without being bound by any theory or hypothesis, ADAR-recruiting RNA (“arRNA”) recognize and bind to target nucleic acids comprising adenosine for ADAR-mediated editing of target adenosine in target nucleic acids, e.g. RNA. The arRNA acts through hybridizing to its target RNA in a sequence-specific fashion to form a double-stranded RNA, which recruits an Adenosine Deaminase Acting on RNA (ADAR) to deaminate a target adenosine in the target RNA.

[0199] ADAR-mediated RNA-editing can offer several advantages over DNA-editing, e.g., delivery is simplified as expression of recombinant proteins like Cas9 is not required. Both AD ARI and ADAR2 are endogenous enzymes, so cellular delivery of arRNAs alone can be sufficient for editing. Off-target effects, if any, are transient as changes are not made to genomic DNA. Additionally, ADAR-mediated editing can be used in post-mitotic cells and it does not require an HDR-template for repair. Three vertebrate ADAR genes have been reported with common functional domains (Nishikura Nat Rev Mol Cell Biol.2016 Feb; 17(2): 83-96.; Nishikura Annu Rev Biochem.2010; 79: 321-349; Thomas and Beal Bioessays.2017 Apr;39(4)). All 3 ADARs contain a dsRNA-binding domains (dsRBD), which can contact dsRNA substrates. Some AD ARI also contains Z-DNA-binding domains. AD ARI has been reported to expressed significantly in brain, lung, kidney, liver, and heart, etc., and may occur in two isoforms. In some embodiments, isoform pl50 can be induced by interferon while isoform pl 10 can be constitutively expressed. In some embodiments, it can be beneficial to utilize pl 10 as it is reported to be ubiquitously and constitutively expressed. ADAR2 can be highly expressed, e.g., in the brain and lungs, and is reported to be exclusively localized to the nucleus. ADAR3 is reported to be catalytically inactive and expressed only in the brain. Potential differences in tissue expression can be taken into consideration when choosing a therapeutic target.

[0200] Use of oligonucleotides for RNA editing by ADAR has been reported. Among other things, the present disclosure recognizes that previously reported technologies generally suffer one or more disadvantages, such as low stability (e.g., oligonucleotides with natural RNA sugars), low editing efficiency, low editing specificity (e.g., a number of As are edited in a portion of a target nucleic acid substantially complementary to an oligonucleotide), specific structures in oligonucleotides for ADAR recognition / recruitment, exogenous proteins (e.g., those engineered to recognize oligonucleotides with specific structures and / or duplexes thereof (e.g., with target nucleic acids) for editing), etc.

[0201] In embodiments, the provided arRNA in ring-shaped format (i.e., RSO-arRNA) can direct a correction of a guanosine (G) to adenosine (A) mutation in a target sequence, or a product thereof. In some embodiments, a correction of a G to A mutation is or comprises conversion of A to inosine (I), which can be read as G during translation or other biological processes. In embodiments, the provided RSO-arRNAs can direct a correction of a G to A mutation in a target sequence or a product thereof via ADAR-mediated deamination. In embodiments, the provided RSO-arRNAs can direct a correction of a G to A mutation in a target sequence or a product thereof via ADAR-mediated deamination by recruiting an endogenous ADAR (e.g., in a target cell) and facilitating the ADAR-mediated deamination. Regardless, however, the present disclosure is not limited to any particular mechanism. In some embodiments, the present disclosure provides RSO-arRNAs, compositions, methods, etc., capable of operating via double-stranded RNA interference, single-stranded RNA interference, RNase H-mediated knock-down, steric hindrance of translation, ADAR- meidated deamination or a combination of two or more such mechanisms.

[0202] In embodiments, the RSO-arRNA can hybridize to a target RNA sequence nucleic acid in any stage of RNA processing, including but not limited to a pre-mRNA or a mature mRNA. In some embodiments, the RSO-arRNA can hybridize to any element of oligonucleotide nucleic acid or its complement, including but not limited to: a promoter region, an enhancer region, a transcriptional stop region, a translational start signal, a translation stop signal, a coding region, a non-coding region, an exon, an intron, an intron / exon or exon / intron junction, the 5' UTR, or the 3' UTR. In some embodiments, the RSO-arRNA hybridizes to two or more variants of transcripts derived from a sense strand of a target site (e.g., a target sequence).

[0203] In some embodiments, a RSO-arRNA or composition is characterized in that, when it is contacted with a target nucleic acid comprising a target adenosine in a system (e.g., an ADAR-mediated deamination system), modification of the target adenosine (e.g., deamimation of the target A) is improved relative to that observed under reference conditions (e.g., selected from the group consisting of absence of the composition, presence of a reference oligonucleotide or composition, and combinations thereof).

[0204] As appreciated by those skilled in the art, structural features of the present disclosure, such as nucleobase modification, sugar modifications, internucleotidic linkage modifications, linkage phosphorus stereochemistry, etc., and combinations thereof may be utilized with various suitable base sequences to provide RSO-arRNAs and compositions with desired properties and / or activities. For example, RSO-arRNAs for adenosine modification (e.g., conversion to I in the presence of ADAR proteins) typically have sequences that are sufficiently complementary to sequences of target nucleic acids that comprise target adenosines. In many embodiments, e.g., for targeting G to A mutations, RSO-arRNAs may selectively target one and only one target adenosine for modification, e.g., by ADAR to convert into I.

[0205] Base sequences of the RSO-arRNA, as appreciated by those skilled in the art, typically have sufficient lengths and complementarity to their target nucleic acids, e.g., RNA transcripts (e.g., pre-mRNA, mature mRNA, etc.) for, e.g., site-directed editing of target adenosines. In some embodiments, the arRNA of the RSO-arRNA is complementary to a portion of a target RNA sequence comprising a target adenosine (as appreciated by those skilled in the art, in many instances target nucleic acids are longer than the arRNA of the RSO-arRNAs of the present disclosure, and complementarity may be properly assessed based on the shorter of the two).

[0206] As appreciated by those skilled in the art, the RSO-arRNAs of the invention may be utilized to improve oligonucleotides in prior technologies (e.g., those described in WO2016097212, WO2017220751, WO2018041973, W02018134301A1, WO2021071858, WO 2022 / 091100, oligonucleotides and oligonucleotide compositions of each of which are independently incorporated by reference). In some embodiments, the present disclosure provides improvements of prior technologies by applying the RSO-arRNAs described herein to prior reported oligonucleotide base sequences. In some embodiments, the present disclosure provides RSO-arRNA compositions of previously reported oligonucleotides that may be useful for adenosine editing. In some embodiments, the present disclosure provides improvements of previously reported adenosine editing oligonucleotide compositions by performing such editing using RSO-arRNA compositions.

[0207] In some embodiments, a RSO-arRNA can form a dsRNA structure with a target mRNA through base pairing. In some embodiments, formed dsRNA structures (optionally with secondary mismatches) contain bulges that promote ADAR binding and therefore, can facilitate ADAR-mediated editing (e.g., deamination of a target adenosine). Various technologies may be utilized to assess / characterize RSO-arRNAs in accordance with the present disclosure.

[0208] In some embodiments, the functional oligonucleotide is a guide RNA that recruit endogenous ADAR (adenosine deaminase acting on RNA) enzymes to edit endogenous transcripts.

[0209] The guide RNA of an ADAR system itself comprises one or more domains. One domain, known as the complementary domain, comprises a region of consecutive nucleotides that are complementary to the target RNA. In embodiments, the complementary domain is from about 15 to about 120 nucleotides in length. In embodiments, the complementary domain is from about 17 to about 60 nucleotides in length. Another domain, known as the recruiting domain, comprises a region of the oligonucleotide that recruits ADAR enzymes.

[0210] CRISPR

[0211] In embodiments, the invention provides a ring-shaped oligonucleotide (RSO) comprising a functional oligonucleotide comprising an antisense oligonucleotide (e.g., guide RNA) of a CRISPR-based system.

[0212] The CRISPR / Cas system (“CRISPR system”) utilizes a short RNA molecule (e.g., a guideRNA) to recognize a specific DNA target and to recruit a Cas enzyme to the specific DNA target.

[0213] In general, a CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (guideRNA), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.

[0214] In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guideRNA is designed to have complementarity, where hybridization between a target sequence and a guideRNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. In an aspect of the invention the recombination is homologous recombination.

[0215] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guideRNA hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guideRNA. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex.

[0216] As with the target sequence, a complete complementarity is not needed, provided there is sufficient hybridization and / or binding to be functional. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when aligned.

[0217] In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guideRNA linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5- with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding “sequence” of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guideRNA, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one in iron, or ail in a single intron). In some embodiments, the CRISPR enzyme, guideRNA, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

[0218] Non-limiting examples of Cas proteins include Casl, Casl B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof or modified versions thereof. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.

[0219] In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863 A. In aspects of the invention, nickases may be used for genome editing via homologous recombination.

[0220] In general, a guideRNA is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

[0221] In some embodiments, a guideRNA is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guideRNA to be tested, may be provided to a host cell having the corresponding target sequence, such, as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guideRNA to be tested and a control guideRNA different from the test guideRNA, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. A guideRNA may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell.

[0222] The terms “gRNA” and “guideRNA” are interchangeable and refer to a specific RNA sequence that recognizes the target DNA or RNA region of interest and directs an endonuclease to that location for gene editing.

[0223] The gRNA is usually made up of two parts: crispr RNA (crRNA), a 17-30 nucleotide sequence complementary to the target DNA, and a tracr RNA, which serves as a binding scaffold for the Cas nuclease. Any suitable engineered gRNA, or crRNA and tracrRNA, can be employed as long as it is effective for recognizing a target DNA or RNA. The design of such gRNA, or crRNA and tracrRNA is within the skill of ordinary artisans.

[0224] The present invention provides a RSO comprising a guideRNA for a CRISPR-based system as the functional domain (“RSO-gRNA”). RSO-gRNAs according to the invention are described herein.

[0225] The RSO-gRNAs according to the invention can be used in a CRISPR system as known to one skilled in the art. For example, suitable CRISPR systems can include, but are not limited to WO 2013 / 176772, WO 2013 / 188638, WO 2014 / 018423, WO 2014 / 022702, WO 2014 / 093661, WO 2014 / 093622, WO 2017 / 004279, WO 2017 / 189308, WO 2018 / 119354, WO 2018 / 208998, WO 2019 / 089804, WO 2019 / 089910, WO 2019 / 089808, WO 2019 / 089796, WO 2019 / 089820, and WO 2021 / 087394, the contents of each of which are herein incorporated by reference in their entireties.

[0226] The functional oligonucleotide is an antisense oligonucleotide that functions as a guideRNA for a CRISPR-based system. The terms “sgRNA” and “guideRNA” are interchangeable and refer to a specific RNA sequence that recognizes the target DNA or RNA region of interest and directs the endonuclease there for editing. The gRNA is usually made up of two parts: crispr RNA (crRNA), a 17-30 nucleotide sequence complementary to the target DNA, and a tracr RNA, which serves as a binding scaffold for the Cas nuclease.

[0227] Prime-editors (PEs) represent another addition to the CRISPR genome-engineering toolkit and represents an approach to expand the scope of donor-free precise DNA editing to not only all transition and transversion mutations, but small insertion and deletion mutations as well (Anzalone et al., Nature. 2019; 576: 149-157). Collectively, DNA base-editing and prime-editing tools enable precise nucleotide substitutions in a programmable manner, without requiring a donor template. Many DNA base-editing and prime-editing techniques are reviewed in Kantor et al. (Int J Mol Sci. 2020 September; 21(17): 6240).

[0228] As with CRISPR-mediated base-editing, prime-editing does not rely on DSBs. Primeeditors use an engineered reverse transcriptase fused to Cas9 nickase and a prime-editing guide RNA (pegRNA). Importantly, the pegRNA differs significantly from regular sgRNAs and plays a major role in the system's function. The pegRNA contains not only (a) the sequence complimentary to the target sites that directs nCas9 to its target sequence, but also (b) an additional sequence spelling the desired sequence changes (Anzalone et al., Nature. 2019; 576:149-157). The 5' of the pegRNA binds to the primer binding site (PBS) region on the DNA, exposing the non-complimentary strand. The unbound DNA of the PAM- containing strand is nicked by Cas9, creating a primer for the reverse transcriptase (RT) that is linked to nCas9. The nicked PAM-strand is then extended by the RT by using the interior of the pegRNA as a template, consequently modifying the target region in a programmable manner. The result of this step is two redundant PAM DNA flaps: the edited 3' flap that was reverse transcribed from the pegRNA and the original, unedited 5' flap. The choice of which flap hybridizes with the non-PAM containing DNA-strand is an equilibrium process, in which the perfectly complimentary 5' would likely be thermodynamically favored. However, the 5' flaps are preferentially degraded by cellular endonucleases that are ubiquitous during lagging-strand DNA synthesis (Hosfield et al., Cell. 1998; 95: 135-146). Finally, the resulting heteroduplex containing the unedited strand and edited 3' flap is resolved and stably integrated into the host genome via cellular replication and repair process.

[0229] In embodiments, the functional oligonucleotide is an antisense oligonucleotide that functions as a prime editing guide RNA (pegRNA).

[0230] Any suitable engineered sgRNA, pegRNA, or crRNA and tracrRNA, can be employed as long as it is effective for recognizing a target DNA or RNA. The design of such sgRNA, pegRNA, or crRNA and tracrRNA is within the skill of ordinary artisans. mRNA

[0231] In embodiments, the invention provides a ring-shaped (RSO) comprising a functional oligonucleotide comprising a nucleic acid molecule, specifically a polynucleotide, primary constructs and / or mRNA which encode one or more polypeptides of interest or fragments thereof and which retains sufficient structural and / or chemical features to allow the polypeptide of interest encoded therein to be translated. Such polypeptides of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more regions or parts or the whole of a polynucleotide. As used herein, the term "polypeptides of interest" refer to any polypeptide which is selected to be encoded within, or whose function is affected by, the polynucleotides of the present invention.

[0232] In preferred embodiments, the nucleic acid molecule is a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0233] In embodiments, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail.

[0234] In embodiments, the mRNA molecule can comprise one or more internal ribosome entry sites (IRES), which are cv.s-acting RNA sequences able to mediate internal entry of the 40S ribosomal subunit on some eukaryotic and viral messenger RNAs upstream of a translation initiation codon.

[0235] In embodiments, the mRNA molecules comprise one or more structural and / or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. As used herein, a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification to the polynucleotide.

[0236] In embodiments, the mRNA functional oligonucleotide comprises a first region of linked nucleotides that is flanked by a first flanking region and a second flaking region. This first region may include, but is not limited to, the encoded polypeptide of interest. The polypeptide of interest may comprise at its 5' terminus one or more signal sequences encoded by a signal sequence region. The first flanking region may comprise a region of linked nucleotides comprising one or more complete or incomplete 5' UTRs sequences. The first flanking region may also comprise a 5' terminal cap. The second flanking region may comprise a region of linked nucleotides comprising one or more complete or incomplete 3' UTRs. The second flanking region may also comprise a 3' tailing sequence.

[0237] In embodiments, a first operation region bridges the 5' terminus of the first region and the first flanking region. This operational region comprises a Start codon. The operational region may alternatively comprise any translation initiation sequence or signal including a Start codon.

[0238] In embodiments, a second operation region bridges the 3' terminus of the first region and the second flanking region. This second operational region comprises a Stop codon. The second operational region may alternatively comprise any translation initiation sequence or signal including a Stop codon. In embodiments, multiple serial stop codons may also be used.

[0239] In embodiments, the shortest length of the first region of the primary construct of the present invention can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10- 20 amino acids. The length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.

[0240] Generally, the length of the first region encoding the polypeptide of interest of the present invention is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). As used herein, the "first region" may be referred to as a "coding region" or "region encoding" or simply the "first region."

[0241] In some embodiments, mRNA includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1 ,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1 ,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

[0242] In embodiments, the first and second flanking regions may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[0243] In embodiments, the tailing sequence may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional. In embodiments, the capping region may comprise a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent.

[0244] In embodiments, the first and second operational regions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and / or Stop codon, one or more signal and / or restriction sequences.

[0245] In embodiments, the mRNA functional oligonucleotide may be designed to encode polypeptides of interest selected from any of several target categories including, but not limited to, biologies, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. In embodiments, the mRNA functional oligonucleotide may encode variant polypeptides which have a certain identity with a reference polypeptide sequence. As used herein, a "reference polypeptide sequence" refers to a starting polypeptide sequence. Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence. The polypeptide variant may have the same or a similar activity as the reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide. Generally, variants of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.

[0246] AAV

[0247] In embodiments, the invention provides a ring-shaped oligonucleotide (RSO) comprising a functional oligonucleotide comprising an adeno-associated virus (AAV) vector. Wild-type AAV is a small, non-enveloped parvovirus (~25 nm) with a ~4.7-kb singlestranded DNA (ssDNA) genome. Recombinant AAV vectors exploited as delivery vehicles generally contain the same capsid components and structure as wild-type AAV but all viral coding sequences are replaced by therapeutic gene expression cassettes, maximizing packing capacity and reducing immunogenicity.

[0248] In some embodiments, the functional oligonucleotide is an AAV vector. In some embodiments, the functional oligonucleotide is a recombinant AAV vector. In embodiments, the AAV vector has been further modified to include a gene expression cassette to express one or more proteins or nucleic acids of interest, such as an antisense oligonucleotide, a microRNA (miRNA), an siRNA, a piRNA, an hnRNA, an ncRNA, an snRNA, a miRNA mimic, an sgRNA, an esiRNA, an shRNA, a IncRNA, an mRNA, a guide RNA for a CRISPR-based system, a guide RNA for prime editing, a guide RNA for an adenosine deaminase acting on RNA (ADAR) system, an immunomodulatory oligonucleotide, immune antagonist oligonucleotide, or a splicing oligonucleotide.

[0249] Immunostimulatory Oligonucleotides

[0250] In embodiments, the invention provides a ring-shaped oligonucleotide (RSO) comprising a functional oligonucleotide comprising an immunostimulatory oligonucleotide.

[0251] In embodiments, the immunostimulatory oligonucleotide is capable of inducing an interferon response in a vertebrate cell.

[0252] In embodiments, the nucleotide sequence of the immunostimulatory oligonucleotide is not complementary to and does not bind to another nucleotide sequence, for example, a target RNA. In this embodiment, the nucleotide sequence of the immunostimulatory oligonucleotide is not, for example, an antisense oligonucleotide and does not have antisense activity.

[0253] In embodiments, the immunostimulatory oligonucleotide is between 11 and 40 nucleotides in length. In embodiments, immunostimulatory oligonucleotide is between 15 and 28 nucleotides in length. In embodiments, immunostimulatory oligonucleotide is between 17 and 25 nucleotides in length.

[0254] In embodiments, the internucleotide linkages of the immunostimulatory oligonucleotide are phosphorothioate, phosphodiester, or combinations thereof.

[0255] In embodiments, the internucleotide linkages of the immunostimulatory oligonucleotide are phosphorothioate intemucleotide linkages.

[0256] In embodiments, the internucleotide linkages of the immunostimulatory oligonucleotide are phosphodiester. In embodiments, the internucleotide linkages of the immunostimulatory oligonucleotide are a combination of phosphorothioate and phosphodiester internucleotide linkages.

[0257] Immunostimulatory oligonucleotides include, but are not limited to, oligonucleotides that induce immunostimulation through endosomal toll-like receptors, RIG like receptors, STING, cGAS and inflammasomes. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) which play a crucial role in the initiation of innate immune response by detecting potential harmful pathogens. Each TLR has a broad range of specificities, for example, TLR1, 2, 4 and 6 recognize bacterial lipids, TLR3, 7 and 8 recognize viral RNA, TLR9 recognizes bacterial DNA comprising a CG motif, and TLR5 and 10 recognize bacterial or parasite proteins. The design of such immunostimulatory oligonucleotides is within the skill of ordinary artisans.

[0258] Exemplary first oligonucleotides comprising an immunostimulatory oligonucleotide functional oligonucleotide include, but are not limited to, 5’-gagagtctggcTCGACTTGAACGATTCGAATACGTAACgagagtcctga-3’ (SEQ ID NO: 61); 5’-gagagtetggcTCGACTTGAACGATTCGAATACGTAACgagagtcctga-3’ (SEQ ID NO: 62); 5 '-gagagtctggcTCGACTTGA ACGATTCGA ATACGTA ACgagagtcctga-3 ’ (SEQ ID NO: 63); 5’-gagagtctggcTCGACTTGAACGATTCGAATACGTAACgagagtcctga-3’; (SEQ ID NO: 64) 5’-aatctgtgatcctTCGACTTGAACGATTCGAATACGTAACgttcagtgcttct-3’; (SEQ ID NO: 65) 5 ’ -aatctgtgatcctTCGACTTGAACGATTCGAATACGTAACgttcagtgcttct-3 ’ (SEQ ID NO: 66); 5 ’-aatctgtgatcctTCGACTTGAACGATTCGAATACGTAACgttcagtgcttct-3 ’ (SEQ ID NO: 67); 5 '-aatctgtgatcctTCGACTTGA ACGATTCGA ATACGTA ACgttcagtgcttct-3 ’ (SEQ ID NO: 68); 5’-gagagtctggcGTTACGTATCGAATCGTTCAAGTCGAgagagtcctga-3’ (SEQ ID NO: 69); 5’-gagagtctggcTCGTAACGTTACGTATTCGAATCGT-gagagtcctga-3’ (SEQ ID NO: 70); 5’-aatctgtgatcctGTTACGTATCGAATCGTTCAAGTCGAgttcagtgcttct-3’ (SEQ ID NO: 71); and 5’ - aatctgtgatcctTCGTAACGTTACGTATTCGAATCGT-gttcagtgcttct-3’ (SEQ ID NO: 72); where Uppercase G / C / A / T- DNA with phosphorothioate linkage; lowercase G / C / A / T - extension segment comprising RNA, DNA or combinations thereof; Underlined lowercase G / C / A / T - extension segment RNA phosphodiester linkage. These first oligonucleotides can duplex with an anchor oligonucleotide, e.g., SEQ ID NOs: 43-46 in Table 2, to form an RSO of the invention.

[0259] It is also contemplated that other unmodified or modified DNA and / or RNA sequences that act as pathogen-associated molecular patterns (PAMPs) for various pattern recognition receptors (PRRs) can be used as the functional oligonucleotide of the invention to modulate an immune response. PRRs for nucleic acid-based PAMPs are well known to one of skill in the art and can include, but are not limited to, toll-like receptors (TLRs), rig-like receptors (RIG-I), inflammasomes, and STING / cGAS. The most relevant TLRs for nucleic acid PAMPs include TLR3, TLR7, TLR8 and TLR9. Each receptor recognizes PAMPs based on nucleotide sequence, structure, and motifs, leading to varying immune modulation.

[0260] In embodiments, ring-shaped PAMPs could consist of DNA and RNA sequences and their modified versions. The functional domain and extension domain can be single-stranded, double-stranded, or combinations of both. The anchor domain itself could be a sequence that acts as a PAMP.

[0261] Immune Antagonist

[0262] In embodiments, the invention provides a ring-shaped oligonucleotide (RSO) comprising a functional oligonucleotide comprising an immune antagonist oligonucleotide.

[0263] In embodiments, the immune antagonist oligonucleotide is capable of blocking an interferon response in a vertebrate cell.

[0264] In embodiments, the nucleotide sequence of the immune antagonist oligonucleotide is not complementary to and does not bind to another nucleotide sequence, for example, a target RNA. In this embodiment, the nucleotide sequence of the immune antagonist oligonucleotide is not, for example, an antisense oligonucleotide and does not have antisense activity.

[0265] In embodiments, the immune antagonist oligonucleotide is between 11 and 30 nucleotides in length. In embodiments, immune antagonist oligonucleotide is between 15 and 28 nucleotides in length. In embodiments, immune antagonist oligonucleotide is between 17 and 25 nucleotides in length.

[0266] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotide are phosphorothioate, phosphodiester, or combinations thereof.

[0267] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotide are phosphorothioate intemucleotide linkages.

[0268] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotide are phosphodiester.

[0269] In embodiments, the internucleotide linkages of the immune antagonist oligonucleotide are a combination of phosphorothioate and phosphodiester internucleotide linkages.

[0270] Immune antagonist oligonucleotides include, but are not limited to, oligonucleotides that block immunostimulation through endosomal toll-like receptors, RIG like receptors, STING, cGAS and inflammasomes. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) which play a crucial role in the initiation of innate immune response by detecting potential harmful pathogens. Each TLR has a broad range of specificities, for example, TLR1, 2, 4 and 6 recognize bacterial lipids, TLR3, 7 and 8 recognize viral RNA, TLR9 recognizes bacterial DNA comprising a CG motif, and TLR5 and 10 recognize bacterial or parasite proteins. The design of such immune antagonist oligonucleotides is within the skill of ordinary artisans.

[0271] Exemplary first oligonucleotides comprising an immune antagonist oligonucleotide functional oligonucleotide include, but are not limited to, 5’-gagagtctggcTCmGACTTGAACmGATTCmGAATACmGTAACgagagtcctga-3’ (SEQ ID NO: 73); 5’-gagagtctggcGTTACmGTATTCmGAATCmGTTCAACmGTTGAgagagtcctga-3’ (SEQ ID NO: 74); 5’-aatctgtgatcctTCmGACTTGAACmGATTCmGAATACmGTAACgttcagtgcttct-3’ (SEQ ID NO: 75); and 5’-aatctgtgatcctGTTACmGTATTCmGAATCmGTTCAACmGTTGAgttcagtgcttct-3’ (SEQ ID NO: 76); where Uppercase G / C / A / T- DNA with phosphorothioate linkage; lowercase G / C / A / T - extension segment comprising RNA, DNA or combinations thereof; Cmis 5-methyl-C. These first oligonucleotides can duplex with an anchor oligonucleotide, e.g., SEQ ID NOs: 43-46 in Table 2, to form an RSO of the invention.

[0272] Pharmaceutical composition

[0273] In certain embodiments, described herein are pharmaceutical compositions comprising one or more RSO compounds of the invention. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more RSO compounds. In certain embodiments, a pharmaceutical composition consists of a sterile saline solution and one or more RSO compounds. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more RSO compounds and sterile water. In certain embodiments, a pharmaceutical composition consists of one RSO compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more RSO compounds and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more RSO compounds and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS. In certain embodiments, pharmaceutical compositions comprise one or more RSO compounds and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

[0274] In certain embodiments, RSO compounds of the invention may be admixed with pharmaceutically acceptable active and / or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0275] Conjugate Groups

[0276] In certain embodiments, the RSOs according to the invention optionally further comprise one or more conjugate groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the one or more conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and / or at any internal position. In certain embodiments, conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3' and / or 5'-end of oligonucleotides. In certain such embodiments, conjugate groups are attached at the 3'-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3'-end of oligonucleotides. In certain embodiments, conjugate groups are attached at the 5'-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5'-end of oligonucleotides.

[0277] In any embodiment herein, the conjugate group comprises a GalNAc cluster comprising 1-3 GalNAc ligands.

[0278] In any embodiment herein, the conjugate linker consists of a single bond.

[0279] In any embodiment herein, the conjugate linker is cleavable.

[0280] In any embodiment herein, the conjugate linker comprises 1-3 linker-nucleosides.

[0281] In any embodiment herein, the conjugate group is attached to the RSO at the terminal end of the first nucleic acid molecule and / or the second nucleic acid molecule. The terminal end of the first or second nucleic acid molecule is the end not bound to the oligonucleotide of the RSO of the invention. In certain embodiments, the RSOs are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the RSO, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the RSO, e.g., fluorophores or reporter groups that enable oligonucleotide detection. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Set. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl- S-trityl thiol (Manoharan et al., Ann. N. Y. Acad. Set., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol 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), a phospholipid, e.g., di-hexadecyl- rac-glycerol or triethyl-ammonium l,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), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734- 740), or a GalNAc cluster (e.g., WO2014 / 179620).

[0282] Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0283] In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (5)- (+)-pranoprofen, carprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, fmgolimod, flufenamic acid, folinic acid, a benzothiadi azide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

[0284] Conjugate moieties are attached to the RSO through conjugate linkers. In certain embodiments, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to the RSO through 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, nucleosides, or amino acid units.

[0285] In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.

[0286] In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on the RSO and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0287] Examples of conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0288] In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides. In certain embodiments, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5 -methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0289] As used herein, linker-nucleosides are not considered part of the RSO in general or part of the 5’ region or 3’ region of the anchor oligonucleotide in particular. Accordingly, the nucleotides of a linker-nucleosides are not counted toward the length of the RSO or the regions thereof and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.

[0290] In certain embodiments, it is desirable for a conjugate group to be cleaved from the RSO. For example, in certain circumstances, RSOs comprising a particular conjugate moiety are better taken up by a particular cell type, but once the RSO has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent RSO. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

[0291] In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.

[0292] Use

[0293] Ring-shaped oligonucleotides of the invention could be useful for various mechanisms of action. These include antisense for mRNA, ncRNA, microRNA, IncRNA and splicing. Also, the double stranded ring-shaped structure could be used to deliver siRNA constructs. In addition, the ring-shaped structure could provide novel approach to deliver antisense for ADAR and CRISPR-based mechanisms. Furthermore, the ring-shaped structure could provide a novel approach to deliver antisense to disrupt structures of RNA to increase translation. Ring-shaped structures also provide defined structures to nucleic acids thereby allowing varying degree of interaction with PRRs and induced immune cascade.

[0294] For example, when the functional oligonucleotide is an antisense oligonucleotide, RSOs according to the invention are also useful in therapeutic approaches in which inhibition of gene expression is desired. This can include, for example, inhibition of an endogenous gene (e.g., an oncogene) or an exogenous gene (e.g., a gene essential for growth and / or metabolism of a pathogen).

[0295] In embodiments, the invention provides a method for inhibiting gene expression comprising administering a ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide.

[0296] In embodiments, the invention provides a method for inhibiting allele-specific gene expression comprising administering a ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide.

[0297] The method according to the invention are useful for treating a subject having disease or disorder wherein inhibiting expression of a gene would be beneficial. In embodiments, the disease or disorder results from abnormal expression or product of a cellular gene.

[0298] In embodiments, the ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide is administered locally.

[0299] In embodiments, the ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide is administered systemically.

[0300] In embodiments, a method for modulating RNA processing comprising administering a RSO compound as described herein wherein the functional oligonucleotide comprises an antisense oligonucleotide comprising 14 to 30 linked nucleotides having at least 12 contiguous nucleobases complementary to an equal length portion of a target RNA, wherein the antisense oligonucleotide comprises 1 to 3 DNA regions each region independently comprising from 2 to 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2 ’-substituted, non-ionic or constrained sugar nucleotides, or combinations thereof. In embodiments, processing of RNA comprises splicing.

[0301] In embodiments, the invention provides a method for selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, the method comprising administering a ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide.

[0302] In embodiments, the invention provides a method of treating a disease or disorder in a subject wherein modulating RNA processing would be beneficial to treat the subject, the method comprising administering a ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide.

[0303] In embodiments, the invention provides a method of inducing nonsense mediated decay of a target RNA comprising administering a ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide.

[0304] In embodiments, the invention provides a method of increasing a level of mRNA encoding a protein or a functional mRNA and increasing expression of the protein or the functional mRNA comprising administering a ring-shaped oligonucleotide as described herein or a composition comprising the ring-shaped oligonucleotide.

[0305] In another aspect, the present disclosure provides a method of treating a subject in need thereof comprising administering a therapeutically effective amount of a composition comprising the circular RNA polynucleotide disclosed herein,

[0306] In another aspect, the present disclosure provides a method of treating a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein.

[0307] In another aspect, the present application provides a method of treating or preventing a disease, disorder, or condition, comprising administering an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the disease, disorder, or condition is associated with aberrant expression, activity, or localization of a polypeptide Also disclosed herein is RNA therapy, along with associated compositions and methods. In some embodiments, the RNA therapy allows for increased RNA stability, expression, and prolonged half-life, among other things. In certain aspects, provided herein is a method of treating and / or preventing a condition, e.g., an autoimmune disorder or cancer.

[0308] Also provided herein are methods comprising administration of a RSO provided herein into cells for therapy or production of useful proteins, for example, when the functional oligonucleotide is a mRNA. In some embodiments, the method is advantageous in providing the production of a desired polypeptide inside eukaryotic cells with a longer halflife than linear RNA, due to the resistance of the circular RNA to ribonucleases. Accordingly, also provided herein are methods of transfecting one or more target cells. Such methods generally comprise the step of contacting the one or more target cells with the RSO compounds and / or pharmaceutical compositions disclosed herein such that the one or more target cells are transfected with the RSO. As used herein, the terms “transfect” or “transfection” refer to the intracellular introduction of one or more RSOs into a cell, or preferably into a target cell. The term “transfection efficiency” refers to the relative amount of such RSOs up-taken by, introduced into and / or expressed by the target cell which is subject to transfection.

[0309] In embodiments where the functional oligonucleotide is a a guide RNA for a CRISPR-based system or a guide RNA for an adenosine deaminase acting on RNA (ADAR) system, the invention also provides a RSO of the invention for use in a method for making a change in a target DNA or RNA sequence in a mammalian, preferably human cell, as described herein. Similarly, the invention provides the use of a RSO of the invention in the manufacture of a medicament for making a change in a target DNA or RNA sequence in a mammalian, preferably human cell, as described herein.

[0310] In embodiments, the method of modifying a target DNA comprises delivering to a cell or a subject a RSO as described herein wherein the functional oligonucleotide is a guide RNA for a CRISPR-based system. In embodiments, the invention comprises a method or use for modulation of a target gene comprising, administering or delivering a RSO wherein the functional oligonucleotide is a guide RNA for a CRISPR-based system, compositions, or pharmaceutical formulations as described herein. In some embodiments, the modulation is editing of the target gene. In some embodiments, the modulation is a change in expression of the protein encoded by the target gene. As used herein, a “gene editing” or “genetic modification” is a change at the DNA level, e.g., induced by a gRNA / Cas complex. A gene editing or genetic modification may comprise an insertion, deletion, or substitution (base substitution, e.g., C-to-T, or point mutation), typically within a defined sequence or genomic locus. A genetic modification changes the nucleic acid sequence of the DNA. A genetic modification may be at a single nucleotide position. A genetic modification may be at multiple nucleotides, e.g., 2, 3, 4, 5 or more nucleotides, typically in close proximity to each other, e.g., contiguous nucleotides. In some embodiments, the method or use results in gene editing. In some embodiments, the method or use results in a double-stranded break (DSB) within the target gene. In some embodiments, the method or use results in formation of indel mutations during non-homologous end joining of the DSB. In some embodiments, the method or use results in an insertion or deletion of nucleotides in a target gene. In some embodiments, the insertion or deletion of nucleotides in a target gene leads to a frameshift mutation or premature stop codon that results in a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in a target gene leads to a knockdown or elimination of target gene expression. In some embodiments, the method or use comprises homology directed repair of a DSB. In some embodiments, the method or use further comprises delivering to the cell a template, wherein at least a part of the template incorporates into a target DNA at or near a double strand break site induced by the nuclease. In some embodiments, the method or use results in a single strand break within the target gene. In some embodiments, the method or use results in a base change, e.g., by deamination, within the target gene. The gene editing typically occurs within or adjacent to the portion of the target gene with which the spacer sequence forms a duplex. In some embodiments, the method or use results in gene modulation. In some embodiments, the gene modulation is an increase or decrease in gene expression, a change in methylation state of DNA, or modification of a histone subunit. In some embodiments, the method or use results in increased or decreased expression of the protein encoded by the target gene.

[0311] In certain other embodiments of the present disclosure, the invention provides a method for introducing a genetic modification in cell. The genetic modification comprises one or more introduced in-frame stop codon mutations or other mutations for disrupting expression (e.g., by base editing splice junctions, start codons, promoter sequence motifs and the like). In some embodiments, the genetic modification is introduced using a gene editing technique, optionally wherein the gene editing technique is selected from the group consisting of CRISPR-Cas9 gene editing, prime editing, and base editing.

[0312] In some embodiments, the invention provides for prime editing, which is a precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. In embodiments, the pegRNA is in RSO format. As used herein, “genetic modification” refers to a site of genomic DNA that has been genetically edited or manipulated using any molecular biological method, e.g., methods described herein, e.g., by delivering to a site of genomic DNA an endonuclease and at least one guide RNA (gRNA). Examples of genetic modifications include insertions, deletions, mutations, duplications, inversions, and translocations, and combinations thereof. In some embodiments, a genetic modification is a deletion. In some embodiments, a genetic modification is an insertion. In other embodiments, a genetic modification is an insertiondeletion mutation (or indel), such that the reading frame of the target gene is shifted leading to an altered gene product or no gene product.

[0313] In some embodiments where the functional oligonucleotide of the RSO of the invention is a guide RNA for an adenosine deaminase acting on RNA (ADAR) system, the invention also relates to a method for the deamination of at least one specific target adenosine present in a target RNA sequence in a cell or subject. In embodiments, said method including the steps of providing said cell or subject with a RSO described herein comprising a guide RNA for an adenosine deaminase acting on RNA (ADAR) system as the functional oligonucleotide; allowing uptake by the cell of the RSO; allowing annealing of the RSO to the target RNA sequence; allowing a mammalian ADAR enzyme including a natural dsRNA binding domain as found in the wild type enzyme to deaminate said target adenosine in the target RNA sequence to an inosine; and optionally identifying the presence of the inosine in the RNA sequence. In embodiments, the invention provides methods to treat disorders for which deamination of an adenosine in an mRNA produces a therapeutic result, e.g., in a subject in need thereof.

[0314] In any of the methods described herein, the ring-shaped oligonucleotide (RSO) of the invention may be administered alone or in combination with any other agent or therapy. Agents or therapies can be co-administered or administered concomitantly. Such agent or therapy may be useful for treating or preventing the disease or condition and does not diminish the gene expression modulation effect of the ring-shaped oligonucleotide according to the invention. Agent(s) useful for treating or preventing the disease or condition includes, but is not limited to, small molecules, peptides vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonist (e.g. antagonists of TLR3 and / or TLR7 and / or antagonists of TLR8 and / or antagonists of TLR9), chemotherapeutic agents (both traditional chemotherapy and modem targeted therapies), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and co-stimulatory molecules (e.g. cytokines, chemokines, protein ligands, trans-activating factors, peptides or peptides comprising modified amino acids), or combinations thereof. Alternatively, the ring-shaped oligonucleotide according to the invention can be administered in combination with other compounds (for example lipids or liposomes) to enhance the specificity or magnitude of the gene expression modulation of the ring-shaped oligonucleotide according to the invention.

[0315] In any of the methods described herein, the ring-shaped oligonucleotide of the invention may be administered by any suitable route, including, without limitation, parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intratumoral, intravenous, subcutaneous, intrathecal, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form. In any of the methods according to the invention, administration of the ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, can be directly to a tissue or organ such as, but not limited to, the bladder, liver, lung or kidney. In certain embodiments, administration of the ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by intramuscular administration. In certain embodiments, administration of ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by mucosal administration. In certain embodiments, administration of ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by oral administration. In certain embodiments, administration of ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by intrarectal administration. In certain embodiments, administration of ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by intrathecal administration. In certain embodiments, administration of ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by intratumoral administration. In certain embodiments, administration of ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by parenteral administration. In certain embodiments, administration of ring-shaped oligonucleotide according to the invention, alone or in combination with any other agent, is by subcutaneous administration.

[0316] In embodiments, any of the ring-shaped oligonucleotide described herein can be conjugated with a moiety that provides for site specific delivery of the RSO. In embodiments, such conjugates include, but are not limited to, an antibody, a peptide, a lipid, or a small molecule. Peptides can be linear or cyclic. Conjugation of these moieties can be to the anchor oligonucleotide or the first oligonucleotide at either the extension segment or the functional oligonucleotide.

[0317] In embodiments, the moiety that provides for site specific delivery of the RSO is an antibody.

[0318] The antibody is not particularly limited as long as it is an antibody that can recognize antigens present on the surface of target cells. It is preferable that the antigen present on the surface of target cells be an antigen specific for the target cells, not expressed or expressed in a small amount in normal cells.

[0319] Here, it is sufficient that the “antibody” be an antibody including at least a heavy chain variable domain and a light chain variable domain, and it may be a complete antibody or a fragment of a complete antibody that is an antigen-binding fragment having an antigenrecognition site. The complete antibody has two full length light chains and two full length heavy chains, and respective light chains and heavy chains are linked by disulfide bonds. The complete antibody includes IgA, IgD, IgE, IgM and IgG, and IgG includes IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4 as subtypes. In addition, it is preferable that the antibody be a monoclonal antibody. The antibody moiety and the linker moiety are linked via a sulfhydryl group obtained by reducing a disulfide bond in the antibody.

[0320] Examples of one aspect of mAb include, but are not limited to, brentuximab, trastuzumab, inotuzumab, gemtuzumab, glembatumumab, labetuzumab, sacituzumab, lifastuzumab, indusatumab, polatuzumab, pinatuzumab, coltuximab, indatuximab, milatuzumab, rovalpituzumab, anetumab, tisotumab, mirvetuximab, lorvotuzumab, rituximab, depatuxizumab, denintuzumab, telisotuzumab, vandortuzumab, sofituzumab, vorsetuzumab, mirvetuximab, naratuximab, cantuzumab, laprituximab, bivatuzumab, vadastuximab, lupartumab, aprutumab, abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atorolimumab, avelumab, azintuxizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camrelizumab, caplacizumab, capromab, carlumab, carotuximab, catumaxomab, cedelizumab, certolizumab, cetuximab, citatuzumab, cixutumumab, clenoliximab, clivatuzumab, codrituzumab, conatumumab, concizumab, cosfroviximab, crenezumab, crizanlizumab, crotedumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab, daratumumab, dectrekumab, demcizumab, denosumab, detumomab, dezamizumab, dinutuximab, diridavumab, domagrozumab, dorlimomab, drozitumab, duligotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enavatuzumab, enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frunevetmab, fulranumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gevokizumab, gilvetmab, girentuximab, golimumab, guselkumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifabotuzumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, inebilizumab, infliximab, inolimomab, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, lacnotuzumab, lampalizumab, lanadelumab, landogrozumab, larcaviximab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifatuzumab, ligelizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab, losatuximab, lucatumumab, lulizumab, lumretuzumab, lutikizumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, minretumomab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, motavizumab, moxetumomab, muromonab, nacolomab, namilumab, naptumomab, narnatumab, natalizumab, navicixizumab, navivumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab, oregovomab, oreticumab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, perakizumab, pertuzumab, pexelizumab, pidilizumab, placulumab, plozalizumab, ponezumab, porgaviximab, prezalumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, refanezumab, regavirumab, remtolumab, reslizumab, rilotumumab, rinucumab, risankizumab, rivabazumab, robatumumab, roledumab, romosozumab, rontalizumab, rosmantuzumab, rovelizumab, rozanolixizumab, ruplizumab, samalizumab, sarilumab, satralizumab, satumomab, secukinumab, selicrelumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sontuzumab, stamulumab, sulesomab, suptavumab, suvizumab, suvratoxumab, tabalumab, tadocizumab, talizumab, tamtuvetmab, tanezumab, taplitumomab, tarextumab, tavolixizumab, fanolesomab, nofetumomab, pintumomab, tefibazumab, telimomab, telisotuzumab, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tezepelumab, tigatuzumab, tildrakizumab, timigutuzumab, timolumab, tocilizumab, tomuzotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vantictumab, vanucizumab, vapaliximab, varisakumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, votumumab, vunakizumab, tacatuzumab, zalutumumab, zanolimumab, ziralimumab, zolimomab or anti-embigin antibody; examples of another aspect thereof include brentuximab, trastuzumab, inotuzumab, gemtuzumab, labetuzumab, polatuzumab, coltuximab, indatuximab, anetumab, rituximab, denintuzumab, laprituximab, vadastuximab, glembatumumab, cetuximab, alemtuzumab, depatuxizumab or anti-embigin antibody; examples of another aspect thereof include brentuximab, trastuzumab, rituximab or anti- embigin antibody; and examples of another aspect thereof include brentuximab or trastuzumab.

[0321] Examples of another aspect of mAb include, but are not limited to, anti-TfR antibody, anti-19A antibody, anti-AXL antibody, anti-BCMA antibody, anti-C4.4a antibody, anti-CA6 antibody, anti-CA9 antibody, anti-CA-125 antibody, anti-cadherin-6 antibody, anti-CD166 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD27 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD40 antibody, anti-CD41 antibody, anti-CD44v6 antibody, anti-CD51 antibody, anti-CD52 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD79 antibody, anti-CD79b antibody, anti-CEACAM5 antibody, anti-c-Met antibody, anti-DLL3 antibody, anti-DPEP3 antibody, anti-EGFR antibody, anti-EGFRvIII antibody, anti-ENPP3 antibody, anti-EpCAM antibody, anti-EphA4 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FTL3 antibody, anti-folate receptor a antibody, anti- gripican 3 antibody, anti-gpNMB antibody, anti-HER2 antibody, anti-HER3 antibody, anti- IL-3RA antibody, anti-LAMPl antibody, anti-LIV-1 antibody, anti-LRRC15 antibody, anti- Ly6E antibody, anti-mesothelin antibody, anti-MUC-16 antibody, anti-NaPi2b antibody, anti- nectin-4 antibody, anti-CD352 antibody, anti-P-cadherin antibody, anti-PMSA antibody, antiprotein tyrosine kinase 7 antibody, anti-SLITRK antibody, anti-STEAPl antibody, anti- CD138 antibody, anti -tissue factor antibody, anti-CD71 antibody, anti-TIM-1 antibody, anti- Trop2 antibody, anti-5T4 antibody, anti-B7-H3 antibody, anti-CD163 macrophage receptor antibody, anti-CD38 antibody, anti-CD48 antibody, anti-cKit antibody, anti -guanyl ate cyclase C antibody, anti-gastrin releasing peptide antibody, anti-solute carrier antibody, antitumor-associated MUC-1 antibody, anti-GD2 antibody, anti- a4p7 integrin antibody or anti- embigin antibody. Examples of another aspect of mAb include anti-CD19 antibody, anti- CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD52 antibody, anti-CD70 antibody, anti-CD79b antibody, anti-CEACAM5 antibody, anti-EGFR antibody, anti-EGFRvIII antibody, anti-gpNMB antibody, anti-HER2 antibody, anti- mesothelin antibody, anti-CD138 antibody, anti-CD38 antibody or anti-GD2 antibody. Examples of another aspect of mAb include anti-CD19 antibody, anti-CD20 antibody, anti- CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD52 antibody, anti-CD79b antibody, anti-CEACAM5 antibody, anti-EGFR antibody, anti-EGFRvIII antibody, anti- gpNMB antibody, anti-HER2 antibody, anti-mesothelin antibody or anti-CD138 antibody.

[0322] In embodiments, any of the ring-shaped oligonucleotide described herein can be encapsulated with a moiety that provides for site specific delivery of the RSO. In embodiments, the RSO can be encapsulated in, for example, a lipid, lipid nanoparticles (LNP), or peptide macrocyclic structures.

[0323] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Administration of the ring-shaped oligonucleotides according to the invention can be carried out using known procedures using an effective amount and for periods of time effective to reduce symptoms or surrogate markers of the disease. For example, an effective amount of a ring-shaped oligonucleotide according to the invention for treating a disease and / or disorder could be that amount necessary to alleviate or reduce the symptoms, or delay or ameliorate a tumor, cancer, or bacterial, viral or fungal infection. In the context of administering a composition that modulates gene expression, an effective amount of a ring-shaped oligonucleotide according to the invention is an amount sufficient to achieve the desired modulation as compared to the gene expression in the absence of the antisense oligonucleotide according to the invention. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular oligonucleotide being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular ringshaped oligonucleotide without necessitating undue experimentation.

[0324] Synthesis

[0325] RSOs described herein can be prepared by any suitable art recognized method including, but not limited to, H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H- phosphonate chemistry for some cycles and phosphoramidite chemistry for other cycles), which can be carried out manually or by an automated synthesizer. The oligonucleotides of the invention may also be modified in a number of ways without compromising their ability to hybridize to their target (see e.g., Agrawal and Gait, Advances in Nucleic Acid Therapeutics, (2019) https: / / doi.org / 10.1039 / 9781788015714).

[0326] Definitions

[0327] Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

[0328] Unless otherwise indicated, the following terms have the following meanings: As used herein, "2'-deoxynucleoside" means a nucleoside comprising 2'-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[0329] As used herein, "2 '-substituted nucleoside" means a nucleoside comprising a 2 '- substituted sugar moiety. As used herein, "2 '-substituted" in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.

[0330] As used herein, “5 -methyl cytosine” means a cytosine modified with a methyl group attached to the 5-position. A 5-methyl cytosine is a modified nucleobase.

[0331] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0332] As used herein, "administering" means providing a pharmaceutical agent to an animal.

[0333] As used herein, "animal" means a human or non-human animal.

[0334] As used herein, "individual in need thereof refers to a human or non-human animal selected for treatment or therapy that is in need of such treatment or therapy.

[0335] As used herein, "antisense activity" means any detectable and / or measurable change attributable to 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 a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound. In certain embodiments, antisense activity is an increase in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.

[0336] As used herein, "antisense compound" means an oligomeric compound capable of achieving at least one antisense activity.

[0337] As used herein, "ameliorate" in reference to a treatment means improvement in at least one symptom relative to the same symptom in the absence of the treatment. In certain embodiments, amelioration is the reduction in the severity or frequency of a symptom or the delayed onset or slowing of progression in the severity or frequency of a symptom. In certain embodiments, the symptom or hallmark is ataxia, neuropathy, and aggregate formation. In certain embodiments, amelioration of these symptoms results in improved motor function, reduced neuropathy, or reduction in number of aggregates.

[0338] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety. As used herein, "bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the 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 comprise a furanosyl moiety.

[0339] As used herein, "chirally enriched population" means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom. Chirally enriched populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the molecules are compounds comprising modified oligonucleotides.

[0340] As used herein, "cleavable moiety" means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.

[0341] As used herein, the term “complementary” refers to a pair of nucleobases (or simply a “base”) that hydrogen bond to each other in preference to other heterocyclic bases under selected (e.g., physiological) conditions (or some degree of complementarity thereof as context may require in the instance of assessing “complementary-ness” of oligonucleotides). When the nucleobases are modified or unmodified, natural or synthetic purines and pyrimidines, the term “complementary” means complementary in the Watson Crick sense. In embodiments, at least 70% of the nucleobases of the oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases refers to nucleobases that are capable of forming hydrogen bonds with one another.

[0342] Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5 -methyl cytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, "fully complementary" or " 100% complementary" in reference to oligonucleotides means that oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide. As used herein, "conjugate group" means a group of atoms that is directly or indirectly attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

[0343] As used herein, "conjugate linker" means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

[0344] As used herein, "conjugate moiety" means a group of atoms that is attached to an oligonucleotide via a conjugate linker.

[0345] As used herein, "contiguous" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to each other in a sequence.

[0346] As used herein, "linker-nucleoside" means a nucleoside that links, either directly or indirectly, the RSO of the invention to a conjugate moiety. Linker-nucleosides are located within the conjugate linker and are not considered part of the RSO compound even if they are contiguous with the RSO.

[0347] As used herein, "gapmer" means a modified oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings." Unless otherwise indicated, "gapmer" refers to a sugar motif. Unless otherwise indicated, the sugar moieties of the nucleosides of the gap of a gapmer are unmodified 2'-deoxyfuranosyl. Thus, the term "MOE gapmer" indicates a gapmer having a sugar motif of 2'-M0E nucleosides in both wings and a gap of 2'-deoxynucleosides. Unless otherwise indicated, a MOE gapmer may comprise one or more modified internucleoside linkages and / or modified nucleobases and such modifications do not necessarily follow the gapmer pattern of the sugar modifications.

[0348] As used herein, "hotspot region" is a range of nucleobases on a target nucleic acid amenable to oligomeric compounds for reducing the amount or activity of the target nucleic acid as demonstrated in the examples herein below.

[0349] As used herein, "hybridization" means the pairing or annealing of complementary oligonucleotides and / or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

[0350] As used herein, the term "internucleoside linkage" is the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein "modified internucleoside linkage" means any internucleoside linkage other than a phosphodiester internucleoside linkage. "Phosphorothioate linkage" is a modified intemucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester internucleoside linkage is replaced with a sulfur atom.

[0351] As used herein, the phrase "inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.

[0352] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

[0353] As used herein, "mismatch" or "non-complementary" means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligomeric compound are aligned.

[0354] As used herein, "MOE" means methoxy ethyl. "2'-M0E" means a 2'-OCH2CH2OCH3 group in place of the 2’ OH group of a ribosyl sugar moiety.

[0355] As used herein, "motif1means the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages, in an oligonucleotide.

[0356] As used herein, "mRNA" means an RNA transcript that encodes a protein and includes pre-mRNA and mature mRNA unless otherwise specified.

[0357] As used herein, "nucleobase" means an unmodified nucleobase or a modified nucleobase. As used herein an "unmodified nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). As used herein, a "modified nucleobase" is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase. A "5 -methylcytosine" is an example of a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. Modified bases, also referred to as heterocyclic base moieties, include other nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, 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-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),

[0358] 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines), 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3 -deazaadenine.

[0359] In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5- hydroxymethyl cytosine, 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, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other

[0360] 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2- amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3- deazaguanine and 3 -deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certain embodiments, the modified nucleobase is a 5-methylcytosine.

[0361] Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2', 3' or 4' positions and sugars having substituents in place of one or more hydrogen atoms of the sugar. In certain embodiments, the sugar is modified by having a substituent group at the 2' position. In additional embodiments, the sugar is modified by having a substituent group at the 3' position. In other embodiments, the sugar is modified by having a substituent group at the 4' position. It is also contemplated that a sugar may have a modification at more than one of those positions, or that an antisense oligonucleotide may have one or more nucleotides with a sugar modification at one position and also one or more nucleotides with a sugar modification at a different position.

[0362] Sugar modifications contemplated in an oligonucleotide include, but are not limited to, a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl. In some embodiments, these groups may be chosen from: O(CH2)xOCH3, O((CH2)xO)yCH3, O(CH2)XNH2, O(CH2)XCH3, O(CH2)XONH2, and O(CH2)xON((CH2)xCH3)2, where x and y are independently from 1 to 10.

[0363] In some embodiments, the modified sugar comprises a substituent group selected from the following: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, Cl, Br, CN, OCN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an antisense oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide, and other substituents having similar properties. In one embodiment, the modification includes 2'- methoxyethoxy (2'-O-CH2CH2OCH3, which is also known as 2'-O-(2 -methoxyethyl) or 2'- MOE) (Martin et al., 1995), that is, an alkoxyalkoxy group. Another modification includes 2'- dimethylaminooxyethoxy, that is, a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE and 2'-dimethylaminoethoxy ethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), that is, 2'-O-CH2-O-CH2-N(CH3)2.

[0364] Additional sugar substituent groups include allyl (-CH2-CH=CH2), -O-allyl CH2- CH=CH2), methoxy (-O-CH3), aminopropoxy (-OCH2CH2CH2NH2), and fluoro (F). Sugar substituent groups on the 2' position (2'-) may be in the arabino (up) position or ribo (down) position. One 2'-arabino modification is 2'-F. Other similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligomeric compounds may also have sugar mimetics, for example, cyclobutyl moieties, in place of the pentofuranosyl sugar. Examples of U.S. patents that disclose the preparation of modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, which are herein incorporated by reference in its entirety.

[0365] Representative sugar substituent groups include groups described in U.S. Patent Application Publication 2005 / 0261218, which is hereby incorporated by reference. In particular embodiments, the sugar modification is a 2'-0-Me modification, a 2' F modification, a 2' H modification, a 2' amino modification, a 4' thioribose modification or a phosphorothioate modification on the carboxy group linked to the carbon at position 6', or combinations thereof.

[0366] In certain embodiments, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2 '-substituent group selected from: F, OCH3, and OCH2CH2OCH3.

[0367] Certain modified sugar moieties comprise a substituent that bridges 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 comprises a bridge between the 4' and the 2' furanose ring atoms. 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' (referred to as “constrained ethyl” or “cEt”), 4’-CH2-O-CH2-2’, 4’-CH2-N(R)-2’, 4'-CH(CH2OCH3)-O-2' (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. 7,399,845, Bhat et al, U.S. 7,569,686, Swayze et al., U.S. 7,741,457, and Swayze et al, U.S. 8,022,193), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,283), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al, U.S. 8,278,425), 4'-CH2-O-N(CH3)-2' (see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al, U.S. 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou, et al, J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see e.g., Seth et al., U.S. 8,278,426), 4’-C(RaRb)-N(R)-O-2’, 4 -C(RaRb)-O-N(R)-2’, 4'-CH2-O-N(R)-2', and 4'-CH2- N(R)-0-2', wherein each R, Ra and Rb is, independently, H, a protecting group, or C1-C12 alkyl (see, e.g. Imanishi et al., U.S. 7,427,672).

[0368] In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, -C(Ra)=C(Rb)-, - C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)X-, and -N(Ra)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Raand Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ1J2, SJi, N3, COOJi, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-Ji), or sulfoxyl (S(=O)-Ji); and each Ji and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0369] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J Am. Chem. Soc, 20017, 129, 8362-8379;Wengel et al., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al., U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133; Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; and Ramasamy et al., U.S. 6,525,191; Torsten et al., WO 2004 / 106356; Wengel et al., WO 1999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750, 131; Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421; Seth et al., U.S. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008 / 0039618 and Migawa et al., US2015 / 0191727.

[0370] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the a-L configuration or in the P-D configuration. a-L-methyleneoxy (4'-CH2-0-2') or a-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the P-D configuration, unless otherwise specified.

[0371] In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5 '-substituted and 4'-2' bridged sugars).

[0372] In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and / or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4'-sulfur atom and a substitution at the 2'-position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and / or the 5' position.

[0373] In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid ("HNA"), anitol nucleic acid ("ANA"), manitol nucleic acid ("MNA") (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:

[0374] F4I A

[0375] ("F-HNA", see e.g., Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803;

[0376] Swayze et al., U.S. 8,796,437; and Swayze et al., U.S. 9,005,906; F-HNA can also be referred to as a F-THP or 3'-fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula: wherein, independently, for each of said modified THP nucleoside:

[0377] Bx is a nucleobase moiety;

[0378] T3 and T4 are each, independently, an intemucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleoside linking group linking the modified THP nucleoside to the remainder of an 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; qi, q2, qs, q4, qs, qe and q? are each, independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and each of Ri and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, S Ji, N3, OC(=X)Ji, OC(=X)NJih, NJ3C(=X)NJiJ2, and CN, wherein X is O, S or NJi, and each Ji, J2, and J3 is, independently, H or Ci-Ce alkyl.

[0379] In certain embodiments, modified THP nucleosides are provided wherein qi, q2, q3, q4, qs, qe and q? are each H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q? is other than H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q? is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of Ri and R2 is F. In certain embodiments, Ri is F and R2 is H, in certain embodiments, Ri is methoxy and R2 is H, and in certain embodiments, Ri is methoxy ethoxy and R2 is H.

[0380] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moi eties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506). As used here, the term "morpholino" means a sugar surrogate having the following structure:

[0381] In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0382] In certain embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid ("PNA"), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011 / 133876.

[0383] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

[0384] The nucleoside residues of the first and / or second oligonucleotides of the RSO can be coupled to each other by any of the numerous known intemucleoside linkages. The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing intemucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond ("P=O") (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S"), and phosphorodithioates ("HS-P=S"). Representative non-phosphorus containing intemucleoside linking groups include but are not limited to methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-Sifb-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)- N(CHs)-). Methods of preparation of phosphorous-containing and non-phosphorous- containing intemucleoside linkages are well known to those skilled in the art.

[0385] Such intemucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone intemucleoside linkages. In some embodiments, the synthetic antisense oligonucleotides of the invention may comprise combinations of internucleotide linkages. In some embodiments, the synthetic antisense oligonucleotides of the invention may comprise combinations of phosphorothioate and phosphodiester internucleotide linkages. In some embodiments more than half but less that all of the internucleotide linkages are phosphorothioate internucleotide linkages. In some embodiments all of the internucleotide linkages are phosphorothioate intemucleotide linkages.

[0386] Modified oligonucleotides comprising intemucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom intemucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise phosphorothioate intemucleoside linkages wherein all of the phosphorothioate intemucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate internucleoside linkages in a particular, independently selected stereochemical configuration.

[0387] In certain embodiments, the phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be made stereoregular or substantially stereoregular in either Rp or Sp form. In embodiments where the linkages are mixed Rp and Sp enantiomers, the Rp and Sp forms may be at defined places within the oligonucleotide or randomly placed throughout the oligonucleotide.

[0388] As used herein, "nucleobase sequence" means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or intemucleoside linkage modification.

[0389] As used herein, "nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, "modified nucleoside" means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase. "Linked nucleosides" are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are presented between those that are linked).

[0390] The term "nucleic acid," in its broadest sense, includes any compound and / or substance that comprise a polymer of nucleotides. These polymers are often referred to as oligonucleotides or polynucleotides. Exemplary nucleic acids of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization) or hybrids thereof.

[0391] As used herein, "oligomeric compound" means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A "singled-stranded oligomeric compound" is an unpaired oligomeric compound. As used herein, "oligonucleotide" means a strand of linked nucleosides of any length, including polynucleotides, connected via intemucleoside linkages, wherein each nucleoside and intemucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides.

[0392] As used herein, "modified oligonucleotide" means an oligonucleotide, wherein at least one nucleoside or intemucleoside linkage is modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not comprise any nucleoside modifications or intemucleoside modifications.

[0393] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.

[0394] As used herein "pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0395] As used herein "pharmaceutical composition" means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an antisense compound and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.

[0396] As used herein, "phosphorus moiety" means a group of atoms comprising a phosphorus atom. In certain embodiments, a phosphorus moiety comprises a mono-, di-, or tri- phosphate, or phosphorothioate.

[0397] As used herein, "polypeptide" means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0398] The term "polypeptide variant" refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.

[0399] As used herein "prodrug" means a therapeutic agent in a form outside the body that is converted to a different form within an animal or cells thereof. Typically, conversion of a prodrug within the animal is facilitated by the action of an enzyme (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and / or by physiologic conditions.

[0400] As used herein, "OMe" means methoxy. "2'-0Me" means a 2'-OCH3 group in place of the 2’ OH group of a ribosyl sugar moiety.

[0401] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0402] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 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.

[0403] As used herein, "reducing or inhibiting the amount or activity" refers to a reduction or blockade of the transcriptional expression or activity relative to the transcriptional expression or activity in an untreated or control sample and does not necessarily indicate a total elimination of transcriptional expression or activity. As used herein, "self-complementary" in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself.

[0404] As used herein, "standard cell assay" means the assay described in Example 1 and reasonable variations thereof.

[0405] As used herein, "stereorandom chiral center" in the context of a population of molecules of identical molecular formula means a chiral center having a random stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules having the (S) configuration of the stereorandom chiral center may be but is not necessarily the same as the number of molecules having the (R) configuration of the stereorandom chiral center. The stereochemical configuration of a chiral center is considered random when it is the result of a synthetic method that is not designed to control the stereochemical configuration. In certain embodiments, a stereorandom chiral center is a stereorandom phosphorothioate internucleoside linkage.

[0406] As used herein, "sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" means a 2'-0H(H) furanosyl moiety, as found in RNA (an "unmodified RNA sugar moiety"), or a 2'-H(H) moiety, as found in DNA (an "unmodified DNA sugar moiety"). Unmodified sugar moieties have 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 a sugar surrogate. As used herein, modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.

[0407] As used herein, "sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.

[0408] As used herein, "target nucleic acid" and "target RNA" mean a nucleic acid that an antisense compound is designed to affect. As used herein, "target region" means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

[0409] As used herein, "terminal group" means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.

[0410] As used herein, "therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal. For example, a therapeutically effective amount improves a symptom of a disease.

[0411] As used herein, "treat", “treatment”, or "treating" refers to administering a compound described herein to effect an alteration or improvement of a disease, disorder, or condition.

[0412] “Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

[0413] The term “co-administration” or “co-administered” generally refers to the administration of at least two different substances. Co-administration refers to simultaneous administration, as well as temporally spaced order of up to several days apart, of at least two different substances in any order, either in a single dose or separate doses.

[0414] The term “in combination with” generally means administering an oligonucleotide- based compound according to the invention and another agent useful for treating a disease or condition that does not abolish the activity of the compound in the course of treating a patient. Such administration may be done in any order, including simultaneous administration, as well as temporally spaced order from a few seconds up to several days apart. Such combination treatment may also include more than a single administration of the compound according to the invention and / or independently the other agent. The administration of the compound according to the invention and the other agent may be by the same or different routes.

[0415] The term “individual” or “subject” or “patient” generally refers to a mammal, such as a human. The term “mammal” is expressly intended to include warm blooded, vertebrate animals, including, without limitation, humans, non-human primates, rats, mice, cats, dogs, horses, cattle, cows, pigs, sheep and rabbits. As used herein, "individual in need thereof refers to a human or non-human animal selected for treatment or therapy that is in need of such treatment or therapy. As used herein, "inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity of a RNA or protein and does not necessarily indicate a total elimination of expression or activity. Examples

[0416] Synthesis of RSO Comprising an Antisense Oligonucleotide Functional Oligonucleotide

[0417] Ring-shaped oligonucleotides according to the invention can be synthesized by procedures that are well known in the art, such as phosphoramidate or H-phosphonate chemistry which can be carried out manually or by an automated synthesizer. For example, the oligonucleotides of the invention may be synthesized by a linear synthesis approach.

[0418] Synthesis protocols using phosphoramidite chemistry are described in detail, for example in pubs. rsc.org / en / content / chapter / bk9781788012096-00453 / 978- 1-78801-209-6, which is incorporated herein by reference.

[0419] An anchoring oligonucleotide is mixed at a 1.5 to 1 molar ratio with the first oligonucleotide, heated, and then allowed to cool to anneal to form the ring shape before using in experiments. The concentration of the mixed oligos is calculated based on the mixture.

[0420] Inhibition of target RNA by RSO Comprising an Antisense Oligonucleotide Functional Oligonucleotide

[0421] Ring-shaped oligonucleotides can be designed targeting a PCSK9 nucleic acid, for example, and tested for their effects on PCSK9 mRNA in vitro.

[0422] Hepa 1-6 cells can be cultured in DMEM medium plus 10% FBS and lOOU / ml Pen / Strep (cells from ATCC). The cells can be seeded and allowed to incubate overnight until they are -70% confluent at the time of transfection - -100,000 cells / ml / 12 well plate. Cell media can be changed and 900 pl can be added to each well. The oligonucleotides can be mixed with Lipofectamine in Opti-MEM medium, added to lipid (1 : 1 ratio) and incubated for 15-20 minutes. lOOpl can be added to each well for an antisense concentration of 100 nM. After a treatment period of about 16 to 48 hours, cells can be harvested for RNA and / or protein analysis.

[0423] Culture supernatants can be assayed for AK release cytotoxicity assay. Taqman probes for mPCSK9 and PPIB or HPRT1 (housekeeping controls) can be used (probes provided by ThermoFisher).

[0424] Ring-shaped oligonucleotides can be designed targeting a PNPLA3 nucleic acid, for example, and tested for their effects on PNPLA3 mRNA in vitro.

[0425] Human HepG2 cells can be cultured following ATCC recommended condition and media (Eagle's Minimum Essential Medium with 10% FBS). Cells can be plated in PDL coated 96 well plates at 50K / well density and reverse transfected with 0.6ul / well RNAiMax and oligonucleotide compounds of indicated concentration. 24 hrs later, cells can be harvested using Cells to CT lysis reagent (ThermoFisher 4391851C). RNA can be reverse transcribed to the cDNA templates using the RT reagent kit (ThermoFisher A39110). Quantitative PCR can be performed using qPCR Master Mix (ThermoFisher 4444964). PNPLA3 expression level can be quantified using the PNPLA3-FAM probe (ThermoFisher 4351368 Assay ID: Hs00228747) and normalized with the housekeeping gene POLR2A (ThermoFisher, 4448491, Assay ID: HsOl 108291). Data can be analyzed in GraphPad Prism.

[0426] To see if a linear ASO with modified RNA in a splitmer format, as described herein, could provide further efficacy in the ring-shaped form, the level of PNPLA3 knockdown using oligonucleotides having the same base sequence in the different splitmer format and chemistry arrangement can be compared.

[0427] Ring-shaped nucleic acids were designed targeting an APOC3 (ApoCIII) nucleic acid and tested for their effects on APOC3 mRNA in vitro. Hep3B cells (human hepatic origin) were cultured following ATCC recommended conditions and media (Eagle's Minimum Essential Medium with 10% FBS). Cells were plated in 96 well plates at 20K / well density and reverse transfected with 0.3ul / well RNAiMax and anchor oligonucleotides, linear first oligonucleotides comprising antisense oligonucleotides targeting ApoC3, or RSOs thereof. The RSOs were prepared by mixing the anchoring oligonucleotide with the first oligonucleotide at a 1.5 to 1 molar ratio using IX PBS. The mixture was heated at 95 degrees C for 2 minutes, and then cooled to 25 degree C to perform the annealing process. The annealed RSOs were transfected at eight different concentrations (25, 5, 1, 0.20, 0.04, 0.008, 0.0016, and 0.00032 nM). The final concentration of the RSOs was calculated based on the concentration of the first (functional) oligonucleotide. Control cells were transfected with 0.3ul / well RNAiMax and media alone and incubated for 48 hours post-transfection. Gene expression was assayed using the Invitrogen™ TaqMan™ Fast Advanced Cells-to-CT™ Kit following the manufacturer’s protocol (A35378, Invitrogen™). In brief, cells were harvested using the Cells to CT lysis reagent and immediately following total RNA was reverse transcribed into cDNA using the RT reagents. Quantitative polymerase chain reaction (qPCR) was performed via multiplexed reactions using qPCR Fast Advanced Master Mix and pre-designed primers and FAM-labelled probes (4351370, Invitrogen™) for the corresponding gene of interest (GOI) and normalized using pre-designed primers and VIC- labeled probes (#4448486, Invitrogen™) for the reference gene Hypoxanthine-guanine phosphoribosyltransferase (HPRT1). Data was plotted and analyzed using the ‘Absolute IC50’ nonlinear regression dose-response model of GraphPad Prism. Results are shown in Fig. 4A through Fig. 4H.

[0428] Ring-shaped nucleic acids were designed targeting a MAPT nucleic acid and tested for their effects on MAPT mRNA in vitro. U-251 MG cells were cultured following ATCC recommended conditions and media (Eagle's Minimum Essential Medium with 10% FBS). Cells were plated in 96 well plates at 20K / well density and reverse transfected with 0.3ul / well RNAiMax and anchor oligonucleotides, linear first oligonucleotides comprising antisense oligonucleotides targeting MAPT, or RSOs thereof. The RSOs were prepared by mixing the anchoring oligonucleotide with the first oligonucleotide at a 1.5 to 1 molar ratio using IX PBS. The mixture was heated at 95 degrees C for 2 minutes, and then cooled to 25 degree C to perform the annealing process. The annealed RSOs were transfected at eight different concentrations (25, 5, 1, 0.20, 0.04, 0.008, 0.0016, and 0.00032 nM). The final concentration of the RSOs was calculated based on the concentration of the first (functional) oligonucleotide. Gene expression was assayed using the Invitrogen™ TaqMan™ Fast Advanced Cells-to-CT™ Kit following the manufacturer’s protocol (A35378, Invitrogen™). In brief, cells were harvested using the Cells to CT lysis reagent and immediately following total RNA was reverse transcribed into cDNA using the RT reagents. Quantitative polymerase chain reaction (qPCR) was performed via multiplexed reactions using qPCR Fast Advanced Master Mix and pre-designed primers and FAM-labelled probes (4351370, Invitrogen™) for the corresponding gene of interest (GOI) and normalized using predesigned primers and VIC-labeled probes (#4448486, Invitrogen™) for the reference gene Hypoxanthine-guanine phosphoribosyltransferase (HPRT1). Data was plotted and analyzed using the ‘Absolute IC50’ nonlinear regression dose-response model of GraphPad Prism. Results are shown in Fig. 5A through Fig. 5D.

[0429] Generation of Immune Response by RSO Comprising an Immunostimulatory Oligonucleotide Functional Oligonucleotide

[0430] Mouse solenocyte restimulation assay

[0431] Spleens from C57BL / 6J mice can be mechanically dispersed into single-cell suspensions, with ammonium chloride lysis buffer (Cat # 420302, BioLegend, San Diego, CA) used to remove erythrocytes. Cell viability can be determined using vital dye stain and an automated cell counting system (Countess 3, Thermo Fisher, Waltham, MA). Following counting, IxlO5viable mouse splenocytes can be seeded into each well of a 96-well flat bottom sterile tissue culture treated plate (Cat # 3596, Corning, Glendale, AZ) in RPMI 1640 (Cat # A1049101, Thermo Fisher) containing 10% Fetal Bovine Serum (Cat # F2442, MilliporeSigma, Burlington, MA).

[0432] Immunostimulatory oligonucleotides (ISO) agonists for toll like receptor 9 (TLR9) and RSOs comprising TLR9 immunostimulatory oligonucleotide (ISO) as the functional oligonucleotide can be synthesized using standard methodologies and provided as lyophilized preparations, which can be reconstituted in annealing buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 7.5) using a heat block incubation step (95°C, 5 minutes). Following annealing, sample concentrations can be determined using the absorbance method for oligonucleotide concentration determination using a NanoDrop spectrophotometer (Thermo Fisher). Control TLR9 agonists can be. Doses of RSOs or control agonists can be added to indicated wells, and cells can be stimulated for 24 hours in a 37°C, 5% CO2 incubator. Following the incubation period, cell culture supernatants can be harvested and analyzed using a custom multiplex cytokine / chemokine assay kit (U-PLEX custom biomarker assay, Cat # K15069M-2, Meso Scale Diagnostics, Rockville, MD) with data collection performed on a MSD S600 bioanalyzer (Meso Scale) and raw data analysis performed through MSD Discovery Workbench (Meso Scale).

[0433] To evaluate the activity of RSOs comprising immune antagonist oligonucleotide as the functional oligonucleotide, such RSOs can be incubated alone or with a TLR9 agonist. Cytokines can be assessed 24 hours later.

[0434] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is Claimed:

1. A ring-shaped oligonucleotide (“RSOs”) comprising a first and a second oligonucleotide, wherein the first oligonucleotide comprises a functional oligonucleotide and the second oligonucleotide comprises a anchor oligonucleotide, wherein the anchor oligonucleotide comprises a 5’ region and a 3’ domain, wherein the 5’ region and the 3’ region are linked, directly or through a linker segment, and wherein the 5’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 5’ end of the first oligonucleotide and the 3’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides at the 3’ end of the first oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ring-shape.

2. A RSO comprising a first and a second oligonucleotide, wherein the first oligonucleotide comprises a functional oligonucleotide and the second oligonucleotide comprises a anchor oligonucleotide, wherein the first oligonucleotide further comprises a first extension segment at the 5’ end and a second extension segment at the 3’ end, wherein the anchor oligonucleotide comprises a 5’ region and a 3’ region, wherein the 5’ region and the 3’ region are linked, directly or through a linker segment, and wherein the 5’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the first extension segment and the 3’ region of the anchor oligonucleotide is complementary to and of opposite polarity to a sequence of nucleotides in the second extension segment, wherein the first oligonucleotide and the second oligonucleotide hybridize to form two double-stranded sections, and wherein the 5’ end of the first oligonucleotide is brought into proximity with the 3’ end of the first oligonucleotide thereby creating a ring-shape.

3. The RSO according to claim 1 or 2, wherein the functional oligonucleotide is selected from an antisense oligonucleotide, a microRNA (miRNA), an siRNA, a piRNA, an hnRNA, an ncRNA, an snRNA, a miRNA mimic, an sgRNA, an esiRNA, an shRNA, a IncRNA, an mRNA, a guide RNA for a CRISPR-based system, a guide RNA for an adenosine deaminaseacting on RNA (ADAR) system, a guide RNA for prime editing, a splicing oligonucleotide, an immunostimulatory oligonucleotide or an immune-inhibitory oligonucleotide.

4. The RSO according to claim 3, wherein the functional oligonucleotide comprises an antisense oligonucleotide between 17 and 25 nucleotides in length and comprising at least 12 contiguous nucleobases complementary to an equal length portion of a target RNA sequence, wherein the antisense oligonucleotide compound comprises a 3’ domain and a 5’ domain, which is contiguous with the 3’ domain, wherein the 3’ domain begins at the terminal nucleotide at the 3’ end and is 10 to 12 nucleotides in length and wherein each nucleotide is independently any deoxyribonucleotide and wherein each internucleotide linkage between adjacent deoxyribonucleotides is a phosphodiester or phosphorothioate internucleotide linkage or combinations thereof; wherein the 5’ domain begins at the first nucleotide following the 3’ domain and continues to the terminal nucleotide at the 5’ end, wherein the 5’ domain comprises unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or combinations thereof, provided that at least 3 nucleotides of the 5’ domain comprise a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone.

5. The RSO according to claim 4, wherein the 3’ domain is 12 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the 3’ end.

6. The RSO according to claim 4, wherein the 3’ domain is 11 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 from the 3’ end.

7. The RSO according to claim 4, wherein the 3’ domain is 10 nucleotides in length and comprises nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the 3’ end.

8. The RSO according to any one of claims 4-7, wherein at least half of the nucleotides of the 5’ domain are a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone.

9. The RSO according to any one of claims 4-8, wherein all of the nucleotides of the 5’ domain are a modified deoxyribonucleotide or modified ribonucleotide comprising a modified sugar and / or backbone.

10. The RSO according to any one of claims 4-9, wherein at least half of the nucleotides of the 5’ domain, preferably wherein all of the nucleotides of the 5’ domain, comprises modified ribonucleotides, wherein the modified ribonucleotides comprise 2 ’-substituted ribonucleotides.

11. The RSO according to claim 10, wherein the 2 ’-substituted ribonucleotides are 2’- OMe ribonucleotides or 2’ -MOE ribonucleotides.

12. The RSO according to any one of claims 1-11, wherein the RSO is conjugated to a moiety that provides for site specific delivery of the RSO.

13. A pharmaceutical composition comprising a RSO according to any one of claims 1-12 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 13, further comprising one or more agents selected from a small molecule, a peptide, a vaccine, an antigen, an antibody, a cytotoxic agent, a kinase inhibitor, an allergen, an antibiotic, an siRNA molecule, an antisense oligonucleotide, a TLR antagonist, a chemotherapeutic agent, a targeted therapeutic agent, an activated cell, a protein, a gene therapy vector, a peptide vaccine, a protein vaccine, a DNA vaccine, an adjuvant, and a co-stimulatory molecule, or combinations thereof.

15. A method for inhibiting gene expression comprising administering a RSO according to any one of claims 1-12 or a composition according to claim 13 or 14.

16. A method of treating a disease or disorder in a subject wherein modulating RNA would be beneficial to treat the subject, the method comprising administering a ring-shaped oligonucleotide according to any one of claims 1-12 or a composition according to claim 13 or 14.

17. A method of treating a disease or disorder in a subject wherein inhibiting gene expression would be beneficial to treat the subject, the method comprising administering a ring-shaped oligonucleotide according to any one of claims 1-12 or a composition according to claim 13 or 14.

18. A method of treating a disease or disorder in a subject wherein inducing an immune response would be beneficial to treat the subject, the method comprising administering a ring-shaped oligonucleotide according to any one of claims 1-12 or a composition according to claim 13 or 14.

19. A method of treating a disease or disorder in a subject wherein inhibiting an immune response would be beneficial to treat the subject, the method comprising administering a ring- shaped oligonucleotide according to any one of claims 1-12 or a composition according to claim 13 or 14.

20. A method of inducing nonsense mediated decay of a target RNA comprising administering a ring-shaped oligonucleotide according to any one of claims 1-12 or a composition according to claim 13 or 14.

21. A method of increasing a level of mRNA encoding a protein or a functional mRNA and increasing expression of the protein or the functional mRNA comprising administering a ring-shaped oligonucleotide according to any one of claims 1-12 or a composition according to claim 13 or 14.

22. The method according to any one of claims 15-21, wherein the RSO is administered locally.

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